US20040110210A1

US20040110210A1 - Bacillus stearothermophilus SSB protein and use thereof

Info

Publication number: US20040110210A1
Application number: US10/673,119
Authority: US
Inventors: Michael O'Donnell; Alexander Yuzhakov; Olga Yurieva; David Jeruzalmi; Irina Bruck; John Kuriyan
Original assignee: Individual
Current assignee: Individual
Priority date: 1997-04-08
Filing date: 2003-09-26
Publication date: 2004-06-10
Also published as: US20040081995A1; US20040038289A1; US20040043415A1; US20050153299A1; US7556958B1; US20050100920A1; US20100003720A1; US20040197796A1; US20040048309A1; US20040106137A1; US20040043414A1; US6897053B1; US20050112580A1; US20040077012A1; US20040038290A1; US7803596B2; US20050048510A1

Abstract

The present invention relates to an isolated DNA molecule from a thermophilic bacterium which encodes a DNA polymerase III-type enzyme subunit. Also encompassed by the present invention are host cells and expression system including the heterologous DNA molecule of the present invention, as well as isolated replication enzyme subunits encoded by such DNA molecules. Also disclosed is a method of producing a recombinant thermostable DNA polymerase III-type enzyme, or subunit thereof, from a thermophilic bacterium, which is carried out by transforming a host cell with at least one heterologous DNA molecule of the present invention under conditions suitable for expression of the DNA polymerase III-type enzyme, or subunit thereof, and then isolating the DNA polymerase III-type enzyme, or subunit thereof.

Description

The present application is a continuation of U.S. patent application Ser. No. 09/716,964, filed Nov. 21, 2000, which is a continuation-in-part of U.S. patent application Ser. No. 09/642,218, filed Aug. 18, 2000, as a continuation of U.S. patent application Ser. No. 09/057,416 filed Apr. 8, 1998, which claims the benefit of U.S. Provisional Patent Application Serial No. 60/043,202 filed Apr. 8, 1997, all of which are hereby incorporated by reference in their entirety.[0001]
[0002] The present invention was made with funding from National Institutes of Health Grant No. GM38839. The United States Government may have certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to thermostable DNA polymerases and, more particularly, to such polymerases as can serve as chromosomal replicases and are derived from thermophilic bacteria. More particularly, the invention extends to DNA polymerase III-type enzymes from thermophilic bacteria, including Aquifex aeolicus, Thermus thermophilus, Thermotoga maritima, and Bacillus stearothermophilus, as well as purified, recombinant or non-recombinant subunits thereof and their use, and to isolated DNA coding for such polymerases and their subunits. Such DNA is obtained from the respective genes (e.g., dnaX, holA, holB, dnaA, dnaN, dnaQ, dnaE, ssb, etc.) of various thermophilic eubacteria, including but not limited to Thermus thermophilus, Aquifex aeolicus, Thermotoga maritima, and Bacillus stearothermophilus.

BACKGROUND OF THE INVENTION

Thermostable DNA polymerases have been disclosed previously as set forth in U.S. Pat. No. 5,192,674 to Oshima et al., U.S. Pat. Nos. 5,322,785 and 5,352,778 to Comb et al., U.S. Pat. No. 5,545,552 to Mathur, and others. All of the noted references recite the use of polymerases as important catalytic tools in the practice of molecular cloning techniques such as polymerase chain reaction (PCR). Each of the references states that a drawback of the extant polymerases are their limited thermostability, and consequent useful life in the participation in PCR. Such limitations also manifest themselves in the inability to obtain extended lengths of nucleotides, and in the instance of Taq polymerase, the lack of 3′ to 5′ exonuclease activity, and the drawback of the inability to excise misinserted nucleotides (Periino, 1990).

More generally, such polymerases, including those disclosed in the referenced patents, are of the Polymerase I variety as they are often 90-95 kDa in size and may have 5′ to 3′ exonuclease activity. They define a single subunit with concomitant limits on their ability to hasten the amplification process and to promote the rapid preparation of longer strands of DNA.

Chromosomal replicases are composed of several subunits in all organisms (Komberg and Baker, 1992). In keeping with the need to replicate long chromosomes, replicases are rapid and highly processive multiprotein machines. Cellular replicases are classically comprised of three components: a clamp, a clamp loader, and the DNA polymerase (reviewed in Kelman and O'Donnell, 1995; McHenmy, 1991). For purposes of the present invention, the foregoing components also serve as a broad definition of a “Pol III-type enzyme”.

DNA polymerase III holoenzyme (Pol III holoenzyme) is the multi-subunit replicase of the E. coli chromosome. Pol III holoenzyme is distinguished from Pol I type DNA polymerases by its high processivity (>50 kbp) and rapid rate of synthesis (750 nts/s) (reviewed in Kornberg and Baker, 1992; Kelman and O'Donnell, 1995). The high processivity and speed is rooted in a ring shaped subunit, called β, that encircles DNA and slides along it while tethering the Pol III holoenzyme to the template (Stukenberg et al., 1991; Kong et al., 1992). The ring shaped, clamp is assembled around DNA by the multisubunit clamp loader, called γ complex. The γ complex couples the energy of ATP hydrolysis to the assembly of the β clamp onto DNA. This γ complex, which functions as a clamp loader, is an integral component of the Pol III holoenzyme particle. A brief overview of the organization of subunits within the holoenzyme and their function follows.

Pol III holoenzyme consists of 10 different subunits, some of which are present in multiple copies for a total of 18 polypeptide chains (Onrust et al., 1995). The organization of these subunits in the holoenzyme particle is illustrated in FIG. 1. As depicted in the diagram, the subunits of the holoenzyme can be grouped functionally into three components: 1) the DNA polymerase III core is the catalytic unit and consists of the a (DNA polymerase), ε (3′-5′ exonuclease), and θ subunits (McHenry and Crow, 1979), 2) the δ “sliding clamp” is the ring shaped protein that secures the core polymerase to DNA for processivity (Kong et al., 1992), and 3) the 5 protein γ complex (γδδ′χψ) is the “clamp loader” that couples ATP hydrolysis to assembly of β clamps around DNA (O'Donnell, 1987; Maki et al., 1988). A dimer of the τ subunit acts as a “macromolecular organizer” holding together two molecules of core (Studwell-Vaughan and O'Donnell, 1991; Low et al., 1976) and one molecule of γ complex forming the Pol III* subassembly (Onrust et al., 1995). This organizing role of τ to form Pol III* is indicated in the center of FIG. 1. Two β dimers associate with the two cores within Pol III* to form the holoenzyme, which is capable of replicating both strands of duplex DNA simultaneously (Maki et al., 1988).

The DNA polymerase III holoenzyme assembles onto a primed template in two distinct steps. In the first step, the γ complex assembles the β clamp onto the DNA. The γ complex and the core polymerase utilize the same surface of the β ring and they cannot both utilize it at the same time (Naktinis et al., 1996). Hence, in the second step the γ complex moves away from β thus allowing access of the core polymerase to the β clamp for processive DNA synthesis. The γ complex and core remain attached to each other during this switching process by the τ subunit organizer.

The γ complex consists of 5 different subunits (γ _2-4δ₁δ′₁χ₁ψ₁). An overview of the mechanism of the clamp loading process follows. The δ subunit is the major touch point to the β clamp and leads to ring opening, but 8 is buried within γ complex such that contact with β is prevented (Naktinis et al., 1995). The γ subunit is the ATP interactive protein but is not an ATPase by itself (Tsuchihashi and Kornberg, 1989). The δ′ subunit bridges the δ and γ subunits resulting in a γδδ′ complex that exhibits DNA dependent ATPase activity and is competent to assemble clamps on DNA (Onrust et al., 1991). Upon binding of ATP to γ, a change in the conformation of the complex exposes δ for interaction with β (Naktinis et al., 1995).

The function of the smaller subunits, χ and ψ, is to contact SSB (through χ) thus promoting clamp assembly and high processivity during replication (Kelman and O'Donnell, 1995).

The three component Pol III-type enzyme in eukaryotes contains a clamp that has the same shape as E. coli β, but instead of a homodimer it is a heterotrimer. This heterotrimeric ring, called PCNA (proliferating cell nuclear antigen), has 6 domains like β, but instead of each PCNA monomer being composed of 3 domains and dimerizing to form a 6 domain ring (e.g., like β), the PCNA monomer has 2 domains and it trimerizes to form a 6 domain ring (Krishna et al., 1994; Kuriyan and O'Donnell, 1993). The chain fold of the domains are the same in prokaryotes (β) and eukaryotes (PCNA); thus, the rings have the same overall 6-domain ring shape. The clamp loader of the eukaryotic Pol III-type replicase is called RFC (Replication factor C) and it consists of subunits having homology to the γ and δ′ subunits of the E. coli γ complex (Cullmann et al., 1995). The eukaryotic DNA polymerase III-type enzyme contains either of two DNA polymerases, DNA polymerase δ and DNA polymerase ε (Bambara and Jessee, 1991; Linn, 1991; Sugino, 1995). It is entirely conceivable that yet other types of DNA polymerases can function with either a PCNA or β clamp to form a Pol III-type enzyme (for example, DNA polymerase II of E. coli functions with the β subunit placed onto DNA by the γ complex clamp loader) (Hughes et al., 1991; Bonner et al., 1992). The bacteriophage, T4 also utilizes a Pol III-type 3-component replicase. The clamp is a homotrimer like PCNA, called gene 45 protein (Young et al., 1992). The gene 45 protein forms the same 6-domain ring structure as β and PCNA (Moarefi et al., 2000). The clamp loader is a complex of two subunits called the gene 44/62 protein complex. The DNA polymerase is the gene 43 protein and it is stimulated by the gene 45 sliding clamp when it is assembled onto DNA by the 44/62 protein clamp loader. The Pol III-type enzyme may be either bound together into one particle (e.g., E. coli Pol III holoenzyme), or its three components may function separately (like the eukaryotic Pol III-type replicases).

There is an early report on separation of three DNA polymerases from T.th. cells, however each polymerase form was reminiscent of the preexisting types of DNA polymerase isolated from thermophiles in that each polymerase was in the 110,000-0.120,000 range and lacked 3′-5′ exonuclease activity (Ruttimann et al., 1985). These are well below the molecular weight of Pol III-type complexes that contain in addition to the DNA polymerase subunit, other subunits such as γ and τ. Although the three polymerases displayed some differences in activity (column elution behavior, and optimum divalent cation, template, and temperatures) it seems likely that these three forms were either different repair type polymerases or derivatives of one repair enzyme (e.g., Pol I) that was modified by post translational modification(s) that altered their properties (e.g. phosphorylation, methylation, proteolytic clipping of residues that alter activity, or association with different ligands such as a small protein or contaminating DNA). Despite this previous work, it remained to be demonstrated that thermophiles harbor a Pol III-type enzyme that contain multiple subunits such as γ and/or τ, functioned with a sliding clamp accessory protein, or could extend a primer rapidly and processively over a long stretch (>5 kb) of ssDNA (Ruttimann et al., 1985).

Previously, it was not known what polymerase thermophilic bacteria used to replicate their chromosome since only Pol I type enzymes have been reported from thermophiles. By distinctions chromosomal replicases, such as Polymerase III, identified in E. coli, if available in a thermostable bacterium, with all its accessory subunits, could provide a great improvement over the Polymerase I type enzymes, in that they are generally much more efficient—about 5 times faster—and much more highly processive. Hence, one may expect faster and longer chain production in PCR, and higher quality of DNA sequencing ladders. Clearly, the ability to practice such synthetic techniques as PCR would be enhanced by these methods disclosed for how to obtain genes and subunits of DNA polymerase III holoenzyme from thermophilic sources.

The present invention is directed to achieving these objectives and overcoming the various deficiencies in the art.

SUMMARY OF THE INVENTION

In accordance with the present invention, DNA Polymerase III-type enzymes as defined herein are disclosed that may be isolated and purified from a thermophilic bacterial source, that display rapid synthesis characteristic of a chromosomal replicase, and that possesses all of the structural and processive advantages sought and recited above. More particularly, the invention extends to thermostable Polymerase III-type enzymes derived from thermophilic bacteria that exhibit the ability to extend a primer over a long stretch (>5 kb) of ssDNA at elevated temperature, the ability to be stimulated by a cognate sliding clamp (e.g., β) of the type that is assembled on DNA by a ‘clamp’ loader. (e.g., γ complex), and have clamp loading subunits that show DNA stimulated ATPase activity at elevated temperature and/or ionic strength. Representative thermophile polymerases include those isolated from the thermophilic eubacteria Aquifex aeolicus ( A.ae. polymerase) and other members of the Aquifex genus; Thermus thermophilus (T.th. polymerase), Thermus favus (Tfl/Tub polymerase), Thermus ruber (Tru polymerase), Thermus brockianus (DYNAZYME™ polymerase), and other members of the Thermus genus; Bacillus stearothermophilus (B.st. polymerase) and other members of the Bacillus genus; Thermoplasma acidophilum (Tac polymerase) and other members of the Thermoplasma genus; and Thermotoga neapolitana (Tne polymerase; see WO 96/10640 to Chattedjee et al.), Thermotoga maritima (Tma polymerase; see U.S. Pat. No. 5,374,553 to Gelfand et al.), and other species of the Thermotoga genus (Tsp polymerase). In a preferred embodiment, the thermophilic bacteria comprise species of Aquifex, Thermus, Bacillus, and Thermotoga, and particularly A.ae., T.th., B.st., and Tma.

A particular Polymerase III-type enzyme in accordance with the invention may include at least one of the following sub-units:

A. a γ subunit having an amino acid sequence corresponding to SEQ. ID. Nos. 4 or 5 ( T.th.);

B. a τ subunit having an amino acid sequence corresponding to SEQ. ID. No. 2 ( T.th.), SEQ. ID. No. 120 (A.ae.), SEQ. ID. No. 142 (T.ma.) or SEQ. ID. No. 182 (B.st.);

C. a ε subunit having an amino acid sequence corresponding to SEQ. ID. No. 95 ( T.th.), SEQ. ID. No. 128 (A.ae.), or SEQ. ID. No. 140 (T.ma.);

D. a α subunit including an amino acid sequence corresponding to SEQ. ID. No. 87 ( T.th.), SEQ. ID. No. 118 (A.ae.), SEQ. ID. No. 138 (T.ma.), or SEQ. ID. Nos. 184 (POIC which has both α and ε activity, B.st.);

E. a β subunit having an amino acid sequence corresponding to SEQ. ID. No. 107 ( T.th.), SEQ. ID. No. 122 (A.ae.), SEQ. ID. No. 144 (T.ma.), or SEQ. ID. No. 174 (B.st.);

F. a δ subunit having an amino acid sequence corresponding to SEQ. ID. No. 158 ( T.th.), SEQ. ID. No. 124 (A.ae.), SEQ. ID. No. 146 (T.ma) or SEQ. ID. No. 178 (B.st.);

G. a δ′ subunit having an amino acid sequence corresponding to SEQ. ID. No. 156 ( T.th.), SEQ. ID. No. 126 (A.ae.), SEQ. ID. No. 148 (T.ma.) or SEQ. ID. No. 180 (B.st.);

variants, including allelic variants, muteins, analogs and fragments of any of subparts (A) through (G), and compatible combinations thereof, capable of functioning in DNA amplification and sequencing.

The invention also extends to the genes that correspond to and can code on expression for the subunits set forth above, and accordingly includes the following: dnax, holA, holB, dnaQ, dnaE, dnaN, and ssb, as well as conserved variants and active fragments thereof.

Accordingly, the Polymerase III-type enzyme of the present invention comprises at least one gene encoding a subunit thereof, which gene is selected from the group consisting of dnax, holA, holb, dnaQ, dnaE and dnaN, and combinations thereof. More particularly, the invention extends to the nucleic acid molecule encoding the γ and τ subunits, and includes the dnaX gene which has a nucleotide sequence as set forth herein, as well as conserved variants, active fragments and analogs thereof. Likewise, the nucleotide sequences encoding the α subunit (dnaE gene), the ε subunit (dnaQ gene), the β subunit (dnaN gene), the δ subunit (holA gene), and the δ′ subunit (holb gene) each comprise the nucleotide sequences as set forth herein, as well as conserved variants, active fragments and analogs thereof. Those nucleotide sequences for T.th. are as follows: dnaX (SEQ. ID. No. 3), dnaE (SEQ. ID. No. 86), dnaQ (SEQ. ID. No. 94), dnaN. SEQ. ID. No. 106), hola (SEQ. ID. No. 157), and holB (SEQ. ID. No. 155). Those nucleotide sequences for A.ae. are as follows: dnaX(SEQ. ID. No. 119), dnaE (SEQ. ID. No. 117), dnaQ (SEQ. ID. No. 127), dnaN (SEQ. ID. No. 121), hola (SEQ. ID. No. 123), and holB (SEQ. ID. No. 125). Those nucleotide sequences for T.ma. are as follows: dnaX (SEQ. ID. No. 141), dnaE (SEQ. ID. No. 137) dnaQ (SEQ. ID. No. 139), dnaN (SEQ. ID. No. 143), holA (SEQ. ID. No. 145), and holb (SEQ. ID. No. 147). Those nucleotide sequences for B.st. are as follows: dnaX (SEQ. ID. No. 181), polC (SEQ. ID. Nos. 183), dnaN (SEQ. ID. No. 173), holA (SEQ. ID. No. 177), and holb (SEQ. ID. No. 179).

The invention also provides methods and products for identifying, isolating and cloning DNA molecules which encode such accessory subunits encoded by the recited genes of the DNA polymerase III-type enzyme hereof.

Yet further, the invention extends to Polymerase III-type enzymes prepared by the purification of an extract taken from, e.g., the particular thermophile under examination, treated with appropriate solvents and then subjected to chromatographic separation on, e.g., an anion exchange column, followed by analysis of long chain synthetic ability or Western analysis of the respective peaks against antibody to at least one of the anticipated enzyme subunits to confirm presence of Pol III, and thereafter, peptide sequencing of subunits that co purify and amplification to obtain the putative gene and its encoded enzyme.

The present invention also relates to recombinant γ, τ, ε, α (as well as POlC), δ, δ′ and β subunits and SSB from thermophiles. In the instance of the γ and τ subunits of T.th., the invention includes the characterization of a frame-shifting sequence that is internal to the gene and specifies relative abundance of the γ and τ gene products of T.th. dnaX. From this characterization, expression of either one of the subunits can be increased at the expense of the other (i.e. mutant frameshift could make all τ, simple recloning at the end of the frameshift could make exclusively γ and no τ).

In a further aspect of the present invention, DNA probes can be constructed from the DNA sequences coding for, e.g., the T.th., A.ae., T.ma., or B.st. dnax, dnaQ, dnaE, dnaA, dnaN, hola, holB, and ssb genes, conserved variants and active fragments thereof, all as defined herein, and may be used to identify and isolate the corresponding genes coding for the subunits of DNA polymerase III holoenzyme from other thermophiles, such as those listed earlier herein. Accordingly, all chromosomal replicases (DNA Polymerase III-type) from thermophilic sources are contemplated and included herein.

The invention also extends to methods for identifying Polymerase III-type enzymes by use of the techniques of long-chain extension and elucidation of subunits with antibodies, as described herein and with reference to the examples.

The invention further extends to the isolated and purified DNA Polymerase III from T.th., A.ae., Tma., and B.st., the amino acid sequences of the γ, τ, ε, α (as well as PolC), δ, δ′, and β subunits and SSB, as set forth herein, and the nucleotide sequences of the corresponding genes from T.th., A.ae., T.ma., or B.st. set forth herein, as well as to active fragments thereof, oligonucleotides and probes prepared or derived therefrom and the transformed cells that may be likewise prepared. Accordingly, the invention comprises the individual subunits enumerated above and hereinafter, corresponding isolated polynucleotides and respective amino acid sequences for each of the γ, τ, ε, α (as well as POIC), δ, δ′, and β subunits and SSB, and to conserved variants, fragments, and the like, as well as to methods of their preparation and use in DNA amplification and sequencing. In a particular embodiment, the invention extends to vectors for the expression of the subunit genes 110 of the present invention.

The invention also includes methods for the preparation of the DNA Polymerase III-type enzymes and the corresponding subunit genes of the present invention, and to the use of the enzymes and constructs having active fragments thereof, in the preparation, reconstitution or modification of like enzymes, as well as in amplification and sequencing of DNA by methods such as PCR, and like protocols, and to the DNA molecules amplified and sequenced by such methods. In this regard, a Pol III-type enzyme that is reconstituted in the absence of ε, or using a mutated c with less 3′-5′ exonuclease activity, may be a superior enzyme in either PCR or DNA sequencing applications, (e.g. Tabor et, al., 1995).

The invention is directed to methods for amplifying and sequencing a DNA molecule, particularly via the polymerase chain reaction (PCR), using the present DNA polymerase III-type enzymes or complexes. In particular, the invention extends to methods of amplifying and sequencing of DNA using thermostable pol III-type enzyme complexes isolated from thermophilic bacteria such as Thermotoga and Thermus species, or recombinant thermostable enzymes. The invention also provides amplified DNA molecules made by the methods of the invention, and kits for amplifying or sequencing a DNA molecule by the methods of the invention.

In this connection, the invention extends to methods for amplification of DNA that can achieve long chain extension of primed DNA, as by the application and use of Polymerase III-type enzymes of the present invention. An illustration of such methods is presented in Examples 15 and 16, infra.

Likewise, kits for amplification and sequencing of such DNA molecules are included, which kits contain the enzymes of the present invention, including subunits thereof, together with other necessary or desirable reagents and materials, and directions for use. The details of the practice of the invention as set forth above and later on herein, and with reference to the patents and literature cited herein, are all expressly incorporated herein by reference and made a part hereof. As stated, and in accordance with a principal object of the present invention, Polymerase III-type enzymes and their sub-units are provided that are derived from thermophiles and that are adapted to participate in improved DNA amplification and sequencing techniques, and the consequent ability to prepare larger DNA strands more rapidly and accurately.

It is a further object of the present invention to provide DNA molecules that are amplified and sequenced using the Polymerase III-type enzymes hereof.

It is a still further object of the present invention to provide enzymes and corresponding methods for amplification and sequencing of DNA that can be practiced without the participation of the clamp-loading component of the enzyme.

It is a still further object of the present invention to provide kits and other assemblies of materials for the practice of the methods of amplification and sequencing as aforesaid, that include and use the DNA polymerase III-type enzymes herein as part thereof.

One goal of this invention is to fully reconstitute the rapid and processive replicase from an extreme thermophilic eubacterium from fully recombinant protein subunits. One might think that the extreme heat in which these bacteria grow may have resulted in a completely different solution to the problem of chromosome replication. Prior to filing of the previously-identified priority applications, it is believed that Pol III had not been identified in any thermophile until the present inventors found that Thermus thermophilus, which grows at a rather high temperature of 70-80° C., would appear to contain a Pol III. Subsequent to this invention, the genome sequence of A. aeolicus was published which shows dnaE, dnaN, and dnaX genes. However, previous work did not fully reconstitute the working replication machinery from fully recombinant subunits. A holA gene and holB has not been identified previously in T. thermophilus or A. aeolicus, and studies in the E. coli system show that delta and delta prime, encoded by holA and holB, respectively, are essential to loading the beta clamp onto DNA and, thus, is essential for rapid and processive holoenzyme function (U.S. Pat. Nos. 5,583,026 and 5,668,004 to O'Donnell, which are hereby incorporated by reference).

This invention fully reconstitutes a functional DNA polymerase III holoenzyme from the extreme thermophiles Thermus thermophilus and Aquifex aeolicus. Aquifex aeolicus grows at an even higher temperature than Thermus thermophilus, up to 85° C. In this invention, the genes of Thermus thermophilus, Aquifex aeolicus, Thermotoga maritima, and Bacillus stearothermophilus that are necessary to reconstitute the complete DNA polymerase III machinery, which acts as a rapid and processive polymerase, are identified. Indeed, a deltaprime (holB) and delta (holA) subunits are needed.

The dnaE, dnaN, dnax, dnaQ, holA, and holB genes are used to express and purify the protein “gears”, and the proteins are used to reassemble the replication machine. The T.th. Pol III is similar to E. coli. The A.ae. Pol III is slightly dissimilar from the machinery of previously studied replicases. The A.ae. dnaX gene encoded only one protein, tau, and in this fashion is similar to the dnaX of the gram positive organism, Staphylococcus aureus. In contrast, the dnaX of the gram negative cell, E. coli, produces two proteins. The Aquifex aeolicus polymerase subunit, alpha (encoded by dnaE) does not contain the 3′-5′ proofreading exonuclease. In this regard, A. aeolicus is similar to E. coli, but dissimilar to the replicase of the gram positive organisms. In Gram positive organisms, the PolC polymerase subunit of the replicase contains the exonuclease activity in the same polypeptide chain as the polymerase (Low et al., 1976, Bames et al., 1994; Pacitti et al., 1995). Further, the polymerase III of thermophilic bacteria retains activity at high temperature.

Thermostable rapid and processive three component DNA polymerases can be applied to several important uses. DNA polymerases currently in use for DNA sequencing and DNA amplification use enzymes that are much slower and thus could be improved upon. This is especially true of amplification as the three component polymerase is capable of speed and high processivity making possible amplification of very long (tens of Kb to Mb) lengths of DNA in a time-efficient, manner. These three component polymerases also function in conjunction with a replicative helicase (DnaB), and thus are capable of amplification at a single temperature, using the helicase to melt the DNA duplex. This property could be useful in some methods of amplification, and in polymerase chain reaction (PCR) methodology. For example, the ατδδ′/β form of the E. coli DNA polymerase III holoenzyme has been shown to function in both DNA sequencing and PCR (U.S. Pat. Nos. 5,583,026 and 5,668,004 to O'Donnell).

Other objects and advantages will become apparent from a review of the ensuing description which proceeds with reference to the following illustrative drawings.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depiction of the structure and components of enzymes of the general family to which the enzymes of the present invention belong. [0046]
FIG. 2 is an alignment of the N-terminal regions of [0047] E. coli (SEQ. ID. No. 19) and B. subtilis (SEQ. ID. No. 20) dnaX gene product. Asterisks indicate identities. The ATP binding consensus sequence is indicated. The two regions used for PCR primer design are shown in bold.
FIG. 3 is an image showing the Southern analysis of [0048] T. thermophilus genomic DNA. Genomic DNA was analyzed for presence of the dnaZ gene using the PCR radiolabeled probe. Enzymes used for digestion are shown above each lane. The numbering to the right corresponds to the length of DNA fragments (kb).
FIGS. 4A and 4B depict the full sequence of the dnaX gene of [0049] T. thermophilus. DNA sequence (upper case, and corresponding to SEQ ID No. 1) and predicted amino acid sequence (lower case, and corresponding to SEQ ID No. 2) yields a 529 amino acid protein (r) of 58.0 kDa. A putative frameshifting sequence containing several A residues 1478-1486 (underlined) may produce a smaller protein (τ) of 49.8 kDa. The potential Shine-Dalgarno (S.D.) signal is bold and underlined. The start codon is in bold, and the stop codon for τ is marked by an asterisk. The potential stop codon for γ is shown in bold after the frameshift site, and two potential Shine-Dalgarno sequences upstream of the frameshift site are indicated. Sequences of the primers used for PCR are shown in italics above the nucleotide sequence of dnax. The ATP binding site is indicated, and the asterisks above the four Cys residues near the ATP site indicate the putative Zn²⁺ finger. The proline rich area is indicated above the sequence. Numbering of the nucleotide sequence is presented to the right. Numbering of the amino acid sequence of τ is shown in parenthesis to the right.
FIG. 4C depicts the isolated DNA coding sequence for the dnax gene (also present in FIGS. 3A and 3B) in accordance with the invention, which corresponds to SEQ. ID. No. 3. [0050]
FIG. 4D depicts the polypeptide sequence of the γ subunit of the Polymerase III of the present invention, which corresponds to SEQ. ID. No. 4. [0051]
FIG. 4E depicts the polypeptide sequence of the γ subunit of the Polymerase III of the present invention defined by a −1 frameshift, which corresponds to SEQ. ID. No. 4. [0052]
FIG. 4F depicts the polypeptide sequence of the γ subunit of the Polymerase III of the present invention defined by a −2 frameshift, which corresponds to SEQ. ID. No. 5. [0053]
FIGS. [0054] 5A-B are alignments of the γ/τ ATP binding domains for different bacteria. Dots indicate those residues that are identical to the E. coli dnax sequence. The ATP consensus site is underlined, and the conserved cysteine residues that form the zinc finger are indicated with asterisks. E. coli, Escherichia coli (SEQ. ID. No. 21); H. inf., Haemophilus influenzae (SEQ. ID. No. 22); B. sub., Bacillus subtilis (SEQ. ID. No. 23); C. cres., Caulobacter crescentus (SEQ. ID. No. 24), M gen., Mycoplasma genitalium (SEQ. ID. No. 25); T.th., Thermus thermophilus (SEQ. ID. No. 26). Alignments were produced using Clustal.
FIG. 6 is a diagram indicating a signal for ribosomal franeshifting in [0055] T.th. dnaX. The diagram shows part of the sequence of the RNA (SEQ. ID. No. 27) around the frameshifting site (SEQ. ID. No. 28), including the suspected slippery sequence A9 (bold italic). The stop codon in the −2 reading frame is indicated. Also indicated are potential step loop structures and the nearest stop codons in the −1 reading frame.
FIG. 7 is an image showing a Western analysis of γ and τ in [0056] T.th. cells. Whole cells were lysed in SDS and electrophoresed on a 10% SDS polyacrylamide gel then transferred to a membrane and probed with polyclonal antibody against E. coli γ/τ as described in Experimental Procedures. Positions of molecular weight size markers are shown to the left. Putative T.th. γ and τ are indicated to the right.
FIGS. [0057] 8A-B are images of E. coli colonies expressing T.th. dnaX −1 and −2 frameshifts. The region of the dnaX gene slippery sequence was cloned into the lacz gene of pUC19 in three reading frames, then transformed into E. coli cells and plated on LB plates containing X-gal. The slippery sequence was also mutated by inserting two G residues into the A9 sequence and then cloned into pUC19 in all three reading frames. Color of colonies observed are indicated by the plus signs. The picture shows the colonies, the type of frameshift required for readthrough (blue color) is indicted next to the sector.
FIG. 9 shows the construction of the [0058] T.th. γ/τ expression vector. A genomic fragment containing a partial sequence of dnaX was cloned into pALTER-1. This fragment was subcloned into pUC19 (pUC19_dnaX). Then the N-terminal section of dnaX was amplified such that the fragment was flanked by NdeI (at the initiating codon) and the internal BamHI site. This fragment was inserted to form the entire coding sequence of the dnaX gene in pUC19 (pUC19dnaX). The dnaX gene was then cloned behind the polyhistidine leader in the T7 based expression vector pET16 to give pET16dnaX. Details are in “Experimental Procedures”.
FIGS. [0059] 10A-C illustrate the purification of recombinant T.th. γ and τ subunits. T.th. γ and τ subunits were expressed in E. coli harboring pET 16dnaX. Molecular size markers are shown to the left of the gels, and the two induced proteins are labeled as g and t to the right of the gel. Panel A) 10% SDS gel of E. coli whole cell lysates before and after induction with IPTG. Panel B) 8% SDS gel of the purification two steps after cell lysis. First lane: the lysate was applied to a HiTrap Nickel chromatography column. Second lane: the T.th. γ/τ subunits were further purified on a Superose 12 gel filtration column. Third lane, the E. coli γ and τ subunits. Panel C) Western analysis of the pure T.th. γ and τ subunits (first lane) and E. coli γ and τ subunits (second lane).
FIGS. [0060] 11A-B show the gel filtration of T.th. γ and γ. T.th. γ and τ were gel filtered on a Superose 12 column. Column fractions were analyzed for ATPase activity and in a Coomassie Blue stained 10% SDS polyacrylamide gel. Positions of molecular weight markers are shown to the left of the gel. The elution position of size standards analyzed in a parallel Superose 12 column under identical conditions are indicated above the gel. Thyroglobin (670 kDa), bovine gamma globin (150 kDa), chicken ovalbumin (44 kDa), equine myoglobin (17 kDa).
FIGS. [0061] 12A-C illustrate the characterization of the T.th. γ and τ ATPase activity. The T.th. γ/τ and E. coli τ subunits are compared in their ATPase activity characteristics. Due to the greater activity of E. coli τ, the values are plotted as percent for ease of comparison. Actual specific activities for 100% values are given below as pmol ATP hydrolyzed/30 min./μmol T.th. γ/τ (or pmol E. coli τ). Panel A) T.th. γ and τ ATPase is stimulated by the presence of ssDNA. T.th. γ/τ was incubated at 65° C. Specific activity was: 11.5 (+DNA); 2.5 (−DNA); E. coli τ was assayed at 37° C. Specific activity values were: 112.5 (+DNA); (7.3−DNA). Panel B) Temperature stability of DNA stimulated ATPase activity. T.th. γ/τ, 11.3 (65° C.); E. coli τ, 97.5 (37° C.). Panel C) Stability of T.th. γ/τ ATPase to NaCl. T.th. γ/τ, 8.1 (100 mM added NaCl and 65° C.); E. coli τ, 52.7 (0 M added NaCl and 37° C.).
FIGS. [0062] 13A-13C are graphs that summarize the purification of the DNA polymerase III from T.th. extracts. Panel A) shows the activity and total protein in column fractions from the Heparin Agarose column. Peak 1 fractions were chromatographed on ATP agarose. Panel B) depicts the ATP-agarose column step, and Panel C) shows the total protein and DNA polymerase activity eluted from the MonoQ column.
FIGS. [0063] 14A-B are SDS polyacrylamide gels of T.th. subunits. FIG. 14A is a 12% SDS polyacrylamide gel stained with Coomassie Blue of the MonoQ column. Load stands for the material loaded onto the column (ATP agarose bound fractions). FT stands for protein that flowed through the MonoQ column. Fractions are indicated above the gel. T.th. subunits in fractions 17-19 are indicated by the labels placed between fractions 18 and 19. Additional small subunits may be present but difficult to visualize, or may have run off the gel. E. coli γ,δ shows a mixture of the α, γ, and δ subunits of DNA polymerase III holoenzyme (they are labeled to the right in the figure). FIG. 14B shows the Western results of an SDS gel of the MonoQ fractions probed with rabbit antiserum raised against the E. coli α subunit. Load and FT are as described in Panel A. Fraction numbers are shown above the gel. The band that comigrates with E. coli α, and the band in the Coomassie Blue stained gel in Panel A, is marked with an arrow. This band was analyzed for microsequence and the results are shown in FIG. 15.
FIGS. [0064] 15A-B show the alignments of the peptides obtained from T.th. α subunit, TTH1 (shown in A) and TTH2 (shown in B) with the amino acid sequences of the α subunits of other organisms. The amino acid number of these regions within each respective protein sequence are shown to the right. The abbreviations of the organisms are as follows. E. coli-Escherichia coli (SEQ ID NOS: 72 and 79 in 15A-B, respectively), V.chol.-Vibrio cholerae (SEQ ID NOS: 73 and 80 in 15A-B, respectively), H.inf-Haemophilus influenzae (SEQ ID NOS: 74 and 81 in 15A-B, respectively), R.prow.-Rickettsia prowazekii (SEQ ID NOS: 75 and 82 in 15A-B, respectively), H.pyl.-Helicobacter pylori (SEQ ID NOS: 76 and 83 in 15A-B, respectively), S.sp. -Synechocystis sp. (SEQ ID NOS: 77 and 84 in 15A-B, respectively), M.tub. -Mycobacterium tuberculosis (SEQ ID NOS: 78 and 85 in 15A-B, respectively), T.th. Thermus thermophilus (SEQ ID NOS: 61 and 60 in 15A-B, respectively).
FIGS. [0065] 16A-C show a nucleotide (Panels A-B, SEQ. ID. No. 86) and amino acid (Panel C, SEQ. ID. No. 87) sequence of the dnaE gene encoding the α subunit of DNA polymerase III replication enzyme.
FIG. 17 shows an alignment of the amino acid sequence of ε subunits encoded by dnaQ of several organisms. The amino acid sequence of the [0066] Thermus thermophilus ε subunit of dnaQ is also shown. T.th., Thermus thermophilus (SEQ. ID. No. 88); D.rad., Deinococcus radiodurans (SEQ. ID. No. 89); Bac.sub., Bacillus subtilis (SEQ. ID. No. 90); H.inf., Haemophilus influenzae (SEQ. ID. No. 91); E.c., Escherichia coli (SEQ. ID. No. 92); H.pyl., Helicobacter pylori (SEQ. ID. No. 93). The regions used to obtain the inner part of the dnaQ gene are shown in bold. The starts used for expression of the T.th. ε subunit are marked.
FIGS. [0067] 18A-B show the nucleotide (Panel A, SEQ. ID. No. 94) and amino acid (Panel B, SEQ. ID. No. 95) sequence of the dnaQ gene encoding the ε subunit of DNA polymerase III replication enzyme.
FIGS. [0068] 19A-B show an alignment of the DnaA protein of several organisms. The amino acid sequence of the Thermus thermophilus DnaA protein is also shown. P.mar., Pseudomonas marcesans (SEQ. ID. No. 96); Syn.sp., Synechocystis sp. (SEQ. ID. No. 97); Bac.sub., Bacillus subtilis (SEQ. ID. No. 98); M. tub; Mycobacterium tuberculosis (SEQ. ID. No. 99); Tth., Thermus thermophilus (SEQ. ID. No. 100); E. coli., Escherichia coli (SEQ. ID. No. 101); T. mar., Thermatoga maritima (SEQ. ID. No. 102); and H.pyl., Helicobacter pylori (SEQ. ID. No. 103).
FIGS. [0069] 20A-B show the nucleotide (Panel A, SEQ. ID. No. 104) and amino acid (Panel B, SEQ. ID. No. 105) sequence of the dnaA gene of Thermus thermophilus.
FIGS. [0070] 21A-B show the nucleotide (Panel A, SEQ. ID. No. 106) and amino acid (Panel B, SEQ. ID. No. 107) sequence of the dnaN gene encoding the p subunit of DNA polymerase III replication enzyme.
FIGS. [0071] 22A-B show an alignment of the β subunit of T.th. to the B3 subunits of other organisms. T.th.; Thermus thermophilus (SEQ. ID. No. 108); E. coli, Escherichia coli (SEQ. ID. No. 109); P. mirab, Proteus mirabilis (SEQ. ID. No. 110); H. infl, Haemophilus influenzae (SEQ. ID. No. 111); P. put., Pseudomonas putida (SEQ. ID. No. 112); and B. cap., Buchnera aphidicola (SEQ. ID. No. 113).
FIG. 23 is a map of the pET24:dnaN plasmid. The functional regions of the plasmid are indicated by arrows and italic, restriction sites are marked with bars and symbols. The hatched parts in the plasmid correspond to [0072] T.th. dnaN.
FIGS. [0073] 24A-B show the induction of T.th. β in E. coli cells harboring the T.th. β expression vector. Panel A is the cell induction. The first lane shows molecular weight markers (MW). The second lane shows uninduced E. coli cells, and the third lane shows induced E. coli. The induced T.th. β is, indicated by the arrow shown to the left. Induced cells were lysed then treated with heat and the soluble portion was chromatographed on MonoQ. Panel β shows the results of MonoQ purification of T.th. β.
FIG. 25A is a schematic depiction of the use of the use of the enzymes of the present invention in accordance with an alternate embodiment hereof. In this scheme the clamp (β or PCNA) slides over the end of linear DNA to enhance the polymerase (Pol III-type such as Pol III, Polβ or Polδ.) In this fashion the clamp loader activity is not needed. [0074]
FIG. 25B graphically demonstrates the results of the practice of the alternate embodiment of the invention described and set forth in Example 15, infra. [0075] Lane 1, E. coli Pol III without β; Lane 2, E. coli with 3; Lane 3, human Polδ without PCNA; Lane 4, human Polδ with PCNA; Lane 5, T.th. Pol III without T.th. β; Lane 6, T.th. Pol III with T.th. β. The respective pmol synthesis in lanes 1-6 are: 6, 35, 2, 24, 0.6 and 1.9.
FIGS. [0076] 26A-B show the use of T.th. Pol III in extending singly primed M13 mp18 to an RFII form. The scheme in FIG. 26A shows the primed template in which a DNA 57mer was annealled to the M13.mp18 ssDNA circle. Then T.th. β subunit (produced recombinantly) and T.th. Pol III were added to the DNA in the presence of radioactive nucleoside triphosphates. In FIG. 26B, the products of the reaction were analyzed in a 0.8% native agarose gel. The position of ssDNA starting material, the RFII product, and of intermediate species, are shown to the sides of the gel. Lane 1, use of Pol III. Lane 2, use of the non-Pol III DNA polymerase.
FIG. 27 is an SDS polyacrylamide gel of the proteins of the [0077] A. aeolicus replication machinery.
FIG. 28 is an SDS polyacrylamide gel analysis of the MonoQ fractions of the method used to reconstitute and purify the [0078] A. aeolicus τδδ′ complex.
FIG. 29 is an SDS polyacrylamide gel analysis of the gel filtration column fractions used in the preparation of the [0079] A. aeolicus ατδδ′ complex. The bottom gel analysis shows the profile obtained using the A. aeolicus α subunit (polymerase) in the absence of the other subunits.
FIG. 30 is an alkaline agarose gel analysis of reaction products for extension of a single primer around a 7.2 kb M13 mp18 circular ssDNA genome that has been coated with [0080] A. aeolicus SSB. The time course on the left are produced by ατδδ′/β, and the time course on the right is produced by ατδδ′ in the absence of β.
FIG. 31 is, a graph illustrating the optimal temperature for activity of the alpha subunit of Thermus replicase using a calf thymus DNA replication assay. Reactions were shifted to the indicated temperature for 5 minutes before detecting the level of DNA synthesis activity. [0081]
FIG. 32 is a graph illustrating the optimal temperature for activity of the alpha subunit of the Aquifex replicase using a calf thymus DNA replication assay. Reactions were shifted to the indicated temperature for 5 minutes before detecting the level of DNA synthesis activity. [0082]
FIGS. [0083] 33A-E illustrate the heat stability of Aquifex components. Assays of either a (FIG. 33A), β (FIG. 33B), τδδ′ complex (FIG. 33C), SSB (FIG. 33D) and ατδδ′ complex (FIG. 33E) were performed after heating samples at the indicated temperatures. Components were heated in buffer containing the following: 0.1% Triton X-100 (filled diamonds); 0.05% Tween-20 and 0.01% NP-40 (filled circles); 4 mM CaCl₂(filled triangles); 40% Glycerol (inverted filled triangles); 0.01% Triton X-100, 0.05% Tween-20, 0.01% NP-40, 4 mM CaCl₂(half-filled square); 40% Glycerol, 0.1% Triton X-100 (open diamonds); 40% Glycerol, 0.05% Tween-20, 0.01% NP-40 (open circles); 40% Glycerol, 4 mM CaCl₂(open triangles); 40% Glycerol, 0.01% Triton X-100, 0.05% Tween-20, 0.01% NP-40, 4 mM CaCl₂(half-filled diamonds).
FIGS. [0084] 34A-B show the nucleotide sequence (SEQ. ID. No. 117) of the dnaE gene of A. aeolicus.
FIG. 35 shows the amino acid sequence (SEQ. ID. No. 118) of the α subunit of [0085] A. aeolicus.
FIG. 36 shows the nucleotide sequence (SEQ. ID. No. 119) of the dnaX gene of [0086] A. aeolicus.
FIG. 37 shows the amino acid sequence (SEQ. ID. No. 120) of the tau subunit of [0087] A. aeolicus.
FIG. 38 shows the nucleotide sequence (SEQ. ID. No. 121) of the dnaN gene of [0088] A. aeolicus.
FIG. 39 shows the amino acid sequence (SEQ. ID. No. 122) of the β subunit of [0089] A. aeolicus.
FIG. 40 shows the partial nucleotide sequence (SEQ. ID. No. 123) of the holA gene of [0090] A. aeolicus.
FIG. 41 shows the partial amino acide sequence (SEQ. ID. No. 124) of the δ subunit of [0091] A. aeolicus.
FIG. 42 shows the nucleotide sequence (SEQ. ID. No. 125) of the holB gene of [0092] A. aeolhcus.
FIG. 43 shows the amino acid sequence (SEQ. ID. No. 126) of the δ′ subunit of [0093] A. aeolicus.
FIG. 44 shows the nucleotide sequence (SEQ. ID. No. 127) of the dnaQ of A. aeolicus. [0094]
FIG. 45 shows the amino acid sequence (SEQ. ID. No. 128) of the ε subunit of [0095] A. aeolicus.
FIG. 46 shows the nucleotide sequence (SEQ. ID. No. 129) of the ssb gene of [0096] A. aeolicus.
FIG. 47 shows the amino acid sequence (SEQ. ID. No. 130) of the single-strand binding protein of [0097] A. aeolicus.
FIG. 48 shows the nucleotide sequence (SEQ. ID. No. 131) of the dnaB gene of [0098] A. aeolicus.
FIG. 49 shows the amino acid sequence (SEQ. ID. No. 132) of the DnaB helicase of [0099] A. aeolicus.
FIG. 50 shows the nucleotide sequence (SEQ. ID. No. 133) of the dnaG gene of [0100] A. aeolicus.
FIG. 51 shows the amino acid sequence (SEQ. ID. No. 134) of the DnaG primase of [0101] A. aeolicus.
FIG. 52 shows the nucleotide sequence (SEQ. ID. No. 135) of the dnaC gene of [0102] A. aeolicus.
FIG. 53 shows the amino acid sequence (SEQ. ID. No. 136) of the DnaC protein of [0103] A. aeolicus.
FIGS. [0104] 54A-B shows the nucleotide sequence (SEQ. ID. No. 137) of the dnaE gene of T. maritima.
FIG. 55 shows the amino acid sequence (SEQ. ID. No. 138) of the α subunit of [0105] T. maritima.
FIG. 56 shows the nucleotide sequence (SEQ. ID. No. 139) of the dnaQ gene of [0106] T. maritima.
FIG. 57 shows the amino acid sequence (SEQ. ID. No. 140) of the ε subunit of [0107] T. maritima.
FIG. 58 shows the nucleotide sequence (SEQ. ID. No. 141) of the dnaX gene of [0108] T. maritima.
FIG. 59 shows the amino acid sequence (SEQ. ID. No. 142) of the tau subunit of [0109] T. maritima.
FIG. 60 shows the nucleotide sequence (SEQ. ID. No. 143) of the dnaN gene of [0110] T. maritima.
FIG. 61 shows the amino acid sequence (SEQ. ID. No. 144) of the β subunit of [0111] T. maritima.
FIG. 62 shows the nucleotide sequence (SEQ. ID. No. 145) of the holA gene of [0112] T. maritima.
FIG. 63 shows the amino acid sequence (SEQ. ID. No. 146) of the δ subunit of [0113] T. maritima.
FIG. 64 shows the nucleotide sequence (SEQ. ID. No. 147) of the holB gene of [0114] T. maritima.
FIG. 65 shows the amino acid sequence (SEQ. ID. No. 148) of the δ′ subunit of [0115] T. maritima.
FIG. 66 shows the nucleotide sequence (SEQ. ID. No. 149) of the ssb gene of [0116] T. maritima.
FIG. 67 shows the amino acid sequence (SEQ. ID. No. 150) of the single-strand binding protein of [0117] T. maritima.
FIG. 68 shows the nucleotide sequence (SEQ. ID. No. 151) of the dnaB gene of [0118] T. maritima.
FIG. 69 shows the amino acid sequence (SEQ. ID. No. 152) of the DnaB helicase of [0119] T. maritima.
FIG. 70 shows the nucleotide sequence (SEQ. ID. No. 153) of the dnaG gene of [0120] T. maritima.
FIG. 71 shows the amino acid sequence (SEQ. ID. No. 154) of the DnaG primase of [0121] T. maritima.
FIG. 72 shows the nucleotide sequence (SEQ. ID. No. 155) of the holB gene of [0122] T. thermophilus.
FIG. 73 shows the amino acid sequence (SEQ. ID. No. 156) of the δ′ subunit of [0123] T. thermophilus.
FIG. 74 shows the nucleotide sequence (SEQ. ID. No. 157) of the holA gene of [0124] T. thermophilus.
FIG. 75 shows the amino acid sequence (SEQ. ID. No. 158) of the δ subunit of [0125] T. thermophilus.
FIG. 76 shows the nucleotide sequence (SEQ. ID. No. 171) of the ssb gene of [0126] T. thermophilus.
FIG. 77 shows the amino acid sequence (SEQ. ID. No. 172) of the single-strand binding protein of [0127] T. thermophilus.
FIG. 78 shows the partial nucleotide sequence (SEQ. ID. No. 173) of the dnaN gene of [0128] B. stearothermophilus.
FIG. 79 shows the partial amino acid sequence (SEQ. ID. No. 174) of the β subunit of [0129] B. stearothermophilus.
FIG. 80 shows the nucleotide sequence (SEQ. ID. No. 175) of the ssb gene of [0130] B. stearothermophilus.
FIG. 81 shows the amino acid sequence (SEQ. ID. No. 176) of the single-strand binding protein of [0131] B. stearothermophilus.
FIG. 82 shows the nucleotide sequence (SEQ. ID. No. 177) of the holA gene of [0132] B. stearothermophilus.
FIG. 83 shows the amino acid sequence (SEQ. ID. No. 178) of the δ subunit of [0133] B. stearothermophilus.
FIG. 84 shows the nucleotide sequence (SEQ. ID. No. 179) of the holB gene of [0134] B. stearothermophilus.
FIG. 85 shows the amino acid sequence (SEQ. ID. No. 180) of the δ′ subunit of [0135] B. stearothermophilus.
FIGS. [0136] 86A-B show the partial nucleotide sequence (SEQ. ID. No. 181) of the dnaX gene of B. stearothermophilus.
FIG. 87 shows the partial amino acid sequence (SEQ. ID. No. 182) of the tau subunit of [0137] B. stearothermbphilus.
FIGS. [0138] 88A-B show the nucleotide sequence (SEQ. ID. No. 183) of the polC gene of B. stearothermophilus.
FIG. 89 shows the amino acid sequence (SEQ. ID. No. 184) of the PolC or α-large subunit of [0139] B. stearothermophilus.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al., “Molecular Cloning: A Laboratory Manual” (1989); “Current Protocols in Molecular Biology” Volumes I-III (Ausubel, R. M., ed.) (1994); “Cell Biology: A Laboratory Handbook” Volumes I-III (Celis, J. E., ed.) (1994); “Current Protocols in Immunology” Volumes I-III (Coligan, J. E., ed.) (1994); “Oligonucleotide Synthesis” (M. J. Gait, ed.) (1984); “Nucleic Acid Hybridization” (B. D. Hames & S. J. Higgins, eds.) (1985); “Transcription And Translation” (B. D. Hames & S. J. Higgins, eds.) (1984); “Animal Cell Culture” (R. I. Freshney, ed.) (1986); “Immobilized Cells And Enzymes” (IRL Press) (1986); B. Perbal, “A Practical Guide To Molecular Cloning” (1984), each of which is hereby incorporated by reference. [0140]
Therefore, if appearing herein, the following terms shall have the definitions set out below. [0141]
The terms “DNA Polymerase III,” “Polymerase III-type enzyme(s)”, “Polymerase III enzyme complex(s)”, “[0142] T.th. DNA Polymerase III”, “A.ae. DNA Polymerase III”, “T.ma.DNA Polymerase III”, and any variants not specifically listed, may be used herein interchangeably, as are β subunit and sliding clamp and clamp as are also γ complex, clamp loader, and RFC, as used throughout the present application and claims refer to proteinaceous material including single or multiple proteins, and extends to those proteins having the amino acid sequence data described herein and presented in the Figures and corresponding Sequence Listing entries, and the corresponding profile of activities set forth herein and in the claims. Accordingly, proteins displaying substantially equivalent or altered activity are likewise contemplated. These modifications may be deliberate, for example, such as modifications obtained through site-directed mutagenesis, or may be accidental, such as those obtained through mutations in hosts that are producers of the complex or its named subunits. Also, the terms “DNA Polymerase III,” “T.th. DNA Polymerase III.” and “γ and τ subunits”, “β subunit”, “α subunit”, “ε subunit”, “δ subunit”, “δ′ subunit”, “SSB protein”, “sliding clamp” and “clamp loader” are intended to include within their scope proteins specifically recited herein as well as all substantially homologous analogs and allelic variations. As used herein γ complex refers to a particular type of clamp loader that includes a γ subunit.
Also as used herein, the term “thermolabile enzyme” refers to a DNA polymerase which is not resistant to inactivation by heat. For example, T5 DNA polymerase, the activity of which is totally inactivated by exposing the enzyme to a temperature of 90° C. for 30 seconds, is considered to be a thermolabile DNA polymerase. As used herein, a thermolabile DNA polymerase is less resistant to heat inactivation than in a thermostable DNA polymerase. A thermolabile DNA polymerase typically will also have a lower optimum temperature than a thermostable DNA polymerase. Thermolabile DNA polymerases are typically isolated from mesophilic organisms, for example mesophilic bacteria or eukaryotes, including certain animals. [0143]
As used herein, the term “thermostable enzyme” refers to an enzyme which is stable to heat and is heat resistant and catalyzes (facilitates) combination of the nucleotides in the proper manner to form the primer extension products that are complementary to each nucleic acid strand. Generally, the synthesis will be initiated at the 3′ end of each primer and will proceed in the 5′ direction along the template strand, until synthesis terminates, producing molecules of different lengths. [0144]
The thermostable enzyme herein must satisfy a single criterion to be effective for the amplification reaction, i.e., the enzyme must not become irreversibly denatured (inactivated) when subjected to the elevated temperatures for the time necessary to effect denaturation of double-stranded nucleic acids. Irreversible denaturation for purposes herein refers to permanent and complete loss of enzymatic activity. The heating conditions necessary for denaturation will depend, e.g., on the buffer salt concentration and the length and nucleotide composition of the nucleic acids being denatured, but typically range from about 90C° to about 96° C. for a time depending mainly on the temperature and the nucleic acid length, typically about 0.5 to four minutes. Higher temperatures may be tolerated as the buffer salt concentration and/or GC composition of the nucleic acid is increased. Preferably, the enzyme will not become irreversibly denatured at about 90°-100° C. [0145]
The thermostable enzymes herein preferably have an optimum temperature at which they function that is higher than about 40° C., which is the temperature below which hybridization of primer to template is promoted, although, depending on (1) magnesium and salt concentrations and (2) composition and length of primer, hybridization can occur at higher temperature (e.g., 45′-70° C.). The higher the temperature optimum for the enzyme, the greater the specificity and/or selectivity of the primer-directed extension process. However, enzymes that are active below 40° C., e.g., at 37° C., are also within the scope of this invention provided they are heat-stable. Preferably, the optimum temperature ranges from about 50° to about 90° C., more preferably about 600 to about 80° C. In this connection, the term “elevated temperature” as used herein is intended to cover sustained temperatures of operation of the enzyme that are equal to or higher than about 60° C. [0146]
The term “template” as used herein refers to a double-stranded or single-stranded DNA molecule which is to be amplified, synthesized, or sequenced. In the case of a double-stranded DNA molecule, denaturation of its strands to form a first and a second strand is performed before these molecules may be amplified, synthesized or sequenced. A primer, complementary to a portion of a DNA template is hybridized underappropriate conditions and the DNA polymerase of the invention may then synthesize a DNA molecule complementary to said template or a portion thereof. The newly synthesized DNA molecule, according to the invention, may be equal or shorter in length than the original DNA template. Mismatch incorporation during the synthesis or extension of the newly synthesized DNA molecule may result in one or a number of mismatched base pairs. Thus, the synthesized DNA molecule need not be exactly complementary to the DNA template. [0147]
The term “incorporating” as used herein means becoming a part of a DNA molecule or primer. [0148]
As used herein “amplification” refers to any in vitro method for increasing the number of copies of a nucleotide sequence, or its complimentary sequence, with the use of a DNA polymerase. Nucleic acid amplification results in the incorporation of nucleotides into a DNA molecule or primer thereby forming a new DNA molecule complementary to a DNA template. The formed DNA molecule and its template can be used as templates to synthesize additional DNA molecules. As used herein, one amplification reaction may consist of many rounds of DNA replication. DNA amplification reactions include, for example, polymerase chain reactions (PCR). One PCR reaction may consist of about 20 to 100 “cycles” of denaturation and synthesis of a DNA molecule. In this connection, the use of the term “long stretches of DNA” as it refers to the extension of primer along DNA is intended to cover such extensions of an average length exceeding 7 kilobases. Naturally, such length will vary, and all such variations are considered to be included within the scope of the invention. [0149]
As used herein, the term “holoenzyme” refers to a multi-subunit DNA polymerase activity comprising and resulting from various subunits which each may have distinct activities but which when contained in an enzyme reaction operate to carry out the function of the polymerase (typically DNA synthesis) and enhance its activity over use of the DNA polymerase subunit alone. For example, [0150] E. coli DNA polymerase III is a holoenzyme comprising three components of one or more subunits each: (1) a core component consisting of a heterotrimer of α, ε and θ subunits; (2) a β component consisting of a β subunit dimer; and (3) a γ complex component consisting of a heteropentamer of γ, δ, δ′, χ and ψ subunits (see Studwell and O'Donnell, 1990). These three components, and the various subunits of which they consist, are linked non-covalently to form the DNA polymerase III holoenzyme complex. However, they also function when not linked in solution.
As used herein, “enzyme complex” refers to a protein structure consisting essentially of two or more subunits of a replication enzyme, which may or may not be identical, noncovalently linked to each other to form a multi-subunit structure. An enzyme complex according to this definition ideally will have a particular enzymatic activity, up to and including the activity of the replication enzyme. For example, a “DNA pol III enzyme complex” as used herein means a multi-subunit protein activity comprising two or more of the subunits of the DNA pol III replication enzyme as defined above, and having DNA polymerizing or synthesizing activity. Thus, this term encompasses the native replication enzyme, as well as an enzyme complex lacking one or more of the subunits of the replication enzyme (e.g., DNA pol III exo-, which lacks the ε subunit). [0151]

The amino acid residues described herein are preferred to be in the “L” isomeric form. However, residues in the “D” isomeric form can be substituted for any L-amino acid residue, as long as the desired functional property of immunoglobulin15 binding is retained by the polypeptide. NH ₂refers to the free amino group present at the amino terminus of a polypeptide. COOH refers to the free carboxy group present at the carboxy terminus of a polypeptide. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243:3552-59 (1969), abbreviations for amino acid residues are shown in the following Table of Correspondence:

TABLE OF CORRESPONDENCE

SYMBOLS

1-Letter	3-Letter	AMINO ACID

Y	Tyr	tyrosine
G	Gly	glycine
F	Phe	phenylalanine
M	Met	methionine
A	Ala	alanine
S	Ser	serine
I	Ile	isoleucine
L	Leu	leucine
T	Thr	threonine
V	Val	valine
P	Pro	proline
K	Lys	lysine
H	His	histidine
Q	Gln	glutamine
E	Glu	glutamic acid
W	Trp	tryptophan
R	Arg	arginine
D	Asp	aspartic acid
N	Asn	asparagine
C	Cys	cysteine

It should be noted that all amino-acid residue sequences are represented herein by formulae whose left and right orientation is in the conventional direction of amino-terminus to carboxy-terminus. Furthermore, it should be noted that a dash at the beginning or end of an amino acid residue sequence indicates a peptide bond to a further sequence of one or more amino-acid residues. The above Table is presented to correlate the three-letter and one-letter notations which may appear alternately herein. [0153]
A “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo; i.e., capable of replication under its own control. [0154]
A “vector” is a replicon, such as plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment. [0155]
A “DNA molecule” refers to the polymeric form of deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in its either single stranded form, or a double-stranded helix. This term refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear DNA molecules (e.g., restriction fragments), viruses, plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences may be described herein according to the normal convention of giving only the sequence in the 5′ to 3′ direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). [0156]
An “origin of replication” refers to those DNA sequences that participate in DNA, synthesis. [0157]
A DNA “coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. A polyadenylation signal and transcription termination sequence will usually be located 3′ to the, coding sequence. [0158]
Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell. [0159]
A “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3′ direction) coding sequence. For purposes of defining the present invention, the promoter sequence is bounded at its 3′ terminus by the transcription initiation site and extends upstream (5′ direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the −10 and −35 consensus sequences. [0160]
An “expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence. A coding sequence is “under the control” of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence. [0161]
A “signal sequence” can be included before the coding sequence. This sequence encodes a signal peptide, N-terminal to the polypeptide, that communicates to the host cell to direct the polypeptide to the cell surface or secrete the polypeptide into the media, and this signal peptide is clipped off by the host cell before the protein leaves the cell. Signal sequences can be found associated with a variety of proteins native to prokaryotes and eukaryotes. [0162]
The term “oligonucleotide,” as used generally herein, such as in referring to probes prepared and used in the present invention, is defined as a molecule comprised of two or more (deoxy)ribonucleotides, preferably more than three. Its exact size will depend upon many factors which, in turn, depend upon the ultimate function and use of the oligonucleotide. [0163]
The term “primer” as used herein refers to an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product, which is complementary to a nucleic acid strand, is induced, i.e., in the presence of nucleotides and an inducing agent such as a DNA polymerase and at a suitable temperature and pH. The primer may be either single-stranded or double-stranded and must be sufficiently long to prime the synthesis of the desired extension product in the presence of the inducing agent. The exact length of the primer will depend upon many factors, including temperature, source of primer and use of the method. For example, for diagnostic applications, depending on the complexity of the target sequence, the oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides. [0164]
The primers herein are selected to be “substantially” complementary to different strands of a particular target DNA sequence. This means that the primers must be sufficiently complementary to hybridize with their respective strands. [0165]
Therefore, the primer sequence need not reflect the exact sequence of the template. For example, a non-complementary nucleotide fragment may be attached to the 5′ end of the primer, with the remainder of the primer sequence being complementary to the strand. Alternatively, non-complementary bases or longer sequences can be interspersed into the primer, provided that the primer sequence has sufficient complementarity with the sequence of the strand to hybridize therewith and thereby form the template for the synthesis of the extension product. [0166]
As used herein, the terms “restriction endonucleases” and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence. [0167]
A cell has been “transformed” by exogenous or heterologous DNA 0.30 when such DNA has been introduced inside the cell. The transforming DNA may or may not be integrated (covalently linked) into chromosomal DNA making up the genome of the cell. In prokaryotes, yeast, and mammalian cells for example, the transforming DNA may be maintained on an episomal element such as a plasmid. [0168]
With respect to eukaryotic cells, a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming. DNA. A “clone” is a population of cells derived from a single cell or common ancestor by mitosis. A “cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations. [0169]
Two DNA sequences are “substantially homologous” when at least about 75% (preferably at least about 80%, and most preferably at least about 90 or 95%) of the nucleotides match over the defined length of the DNA sequences. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Suitable conditions include those characterized by a hybridization buffer comprising 0.9M sodium citrate (“SSC”) buffer at a temperature of about 37° C. and washing in SSC buffer at a temperature of about 37° C.; and preferably in a hybridization buffer comprising 20% formamide in 0.9M SSC buffer at a temperature, of about 42° C. and washing with 0.2×SSC buffer at about 42° C. Stringency conditions can be further varied by modifying the temperature and/or salt content of the buffer, or by modifying the length of the hybridization probe as is known to those of skill in the art. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis et al., 1982; Glover, 1985; Hames and Higgins, 1984. [0170]

It should be appreciated that also within the scope of the present invention are degenerate DNA sequences. By “degenerate” is meant that a different three-letter codon is used to specify a particular amino acid. It is well known in the art that the following codons can be used interchangeably to code for each specific amino acid:


Phenylalanine	(Phe or F)	UUU or UUC

Leucine	(Leu or L)	UUA or UUG or

		CUU or CUC or

		CUA or CUG

Isoleucine	(Ile or I)	AUU or AUC or AUA

Methionine	(Met or M)	AUG

Valine	(Val or V)	GUU or GUC or

		GUA or GUG

Serine	(Ser or S)	UCU or UCC or

		UCA or UCG or

		AGU or AGC

Proline	(Pro or P)	CCU or CCC or

		CCA or CCG

Threonine	(Thr or T)	ACU or ACC or

		ACA or ACG

Alanine	(Ala or A)	GCU or GCG or

		GCA or GCG

Tyrosine	(Tyr or Y)	UAU or UAC

Histidine	(His or H)	CAU or CAC

Glutamine	(Gln or Q)	CAA or GAG

Asparagine	(Asn or N)	AAU or AAC

Lysine	(Lys or K)	AAA or AAG

Aspartic Acid	(Asp or D)	GAU or GAG

Glutamic Acid	(Glu or E)	GAA or GAG

Cysteine	(Cys or C)	UGU or UGC

Arginine	(Arg or R)	CGU or CGC or

		CGA or CGG or

		AGA or AGG

Glycine	(Gly or G)	GGU or GGC or

		GGA or GGG

Tryptophan	(Trp or W)	UGG

Termination codon		UAA (ochre) or

		UAG (amber) or

		UGA (opal)

It should be understood that the codons specified above are for RNA sequences. The corresponding codons for DNA have a T substituted for U. [0172]
Mutations can be made, e.g., in SEQ. ID. No. 1, or any of the nucleic acids set forth herein, such that a particular codon is changed to a codon which codes for a different amino acid. Such a mutation is generally made by making the fewest nucleotide changes possible. A substitution mutation of this sort can be made to change an amino acid in the resulting protein in a non-conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to another grouping) or in a conservative manner (i.e., by changing the codon from an amino acid belonging to a grouping of amino acids having a particular size or characteristic to an amino acid belonging to the same grouping). Such a conservative change generally leads to less change in the structure and function of the resulting protein. A non-conservative change is more likely to alter the structure, activity or function of the resulting protein. The present invention should be considered to include sequences containing conservative changes which do not significantly alter the activity or binding characteristics of the resulting protein. [0173]
The following is one example of various groupings of amino acids: [0174]
Amino Acids with Nonpolar R Groups [0175]
Alanine [0176]
Valine [0177]
Leucine [0178]
Isoleucine [0179]
Proline [0180]
Phenylalanine [0181]
Tryptophan [0182]
Methionine [0183]
Amino Acids with Uncharged Polar R Groups [0184]
Glycine [0185]
Serine [0186]
Threonine [0187]
Cysteine [0188]
Tyrosine [0189]
Asparagine [0190]
Glutamine [0191]
Amino Acids with Charged Polar R Groups (Negatively Charged at pH 6.0) [0192]
Aspartic acid [0193]
Glutamic acid [0194]
Basic Amino Acids (Positively Charged at pH 6.0) [0195]
Lysine [0196]
Arginine [0197]
Histidine (at pH 6.0) [0198]
Amino Acids with Phenyl Groups: [0199]
Phenylalanine [0200]
Tryptophan [0201]
Tyrosine [0202]

Another grouping may be according to molecular weight (i.e., size of R groups):



	Glycine	75
	Alanine	89
	Serine	105
	Proline	115
	Valine	117
	Threonine	119
	Cysteine	121
	Leucine	131
	Isoleucine	131
	Asparagine	132
	Aspartic acid	133
	Glutamine	146
	Lysine	146
	Glutamic acid	147
	Methionine	149
	Histidine (at pH 6.0)	155
	Phenylalanine	165
	Arginine	174
	Tyrosine	181
	Tryptophan	204

Particularly preferred substitutions are: [0204]
Lys for Arg and vice versa such that a positive charge may be maintained; [0205]
Glu for Asp and vice versa such that a negative charge may be maintained; [0206]
Ser for Thr such that a free —OH can be maintained; and [0207]
Gln for Asn such that a free NH[0208] ₂can be maintained.
Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, a Cys may be introduced into a potential site for disulfide bridges with another Cys. A His may be introduced as a particularly “catalytic” site (i.e., His can act as an acid or base and is the most common amino acid in biochemical catalysis). Pro may be introduced because of its particularly planar structure, which induces P-turns in the protein's structure. [0209]
Two amino acid sequences are “substantially homologous” when at least about 70% of the amino acid residues (preferably at least about 80%, and most preferably at least about 90 or 95%) are identical, or represent conservative substitutions. [0210]
A “heterologous” region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature. Thus, when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein. [0211]
An “antibody” is any inumunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies, the last mentioned described in further detail in U.S. Pat. Nos. 4,816,397 to Boss et al. and 4,816,567 to Cabilly et al. [0212]
An “antibody combining site” is that structural portion of an antibody molecule comprised of heavy and light chain variable and hypervariable regions that specifically binds antigen. [0213]
The phrase “antibody molecule” in its various grammatical forms as used herein contemplates both an intact immunoglobulin molecule and an immunologically active portion of an immunoglobulin molecule. Exemplary antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and those portions of an immunoglobulin molecule that contains the paratope, including those portions known in the art as Fab, Fab′, F(ab′)[0214] ₂and F(v), which portions are preferred for use in the therapeutic methods described herein. Fab and F(ab′)₂portions of antibody molecules are prepared by the proteolytic reaction of papain and pepsin, respectively, on substantially intact antibody molecules by methods that are well-known. See for example, U.S. Pat. No. 4,342,566 to Theofilopolous et al. Fab′ antibody molecule portions are also well-known and are produced from F(ab′)₂portions followed by reduction of the disulfide bonds linking the two heavy chain portions as with mercaptoethanol, and followed by alkylation of the resulting protein mercaptan with a reagent such as iodoacetamide. An antibody containing intact antibody molecules is preferred herein.
The phrase “monoclonal antibody” in its various grammatical forms refers to an antibody having only one species of antibody combining site capable of immunoreacting with a particular antigen. A monoclonal antibody thus typically displays a single binding affinity for any antigen with which it immunoreacts. A monoclonal antibody may therefore contain an antibody molecule having a plurality of antibody combining sites, each immnunospecific for a different antigen; e.g., a bispecific (chimeric) monoclonal antibody. [0215]
A DNA sequence is “operatively linked” to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence. The term “operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert: into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene. [0216]
The term “standard hybridization conditions” refers to salt and temperature conditions substantially equivalent to 5×SSC and 65° C. for, both hybridization and wash. However, one skilled in the art will appreciate that such “standard hybridization conditions” are dependent on particular conditions including the concentration of sodium and magnesium in the buffer, nucleotide sequence length and concentration, percent mismatch, percent formamide, and the like. Also important in the determination of “standard hybridization conditions” is whether the two sequences hybridizing are RNA-RNA, DNA-DNA or RNA-DNA. Such standard hybridization conditions are easily determined by one skilled in the art according to well known formulae, wherein hybridization is typically 10-20° C. below the predicted or determined T[0217] _mwith washes of higher stringency, if desired.
In its primary aspect, the present invention concerns the identification of a class of DNA Polymerase III-type enzymes or complexes found in thermophilic bacteria such as [0218] Thermus thermophilus (T.th.), Aquifex aeolicus (A.ae.), Thermotoga maritima (T.ma.), Bacillus stearothermophilus (B. st.) and other eubacteria which exhibit the following characteristics, among their properties: the ability to extend a primer over a long stretch of ssDNA at elevated temperature, stimulation by its cognate sliding clamp of the type that is assembled on DNA by a clamp loader, accessory subunits that exhibit DNA-stimulated ATPase activity at elevated temperature and/or ionic strength, and an associated 3′-5′ exonuclease activity. In a particular aspect, the invention extends to Polymerase III-type enzymes derived from a broad class of thermophilic eubacteria that include polymerases isolated from the thermophilic bacteria Aquifex aeolicus (A. ae. polymerase) and other members of the Aquifex genus; Thermus thermophilus (T.th. polymerase), Thermus favus (Tfl/Tub polymerase), Thermus ruber (Tru polymerase), Thermus brockianus (DYNAZYME™ polymerase) and other members of the Thermus genus; Bacillus stearothermophilus (Bst polymerase) and other members of the Bacillus genus; Thermoplasma acidophilum (Tac polymerase) and other members of the Thermoplasma genus; and Thermotoga neapolitana (Tne polymerase; See WO 96/10640 to Chattejee et al.), Thermotoga maritima (Tma polymerase; See U.S. Pat. No. 5,374,553 to Gelfand et al.), and other members of the Thermotoga genus. The particular polymerase discussed herein by way of illustration and not limitation, is the enzyme derived from T.th., A. ae., T.ma., or B.st.
Polymerase III-type enzymes covered by the invention include those that may be prepared by purification from cellular material, as described in detail in the Examples infra, as well as enzyme assemblies or complexes that comprise the combination of individually prepared enzyme subunits or components. Accordingly, the entire enzyme may be prepared by purification from cellular material, or may be constructed by the preparation of the individual components and their assembly into the functional enzyme. A representative and non-limitative protocol for the preparation of an enzyme by this latter route is set forth in U.S. Pat. No. 5,583,026 to O'Donnell, and the disclosure thereof is incorporated herein in its entirety for such purpose. [0219]
Likewise, individual subunits may be modified, e.g. as by incorporation therein of single residue substitutions to create active sites therein, for the purpose of imparting new or enhanced properties to enzymes containing the modified subunits. (see, e.g., Tabor, 1995). Likewise, individual subunits prepared in accordance with the invention, may be used individually and for example, may be substituted for their counterparts in other enzymes, to improve or particularize the properties of the resultant modified enzyme. Such modifications are within the skill of the art and are considered to be included within the scope of the present invention. [0220]
Accordingly, the invention includes the various subunits that may comprise the enzymes, and accordingly extends to the genes and corresponding proteins that may be encoded thereby, such as the a (as well as PolC), β, γ, γ, τ, δ and δ′ subunits, respectively. More particularly, in [0221] Thermus thermophilus the α subunit corresponds to dnaE, the β subunit corresponds to dnaN, the ε subunit corresponds to dnaQ, and the γ and τ subunits correspond to dnax, the δ subunit corresponds to holA, and the δ′ subunit corresponds to holB. In Aquifex aeolicus and Thermotoga maritima, the α subunit corresponds to dnaE, the β subunit corresponds to dnaN, the ε subunit corresponds to dnaQ, the τ subunit corresponds to dnaX, the δ subunit corresponds to hola, and the δ′ subunit corresponds to holB. In Bacillus stearothermophilus, the PolC which has both α and ε activities corresponds to polC, the β subunit corresponds to dnaN, the ε subunit corresponds to dnaQ, the τ subunit corresponds to dnax, the δ subunit corresponds to holA, and the δ′ subunit corresponds to holB.
Accordingly, the Polymerase III-type enzyme of the present invention comprises at least one gene encoding a subunit thereof, which gene is selected from the group consisting of dnaX, dnaQ, dnaE, dnan, hola, holB, and combinations thereof. More particularly, the invention extends to the nucleic acid molecule encoding them and their encoded subunits. [0222]
In the [0223] T.th. Pol III enzyme, this includes the following nucleotide sequences: dnaX(SEQ. ID. No. 3), dnaE (SEQ. ID. No. 86), dnaQ (SEQ. ID. No. 94), dnaN (SEQ. ID. No. 106), hola (SEQ. ID. No. 157), and holB (SEQ. ID. No. 155).
In the [0224] A.ae. Pol III enzyme, this includes the following nucleotide sequences: dnaX (SEQ. ID. No. 119), dnaE (SEQ. ID. No. 117), dnaQ (SEQ. ID. No. 127), dnan (SEQ. ID. No. 121), hola (SEQ. ID. No. 123), and holB (SEQ. ID. No. 125).
In the [0225] T.ma. Pol III enzyme, this includes the following nucleotide sequences: dnaX (SEQ. ID. No. 141), dnaE (SEQ. ID. No. 137), dnaQ (SEQ. ID. No. 139), dnaN(SEQ. ID. No. 143), holA (SEQ. ID. No. 145), and holB (SEQ. ID. No. 147).
In the [0226] B.st. Pol III enzyme, this includes the following nucleotide sequences: dnaX (SEQ. ID. No. 181), dnaN (SEQ. ID. No. 173), holA (SEQ. ID. No. 177), holB (SEQ. ID. No. 179), and polC (SEQ. ID. Nos. 183).
In each of the Pol III type enzymes of the present invention, not only are each of the above-identified coding sequences contemplated, but also conserved variants, active fragments and analogs thereof. [0227]
A particular [0228] T.th. Polymerase III-type enzyme in accordance with the invention may include at least one of the following sub-units: a γ subunit having an amino acid sequence corresponding to SEQ. ID. Nos. 4 and 5; a τ subunit having an amino acid sequence corresponding to SEQ. ID. No. 2; a ε subunit having an amino acid sequence corresponding to SEQ. ID. No. 95; a α subunit including an amino acid sequence corresponding SEQ. ID. No. 87; a β subunit having an amino acid sequence corresponding to SEQ. ID. No. 107; a δ subunit having an amino acid sequence corresponding to SEQ. ID. No. 158; a δ′ subunit having an amino acid sequence corresponding to SEQ. ID. No. 156; as well as variants, including allelic variants, muteins, analogs and fragments of any of the subunits, and compatible combinations thereof, capable of functioning in DNA amplification and sequencing.
A particular [0229] A.ae. Polymerase III-type enzyme in accordance with the invention may include at least one of the following sub-units: a τ subunit having an amino acid sequence corresponding to SEQ. ID. No. 120; a ε subunit having an amino acid sequence corresponding to SEQ. ID. No. 128; a α subunit including an amino acid sequence corresponding to SEQ. ID. No. 118; a β subunit having an amino acid sequence corresponding to SEQ. ID. No. 122; a δ subunit having an amino acid sequence corresponding to SEQ. ID. No. 124; a δ′ subunit having an amino acid sequence corresponding to SEQ. ID. No. 126; as well as variants, including allelic variants, muteins, analogs and fragments of any of the subunits, and compatible combinations thereof, capable of functioning in DNA amplification and sequencing.
A particular [0230] T.ma. Polymerase III-type enzyme in accordance with the invention may include at least one of the following sub-units: a τ subunit having an amino acid sequence corresponding to SEQ. ID. No. 142; a ε subunit having an amino acid sequence corresponding to SEQ. ID. No. 140; a α subunit including an amino acid sequence corresponding to SEQ. ID. No. 138; a β subunit having an amino acid sequence corresponding to SEQ. ID. No. 144; a δ subunit having an amino acid sequence corresponding to SEQ. ID. No. 146; a δ′ subunit having an amino acid sequence corresponding to SEQ. ID. No. 148; as well as variants, including allelic variants, muteins, analogs and fragments of any of the subunits, and compatible combinations thereof, capable of functioning in DNA amplification and sequencing.
A particular [0231] B.st. Polymerase III-type enzyme in accordance with the invention may include at least one of the following subunits: a τ subunit having a partial amino acid sequence corresponding to SEQ. ID. No. 182; a β subunit having an amino acid sequence corresponding to SEQ ID. No. 174; a δ subunit having an amino acid sequence corresponding to SEQ. ID. No. 178; a δ′ subunit having an amino acid sequence corresponding to SEQ. ID. No. 180; a PolC subunit having an amino acid sequence corresponding to SEQ. ID. Nos. 184; as well as variants, including allelic variants, muteins, analogs and fragments of any of the subunits, and compatible combinations thereof, capable of functioning in DNA amplification and sequencing.
The invention also includes and extends to the use and application of the enzyme and/or one or more of its components for DNA molecule amplification and sequencing by the methods set forth hereinabove, and in greater detail later on herein. [0232]
One of the subunits of the invention is the [0233] T.th. γ/τ subunit encoded by a dnaX gene, which frameshifts as much as −2 with high efficiency, and that, upon frameshifting, leads to the addition of more than one extra amino acid residue to the C-terminus (to form the γ subunit). Further, the invention likewise extends to a dnax gene derived from a thermophile such as T.th., that possesses the frameshift defined herein and that codes for expression of the γ and τ subunits of DNA Polymerase III.
The present invention provides methods for amplifying or sequencing a nucleic acid molecule comprising contacting the nucleic acid molecule with a composition comprising a DNA polymerase III enzyme (DNA pol III) complex (for sequencing, preferably a DNA pol III complex that is substantially reduced in 3′-5′ exonuclease activity). DNA pol III complexes used in the methods of the present invention are thermostable. [0234]
The invention also provides DNA molecules amplified by the present methods, methods of preparing a recombinant vector comprising inserting a DNA molecule amplified by the present methods into a vector, which is preferably an expression vector, and recombinant vectors prepared by these methods. [0235]
The invention also provides methods of preparing a recombinant host cell comprising inserting a DNA molecule amplified by the present methods into a host cell, which preferably a bacterial cell, most preferably an [0236] Escherichia coli cell; a yeast cell; or an animal cell, most preferably an insect cell, a nematode cell or a mammalian cell. The invention also provides and recombinant host cells prepared by these methods.
In additional preferred embodiments, the present invention provides kits for amplifying or sequencing a nucleic acid molecule. DNA amplification kits according to the invention comprise a carrier means having in close confinement therein two or more container means, wherein a first container means contains a DNA polymerase III enzyme complex and a second container means contains a deoxynucleoside triphosphate. DNA sequencing kits according to the present invention comprise a multi-protein Pol III-type enzyme complex and a second container means contains a dideoxynucleoside triphosphate. The DNA pol III contained in the container means of such kits is preferably substantially reduced in 5′-3′ exonuclease activity, may be thermostable; and may be isolated from the thermophilic cellular sources described above. [0237]
DNA pol III-type enzyme complexes for use in the present invention may be isolated from any organism that produced the DNA pol III-type enzyme complexes naturally or recombinantly. Such enzyme complexes may be thermostable, isolated from a variety of thermophilic organisms. [0238]
The thermostable DNA polymerase III-type enzymes or complexes that are an important aspect of this invention, may be isolated from a variety of thermophilic bacteria that are available commercially (for examples from American Type Culture Collection, Rockville, Md.). Suitable for use as sources of thermostable enzymes are the thermophilic eubacteria [0239] Aquifex aeolicus and other species of the Aquifex genus; Thermus aquaticus, Thermus thermophilus, Thermus flavus, Thermus ruber, Thermus brockianus, and other species of the Thermus genus; Bacillus stearothermophilus, Bacillus subtilis, and other species of the Bacillus genus; Thermoplasma acidophilum and other species of the Thermoplasma genus; Thermotoga neapolitana, Thermotoga maritima and other species of the Thermotoga genus; and mutants of each of these species. It will be understood by one of ordinary skill in the art, however, that any thermophilic microorganism might be used as a source of thermostable DNA pol III-type enzymes and polypeptides for use in the methods of the present invention. Bacterial cells may be grown according to standard microbiological techniques, using culture media and incubation conditions suitable for growing active cultures of the particular thermophilic species that are well-known to one of ordinary skill in the art (see, e.g., Brock et al., 1969; Oshima et al., 1974). Thermostable DNA pol III complexes may then be isolated from such thermophilic cellular sources as described for thermolabile complexes above.
Several methods are available for identifying homologous nucleic acids and protein subunits in other thermophilic eubacteria, either those listed above or otherwise. These methods include the following: [0240]
(1) The following procedure was used to obtain the genes encoding [0241] T.th. ε (dnaQ), τ/γ (dnaX), DnaA (dnaA), and, (dnaN). Protein sequences encoded by genes of non-thermophilic bacteria (i.e., mesophiles) are aligned to identify highly conserved amino acid sequences. PCR primers at conserved positions are designed using the codon usage of the organism of interest to amplify an internal section of the gene from genomic DNA extracted from the organism. The PCR product is sequenced. New primers are designed near the ends of the sequence to obtain new sequence that flanks the ends using circular PCR (also called inversed PCR) on genomic DNA that has been cut with the appropriate restriction enzyme and ligated into circles. These new PCR products are sequenced. The procedure is repeated until the entire gene sequence has been obtained. Also, dnaN (encoding β) is located next to dnaA in bacteria and, therefore, dnaN can be obtained by cloning DNA flanking the dnaA gene by the circular PCR procedure starting within dnaA. Once the gene is obtained, it is cloned into an expression vector for protein production.
(2) The following procedure was used to obtain the genes encoding [0242] T.th α polymerase (dnaE gene). The DNA polymerase III can be purified directly from the organism of interest and amino acid sequence of the subunit(s) obtained directly. In the case of T.th., T.thcells were lysed and proteins were fractionated. An antibody against E. coli a was used to probe column fractions by Western analysis, which reacted with T.th. α. The T.th. α was transferred to a membrane, proteolyzed, and fragments were sequenced. The sequence was used to design PCR primers for amplification of an internal section of the dnaE gene. Remaining flanking sequences are then obtained by circular PCR.
(3) The following procedure can be used to identify published nucleotide sequences which have not yet been identified as to their function. This method was used to obtain [0243] T.th. δ (holA) and δ′ (holB), although they could presumably also have been obtained via Methods 1 and 2 above. Discovery of T.th. dnaE (α), dnaN (β) and dnaX (τ/γ) indicates that thermophiles use a class III type of DNA polymerase (α) that utilize a clamp (β) and must also use a clamp loader since they have τ/γ. Also, the biochemical experiments in the Examples infra show that the T.th. polymerase functions with the T.th. β clamp. Having demonstrated that a thermophile (e.g., T.th.) does indeed utilize a class III type of polymerase with a clamp and clamp loader, it can be assumed that they may have δ and δ′ subunits needed to form a complex with τ/γ for functional clamp loading activity (i.e., as shown in E. coli, δand δ′ bind either τ or γ to form τδδ′ or γδδ′ complex, both of which are functional clamp loaders). The δ subunit is not very well conserved, but does give a match in the sequence databases for A.ae., T.ma, and T.th. The T.th. database provided limited information on the amino acid sequence of δ subunit, although one can easily obtain the complete sequence of T.th. hola by PCR and circular PCR as outlined above in Method 1. The A.ae. and T.ma. databases are complete and, therefore, the entire holA sequence from these genomes are identified. Neither database recognized these sequences as. 8 encoded by holA. The δ′ subunit (holB) is fairly well conserved. Again the incomplete T.th. database provided limited δ′ sequence, but as with δ, it is a straight forward process for anyone experienced in the area to obtain the rest of the holB sequence using PCR and circular PCR as described in Method 1. Neither the A.ae. nor T.ma. databases recognized holB encoding δ′. Nevertheless, holb was identified as encoding δ′ by searching the databases with δ′ sequence. In each case, the Thermatoga maritima and Aquifex aeolicus holB gene and δ′ sequence were obtained in their entirety. Neither database had previously annotated holA or holb encoding δ and δ′.
As stated above and in accordance with the present invention, once nucleic acid molecules have been obtained, they may be amplified according to any of the literature-described manual or automated amplification methods. Such methods includes, but are not limited to, PCR (U.S. Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis), Strand Displacement Amplification (SDA) (U.S. Pat. No. 5,455,166 to Walker), and Nucleic Acid Sequence-Based. Amplification, (NASBA) (U.S. Pat. No. 5,409,818 to Davey et al.; EP 329,822 to Davey et al.). Most preferably, nucleic acid molecules are amplified by the methods of the present invention using PCR-based amplification techniques. [0244]
In the initial steps of each of these amplification methods, the nucleic acid molecule to be, amplified is contacted with a composition comprising a DNA polymerase belonging to the evolutionary “family A” class (e.g., Taq DNA pol I or [0245] E. coli pol I) or the “family “B” class (e.g., Vent and Pfu DNA polymerases—see Ito and Braithwaite, 1991). All of these DNA polymerases are, present as single subunits and are primarily involved in DNA repair. In contrast, the DNA pol III-type enzymes are multisubunit complexes that mainly function in the replication of the chromosome, and the subunit containing the DNA polymerase activity is in the “family C” class.
Thus, in amplifying a nucleic acid molecule according to the methods of the present invention, the nucleic acid molecule is contacted with a composition comprising a thermostable DNA pol III-type enzyme complex. [0246]
Once the nucleic acid molecule to be amplified is contacted with the DNA pol III-type complex, the amplification reaction may proceed according to standard protocols for each of the above-described techniques. Since most of these techniques comprise a high-temperature denaturation step, if a thermolabile. DNA pol III-type enzyme complex is used in nucleic acid amplification by any of these techniques the enzyme would need to be added at the start of each amplification cycle, since it would be heat-inactivated at the denaturation step. However, a thermostable DNA pol III-type complex used in these methods need only be added once at the start of the amplification (as for Taq DNA polymerase in traditional PCR amplifications), as its activity will be unaffected by the high temperature of the denaturation step. It should be noted, however, that because DNA pol III-type enzymes may have a much more rapid rate of nucleotide incorporation than the polymerases commonly used in these amplification techniques, the cycle times may need to be adjusted to shorter intervals than would be standard. [0247]
In an alternative preferred embodiment, the invention provides methods of extending primers for several kilobases, a reaction that is central to amplifying large nucleic acid molecules, by a technique commonly referred to as “long chain PCR” (Barnes, 1994; Cheng, 1994). [0248]
In such a method the target primed DNA can contain a single strand stretch of DNA to be copied into the double strand form of several or tens of kilobases. The reaction is performed in a suitable buffer, preferably Tris, at a pH of between 5.5-9.5, preferably 7.5. The reaction also contains MgCl[0249] ₂in the range 1 mM to 10 mM, preferably 8 mM, and may contain a suitable salt such as NaCl, KCl or sodium or potassium acetate. The reaction also contains ATP in the range of 20 μM to 1 mM, preferably 0.5 mM, that is needed for the clamp loader to assemble the clamp onto the primed template, and a sufficient concentration of deoxynucleoside triphosphates in the range of 50 μM to 0.5 mM, preferably 60 μM for chain extension. The reaction contains a sliding clamp, such as the β subunit, in the range of 20 ng to 200 ng, preferably 100 ng, for action as a clamp to stimulate the DNA polymerase. The chain extension reaction contains a DNA polymerase and a clamp loader: that could be added either separately or as a single Pol III*-like particle, preferably as a Pol III* like particle that contains the DNA polymerase and clamp loading activities. The Pol III-type enzyme is added preferably at a concentrations of about 0.0002-200 units per milliliter, about 0.002-100 units per milliliter, about 0.2-50 units per milliliter, and most preferably about 2-50 units per milliliter. The reaction is incubated at elevated temperature, preferably 60° C. or more, and could include other proteins to enhance activity such as a single strand DNA binding protein.
In another preferred embodiment, the invention provides methods of extending primers on linear templates in the absence of the clamp loader. In this reaction, the primers are annealled to the linear DNA, preferably at the ends such as in standard PCR applications. The reaction is performed in a suitable buffer, preferably Tris, at a pH of between 5.5-9.5, preferably 7.5. The reaction also contains MgCl[0250] ₂in the range of 1 mM to 10 mM, preferably 8 mM, and may contain a suitable salt such as NaCl, KCl or sodium or potassium acetate. The reaction also contains a sufficient concentration of deoxynucleoside triphosphates in the range of 50 μM to 0.5 mM, preferably 60 μM for chain extension. The reaction contains a sliding clamp, such as the β subunit, in the range of 20 ng to 20 μg, preferably about 2 μg, for ability to slide on the end of the DNA and associate with the polymerase for action as a clamp to stimulate the DNA polymerase. The chain extension reaction also contains a Pol III-type polymerase subunit such as a, core, or a Pol III*-like particle. The Pol III-type enzyme is added preferably at a concentrations of about 0.0002-200 units per milliliter, about 0.002-100 units per milliliter, about 0.2-50 units per milliliter, and most preferably about 2-50 units per milliliter. The reaction is incubated at elevated temperature, preferably 60° C. or more, and could include other proteins to enhance activity such as a single strand DNA binding protein.
The methods of the present invention thus will provide high-fidelity amplified copies of a nucleic acid molecule in a more rapid fashion than traditional amplification methods using the repair-type enzymes. [0251]
These amplified nucleic acid molecules may then be manipulated according to standard recombinant DNA techniques. For example, a nucleic acid molecule amplified according to the present methods may be inserted into a vector, which is preferably an expression vector, to produce a recombinant vector comprising the amplified nucleic acid molecule. This vector may then be inserted into a host cell, where it may, for example, direct the host cell to produce a recombinant polypeptide encoded by the amplified nucleic acid molecule. Methods for inserting nucleic acid molecules into vectors, and inserting these vectors into host cells, are well-known to one of ordinary skill in the art (see, e.g., Maniatis, 1992). [0252]
Alternatively, the amplified nucleic acid molecules may be directly inserted into a host cell, where it may be incorporated into the host cell genome or may exist as an extrachromosomal nucleic acid molecule, thereby producing a recombinant host cell. Methods for introduction of a nucleic acid molecule into a host cell, including calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods, are described in many standard laboratory manuals (see, e.g., Davis, 1986). [0253]
For each of the above techniques wherein an amplified nucleic acid molecule is introduced into a host cell via a vector or via direct introduction, preferred host cells include but are not limited to a bacterial cell, a yeast cell, or an animal cell. Bacterial host cells preferred in the present invention are [0254] E. coli, Bacillus spp., Streptomyces, spp., Erwinia spp., Klebsiella spp. and Salmonella typhimurium. Preferred as a host cell is E. coli, and particularly preferred are E. coli strains DH 10B and Stb12, which are available commercially (Life. Technologies, Inc. Gaithersburg, Md.). Preferred animal host cells are insect cells, nematode cells and mammalian cells. Insect host cells preferred in the present invention are Drosophila spp. cells, Spodoptera Sf9 and Sf21 cells, and Trichoplusa High-Five cells, each of which is available commercially (e.g., from Invitrogen; San Diego, Calif.). Preferred nematode host cells are those derived from C. elegans, and preferred mammalian host cells are those derived from rodents, particularly rats, mice or hamsters, and primates, particularly monkeys and humans. Particularly preferred as mammalian host cells are CHO cells, COS cells and VERO cells.
By the present invention, nucleic acid molecules may be sequenced according to any of the literature-described manual or automated sequencing methods. Such methods include, but are not limited to, dideoxy sequencing methods such as “Sanger sequencing” (Sanger and Coulson, 1975; Sanger et al., 1977; U.S. Pat. No. 4,962,022 to Fleming et al.; and U.S. Pat. No. 5,498,523 to Tabor et al.), as well as more complex PCR-based nucleic acid fingerprinting techniques such as Random Amplified Polymorphic DNA (RAPD) analysis (Williams et al., 1990). Arbitrarily Primed PCR (AP-PCR) (Welsh and McClelland, 1990), DNA Amplification Fingerprinting (DAF) (Caetano-Anolles, 1991), microsatellite PCR or Directed Amplification of Minisatellite-region DNA (DAMD) (Heath et al., 1993), and Amplification Fragment Length Polymorphism (AFLP) analysis (EP 534,858 to Vos et al.; Vos et al., 1995; Lin and Kuo, 1995). [0255]
As described above for amplification methods, the nucleic acid molecule to be sequenced by these methods is typically contacted with a composition comprising a type A or type β DNA polymerase. By contrast, in sequencing a nucleic acid molecule according to the methods of the present invention, the nucleic acid molecule is contacted with a composition comprising a thermostable DNA pol III-type enzyme complex instead of necessarily using a DNA polymerase of the family A or β classes. As for amplification methods, the DNA pol III-type complexes used in the nucleic acid sequencing methods of the present invention are preferably substantially reduced in 3′-5′ exonuclease activity; most preferable for use in the present methods is a DNA polymerase III-type complex which lacks the E subunit. DNA pol III-type complexes used for nucleic acid sequencing according to the present methods are used at the same preferred concentration ranges described above for long chain extension of primers. [0256]
Once the nucleic acid molecule to be sequenced is contacted with the DNA pol III complex, the sequencing reactions may proceed according to the protocols disclosed in the above-referenced techniques. [0257]
As discussed above, the invention extends to kits for use in nucleic acid amplification or sequencing utilizing DNA polymerase Ill-type enzymes according to the present methods. A DNA amplification kit according to the present invention may comprise a carrier means, such as vials, tubes, bottles and the like. A first such container means may contain a DNA polymerase III-type enzyme complex, and a second such container means may contain a deoxynucleoside triphosphate. The amplification kit encompassed by this aspect of the present invention may further comprise additional reagents and compounds necessary for carrying out standard nucleic amplification protocols (See U.S. Pat. No. 4,683,195 to Mullis et al. and U.S. Pat. No. 4,683,202 to Mullis, which are directed to methods of DNA amplification by PCR). [0258]
Similarly, a DNA sequencing kit according to the present invention comprises a carrier means having in close confinement therein two or more container means, such as vials, tubes, bottles and the like. A first such container means may contain a DNA polymerase III-type enzyme complex, and a second such container means may contain a dideoxynucleoside triphosphate. The sequencing kit may further comprise additional reagents and compounds necessary for carrying out standard nucleic sequencing protocols, such as pyrophosphatase, agarose or polyacrylamide media for formulating sequencing gels, and other components necessary for detection of sequenced nucleic acids (See U.S. Pat. No. 4,962,020 to Fleming et al. and U.S. Pat. No. 5,498,523 to Tabor et al., which are directed to methods of DNA sequencing). [0259]
The DNA polymerase III-type complex contained in the first container means of the amplification and sequencing kits provided by the invention is preferably a thermostable DNA polymerase III-type enzyme complex and more preferably a DNA polymerase III-type enzyme complex that is reduced in 3-5′ exonuclease activity. Naturally, the foregoing methods and kits are presented as illustrative and not restrictive of the use and application of the enzymes of the invention for DNA molecule amplification and sequencing., Likewise, the applications of specific embodiments of the enzymes, including conserved variants and active fragments thereof are considered to be disclosed and included within the scope of the invention. [0260]
As discussed earlier, individual subunits could be modified to customize enzyme construction and corresponding use and activity. For example, the region of a that interacts with β could be subcloned onto another DNA polymerase, thereby causing β to enhance the activity of the recombinant polymerase. Alternatively, the β clamp could be modified to function with another protein or enzyme thereby enhancing its activity or acting to localize its action to a particular targeted DNA. Finally, the polymerase active site could be modified to enhance its action, for example changing Tyrosine enabling more equal site stoppage with the four ddNTPs (Tabor et al., 1995). This represents a particular non-limiting illustration of the scope and practice of the present invention with reference to the utility of individual subunits hereof. [0261]
Accordingly and as stated above, the present invention also relates to a recombinant DNA molecule or cloned gene, or a degenerate variant thereof, which encodes any one or all of the subunits of the DNA Polymerase III-type enzymes of the present invention, or active fragments thereof. In the instance of the τ subunit, a predicted molecular weight of about 58 kD and an amino acid sequence set forth in SEQ ID Nos. 4 or 5 is comprehended; preferably a nucleic acid molecule, in particular a recombinant DNA molecule or cloned gene, encoding the 58 kD subunit of the Polymerase III of the invention, that has a nucleotide sequence or is complementary to a DNA sequence shown in FIGS. 4A and 4B (SEQ ID No. 1), and the coding region for dnaX set forth in FIG. 4C (SEQ ID No. 3). The γ subunit is smaller, and is approximately 50 kD, depending upon the extent of the frameshift that occurs. More particularly, and as set forth in FIG. 4E (SEQ ID No. 4), the γ subunit defined by a −1 frameshift possesses a molecular weight of 50.8 kD, while the γ subunit defined by a −2 frameshift, set forth in FIG. 4F (SEQ ID No. 5), possesses a molecular weight of 49.8 kD. [0262]
As discussed above, the invention also extends to the genes including holA, holB, dnax, dnaQ, dnaE, and dnaN from thermophilic eubacteria (i.e., [0263] T.th. and A.ae.) that have been isolated and/or purified, to corresponding vectors for the genes, and particularly, to the vectors disclosed herein, and to host cells including such vectors. In this connection, probes, have been prepared which hybridize to the DNA polymerase III-type enzymes of the present invention, and which are selected from the various oligonucleotide probes or primers set forth in the present application. These include, without limitation, the oligonucleotide defined in SEQ ID No. 6 the oligonucleotide defined in SEQ ID No. 8 the oligonucleotide defined in SEQ ID No. 10 the oligonucleotide defined in SEQ ID No. 11 the oligonucleotide defined in SEQ ID No. 12 the oligonucleotide defined in SEQ ID No. 13 the oligonucleotide defined in SEQ ID No. 14 the oligonucleotide defined in SEQ ID No. 15, and the oligonucleotide defined in SEQ ID No. 16.
The methods of the invention include a method for producing a recombinant thermostable DNA polymerase III-type enzyme from a thermophilic bacterium, such as [0264] T.th., A.ae., Th.ma., or B.st. which comprises culturing a host cell transformed with a vector of the invention under conditions suitable for the expression of the present DNA polymerase III. Another method includes a method for isolating a target DNA fragment consisting essentially of a DNA coding for a thermostable DNA polymerase III-type enzyme from a thermophilic bacterium comprising the steps of:
(a) forming a genomic library from the bacterium; [0265]
(b) transforming or transfecting an appropriate host cell with the library of step (a); [0266]
(c) contacting DNA from the transformed or transfected host cell with a DNA probe which hybridizes to a DNA fragment selected from the group consisting of the DNA fragments defined in SEQ ID No. 6 and the DNA fragments defined in SEQ ID No. 8 or the oligonucleotides set forth above; wherein hybridization is conducted under the following conditions: [0267]
i) hybridization: 1% crystalline BSA (fraction V) (Sigma), 1 mM EDTA, 0.5 M NaHPO[0268] ₄(pH 7.2), 7% SDS at 65° C. for 12 hours and;
ii) wash: 5×20 minutes with wash buffer consisting of 0.5% BSA, fraction V), 1 mM Na[0269] ₂EDTA, 40 mM NaHPO₄(pH 7.2), and 5% SDS;
(d) assaying the transformed or transfected cell of step (c) which hybridizes to the DNA probe for DNA polymerase III-type activity; and [0270]
(e) isolating a target DNA fragment which codes for the thermostable DNA polymerase III-type enzyme. [0271]
Also, antibodies including both polyclonal and monoclonal antibodies, and the DNA Polymerase III-like enzyme complex and/or their γ and τ subunits, α subunit(s), δ subunit, δ′ subunit, β subunit, ε subunit may be used in the preparation of the enzymes of the present invention as well as other enzymes of similar thermophilic origin. For example, the DNA Polymerase III-type complex or its subunits may be used to produce both polyclonal and monoclonal antibodies to themselves in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells. [0272]
The general methodology for making monoclonal antibodies by hybridomas is well known. Immortal, antibody-producing cell lines can also be created by techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus. See, e.g., Schreier et al., 1980; Hammerling et al., 1981; Kennett et al., 1980; see also: U.S. Pat. No. 4,341,761 to Ganfield et al.; U.S. Pat. No. 4,399,121 to Albarella et al.; U.S. Pat. No. 4,427,783 to Newman et al.; U.S. Pat. No. 4,444,887 to Hoffman; U.S. Pat. No. 4,451,570 to Royston et al.; U.S. Pat. No. 4,466,917 to Nussenzweig et al.; U.S. Pat. No. 4,472,500 to Milstein et al.; U.S. Pat. No. 4,491,632 to Wands et al.; and U.S. Pat. No. 4,493,890 to Morris. [0273]
Methods for producing polyclonal anti-polypeptide antibodies are well-known in the art. See U.S. Pat. No. 4,493,795 to Nestor et al. A monoclonal antibody, typically containing Fab and/or F(ab′)[0274] ₂portions of useful antibody molecules, can be prepared using the hybridoma technology described in Antibodies—A Laboratory Manual, Harlow and Lane, eds., Cold Spring Harbor Laboratory, New York (1988), which is incorporated herein by reference. Briefly, to form the hybridoma from which the monoclonal antibody composition is produced, a myeloma or other self-perpetuating cell line is fused with lymphocytes obtained from the spleen of a mammal hyperimmunized with an elastin-binding portion thereof.
A monoclonal antibody useful in practicing the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of the appropriate antigen specificity. The culture is maintained under conditions and for a time period sufficient for the hybridoma to secrete the antibody molecules into the medium. The antibody-containing medium is then collected. The antibody molecules can then be further isolated by well-known techniques. [0275]
Media useful for the preparation of these compositions are both well-known in the art and commercially available and include synthetic culture media, inbred mice and the like. An exemplary synthetic medium is Dulbecco's minimal essential medium (DMEM) (Dulbecco et al., 1959) supplemented with 4.5 gm/l glucose, 20 mm glutamine, and 20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c. [0276]
Another feature of this invention is the expression of the DNA sequences disclosed herein. As is well known in the art, DNA sequences may be expressed by operatively linking them to an expression control sequence in an appropriate expression vector and employing that expression vector to transform an appropriate unicellular host. [0277]
Such operative linking of a DNA sequence of this invention to an expression control sequence, of course, includes, if not already part of the DNA sequence, the provision of an initiation codon, ATG, in the correct reading frame upstream of the DNA sequence. [0278]
A wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention. Useful expression vectors, for example, may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences. Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., [0279] E. coli plasmids col E1, pCR1, pBR322, pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage λ, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
Any of a wide variety of expression control sequences—sequences that control the expression of a DNA sequence operatively linked to it—may be used in these vectors to express the DNA sequences of this invention. Such useful expression control sequences include, for example, the early or late promoters of SV40, CMV, vaccinia, polyoma or adenovirus, the lac system, the trp system, the TAC system, the TRC system, the LTR system, the major operator and promoter regions of phage λ, the control regions of fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase (e.g., Pho5), the promoters of the yeast α-mating factors, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. [0280]
A wide variety of unicellular host cells are also useful in expressing the DNA sequences of this invention. These hosts may include well known eukaryotic and prokaryotic hosts, such as strains of [0281] E. coli, Pseudomonas, Bacillus, Streptomyces, fungi such as yeasts, and animal cells, such as CHO, R1.1, B-W and L-M cells, African Green Monkey kidney cells (e.g., COS 1, COS 7, BSC1, BSC40, and BMT10), insect cells (e.g., Sf9), and human cells and plant cells in tissue culture.
It will be understood that not all vectors, expression control sequences and hosts will function equally well to express the DNA sequences of this invention. Neither will all hosts function equally well with the same expression system. However, one skilled in the art will be able to select the proper vectors, expression control sequences, and hosts without undue experimentation to accomplish the desired, expression without departing from the scope of this invention. For example, in selecting a vector, the, host must be considered because the vector must function in it. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, will also be considered. [0282]
In selecting an expression control sequence, a variety of factors will normally be considered. These include, for example, the relative strength of the system, its controllability, and its compatibility with the particular DNA sequence or gene to be expressed, particularly with regard to potential secondary structures. Suitable unicellular hosts will be selected by consideration of, e.g., their compatibility with the chosen vector, their secretion characteristics, their ability to fold proteins correctly, and their fermentation requirements, as well as the toxicity to the host of the product encoded by the DNA sequences to be expressed, and the ease of purification of the expression products. [0283]
Considering these and other factors a person skilled in the art will be able to construct a variety of vector/expression control sequence/host combinations that will express the DNA sequences of this invention on fermentation or in large scale animal culture. [0284]
It is further intended that analogs may be prepared from nucleotide sequences of the protein complex/subunit derived within the scope of the present invention. Analogs, such as fragments, may be produced, for example, by pepsin digestion of bacterial material. Other analogs, such as muteins, can be produced by standard site-directed mutagenesis of dnax, dnaE, dnaQ, dnaN, holA, or holb coding sequences. Especially useful may be a mutation in dnaE that provides the polymerase with the ability to incorporate all four ddNTPs with equal efficiency thereby producing an even binding pattern in sequencing gels, as discussed above and with reference to Tabor et al., 1995. [0285]
As mentioned above, a DNA sequence corresponding to dnaX, dnaQ, holA, holB, dnaE, or dnaN, or encoding the subunits of the DNA Polymerase III of the invention can be prepared synthetically rather than cloned. The DNA sequence can be designed with the appropriate codons for the amino acid sequence of the subunit(s) of interest. In general, one will select preferred codons for the intended host if the sequence will be used for expression. The complete sequence is assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence (Edge, 1981; Nambair et al., 1984; Jay et al., 1984). [0286]
Synthetic DNA sequences allow convenient construction of genes which will express DNA Polymerase III analogs or “muteins”. Alternatively, DNA encoding muteins can be made by site-directed mutagenesis of native dnaX, dnaQ, holA, holB, dnaE or dnaN genes or their corresponding cDNAs, and muteins can be made directly using conventional polypeptide synthesis. [0287]
A general method for site-specific, incorporation of unnatural amino acids into proteins is described in Noren et al., 1989. This method may be used to create analogs with unnatural amino acids. [0288]

GENERAL DESCRIPTION OF THE INVENTION

As discussed above, the present invention has as one of its characterizing features, that a Polymerase III-type enzyme as defined hereinabove, has been discovered in a thermophile, that has the structure and function of a chromosomal replicase. This structure and function confers significant benefit when the enzyme is employed in procedures such as PCR where speed and accuracy of DNA reconstruction is crucial. [0289]
Chromosomal replicases are composed of several subunits in all organisms (Kornberg and Baker, 1992). In keeping with the need to replicate long chromosomes, replicases are rapid and highly processive multiprotein machines. All cellular replicases examined to date derive their processivity from one subunit that is shaped like a ring and completely encircles DNA (Kuriyan and O'Donnell, 1993; Kelman and O'Donnell, 1994). This “sliding clamp” subunit acts as a mobile tether for the polymerase machine (Stukenberg et al., 1991). The sliding clamp does not assemble onto the DNA by itself, but, requires a complex of several proteins, called a “clamp loader” which couples ATP hydrolysis to the assembly of sliding clamps onto DNA (O'Donnell et al., 1992). Hence, Pol III-type cellular replicases are comprised of three components: a clamp, a clamp loader, and the DNA polymerase. [0290]
An overall goal is to identify and isolate all of the genes encoding the replicase subunits from a thermophile for expression and purification in large quantity. Following this, the replication apparatus can be reassembled from individual subunit components for use in kits, PCR, sequencing and diagnostic applications (Onrust et al., 1995). [0291]
As a beginning to identify and characterize the replicase of a thermophile, we started by looking for a homologue to the prokaryotic dnaX gene which encode subunits (γ and τ) of the replicase. The dnaX gene has another homologue, holB, which encodes yet another subunit (δ′) of the replicase. The amino acid sequence of δ′ (encoded by holA) and τ/γ subunits (encoded by dnaX) are particularly highly conserved in evolution from prokaryotes to eukaryotes (Chen et al., 1992; O'Donnell et al., 1993; Onrust et al., 1993; Carter et al., 1993; Cullman et al., 1995). [0292]
One organism chosen for study and exposition herein is the exemplary extreme thermophile [0293] Thermus thermophilus (T.th.). It is understood that other members: of the class such as the eubacterium Thermatoga are expected to be analogous in both structure and function. Thus, the investigation of T.th. proceeded and initially, a T.th. homologue of dnaX was identified. The gene encodes a full length protein of 529 amino acids. The amino terminal third of the sequence shares over 50% homology to dnaX genes as divergent as E. coli (gram negative) and B. subtilis (gram positive). The T.th. dnaX gene contains a DNA sequence that provides a translational frameshift signal for production of two proteins from the same gene. Such frameshifting has been documented only in the case of E. coli (Tsuchihashi and Kornberg, 1990; Flower and McHenry, 1990; Blinkowa and Walker, 1990). No frameshifting has been documented to occur in the dnaX homologues (RFC subunit genes) of yeast and humans (Eukaryotic kingdom).
The presence of a dnaX gene that produces two subunits implies that [0294] T.th. has a clamp loader (γ) and may be organized by τ into a PolIII*-type replicase like the replicative DNA polymerase of Escherichia coli, DNA polymerase III holoenzyme. The E. coli DNA polymerase III holoenzyme contains 10 different subunits, some in copies of two or more for a total composition of 18 polypeptide chains (Kornberg and Baker, 1992; Onrust et al., 1995). The holoenzyme is composed of three major activities: the 3-subunit DNA polymerase core (αεθ), the β subunit DNA sliding clamp, and the 5-subunit γ complex clamp loader (γδδ′χψ). This 3 component strategy generalizes to eukaryotes which utilize a clamp (PCNA) and a 5-subunit RFC clamp loader (RFC) which provide processivity to DNA polymerase δ (reviewed in Kelman and O'Donnell, 1994).
In [0295] E. coli, the polymerase and clamp loader components are organized into one PolIII* particle by the τ subunit, that acts as a “glue” protein (Onrust et al., 1995). One dimer of τ holds together two core polymerases in the particle which are utilized for the coordinated and simultaneous replication of both strands of duplex DNA (McHenry, 1982; Maki et al., 1988; Yuzhakov et al., 1996). The “glue” protein τ subunit also binds one clamp loader (called γ complex) thereby acting as a scaffold for a large superstructure assembly called DNA polymerase III*. The gene encoding τ, called dnax, also encodes the γ subunit of DNA polymerase III. The subunit then associates with Pol III* to form the DNA polymerase III holoenzyme. The γ subunit is approximately ⅔ the length of τ. γ shares the N-terminus of τ, but is truncated by a translational frameshifting mechanism that, after the shift, encounters a stop codon within two amino acids (Tsuchihashi and Komberg, 1990; Flower and McHenry, 1990; Blinkowa and Walker, 1990). Hence, γ is the N-terminal 453 amino acids of τ, but contains one unique residue at the C-terminus (the penultimate codon encodes a Lys residue which is the same sequence as if the frameshift did not take place). This frameshift is highly efficient and occurs approximately 50% of the time.
The sequence of the γ and τ subunits encoded by the dnaX gene are homologous to the clamp loading subunits in all other organisms extending from gram negative bacteria through gram positive bacteria, the Archeae Kingdom and the Eukaryotic Kingdom from yeast to humans (O'Donnell et al., 1993). All of these organisms utilize a three component replicase (DNA polymerase, clamp and clamp loader) and in these cases the 3 components appear to behave as independent units in solution rather than forming a large holoenzyme superstructure. For example, in eukaryotes from yeast to humans, the clamp loader is the five subunit RFC, the clamp is PCNA, and the polymerases δ and ε are all stimulated by the PCNA clamp assembled onto primed DNA by RFC (reviewed in Kelman and O'Donnell 1994). [0296]
The discovery of a dnaX gene in [0297] T.th. provided confidence that thermophilic bacteria would contain a three component Pol III-type enzyme. Hence, we proceeded to identify the dnaQ and dnaN genes encoding, respectively, the proofreading 3′-5′ exonuclease, and the β DNA sliding clamp subunits of a Pol III-type enzyme. Following this, we purified from extracts of T.th. cells, a Pol III-type enzyme. This enzyme preparation had the unique property of extending a single primer around a long 7.2 kb single strand DNA genome of M13 mp18 bacteriophage. Such a primer extension assay serves as a tool to detect and identify the Pol III-type of enzyme in cell extracts. The enzyme was confirmed to be a Pol III-type enzyme based on its reactivity with antibody directed against the E. coli α subunit (the DNA polymerase subunit) and antibody directed against E. coli γ subunit. Proteins corresponding to α, τ, γ, δ and δ′ were easily visible and lend themselves to identification of the genes through use of peptide microsequencing followed by primer design for PCR amplification. For example, from this DNA pol III-type preparation, the peptide sequence of the α subunit was obtained, which then allowed the dnaE gene encoding the α subunit (DNA polymerase) of the Pol III-type enzyme to be obtain.
These methods should be widely applicable to other thermophilic bacteria. Additional antibody reagents against other Pol III-type enzyme components, such as RFC subunits, DNA polymerase delta, epsilon or beta, and the PCNA clamp from known organisms can be made quite easily as polyclonal or monoclonal antibody preparations using as antigen either naturally purified sequence, recombinant sequence, or synthetic peptide sequence. Examples of known sequences of these Pol III-type enzymes are to be found in: DNA polymerases (Braithwaite and Ito, 1993), RFC clamp loaders (Cullman et al., 1995) and PCNA (Kelman and O'Donnell, 1995). [0298]
The remaining genes of [0299] T.th. Pol III needed for efficient extension of primed templates, holA and holB, are now identified. The holA coding sequence (SEQ. ID. No. 157) encodes the δ subunit (SEQ. ID. No. 158) and the holB coding sequence (SEQ. ID. No. 155) encodes the δ′ subunit (SEQ. ID. No. 156). The hola and holB coding sequences and the δ and δ′ subunits were identified via BLAST search (Altschul et al., 1997), and subsequently isolated following circular PCR. These genes will provide the subunit preparations through use of standard recombinant techniques and protein purification protocols. The protein subunits can then be used to reconstitute the enzyme complexes as they exist in the cell. This type of reconstitution of Pol III has been demonstrated using the protein subunits of DNA polymerase III holoenzyme from E. coli to assemble the entire particle. See, e.g., U.S. Pat. Nos. 5,583,026 and 5,668,004 to O'Donnell; and Onrust et al., 1995. The disclosures of these references are incorporated herein in their entireties.
Another organism chosen for study and exposition herein is the extreme thermophile [0300] Aquifex aeolicus. Thus, the present invention also relates to various isolated DNA molecules from Aquifex aeolicus, in particular the DNA molecules encoding various replication proteins. These include dnaE, dnaX, dnaN, hola, holB, ssb DNA molecules from A. aeolicus. These DNA molecules can be inserted into an expression system or used to transform host cells from which isolated proteins can be obtained. The isolated proteins encoded by these DNA molecules are also disclosed.
Unless otherwise indicated below, the [0301] Aquifex aeolicus sequences were obtained by sequence comparisons using the Thermus thermophilus counterparts as query against the genome of Aquifex aeolicus (Deckert et al., 1998).
The [0302] A. aeolicus dnaE gene has a nucleotide coding sequence according to SEQ. ID. No. 117 and encodes the α subunit of the of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 118. The A.ae. α subunit has approximately 41% aa identity to the T.th. α subunit.
The [0303] A. aeolicus dnaX gene has a nucleotide coding sequence according to SEQ. ID. No. 119 and encodes the τ subunit of the of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 120. The A.ae. τ subunit has approximately 51% aa identity to the T.th. τ subunit.
The [0304] A. aeolicus dnaN gene has a nucleotide coding sequence according to SEQ. ID. No. 121 and encodes the, β subunit of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 122. The A.ae. β subunit has approximately 27% aa identity to the. T.th. β subunit.
The [0305] A. aeolicus dnaQ gene has a nucleotide coding sequence according to SEQ. ID. No. 127 and encodes the ε subunit of the of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 128. The A.ae. ε subunit has approximately 26% aa identity to the T.th. ε subunit.
The [0306] A. aeolicus ssb gene has a nucleotide coding sequence according to SEQ. ID. No. 129 and encodes the SSB protein, which has an amino acid sequence according to SEQ. ID. No. 130. The A.ae SSB protein has approximately 22% aa identity to the T.th. SSB protein.
Further, the coding sequences of [0307] A. aeolicus genes encoding the helicase (dnaB), helicase loader (dnaC), and primase (dnaG) are also disclosed. The A. aeolicus dnaB gene has a nucleotide coding sequence according to SEQ. ID. No. 131 and encodes the DnaB protein, which functions as a helicase and has an amino acid sequence according to SEQ. ID. No. 132. The A. aeolicus dnaG gene has a nucleotide coding sequence according to SEQ. ID. No. 133 and encodes the DnaG protein, which functions as a primase and has an amino acid sequence according to SEQ. ID. No; 134. The A. aeolicus dnaC gene has a nucleotide coding sequence according to SEQ. ID. No. 135 and encodes the DnaC protein, which functions as a helicase loader and has an amino acid sequence according to SEQ. ID. No. 136.
The [0308] A. aeolicus holA and holB genes were previously unidentified by Deckert et al., 1998. Using Thermus thermophilus δ′ subunit amino acid sequence and the Thermatoga maritima δ subunit amino acid sequence (SEQ. ID. No. 146 which itself was obtained using the T.th. δ subunit amino acid sequence of SEQ. ID. No. 158) in separate BLAST searches (Altschul et al., 1997), corresponding polypeptide products in Aquifex aeolicus were identified. The A. aeolicus holA gene has a nucleotide coding sequence according to SEQ. ID. No. 123 and encodes the 6 subunit of the of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 124. The A.ae. δ subunit has approximately 21% aa identity to the T.m. δ subunit. The A. aeolicus holB gene has a nucleotide coding sequence according to SEQ. ID. No. 125 and encodes the δ′ subunit of the of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 126. The A.ae. δ′ subunit has approximately 24% aa identity to the T.th. δ′ subunit.
This invention also clones at least the coding regions of a set of [0309] A. aeolicus genes which encode proteins that assemble into an A. aeolicus DNA polymerase III replication enzyme. These genes (dnaE, dnaN, dnaX, dnaQ, holA, holB, ssb) were cloned into expression vectors, the proteins were expressed in E. coli, and the corresponding protein subunits were purified (alpha, beta, tau, delta, delta prime, SSB). This invention identifies the major protein-protein contacts among these subunits, shows how these proteins can be assembled into higher order multi-protein complexes, and how to form a rapid and processive DNA polymerase III holoenzyme.
In contrast to the [0310] E. coli and T. thermophilus dnaX genes which encode both τ and γ subunits, the A. aeolicus dnaX gene produces only the full length τ subunit when expressed in E. coli. The A. aeolicus τ is, intermediate in length between the γ and τ subunits of E. coli DNA polymerase III holoenzyme. The E. coli τ binds α, the γ subunit does not bind α. Due to the intermediate size of A. aeolicus τ, it was not known whether the A. aeolicus τ would bind the cc subunit. This invention shows that indeed, the A. aeolicus τ binds to α, as well as δ and δ′, thereby forming an A. aeolicus ατδδ′ complex. Until the identification of the δ and δ′ subunits by the present invention, their existence, let alone their interaction with τ and α, was not even known.
The [0311] A. aeolicus ατδδ′/β Pol III can be applied in several useful DNA handling techniques. For example, the thermophilic Pol III will be useful in DNA sequencing, especially at high temperature. Also, use of a thermal resistant rapid and processive Pol III is an important improvement to polymerase chain reaction technology. The ability of the A. aeolicus Pol III to extend primers for multiple kilobases makes possible the amplification of very long segments of DNA (long chain PCR).
Another organism chosen for study and exposition herein is the extreme thermophile [0312] Thermotoga maritima. Thus, the present invention also relates to various isolated DNA molecules from Thermotoga maritima, in particular the DNA molecules encoding various replication proteins. These include dnaE, dnax, dnaN, dnaQ, holA, holB, ssb DNA molecules from Thermotoga maritima. These DNA molecules can be inserted into an expression system or used to transform host cells from which isolated proteins can be obtained. The isolated proteins encoded by these DNA molecules are also disclosed.
Unless otherwise indicated below, the [0313] Thermotoga maritima sequences were obtained by sequence comparisons using the Thermus thermophilus counterparts as query against the genome of Thermotoga maritima (Nelson et al., 1999).
The [0314] T. maritima dnaE gene has a nucleotide coding sequence according to SEQ. ID. No. 137 and encodes the α subunit of the of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 138. The T.m. α subunit has approximately 33% aa identity to the T.th. α subunit.
The [0315] T. maritima dnaQ gene has a nucleotide coding sequence according to SEQ. ID. No. 139 and encodes the ε subunit of the of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 140. The T.m. ε subunit has approximately 34% aa identity to the T.th. ε subunit.
The [0316] T. maritima dnaX gene has a nucleotide coding sequence according to SEQ. ID. No. 141 and encodes the τ subunit of the of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 142. The T.m. τ subunit has approximately 48% aa identity to the T.th. τ subunit.
The [0317] T. maritima dnaN gene has a nucleotide coding sequence according to SEQ. ID. No. 143 and encodes the β subunit of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 144. The T.m. β subunit has approximately 28% aa identity to the T.th. β subunit.
The [0318] T. maritima ssb gene has a nucleotide coding sequence according to SEQ. ID. No. 149 and encodes the SSB protein, which has an amino acid sequence according to SEQ. ID. No. 150. The T.m. SSB protein has approximately 18% aa identity to the T.th. SSB protein.
Further, the coding sequences of [0319] T. maritima genes encoding the helicase (dnaB) and primase (dnaG) are also disclosed. The T. maritima dnaB gene has a nucleotide coding sequence according to SEQ. ID. No. 151 and encodes the DnaB protein, which functions as a helicase and has an amino acid sequence according to SEQ. ID. No. 152. The T. maritima dnaG gene has a nucleotide coding sequence according to SEQ. ID. No. 153 and encodes the DnaG protein, which functions as a primase and has an amino acid sequence according to SEQ. ID. No. 154.
The [0320] T. maritima holA and holB genes were previously unidentified by Nelson et al., 1999). Using the Thermus thermophilus δ and δ′ subunit amino acid sequences (SEQ. ID. Nos. 158 and 156, respectively) in separate BLAST searches (Altschul et al., 1997), corresponding polypeptide products in T. maritima were identified. The T. maritima holA gene has a nucleotide coding sequence according to SEQ. ID. No. 145 and encodes the δ subunit of the of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 146. The T.m. δ subunit has approximately 37% aa identity to the T.th. δ subunit. The T.m. holB gene has a nucleotide coding sequence according to SEQ. ID. No. 147 and encodes the δ′ subunit which has an amino acid sequence according to SEQ. ID. No. 148. The T.m. δ′ subunit has approximately 25% aa identity to the T.th. δ′ subunit.
Yet another organism chosen for study and exposition herein is the extreme thermophile [0321] Bacillus stearothermophilus. Thus, the present invention also relates to various isolated DNA molecules from Bacillus stearothermophilus, in particular the DNA molecules encoding various replication proteins. These include dnaE, dnax, dnaN, dnaQ, holA, holB; ssb DNA molecules from Bacillus stearothermophilus. These DNA molecules can be inserted into an expression system or used to transform host cells from which isolated proteins can be obtained. The isolated proteins encoded by these DNA molecules are also disclosed.
Unless otherwise indicated below, the [0322] Bacillus stearothermophilus sequences were obtained by searching the database of this organism (at http://www.genome.ou.edu).
The [0323] B. stearothermophilus polC gene has a nucleotide coding sequence according to SEQ. ID. No. 183 and encodes the PolC or α-large subunit of the DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 184. The B.st. PolC subunit, like the PolC subunits of other Gram positive organisms, contains both polymerase and 3′-5′ exonuclease activity. This subunit, therefore, is essentially a fusion of α and ε.
The [0324] B. stearothermophilus dnaX gene has a partial nucleotide coding sequence according to SEQ. ID. No. 181 and encodes the τ subunit of the of DNA Polymerase III, which has a partial amino acid sequence according to SEQ. ID. No. 182. The B.st. τ subunit has approximately 31% aa identity to the T.th. τ subunit.
The [0325] B. stearothermophilus dnaN gene has a partial nucleotide coding sequence according to SEQ. ID. No. 173 and encodes the β subunit of DNA Polymerase III, which has a partial amino acid sequence according to SEQ. ID. No. 174. The B.st β subunit has approximately 21% aa identity to the T.th. β subunit.
The [0326] B. stearothermophilus ssb gene has a nucleotide coding sequence according to SEQ. ID. No. 175 and encodes the SSB protein, which has an amino acid sequence according to SEQ. ID. No. 176. The B.st. SSB protein has approximately 23% aa identity to the T.th. SSB protein.
The [0327] B. stearothermophilus holA gene has a nucleotide coding sequence according to SEQ. ID. No. 177 and encodes the δ subunit of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 178. The B.st. δ subunit has approximately 26% aa identity to the T.th. 8 subunit.
The [0328] B. stearothermophilus holB gene has a nucleotide coding sequence according to SEQ. ID. No. 179 and encodes the δ′ subunit of DNA Polymerase III, which has an amino acid sequence according to SEQ. ID. No. 180. The B.st. δ subunit has approximately 25% aa identity to the T.th; δ′ subunit.
By conducting BLAST searches of unidentified genomic DNA from other thermophilic eubacteria, it is possible to identify coding regions which encode various functional subunits of other Pol III replicative machinery. [0329]
Although it is generally appreciated that proteins isolated from a thermophile should retain activity at high temperature, there is no guarantee that they will retain temperature resistance when isolated in pure form. This invention shows that the [0330] A. aeolicus Pol III, like the T. thermophilus Pol III, is resistant to high temperature. It is expected that the Th. maritima and B. stearothermophilus Pol III enzymes will similarly be resistant to high temperature.
The following experiments illustrate the identification and characterization of the enzymes and constructs of the present invention. Accordingly, in Examples 1-8 below, the identification and expression of the γ and τ is presented, as the first step in the elucidation of the [0331] Thermus thermophilus Polymerase III reflective of the present invention. Examples 9-12 which follow set forth the protocol for the purification of the remainder of the sub-units of the enzyme that represent substantial entirety of the functional replicative machinery of the enzyme. Examples 1.8-30 demonstrate the preparation of isolated A. aeolicus sequences Pol III subunits and their thermostable use.

EXAMPLE 1

Experimental Procedures

Materials [0332]
DNA modification enzymes were from New England Biolabs. Labelled nucleotides were from Amersham, and unlabeled nucleotides were from New England Biolabs The Alter-1 vector was from Promega. pET plasmids and [0333] E. coli strains, BL21(DE3) and BL21 (DE3)pLysS were from Novagen. Oligonucleotides were from Operon. Buffer A is 20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mMDTT, and 10% glycerol.
Genomic DNA [0334]
[0335] Thermus thermophilus (strain HB8) was obtained from the American Type Tissue Collection. Genomic DNA was prepared from cells grown in 0.1 μl of Thermus medium N697 (ATCC: 4 g yeast extract, 8.0 g polypeptone (BBL 11910), 2.0 g NaCl, 30.0 g agar, 1.0 L distilled water) at 75° C. overnight. Cells were collected by centrifugation at 4° C. and the cell pellet was resuspended in 25 ml of 100 mM Tris-HCl (pH 8.0), 0.05 M EDTA, 2 mg/ml lysozyme and incubated at room temperature for 10 min. Then 25 ml 0.10 M EDTA (pH 8.0), 6% SDS was added and mixed followed by 60 ml of phenol. The mixture was shaken for 40 min. followed by centrifugation at 10,000×G for 10 min. at room temperature. The upper phase (50 ml) was removed and mixed with 50 ml of phenol:chloroform (50:50 v/v) for 30 min. followed by centrifugation for 10 min. at room temperature. The upper phase was decanted and the DNA was precipitated upon addition of {fraction (1/10)}th volume 3 M sodium acetate (pH 6.5) and 1 volume ethanol. The precipitate was collected by centrifugation and washed twice with 2 ml of 80% ethanol, dried and resuspended in 1 ml T.E. buffer (10 mM Tris HCl (pH 7.5), 1 mM EDTA).
Cloning of dnaX [0336]
DNA oligonucleotides for amplification of [0337] T.th. genomic DNA were as follows. The upstream 32mer (5′-CGCAAGCTTCACGCSTACCTSTTCTCCGGSAC -3′, S indicating a mixture of G and C) (SEQ. ID. No. 6) consists of a Hind III site within the first 9 nucleotides (underlined) followed by codons (SEQ. ID. No. 29) encoding the following amino acid sequence (HAYLFSGT) (SEQ. ID. No. 7). The downstream 34 mer (5′-CGCGAATTCGTGCTCSGGSGGCTCCTCSAGSGTC-3′) (SEQ. ID. No. 8) consists of an EcoRI site (underlined) followed by codons (SEQ. ID. No. 30) encoding the sequence KTLEEPPEH (SEQ. ID. No. 9) on the complementary strand. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture according to the manufacturers instructions (10 μl ThermoPol Buffer, 0.5 mM each dNTP and 0.5 mM MgSO₄). Amplification was performed using the following cycling scheme: 5 cycles of: 30 sec. at 95.5° C., 30 sec. at 40° C., 2 min. at 72° C.; 5 cycles of: 30 sec. at 95.5° C., 30 sec. at 45° C., and 2 min. at 72° C.; and 30 cycles of: 30 sec. at 95.5° C., 30 sec. at 50° C., and 30 sec. at 72° C. Products were visualized in a 1.5% native agarose gel.
Genomic DNA was digested with either XhoI, XbaI, StuI, PstI, NcoI, MluI, KpnI, HindIII, EcoRI, EagI, BglI, or BamHI, followed by Southern analysis in a native agarose gel (Maniatis et al., 1982). Approximately 0.5 μg of digest was analyzed in each lane of a 0.8% native agarose gel followed by transfer to an MSI filter (Micron Separations Inc.). The transfer included the following steps: [0338]
1. The agarose gel was soaked in 500 ml of 1% HCl with gentle shaking for 10 min. [0339]
2. Then the gel was soaked in 500 ml of 0.5 M NaOH+1.5 M NaCl for 40 min. [0340]
3. After that the gel was soaked in 500 ml of 1M ammonium acetate for 1 h. [0341]
4. The DNA was transferred to the MSI filter with the use of blotting paper for 4 h. [0342]
5. The filter was kept at 80° C. for 15 min. in the oven. [0343]
6. The pre-hybridization step was run in 10 ml of Hybridization solution (1% crystalline BSA (fraction V) (Sigma), 1 mM EDTA, 0.5 M NaHPO[0344] ₄(pH 7.2), 7% SDS) at 65° C. for 30 min.
7. The probe, radiolabelled by the random priming method (see below), was added to the pre-hybridization solution and kept at 65° C. for 12 h. [0345]
8. The filter was washed with low stringency with 200 ml of the wash buffer (0.5% BSA, fraction V), 1 mM Na[0346] ₂EDTA, 40 mM NaHPO₄(pH 7.2), 5% SDS with gentle shaking for 20 min. This step was repeated 5 times, followed by exposure to X-ray film (XAR-5. Kodak).
As a probe, the PCR product was radiolabelled by random as follows. [0347]
1. 14 ml of the mixture containing 0.2 μg of PCR product DNA, 1 μg of the pd(N6) (Promega) and 2.5 ml of the 10× Klenow reaction buffer (100 mM Tris-HCl (pH 7.5), 50 mM MgCl[0348] ₂, 75 mM dithiothreitol) were boiled for 10 min. and then kept at 4° C.
2. The reaction volume was increased up to 25 μl, containing in [0349] addition 33 μM of each dNTP, except DATP, 10 μi [α-³²P] dATP (800 Ci/mM), and 2 units of Klenow enzyme. The reaction mixture was incubated 1.5 h.
3. 2 mg of sonicated herring sperm DNA (GibcoBRL) was added to the reaction and the volume was increased to 2 ml using hybridization solution. The sample was then boiled for 10 min. [0350]
A genomic library of XbaI digested DNA was prepared upon treating 1 μg genomic [0351] T.th. DNA with 10 units of XbaI in 100 μl of NE Buffer N2 (50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl₂, 1 mM DTT) for 2 h at 37° C. The digested DNA was purified by phenol chloroform extraction and ethanol precipitation. The Alter-1 vector (0.51 g)(Promega) was digested with 1 unit of XbaI in NE Buffer N2 and then purified by phenol/chloroform extraction and ethanol precipitation. One microgram of genomic digest was incubated with 0.05 μg of digested Alter-1 and 20 U of T4 ligase in 30 μl of ligase buffer (50 mM, Tris-HCl (pH 7.8), 10 mM MgCl₂, 10 mM DTT and 1 mM ATP) at 15° C. for 12 h. The ligation reaction was transformed into the DH5α strain of E. Coli and transformants were plated on LB plates containing ampicillin and screened for the dnaX insert using the radiolabelled PCR probe as follows:
1. The colonies tested were lifted onto MSI filters, approximately 100 colonies to each filter. [0352]
2. The filters, removed from the LB/Tc plates, were placed side up on a sheet of [0353] Whatman 3 μM paper soaked with 0.5 M NaOH for 5 min.
3. The filters were transferred to a sheet of paper soaked with 1 M Tris-HCl (pH 7.5) for 5 min. [0354]
4. The filters were placed on a sheet of paper soaked in 0.5 M Tris-HCl (pH 7.5), 1.25 M NaCl for 5 min. [0355]
5. After drying by air, the filters were heated in the oven 80° C. for 15 min. and then were analyzed by Southern hybridization. [0356]
Plasmid DNA was prepared from 20 positive colonies; of these 6 contained the expected 4 kb insert when digested with XbaI. Sequencing of the insert was performed by the Sanger method using the Vent polymerase sequencing kit according to the manufacturers instructions (New England Biolabs). [0357]
Identification of the dnaX Gene [0358]
The dnaX genes of the gram negative [0359] E. coli and the gram positive B. subtilis share more than 50% identity in amino acid sequence within the N-terminal 180 residues containing the ATP-binding domain (FIG. 2). Two highly conserved regions (shown in bold in FIG. 2) were used to design oligonucleotide primers for application of the polymerase chain reaction to T.th. genomic DNA. The expected PCR product, including the restriction sites (i.e. before cutting) is 345 nucleotides. Use of these primers with genomic T.th. DNA resulted in a product of the expected size. The PCR product was then radiolabelled and used to probe genomic DNA in a Southern analysis (FIG. 3). Genomic DNA was digested with several different restriction endonucleases, electrophoresed in a native agarose gel and then probed with the PCR fragment. The Southern analysis showed an XbaI fragment of approximately 4 kb, more than sufficient length to encode the dnaX gene. Other restriction nucleases produced fragments that were significantly longer, or produced two or more fragments indicating presence of a site within the coding sequence of dnax.
To obtain full length dnax, genomic DNA was digested with XbaI and ligated into XbaI digested Alter-1 vector. Ligated DNA was transformed into DH5 alpha cells, and colonies were screened with the labeled PCR probe. Plasmid DNA was prepared from 20 positive colonies and analyzed for the appropriate sized insert using XbaI. Six of the twenty clones contained the expected 4 kb XbaI fragment as an insert, the sequence of which is shown in FIGS. 4A and 4B. [0360]
The Frameshift Site [0361]
The dnaX gene of [0362] E. coli produces two proteins, the γ and τ subunits, by a −1 frameshift (Tsuchihashi and Kornberg, 1990; Flower and McHenry, 1990; Blinkowa and Walker, 1990). The full length product yields a, and the frameshift results in addition of one amino acid before encountering a stop codon to produce y. The −1 frameshift site in the E. coli dnaX gene contains the sequence, A AAA AAG, which follows the X XXY YYZ rule found in retroviral genes (Jacks et al., 1988). This “slippery sequence” preserves the initial two residues of the tRNAs in the aminoacyl and peptidyl sites both before and after the frameshift. Mutagenesis of the E. coli dnaX frameshifting site has shown that the first three residues can be nucleotides other than A, but that A's in the second set of three nucleotides is important to frameshifting (Tsuchihashi and Brown, 1992).
Immediately downstream of the stop codon is a potential stem-loop structure which enhances frameshifting, presumably by causing the ribosome to pause. Further, the AAG codon lacks a cognate tRNA in [0363] E. coli and thus the G residue may facilitate the pause, and has been shown to aid the vigorous frameshifting observed in the E. coli dnaX gene (Tsuchihashi and Brown, 1992). A fourth component of frameshifting in the E. coli dnaX gene is presence of an upstream Shine-Dalgarno sequence which is thought to pair with the 16S rRNA to increase the frequency of frameshifting still further (Larsen et al., 1994).
Examination of the [0364] T.th. dnaX sequence reveals a single site that fulfills the X XXY YYZ rule in which positions 4-7 are A residues. The site is unique from that in E. coli as all seven residues are A, and the heptanucleotide sequence is flanked by another A residue on each side (i.e. A9). Surprisingly, the stop codon immediately downstream of this site is in the −2 frame, although there is a stop codon in the −1 frame 28 nucleotides downstream of the −2 stop codon. Indeed, a −2 frameshift would fulfill the requirement that the first two nucleotides of each codon in the peptidyl and aminoacyl sites be conserved during either a −1 or a −2 frameshift. As with the case of E. coli dnaX, there are secondary structure, step loop structures immediately downstream. Finally, there is a Shine-Dalgarno sequence immediately adjacent to the frameshift site, as well as another Shine-Dalgarno sequence 22 nucleotides upstream of the frameshift site.
Assuming the first stop codon is utilized (i.e. −2 frameshift), the predicted size of the γ subunit in [0365] T.th. is 454 amino acids for a mass of 49.8 kDa, over 2 kDa larger than the 431 residue γ subunit (47.5 kDa) of E. coli. This would result in 2 residues after the −2 frameshift (i.e. after the GlutysLys, the residues LysAla would be added) to be compared to the result of the −1 frameshift in E. coli which also results in 2 residues (LysGlu). In the event that a −1 frameshift were utilized in the T.th. dnaX gene, then an additional 12 residues would be added following the frameshift for a molecular mass of 50.8 kDa (i.e. after the GluLysLys, the residues LysProAspProLysAlaProProGlyProThrSer would be added at aa 453-464 of SEQ. ID. No. 4). As explained later, this nucleotide sequence was found to promote both −1 and −2 frameshifting in E. coli (FIG. 8). But first, we examined T.th. cells by Western analysis for the presence of two subunits homologous to E. coli γ and τ.

EXAMPLE 2

Frameshifting Analysis of the [0366] T.th. dnaX Gene
Frameshifting was analyzed by inserting the frameshift site into lacZ in the three different reading frames, followed by plating on X-gal and scoring for blue or white colony formation (Weiss et al., 1987). The frameshifting region within [0367] T.th dnaX was subcloned into the EcoRI/BamHI sites of pUC19. These sites are within the polylinker inside of the β-galactosidase gene. Three constructs were produced such that the insert was either in frame with the downstream coding sequence of β-galactosidase, or were out of frame (either −1 or −2). An additional three constructs were designed by mutating the frameshift sequence and then placing this insert into the three reading frames of the β-galactosidase gene. These six plasmids were constructed as described below.
The upstream primer for the shifty sequences was 5′-gcg cgg atc cgg agg gag aaa aaa aaa gcc tca gcc α-3′ (SEQ. ID. No. 10). The BamHI site for cloning into pUC is underlined. Also, the stop codon, tga, has been mutated to tca (also underlined). The upstream primer for the mutant shifty sequence was: 5′-gcg cgg atc cgg agg gag aga aga aaa gcc tca gcc α-3′ (SEQ. ID. No. 11). The mutant sequence contains two substitutions of a G for an A residue in the polyA stretch (underlined). Three downstream primers were utilized with each upstream primer to create two sets of three inserts in the 0 frame, −1 frame and −2 frame. The sequence of these primers, and the length of insert (after cutting with EcoRI and BamHI and inserting into pUC19) are as follows: 5′-gaa tta aat tcg cgc ttc ggg agg tgg g-3′ (0 frameshift, total 58 nucleotide insert) (SEQ. ID. No. 12); 5′-gcg cga att cgc gct tcg gga ggt ggg-3′ (−1 frame, 54mer insert) (SEQ. ID. No. 13); and 5′-gcg cga att cgg gcg ctt cag gag gtg gg-3′ (−2 frame, 56mer insert) (SEQ. ID. No. 14). The downstream primers have an EcoRI site (underlined); the EcoRI site of the 0 frame insert was blunt ended to produce the greater length insert (converting the EcoRI site to an aattaatt sequence). Also, the tcg sequence, which produces the tga stop codon (underlined) was mutated to tca in the −2 downstream primer so that readthrough would be allowed after the frameshift occurred. [0368]
In summary, a region surrounding the frameshift site and ending at least 5 nucleotides past the −1 frameshift stop codon was inserted into the β-galactosidase gene of pUC19 in the three different reading frames (stop codons were mutated to prevent stoppage following a frameshift). These three plasmids were introduced into [0369] E. coli and plated with X-gal. The results, in FIG. 8, show that blue colonies were observed after 24 h incubation with all three plasmids and therefore both −1 and −2 frameshifting had occurred.
To further these results, two γ residues were introduced into the polyA tract which should disrupt the ability of this sequence to direct frameshifts. The mutated slippery sequence was inserted into pUC19 followed by transformation into [0370] E. coli and plating on X-gal. The results showed that both −1 and −2 frameshifting was prevented, further supporting the fact that frameshifting requires the polyA tract as expected (FIG. 8).

EXAMPLE 3

Expression Vector for [0371] T.th. γ and τ
The dnaX gene was cloned into the pET16 expression vector in the steps shown in FIG. 9. First, the bulk of the gene was cloned into pET16 by removing the PmlI/XbaI fragment from pAlter dnaX, and placing it in to SmaI/XbaI digested Puc19 to yield Puc19dnaXCterm. The N-terminal sequence of the dnaX gene was then reconstructed to position an NdeI site at the N-terminus. This was performed by amplifying the 5′ region encoding the N-terminal section of Wγ/τ using an upstream primer containing an NdeI site that hybridizes to the dnaX gene at the initiating gtg codon (i.e. to encode Met where the Met is created by the PCR primer, and the Val is the initiating gtg start codon of dnaX). The primer sequence for this 5′ end was: 5′-gtggtg[0372] catatg gtg agc gcc ctc tac cgc c-3′ (SEQ. ID. No. 15) (where the NdeI site is underlined, and the coding sequence of dnaX follows). The downstream primer hybridizes past the PmlI site at nucleotide positions 987-1004 downstream of the initiating gtg (primer sequence: 5′-gtggtggtcgac cca gga ggg cca cct cca g-3′ (SEQ. ID. No. 16) where the initial 12 nucleotides contain a SalGI restriction site, followed by the sequence from the region downstream the stop codon). The 1.1 kb nucleotide PCR product was digested with PmlI/NdeI and the PmlI/NdeI fragment was ligated into NdeI/PmlI digested PUC19dnaXCterm to form PUC19dnaX. The PUC19dnaX plasmid was then digested with NdeI and SalI and the 1.9 kb fragment containing the dnaX gene was purified using the Sephaglas BandPrep Kit (Pharmacia-LKB). pET16b was digested with NdeI and XhoI. Then the full length dnaX gene was ligated into the digested pET16b to form pET dnaX.

EXAMPLE 4

Expression of [0373] T.th. γ and τ
As discussed in the previous example, the dnaX gene was engineered into the T7 based IPTG inducible pET16 vector such that the initiation codon was placed precisely following the Met residue N-terminal leader sequence (FIG. 9). This should produce a protein containing the entire sequence of γ and τ, along with a 21 residue leader containing 10 contiguous His residues (tagged-τ=60.6 kDa; tagged-γ=52.4 kDa for −2 frameshift). The pETdnaX plasmid was introduced into BL21 (DE3)pLysS cells harboring the gene encoding T7 RNA polymerase under control of the lac repressor. Log phase cells were induced with IPTG and analyzed before and after induction in an SDS polyacrylamide gel (FIG. 10, [0374] lanes 1 and 2). The result shows that upon induction, two new proteins are expressed with the approximate sizes expected of the T.th. γ and τ subunits (larger than E. coli γ, and smaller than E. coli τ). The two proteins are produced in nearly equal amounts, similar to the case of the E. coli γ and τ subunits. Western analysis using antibodies against the E. coli γ and τ subunits cross-reacted with the induced proteins further supporting their identity as T.th. γ and τ (data not shown, but repeated with the pure subunits shown in FIG. 10, lane 6).

EXAMPLE 5

Purification of [0375] T.th. γ and τ The His-tagged T.th. γ and τ proteins were purified from 6 L of induced E. coli cells containing the pET dnaX plasmid. Cells were lysed, clarified from cell debris by centrifugation and the supernatant was applied to a HiTrap chelate affinity column. Elution of the chelate affinity column yielded approximately 35 mg of protein in which the two predominant bands migrated in a region consistent with the molecular weight predicted from the dnaX gene (FIG. 10, lane 3), and produced a positive signal by Western analysis using polyclonal antibody directed against the E. coli γ and τ subunits lane 4). The γ and τ subunits are present in nearly equal amounts consistent with the nearly equal expression of these proteins in E. coli cells harboring the pETdnaX plasmid.
The γ and τ subunits were further purified by gel filtration on a Superose 12 column (FIG. 10, [0376] lane 4; FIG. 11). Recovery of T.th. γ and τ subunits through gel filtration was 81%. The. E. coli γ and τ subunits, when separated from one another, elute during gel filtration as tetramers. A mixture of E. coli γ/τ results in a mixed tetramer of y22 along with y4 and r4 tetramers (Onrust et al., 1995). The mixture of T.th. γ/τ elutes ahead of the 150 kDa marker, and thus is consistent with the expected mass of a γ2τ2 tetramer (225 kDa) and γ4 and τ4 tetramers.
As described earlier, the dnaX frameshifting sequence could produce either a −1 or −2 frameshift to yield a His-tagged γ subunit of mass either 53.3 kDa or 52.4 kDa, respectively. The difference in these two possible products is too close to determine from migration in SDS gels. It also remains possible that two γ products are present and do not resolve, under the conditions used. The exact protocol for this purification is described below. [0377]
Six liters of BL21 (DE3)pLysS pETdnaX cells were grown in LB media containing 50 μg/ml ampicillin and 25 μg/ml chloramphenicol at 37° C. to an O.D. of 0.8 and then IPTG was added to a concentration of 2 mM. After a further 2 h at 37° C., cells were harvested by centrifugation and stored at −70° C. The following steps were performed at 4° C. Cells (15 g wet weight) were thawed and resuspended in 45 [0378] ml 1× binding buffer (5 mM imidizole, 0.5 M NaCl, 20 mM Tris HCl (final pH 7.5)) using a ounce homogenizer to complete cell lysis and 450 ml of 5% polyamine P (Sigma) was added. Cell debris was removed by centrifugation at 18,000 rpm for 30 min. in a Sorvall SS24 rotor at 4° C. The supernatant (Fraction I, 40 ml, 376 mg protein) was applied to a 5 ml HiTrap Chelating Separose column (Pharmacia-LKB). The column was washed with 25 ml of binding buffer, then with 30 ml of binding buffer containing 60 mM imidizole, and then eluted with 30 ml of 0.5 M imidizole, 0.5 M NaCl, 20 mM Tris-HCl (pH 7.5). Fractions of 1 ml were collected and analyzed on an 8% Coomassie Blue stained SDS polyacrylamide gel. Fractions containing subunits migrating at the T.th γ and c positions, and exhibiting cross reactivity with antibody to E. coli γ and τ in a Western analysis, were pooled and dialyzed against buffer A (20 mM Tris-HCl (pH 7.5), 0.1 mM EDTA, 5 mM DTT and 10% glycerol) containing 0.5 M NaCl (Fraction II, 36 mg in 7 ml). Fraction II was diluted 2-fold with buffer A and passed through a 2 ml ATP agarose column equilibrated in buffer A containing 0.2 M NaCl to remove any E. coli γ complex contaminant. Then 0.18 mg (300 ml) Fraction II was gel filtered on a 24 ml Superose 12 column (Pharmacia-LKB) in buffer A containing 0.5 M NaCl. After the first 216 drops, fractions of 200 μl were collected (Fraction III) and analyzed by Western analysis (by procedures similar to those described in Example 6), by ATPase assays and by Coomassie Blue staining of an 8% Coomassie Blue stained SDS polyacrylamide gel. The Coomassie stained gels and Western analysis of recombinant T.th. gamma and tau for these purification steps are summarized in FIG. 10.

EXAMPLE 6

Western Analysis of [0379] T.th. Cells for Presence of γ and τ subunits
Polyclonal antibody to [0380] E. coli γ/τ—E. coli γ subunit was prepared as described (Studwell-Vaughan and O'Donnell, 1991). Pure γ subunit (100 μg) was brought up in Freund's adjuvant and injected subcutaneously into a New Zealand Rabbit (Poccono Rabbit Farms). After two weeks, a booster consisting of 50 μg γ in Freund's adjuvant was administered, followed after two weeks by a third injection (50 μg).
The homology between the amino terminal regions of [0381] T.th. and E. coli γ/τ subunits suggested that there may be some epitopes in common between them. Hence, polyclonal antibody directed against the E. coli γ/τ subunits was raised in rabbits for use in probing T.th. cells by Western analysis. FIG. 7 shows the results of a Western analysis of whole T.th. cells lysed in SDS. The results show that in T.th. cells, the antibody is rather specific for two high molecular proteins which migrate in the vicinity of the molecular masses of E. coli γ and τ subunits.
Procedure for Western Analysis [0382]
Samples were, analyzed in duplicate 10% SDS polyacrylamide gels by the Western method (Towbin et al. 1979). One gel was Coomassie stained to evaluate the pattern of proteins present, and the other gel was then electroblotted onto a nitrocellulose membrane (Schleicher and Schuell). For molecular size markers, the kaliedoscope molecular weight markers (Bio-Rad) were used to verify by visualization that transfer of proteins onto the blotted membrane had occurred. The gel used in electroblotting was also stained after electroblotting to confirm that efficient transfer of protein had occured. Membranes were blocked using 5% non-fat milk, washed with 0.05%[0383] ₀Tween in TBS (TBS-T) and then incubated for over 1 h with a {fraction (1/5000)} dilution of rabbit polyclonal antibody directed against E. coli γ and τ in 1% gelatin in TBS-T at room temperature. Membranes were washed using TBS-T buffer and then antibody was detected on X-ray film (Kodak) by using the ECL kit from (Amersham) and the manufactures recommended procedures.
Samples included: 1) a mixture of [0384] E. coli γ (15 ng) and τ (15 ng) subunits; 2) T.th. whole cells (100 μl) suspended in cracking buffer; and 3) purified T.th. γ and τ fraction II (0.6 fig as a mixture).

EXAMPLE 7

Characterization of the ATPase Activity of γ/τ[0385]
The [0386] E. coli τ subunit is a DNA dependent ATPase (Lee and Walker, 1987; Tsuchihashi and Kornberg, 1989). The γ subunit binds ATP but does not hydrolyze it even in the presence of DNA unless other subunits of the DNA polymerase III holoenzyme are also present (Onrust et al., 1991). Next we examined the T.th. γ/τ subunits for DNA dependent ATPase activity. The γ/τ preparation was, in fact, a DNA stimulated ATPase (FIG. 11, top panel). The specific activity of the T.th. γ/τ was 11.5 mol ATP hydrolyzed/mol γ/τ (as monomer and assuming an equal mixture of the two). Furthermore, analysis of the gel filtration column fractions shows that the ATPase activity coelutes with the T.th. γ/τ subunits, supporting evidence that the weak ATPase activity is intrinsic to the γ/τ subunits (FIG. 11). The specific activity of the γ/τ preparation before gel filtration was the same as after gel filtration (within 10%), further indicating that the DNA stimulated ATPase is an inherent activity of the γ/τ subunits. Presumably, only the τ subunit contains ATPase activity, as in the case of E. coli. Assuming only T.th. τ contains ATPase activity, its specific activity is twice the observed rate (after factoring out the weight of y). This rate is still only one-fifth that of E. coli τ.
The [0387] T.th. γ/τ ATPase activity is lower at 37° C. than at 65° C. (middle panel), consistent with the expected behavior of protein activity from a thermophilic source. However, there is no apparent increase in activity in proceeding from 50° C. to 65° C. (the rapid breakdown of ATP above 65° C. precluded measurement of ATPase activity at temperatures above 65° C.). In contrast, the E. coli τ subunit lost most of its ATPase activity upon elevating the temperature to 50° C. (middle panel). These reactions contain no stabilizers such as a nonionic detergent or gelatin, nor did they include substrates such as ATP, DNA or magnesium.
Last, the relative stability of [0388] T.th. γ/τ and E. coli γ/τ to addition of NaCl (FIG. 12, bottom panel) was examined. Whereas the E. coli τ subunit rapidly lost activity at even 0.2 M NaCl, the T.th. γ/τ retained full activity in 1.0 M NaCl and was still 80% active in 1.5 M NaCl. The detailed procedure for the ATPase activity assay is described below.
ATPase assays [0389]
ATPase assays were performed in 20 μl of 20 mM Tris-HCl (pH 7.5), 8 mM MgCl[0390] ₂containing 0.72 μg of M13mp18 ssDNA (where indicated), 100 mM [γ-³²P]-ATP (specific activity of 2000-4000 cpm/pmol), and the indicated protein. Some reactions contained additional NaCl where indicated. Reactions were incubated at the temperatures indicated in the figure legends for 30 min. and then were quenched with an equal volume of 25 mM EDTA (final). The aliquots were analyzed by spotting them (1 ill each) onto thin layer chromatography (TLC) sheets coated with Cel-300 polyethyleneimine (Brinkmann Instruments Co.). TLC sheets were developed in 0.5 M lithium chloride, 1 M formic acid. An autoradiogram of the TLC chromatogram was used to visualize Pi at the solvent front and ATP near the origin which were then cut from the TLC sheet and quantitated by liquid scintillation. The extent of ATP hydrolyzed was used to calculate the mol of Pi released per mol of protein per min. One mol of E. coli τ was calculated assuming a mass of 71 kDa per monomer. The T.th. γ and τ preparation was treated as an equal mixture and thus one mole of protein as monomer was the average of the predicted masses of the γ and τ subunits (54 kDa).

EXAMPLE 8

Homolog of [0391] T.th. γ/τ to dnaX Gene Products of Other Organism
The XbaI insert encoded an open reading frame, starting with a GTG codon, of 529 amino acids in length (58.0 kDa), closer to the predicted length of the [0392] B. subtilis τ subunit (563 amino acids, 62.7 kDa mass)(Alonso et al., 1990) than the E. coli τ subunit (71.1 kDa)(Yin et al., 1986). The dnaX gene encoding the γ/τ subunits of E. coli DNA polymerase III holoenzyme is homologous to the holB gene encoding the δ′ subunit of the γ complex clamp loader, and this homology extends to all 5 subunits of the eukaryotic RFC clamp loader as well as the bacteriophage gene protein 44 of the gp44/62 clamp loading complex (O'Donnell et al., 1993). These gene products show greatest homology over the N-terminal 166 amino acid residues (of E. coli dnaX); the C-terminal regions are more divergent. FIG. 4 shows an alignment of the amino acid sequence of the N-terminal regions of the T.th. dnaX gene product to those of several other bacteria. The consensus GXXGXGKT (SEQ. ID. No. 17) motif for nucleotide binding is conserved in all these protein products. Further, the E. coli δ′ crystal structure reveals one atom of zinc coordinated to four Cys residues (Guenther, 1996). These four Cys residues are conserved in the E. coli dnaX gene, and the γ and τ subunits encoded by E. coli dnaX bind one atom of zinc. These Cys residues are also conserved in T.th. dnaX (shown in FIG. 4). Overall, the level of amino acid identity relative to E. coli dnaX in the N-terminal 165 residues of T.th. dnaX is 53%. The T.th. dnaX gene is just as homologous to the B. subtilis dnaX (53% identity) gene relative to E. coli dnaX. After this region of homology, the C-terminal region of T.th. dnaX shares 26% and 20% identity to E. coli and B. subtilis dnaX, respectively. A proline rich region, downstream of the conserved region, is also present in T.th. dnaX (residues 346-375), but not in the B. subtilis dnaX (see FIGS. 3A and 3B). The overall identity between E. coli dnaX and T.th. dnaX over the entire gene is 34%. Identity of T.th. dnaX to B. subtilis dnaX over the entire gene is 28%.
Comparison of dnaX Genes from [0393] T.th. and E. coli
The above identifies a homologue of the dnaX gene of [0394] E. coli in Thermus thermophilus. Like the E. coli gene, T.th. dnaX encodes two related proteins through use of a highly efficient translational frameshift. The T.th. γ/τ subunits are tetramersi or mixed tetramers, similar to the γ and τ subunits of E. coli. Further, the γ/τ subunit is a DNA stimulated ATPase like its E coli counterpart. As expected for proteins from a thermophile, the T.th. γ/τ ATPase activity is thermostabile and resistant to added salt.
In [0395] E. coli, γ is a component of the clamp loader, and the τ subunit serves the function of holding the clamp loading apparatus together with two DNA polymerases for coordinated replication of duplex DNA. The presence of γ in T.th. suggests it has a clamp loading apparatus and thus a clamp as well. The presence of the τ subunit of T.th. implies that T.th. contains a replicative polymerase with a structure similar to that of E. coli DNA polymerase III holoenzyme.
A significant difference between [0396] E. coli and T.th. dnaX genes is in the translational frameshift sequence. In E. coli, the heptamer frameshift site contains six A residues followed by a G residue in the context A AAA AAG. This sequence satisfies the X XXY YYZ rule for −1 frameshifting. The frameshift is made more efficient by the absence of the AAG tRNA for Lys which presumably leads to stalling of the ribosome at the frameshift site and increases the efficiency of frameshifting (Tsuchihashi and Brown, 1992). Two additional aids to frameshifting include a downstream hairpin and an upstream Shine-Dalgarno sequence (Tsuchihashi and Kornberg, 1990; Larsen et al., 1994). The −1 frameshift leads to incorporation of one unique residue at the C-terminus of E. coli γ before encounter with a stop codon.
In [0397] T.th., the dnaX frameshifting heptamer is A AAA AAA, and it is flanked by two other A residues, one on each side. There is also a downstream region of secondary structure. The nearest downstream stop codon is positioned such that gamma would contain only one unique amino acid, as in E. coli. However, the T.th. stop codon is in the −2 reading frame thus requires a −2 frameshift. No precedent exists in nature for −2 frameshifting, although −2 frameshifting has been shown to occur in test cases (Weiss et al., 1987). In vivo analysis of the T.th. frameshift sequence shows that this natural sequence promotes both −1 and −2 frameshifting in E. coli. Whereas the −2 frameshift results in only one unique C-terminal residue, a −1 frameshift would result in an extension of 12 C-terminal residues. At present, the results do not discriminate which path occurs in T.th., a −1 or −2 frameshift, or a combination of the two.
There are two Shine-Dalgarno sequences just upstream of the frameshift site in [0398] T.th. dnaX. In two cases of frameshifting in E. coli, an upstream Shine-Dalgarno sequence has been shown to stimulate frameshifting (reviewed in Weiss et al., 1897). In release factor 2 (RF2), the Shine-Dalgarno is 3 nucleotides upstream of the shift site, and it stimulates a +1 frameshift event. In the case of E. coli dnaX; a Shine-Dalgarno sequence 10 nucleotides upstream of the shift sequence stimulates the −1 frameshift. One of the T.th. dnaX Shine-Dalgarno sequences is immediately adjacent to the frameshift sequence with no extra space, the other is 22 residues upstream of the frameshift site. Which of these Shine-Dalgarno sequences plays a role in T.th. dnaX frameshifting, if any, will require future study.
In [0399] E. coli, efficient separation of the two polypeptides, γ and τ, is achieved by mutation of the frameshift site such that only one polypeptide is produced from the gene (Tsuchihashi and Kornberg, 1990). Substitution of G-to-A in two positions of the heptamer of T.th. dnaX eliminates frameshifting and thus should be a source to obtain τ subunit free of γ. To produce pure γ subunit free of τ, the frameshifting site and sequence immediately downstream of it can be substituted for an in-frame sequence with a stop codon.
Examination of the [0400] B. subtilis dnaX gene shows no frameshift sequence that satisfies the X XXY YYZ rule. Hence, it would appear that dnaX does not make two proteins in this gram positive organism.
Rapid thermal motions associated with high temperature may make coordination of complicated processes more difficult. It seems possible that organizing the components of the replication apparatus may become yet more important at higher temperature. Hence, production of a τ subunit that could be used to crosslink two polymerases and a clamp loader into one organized particle may be most useful at elevated temperature. [0401]
As stated above, the following examples describe the continued isolation and purification of the substantial entirety of the Polymerase III from the extreme thermophile [0402] Thermus thermophilus. It is to be understood that the following exposition is reflective of the protocol and characteristics, both morphological and functional, of the Polymerase III-type enzymes that are the focus of the present invention, and that the invention is hereby illustrated and comprehends the entire class of enzymes of thermophilic origin.

EXAMPLE 9

Purification of the [0403] Thermus thermophilus DNA Polymerase III
All steps in the purification assay were performed at 4° C. The following assay was used in the purification of DNA polymerase from [0404] T.th. cell extracts. Assays contained 2.5 mg activated calf thymus DNA (Sigma Chemical Company) in a final volume of 25 ml of 20 mM Tris-Cl (pH 7.5), 8 mM MgCl₂, 5 mM DTT, 0.5 Ii EDTA, 40 mg/ml BSA, 4% glycerol, 0.5 mM ATP, 3 mM each dCTP, dGTP, DATP, and 20 mM [α-³²P]dTTP. An aliquot of the fraction to be assayed was added to the assay mixture on ice followed by incubation at 60° C. for 5 min. DNA synthesis was quantitated using DE81 paper followed by washing off unincorporated nucleotide. Incorporated nucleotide was determined by scintillation counting of the filters.
[0405] Thermus thermophilus cell extracts were prepared by suspending 35 grams of cell paste in 200 ml of 50 mM TRIS-HCl, pH=7.5, 30 mM spermidine, 100 mM NaCl, 0.5 mM EDTA, 5 mM DTT, 5% glycerol, followed by disruption by passage through a French pressure cell (15,000 PSI). Cell debris was removed by centrifugation (12,000 RPM, 60 min). DNA polymerase III in the clarified supernatant was precipitated by treatment with ammonium sulphate (0.226 gm/liter) and recovered by centrifugation. This fraction was then backwashed with the same buffer (but lacking spermidine) containing 0.20 gm/l ammonium sulfate. The pellet was then resuspended in buffer A and dialyzed overnight against 2 liters of buffer A; a precipitate which formed during dialysis was removed by centrifugation (17,000 RPM, 20 min).
The clarified dialysis supernatant, containing approximately 0.336 mg of protein, was applied onto a 60 ml heparin agarose column equilibrated in buffer A which was washed with the same buffer until A280 reached baseline. The column was developed with a 500 ml linear gradient of buffer A from 0 to 500 mM NaCl. More tightly adhered proteins were washed off the column by treatment with buffer A (20 mM Tris HCl, pH=7.5, 0.1 mM EDTA, 5 mM DTT, and 10% glycerol) and 1M NaCl. Some DNA polymerase activity flowed through the column. Two peaks (HEP.P1 and HEP.P2) of DNA polymerase activity eluted from the heparin agarose column containing 20 mg and 2 mg of total protein respectively (FIG. 13A). These were kept separate throughout the remainder of the purification protocol. [0406]
The Pol III resided in HEP.P1 as indicated by the following criteria: 1) Western analysis using antibody directed against the α subunit of [0407] E. coli Pol III indicated presence of Pol III in HEP.P1; 2). Only the HEP.P1 fraction was capable of extending a single primer around an M13 mp187.2 kb ssDNA circle (explained later in Example 16), such long primer extension being a characteristic of Pol III type enzymes; and 3) Only the HEP.P1 provided DNA polymerase activity that was retained on an ATP-agarose affinity column, which is indicative of a Pol III-type DNA polymerase since the γ and τ subunits are ATP interactive proteins.
The first peak of the heparin agarose column (HEP.P1: 20 mg in 127.5 ml) was dialyzed against buffer A and applied onto a 2 ml N6-linkage ATP agarose column pre-equilibrated in the same buffer. Bound protein was eluted by a slow (0.05 ml/min) wash with buffer A +2M NaCl and collected into 200 μl fractions. Chromatography of peak HEP.P1 yielded a flow-through (HEP.P1-ATP-FT) and a bound fraction (HEP.P1-ATP-Bound) (FIG. 13B). Binding of peak HEP.P2 to the ATP column could not be detected, though DNA polymerase activity was recovered in the flow-through. [0408]
The HEP.P1-ATP-Bound fractions from the ATP agarose chromatographic step were further purified by anion exchange over monoQ. The HEP.P1-ATP-Bound fractions were diluted with buffer A to approximately the conductivity of buffer A plus 25 mM NaCl and applied to a 1 ml monoQ column equilibrated in Buffer A. DNA polymerase activity eluted in the flow-through and in two resolved chromatographic peaks (MONOQ peak1 and peak2) (FIG. 13C). Peak 2 was by far the major source of DNA polymerase activity. Western analysis using rabbit antibody directed against the [0409] E. coli α subunit confirmed presence of the α subunit in the second peak (see the Western analysis in FIG. 14B). Antibody against the E. coli τ subunit also confirmed the presence of the τ subunit in the second peak. Some reaction against α and τ was also present in the minor peak (first peak). The Coomassie Blue SDS polyacrylamide gel of the MonoQ fractions (FIG. 14A) showed a band that co-migrated with E. coli a and was in the same post1 on as the antibody reactive material (antibody against E. coli α). Also present are bands corresponding to τ, γ, δ, and δ′. These subunits, along with 1, are all that is necessary for rapid and processive synthesis and primer extension over a long (>7 kb) stretch of ssDNA in the case of E. coli DNA Polymerase III holoenzyme.
The Pol III-type enzyme purified from [0410] T.th. may be a Pol III*-like enzyme that contains the DNA polymerase and clamp loader subunits (i.e., like the Pol III* of E. coli). The evidence for this is: 1) the presence of dnaX and dnaE gene products in the same column fractions as indicated by Western analysis (see above); 2) the ability of this enzyme to extend a primer around a 7.2 kb circular ssDNA upon adding only β (see Example 16); 3) stimulation of Pol III by adding β on linear DNA, indicating β subunit is not present in saturating amounts (see Example 15); and 4) the presence of τ in T.th. which may glue the polymerase and clamp loader into a Pol III* as in E. coli; and 5) the comigration of a with subunits τ, γ, δ and δ′ of the clamp loader in the column fractions of the last chromatographic step (MonoQ, FIG. 14A). Micro-sequencing of T.th DNA Polymerase III cc subunit The α subunit from the purified T.th DNA polymerase III (HEP.P1.ATP-Bound.MONOQ peak2) was blotted onto PVDF membrane and was cut out of the SDS-PAGE gel and submitted to the Protein-Nucleic Acid Facility at Rockefeller University for N-terminal sequencing and proteolytic digestion, purification and microsequencing of the resultant peptides. Analysis of the a candidate band (Mw 130 kD) yielded four peptides, two of which (TTH1, TTH2) showed sequence similarity to α subunits from various bacterial sources (see FIG. 15).

EXAMPLE 10

Identification of the [0411] Thermus thermophilus dnaE Gene Encoding the a Subunit of DNA Polymerase III Replication Enzyme
Cloning of the dnaE gene was started with the sequence of the TTH1 peptide from the purified α subunit (FFIEIQNHGLSEQK) (SEQ. ID. No. 61). The fragment was aligned to a region at approximately 180 amino acids downstream of the N-termini of several other known α subunits as shown in FIG. 15. The upstream 33mer (5′-GTG[0412] GGATCCGTGGTTCTGGATCTCGATGAAGAA-3′) (SEQ. ID. No. 31) consists of a BamHI site within the first 9 nucleotides (underlined) and the sequence coding for the following peptide HGLSEQK on the complementary strand. The downstream 29mer (5′-GTGGGATCCACGGSCTSTCSGAGCAGAAG-3′) (SEQ. ID. No. 32) consists of a BamHI site within the first 9 nucleotides (underlined) and the following sequence coding for the peptide FFIEIQNH (SEQ. ID. No. 62).
These two primers were directed away from each other for the purpose of performing inverse PCR (also called circular PCR). The amplification reactions contained 10 ng [0413] T.th. genomic DNA (that had been cut and religated with XmaI), 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl The moPol Buffer, 0.5 mM of each dNTP and 0.25 mM MgSO₄. Amplification was performed using the following cycling scheme:
1. 4 cycles of: 95.5° C.—30 sec., 45° C.—30 sec., 75° C.—8 min. [0414]
2. 6 cycles of: 95.5° C.—30 sec., 50° C.—30 sec., 75° C.—6 min. [0415]
3.30 cycles of: 95.5° C.—30 sec., 52.5° C.—30 sec., 75° C.—:5 min. [0416]
A 1.4 kb fragment was obtained and cloned into pBS-SK:BamHI (i.e. pBS-SK (Stratragene) was cut with BamHI). This sequence was bracketted by the 29mer primer on both sides and contained the sequence coding for the N-terminal part of the subunit up to the peptide used for primer design. [0417]
To obtain further dnaE gene sequence, the TTH2 peptide was used. It was aligned to a region about 600 amino acids from the N-termini of the other known subunits (FIG. 15B). [0418]
The upstream 34mer (5′-GCGG[0419] GATCCTCAACGAGGACCTCTCCATCTTCAA-3′) (SEQ. ID. No. 33) consists of a BamHI site within the first 9 nucleotides (underlined) and the sequence from the end of the fragment previously obtained. The downstream 35mer (5′-GCGGGATCCTTGTCGTCSAGSGTSAGSGCGTCGTA-3′) (SEQ. ID. No. 34) consists of a BamHI site within the first 9 nucleotides (underlined) and the following sequence coding for the peptide YDALTLDD (SEQ. ID. No. 63) on the complementary strand. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 0.5 mM of each dNTP and 0.25 mM MgSO₄. Amplification was performed using the following cycling scheme:
1. 4 cycles of: 95.5° C.—30 sec., 45° C.—30 sec., 75° C.—8 min.: [0420]
2.6 cycles of: 95.5° C. 30 sec., 50° C.—30 sec., 75° C.—6 min. [0421]
3. 30 cycles of: 95.5° C.—30 sec., 55° C. 30 sec., 75° C.—5 min. [0422]
A 1.2 kb PCR fragment was obtained and cloned into pUC19:BamHI. The fragment was bracketted by the downstream primer on both sides and contained the region overlapping in 56 bp with the fragment previously cloned. [0423]
To obtain yet more dnaE sequence, the following primers were used. The upstream 39mer (3′-GTGT[0424] GGATCCTCGTCCCCCTCATGCGCGACCAGGAAGGG-5′) (SEQ. ID. Nos. 35 and 114) consists of a BamHI site within the first 10 nucleotides (underlined) and the sequence from the end of the fragment previously obtained. The downstream 27mer (5′-GTGTGGATCCTTCTTCTTSCCCATSGC-3′) (SEQ. ID. No. 36) consists of a BamHI site within the first 10 nucleotides (underlined), and the sequence coding for the peptide AMGKKK (SEQ. ID. No. 64) (at position approximately 800 residues from the N terminus) on the complementary strand. The AMGKKK (SEQ. ID. No. 64) sequence was chosen for primer design as it is highly conserved among the known gram-negative α subunits. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Taq polymerase reaction mixture containing 10 μl PCR Buffer, 0.5 mM of each dNTP and 2.5 mM MgCl₂. Amplification was performed using the following cycling scheme:
1.3 cycles of: 95.5° C.—30 sec., 45° C.—30 sec., 72° C.—8 min. [0425]
2.6 cycles of: 94.5° C.—30 sec., 55° C.—30 sec., 72° C.—6 min. [0426]
3.32 cycles of: 94.5° C.—30 sec., 50° C.—30 sec., 72° C.—5 min. [0427]
A 2.3 kb PCR fragment was obtained instead of the expected 0.6 kb fragment. BamHI digestion of the PCR product resulted in three fragments of 1.1 kb, 0.7 kb and 0.5 kb. The 1.1 kb fragment was cloned into pUC19:BamHI. It turned out to be the one adjacent to the fragment previously obtained and contained the dnaE sequence right up to the region coding for the AMGKKK (SEQ. ID. No. 64) peptide, but was disrupted by an intron just upstream of this region. The sequence that follows this was amplified from the 2.3 kb original PCR product using the same conditions and cycling scheme as for the 2.3 kb fragment. The downstream primer was the same as in the previous step. The upstream 27mer (3′-GTGTGGATCCGTGGTGACCTTAGCCAC-5′) (SEQ. ID. Nos. 37 and 115) consisted of a BamHI site within the first 9 nucleotides (underlined) and the sequence from the end of the 1.1 kb fragment previously described. [0428]
The expected 1.2 kb PCR fragment was obtained and cloned into pUC19:SmaI. This fragment coded for the rest of the intein and the end of it was used to obtain the next sequence of dnaE downstream of this region. The upstream 30mer (3′-TTCGTGTCCGAGGACCTTGTGGTCCACAAC-5′) (SEQ. ID. Nos. 38 and 116) was a sequence from the end of the intron. The downstream 23mer (5′-CCAGAATCGTCTGCTGGTCGTAG-3′) (SEQ. ID. No. 39) was the sequence from the end of the dhaE gene of [0429] D.rad. (coding on the complementary strand for the region slightly homologous in the distantly related α subunits and possibly highly homologous between T.th. and D.rad. α subunits). The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl Thermo Pol Buffer, 0.5 mM of each dNTP and 0.1 mM MgSO₄. Amplification was performed using the following cycling scheme:
1. 3 cycles of: 95.5° C.—30 sec., 55° C.—30 sec., 75° C.—8 min. [0430]
2. 32 cycles of: 94.5° C.—30 sec., 50° C.—30 sec., 75° C.—5 min. [0431]
A 2.5 kb PCR fragment was obtained and cloned into pUC19:SmaI. This fragment contained the dnaE sequence coding for the 300 amino acids next to the AMGKKK (SEQ. ID. No. 64) region disrupted by yet a second intein inside another sequence that is conserved among the known α subunits (FNKSHSAAY) (SEQ. ID. No. 65). [0432]
To obtain the rest of the dnaE gene the upstream 19mer (5′-AGCACCCTGoGAGGAGCTTC-3′) (SEQ. ID. No. 40) from the end of the known dnaE sequence was used. The downstream primer was: 5′-CATGTCGTACTGGGTGTAC-3′ (SEQ. ID. No. 41). The amplification reactions contained 10 ng [0433] T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl Thermo P61 Buffer, 0.5 mM of each dNTP and 0.1 mM MgSO₄. Amplification was performed using the following cycling scheme:
1. 3 cycles of: 95.5° C.—30 sec., 55° C.—30 sec., 75° C.—8 min. [0434]
2. 32 cycles of: 94.5° C.—30 sec., 50° C.—30 sec., 75° C.—5 min. [0435]
A 1.0 kb fragment bracketed by this upstream primer was obtained. It contained the 3′ end of the dnaE gene. [0436]

EXAMPLE 11

Cloning and Expression of the [0437] Thermus thermophilus dnaQ Gene Encoding the ε Subunit of DNA Polymerase III Replication Enzyme
Cloning of dnaQ [0438]
The dnaQ gene of [0439] E. coli and the corresponding region of POlC of B. subtilis, evolutionary divergent organisms, share approximately 30% identity. Comparison of the predicted amino acid sequences for DnaQ (e) of E. coli and PolC of B. subtilis revealed two highly conserved regions (FIG. 17). Within each of these regions, a nine amino acid sequence was used to design two oligonucleotide primers for use in the polymerase chain reaction.
The regions highly conservative among Pol III exonucleases were chosen to design the degenerate primers for the amplification of a [0440] T.th. dnaQ internal fragment (see FIG. 17). DNA oligonucleotides for amplification of T.th. genomic DNA were as follows. The upstream 27mer (5′-GTSGTSNNSGACNNSGAGACSACSGGG-3′ (SEQ. ID. No. 42)) encodes the following sequence (VVXDXETTG) (SEQ. ID. No. 66). The downstream 27mer (5′-GAASCCSNNGTCGAASNNGGCGTTGTG-3′) (SEQ. ID. No. 43) encodes the sequence HNAXFDXGF (SEQ. ID. No. 67) on the complementary strand. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 0.5 mM of each dNTP and 0.5 mM MgSO₄. Amplification was performed using the following cycling scheme:
1. 5 cycles of: 95.5° C.—30 sec., 40° C.—30 sec., 72° C.—2 min. [0441]
2. 5 cycles of: 95.5° C.—30 sec., 45° C.—30 sec., 72° C.—2 min. [0442]
3. 30 cycles of: 95.5° C.—30 sec., 50° C.—30 sec., 72° C.—30 min. [0443]
Products were visualized in a 1.5% native agarose gel. A fragment of the expected size of 270 bp was cloned into the SmaI site of pUC19 and sequenced with the Circumvent Thermal Cycle DNA sequencing kit according to the manufacturer's instructions (New England Biolabs). [0444]
To obtain further sequence of the dnaQ gene, genomic DNA was digested with either mhol, BamHI, KpnI or NcoI. These restriction enzymes were chosen because they cut [0445] T.th. genomic DNA frequently. Approximately 0.1 μg of DNA for each digest was ligated by T4 DNA ligase in 50 μl of ligation buffer (50 mM Tris-HCl (pH 7.8), 10 mM MgCl₂, 10 mM dithiothreitol, 1 mM ATP, 25 mg/ml bovine serum albumin) overnight at 20° C. The ligation mixtures were used for circular PCR.
DNA oligonucleotides for amplification of [0446] T.th. genomic DNA were the following. The upstream 27mer (5′-CGGGGATCCACCTCAATCACCTCGTGG-3′) (SEQ. ID. No. 44) consists of a BamHI site within the first 9 nucleotides (underlined) and the sequence complementary to 42-61 bp region of the previously cloned dnaQ fragment. The downstream 30mer (5′-CGGGGATCCGCCACCTTGCGGCTCCGGGTG-3′) (SEQ. ID. No. 45) consists of a BamHI site within the first 9 nucleotides (underlined) and the sequence corresponding to 240-261 bp region of the dnaQ fragment (see FIG. 17).
The amplification reactions contained 1 ng [0447] T.th. genomic DNA (that had been cut with NcoI and religated into circular DNA for circular PCR), 0.4 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 0.5 mM of each dNTP, 0.5 mM MgSO₄, and 10% DMSO. Circular amplification was performed using the following cycling scheme:
1. 5 cycles of: 95.5° C.—30 sec., 50° C.—30 sec., 72° C.—8 min. [0448]
2. 35 cycles of: 95.5° C.—30 sec., 55° C.—30 sec., 72° C.—6 min. [0449]
3. 72° C.—10 min. [0450]
A 1.5 kb fragment was obtained and cloned into the BamHI site of the pUC19 vector. Partial sequencing of the fragment reveiled that it contained the dnaQ regions adjacent to sequences corresponding to the PCR primers and hence contained the sequences both upstream and downstream of the previously cloned dnaQ fragment. One of NcoI sites turned out to be approximately 300 bp downstream of the end of the first cloned dnaQ sequence and hence did not include the 3′ end of dnaQ. To obtain the 3′ end, another inverse PCR reaction was performed. Since an ApaI restiction site was recognized within this newly sequenced dnaQ fragment, the circular PCR procedure was performed using as template an ApaI digest of [0451] T.th. genomic DNA that was ligated (circularized) under the same conditions as described above.
DNA oligonucleotides for amplification of the ApaI/religated [0452] T.th. genomic DNA were as follows. The upstream 31 mer (5′-GCGCTCTAGACGAGTTCCCAAAGCGTGCGGT-3′) (SEQ. ID. No. 46) consists of a mbaI site within the first 10 nucleotides (underlined) and the sequence complementary to the region downstream of the ApaI restriction site in the newly sequenced dnaQ fragment. The downstream 25 mer (5′-CGCGTCTAGATCACCTGTATCCAGA-3′) (SEQ. ID.:No. 47) consists of a XbaI site within the first 10 nucleotides (underlined) and the sequence corresponding to another region downstream of the ApaI restriction site in the newly sequenced dnaQ fragment. The 1.7 kb PCR fragment was cloned into the XbaI site of the pUC19 vector and partially sequenced. The sequence of dnaQ, and the protein sequence of the ε subunit encoded by it, is shown in FIG. 18.
The dnaQ gene is encoded by an open reading frame of 209 (or 190 depending on which Val is used as the initiating residue) amino acids in length (23598.5 kDa- or 21383.8 kDa for shorter version), similar to the length of the [0453] E. coli ε subunit (243 amino acids, 27099.1 kDa mass) (see FIG. 17).
The entire amino acid sequence of the ε subunit predicted from the [0454] T.th. dnaQ gene aligns with the predicted amino acid sequence of the dnaQ genes of other organisms with only a few gaps and insertions (the first two amino acids, and four positions downstream) (FIG. 17). The consensus motifs VVXDXETTG (SEQ. ID. Nos. 66 and 68), HNAXFDXGF (SEQ. ID. No. 67), and HRALYD (SEQ. ID. No. 70), characteristic for exonucleases, are conserved. Overall, the level of amino acid identity relative to most of the known ε subunits, or corresponding proofreading exonuclease domains of gram positive PolC genes is approximately 30%. Upstream of start 1 (FIG. 17) there were stop codons in all three reading frames.
Expression of dnaQ [0455]
The dnaQ gene was cloned gene into the pET24-a expression vector in two steps. First, the PCR fragment encoding the N-terminal part of the gene was cloned into the pUC19 plasmid, containing the ApaI inverse PCR fragment into NdeI/ApaI sites. DNA oligonucleotides for amplification of [0456] T.th. genomic DNA were as follows. The upstream 33mer (5′-GCGGCGCATATGGTGGTGGTCCTGGACCTGGAG-3′) (SEQ. ID. No. 48) consists of an NdeI site within the first 12 nucleotides (underlined) and the beginning of the dnaQ gene. The downstream 25 mer (5′-CGCGTCTAGATCACCTGTATCCAGA-3′) (SEQ. ID. No. 49), already used for ApaI circular PCR, consists of an XbaI site within the first 10 nucleotides (underlined) and the sequence corresponding to the region downstream of the ApaI restriction site., The 2.2 kb NdeI/SalI fragment was then cloned into the NdeI/XhoI sites of the pET16 vector to produce pET24-a:dnaQ. The ε subunit was expressed in the BL21/LysS strain transformed by the pET24-a:dnaQ plasmid.

EXAMPLE 12

The [0457] Thermus thermophilus dnaN Gene Encoding the β Subunit of DNA Polymerase III Replication Enzyme
Strategy of Cloning dnaN by Use of DnaN [0458]
DnaN proteins are highly divergent in bacteria making it difficult to clone them by homology. The level of identity between DnaN representatives from [0459] E. coli and B. subtilis is as low as 18%. These 18% of identical amino acid residues are dispersed through the proteins rather then clustering together in conservative regions, further complicating use of homology to design PCR primers. However, one feature of dnaN genes among widely different bacteria is their location in the chromosome. They appear to be near the origin, and immediately adjacent to the dnaa gene. The dnaa genes show good homology among different bacteria and, thus, dnaa was first cloned in order to obtain a DNA probe that is likely near dnaN.
Identification of dnaA and dnaN [0460]
The dnaA genes of [0461] E. coli and B. subtilis share 58% identity at the amino acid sequence level within the ATP-binding domain (or among the representatives of gram-positive and gram-negative bacteria, evolutionary divergent organisms). Comparison of the predicted amino acid sequences encoded by dnaA of E. coli and B. subtilis revealed two highly conserved regions (FIG. 19). Within each of these regions, a seven amino acid sequence was used to design two oligonucleotide primers for use in the polymerase chain reaction. The DNA oligonucleotides for amplification of T.th. genomic DNA were as follows. The upstream 20mer (5′-GTSCTSGTSAAGACSCACTT-3′) (SEQ. ID. No. 50) encodes the following sequence: VLVKTHL (SEQ. ID. No. 69). The downstream 21mer (5′-SAGSAGSGCGTTGAASGTGTG-3′, where S is G or C) (SEQ. ID. No. 51) encodes the sequence: HTFNALL (SEQ. ID. No. 71), on the complementary strand. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 mM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl TherinoPol Buffer, 0.5 mM of each dNTP and 0.5 mM MgSO₄. Amplification was performed using the following cycling scheme:
1. 5 cycles of: 95.5° C.—30 sec., 45° C.—30 sec., 75° C.—2 min. [0462]
2. 5 cycles of: 95.5° C.—30 sec., 50° C.—30 sec., 75° C.—2 min. [0463]
3. 30 cycles of: 95.5° C.—30 sec., 52° C.—30 sec., 75° C.—30 min. [0464]
Products were visualized in a 1.5% native agarose gel. A fragment of the expected size of 300 bp was cloned into the SmaI site of pUC19 and sequenced with the CircumVent Thermal Cycle DNA sequencing kit (New England Biolabs). [0465]
To obtain a larger section of the [0466] T.th. dnaA gene, genomic DNA was digested with either HaeII, HindIII, KasI, KpnI, MluI, NcoI, NgoMI, NheI, NsiI, PaeR7I, PstI, SacI, SalI, SpeI, SphI, StuI, or XhoI, followed by Southern analysis in a native agarose gel. The filter was probed with the 300 bp PCR product radiolabeled by random priming. Four different restriction digests showed a single fragment of reasonable size for further cloning. These were, KasI, NgoMI, and StuI, all of which produced fragments of about 3 kb, and NcoI that produced a 2 kb fragment. Also, a KpnI digest resulted in two fragments of about 1.5 kb and 10 kb.
Genomic DNA digests using either NgoMI and StuI were used to obtain the dnaA gene by inverse PCR (also referred to as circular PCR). In this procedure, 0.1 μg of DNA from each digest was treated separately with T4 DNA ligase in 50 μl of ligation buffer (50 mM Tris-HCl (pH 7.8), 10 mM MgCl[0467] ₂, 10 mM dithiothreitol, 1 mM ATP, 25 mg/ml bovine serum albumin) overnight at 20° C. This results in circularizing the genomic DNA fragments. The ligation mixtures were used as substrate in inverse PCR.
DNA oligonucleotides for amplification of recircularized [0468] T.th. genomic DNA were as follows. The upstream 22mer was (5′-CTCGTTGGTGAAAGTTTCCGTG-3′) (SEQ. ID. No. 52), and the downstream 24mer was (5′-CGTCCAGTTCATCGCCGGAAAGGA-3′) (SEQ. ID. No. 53). The amplification reactions contained 5 ng T.th. genomic DNA, 0.5 μM of each primer, in a volume of 100 μl of Taq polymerase reaction mixture containing 10 μl PCR Buffer, 0.5 mM of each dNTP and 2.5 mM MgCl₂. Amplification was performed using the following cycling scheme:
1. 5 cycles of: 95.0° C.—30 sec., 55° C.—30 sec., 72° C.—10 min. [0469]
2. 35 cycles of: 95.5° C.—30 sec., 50° C.—30 sec., 72° C.—8 min. [0470]
The PCR fragments of the expected length for NgoMI and StuI treated and then ligated chromosomal DNA were digested with either BamHI or Sau3a and cloned into pUC19:BamHI and pUCC19:(BamHI+SmaI) and sequenced with CircumVent Thermal Cycle DNA sequencing kit. The 1.6 kb (BamHI+BamH) fragment from the NgoMI PCR product contained a sequence coding for the N-terminal part of dnaN, followed by the gene for enolase. The 1 kb (Sau3a+Sau3a) fragment from the samne PCR product included the start of dnaN gene and sequence characteristic of the origin of replication (i.e., 9mer DnaA-binding site sequences). The 0.6 kb (BamHI+BamHI) fragment from the StuI PCR reaction contained starts for dnaA and gidA genes in inverse orientation to each other. The 0.4 kb (Sau3a+Sau3a) fragment from the same PCR product contained the 3′ end of the dnaA gene and DNA sequence characteristic for the origin of replication. [0471]
This sequence information provided the beginning and end of both the dnaA and the dnaN genes. Hence, these genes were easily cloned from this information. Further, the dnaN gene was readily cloned and expressed in a pET24-a vector. These steps are described below. [0472]
Cloning and Sequence of the dnaA Gene [0473]
The dnaA gene was cloned for sequencing in two parts: from the potential start of the gene up to its middle and from the middle up to the end. For the N-terminal part, the upstream 27mer (5′-TCTGGCAACACGTTCTGGAGCACATCC-3′) (SEQ. ID. No. 54) was 20 bp downsteam of the potential start codon of the gene. The downstream 23mer (5′-TGCTGGCGTTCATCTTCAGGATG-3′) (SEQ. ID. No. 55) was approximately from the middle of the dnaA gene. For the C-terminal part, the upstream 23mer (5′-CATCCTGAAGATGAACGCCAGCA-3′) (SEQ. ID. No. 56) was complementary to the previous primer. The downstream 25mer (5′-AGGTTATCCACAGGGGTCATGTGCA-3′) (SEQ. ID. No. 57) was 20 bp upstream the potential stop codon for the dnaA gene. The amplification reactions contained 10 ng [0474] T.th. genomic DNA, 0.5 μM of each primer, in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 0.5 mM of each dNTP and 0.5 mM MgSO₄. Amplification was performed using the following cycling scheme:
1. 5 cycles of: 95.5° C.—30 sec., 55° C.—30 sec., 75° C.—3 min. [0475]
2. 30 cycles of: 95.5° C.—30 sec., 50° C.—30 sec., 75° C.—2 min. [0476]
Products were visualized in a 1.0% native agarose gel. Fragments of the expected sizes of 750 bp and 650 bp were produced, and were sequenced using CircumVent Thermal Cycle DNA sequencing method (New England Biolabs). The nucleotide and amino acid sequences of dnaA and its protein product are shown in FIG. 20. The DnaA protein is homologous to the DnaA proteins of several other bacteria as shown in FIG. 19. [0477]
Cloning and Expression of dnaN [0478]
The full length dnaN gene was obtained by PCR from [0479] T.th. total DNA. DNA oligonucleotides for amplification of T.th. dnaN were the following: the upstream 29mer (5′-GTGTGTCATATGAACATAACGGTTCCCAA-3′) (SEQ. ID. No. 58) consists of an NdeI site within first 11 nucleotides (underlined), followed by the sequence for the start of the dnaN gene; the downstream 29mer (5′-GCGCGAATTCTCCCTTGTGGAAGGCTTAG-3′) (SEQ. ID. No. 59) consists of an EcoRI site within the first 10 nucleotides (underlined), followed by the sequence complementary to a section just downstream of the dnaN stop codon. The amplification reactions contained 10 ng T.th. genomic DNA, 0.5 μM of each primer, in a volume of 100 μl of Vent p lymerase reaction mixture containing 10 μl Thermopol Buffer, 0.5 mM of each dNTP and 0.2 mM MgSO₄. Amplification was performed using the following cycling scheme:
1. 5 cycles of: 95.0° C.—30 sec., 55° C.—30 sec., 75° C.—5 min. [0480]
2. 35 cycles of: 95.5° C.—30 sec., 50° C.—30 sec., 75° C.—4 min. [0481]
The nucleotide and amino acid sequences of dnaN and the β subunit, respectively, are shown in FIG. 21. The [0482] T.th. β subunit shows limited homology to the β subunit sequences of several other bacteria over its entire length (FIG. 22).
The approximately 1 kb dnaN gene was cloned into the pET24-a expression vector using the NdeI and EcoRI restriction sites both in the dnaN containing PCR product and in pEt24-a (FIG. 23). Expression of [0483] T.th. β subunit was obtained under the following conditions: a fresh colony of B121 (DE3) E. coli strain was transformed by the pET24-a:dnaN plasmid, and then was grown in LB broth containing 50 mg/ml kanamycin at 37° C. until the cell density reached 0.4 OD₆₀₀The cell culture was then induced for dnaN expression upon addition of 2 mM IPTG. Cells were harvested after 4 additional hours of growth under 37° C. The induction of the T th. β subunit is shown in FIG. 24.
Two liters of BL21(DE3)pETdnaN cells were grown in LB media containing 50 mg/ml ampicillin at 37° C. to an O.D. of 0.8 and then IPTG was added to a concentration of 2 mM. After a further 2 h at 37° C., cells were harvested by centrifugation and stored at −70° C. The following steps were performed at 4° C. Cells were thawed and resuspended in 40 ml of 5 mM Tris-HCl (pH 8.0), 1% sucrose, 1M NaCl, 5 mM DTT, and 30 mM spermidine. Cells were lysed using a French Pressure cell at 20,000 psi. The lysate was allowed to sit at 4° C. for 30 min. and then cell debris was removed by centrifugation (Sorvall SS-34 rotor, 45 min. 18,000 rpm). The supernatant was incubated at 65° C. for 20 minutes with occasional stirring. The resulting protein precipitate was removed by centrifugation as described above. The supernatant was dialyzed against 4 liters of buffer A containing 50 mM NaCl overnight. The dialyzed supernatant was clarified by centrifugation (35 ml, 150 mg total) and then loaded onto an 8 ml MonoQ column equilibrated in buffer A containing 50 mM NaCl. The column was washed with 5 column volumes of the same buffer and then eluted with a 120 ml gradient of buffer A plus 50 mM NaCl to buffer A plus 500 mM NaCl. Fractions of 2 ml were collected. Over 50 mg of [0484] T.th. B was recovered in fractions 5-21.

EXAMPLE 13

Identification and Cloning of [0485] T. thermophilus holA

A search of the incomplete T.th. genome database (www.g21.bio.unigoettingen.de) showed a match to E. coli δ encoded by holA. The sequence obtained from the database was as follows (SEQ. ID. No. 185):


TPKGKDLVRHLENRAKRLGLRLPGGVAQYLA-SLEGDLEALERELEKLAL

LSP-PLTLEKVEKVVALRPPLTGFDLVRSVLEKDPKEALLRLGRLKEEGE

EPLRLLGALSWQFALLARAFFLLREMPRPKEEDLARLEAHPYAAKKALL-

EAARRLTEEALKEALDALMEAEKRAKG-GKDPQLALEAAVLRLAR-PAGQ

PRVD

Next, the following PCR primers were designed from the codon usage of T.th.: upstream 27mer (5′-GCC CAG TAC CTC GCC TCC CTC GAG GGG-3′) (SEQ. ID. No. 186) and downstream 27mer (5′-GGC CCC CTT GGC CTT CTC GGC CTC CAT-3′ (SEQ. ID. No. 187) to obtain a partial holA nucleotide sequence (SEQ. ID. No. 188):


AGACTCGAGG CCCTGGAGCG GGAGCTGGAG AAGCTTGCCC TCCTCTCCCC ACCCCTCACC	60

CTGGAGAAGG TGGAGAAGGT GGTGGCCCTG AGGCCCCCCC TCACGGGCTT TGACCTGGTG	120

CGCTCCGTCC TGGAGAAGGA CCCCAAGGAG GCCCTCCTGC GCCTCAGGCG CCTCAGGGAG	180

GAGGGGGAGG AGCCCCTCAG GCTCCTCGGG GCCCTCTCCT GGCAGTTCGC CCTCCTCGCC	240

CGGGCCTTCT TCCTCCTCCG GGAAAACCCC AGGCCCAAGG AGGAGGACCT CGCCCGCCTC	300

GAGGCCCACC CCTACGCCGC CAAGAAGGCC A 331

This sequence codes for a partial amino acid sequence of the [0488] T.th. δ subunit (SEQ. ID. No. 189):

RLEALERELEKLALLSPPLTLEKVEKVVALRPPLTGFDLVRSVLEKDPKE

ALLRLRRLREEGEEPLRLLGALSWQFALLARAFFLLRENPRPKEEDLARL

EAHPYAAKKA
The DNA sequence obtained by PCR (SEQ. ID. No. 188) was used to design internal primers for inverted PCR. The upstream 31mer (5′GTGGTGTCTAGACATCATAACGGTTCTGGCA-3′) (SEQ. ID. NO. 190) introduced an XbaI site for cloning holA into a pGEX vector. The downstream 27mer (5′-GAGGGCCACCACCTTCTCCACCTTCTC-3′) (SEQ. ID. No. 191) encodes holA sequence EKVEKVVAL (aa residues 159-167 of SEQ. ID. No. 158) on the complementary strand. The amplification reactions contained 50 ng [0489] T.th. genomic DNA and 0.1 μM of each primer in a volume of 10011 of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 2.5 mM of each dNTP, 2 mM MgSO₄, and 10 μl of formamide. Amplification was performed using the following cycling scheme:
1. 5 cycles of: 95° C.—30 sec., 65° C.—20 sec., 75°C.—5 min. [0490]
2. 5 cycles of: 95° C.—20 sec., 58° C.—10 sec., 75° C.—5 min. [0491]
3.35 cycles of: 95° C.—20 sec., 50° C.—5 sec., 75° C.—4 min. [0492]
Products were visualized in a 1.0% native agarose gel. A fragment of 1.5 Kb was gel purified and partially sequenced. [0493]
A different set of primers were used to obtain the 3′-end of [0494] T.th. holA, including an upstream 25mer (5′-CTCCGTCCTGGAGAAGGACCCCAAG-3′) (SEQ. ID. No. 192) which encoded the amino acid sequence SVLEKDPK from T.th. holA (aa residues 179-186. of SEQ. ID. No. 158), and a downstream 29mer (5′CGCGAATTCAACGCSCTCCTCAAGACSCT-3′ where S═C or G) (SEQ. ID. No. 193) was not related to the holA sequence. The amplification reactions contained 50 ng T.th. genomic DNA and 0.1 μM of each primer in a volume of 100 μl of Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 2.5 mM of each dNTP, and 1-2 mM MgSO₄, and 10 μl of formamide. Amplification was performed using the following cycling scheme:
1.5 cycles of: 95° C.—30 sec., 65° C.—20 sec., 75° C.—5 min. [0495]
2. 5 cycles of: 95° C.—20 sec., 55° C.—10 sec., 75° C.—5 min. [0496]
3.35 cycles of: 95° C.—20 sec., 50° C.—5 sec., 75° C.—4 min. [0497]
Products were visualized in a 1.0% native agarose gel. A fragment of 1.2 Kb was gel purified and partially sequenced to obtain the remainder of the [0498] T.th. holA gene.
The [0499] T.th. holA gene was cloned into the NdeI/EcoRI sites in the pET24 vector using a pair of primers. The upstream 31 mer (5′GACACTTAACATATGGTCATCGCCTTCACCG-3′) (SEQ. ID. No. 194) contains a NdeI site within the first 15 nucleotides (underlined) and has a sequence corresponding to 5′ region of T.th. holA. The downstream 38 mer (5′GTGTGTGAATTCGGGTCAACGGGCGAGGCGGAGGACCG-3′) (SEQ. ID. No. 195) contains a EcoRI site within the first 12 nucleotides (underlined) and has a sequence complementary to the 3′ end of holA gene.

EXAMPLE 14

Identification of [0500] T.th. holB Encoding δ′ Subunit

To clone the ends of T.th. holB gene, it was assumed that the order of genes in Thermus thermophilis could be the same as in related Deinococcus radiodurance. Multiple alignment of the upstream neighbor (probable phosphoesterase, DNA repair Rad24c related protein) revealed a conservative region close to the C-terminus of the protein sequence:


	Deinococcus radiodurance
	VILNPGSVGQ	(SEQ. ID. No. 196)

	Methanococcus janaschii
	VLINPGSVGQ	(SEQ. ID. No. 197)

	Thermotoga maritima
	LVLNPGSAGR	(SEQ. ID. No. 198)

The [0502] Drad. sequence was used to design an upstream 28mer primer (5′-CTGGTGAACCCGGGCTCCGTGGGCCAGC-3′) (SEQ. ID. No. 199) that encodes the amino acid sequence LLVNPGSVGQ (SEQ. ID. No. 200) and a downstream 27mer (5′-CTCGAGGAGCTTGAGGAGGGTGTTGGC-3′) (SEQ. ID. No. 201) encodes the sequence ANTLLKLLE (SEQ. ID. No. 202) on the complementary strand. The amplification reactions contained 50 ng T.th. genomic DNA and 0.1 μM of each primer in a volume of 100 μl of Deep Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 2.5 mM of each dNTP, 1.5 mM MgSO₄, and 10 μl formamide. Amplification was performed using the following cycling scheme:
1. 5 cycles of: 95° C.—30 sec., 68° C.—20 sec., 75° C.—3 min. [0503]
2. 5 cycles of: 95° C.—20 sec., 63° C.—20 sec., 75° C.—3 min. [0504]
3. 35 cycles of: 95° C.—20 sec., 55° C.—10 sec., 75° C.—3 min. [0505]
Product was visualized in a 1.0% native agarose gel as a single band of 0.7 Kb. The fragment was purified and partially sequenced. [0506]

Multiple alignment of the gene downstream of D.rad. identified the following conservative region:


Deinococcus radiodurans
GFGGVQLHAAHGYLLSQFLSPRHNVREDEYGG	(SEQ. ID. No. 203)

Caenorhabditis elegans
GFDGIQLHGAHGYLLSQFTSPTTNKRVDKYGG	(SEQ. ID. No. 204)

Pseudomonas aeruginosa
GFSGVEIHAAHGYLLSQFLSPLSNRRSDAWGG	(SEQ. ID. No. 205)

Archaeoglobus fulgidus
GFDAVQLHAAHGYLLSEFISPHVNRRKDEYGG	(SEQ. ID. No. 206)

The fragment in bold was used to design primers, specifically the downstream primer, for cloning of the 3′ region of the [0508] T.th. holB gene. The upstream 30mer (5′-CATCCTGGACTCGGCCCACCTCCTCACCGA-3′) (SEQ. ID. No. 207) encodes the amino acid sequence ILDSAHLLT (SEQ. ID. No. 208). The downstream 33mer (5′-GAGGAGGTAGCCGTGGGCCGCGTGGAGCTCCAC-3′) (SEQ. ID. No. 209) encodes the sequence VELHAAHGYLL (SEQ. ID. No. 210) on the complementary strand. The amplification reactions contained 50 ng T.th. genomic DNA and 0.1 μM of each primer in a volume of 100 μl of Deep Vent polymerase reaction mixture containing 10 μl ThermoPol Buffer, 2.5 mM of each dNTP, 2 mM MgSO₄, and 10 μl DMSO. Amplification was performed using the following cycling scheme:
1. 5 cycles of: 95° C.—30 sec., 70° C.—20 sec., 75° C.—4 min. [0509]
2. 5 cycles of: 95° C.—20 sec., 66° C.—20 sec., 75° C.—4 min. [0510]
3.30 cycles of: 95° C.—20 sec., 60° C.—10 sec., 77° C.—4 min. [0511]
Products were visualized in a 1.0% native agarose gel as a single band of 1.1 kb. The Kb fragment was gel purified and sequenced to provide the remainder of the holB gene encoding [0512] T.th. δ′.
For protein expression, the [0513] T.th. holB gene was cloned into the pET24 vector at the Nde:EcoR sites using a pair of primers. The upstream 32mer (5′-GGCTTTCCCATATGGCTCTACACCCGGCTCAC-3′) (SEQ. ID. No. 211) contains a NdeI site within the first 15 nucleotides (underlined) and the sequence corresponding to the 5′ region of T.th. holB. The downstream 29 mer (5′-GCGTGGATCCACGGTCATGTCTCTAAGTC-3′) (SEQ. ID. No. 212) contains a BamHI site within the first 10 nucleotides (underlined) and a sequence complementary to the 3′ end of the holB gene.

EXAMPLE 15

Alternate Synthetic Path in Absence of Clamp Loader Activity [0514]
As discussed earlier, the Pol III-type enzyme of the present invention is capable of application and use in a variety of contexts, including a method wherein the clamp loader component that is traditionally involved in the initiation of enzyme activity, is not required. The clamp loader generally functions to increase the efficiency of ring assembly onto circular primed DNA, because both the ring and the DNA are circles and one must be broken transiently for them to become interlocked rings. In such a reaction, the clamp loader increases the efficiency of opening the ring. [0515]
The procedure described below illustrates the instance where the clamp loader need not be present. For example, the β clamp can be assembled onto DNA in the absence of the clamp loader. Particularly, the bulk of primed templates in PCR reactions are linear ssDNA fragments that are primed at the ends. On linear primed DNA, the ring need not open at all. Instead, the ring can simply thread onto the end of the linear primed template (Bauer and Burgers, 1988; Tan et al, 1986; O'Day et al., 1992; Burgers and Yoder, 1993). Hence, on linear primed templates, such as those generated in PCR, the beta clamp can simply slide over the DNA end. After the ring slides onto the end, the DNA polymerase can associate with the ring for enhanced DNA synthesis. [0516]
Such “end assembly” is common among Pol III-type enzymes and has been demonstrated in yeast and human systems. Rings assembling onto linear DNA for use by their respective DNA polymerases are shown in the following example demonstrated in the [0517] E. coli bacterial system, in the human system, and in the T.th. system.
The bulk of the primed templates in PCR reactions are linear ssDNA fragments that are primed at their ends. However, these end primed linear fragments are not generated until after the first step of PCR has already been performed. In the very first step, PCR primers generally anneal at internal sites in a heat denatured ssDNA template. Primed linear templates are then generated in subsequent steps enabling use of this alternate path. For this first step, the clamp may be assembled onto an internal site in the absence of the clamp loader using special conditions that allow clamp assembly in the absence of a clamp loader. [0518]
For example, a set of conditions that lead to assembly of the clamp onto circular DNA (i.e., internal primed sites) have been described in the protocol for the use of the bacteriophage T4 ring shaped clamp (gene 45 protein) without the clamp loader (Reddy et al., 1993). In this case, polyethylene glycol leads to “macromolecular crowding” such that the clamp and DNA are pushed together in close proximity, leading to the ring self assembling onto internal primed sites on circular DNA. Other possible conditions that may lead to assembly of rings onto internal sites include use of a high concentration of beta such that use of heat or denaturant to break the dimeric ring into two half rings (crescents) followed by lowering the heat (or dilution or removal of denaturant) leading to rings assembling around the DNA. [0519]
The ring shaped sliding clamps of [0520] E. coli and human slide over the end of linear DNA to activate their respective DNA polymerase in the absence of the clamp loader. This clamp loader independent assay is performed in the bacterial system in FIG. 25A. For this assay, the linear template is polydA primed with oligodT. The polydA is of average length 4500 nucleotides and was purchased from SuperTecs. OligodT35 was synthesized by Oligos etc. The template wasprepared using 145 μl of 5.2 mM (as nucleotide) polydA and 22 μl of 1.75 mM (as nucleotide) oligodT. The mixture was incubated in a final volume of 2100 μl T.E. buffer (ratio as nucleotide was 21:1 polydA to oligodT). The mixture was heated to boiling in a 1 ml Eppendorf tube, then removed and allowed to cool to room temperature. Assays were performed in a final volume of 25 μl 20 mM Tris-Cl (pH 7.5), 8 mM MgCl₂, 5 mM DTT, 0.5 mM EDTA, 40 mg/ml BSA, 4% glycerol, containing 20 μM [α-³²P]dTTP, 0.1 μg polydA-oligodT, 25 ng Pol III and, where present, 5 μg of β subunit. Proteins were added to the reaction on ice, then shifted to 37° C. for 5 min. DNA synthesis was quantitated using DE81 paper as described (Rowen and Komberg, 1978).
In the linear template assay, no ATP or dATP is provided and therefore, a clamp loader, even if present, is not active. Thus, the clamp (e.g., β) can only stimulate the DNA polymerase provided the clamp threads onto the DNA (see diagram in FIG. 25). Hence, threading of the clamp is shown by a stimulation of the DNA polymerase. In [0521] lane 1 of FIG. 25A, the DNA polymerase is incubated with the the linear DNA in the absence of the clamp, and lane 2 shows the result of adding the clamp. The results show that the clamp is able to thread onto the DNA ends and stimulate the DNA polymerase in the absence of ATP and thus, in the absence of clamp loading as well.
This clamp loader independent assay is performed in the human system in FIG. 25B. The assay reaction (2511) contains 50 mM Tris-HCl (pH=7.8), 8 mM MgCl[0522] ₂, 1 mM DTT, 1 mM creatine phosphate, 40 μg/ml bovine serum albumin, 0.55 μg human SSB, 100 ng PCNA (where present), 7 units DNA polymerase delta (1 unit incorporates 1 pmol dTMP in 60 min.), 40-mM [α-³²P]dTTP and 0.1 μg polydA-oligodT. Proteins were added to the reaction on ice, then shifted to 37° C. for 60 min. DNA synthesis was quantitated using DE81 paper as described (Rowen and Kornberg, 1978). In lane 3, (FIG. 25) the DNA polymerase 6 is incubated with the linear. DNA in the absence of the clamp, and lane 4 shows the result of adding the PCNA clamp. The results demonstrate that the clamp is able to thread onto the DNA ends and stimulate the DNA polymerase in the absence of ATP and thus, the absence of clamp loading.
This clamp loader independent assay is performed in the [0523] T.th. system in FIG. 25C. The assay reaction is exactly as described above for use of the E. coli Pol III and beta system except the temperature is 60° C. and here the Pol III is HEP.P1 T.th. Pol III (0.5 μl, providing 0.1 units where one unit is equal to 1 pmol of dTTP incorporated in 1 minute under these conditions and in the absence of beta), and the beta subunit is 7 μg T.th. β (from the MonoQ column). Proteins were added to the reaction on ice, then shifted to 37° C. for 60 min. DNA synthesis was quantitated using DE81 paper as described (Rowen and Komberg, 1978). In lane 3 (FIG. 25C), the T.Th. Pol III is incubated with the linear DNA in the absence of the clamp, and lane 4 shows the result of adding the T.th. β clamp. The results demonstrate that the clamp is able to thread onto the DNA ends and stimulate the DNA polymerase in the absence of clamp loader activity.

EXAMPLE 16

Use of [0524] T.th. Pol III in Long Chain Primer Extension
A characteristic of Pol III-type enzymes is their ability to extend a single primer for several kilobases around a long (e.g. 7 kb) circular single stranded DNA genome of a bacteriophage. This reaction uses the circular β clamp protein. For the circular β to be assembled onto a circular DNA genome, the circular β must be opened, positioned around the DNA, and then closed. This assembly of the circular beta around DNA requires the action of the clamp loader, which uses ATP to open and close the ring around DNA. In this example, the 7.2 kb circular single strand DNA genome of bacteriophage M13mp18 was used as a template. This template was primed with a single DNA 57mer oligonucleotide and the Pol III enzyme was tested for conversion of this template to a double strand circular form (RFII). The reaction was supplemented with recombinant [0525] T.th. β produced in E. coli. This assay is summarized in the scheme at the top of FIG. 26. M13mp18 ssDNA was phenol extracted from phage purified as described (Turner and O'Donnell, 1995). M13mp18 ssDNA was primed with a 57mer DNA oligomer synthesized by Oligos etc. The replication assays contained 73 ng singly primed M13mp18 ssDNA and 100 ng T.th. β subunit in a 25 μl reaction containing 20 mM Tris-HCl (pH 7.5), 8 mM MgCl₂, 40 μg/ml BSA, 0.1 mM EDTA, 4% glycerol, 0.5 mM ATP, 60 M each of dCTP, dGTP, dATP and 20 μM α-³²P-TTP (specific activity 2,000-4,000 cpm/μmol). Either T.th. Pol III from the Heparin, peak 1 (HEP.P1; 5 μl, 0.21 units where 1 unit equals 1 pmol nucleotide incorporated in 1 min.) or a non-Pol III from the Heparin peak 2 (HEP.P2; 5 μl, 2.6 units) were added to the reaction. Reactions were shifted to 60° C. for 5 min., and then DNA synthesis was quenched upon adding 25 μl of 1% SDS, 40 mM EDTA. One half of the reaction was analyzed in a 0.8% native agarose gel, and the other half was quantitated using DE81 paper as described (Studwell and O'Donnell, 11990).
The results of the assay are shown in FIG. 26. [0526] Lane 1 is the result obtained using the T.th. Pol III (HEP.P1) which was capable of extending the primer around the ssDNA circle to form RFII. Lane 2 shows the result of using the non-Pol III (HEP.P2) which was not capable of this extension and produced only incomplete DNA products (the result shown included 0.8 μg E. coli SSB which did not increase the chain length of the product). In the absence of SSB, the same product was observed, although the band contained more counts. The greater amount of total synthesis observed in lane 2 is due to the build up of immature products in a small region of the gel. The presence of immature products in lane 1 is likely due to a contaminating polymerase in the preparation that can not convert the single primer to the full length RFII form. Alternatively, the presence of incomplete products in lane 1 (Pol III type enzyme) is due to secondary structure in the DNA which causes the Pol III to pause. In this case it may be presumed that performing the reaction at higher temperature could remove the secondary structure barrier. Alternatively, SSB could be added to the assay (although T.th. SSB would be needed, because addition of E. coli SSB was tried and did not alter the quality of the product profile). Generally, SSB is needed to remove secondary structure elements from ssDNA at 37° C. for complete extension of primers by mesophilic Pol III-type enzymes.
The assay described above was performed at 60° C. The [0527] T.th. Pol III HEP.P1 gained activity as the temperature was increased from 37° C. to 60° C., as expected for an enzyme from a thermophilic source. The E. coli Pol III lost activity at 60° C. compared to 37° C., as expected for an enzyme from a mesophilic source.

EXAMPLE 17

Materials Used in Examples 18-29 [0528]
Radioactive nucleotide were from Dupont NEN; unlabeled nucleotides were from Pharmacia Upjohn. DNA oligonucleotides were synthesized by Gibco BRL. M13mp18 ssDNA was purified from phage that was isolated by two successive bandings in cesium chloride gradients. M13mp18 ssDNA was primed with a 30-mer (map position 6817-6846) as described. The pET protein expression vectors and BL21 (DE3) protein expression strain of [0529] E. coli were purchased from Novagen. DNA modification enzymes were from New England Biolabs. Aquifex aeolicus genomic DNA was a gift of Dr. Robert Huber and Dr. Karl Stetter (Regensburg University, Germany). Protein concentrations were determined by absorbance at 280 nm using extention coefficients calculated from their known Trp and Tyr content using the equation ε₂₈₀=Trp_m(5690 M⁻¹cm⁻)+Tyr, (1280 M⁻¹cm⁻).

EXAMPLE 18

Purification of α Encoded by dnaE [0530]
The [0531] Aquifex aeolicus dnaE gene was previously identified (Deckert et al., 1998). The dnaE was obtained by searching the Aquifex aeolicus genome, with the amino acid sequence of T.th α subunit (encoded by dnaE). The dnaE gene was amplified from Aquifex aeolicus genomic DNA by PCR using the following primers: the upstream 37mer (5′-GTGTGTCATATGAGTAAG GATTTCGTCCACCTTCACC-3′) (SEQ. ID. No. 157) contains a nNdeI site (underlined); the downstream 34mer (5′-GTGTGTGGATCCGGGGACTACTCGGAAGTAAGGG-3′) (SEQ. ID. No. 158) contains a BamHI site (underlined). The PCR product was digested with NdeI and BamHI, purified, and ligated into the pET24 NdeI and BamHI sites to produce pETAadnaE.
The pETAadnaE plasmid was transformed into the BL21 (DE3) strain of [0532] E. coli. Cells were grown in 50L of LB containing 100 μg/ml of kanamycin, 5 mM MgSO₄at 37° C. to OD₆₀₀=2.0, induced with 2 mM IPTG for 20 h at 20° C., then collected by centrifugation. Cells were resuspended in 400 ml 50 mM Tris-HCl (pH 7.5), 10% sucrose, 1M NaCl, 30 mM spermidine, 5 mM DTT and 2 mM EDTA. The following procedures were performed at 4° C. Cells were lysed by passing them twice through a French Press (15,000 psi) followed by centrifugation at 13,000 rpm for 90 min at 4° C. In this protein preparation, as well as each of those that follow, the induced Aquifex aeolicus protein was easily discernible as a large band in an SDS polyacrylamide gel stained with Coomassie Blue. Hence, column fractions were assayed for the presence of the Aquifex aeolicus protein by SDS PAGE analysis, which forms the basis for pooling column fractions.
The clarified cell lysate was heated to. 65° C. for 30 min and the precipitate was removed by centrifugation at 13,000 rpm in a GSA rotor for 1 h. The supernatant (1.4 gm, 280 ml) was dialyzed against buffer A (20 mM Tris-HCl (pH 7.5)), 10% glycerol, 0.5 mM EDTA, 5 mM DTT) overnight, then diluted to 320 ml with buffer A to a conductivity equal to 100 mM NaCl. The dialysate was applied to a 150 ml Fast Flow Q (FFQ) Sepharose column (Pharmacia) equilibrated in buffer A, and eluted with a 1.5L linear gradient of 0-500 mM NaCl in buffer A. Eighty fractions were collected. Fractions 38-58 (1 g, 390 ml) were pooled, dialyzed versus buffer A overnight, and applied to a 250 ml Heparin Agarose column (Bio-Rad) equilibrated with buffer A. Protein was eluted with a 1L linear 0-5 mM NaCl gradient in buffer A. One hundred fractions were collected. Fractions 69-79 (320 mg in 200 ml) were pooled and dialyzed against buffer A containing 100 mM NaCl. The a preparation was aliquoted and stored frozen at −80° C. (see FIG. 27). [0533]

EXAMPLE 19

Purification of 6 Encoded by hoM [0534]
The [0535] Aquifex aeolicus holM gene was not previously identified by the genome sequencing group at Diversa (Deckert et al., 1998). Aquifex aeolicus holA was identified by searching the Aquifex aeolicus genome with the amino acid sequence of the T.th. δ subunit (encoded by hola). The Aquifex aeolicus hola was amplified by PCR using the following primers: the upstream 36mer (5′-GTGTGTCATATGGAAACCACAATATTCCAGTTCCAG-3′) (SEQ. ID. No. 159) contains an NdeI site (underlined); the downstream 39mer (5′-GTGTGTGGATCCTTATCCACCATGAGAAGTATTTTTCAC-3′) (SEQ. ID. No. 160) contains a Bamin site (underlined). The PCR product was digested with NdeI and BamHI, purified, and ligated into the pET24 NdeI and BamHI sites to produce pETAaholA.
The pETAaholA plasmid was transformed into [0536] E. coli strain BL21 (DE3). Cells were grown in 50L of LB media containing 100 μg/ml kanamycin. Cells were grown at 37° C. to OD₆₀₀=2.0, induced for 20 h upon addition of 2 mM IPTG, then collected by centrifugation. Cells from 25L of culture were lysed as described in Example 18.
The cell lysate was heated to 65° C. for 30 min and the precipatate was removed by centrifugation. The supernatant (650 mg, 240 ml) was dialyzed against buffer A, adjusted to a conductivity equal to 160 mM NaCl by addition of 40 ml of buffer A, and applied to a 220 ml Heparin Agarose column equilibrated in buffer A containing 100 mM NaCl. The column was eluted with 1.0L linear gradient of 150-700 mM NaCl in buffer A. One hundred and four fractions were collected. Fractions 45-56 were pooled (250 mg, 210 ml), diluted with 230 ml buffer A to a conductivity equal to 230 mM, NaCl, then loaded onto a 100 ml FFQ Sepharose column equilbrated in buffer A containing 150 mM NaCl. The column was eluted with 200 ml linear gradient of 150-750 mM NaCl in buffer A; seventy-three fractions were collected. Fractions 16-38 were pooled (95 mg, 40 ml), aliquoted, and stored at −80° C. (see FIG. 27). [0537]

EXAMPLE 20

Purification of δ′ Encoded by holb [0538]
The [0539] Aquifex aeolicus holb gene was previously identified by the genome sequencing facility at Diversa (Deckert et al., 1998). The Aquifex aeolicus holb sequence was obtained by searching the Aquifex aeolicus genome with the sequence of the T.th. δ′ (encoded by holB). The Aquifex aeolicus holB gene was amplified by PCR using the following primers: the upstream 39mer (5′-GTGTGTCATATGGAAAAAGTTTTTTTTGGAAAAAACTCCAG-3′) (SEQ. ID. No. 161) contains an NdeI site (underlined); the downstream 35mer (5′-GTGTGTGGATCCTTAATCCGCCTGAACGGCTAACG-3′) (SEQ. ID. No. 162) contains a BamHI site (underlined). The PCR product was digested with NdeI and BamHI, purified, and ligated into the pET24 NdeI and BamHI site to produce pETAaholB.
The pETAaholB plasmid was transformed into [0540] E. coli strain BL21 (DE3). Cells were grown at 37° C. in 50L media containing 100 μg/ml kanamycin to OD₆₀₀2.0, then induced for 3 h upon addition of 0.2 mM IPTG. Cells were collected by centrifugation and were lysed using lysozyme by the heat lysis procedure (Wickner and Komberg, 1974). The cell lystate was heated to 65° C. for 30 min and precipatate was removed by centrifugation. The supernatant (2.4 g, 400 ml) was dialyzed versus buffer A, then applied to a 220 ml FFQ Sepharose column equilibrated in buffer A. Protein was eluted with a 1L linear gradient of 0-500 mM NaCl in buffer A; eighty fractions were collected. Fractions 23-30 were pooled and diluted 2-fold with buffer A to a conductivity equal to 100 mM NaCl, then loaded onto a 200 ml Heparin Agarose column equilibrated in buffer A. Protein was eluted with a 1L linear gradient of O-1.0M NaCl in buffer A; eighty-four fractions were collected. Fractions 46-66 were pooled (1.3 g, 395 ml), dialyzed versus buffer A containing 100 mM NaCl, then aliquoted and stored frozen at −80° C. (see FIG. 27)

EXAMPLE 21

Purification of τ Encoded by dnaX [0541]
The [0542] Aquifex aeolicus dnaX gene was previously identified (Deckert et al., 1998). The dnaX gene sequence was obtained by searching the Aquifex aeolicus genome with the sequence of T.th. τ subunit (encoded by dnaX). The Aquifex aeolicus dnaX was amplified by PCR using the following primers: the upstream 41 mer (5′-GTGTGTCATATGAACTACGTTCCCTTCGCGAGAAAGTACAG-3′) (SEQ. ID. No. 163) contains an NdeI site (underlined); the downstream 36mer (5′-GTGTGTGGATCCTTAAAACAGCCTCGTCCCGCTGGA-3′) (SEQ. ID. No. 164) contains a BamHI site (underlined). The PCR product was digested with NdeI and BamHI, purified, and ligated into the pET24 NdeI and BamHI sites to produce pETAadnaX.
The pETAadnaX plasmid was transformed into [0543] E. coli strain BL21 (DE3). Cells were grown in 50L LB containing 100 μg/ml kanamycin at 37° C. to OD₆₀₀=0.6, then induced for 20 h at 20° C. upon addition of IPTG to 0.2 mM. Cells were collected by centrifugation and lysed as described in Example 18. The clarified cell lysate was heated to 65° C. for 30 min and the protein precipitate was removed by centrifugation. The supernatant (1.1 g in 340 ml) was treated with 0.228 g/ml ammonium sulfate followed by centrifugation. The τ subunit remained in the pellet which was dissolved in buffer β (20 mM Hepes (pH 7.5), 0.5 mM EDTA, 2 mM DTT, 10% glycerol) and dialyzed versus buffer β to a conductivity equal to 87 mM NaCl. The dialysate (1073 mg, 570 ml) was applied to a 200 ml FFQ Sepharose column equilibrated in buffer A. The column was eluted with a 1.5L linear gradient of 0500 mM NaCl in buffer A; eighty fractions were collected. Fractions 28-37 were pooled (289 mg, 138 ml), dialyzed against buffer A to a conductivity equal to 82 mM NaCl, then loaded onto a 150 ml column of Heparin Agarose equilibrated in buffer A. The column was eluted with a 900 ml linear gradient of 0-500 mM NaCl in buffer A; thirty-two fractions were collected. Fractions 15-18 (187 mg, 110 ml) were dialyzed versus buffer. A, then aliquoted and stored at −80° C. (see FIG. 27).

EXAMPLE 22

Purification of β Encoded by dnaN [0544]
The [0545] Aquifex aeolicus dnaN gene was previously identified (Deckert et al., 1998). The dnaN sequence was obtained by searching the Aquifex aeolicus genome with the sequence of T.th. β subunit (encoded by dnaN). The Aquifex aeolicus dnaN gene was amplified by PCR using the following primers: the upstream 33mer (5′-GTGTGTCATATG CGCGTTAAGGTGGACAGGGAG-3′) (SEQ. ID. No. 165) contains an NdeI site (underlined); the downstream 36mer (5′-TGTGTCTCGAG TCATGGCTACACCCTCATCGGCAT-3′) (SEQ. ID. No. 166) contains a XhoI site (underlined). The PCR product was digested with NdeI and BamHI, purified, and ligated into the pET24 NdeI and BamHI sites to produce pETAadnaN.
The pETAadnaN plasmid was transformed into [0546] E. coli strain BL21 (DE3). Cells were grown in 1L LB containing 100 mg/ml kanamycin at 37° C. to OD₆₀₀=1.0, then induced for 6 h upon addition of 2 mM IPTG. Cells were collected (7 g) and lysed as described in Example 18. The cell lysate was heated to 65° C. for 30 min and the protein precipitate was removed by centrifugation. The supernatant (39 mg, 45 ml) was applied to a 10 ml DEAE Sephacel column (Pharmacia) equilibrated in buffer A. The column was eluted with a 100 ml linear gradient of 0500 mM NaCl in buffer A; seventy-five fractions were collected. Fractions 45-57 were pooled (18.7 mg), dialyzed versus buffer A, and applied to a 30 ml Heparin Agarose column equilibrated in buffer A. The column was eluted with a 300 ml linear gradient of 0-500 mM NaCl in buffer A; sixty-five fractions were collected. Fractions 27-33 were pooled (11 mg, 28 ml) and stored at −80° C. (see FIG. 27).

EXAMPLE 23

Purification of SSB Encoded by ssb [0547]
The [0548] Aquifex aeolicus ssb gene was previously identified (Deckert et al., 1998 g). The ssb gene sequence was obtained by searching the Aquifex aeolicus genome with the sequence of T.th. SSB (encoded by ssb). The Aquifex aeolicus ssb gene was amplified by PCR using the following primers: the upstream 47mer (5′-GTGTGTCATATGCTCAA TAAGGTTTTTATAATAGGAAGACTTACGGG-3′) (SEQ. ID. No. 167) contains an NdeI site (underlined); the downstream 39mer (5′-GTGTGGATCCTTA AAAAGGTATTTCGTCCTCTTCATCGG-3′) (SEQ. ID. No. 168) contains a BamHI site (underlined). The PCR product was digested with NdeI and BamHI, purified, and ligated into the pET16 NdeI and BamHI sites to produce pETAassb.
The pETAassb plasmid was transformed into [0549] E. coli strain BL21 (DE3). Cells were grown in 6L of LB media containing 200 μg/ml ampicillin. Cells were grown at 37° C. to OD₆₀₀=0.6, then induced at 15° C. overnight in the presence of 2 mM IPTG and collected by centrifugation. Cells were lysed as described above in Example 18, except cells were resuspended in buffer C (20 mM Tris-HCl (pH 7.9), 500 mM NaCl).
The cell lysate was heated to 65° C. for 30 min, then the precipitate was removed by centrifugation. The supernatant (1.4 g, 190 ml) was applied to 25 ml Chelating Sepharose column (Pharmacia-Biotech) charged with 50 mM Nickel Sulfate and then equilibrated in buffer C containing 5 mM Imidazole. The column was eluted with a 300 ml linear gradient of 5-100 mM Imidazole in buffer C. Fractions of 4 ml were collected. Fractions 81-92 were pooled (˜240 mg in 48 ml) and dialyzed overnight against 2L of buffer β containing 200 mM NaCl. The dialysate was diluted to a conductivity equal to 92 mM NaCl using buffer A and then loaded onto an 8 ml MonoQ column equilibrated in buffer A containing 100 mM NaCl. The column was eluted with a 120 ml linear gradient of 100-500 mM Imidazole in buffer A. Seventy-four fractions were collected. Fractions 57-70 were pooled (100 mg, 25 ml), aliquoted, and stored at −80° C. (see FIG. 27). [0550]

EXAMPLE 24

MonoQ Preparation of τδδ′[0551]
The δ subunit (0.29 mg) purified in Example 1.9 and δ′ subunit (0.31 mg) purified in Example 20 were mixed in a volume of 2.8 ml of buffer A at 15° C. After 30 min, the τ subunit (0.5 mg in 1.4 ml), purified in Example 21, was added and the reaction was incubated a further 1 h at 15° C. The reaction was applied to a 1 ml MonoQ column equilibrated in buffer A. The τδδ′ complex elutes later than either τ, δ or δ′ alone. Protein was eluted with a 32 ml linear gradient of 100-500 mM NaCl in buffer A; eighty fractions were collected. Analysis of the MonoQ fractions in a SDS polyacylamide gel shows a peak of τδδ′ complex that elutes in fractions of 32-38 (see FIG. 28). The peak fractions 850 μg were stored at −80° C. This procedure can easily be scaled up. For example, a much larger amount of τδδ′ was constituted by following a similar protocol and using a 8 ml MonoQ column, which yielded 9.6 mg of τδδ′. [0552]

EXAMPLE 25

Constitution of ατδδ′ Complex [0553]
The reaction mixture contained 1.2 mg αsubunit (9 mmol; 133,207 da) purified in Example 18, 0.41 mg τ subunit (7.5 nmol; 54,332 da) purified in Example 21, 0.41 mg δ subunit (10 mmol; 40,693 da) purified in Example 19, and 0.2 mg δ′ subunit (9 nmol; 29,000 da) purified in Example 20 in 1. 1 ml buffer A. The α and τ subunit solutions were premixed in 871 μl for 2 h at 15° C. before adding δ and δ′ subunit solution, then the complete mixture was allowed to incubate an additional 12 h at 15° C. The reaction may not require an order of addition, or these extended incubation times. The reaction mixture was concentrated to 200 μl using a Centricon at 4° C., then applied to an [0554] FPLC Superose 6 HR 10/30 column (25 ml) at 4° C. developed with a continuous flow of buffer A containing 100 mM NaCl. After the first 216 drops (6.6 ml), fractions of 7 drops each were collected. Fractions were analyzed on a SDS polyacrylamide gel stained with Coomassie Blue (FIG. 29). The analysis was repeated using the α subunit alone (FIG. 29). The results show that the peak fractions of a shift to a considerably earlier position when τ, δ and δ′ are present and a comigrates with τ, δ, and δ′, when compared to the elution position of a alone, indicating that a assembles with τ, δ and δ′ into a ατδδ′ complex.

EXAMPLE 26

ατδδ′ Functions with the β Clamp [0555]
Replication reactions were performed using circular M13mp18 ssDNA primed with a [0556] synthetic DNA 90 mer oligonucleotide. Reactions contained 8.6 μg primed M13mp18 ssDNA, 9.4 μg SSB purified in Example 23, 1.0 μg ατδδ′ prepared in Example 25, and 2.0 μg β subunit purified in Example 22 (when present), in 230 μl of 20 mM Tris-HCl (pH 7.5), 5 mM DTT, 4% glycerol, 8 mM MgCl₂, 0.5 mM ATP, 60 μM each dATP and dGTP (buffer composition is for a final volume of 250 μl). Reactions were mixed on ice, then aliquoted into separate tubes containing 25 μl each. For each timed reaction, the mixture was brought to 65° C. for 2 min before initiating syntheses upon addition of 2 μl of dCTP and α³²P-dTTP (final centrations, 60 and 40 μM, respectively). Aliquots were quenched at the times indicated in FIG. 30 upon adding 4 μl of 0.25M EDTA, 1% SDS. Quenched reactions were then analyzed in a 0.8% alkaline agarose gel. The results, illustrated in FIG. 30, demonstrate that efficient synthesis requires addition of the β subunit. Comparison with size standards in the same gel indicates an average speed of ˜125 nucleotides; the leading edge of the product smear indicates a maximum speed of 375 nucleotides/s.

EXAMPLE 27

Purification of [0557] T.th. α subunit
To obtain [0558] T.th. α subunit, 8 L of E. coli BL21(DE3) cells harboring pETtthalpha were grown to O.D. =0.3 and induced upon adding IPTG. Cells were collected by centrifugation and resuspended in 200 ml 50 mM Tris-HCl (pH 7.5), 10% sucrose, 1M NaCl, 30 mM spermidine, 5 mM DTT and 2 mM EDTA. The following procedures were performed at 4° C. Cells were lysed by passing them three times through a French Press (20,000 psi) followed by incubation at 4° C. for 30 min and then centrifugation at 18,000 rpm in an SS-34 rotor for 45 min at 4° C. Induced protein was less that 1% total cell protien but was discernible as a band that migrated in the appropriate position for its predicted molecular weight in an SDS polyacrylamide gel stained with Coomassie Blue. Hence, column fractions were assayed for the presence of the protein by SDS PAGE analysis, which forms the basis for pooling column fractions.
The clarified cell lysate was heated to 65° C. for 30 min and the precipitate was removed by centrifugation. The supernatant (1.4 μm, 280 ml) was dialyzed against buffer A (20 mM Tris-HCl (pH 7.5), 10% glycerol, 0.5 mM EDTA, 5 mM DTT) overnight, then diluted to 320 ml with buffer A to a conductivity equal to 100 mM NaCl. The dialysate (approximately 150 mg) was applied to a 60 ml DEAE Fast Flow Q (FFQ) Sepharose column (Pharmacia) equilibrated in buffer A, and eluted with a 600 ml linear gradient of 0-500 mM NaCl in buffer A. Fractions of 8 ml each were collected. The [0559] T.th. α subunit could be seen as a major band in several fractions, especially in fractions 26-30. In these peak fractions the T.th. α subunit was approximately 20-30 percent pure.

EXAMPLE 28

Purification of [0560] T.th. ε subunit
The dnaQ gene was cloned into the pET16 expression plasmid using the Val within the context “VGLWEW . . . ” and transformed into [0561] E. coli (BL21(DE3). This pET plasmid places an N-terminal leader containing six histidines onto the expressed protein to facilitate purification via use of chelate affinity chromatography. Twelve liters of cells were grown to an OD of 0.7 and induced with IPTG. Induced cells were collected by centrifugation and resuspended in 150 ml of buffer C (20 mM Tris-HCl (pH 7.9), 500 mM NaCl). Cells were lysed by passing them two times through a French Press (20,000 psi) followed by incubation at 4° C. for 30 min and then centrifugation at 13,800 rpm in an SLA-1500 rotor for 45 min at 4° C. Induced protein appeared greater than 5% total cell protien and was easily discernible as a band that migrated in the appropriate position for its predicted molecular weight in an SDS polyacrylamide gel stained with Coomassie Blue. Hence, column fractions were assayed for the presence of the protein by SDS PAGE analysis, which forms the basis for pooling column fractions.
Upon analyzing the precipitate from the cell lysis, and the supernatent, it was determined that the epsilon subunit was insoluble and appeared in the precipitate. Therefore the cell pellet was resuspended in 100 ml of binding buffer containing 6M freshly deionized urea. This resuspension was then placed in centrifuge bottles and spun at 13,800 rpm for 45 min in the SLA-1500 rotor. The epsilon was in the supernatent and was applied to a. 25 ml Chelating Sepharose column (Pharmacia-Biotech) charged with 50 mM Nickel Sulfate and then equilibrated in buffer C containing 5 mM Imidazole. The column was washed with two column volumes of buffer C, then washed with 5 column volumes of buffer C containing 80 mM Imidazole (final). Then the Tth epsilon was eluted with a 250 ml linear gradient of 60-1000 mM Imidazole in buffer C. Fractions of 4 ml were collected. Fractions 15-24 were pooled (˜131 mg) and dialyzed overnight against 2L of buffer A containing 6M urea, but no NaCl or glycerol. The dialysate was then loaded onto an 8 ml MonoQ column equilibrated in buffer A containing 6M urea. The column was eluted with a 120 ml linear gradient of 0-500 mM NaCl in buffer A containing urea. Sixty five fractions were collected. The epsilon is approximately 80-90 percent pure at this stage. Fractions 13-17 were stored at −80° C. The epsilon is in urea but is at a concentration of 5-10 mg/ml, and thus can be used with other proteins by diluting it such that the final urea concentration is less than 0.5 M. This level of urea does not generally denature protein, and should allow epsilon to renature for catalytic activity. [0562]

EXAMPLE 29

Temperature Optimum of Aguifex and Thermus α Subunit DNA Polymerases [0563]
The temperature optimum of the alpha subunits of the Aquifex and Thermus replicases was tested in the calf thymus DNA replication assay. In this experiment, the reactions were assembled on ice in 25 μl containing 2.5 μg calf thymus activated DNA, and either 0.88 μg Aquifex α, or 0.6 μg of the Thermus α DEAE pool of peak fractions (obtained from Examples 18 and 28, respectively) in 20 mM Tris-HCl (pH 8.8), 8 mM MgCl[0564] ₂, 10 mM KCl, 10 mM (NH₄)SO₄, 2 mM MgSO₄, 0.1% Triton X-100, 60 μM each dATP, dCTP, dGTP, and 20 μM a³²pdTTP. Reactions were shifted to either 30, 40, 50, 60, 70, 80, or 90° C., then stopped after 5 minutes and spotted onto DE81 filters to quantitate DNA synthesis. The results, illustrated in FIGS. 31-32, show that these enzymes increase in activity as the temperature is raised. The Thermus α has a broad peak of activity from 70-80° C. (FIG. 31), while the Aquifex α is maximal at 80° C. (FIG. 32). The Aquifex α retains considerable activity at 90° C., whereas the Thermus a is nearly inactive at 90° C., a result that is consistent with the higher temperature at which the Aquifex aeolicus may live relative to the Thermus bacterium.

EXAMPLE 30

Temperature optimum of Aguifex ατδδ′/β[0565]
Aquifex α, β, τδδ′, SSB and ατδδ′ were tested for stability at different temperatures by incubating the protein in a solution, followed by performing a replication assay of the protein. Incubation was performed in 0.4 ml tubes under mineral oil. The 5 μl reaction mixture contained: buffer β (20 mM Tris-HCl (pH 7.5), 5 mM DTT, 5 mM EDTA), and either: 0.352 μg of α (FIG. 33A), 0.2 μg of β (FIG. 33B), 0.125 μg τ complex (FIG. 33C), 0.32 μg SSB and 0.042 μg primed M13mp18 ssDNA (FIG. 33D), 0.82 μg Pol III* (FIG. 33E). Reactions were incubated for 2 min. at either 70, 80, 85, or 90° C. in the presence of either 0.1% Triton X-100 (filled diamonds); 0.05% Tween-20 and 0.01% NP-40 (filled circles); 4 mM CaCl[0566] ₂(filled triangles); 40% Glycerol (inverted filled triangles); 0.01% Triton X-100, 0.05% Tween-20, 0.01% NP-40, 4 mM CaCl₂(half-filled square); 40% Glycerol, 0.10% Triton X-100 (open diamonds); 40% Glycerol, 0.05% Tween-20, 0.01% NP-40 (open circles); 40% Glycerol, 4 mM CaCl₂(open triangles); 40% Glycerol, 0.01% Triton X-100, 0.05% Tween-20, 0.01% NP-40, 4 mM CaCl₂(half-filled diamonds). After heating, reactions were shifted to ice and 20 μl of replication assay buffer was added followed by incubation for 1.5 min at 70° C.; 15 μl was then spotted onto a DE81 filter and DNA synthesis was quantitated. The replication assay buffer contained: 60 mM Tris-HCl (pH 9.1 at 25° C.), 8 mM MgCl₂, 18 mM (NH₄)₂SO₄, 2 mM ATP, 60 μM each of dATP, dCTP, dGTP, and 20 μM [α⁻³²P] TTP (specific activity 10,000 cpm/pmol), and 0.264 μg primed M13mp18 ssDNA. To assay for β, 0.1 ng ατδδ′ was added to the reaction. To assay τδδ′, 0.9 ng β and 0.17 ng a were added to the reaction. To assay for SSB, 0.17 ng E. coli β and 0.1 ng E. coli ατδδ′ were added to the reaction followed by incubation for 1.5 min at 37° C. To assay for ατδδ′, 0.9 ng β was added to the reaction. To assay α, the calf thymus DNA replication assay was performed in the buffer as described above but 2.5 μg activated calf thymus DNA was used instead of primed M13mp18 ssDNA, no other replication proteins were added, and incubation was for 8 min at 70° C.

REFERENCES

The following is a list of documents related to the above disclosure and particularly to the experimental procedures and discussions. The documents should be considered as incorporated by reference in their entirety. [0567]
Alonso, J. C., Shirahige, K., and Ogasawara, N. (1990) Molecular cloning, genetic characterization and DNA sequence analysis of the recM region of [0568] Bacillus subtilis. Nuc. Acids Res. 18:6771-6777.
Altschul et al., (1997) Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. [0569] Nucl. Acids Res. 25:3389-3402.
Ausubel, R. M., ed., [0570] Current Protocols in Molecular Biology, Vol. I-III (1994).
Bambara, R. A., and Jessee, C. B. (1991) Properties of [0571] DNA polymerases 5 and c, and their role in eukaryotic DNA replication. Biochimica et Biophysica Acta 1088:11-24.
Barnes, M. H., Tarantino, Jr., P. M., Spaccioappoli, P., Brown, N. C., Yu, H., and Dybvig, K. (1994) DNA polymerase III of Mycoplasma pulmonis: isolation of characterization of the enzyme and its structural gene, polC. [0572] Molec Microbiol. 13:843-854.
Barnes, W. M., (1994) PCR amplification of up to 35-kb DNA with high fidelity and high yield from lambda bacteriophage templates. [0573] Proc. Natl. Acad. Sci. USA 91:2216-2220.
Bauer, G. A., and Burgers, P. M. (1988) Protein-protein interactions of yeast DNA polymerase III with mammalian and yeast proliferating cell nuclear antigen (PCNA)/cyclin. [0574] Biochim. Biophys. Acta 951(2-3):274-9.
Blinkowa, A. L., and Walker, J. R., (1990) Programmed ribosomal frameshifting generates the [0575] Escherichia coli DNA polymerase III gamma subunit from within the tau subunit reading frame. Nucl. Acids Res. 18(7):1725-1729.
Bonner, C. A., Stukenberg, P. T., Rajagopalan, M., Eritja, R., O'Donnell, M., McEntee, K., Echols, H., and Goodman, M. F. (1992) Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins. [0576] J. Biol. Chem., 267:11431-11438.
Braithwaite, D. K., and Ito, J. (1993) Compilation, alignment, and phylogenetic relationships of DNA polymerases. [0577] Nucl. Acids Res. 21(4):787-802.
Brock, T. D., and Freeze, H., (1969) [0578] Thermus aquaticus gen. n. and sp. n., a nonsporulating extreme thermophile. J. Bacteriol. 98(1):289-297.
Burgers, P. M., Yoder, B. L. (1993) ATP-independent loading of the proliferating cell nuclear antigen requires DNA ends. [0579] J. Biol. Chem. 268(27):19923-19926.
Caetano-Anollés et al., (1991) DNA amplification fingerprinting using very short arbitrary oligonucleotide primers. [0580] Bio/Technology 9:553-557.
Carter, J. R., Franden, M. A., Aebersold, R., and McHenry, C. S. (1993) Identification, isolation, and characterization of the structural gene encoding the δ′ subunit of [0581] E. coli DNA polymerase III holoenzyme. J. Bacteriol. 175:3812-3822.
Celis, J. E., ed., [0582] Cell Biology: A Laboratory Handbook, Vol. I-III (1994).
Chen, M., Pan; Z.-Q., and Hurwitz, J. (1992) Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells. [0583] Proc. Natl. Acad. Sci. USA 89(12):5211-5215.
Cheng, S., Fockler, C., Barnes, W. M., and Higuchi, R., (1994) Effective amplification of long targets from cloned inserts and human genomic DNA. [0584] Proc. Natl. Acad. Sci. USA 91:5695-5699.
Coligan, J. E., ed., [0585] Current Protocols in Immunology, Vol, I-III (1994).
Cullman, G., Fien, K., Kobayashi, R., and Stillman, B. (1995) Characterization of the five replication factor C genes of [0586] Saccharomyces cerevesiae. Mol. and Cell. Biol. 15:4661-4671.
Davis, L. G., [0587] Basic Methods In Molecular Biology, Elsevier Edit., New York (1986).
Decket et al., (1998) The complete genome of the hyperthermophilic bacterium [0588] Aquifex aeolicus. Nature 392:353-358.
Dulbecco, R., et al. (1959) Plaque production by the polyoma virus. [0589] Virol. 8:396-397.
Edge, M. D., et al., (1981) Total synthesis of a human leukocyte interferon gene. [0590] Nature 292:756.
Flower, A. M. and McHenry, C. S. (1990) The γ subunit of DNA polymerase III holoenzyme of [0591] Escherichia coli is produced by ribosomal frameshifting. Proc. Natl. Acad. Sci. USA 87:3713-3717.
Freshney, R. I., ed., [0592] Animal Cell Culture (1986).
Gait, M. J., ed., [0593] Oligonucleotide Synthesis (1984).
Glover, ed., DNA Cloning: A Practical Approach, Vol. I & II, MRL Press, Ltd., Oxford, U.K. (1985). [0594]
Guenther, B. D. (1996) Structural studies on the DNA replication apparatus: X-ray crystal structure of the δ′ subunit of [0595] Escherichia coli DNA Pol III. Ph.D. Thesis, Rockefeller University.
Guibus, J. M., Kelman, Z., Hurwitz, J., O'Donnell, M., and Kuriyan, J. (1996) Structure of the C-terminal region of p21wafl/cipl complexed with human PCNA. [0596] Cell 87:297-306.
Hames, B. D., and Higgins, S. J., eds., [0597] Nucleic Acid Hybridization (1985).
Hames, B. D., and Higgins, S. J., eds., [0598] Transcription and Translation (1984).
Hammerling et al., [0599] Monoclonal Antibodies and T-cell Hybridomas (1981).
Harlow and Lane, eds., [0600] Antibodies—A Laboratory Manual, Cold Spring Harbor, N.Y. (1988).
Heath, D. D., Iwama, G. K., and Devlin, R. H., (1993) PCR primed with VNTR core sequences yields species specific patterns and hypervariable probes. [0601] Nucl. Acids Res. 21(24):5782-5785.
Hughes, Jr., A. J., Bryan, S. K., Chen, H., Moses, R. E., and McHenry, C. S. (1991) [0602] Escherichia coli DNA polymerase II is stimulated by DNA polymerase III holoenzyme auxiliary subunits. J. Biol. Chem. 266:4568-4573.
IRL Press, Publ., [0603] Immobilized Cells and Enzymes (1986).
Ito, J., and Braithwaite, D., (1991) Compilation and alignment of DNA polymerase sequences. [0604] Nucl. Acids Res. 19(15):4045-4057 (1991).
Jacks, T., Madhami, H. D., Masiarz, F. R., and Varmus, H. E. (1988) Signals for ribosomal frameshifting in the Rous sarcoma virus gag-pol region. [0605] Cell 55:447-458.
Jay, E., et al., (1984) Chemical synthesis of a biologically active gene for human immune interferon-gamma. Prospect for site-specific mutagenesis and structure function studies. [0606] J. Biol. Chem. 259:6311-6317.
Kelman Z., and O'Donnell, M. (1995) DNA Polymerase III holoenzyme: Structure and function of a chromosomal replicating machine, [0607] Annu. Rev. Biochem., 64:171-200.
Kelman, Z., and O'Donnell, M. (1994) DNA replication: enzymology and mechanisms. [0608] Current Opinions in Genetics and Development 4:185-195.
Kennett et al., [0609] Monoclonal Antibodies (1980).
Kong, X.-P., Onrust, R., O'Donnell, M., and Kuriyan, J. (1992). Three dimensional structure of the β subunit of [0610] Escherichia coli DNA polymerase III holoenzyme: a sliding DNA clamp. Cell 69:425-437.
Kornberg, A., and Baker, T. (1992). [0611] DNA Replication, second edition. (New York: W. H. Freeman and Company), pp. 165-194.
Krishna, T. S., Kong, X.-P., Gary, S., Burgers, P. M., and Kuriyan, J. (1994) Crystal structure of the eukaryotic DNA polymerase processivity factor PCNA. Cell 79(7): 1233-1243. [0612]
Kuriyan, J. and O'Donnell, M. (1993) Sliding clamps of DNA polymerases.[0613] J. Mol. Biol. 234:915-925.
Larsen, B., Wills, N. M., Gesteland, R. F., and Atkins, J. F. (1994) rRNA-mRNA base pairing stimulates a programmed −1 ribosomal frameshift. [0614] J. Bact. 176: 6842-6851.
Lin, J. J., and Kuo, J. (1995) [0615] Focus 17(2):66-70.
Linn, S. (1991) How many pols does it take to replicate nuclear DNA? [0616] Cell 66:185-187.
Lee, S. H. and Walker, J. R. (1987) [0617] Escherichia coli dnaX product, the τ subunit of DNA polymerase III, is a multifunctional protein with single-stranded DNA-dependent ATPase activity. Proc. Natl. Acad. Sci. USA 84:2713-2717.
Low, R. L., Rashbaum, S. A., and Cozzarelli, N. R. (1976) Purification and characterization of DNA polymerase III from [0618] Bacillus subtilis. J. Biol. Chem., 251:1311-1325.
Maki, H., Maki, S., and Kornberg, A. (1988) DNA polymerase III holoenzyme of [0619] Escherichia coli IV. The holoenzyme is an asymmetric dimer with twin active sites. J. Biol. Chem. 263:6570-6578.
Maniatis, T., Fritsch, E. F., and Sambrook, J. (1992) [0620] Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
McHenry, C. S. (1991) DNA Polymerase III Holoenzyme. [0621] J. Biol. Chem., 266:19127-19130.
McHenry, C. S., and Crow, W. (1979) DNA polymerase III of [0622] Escherichia coli: Purification and identification of Subunits. J. Biol. Chem. 254(5):1748-1753.
McHenry, C. S. (1982) Purification and characterization of DNA polymerase III′. Identification of τ as a subunit of the DNA polymerase III holoenzyme. [0623] J. Biol. Chem. 257:2657-2663.
Moarefi, I., Jeruzalmi, D., Turner, J., O'Donnell, M., and Kuriyan, J. (2000). Crystal structure of the DNA polymerase processivity factor of T4 bacteriophage. [0624] J. Molec. Biol., 296:1215-1223.
Naktinis, V., Turner, J., and O'Donnell, M. (1996) A molecular switch in a replication machine defined by an internal competition for protein rings. [0625] Cell 84(1):137-145.
Naktinis, V., Onrust, R., Fang, L., O'Donnell, M. (1995) Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader, and sliding clamps in one holoenzyme particle. II. Intermediate complex between the clamp loader and its clamp. [0626] J. Biol. Chem. 270:13358-13365.
Nambair, K. P., et al., (1984) Total synthesis and cloning of a gene coding for the ribonuclease S protein. [0627] Science 223:1299-1300.
Nelson, K. E., et al., (1999) Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of [0628] Thermotoga maritima. Nature 399:323-329.
Noren, C. J., et al., (1989) A general method for site-specific incorporation of unnatural amino acids into proteins. [0629] Science 244:182-188.
O'Day, C. L., Burgers, P. M., Taylor, J. S. (1992) PCNA-induced DNA synthesis past cis-syn and trans-syn-I thymine dimers by calf thymus DNA polymerase delta in vitro. [0630] Nucl. Acids Res. 20(20):5403-6.
O'Donnell, M., (1987) Accessory proteins bind a primed template and Mediate Rapid Cycling of DNA polymerase III Holoenzyme from [0631] E. coli. J. Bibl. Chem. 262:16558-16565.
O'Donnell, M., Kuriyan, J., Kong, X-P., Stukenberg, P. T. and Onrust, R. (1992) The sliding clamp of DNA polymerase III holoenzyme encircles DNA. [0632] Molec. Biol. Cell 3:953-957.
O'Donnell, M., Onrust, R., Dean, F. B., Chen, M., and Hurwitz, J. (1993) Homology in accessory proteins of replicative polymerases-[0633] E. coli to humans. Nucl. Acids Res. 21:1-3.
Onrust, R., Finkelstein, J., Turner, J., Naktinis; V., and O'Donnell, M. (1995) Assembly of a chromosomal replication machine: two DNA polymerases, a clamp loader and sliding clamps in one holoenzyme particle. III) Interface between two polymerases and the clamp loader. [0634] J. Biol. Chem. 270:13366-13377.
Onrust, R. and O'Donnell, M. (I 993) DNA polymerase III accessory proteins. I) holA and holB encoding δ and δ′. [0635] J. Biol. Chem. 268:11758-11765.
Onrust, R., Stukenberg, P. T., and O'Donnell, M. (1991) Analysis of the ATPase subassembly which initiates processive DNA synthesis by DNA polymerase III holoenzyme. [0636] J. Biol. Chem. 266:21681-21686.
Oshima, T., and Imahori, K, (1974) Description of [0637] Thermus thermophilus (Yoshida and Oshima) comb-nov, a nonsporulating bacterium from a Japanese spa. Int. J. Syst. Bacteriol. 24(1):102-112.
Pacitti, D. F., Barnes, M. H., Li, D. H., and Brown, N. C. (1995) Characterization and overexpression of the gene encoding [0638] Staphylococcus aureus DNA polymnerase III. Gene, 1165:51-56.
Perbal, B., A Practical Guide to Molecular Cloning (1984). [0639]
Perrino, F. W., and Loeb, L. A. (1990) Hydrolysis of 3′-terminal mispairs in vitro by the 3′ 5′ exonuclease of DNA polymerase δ permits subsequent extension by DNA polymerase α. [0640] Biochem. 29:5226-5231.
Reddy et al., (1993) Assembly of a functional replication complex without ATP hydrolysis: a direct interaction of bacteriophage T4 gp45 with T4 DNA polymerase. [0641] Proc. Natl. Acad. Sci. USA 90(8):3211-3215.
Rowen, L., and Komberg, A. (1978) Primase, the DnaG protein of [0642] Escherichia coli. An enzyme which starts DNA chains. J. Biol. Chem. 253:758-764.
Ruttimann, C., Cotoras, M., Zaldivar, J., and Vicuna, R. (1985) DNA polymerases from the extremely thermophilic bacterium [0643] Thermus thermophilus HB-8. European J. Biochem. 149:41-46.
Sambrook et al., [0644] Molecular Cloning: A Laboratory Manual (1989).
Sanger, F., and Coulson, A. R., (1975) A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. [0645] J. Mol. Biol. 94:441-448.
Sanger, F., et al., (1977) DNA sequencing with chain-terminating inhibitors. [0646] Proc. Natl. Acad. Sci. USA 74:5463-5467
Schreier, M., et al., [0647] Hybridoma Techniques (11980).
Studwell-Vaughan, P. S. and O'Donnell, M. (1991) Constitution of the twin polymerase of DNA polymerase III holoenzyme. [0648] J. Biol. Chem. 266:19833-19841.
Studwell-Vaughan, P. S. and O'Donnell, M. (1990) Processive replication is contingent on the exonuclease subunit of DNA polymerase III holoenzyme. [0649] J. Biol. Chem. 265(2):1171-1178.
Stukenberg, P. T., Studwell-Vaughan, P. S., and O'Donnell, M. (1991) Mechanism of the sliding β-clamp of DNA polymerase III holoenzyme. [0650] J. Biol. Chem. 266:11328-11334.
Sugino, A. (1995) Yeast DNA polymerases and their role at the replication fork. Elsevier Science Ltd., 319-323. [0651]
Tabor, S. and Richardson, C. C. (1995) A single residue in DNA polymerases of the [0652] Escherichia coli DNA polymerase I family is critical for distinguishing between deoxy- and dideoxyribonucleotides. Proc. Natl. Acad. Sci. USA, 92(14):6339-6343.
Tan, C. K., Castillo, C., So, A. G., Downey, K. M. (1986) An auxiliary protein for DNA polymerase-delta from fetal calf thymus. J. Biol. Chem. 261(26):12310-6. [0653]
Towbin, H., Staehelin, T., Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. [0654] Proc. Natl. Acad. Sci. USA 76(9):4350-4354.
Tsuchihashi, Z., and Kornberg, A. (1989) ATP interactions of the tau and gamma subunits of DNA polymerase III holoenzyme of [0655] Escherichia coli. J. Biol. Chem. 264:17790-95.
Tsuchihashi, Z. and Kornberg, A. (1990) Translational frameshifting generates they subunit of DNA polymerase III holoenzyme. [0656] Proc. Natl. Acad. Sci. USA 87:2516-2520.
Tsuchihashi, Z., and Brown, P. O. (1992) Sequence requirements for efficient translational frameshifting in the [0657] Escherichia coli dnaX gene and the role of an unstable interaction between tRNALys and an AAG lysine codon. Genes and Dev. 6:511-519.
Turner, J., and O'Donnell, M. (1995) Cycling of [0658] Escherichia coli DNA polymerase III from one sliding clamp to another: model for lagging strand. Methods Enzymol. 262:442-449.
Vos, P., et al., (1995) AFLP: a new technique for DNA fingerprinting. [0659] Nucl. Acids Res. 23(21):4407-4414.
Weiss, R. B., Dunn, D. M., Atkins, J. F., and Gesteland, R. F., (1987) Slippery runs, shifty stops, backward steps, and forward hops: −2, −1, +2, +5 and +6 ribosomal frameshifting in Cold Spring Harbor Symposia on Quantitative Biology 52: 687-693. [0660]
Welsh, J., and McClelland, M., (1990) Fingerprinting genomes using PCR with arbitrary primers. [0661] Nucl. Acids Res. 18(24):7213-7218.
Wickner, W., and Komberg, A., (1974) A holoenzyme form of DNA Polymerase III. Isolation and Properties. [0662] J. Biol. Chem. 249(19):6244-6249.
Williams, J. G., et al., (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. [0663] Nucl. Acids Res. 18(22):6531-6535.
Yin, K-C., Blinkowa, A., and Walker, J. R. (1986) Nucleotide sequence of the Escherichia replication gene dnaZX. [0664] Nuc. Acids. Res. 14:6541-6549.
Young, M. C., Reddy, M. K., and von Hippel, P. H. (1992) Structure and function of the bacteriophage T4 DNA polymerase holoenzyme. [0665] Biochem., 31:8675-8690.
Yuzhakov, A., Turner, J. and O'Dotnell, M. (1996) Replisome assembly reveals the basis for asymmetric function in leading and lagging strand replication. [0666] Cell 86:877-886.
U.S. Pat. No. 5,668,004 to O'Donnell. [0667]
U.S. Pat. No. 5,583,026 to O'Donnell. [0668]
U.S. Pat. No. 5,545,552 to Mathur. [0669]
U.S. Pat. No. 5,498,523 to Tabor et al. [0670]
U.S. Pat. No. 5,455,166 to Walker. [0671]
U.S. Pat. No. 5,409,818 to Davey et al. [0672]
U.S. Pat. No. 5,374,553 to Gelfand et al. [0673]
U.S. Pat. No. 5,352,778 to Comb et al. [0674]
U.S. Pat. No. 5,322,785 to Comb et al. [0675]
U.S. Pat. No. 5,192,674 to Oshima et al. [0676]
U.S. Pat. No. 4,962,022 to Fleming et al. [0677]
U.S. Pat. No. 4,816,567 to Cabilly et al. [0678]
U.S. Pat. No. 4,816,397 to Boss et al. [0679]
U.S. Pat. No. 4,683,202 to Mullis. [0680]
U.S. Pat. No. 4,683,195 to Mullis et al. [0681]
U.S. Pat. No. 4,493,890 to Morris. [0682]
U.S. Pat. No. 4,493,795 to Nestor et al. [0683]
U.S. Pat. No. 4,491,632 to Wands et al. [0684]
U.S. Pat. No. 4,472,500 to Milstein et al. [0685]
U.S. Pat. No. 4,466,917 to Nussenzweig et al. [0686]
U.S. Pat. No. 4,451,570 to Royston et al. [0687]
U.S. Pat. No. 4,444,887 to Hoffman. [0688]
U.S. Pat. No. 4,427,783 to Newman et al. [0689]
U.S. Pat. No. 4,399,121 to Albarella et al. [0690]
U.S. Pat. No. 4,342,566 to Theofilopous et al. [0691]
U.S. Pat. No. 4,341,761 to Ganfield et al. [0692]
WO 96/10640 to Chatteijee et al. [0693]
EP 329,822 to Davey et al. [0694]
EP 534,858 to Vos et al. [0695]
This invention may be embodied in other forms or carried out in other ways without departing from the spirit or essential characteristics thereof. The present disclosure is therefore to be considered as in all respects illustrative and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein. [0696]
1 212 1 2007 DNA Thermus thermophilus 1 tccgggggtg gggttcccag gtagaccccg gcccctcccg tgagcccctt tacccaggcc 60 gccacctcct ccaggggggc caaggcgtgc aaggagagga acgtccgcac cacgccctat 120 actagccttg tgagcgccct ctaccgccgc ttccgccccc tcaccttcca ggaggtggtg 180 gggcaggagc acgtgaagga gcccctcctc aaggccatcc gggaggggag gctcgcccag 240 gcctacctct tctccgggcc caggggcgtg ggcaagacca ccacggcgag gctcctcgcc 300 atggcggtgg ggtgccaggg ggaagacccc ccttgcgggg tctgccccca ctgccaggcg 360 gtgcagaggg gcgcccaccc ggacgtggtg gacattgacg ccgccagcaa caactccgtg 420 gaggacgtgc gggagctgag ggaaaggatc cacctcgccc ccctctctgc ccccaggaag 480 gtcttcatcc tggacgaggc ccacatgctc tccaaaagcg ccttcaacgc cctcctcaag 540 accctggagg agcccccgcc ccacgtcctc ttcgtcttcg ccaccaccga gcccgagagg 600 atgcccccca ccatcctctc ccgcacccag cacttccgct tccgccgcct cacggaggag 660 gagatcgcct ttaagctccg gcgcatcctg gaggccgtgg ggcgggaggc ggaggaggag 720 gccctcctcc tcctcgcccg cctggcggac ggggccctta gggacgcgga aagcctcctg 780 gagcgcttcc tcctcctgga aggccccctc acccggaagg aggtggagcg cgccctaggc 840 tcccccccag ggaccggggt ggccgagatc gccgcctccc tcgcgagggg gaaaacggcg 900 gaggccctgg gcctcgcccg gcgcctctac ggggaagggt acgccccgag gagcctggtc 960 tcgggccttt tggaggtgtt ccgggaaggc ctctacgccg ccttcggcct cgcgggaacc 1020 ccccttcccg ccccgcccca ggccctgatc gccgccatga ccgccctgga cgaggccatg 1080 gagcgcctcg cccgccgctc cgacgcctta agcctggagg tggccctcct ggaggcggga 1140 agggccctgg ccgccgaggc cctaccccag cccacgggcg ctccttcccc agaggtcggc 1200 cccaagccgg aaagcccccc gaccccggaa cccccaaggc ccgaggaggc gcccgacctg 1260 cgggagcggt ggcgggcctt cctcgaggcc ctcaggccca ccctacgggc cttcgtgcgg 1320 gaggcccgcc cggaggtccg ggaaggccag ctctgcctcg ctttccccga ggacaaggcc 1380 ttccactacc gcaaggcctc ggaacagaag gtgaggctcc tccccctggc ccaggcccat 1440 ttcggggtgg aggaggtcgt cctcgtcctg gagggagaaa aaaaaagcct gagcccaagg 1500 ccccgcccgg ccccacctcc tgaagcgccc gcacccccgg gccctcccga ggaggaggta 1560 gaggcggagg aagcggcgga ggaggccccg gaggaggcct tgaggcgggt ggtccgcctc 1620 ctgggggggc gggtgctctg ggtgcggcgg cccaggaccc gggaggcgcc ggaggaggaa 1680 cccctgagcc aagacgagat agggggtact ggtatataat gggggcatga cgcggaccac 1740 cgacctcgga caagagaccg tggacaacat cctcaagcgc ctccgccgta ttgagggcca 1800 ggtgcggggg ctccagaaga tggtggccga gggccgcccc tgcgacgagg tcctcaccca 1860 gatgaccgcc accaagaagg ccatggaggc ggcggccacc ctgatcctcc acgagttcct 1920 gaacgtctgc gccgccgagg tctccgaggg caaggtgaac cccaagaagc ccgaggagat 1980 cgccaccatg ctgaagaact tcatcta 2007 2 529 PRT Thermus thermophilus 2 Met Ser Ala Leu Tyr Arg Arg Phe Arg Pro Leu Thr Phe Gln Glu Val 1 5 10 15 Val Gly Gln Glu His Val Lys Glu Pro Leu Leu Lys Ala Ile Arg Glu 20 25 30 Gly Arg Leu Ala Gln Ala Tyr Leu Phe Ser Gly Pro Arg Gly Val Gly 35 40 45 Lys Thr Thr Thr Ala Arg Leu Leu Ala Met Ala Val Gly Cys Gln Gly 50 55 60 Glu Asp Pro Pro Cys Gly Val Cys Pro His Cys Gln Ala Val Gln Arg 65 70 75 80 Gly Ala His Pro Asp Val Val Asp Ile Asp Ala Ala Ser Asn Asn Ser 85 90 95 Val Glu Asp Val Arg Glu Leu Arg Glu Arg Ile His Leu Ala Pro Leu 100 105 110 Ser Ala Pro Arg Lys Val Phe Ile Leu Asp Glu Ala His Met Leu Ser 115 120 125 Lys Ser Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu Glu Pro Pro Pro 130 135 140 His Val Leu Phe Val Phe Ala Thr Thr Glu Pro Glu Arg Met Pro Pro 145 150 155 160 Thr Ile Leu Ser Arg Thr Gln His Phe Arg Phe Arg Arg Leu Thr Glu 165 170 175 Glu Glu Ile Ala Phe Lys Leu Arg Arg Ile Leu Glu Ala Val Gly Arg 180 185 190 Glu Ala Glu Glu Glu Ala Leu Leu Leu Leu Ala Arg Leu Ala Asp Gly 195 200 205 Ala Leu Arg Asp Ala Glu Ser Leu Leu Glu Arg Phe Leu Leu Leu Glu 210 215 220 Gly Pro Leu Thr Arg Lys Glu Val Glu Arg Ala Leu Gly Ser Pro Pro 225 230 235 240 Gly Thr Gly Val Ala Glu Ile Ala Ala Ser Leu Ala Arg Gly Lys Thr 245 250 255 Ala Glu Ala Leu Gly Leu Ala Arg Arg Leu Tyr Gly Glu Gly Tyr Ala 260 265 270 Pro Arg Ser Leu Val Ser Gly Leu Leu Glu Val Phe Arg Glu Gly Leu 275 280 285 Tyr Ala Ala Phe Gly Leu Ala Gly Thr Pro Leu Pro Ala Pro Pro Gln 290 295 300 Ala Leu Ile Ala Ala Met Thr Ala Leu Asp Glu Ala Met Glu Arg Leu 305 310 315 320 Ala Arg Arg Ser Asp Ala Leu Ser Leu Glu Val Ala Leu Leu Glu Ala 325 330 335 Gly Arg Ala Leu Ala Ala Glu Ala Leu Pro Gln Pro Thr Gly Ala Pro 340 345 350 Ser Pro Glu Val Gly Pro Lys Pro Glu Ser Pro Pro Thr Pro Glu Pro 355 360 365 Pro Arg Pro Glu Glu Ala Pro Asp Leu Arg Glu Arg Trp Arg Ala Phe 370 375 380 Leu Glu Ala Leu Arg Pro Thr Leu Arg Ala Phe Val Arg Glu Ala Arg 385 390 395 400 Pro Glu Val Arg Glu Gly Gln Leu Cys Leu Ala Phe Pro Glu Asp Lys 405 410 415 Ala Phe His Tyr Arg Lys Ala Ser Glu Gln Lys Val Arg Leu Leu Pro 420 425 430 Leu Ala Gln Ala His Phe Gly Val Glu Glu Val Val Leu Val Leu Glu 435 440 445 Gly Glu Lys Lys Ser Leu Ser Pro Arg Pro Arg Pro Ala Pro Pro Pro 450 455 460 Glu Ala Pro Ala Pro Pro Gly Pro Pro Glu Glu Glu Val Glu Ala Glu 465 470 475 480 Glu Ala Ala Glu Glu Ala Pro Glu Glu Ala Leu Arg Arg Val Val Arg 485 490 495 Leu Leu Gly Gly Arg Val Leu Trp Val Arg Arg Pro Arg Thr Arg Glu 500 505 510 Ala Pro Glu Glu Glu Pro Leu Ser Gln Asp Glu Ile Gly Gly Thr Gly 515 520 525 Ile 3 1590 DNA Thermus thermophilus 3 gtgagcgccc tctaccgccg cttccgcccc ctcaccttcc aggaggtggt ggggcaggag 60 cacgtgaagg agcccctcct caaggccatc cgggagggga ggctcgccca ggcctacctc 120 ttctccgggc ccaggggcgt gggcaagacc accacggcga ggctcctcgc catggcggtg 180 gggtgccagg gggaagaccc cccttgcggg gtctgccccc actgccaggc ggtgcagagg 240 ggcgcccacc cggacgtggt ggacattgac gccgccagca acaactccgt ggaggacgtg 300 cgggagctga gggaaaggat ccacctcgcc cccctctctg cccccaggaa ggtcttcatc 360 ctggacgagg cccacatgct ctccaaaagc gccttcaacg ccctcctcaa gaccctggag 420 gagcccccgc cccacgtcct cttcgtcttc gccaccaccg agcccgagag gatgcccccc 480 accatcctct cccgcaccca gcacttccgc ttccgccgcc tcacggagga ggagatcgcc 540 tttaagctcc ggcgcatcct ggaggccgtg gggcgggagg cggaggagga ggccctcctc 600 ctcctcgccc gcctggcgga cggggccctt agggacgcgg aaagcctcct ggagcgcttc 660 ctcctcctgg aaggccccct cacccggaag gaggtggagc gcgccctagg ctccccccca 720 gggaccgggg tggccgagat cgccgcctcc ctcgcgaggg ggaaaacggc ggaggccctg 780 ggcctcgccc ggcgcctcta cggggaaggg tacgccccga ggagcctggt ctcgggcctt 840 ttggaggtgt tccgggaagg cctctacgcc gccttcggcc tcgcgggaac cccccttccc 900 gccccgcccc aggccctgat cgccgccatg accgccctgg acgaggccat ggagcgcctc 960 gcccgccgct ccgacgcctt aagcctggag gtggccctcc tggaggcggg aagggccctg 1020 gccgccgagg ccctacccca gcccacgggc gctccttccc cagaggtcgg ccccaagccg 1080 gaaagccccc cgaccccgga acccccaagg cccgaggagg cgcccgacct gcgggagcgg 1140 tggcgggcct tcctcgaggc cctcaggccc accctacggg ccttcgtgcg ggaggcccgc 1200 ccggaggtcc gggaaggcca gctctgcctc gctttccccg aggacaaggc cttccactac 1260 cgcaaggcct cggaacagaa ggtgaggctc ctccccctgg cccaggccca tttcggggtg 1320 gaggaggtcg tcctcgtcct ggagggagaa aaaaaaagcc tgagcccaag gccccgcccg 1380 gccccacctc ctgaagcgcc cgcacccccg ggccctcccg aggaggaggt agaggcggag 1440 gaagcggcgg aggaggcccc ggaggaggcc ttgaggcggg tggtccgcct cctggggggg 1500 cgggtgctct gggtgcggcg gcccaggacc cgggaggcgc cggaggagga acccctgagc 1560 caagacgaga tagggggtac tggtatataa 1590 4 464 PRT Thermus thermophilus 4 Met Ser Ala Leu Tyr Arg Arg Phe Arg Pro Leu Thr Phe Gln Glu Val 1 5 10 15 Val Gly Gln Glu His Val Lys Glu Pro Leu Leu Lys Ala Ile Arg Glu 20 25 30 Gly Arg Leu Ala Gln Ala Tyr Leu Phe Ser Gly Pro Arg Gly Val Gly 35 40 45 Lys Thr Thr Thr Ala Arg Leu Leu Ala Met Ala Val Gly Cys Gln Gly 50 55 60 Glu Asp Pro Pro Cys Gly Val Cys Pro His Cys Gln Ala Val Gln Arg 65 70 75 80 Gly Ala His Pro Asp Val Val Asp Ile Asp Ala Ala Ser Asn Asn Ser 85 90 95 Val Glu Asp Val Arg Glu Leu Arg Glu Arg Ile His Leu Ala Pro Leu 100 105 110 Ser Ala Pro Arg Lys Val Phe Ile Leu Asp Glu Ala His Met Leu Ser 115 120 125 Lys Ser Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu Glu Pro Pro Pro 130 135 140 His Val Leu Phe Val Phe Ala Thr Thr Glu Pro Glu Arg Met Pro Pro 145 150 155 160 Thr Ile Leu Ser Arg Thr Gln His Phe Arg Phe Arg Arg Leu Thr Glu 165 170 175 Glu Glu Ile Ala Phe Lys Leu Arg Arg Ile Leu Glu Ala Val Gly Arg 180 185 190 Glu Ala Glu Glu Glu Ala Leu Leu Leu Leu Ala Arg Leu Ala Asp Gly 195 200 205 Ala Leu Arg Asp Ala Glu Ser Leu Leu Glu Arg Phe Leu Leu Leu Glu 210 215 220 Gly Pro Leu Thr Arg Lys Glu Val Glu Arg Ala Leu Gly Ser Pro Pro 225 230 235 240 Gly Thr Gly Val Ala Glu Ile Ala Ala Ser Leu Ala Arg Gly Lys Thr 245 250 255 Ala Glu Ala Leu Gly Leu Ala Arg Arg Leu Tyr Gly Glu Gly Tyr Ala 260 265 270 Pro Arg Ser Leu Val Ser Gly Leu Leu Glu Val Phe Arg Glu Gly Leu 275 280 285 Tyr Ala Ala Phe Gly Leu Ala Gly Thr Pro Leu Pro Ala Pro Pro Gln 290 295 300 Ala Leu Ile Ala Ala Met Thr Ala Leu Asp Glu Ala Met Glu Arg Leu 305 310 315 320 Ala Arg Arg Ser Asp Ala Leu Ser Leu Glu Val Ala Leu Leu Glu Ala 325 330 335 Gly Arg Ala Leu Ala Ala Glu Ala Leu Pro Gln Pro Thr Gly Ala Pro 340 345 350 Ser Pro Glu Val Gly Pro Lys Pro Glu Ser Pro Pro Thr Pro Glu Pro 355 360 365 Pro Arg Pro Glu Glu Ala Pro Asp Leu Arg Glu Arg Trp Arg Ala Phe 370 375 380 Leu Glu Ala Leu Arg Pro Thr Leu Arg Ala Phe Val Arg Glu Ala Arg 385 390 395 400 Pro Glu Val Arg Glu Gly Gln Leu Cys Leu Ala Phe Pro Glu Asp Lys 405 410 415 Ala Phe His Tyr Arg Lys Ala Ser Glu Gln Lys Val Arg Leu Leu Pro 420 425 430 Leu Ala Gln Ala His Phe Gly Val Glu Glu Val Val Leu Val Leu Glu 435 440 445 Gly Glu Lys Lys Lys Pro Glu Pro Lys Ala Pro Pro Gly Pro Thr Ser 450 455 460 5 454 PRT Thermus thermophilus 5 Met Ser Ala Leu Tyr Arg Arg Phe Arg Pro Leu Thr Phe Gln Glu Val 1 5 10 15 Val Gly Gln Glu His Val Lys Glu Pro Leu Leu Lys Ala Ile Arg Glu 20 25 30 Gly Arg Leu Ala Gln Ala Tyr Leu Phe Ser Gly Pro Arg Gly Val Gly 35 40 45 Lys Thr Thr Thr Ala Arg Leu Leu Ala Met Ala Val Gly Cys Gln Gly 50 55 60 Glu Asp Pro Pro Cys Gly Val Cys Pro His Cys Gln Ala Val Gln Arg 65 70 75 80 Gly Ala His Pro Asp Val Val Asp Ile Asp Ala Ala Ser Asn Asn Ser 85 90 95 Val Glu Asp Val Arg Glu Leu Arg Glu Arg Ile His Leu Ala Pro Leu 100 105 110 Ser Ala Pro Arg Lys Val Phe Ile Leu Asp Glu Ala His Met Leu Ser 115 120 125 Lys Ser Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu Glu Pro Pro Pro 130 135 140 His Val Leu Phe Val Phe Ala Thr Thr Glu Pro Glu Arg Met Pro Pro 145 150 155 160 Thr Ile Leu Ser Arg Thr Gln His Phe Arg Phe Arg Arg Leu Thr Glu 165 170 175 Glu Glu Ile Ala Phe Lys Leu Arg Arg Ile Leu Glu Ala Val Gly Arg 180 185 190 Glu Ala Glu Glu Glu Ala Leu Leu Leu Leu Ala Arg Leu Ala Asp Gly 195 200 205 Ala Leu Arg Asp Ala Glu Ser Leu Leu Glu Arg Phe Leu Leu Leu Glu 210 215 220 Gly Pro Leu Thr Arg Lys Glu Val Glu Arg Ala Leu Gly Ser Pro Pro 225 230 235 240 Gly Thr Gly Val Ala Glu Ile Ala Ala Ser Leu Ala Arg Gly Lys Thr 245 250 255 Ala Glu Ala Leu Gly Leu Ala Arg Arg Leu Tyr Gly Glu Gly Tyr Ala 260 265 270 Pro Arg Ser Leu Val Ser Gly Leu Leu Glu Val Phe Arg Glu Gly Leu 275 280 285 Tyr Ala Ala Phe Gly Leu Ala Gly Thr Pro Leu Pro Ala Pro Pro Gln 290 295 300 Ala Leu Ile Ala Ala Met Thr Ala Leu Asp Glu Ala Met Glu Arg Leu 305 310 315 320 Ala Arg Arg Ser Asp Ala Leu Ser Leu Glu Val Ala Leu Leu Glu Ala 325 330 335 Gly Arg Ala Leu Ala Ala Glu Ala Leu Pro Gln Pro Thr Gly Ala Pro 340 345 350 Ser Pro Glu Val Gly Pro Lys Pro Glu Ser Pro Pro Thr Pro Glu Pro 355 360 365 Pro Arg Pro Glu Glu Ala Pro Asp Leu Arg Glu Arg Trp Arg Ala Phe 370 375 380 Leu Glu Ala Leu Arg Pro Thr Leu Arg Ala Phe Val Arg Glu Ala Arg 385 390 395 400 Pro Glu Val Arg Glu Gly Gln Leu Cys Leu Ala Phe Pro Glu Asp Lys 405 410 415 Ala Phe His Tyr Arg Lys Ala Ser Glu Gln Lys Val Arg Leu Leu Pro 420 425 430 Leu Ala Gln Ala His Phe Gly Val Glu Glu Val Val Leu Val Leu Glu 435 440 445 Gly Glu Lys Lys Lys Ala 450 6 32 DNA Artificial Sequence Description of Artificial Sequence primer 6 cgcaagcttc acgcstacct sttctccggs ac 32 7 8 PRT Artificial Sequence Description of Artificial Sequence peptide 7 His Ala Tyr Leu Phe Ser Gly Thr 1 5 8 34 DNA Artificial Sequence Description of Artificial Sequence primer 8 cgcgaattcg tgctcsggsg gctcctcsag sgtc 34 9 9 PRT Artificial Sequence Description of Artificial Sequence peptide 9 Lys Thr Leu Glu Glu Pro Pro Glu His 1 5 10 38 DNA Artificial Sequence Description of Artificial Sequence primer 10 gcgcggatcc ggagggagaa aaaaaaagcc tcagccca 38 11 38 DNA Artificial Sequence Description of Artificial Sequence primer 11 gcgcggatcc ggagggagag aagaaaagcc tcagccca 38 12 28 DNA Artificial Sequence Description of Artificial Sequence primer 12 gaattaaatt cgcgcttcgg gaggtggg 28 13 27 DNA Artificial Sequence Description of Artificial Sequence primer 13 gcgcgaattc gcgcttcggg aggtggg 27 14 29 DNA Artificial Sequence Description of Artificial Sequence primer 14 gcgcgaattc gggcgcttca ggaggtggg 29 15 31 DNA Artificial Sequence Description of Artificial Sequence primer 15 gtggtgcata tggtgagcgc cctctaccgc c 31 16 31 DNA Artificial Sequence Description of Artificial Sequence primer 16 gtggtggtcg acccaggagg gccacctcca g 31 17 8 PRT Artificial Sequence Description of Artificial Sequence peptide 17 Gly Xaa Xaa Gly Xaa Gly Lys Thr 1 5 18 12 PRT Artificial Sequence Description of Artificial Sequence peptide 18 Lys Pro Asp Pro Lys Ala Pro Pro Gly Pro Thr Ser 1 5 10 19 180 PRT Escherichia coli 19 Met Ser Tyr Gln Val Leu Ala Arg Lys Trp Arg Pro Gln Thr Phe Ala 1 5 10 15 Asp Val Val Gly Gln Glu His Val Leu Thr Ala Leu Ala Asn Gly Leu 20 25 30 Ser Leu Gly Arg Ile His His Ala Tyr Leu Phe Ser Gly Thr Arg Gly 35 40 45 Val Gly Lys Thr Ser Ile Ala Arg Leu Leu Ala Lys Gly Leu Asn Cys 50 55 60 Glu Thr Gly Ile Thr Ala Thr Pro Cys Gly Val Cys Asp Asn Cys Arg 65 70 75 80 Glu Ile Glu Gln Gly Arg Phe Val Asp Leu Ile Glu Ile Asp Ala Ala 85 90 95 Ser Arg Thr Lys Val Glu Asp Thr Arg Asp Leu Leu Asp Asn Val Gln 100 105 110 Tyr Ala Pro Ala Arg Gly Arg Phe Lys Val Tyr Leu Ile Asp Glu Val 115 120 125 His Met Leu Ser Arg His Ser Phe Asn Ala Leu Leu Lys Thr Leu Glu 130 135 140 Glu Pro Pro Glu His Val Lys Phe Leu Leu Ala Thr Thr Asp Pro Gln 145 150 155 160 Lys Leu Pro Val Thr Ile Leu Ser Arg Cys Leu Gln Phe His Leu Lys 165 170 175 Ala Leu Asp Val 180 20 180 PRT Bacillus subtilis 20 Met Ser Tyr Gln Ala Leu Tyr Arg Val Phe Arg Pro Gln Arg Phe Glu 1 5 10 15 Asp Val Val Gly Gln Glu His Ile Thr Lys Thr Leu Gln Asn Ala Leu 20 25 30 Leu Gln Lys Lys Phe Ser His Ala Tyr Leu Phe Ser Gly Pro Arg Gly 35 40 45 Thr Gly Lys Thr Ser Ala Ala Lys Ile Phe Ala Lys Ala Val Asn Cys 50 55 60 Glu His Ala Pro Val Asp Glu Pro Cys Asn Glu Cys Ala Ala Cys Lys 65 70 75 80 Gly Ile Thr Asn Gly Ser Ile Ser Asp Val Ile Glu Ile Asp Ala Ala 85 90 95 Ser Asn Asn Gly Val Asp Glu Ile Arg Asp Ile Arg Asp Lys Val Lys 100 105 110 Phe Ala Pro Ser Ala Val Thr Tyr Lys Val Tyr Ile Ile Asp Glu Val 115 120 125 His Met Leu Ser Ile Gly Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu 130 135 140 Glu Pro Pro Glu His Cys Ile Phe Ile Leu Ala Thr Thr Glu Pro His 145 150 155 160 Lys Ile Pro Leu Thr Ile Ile Ser Arg Cys Gln Arg Phe Asp Phe Lys 165 170 175 Arg Ile Thr Ser 180 21 294 PRT Escherichia coli 21 Met Ser Tyr Gln Val Leu Ala Arg Lys Trp Arg Pro Gln Thr Phe Ala 1 5 10 15 Asp Val Val Gly Gln Glu His Val Leu Thr Ala Leu Ala Asn Gly Leu 20 25 30 Ser Leu Gly Arg Ile His His Ala Tyr Leu Phe Ser Gly Thr Arg Gly 35 40 45 Val Gly Lys Thr Ser Ile Ala Arg Leu Leu Ala Lys Gly Leu Asn Cys 50 55 60 Glu Thr Gly Ile Thr Ala Thr Pro Cys Gly Val Cys Asp Asn Cys Arg 65 70 75 80 Glu Ile Glu Gln Gly Arg Phe Val Asp Leu Ile Glu Ile Asp Ala Ala 85 90 95 Ser Arg Thr Lys Val Glu Asp Thr Arg Asp Leu Leu Asp Asn Val Gln 100 105 110 Tyr Ala Pro Ala Arg Gly Arg Phe Lys Val Tyr Leu Ile Asp Glu Val 115 120 125 His Met Leu Ser Arg His Ser Phe Asn Ala Leu Leu Lys Thr Leu Glu 130 135 140 Glu Pro Pro Glu His Val Lys Phe Leu Leu Ala Thr Thr Asp Pro Gln 145 150 155 160 Lys Leu Pro Val Thr Ile Leu Ser Arg Cys Leu Gln Phe His Leu Lys 165 170 175 Ala Leu Asp Val Glu Gln Ile Arg His Gln Leu Glu His Ile Leu Asn 180 185 190 Glu Glu His Ile Ala His Glu Pro Arg Ala Leu Gln Leu Leu Ala Arg 195 200 205 Ala Ala Glu Gly Ser Leu Arg Asp Ala Leu Ser Leu Thr Asp Gln Ala 210 215 220 Ile Ala Ser Gly Asp Gly Gln Val Ser Thr Gln Ala Val Ser Ala Met 225 230 235 240 Leu Gly Thr Leu Asp Asp Asp Gln Ala Leu Ser Leu Val Glu Ala Met 245 250 255 Val Glu Ala Asn Gly Glu Arg Val Met Ala Leu Ile Asn Glu Ala Ala 260 265 270 Ala Arg Gly Ile Glu Trp Glu Ala Leu Leu Val Glu Met Leu Gly Leu 275 280 285 Leu His Arg Ile Ala Met 290 22 294 PRT Haemophilus influenzae 22 Met Ser Tyr Gln Val Leu Ala Arg Lys Trp Arg Pro Lys Thr Phe Ala 1 5 10 15 Asp Val Val Gly Gln Glu His Ile Ile Thr Ala Leu Ala Asn Gly Leu 20 25 30 Lys Asp Asn Arg Leu His His Ala Tyr Leu Phe Ser Gly Thr Arg Gly 35 40 45 Val Gly Lys Thr Ser Ile Ala Arg Leu Phe Ala Lys Gly Leu Asn Cys 50 55 60 Val His Gly Val Thr Ala Thr Pro Cys Gly Glu Cys Glu Asn Cys Lys 65 70 75 80 Ala Ile Glu Gln Gly Asn Phe Ile Asp Leu Ile Glu Ile Asp Ala Ala 85 90 95 Ser Arg Thr Lys Val Glu Asp Thr Arg Glu Leu Leu Asp Asn Val Gln 100 105 110 Tyr Lys Pro Val Val Gly Arg Phe Lys Val Tyr Leu Ile Asp Glu Val 115 120 125 His Met Leu Ser Arg His Ser Phe Asn Ala Leu Leu Lys Thr Leu Glu 130 135 140 Glu Pro Pro Glu Tyr Val Lys Phe Leu Leu Ala Thr Thr Asp Pro Gln 145 150 155 160 Lys Leu Pro Val Thr Ile Leu Ser Arg Cys Leu Gln Phe His Leu Lys 165 170 175 Ala Leu Asp Glu Thr Gln Ile Ser Gln His Leu Ala His Ile Leu Thr 180 185 190 Gln Glu Asn Ile Pro Phe Glu Asp Pro Ala Leu Val Lys Leu Ala Lys 195 200 205 Ala Ala Gln Gly Ser Ile Arg Asp Ser Leu Ser Leu Thr Asp Gln Ala 210 215 220 Ile Ala Met Gly Asp Arg Gln Val Thr Asn Asn Val Val Ser Asn Met 225 230 235 240 Leu Gly Leu Leu Asp Asp Asn Tyr Ser Val Asp Ile Leu Tyr Ala Leu 245 250 255 His Gln Gly Asn Gly Glu Leu Leu Met Arg Thr Leu Gln Arg Val Ala 260 265 270 Asp Ala Ala Gly Asp Trp Asp Lys Leu Leu Gly Glu Cys Ala Glu Lys 275 280 285 Leu His Gln Ile Ala Leu 290 23 294 PRT Bacillus subtilis 23 Met Ser Tyr Gln Ala Leu Tyr Arg Val Phe Arg Pro Gln Arg Phe Glu 1 5 10 15 Asp Val Val Gly Gln Glu His Ile Thr Lys Thr Leu Gln Asn Ala Leu 20 25 30 Leu Gln Lys Lys Phe Ser His Ala Tyr Leu Phe Ser Gly Pro Arg Gly 35 40 45 Thr Gly Lys Thr Ser Ala Ala Lys Ile Phe Ala Lys Ala Val Asn Cys 50 55 60 Glu His Ala Pro Val Asp Glu Pro Cys Asn Glu Cys Ala Ala Cys Lys 65 70 75 80 Gly Ile Thr Asn Gly Ser Ile Ser Asp Val Ile Glu Ile Asp Ala Ala 85 90 95 Ser Asn Asn Gly Val Asp Glu Ile Arg Asp Ile Arg Asp Lys Val Lys 100 105 110 Phe Ala Pro Ser Ala Val Thr Tyr Lys Val Tyr Ile Ile Asp Glu Val 115 120 125 His Met Leu Ser Ile Gly Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu 130 135 140 Glu Pro Pro Glu His Cys Ile Phe Ile Leu Ala Thr Thr Glu Pro His 145 150 155 160 Lys Ile Pro Leu Thr Ile Ile Ser Arg Cys Gln Arg Phe Asp Phe Lys 165 170 175 Arg Ile Thr Ser Gln Ala Ile Val Gly Arg Met Asn Lys Ile Val Asp 180 185 190 Ala Glu Gln Leu Gln Val Glu Glu Gly Ser Leu Glu Ile Ile Ala Ser 195 200 205 Ala Ala His Gly Gly Met Arg Asp Ala Leu Ser Leu Leu Asp Gln Ala 210 215 220 Ile Ser Phe Ser Gly Asp Ile Leu Lys Val Glu Asp Ala Leu Leu Ile 225 230 235 240 Thr Gly Ala Val Ser Gln Leu Tyr Ile Gly Lys Leu Ala Lys Ser Leu 245 250 255 His Asp Lys Asn Val Ser Asp Ala Leu Glu Thr Leu Asn Glu Leu Leu 260 265 270 Gln Gln Gly Lys Asp Pro Ala Lys Leu Ile Glu Asp Met Ile Phe Tyr 275 280 285 Phe Arg Asp Met Leu Leu 290 24 300 PRT Caulobacter crescentus 24 Asp Ala Tyr Thr Val Leu Ala Arg Lys Tyr Arg Pro Arg Thr Phe Glu 1 5 10 15 Asp Leu Ile Gly Gln Glu Ala Met Val Arg Thr Leu Ala Asn Ala Phe 20 25 30 Ser Thr Gly Arg Ile Ala His Ala Phe Met Leu Thr Gly Val Arg Gly 35 40 45 Val Gly Lys Thr Thr Thr Ala Arg Leu Leu Ala Arg Ala Leu Asn Tyr 50 55 60 Glu Thr Asp Thr Val Lys Gly Pro Ser Val Asp Leu Thr Thr Glu Gly 65 70 75 80 Tyr His Cys Arg Ser Ile Ile Glu Gly Arg His Met Asp Val Leu Glu 85 90 95 Leu Asp Ala Ala Ser Arg Thr Lys Val Asp Glu Met Arg Glu Leu Leu 100 105 110 Asp Gly Val Arg Tyr Ala Pro Val Glu Ala Arg Tyr Lys Val Tyr Ile 115 120 125 Ile Asp Glu Val His Met Leu Ser Thr Ala Ala Phe Asn Ala Leu Leu 130 135 140 Lys Thr Leu Glu Glu Pro Pro Pro His Ala Lys Phe Ile Phe Ala Thr 145 150 155 160 Thr Glu Ile Arg Lys Val Pro Val Thr Ile Leu Ser Arg Cys Gln Arg 165 170 175 Phe Asp Leu Arg Arg Val Glu Pro Asp Val Leu Val Lys His Phe Asp 180 185 190 Arg Ile Ser Ala Lys Glu Gly Ala Arg Ile Glu Met Asp Ala Leu Ala 195 200 205 Leu Ile Ala Arg Ala Ala Glu Gly Ser Val Arg Asp Gly Leu Ser Leu 210 215 220 Leu Asp Gln Ala Ile Val Gln Thr Glu Arg Gly Gln Thr Val Thr Ser 225 230 235 240 Thr Val Val Arg Asp Met Leu Gly Leu Ala Asp Arg Ser Gln Thr Ile 245 250 255 Ala Leu Tyr Glu His Val Met Ala Gly Lys Thr Lys Asp Ala Leu Glu 260 265 270 Gly Phe Arg Ala Leu Trp Gly Phe Gly Ala Asp Pro Ala Val Val Met 275 280 285 Leu Asp Val Leu Asp His Cys His Ala Ser Ala Val 290 295 300 25 260 PRT Mycoplasma genitalium 25 Met His Gln Val Phe Tyr Gln Lys Tyr Arg Pro Ile Asn Phe Lys Gln 1 5 10 15 Thr Leu Gly Gln Glu Ser Ile Arg Lys Ile Leu Val Asn Ala Ile Asn 20 25 30 Arg Asp Lys Leu Pro Asn Gly Tyr Ile Phe Ser Gly Glu Arg Gly Thr 35 40 45 Gly Lys Thr Thr Phe Ala Lys Ile Ile Ala Lys Ala Ile Asn Cys Leu 50 55 60 Asn Trp Asp Gln Ile Asp Val Cys Asn Ser Cys Asp Val Cys Lys Ser 65 70 75 80 Ile Asn Thr Asn Ser Ala Ile Asp Ile Val Glu Ile Asp Ala Ala Ser 85 90 95 Lys Asn Gly Ile Asn Asp Ile Arg Glu Leu Val Glu Asn Val Phe Asn 100 105 110 His Pro Phe Thr Phe Lys Lys Lys Val Tyr Ile Leu Asp Glu Ala His 115 120 125 Met Leu Thr Thr Gln Ser Trp Gly Gly Leu Leu Lys Thr Leu Glu Glu 130 135 140 Ser Pro Pro Tyr Val Leu Phe Ile Phe Thr Thr Thr Glu Phe Asn Lys 145 150 155 160 Ile Pro Leu Thr Ile Leu Ser Arg Cys Gln Ser Phe Phe Phe Lys Lys 165 170 175 Ile Thr Ser Asp Leu Ile Leu Glu Arg Leu Asn Asp Ile Ala Lys Lys 180 185 190 Glu Lys Ile Lys Ile Glu Lys Asp Ala Leu Ile Lys Ile Ala Asp Leu 195 200 205 Ser Gln Gly Ser Leu Arg Asp Gly Leu Ser Leu Leu Asp Gln Leu Ala 210 215 220 Ile Ser Leu Ile Val Lys Lys Leu Val Leu Leu Met Leu Lys Lys His 225 230 235 240 Leu Ile Ser Leu Ile Glu Met Gln Asn Leu Leu Leu Leu Lys Gln Phe 245 250 255 Tyr Gln Glu Ile 260 26 289 PRT Thermus thermophilus 26 Val Ser Ala Leu Tyr Arg Arg Phe Arg Pro Leu Thr Phe Gln Glu Val 1 5 10 15 Val Gly Gln Glu His Val Lys Glu Pro Leu Leu Lys Ala Ile Arg Glu 20 25 30 Gly Arg Leu Ala Gln Ala Tyr Leu Phe Ser Gly Pro Arg Gly Val Gly 35 40 45 Lys Thr Thr Thr Ala Arg Leu Leu Ala Met Ala Val Gly Cys Gln Gly 50 55 60 Glu Asp Pro Pro Cys Gly Val Cys Pro His Cys Gln Ala Val Gln Arg 65 70 75 80 Gly Ala His Pro Asp Val Val Asp Ile Asp Ala Ala Ser Asn Asn Ser 85 90 95 Val Glu Asp Val Arg Glu Leu Arg Glu Arg Ile His Leu Ala Pro Leu 100 105 110 Ser Ala Pro Arg Lys Val Phe Ile Leu Asp Glu Ala His Met Leu Ser 115 120 125 Lys Ser Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu Glu Pro Pro Pro 130 135 140 His Val Leu Phe Val Phe Ala Thr Thr Glu Pro Glu Arg Met Pro Pro 145 150 155 160 Thr Ile Leu Ser Arg Thr Gln His Phe Arg Phe Arg Arg Leu Thr Glu 165 170 175 Glu Glu Ile Ala Phe Lys Leu Arg Arg Ile Leu Glu Ala Val Gly Arg 180 185 190 Glu Ala Glu Glu Glu Ala Leu Leu Leu Leu Ala Arg Leu Ala Asp Gly 195 200 205 Ala Leu Arg Asp Ala Glu Ser Leu Leu Glu Arg Phe Leu Leu Leu Glu 210 215 220 Gly Pro Leu Thr Arg Lys Glu Val Glu Arg Ala Leu Gly Ser Pro Pro 225 230 235 240 Gly Thr Gly Val Ala Glu Ile Ala Ala Ser Leu Ala Arg Gly Lys Thr 245 250 255 Ala Glu Ala Leu Gly Leu Ala Arg Arg Leu Tyr Gly Glu Gly Tyr Ala 260 265 270 Pro Arg Ser Leu Val Ser Gly Leu Leu Glu Val Phe Arg Glu Gly Leu 275 280 285 Tyr 27 94 DNA Thermus thermophilus 27 gccggaggga gaaaaaaaaa gccgagccca aggccccgcc cggccccacc ccgaagcgcc 60 cgcacccccg ggccccccga ggaggaggag aggc 94 28 11 PRT Thermus thermophilus 28 Val Leu Glu Gly Glu Lys Lys Ser Leu Ser Pro 1 5 10 29 23 DNA Artificial Sequence Description of Artificial Sequence primer 29 cacgcntacc tnttctccgg nac 23 30 25 DNA Artificial Sequence Description of Artificial Sequence primer 30 gtgctcnggn ggctcctcnt cngtc 25 31 33 DNA Artificial Sequence Description of Artificial Sequence primer 31 gtgggatccg tggttctgga tctcgatgaa gaa 33 32 29 DNA Artificial Sequence Description of Artificial Sequence primer 32 gtgggatcca cggsctstcs gagcagaag 29 33 34 DNA Artificial Sequence Description of Artificial Sequence primer 33 gcgggatcct caacgaggac ctctccatct tcaa 34 34 35 DNA Artificial Sequence Description of Artificial Sequence primer 34 gcgggatcct tgtcgtcsag sgtsagsgcg tcgta 35 35 39 DNA Artificial Sequence Description of Artificial Sequence primer 35 gggaaggacc agcgcgtact ccccctgctc ctaggtgtg 39 36 27 DNA Artificial Sequence Description of Artificial Sequence primer 36 gtgtggatcc ttcttcttsc ccatsgc 27 37 27 DNA Artificial Sequence Description of Artificial Sequence primer 37 caccgattcc agtggtgcct aggtgtg 27 38 30 DNA Artificial Sequence Description of Artificial Sequence primer 38 caacacctgg tgttccagga gcctgtgctt 30 39 23 DNA Artificial Sequence Description of Artificial Sequence primer 39 ccagaatcgt ctgctggtcg tag 23 40 19 DNA Artificial Sequence Description of Artificial Sequence primer 40 agcaccctgg aggagcttc 19 41 19 DNA Artificial Sequence Description of Artificial Sequence primer 41 catgtcgtac tgggtgtac 19 42 27 DNA Artificial Sequence Description of Artificial Sequence primer 42 gtsgtsnnsg acnnsgagac sacsggg 27 43 27 DNA Artificial Sequence Description of Artificial Sequence primer 43 gaasccsnng tcgaasnngg cgttgtg 27 44 27 DNA Artificial Sequence Description of Artificial Sequence primer 44 cggggatcca cctcaatcac ctcgtgg 27 45 30 DNA Artificial Sequence Description of Artificial Sequence primer 45 cggggatccg ccaccttgcg gctccgggtg 30 46 31 DNA Artificial Sequence Description of Artificial Sequence primer 46 gcgctctaga cgagttccca aagcgtgcgg t 31 47 25 DNA Artificial Sequence Description of Artificial Sequence primer 47 cgcgtctaga tcacctgtat ccaga 25 48 33 DNA Artificial Sequence Description of Artificial Sequence primer 48 gcggcgcata tggtggtggt cctggacctg gag 33 49 25 DNA Artificial Sequence Description of Artificial Sequence primer 49 cgcgtctaga tcacctgtat ccaga 25 50 20 DNA Artificial Sequence Description of Artificial Sequence primer 50 gtsctsgtsa agacscactt 20 51 21 DNA Artificial Sequence Description of Artificial Sequence primer 51 sagsagsgcg ttgaasgtgt g 21 52 22 DNA Artificial Sequence Description of Artificial Sequence primer 52 ctcgttggtg aaagtttccg tg 22 53 22 DNA Artificial Sequence Description of Artificial Sequence primer 53 ctcgttggtg aaagtttccg tg 22 54 27 DNA Artificial Sequence Description of Artificial Sequence primer 54 tctggcaaca cgttctggag cacatcc 27 55 23 DNA Artificial Sequence Description of Artificial Sequence primer 55 tgctggcgtt catcttcagg atg 23 56 23 DNA Artificial Sequence Description of Artificial Sequence primer 56 catcctgaag atgaacgcca gca 23 57 25 DNA Artificial Sequence Description of Artificial Sequence primer 57 aggttatcca caggggtcat gtgca 25 58 29 DNA Artificial Sequence Description of Artificial Sequence primer 58 gtgtgtcata tgaacataac ggttcccaa 29 59 29 DNA Artificial Sequence Description of Artificial Sequence primer 59 gcgcgaattc tcccttgtgg aaggcttag 29 60 13 PRT Thermus thermophilus 60 Arg Val Glu Leu Asp Tyr Asp Ala Leu Thr Leu Asp Asp 1 5 10 61 14 PRT Thermus thermophilus 61 Phe Phe Ile Glu Ile Gln Asn His Gly Leu Ser Glu Gln Lys 1 5 10 62 8 PRT Thermus thermophilus 62 Phe Phe Ile Glu Ile Gln Asn His 1 5 63 8 PRT Thermus thermophilus 63 Tyr Asp Ala Leu Thr Leu Asp Asp 1 5 64 6 PRT Thermus thermophilus 64 Ala Met Gly Lys Lys Lys 1 5 65 9 PRT Thermus thermophilus 65 Phe Asn Lys Ser His Ser Ala Ala Tyr 1 5 66 9 PRT Artificial Sequence Description of Artificial Sequence peptide 66 Val Val Xaa Asp Xaa Glu Thr Thr Gly 1 5 67 9 PRT Artificial Sequence Description of Artificial Sequence peptide 67 His Asn Ala Xaa Phe Asp Xaa Gly Phe 1 5 68 9 PRT Artificial Sequence Description of Artificial Sequence peptide 68 Val Val Xaa Asp Xaa Glu Thr Thr Gly 1 5 69 7 PRT Thermus thermophilus 69 Val Leu Val Lys Thr His Leu 1 5 70 6 PRT Artificial Sequence Description of Artificial Sequence peptide 70 His Arg Ala Leu Tyr Asp 1 5 71 7 PRT Thermus thermophilus 71 His Thr Phe Asn Ala Leu Leu 1 5 72 34 PRT Escherichia coli 72 Asp Arg Tyr Phe Leu Glu Leu Ile Arg Thr Gly Arg Pro Asp Glu Glu 1 5 10 15 Ser Tyr Leu His Ala Ala Val Glu Leu Ala Glu Ala Arg Gly Leu Pro 20 25 30 Val Val 73 34 PRT Vibrio cholerae 73 Asp His Phe Tyr Leu Glu Leu Ile Arg Thr Gly Arg Ala Asp Glu Glu 1 5 10 15 Ser Tyr Leu His Phe Ala Leu Asp Val Ala Glu Gln Tyr Asp Leu Pro 20 25 30 Val Val 74 34 PRT Haemophilus influenzae 74 Asp His Phe Tyr Leu Ala Leu Ser Arg Thr Gly Arg Pro Asn Glu Glu 1 5 10 15 Arg Tyr Ile Gln Ala Ala Leu Lys Leu Ala Glu Arg Cys Asp Leu Pro 20 25 30 Leu Val 75 34 PRT Rickettsia prowazekii 75 Asp Arg Phe Tyr Phe Glu Ile Met Arg His Asp Leu Pro Glu Glu Gln 1 5 10 15 Phe Ile Glu Asn Ser Tyr Ile Gln Ile Ala Ser Glu Leu Ser Ile Pro 20 25 30 Ile Val 76 34 PRT Helicobacter pylori 76 Asp Asp Phe Tyr Leu Glu Ile Met Arg His Gly Ile Leu Asp Gln Arg 1 5 10 15 Phe Ile Asp Glu Gln Val Ile Lys Met Ser Leu Glu Thr Gly Leu Lys 20 25 30 Ile Ile 77 34 PRT Synechocystis sp. 77 Asp Asp Tyr Tyr Leu Glu Ile Gln Asp His Gly Ser Val Glu Asp Arg 1 5 10 15 Leu Val Asn Ile Asn Leu Val Lys Ile Ala Gln Glu Leu Asp Ile Lys 20 25 30 Ile Val 78 34 PRT Mycobacterium tuberculosis 78 Asp Asn Tyr Phe Leu Glu Leu Met Asp His Gly Leu Thr Ile Glu Arg 1 5 10 15 Arg Val Arg Asp Gly Leu Leu Glu Ile Gly Arg Ala Leu Asn Ile Pro 20 25 30 Pro Leu 79 46 PRT Escherichia coli 79 Asn Lys Arg Arg Ala Lys Asn Gly Glu Pro Pro Leu Asp Ile Ala Ala 1 5 10 15 Ile Pro Leu Asp Asp Lys Lys Ser Phe Asp Met Leu Gln Arg Ser Glu 20 25 30 Thr Thr Ala Val Phe Gln Leu Glu Ser Arg Gly Met Lys Asp 35 40 45 80 46 PRT Vibrio cholerae 80 Asn Pro Arg Leu Lys Lys Ala Gly Lys Pro Pro Val Arg Ile Glu Ala 1 5 10 15 Ile Pro Leu Asp Asp Ala Arg Ser Phe Arg Asn Leu Gln Asp Ala Lys 20 25 30 Thr Thr Ala Val Phe Gln Leu Glu Ser Arg Gly Met Lys Glu 35 40 45 81 46 PRT Haemophilus influenzae 81 Asn Val Arg Met Val Arg Glu Gly Lys Pro Arg Val Asp Ile Ala Ala 1 5 10 15 Ile Pro Leu Asp Asp Pro Glu Ser Phe Glu Leu Leu Lys Arg Ser Glu 20 25 30 Thr Thr Ala Val Phe Gln Leu Glu Ser Arg Gly Met Lys Asp 35 40 45 82 46 PRT Rickettsia prowazekii 82 Cys Lys Lys Leu Leu Lys Glu Gln Gly Ile Lys Ile Asp Phe Asp Asp 1 5 10 15 Met Thr Phe Asp Asp Lys Lys Thr Tyr Gln Met Leu Cys Lys Gly Lys 20 25 30 Gly Val Gly Val Phe Gln Phe Glu Ser Ile Gly Met Lys Asp 35 40 45 83 45 PRT Helicobacter pylori 83 Leu Lys Ile Ile Lys Thr Gln His Lys Ile Ser Val Asp Phe Leu Ser 1 5 10 15 Leu Asp Met Asp Asp Pro Lys Val Tyr Lys Thr Ile Gln Ser Gly Asp 20 25 30 Thr Val Gly Ile Phe Gln Ile Glu Ser Gly Met Phe Gln 35 40 45 84 46 PRT Synechocystis sp. 84 Gln Glu Arg Lys Ala Leu Gln Ile Arg Ala Arg Thr Gly Ser Lys Lys 1 5 10 15 Leu Pro Asp Asp Val Lys Lys Thr His Lys Leu Leu Glu Ala Gly Asp 20 25 30 Leu Glu Gly Ile Phe Gln Leu Glu Ser Gln Gly Met Lys Gln 35 40 45 85 46 PRT Mycobacterium tuberculosis 85 Ile Asp Asn Val Arg Ala Asn Arg Gly Ile Asp Leu Asp Leu Glu Ser 1 5 10 15 Val Pro Leu Asp Asp Lys Ala Thr Tyr Glu Leu Leu Gly Arg Gly Asp 20 25 30 Thr Leu Gly Val Phe Gln Leu Asp Gly Gly Pro Met Arg Asp 35 40 45 86 3729 DNA Thermus thermophilus 86 atgggccggg agctccgctt cgcccacctc caccagcaca cccagttctc cctcctggac 60 ggggcggcga agctttccga cctcctcaag tgggtcaagg agacgacccc cgaggacccc 120 gccttggcca tgaccgacca cggcaacctc ttcggggccg tggagttcta caagaaggcc 180 accgaaatgg gcatcaagcc catcctgggc tacgaggcct acgtggcggc ggaaagccgc 240 tttgaccgca agcggggaaa gggcctagac gggggctact ttcacctcac cctcctcgcc 300 aaggacttca cggggtacca gaacctggtg cgcctggcga gccgggctta cctggagggg 360 ttttacgaaa agccccggat tgaccgggag atcctgcgcg agcacgccga gggcctcatc 420 gccctctcgg ggtgcctcgg ggcggagatc ccccagttca tcctccagga ccgtctggac 480 ctggccgagg cccggctcaa cgagtacctc tccatcttca aggaccgctt cttcatcgag 540 atccagaacc acggcctccc cgagcagaaa aaggtcaacg aggtcctcaa ggagttcgcc 600 cgaaagtacg gcctggggat ggtggccacc aacgacggcc attacgtgag gaaggaggac 660 gcccgcgccc acgaggtcct cctcgccatc cagtccaaga gcaccctgga cgaccccggg 720 cgctggcgct tcccctgcga cgagttctac gtgaagaccc ccgaggagat gcgggccatg 780 ttccccgagg aggagtgggg ggacgagccc tttgacaaca ccgtggagat cgcccgcatg 840 tgcaacgtgg agctgcccat cggggacaag atggtctacc gaatcccccg cttccccctc 900 cccgaggggc ggaccgaggc ccagtacctc atggagctca ccttcaaggg gctcctccgc 960 cgctacccgg accggatcac cgagggcttc taccgggagg tcttccgcct tttggggaag 1020 cttccccccc acggggacgg ggaggccttg gccgaggcct tggcccaggt ggagcgggag 1080 gcttgggaga ggctcatgaa gagcctcccc cctttggccg gggtcaagga gtggacggcg 1140 gaggccattt tccaccgggc cctttacgag ctttccgtga tagagcgcat ggggtttccc 1200 ggctacttcc tcatcgtcca ggactacatc aactgggccc ggagaaacgg cgtctccgtg 1260 gggcccggca gggggagcgc cgccgggagc ctggtggcct acgccgtggg gatcaccaac 1320 attgaccccc tccgcttcgg cctcctcttt gagcgcttcc tgaacccgga gagggtctcc 1380 atgcccgaca ttgacacgga cttctccgac cgggagcggg accgggtgat ccagtacgtg 1440 cgggagcgct acggcgagga caaggtggcc cagatcggca ccctgggaag cctcgcctcc 1500 aaggccgccc tcaaggacgt ggcccgggtc tacggcatcc cccacaagaa ggcggaggaa 1560 ttggccaagc tcatcccggt gcagttcggg aagcccaagc ccctgcagga ggccatccag 1620 gtggtgccgg agcttagggc ggagatggag aaggacccca aggtgcggga ggtcctcgag 1680 gtggccatgc gcctggaggg cctgaaccgc cacgcctccg tccacgccgc cggggtggtg 1740 atcgccgccg agcccctcac ggacctcgtc cccctcatgc gcgaccagga agggcggccc 1800 gtcacccagt acgacatggg ggcggtggag gccttggggc ttttgaagat ggactttttg 1860 ggcctccgca ccctcacctt cctggacgag gtcaagcgca tcgtcaaggc gtcccagggg 1920 gtggagctgg actacgatgc cctccccctg gacgacccca agaccttcgc cctcctctcc 1980 cggggggaga ccaagggggt cttccagctg gagtcggggg ggatgaccgc cacgctccgc 2040 ggcctcaagc cgcggcgctt tgaggacctg atcgccatcc tctccctcta ccgccccggg 2100 cccatggagc acatccccac ctacatccgc cgccaccacg ggctggagcc cgtgagctac 2160 agcgagtttc cccacgccga gaagtaccta aagcccatcc tggacgagac ctacggcatc 2220 cccgtctacc aggagcagat catgcagatc gcctcggccg tggcggggta ctccctgggc 2280 gaggcggacc tcctgcggcg gtccatgggc aagaagaagg tggaggagat gaagtcccac 2340 cgggagcgct tcgtccaggg ggccaaggaa aggggcgtgc ccgaggagga ggccaaccgc 2400 ctctttgaca tgctggaggc cttcgccaac tacggcttca acaaatccca cgctgccgcc 2460 tacagcctcc tctcctacca gaccgcctac gtgaaggccc actaccccgt ggagttcatg 2520 gccgccctcc tctccgtgga gcggcacgac tccgacaagg tggccgagta catccgcgac 2580 gcccgggcca tgggcataga ggtccttccc ccggacgtca accgctccgg gtttgacttc 2640 ctggtccagg gccggcagat ccttttcggc ctctccgcgg tgaagaacgt gggcgaggcg 2700 gcggcggagg ccattctccg ggagcgggag cggggcggcc cctaccggag cctcggcgac 2760 ttcctcaagc ggctggacga gaaggtgctc aacaagcgga ccctggagtc cctcatcaag 2820 gcgggcgccc tggacggctt cggggaaagg gcgcggctcc tcgcctccct ggaagggctc 2880 ctcaagtggg cggccgagaa ccgggagaag gcccgctcgg gcatgatggg cctcttcagc 2940 gaagtggagg agccgccttt ggccgaggcc gcccccctgg acgagatcac ccggctccgc 3000 tacgagaagg aggccctggg gatctacgtc tccggccacc ccatcttgcg gtaccccggg 3060 ctccgggaga cggccacctg caccctggag gagcttcccc acctggcccg ggacctgccg 3120 ccccggtcta gggtcctcct tgccgggatg gtggaggagg tggtgcgcaa gcccacaaag 3180 agcggcggga tgatggcccg cttcgtcctc tccgacgaga cgggggcgct tgaggcggtg 3240 gcattcggcc gggcctacga ccaggtctcc ccgaggctca aggaggacac ccccgtgctc 3300 gtcctcgccg aggtggagcg ggaggagggg ggcgtgcggg tgctggccca ggccgtttgg 3360 acctacgagg agctggagca ggtcccccgg gccctcgagg tggaggtgga ggcctccctc 3420 ctggacgacc ggggggtggc ccacctgaaa agcctcctgg acgagcacgc ggggaccctc 3480 cccctgtacg tccgggtcca gggcgccttc ggcgaggccc tcctcgccct gagggaggtg 3540 cgggtggggg aggaggctgt aggcggccgc gtggttccgg gcctacctcc tgcccgaccg 3600 ggaggtcctt ctccagggcg gccaggcggg ggaggcccag gaggcggtgc ccttctaggg 3660 ggtgggccgt gagacctagc gccatcgttc tcgccggggg caaggaggcc tgggcccgac 3720 cccttttgg 3729 87 1245 PRT Thermus thermophilus 87 Met Gly Arg Glu Leu Arg Phe Ala His Leu His Gln His Thr Gln Phe 1 5 10 15 Ser Leu Leu Asp Gly Ala Pro Lys Leu Ser Asp Leu Leu Lys Trp Val 20 25 30 Glu Glu Thr Thr Pro Glu Asp Pro Ala Leu Ala Met Thr Asp His Gly 35 40 45 Asn Leu Phe Gly Ala Val Glu Phe Tyr Lys Lys Ala Thr Glu Met Gly 50 55 60 Ile Lys Pro Ile Leu Gly Tyr Glu Ala Tyr Val Ala Ala Glu Ser Arg 65 70 75 80 Phe Asp Arg Lys Arg Gly Lys Gly Leu Asp Gly Gly Tyr Phe His Leu 85 90 95 Thr Leu Leu Ala Lys Asp Phe Thr Gly Tyr Gln Asn Leu Val Arg Leu 100 105 110 Ala Ser Arg Ala Tyr Leu Glu Gly Phe Tyr Glu Lys Pro Arg Ile Asp 115 120 125 Arg Glu Ile Leu Arg Glu His Ala Glu Gly Leu Ile Ala Leu Ser Gly 130 135 140 Cys Leu Gly Ala Glu Ile Pro Gln Phe Ile Leu Gln Asp Arg Leu Asp 145 150 155 160 Leu Ala Glu Ala Arg Leu Asn Glu Tyr Leu Ser Ile Phe Lys Asp Arg 165 170 175 Phe Phe Ile Glu Ile Gln Asn His Gly Leu Pro Glu Gln Lys Lys Val 180 185 190 Asn Glu Val Leu Lys Glu Phe Ala Arg Lys Tyr Gly Leu Gly Met Val 195 200 205 Ala Thr Asn Asp Gly His Tyr Val Arg Lys Glu Asp Ala Arg Ala His 210 215 220 Glu Val Leu Leu Ala Ile Gln Ser Lys Ser Thr Leu Asp Asp Pro Gly 225 230 235 240 Ala Leu Ala Leu Pro Cys Glu Glu Phe Tyr Val Lys Thr Pro Glu Glu 245 250 255 Met Arg Ala Met Phe Pro Glu Glu Glu Val Gly Gly Arg Ser Pro Leu 260 265 270 Thr Thr Pro Trp Arg Ser Pro His Val Gln Arg Gly Ala Ala Ile Gly 275 280 285 Thr Arg Trp Ser Thr Arg Ile Pro Arg Phe Pro Leu Pro Glu Gly Arg 290 295 300 Thr Glu Ala Gln Tyr Leu Met Glu Leu Thr Phe Lys Gly Leu Leu Arg 305 310 315 320 Arg Tyr Pro Asp Arg Ile Thr Glu Gly Phe Tyr Arg Glu Val Phe Arg 325 330 335 Leu Ser Gly Lys Leu Pro Pro His Gly Asp Gly Glu Ala Leu Ala Glu 340 345 350 Ala Leu Ala Gln Val Glu Arg Glu Ala Trp Glu Arg Leu Met Lys Ser 355 360 365 Leu Pro Pro Leu Ala Gly Val Lys Glu Trp Thr Ala Glu Ala Ile Phe 370 375 380 His Arg Ala Leu Tyr Glu Leu Ser Ala Ile Glu Arg Met Gly Phe Pro 385 390 395 400 Gly Leu Leu Pro His Arg Pro Gly Leu His Gln Leu Gly Pro Glu Lys 405 410 415 Gly Val Ser Val Gly Pro Gly Arg Gly Gly Ala Ala Gly Ser Leu Val 420 425 430 Ala Tyr Ala Val Gly Ile Thr Asn Ile Asp Pro Leu Arg Phe Gly Leu 435 440 445 Leu Phe Glu Arg Phe Leu Asn Pro Glu Arg Val Ser Met Pro Asp Ile 450 455 460 Asp Thr Asp Phe Ser Asp Arg Glu Arg Asp Arg Val Ile Gln Tyr Val 465 470 475 480 Arg Glu Arg Tyr Gly Glu Asp Lys Val Ala Gln Ile Gly Thr Leu Gly 485 490 495 Ser Leu Ala Ser Lys Ala Ala Leu Lys Glu Val Ala Arg Val Tyr Gly 500 505 510 Ile Pro Arg Lys Lys Ala Glu Glu Leu Ala Lys Leu Ile Pro Val Gln 515 520 525 Phe Gly Lys Pro Lys Pro Leu Gln Glu Ala Ile Gln Val Val Pro Glu 530 535 540 Leu Arg Ala Glu Met Glu Lys Asp Pro Lys Val Arg Glu Val Leu Glu 545 550 555 560 Val Ala Met Arg Leu Glu Gly Leu Asn Arg His Ala Ser Val His Ala 565 570 575 Gly Arg Gly Gly Val Phe Ser Glu Pro Leu Thr Asp Leu Val Pro Leu 580 585 590 Cys Ala Thr Arg Lys Gly Gly Pro Tyr Thr Gln Tyr Asp Met Gly Ala 595 600 605 Val Glu Ala Leu Gly Leu Leu Lys Met Asp Phe Leu Gly Leu Arg Thr 610 615 620 Leu Thr Phe Leu Asp Glu Val Lys Arg Ile Val Lys Ala Ser Gln Gly 625 630 635 640 Val Glu Leu Asp Tyr Asp Ala Leu Pro Leu Asp Asp Pro Lys Thr Phe 645 650 655 Ala Leu Leu Ser Arg Gly Glu Thr Lys Gly Val Phe Gln Leu Glu Ser 660 665 670 Gly Gly Met Thr Ala Thr Leu Arg Gly Leu Lys Pro Arg Arg Phe Glu 675 680 685 Asp Leu Ile Ala Ile Leu Ser Leu Tyr Arg Pro Gly Pro Met Glu His 690 695 700 Ile Pro Thr Tyr Ile Arg Arg His His Gly Leu Glu Pro Val Ser Tyr 705 710 715 720 Ser Glu Phe Pro His Ala Glu Lys Tyr Leu Lys Pro Ile Leu Asp Glu 725 730 735 Thr Tyr Gly Ile Pro Val Tyr Gln Glu Gln Ile Met Gln Ile Ala Ser 740 745 750 Ala Val Ala Gly Tyr Ser Leu Gly Glu Ala Asp Leu Leu Arg Arg Ser 755 760 765 Met Gly Lys Lys Lys Val Glu Glu Met Lys Ser His Arg Glu Arg Phe 770 775 780 Val Gln Gly Ala Lys Glu Arg Gly Val Pro Glu Glu Glu Ala Asn Arg 785 790 795 800 Leu Phe Asp Met Leu Glu Ala Phe Ala Asn Tyr Gly Phe Asn Lys Ser 805 810 815 His Ala Ala Ala Tyr Ser Leu Leu Ser Tyr Gln Thr Ala Tyr Val Lys 820 825 830 Ala His Tyr Pro Val Glu Phe Met Ala Ala Leu Leu Ser Val Glu Arg 835 840 845 His Asp Ser Asp Lys Val Ala Glu Tyr Ile Arg Asp Ala Arg Ala Met 850 855 860 Gly Ile Glu Val Leu Pro Pro Asp Val Asn Arg Ser Gly Phe Asp Phe 865 870 875 880 Leu Val Gln Gly Arg Gln Ile Leu Phe Gly Leu Ser Ala Val Lys Asn 885 890 895 Val Gly Glu Ala Ala Ala Glu Ala Ile Leu Arg Glu Arg Glu Arg Gly 900 905 910 Gly Pro Tyr Arg Ser Leu Gly Asp Phe Leu Lys Arg Leu Asp Glu Lys 915 920 925 Val Leu Asn Lys Arg Thr Leu Glu Ser Leu Ile Lys Ala Gly Ala Leu 930 935 940 Asp Gly Phe Gly Glu Arg Ala Arg Leu Leu Ala Ser Leu Glu Gly Leu 945 950 955 960 Leu Lys Trp Ala Ala Glu Asn Arg Glu Lys Ala Arg Ser Gly Met Met 965 970 975 Gly Leu Phe Ser Glu Val Glu Glu Pro Pro Leu Ala Glu Ala Ala Pro 980 985 990 Leu Asp Glu Ile Thr Arg Leu Arg Tyr Glu Lys Glu Ala Leu Gly Ile 995 1000 1005 Tyr Val Ser Gly His Pro Ile Leu Arg Tyr Pro Gly Leu Arg Glu Thr 1010 1015 1020 Ala Thr Cys Thr Leu Glu Glu Leu Pro His Leu Ala Arg Asp Leu Pro 1025 1030 1035 1040 Pro Arg Ser Arg Val Leu Leu Ala Gly Met Val Glu Glu Val Val Arg 1045 1050 1055 Lys Pro Thr Lys Ser Gly Gly Met Met Ala Arg Phe Val Leu Ser Asp 1060 1065 1070 Glu Thr Gly Ala Leu Glu Ala Val Ala Phe Gly Arg Ala Tyr Asp Gln 1075 1080 1085 Val Ser Pro Arg Leu Lys Glu Asp Thr Pro Val Leu Val Leu Ala Glu 1090 1095 1100 Val Glu Arg Glu Glu Gly Gly Val Arg Val Leu Ala Gln Ala Val Trp 1105 1110 1115 1120 Thr Tyr Gln Glu Leu Glu Gln Val Pro Arg Ala Leu Glu Val Glu Val 1125 1130 1135 Glu Ala Ser Leu Pro Asp Asp Arg Gly Val Ala His Leu Lys Ser Leu 1140 1145 1150 Leu Asp Glu His Ala Gly Thr Leu Pro Leu Tyr Val Arg Val Gln Gly 1155 1160 1165 Ala Phe Gly Glu Ala Leu Leu Ala Leu Arg Glu Val Arg Val Gly Glu 1170 1175 1180 Glu Ala Leu Gly Ala Leu Glu Ala Ala Gly Phe Pro Ala Tyr Leu Leu 1185 1190 1195 1200 Pro Asn Arg Glu Val Ser Pro Arg Leu Thr Gly Ser Gly Gly Pro Arg 1205 1210 1215 Gly Arg Ala Leu Ser Thr Gly Leu Ala Leu Lys Thr Tyr Pro Ile Ala 1220 1225 1230 Leu Pro Gly Gly Asn Glu Ala Leu Ala Arg Pro Leu Leu 1235 1240 1245 88 198 PRT Thermus thermophilus 88 Val Glu Arg Val Val Arg Thr Leu Leu Asp Gly Arg Phe Leu Leu Glu 1 5 10 15 Glu Gly Val Gly Leu Trp Glu Trp Arg Tyr Pro Phe Pro Leu Glu Gly 20 25 30 Glu Ala Val Val Val Leu Asp Leu Glu Thr Thr Gly Leu Ala Gly Leu 35 40 45 Asp Glu Val Ile Glu Val Gly Leu Leu Arg Leu Glu Gly Gly Arg Arg 50 55 60 Leu Pro Phe Gln Ser Leu Val Arg Pro Leu Pro Pro Ala Glu Ala Arg 65 70 75 80 Ser Trp Asn Leu Thr Gly Ile Pro Arg Glu Ala Leu Glu Glu Ala Pro 85 90 95 Ser Leu Glu Glu Val Leu Glu Lys Ala Tyr Pro Leu Arg Gly Asp Ala 100 105 110 Thr Leu Val Ile His Asn Ala Ala Phe Asp Leu Gly Phe Leu Arg Pro 115 120 125 Ala Leu Glu Gly Leu Gly Tyr Arg Leu Glu Asn Pro Val Val Asp Ser 130 135 140 Leu Arg Leu Ala Arg Arg Gly Leu Pro Gly Leu Arg Arg Tyr Gly Leu 145 150 155 160 Asp Ala Leu Ser Glu Val Leu Glu Leu Pro Arg Arg Thr Cys His Arg 165 170 175 Ala Leu Glu Asp Val Glu Arg Thr Leu Ala Val Val His Glu Val Tyr 180 185 190 Tyr Met Leu Thr Ser Gly 195 89 182 PRT Deinococcus radiodurans PEPTIDE (79) X at position 79 is undefined 89 Pro Trp Pro Gln Asp Val Val Val Phe Asp Leu Glu Thr Thr Gly Phe 1 5 10 15 Ser Pro Ala Ser Ala Ala Ile Val Glu Ile Gly Ala Val Arg Ile Val 20 25 30 Gly Gly Gln Ile Asp Glu Thr Leu Lys Phe Glu Thr Leu Val Arg Pro 35 40 45 Thr Arg Pro Asp Gly Ser Met Leu Ser Ile Pro Trp Gln Ala Gln Arg 50 55 60 Val His Gly Ile Ser Asp Glu Met Val Arg Arg Ala Pro Ala Xaa Lys 65 70 75 80 Asp Val Leu Pro Asp Phe Phe Asp Phe Val Asp Gly Ser Ala Val Val 85 90 95 Ala His Asn Val Ser Phe Asp Gly Gly Phe Met Arg Ala Gly Ala Glu 100 105 110 Arg Leu Gly Leu Ser Trp Ala Pro Glu Arg Glu Leu Cys Thr Met Gln 115 120 125 Leu Ser Arg Arg Ala Phe Pro Arg Glu Arg Thr His Asn Leu Thr Val 130 135 140 Leu Ala Glu Arg Leu Gly Leu Glu Phe Ala Pro Gly Gly Arg His Arg 145 150 155 160 Ser Tyr Gly Asp Val Gln Val Thr Ala Gln Ala Tyr Leu Arg Leu Leu 165 170 175 Glu Leu Leu Gly Glu Arg 180 90 201 PRT Bacillus subtilis 90 His Gly Ile Lys Met Ile Tyr Gly Met Glu Ala Asn Leu Val Asp Asp 1 5 10 15 Gly Val Pro Ile Ala Tyr Asn Ala Ala His Arg Leu Leu Glu Glu Glu 20 25 30 Thr Tyr Val Val Phe Asp Val Glu Thr Thr Gly Leu Ser Ala Val Tyr 35 40 45 Asp Thr Ile Ile Glu Leu Ala Ala Val Lys Val Lys Gly Gly Glu Ile 50 55 60 Ile Asp Lys Phe Glu Ala Phe Ala Asn Pro His Arg Pro Leu Ser Ala 65 70 75 80 Thr Ile Ile Glu Leu Thr Gly Ile Thr Asp Asp Met Leu Gln Asp Ala 85 90 95 Pro Asp Val Val Asp Val Ile Arg Asp Phe Arg Glu Trp Ile Gly Asp 100 105 110 Asp Ile Leu Val Ala His Asn Ala Ser Phe Asp Met Gly Phe Leu Asn 115 120 125 Val Ala Tyr Lys Lys Leu Leu Glu Val Glu Lys Ala Lys Asn Pro Val 130 135 140 Ile Asp Thr Leu Glu Leu Gly Arg Phe Leu Tyr Pro Glu Phe Lys Asn 145 150 155 160 His Arg Leu Asn Thr Leu Cys Lys Lys Phe Asp Ile Glu Leu Thr Gln 165 170 175 His His Arg Ala Ile Tyr Asp Thr Glu Ala Thr Ala Tyr Leu Leu Leu 180 185 190 Lys Met Leu Lys Asp Ala Ala Glu Lys 195 200 91 188 PRT Haemophilus influenzae PEPTIDE (47) X at position 47 is undefined 91 Met Ile Asn Pro Asn Arg Gln Ile Val Leu Asp Thr Glu Thr Thr Gly 1 5 10 15 Met Asn Gln Leu Gly Ala His Tyr Glu Gly His Cys Ile Ile Glu Ile 20 25 30 Gly Ala Val Glu Leu Ile Asn Arg Arg Tyr Thr Gly Asn Asn Xaa His 35 40 45 Ile Tyr Ile Lys Pro Asp Arg Pro Xaa Asp Pro Asp Ala Ile Lys Val 50 55 60 His Gly Ile Thr Asp Glu Met Leu Ala Asp Lys Pro Glu Phe Lys Glu 65 70 75 80 Val Ala Gln Asp Phe Leu Asp Tyr Ile Asn Gly Ala Glu Leu Leu Ile 85 90 95 His Asn Ala Pro Phe Asp Val Gly Phe Met Asp Tyr Glu Phe Arg Lys 100 105 110 Leu Asn Leu Asn Val Lys Thr Asp Asp Ile Cys Leu Val Thr Asp Thr 115 120 125 Leu Gln Met Ala Arg Gln Met Tyr Pro Gly Lys Arg Asn Asn Leu Asp 130 135 140 Ala Leu Cys Asp Arg Leu Gly Ile Asp Asn Ser Lys Arg Thr Leu His 145 150 155 160 Gly Ala Leu Leu Asp Ala Glu Ile Leu Ala Asp Val Tyr Leu Met Met 165 170 175 Thr Gly Gly Gln Thr Asn Leu Phe Asp Glu Glu Glu 180 185 92 189 PRT Escherichia coli 92 Met Ser Thr Ala Ile Thr Arg Gln Ile Val Leu Asp Thr Glu Thr Thr 1 5 10 15 Gly Met Asn Gln Ile Gly Ala His Ser Glu Gly His Lys Ile Ile Glu 20 25 30 Ile Gly Ala Val Glu Val Val Asn Arg Arg Leu Thr Gly Asn Asn Phe 35 40 45 His Val Tyr Leu Lys Asp Arg Leu Val Asp Pro Glu Ala Phe Gly Val 50 55 60 His Gly Ile Ala Val Asp Phe Leu Leu Asp Lys Pro Thr Phe Ala Glu 65 70 75 80 Val Ala Val Glu Phe Met Asp Tyr Ile Arg Gly Ala Glu Leu Val Ile 85 90 95 His Asn Ala Ala Phe Asp Ile Gly Phe Met Asp Tyr Glu Phe Ser Leu 100 105 110 Leu Lys Arg Asp Ile Ala Lys Thr Asn Thr Phe Cys Lys Val Thr Asp 115 120 125 Ser Leu Ala Val Ala Arg Lys Met Phe Pro Gly Lys Arg Asn Ser Leu 130 135 140 Asp Ala Leu Cys Ala Arg Tyr Glu Ile Asp Asn Ser Lys Arg Thr Leu 145 150 155 160 His Gly Ala Leu Leu Asp Ala Gln Ile Leu Ala Glu Val Tyr Leu Ala 165 170 175 Met Thr Gly Gly Gln Thr Ser Met Ala Phe Ala Met Glu 180 185 93 201 PRT Helicobacter pylori 93 Asn Leu Glu Tyr Leu Lys Ala Cys Gly Leu Asn Phe Ile Glu Thr Ser 1 5 10 15 Glu Asn Leu Ile Thr Leu Lys Asn Leu Lys Thr Pro Leu Lys Asp Glu 20 25 30 Val Phe Ser Phe Ile Asp Leu Glu Thr Thr Gly Ser Cys Pro Ile Lys 35 40 45 His Glu Ile Leu Glu Ile Gly Ala Val Gln Val Lys Gly Gly Glu Ile 50 55 60 Ile Asn Arg Phe Glu Thr Leu Val Lys Val Lys Ser Val Pro Asp Tyr 65 70 75 80 Ile Ala Glu Leu Thr Gly Ile Thr Tyr Glu Asp Thr Leu Asn Ala Pro 85 90 95 Ser Ala His Glu Ala Leu Gln Glu Leu Arg Leu Phe Leu Gly Asn Ser 100 105 110 Val Phe Val Ala His Asn Ala Asn Phe Asp Tyr Asn Phe Leu Gly Arg 115 120 125 Tyr Phe Val Glu Lys Leu His Cys Pro Leu Leu Asn Leu Lys Leu Cys 130 135 140 Thr Leu Asp Leu Ser Lys Arg Ala Ile Leu Ser Met Arg Tyr Ser Leu 145 150 155 160 Ser Phe Leu Lys Glu Leu Leu Gly Phe Gly Ile Glu Val Ser His Arg 165 170 175 Ala Tyr Ala Asp Ala Leu Ala Ser Tyr Lys Leu Phe Glu Ile Cys Leu 180 185 190 Leu Asn Leu Pro Ser Tyr Ile Lys Thr 195 200 94 630 DNA Thermus thermophilus 94 atggtggagc gggtggtgcg gacccttctg gacgggaggt tcctcctgga ggagggggtg 60 gggctttggg agtggcgcta cccctttccc ctggaggggg aggcggtggt ggtcctggac 120 ctggagacca cggggcttgc cggcctggac gaggtgattg aggtgggcct cctccgcctg 180 gaggggggga ggcgcctccc cttccagagc ctcgtccggc ccctcccgcc cgccgaagcc 240 cgttcgtgga acctcaccgg catcccccgg gaggccctgg aggaggcccc ctccctggag 300 gaggttctgg agaaggccta ccccctccgc ggcgacgcca ccttggtgat ccacaacgcc 360 gcctttgacc tgggcttcct ccgcccggcc ttggagggcc tgggctaccg cctggaaaac 420 cccgtggtgg actccctgcg cttggccaga cggggcttac caggccttag gcgctacggc 480 ctggacgccc tctccgaggt cctggagctt ccccgaagga cctgccaccg ggccctcgag 540 gacgtggagc gcaccctcgc cgtggtgcac gaggtatact atatgcttac gtccggccgt 600 ccccgcacgc tttgggaact cgggaggtag 630 95 210 PRT Thermus thermophilus 95 Met Val Glu Arg Val Val Arg Thr Leu Leu Asp Gly Arg Phe Leu Leu 1 5 10 15 Glu Glu Gly Val Gly Leu Trp Glu Trp Arg Tyr Pro Phe Pro Leu Glu 20 25 30 Gly Glu Ala Val Val Val Leu Asp Leu Glu Thr Thr Gly Leu Ala Gly 35 40 45 Leu Asp Glu Val Ile Glu Val Gly Leu Leu Arg Leu Glu Gly Gly Arg 50 55 60 Arg Leu Pro Phe Gln Ser Leu Val Arg Pro Leu Pro Pro Ala Glu Ala 65 70 75 80 Arg Ser Trp Asn Leu Thr Gly Ile Pro Arg Glu Ala Leu Glu Glu Ala 85 90 95 Pro Ser Leu Glu Glu Val Leu Glu Lys Ala Tyr Pro Leu Arg Gly Asp 100 105 110 Ala Thr Leu Val Ile His Asn Ala Ala Phe Asp Leu Gly Phe Leu Arg 115 120 125 Pro Ala Leu Glu Gly Leu Gly Tyr Arg Leu Glu Asn Pro Val Val Asp 130 135 140 Ser Leu Arg Leu Ala Arg Arg Gly Leu Pro Gly Leu Arg Arg Tyr Gly 145 150 155 160 Leu Asp Ala Leu Ser Glu Val Leu Glu Leu Pro Arg Arg Thr Cys His 165 170 175 Arg Ala Leu Glu Asp Val Glu Arg Thr Leu Ala Val Val His Glu Val 180 185 190 Tyr Tyr Met Leu Thr Ser Gly Arg Pro Arg Thr Leu Trp Glu Leu Gly 195 200 205 Arg Glx 210 96 461 PRT Pseudomonas marcesans 96 Met Leu Glu Ala Ser Trp Glu Lys Val Gln Ser Ser Leu Lys Gln Asn 1 5 10 15 Leu Ser Lys Pro Ser Tyr Glu Thr Trp Ile Arg Pro Thr Glu Phe Ser 20 25 30 Gly Phe Lys Asn Gly Glu Leu Thr Leu Ile Ala Pro Asn Ser Phe Ser 35 40 45 Ser Ala Trp Leu Lys Asn Asn Tyr Ser Gln Thr Ile Gln Glu Thr Ala 50 55 60 Glu Glu Ile Phe Gly Glu Pro Val Thr Val His Val Lys Val Lys Ala 65 70 75 80 Asn Ala Glu Ser Ser Asp Glu His Tyr Ser Ser Ala Pro Ile Thr Pro 85 90 95 Pro Leu Glu Ala Ser Pro Gly Ser Val Asp Ser Ser Gly Ser Ser Leu 100 105 110 Arg Leu Ser Lys Lys Thr Leu Pro Leu Leu Asn Leu Arg Tyr Val Phe 115 120 125 Asn Arg Phe Val Val Gly Pro Asn Ser Arg Met Ala His Ala Ala Ala 130 135 140 Met Ala Val Ala Glu Ser Pro Gly Arg Glu Phe Asn Pro Leu Phe Ile 145 150 155 160 Cys Gly Gly Val Gly Leu Gly Lys Thr His Leu Met Gln Ala Ile Gly 165 170 175 His Tyr Arg Leu Glu Ile Asp Pro Gly Ala Lys Val Ser Tyr Val Ser 180 185 190 Thr Glu Thr Phe Thr Asn Asp Leu Ile Leu Ala Ile Arg Gln Asp Arg 195 200 205 Met Gln Ala Phe Arg Asp Arg Tyr Arg Ala Ala Asp Leu Ile Leu Val 210 215 220 Asp Asp Ile Gln Phe Ile Glu Gly Lys Glu Tyr Thr Gln Glu Glu Phe 225 230 235 240 Phe His Thr Phe Asn Ala Leu His Asp Ala Gly Ser Gln Ile Val Leu 245 250 255 Ala Ser Asp Arg Pro Pro Ser Gln Ile Pro Arg Leu Gln Glu Arg Leu 260 265 270 Met Ser Arg Phe Ser Met Gly Leu Ile Ala Asp Val Gln Ala Pro Asp 275 280 285 Leu Glu Thr Arg Met Ala Ile Leu Gln Lys Lys Ala Glu His Glu Arg 290 295 300 Val Gly Leu Pro Arg Asp Leu Ile Gln Phe Ile Ala Gly Arg Phe Thr 305 310 315 320 Ser Asn Ile Arg Glu Leu Glu Gly Ala Leu Thr Arg Ala Ile Ala Phe 325 330 335 Ala Ser Ile Thr Gly Leu Pro Met Thr Val Asp Ser Ile Ala Pro Met 340 345 350 Leu Asp Pro Asn Gly Gln Gly Val Glu Val Thr Pro Lys Gln Val Leu 355 360 365 Asp Lys Val Ala Glu Val Phe Lys Val Thr Pro Asp Glu Met Arg Ser 370 375 380 Ala Ser Arg Arg Arg Pro Val Ser Gln Ala Arg Gln Val Gly Met Tyr 385 390 395 400 Leu Met Arg Gln Gly Thr Asn Leu Ser Leu Pro Arg Ile Gly Asp Thr 405 410 415 Phe Gly Gly Lys Asp His Thr Thr Val Met Tyr Ala Ile Glu Gln Val 420 425 430 Glu Lys Lys Leu Ser Ser Asp Pro Gln Ile Ala Ser Gln Val Gln Lys 435 440 445 Ile Arg Asp Leu Leu Gln Ile Asp Ser Arg Arg Lys Arg 450 455 460 97 447 PRT Synechocystis sp. 97 Met Val Ser Cys Glu Asn Leu Trp Gln Gln Ala Leu Ala Ile Leu Ala 1 5 10 15 Thr Gln Leu Thr Lys Pro Ala Phe Asp Thr Trp Ile Lys Ala Ser Val 20 25 30 Leu Ile Ser Leu Gly Asp Gly Val Ala Thr Ile Gln Val Glu Asn Gly 35 40 45 Phe Val Leu Asn His Leu Gln Lys Ser Tyr Gly Pro Leu Leu Met Glu 50 55 60 Val Leu Thr Asp Leu Thr Gly Gln Glu Ile Thr Val Lys Leu Ile Thr 65 70 75 80 Asp Gly Leu Glu Pro His Ser Leu Ile Gly Gln Glu Ser Ser Leu Pro 85 90 95 Met Glu Thr Thr Pro Lys Asn Ala Thr Ala Leu Asn Gly Lys Tyr Thr 100 105 110 Phe Ser Arg Phe Val Val Gly Pro Thr Asn Arg Met Ala His Ala Ala 115 120 125 Ser Leu Ala Val Ala Glu Ser Pro Gly Arg Glu Phe Asn Pro Leu Phe 130 135 140 Leu Cys Gly Gly Val Gly Leu Gly Lys Thr His Leu Met Gln Ala Ile 145 150 155 160 Ala His Tyr Arg Leu Glu Met Tyr Pro Asn Ala Lys Val Tyr Tyr Val 165 170 175 Ser Thr Glu Arg Phe Thr Asn Asp Leu Ile Thr Ala Ile Arg Gln Asp 180 185 190 Asn Met Glu Asp Phe Arg Ser Tyr Tyr Arg Ser Ala Asp Phe Leu Leu 195 200 205 Ile Asp Asp Ile Gln Phe Ile Lys Gly Lys Glu Tyr Thr Gln Glu Glu 210 215 220 Phe Phe His Thr Phe Asn Ser Leu His Glu Ala Gly Lys Gln Val Val 225 230 235 240 Val Ala Ser Asp Arg Ala Pro Gln Arg Ile Pro Gly Leu Gln Asp Arg 245 250 255 Leu Ile Ser Arg Phe Ser Met Gly Leu Ile Ala Asp Ile Gln Val Pro 260 265 270 Asp Leu Glu Thr Arg Met Ala Ile Leu Gln Lys Lys Ala Glu Tyr Asp 275 280 285 Arg Ile Arg Leu Pro Lys Glu Val Ile Glu Tyr Ile Ala Ser His Tyr 290 295 300 Thr Ser Asn Ile Arg Glu Leu Glu Gly Ala Leu Ile Arg Ala Ile Ala 305 310 315 320 Tyr Thr Ser Leu Ser Asn Val Ala Met Thr Val Glu Asn Ile Ala Pro 325 330 335 Val Leu Asn Pro Pro Val Glu Lys Val Ala Ala Ala Pro Glu Thr Ile 340 345 350 Ile Thr Ile Val Ala Gln His Tyr Gln Leu Lys Val Glu Glu Leu Leu 355 360 365 Ser Asn Ser Arg Arg Arg Glu Val Ser Leu Ala Arg Gln Val Gly Met 370 375 380 Tyr Leu Met Arg Gln His Thr Asp Leu Ser Leu Pro Arg Ile Gly Glu 385 390 395 400 Ala Phe Gly Gly Lys Asp His Thr Thr Val Met Tyr Ser Cys Asp Lys 405 410 415 Ile Thr Gln Leu Gln Gln Lys Asp Trp Glu Thr Ser Gln Thr Leu Thr 420 425 430 Ser Leu Ser His Arg Ile Asn Ile Ala Gly Gln Ala Pro Glu Ser 435 440 445 98 446 PRT Bacillus subtilis 98 Met Glu Asn Ile Leu Asp Leu Trp Asn Gln Ala Leu Ala Gln Ile Glu 1 5 10 15 Lys Lys Leu Ser Lys Pro Ser Phe Glu Thr Trp Met Lys Ser Thr Lys 20 25 30 Ala His Ser Leu Gln Gly Asp Thr Leu Thr Ile Thr Ala Pro Asn Glu 35 40 45 Phe Ala Arg Asp Trp Leu Glu Ser Arg Tyr Leu His Leu Ile Ala Asp 50 55 60 Thr Ile Tyr Glu Leu Thr Gly Glu Glu Leu Ser Ile Lys Phe Val Ile 65 70 75 80 Pro Gln Asn Gln Asp Val Glu Asp Phe Met Pro Lys Pro Gln Val Lys 85 90 95 Lys Ala Val Lys Glu Asp Thr Ser Asp Phe Pro Gln Asn Met Leu Asn 100 105 110 Pro Lys Tyr Thr Phe Asp Thr Phe Val Ile Gly Ser Gly Asn Arg Phe 115 120 125 Ala His Ala Ala Ser Leu Ala Val Ala Glu Ala Pro Ala Lys Ala Tyr 130 135 140 Asn Pro Leu Phe Ile Tyr Gly Gly Val Gly Leu Gly Lys Thr His Leu 145 150 155 160 Met His Ala Ile Gly His Tyr Val Ile Asp His Asn Pro Ser Ala Lys 165 170 175 Val Val Tyr Leu Ser Ser Glu Lys Phe Thr Asn Glu Phe Ile Asn Ser 180 185 190 Ile Arg Asp Asn Lys Ala Val Asp Phe Arg Asn Arg Tyr Arg Asn Val 195 200 205 Asp Val Leu Leu Ile Asp Asp Ile Gln Phe Leu Ala Gly Lys Glu Gln 210 215 220 Thr Gln Glu Glu Phe Phe His Thr Phe Asn Thr Leu His Glu Glu Ser 225 230 235 240 Lys Gln Ile Val Ile Ser Ser Asp Arg Pro Pro Lys Glu Ile Pro Thr 245 250 255 Leu Glu Asp Arg Leu Arg Ser Arg Phe Glu Trp Gly Leu Ile Thr Asp 260 265 270 Ile Thr Pro Pro Asp Leu Glu Thr Arg Ile Ala Ile Leu Arg Lys Lys 275 280 285 Ala Lys Ala Glu Gly Leu Asp Ile Pro Asn Glu Val Met Leu Tyr Ile 290 295 300 Ala Asn Gln Ile Asp Ser Asn Ile Arg Glu Leu Glu Gly Ala Leu Ile 305 310 315 320 Arg Val Val Ala Tyr Ser Ser Leu Ile Asn Lys Asp Ile Asn Ala Asp 325 330 335 Leu Ala Ala Glu Ala Leu Lys Asp Ile Ile Pro Ser Ser Lys Pro Lys 340 345 350 Val Ile Thr Ile Lys Glu Ile Gln Arg Val Val Gly Gln Gln Phe Asn 355 360 365 Ile Lys Leu Glu Asp Phe Lys Ala Lys Lys Arg Thr Lys Ser Val Ala 370 375 380 Phe Pro Arg Gln Ile Ala Met Tyr Leu Ser Arg Glu Met Thr Asp Ser 385 390 395 400 Ser Leu Pro Lys Ile Gly Glu Glu Phe Gly Gly Arg Asp His Thr Thr 405 410 415 Val Ile His Ala His Glu Lys Ile Ser Lys Leu Leu Ala Asp Asp Glu 420 425 430 Gln Leu Gln Gln His Val Lys Glu Ile Lys Glu Gln Leu Lys 435 440 445 99 507 PRT Mycobacterium tuberculosis 99 Met Thr Asp Asp Pro Gly Ser Gly Phe Thr Thr Val Trp Asn Ala Val 1 5 10 15 Val Ser Glu Leu Asn Gly Asp Pro Lys Val Asp Asp Gly Pro Ser Ser 20 25 30 Asp Ala Asn Leu Ser Ala Pro Leu Thr Pro Gln Gln Arg Ala Trp Leu 35 40 45 Asn Leu Val Gln Pro Leu Thr Ile Val Glu Gly Phe Ala Leu Leu Ser 50 55 60 Val Pro Ser Ser Phe Val Gln Asn Glu Ile Glu Arg His Leu Arg Ala 65 70 75 80 Pro Ile Thr Asp Ala Leu Ser Arg Arg Leu Gly His Gln Ile Gln Leu 85 90 95 Gly Val Arg Ile Ala Pro Pro Ala Thr Asp Glu Ala Asp Asp Thr Thr 100 105 110 Val Pro Pro Ser Glu Asn Pro Ala Thr Thr Ser Pro Asp Thr Thr Thr 115 120 125 Asp Asn Asp Glu Ile Asp Asp Ser Ala Ala Ala Arg Gly Asp Asn Gln 130 135 140 His Ser Trp Pro Ser Tyr Phe Thr Glu Arg Pro His Asn Thr Asp Ser 145 150 155 160 Ala Thr Ala Gly Val Thr Ser Leu Asn Arg Arg Tyr Thr Phe Asp Thr 165 170 175 Phe Val Ile Gly Ala Ser Asn Arg Phe Ala His Ala Ala Ala Leu Ala 180 185 190 Ile Ala Glu Ala Pro Ala Arg Ala Tyr Asn Pro Leu Phe Ile Trp Gly 195 200 205 Glu Ser Gly Leu Gly Lys Thr His Leu Leu His Ala Ala Gly Asn Tyr 210 215 220 Ala Gln Arg Leu Phe Pro Gly Met Arg Val Lys Tyr Val Ser Thr Glu 225 230 235 240 Glu Phe Thr Asn Asp Phe Ile Asn Ser Leu Arg Asp Asp Arg Lys Val 245 250 255 Ala Phe Lys Arg Ser Tyr Arg Asp Val Asp Val Leu Leu Val Asp Asp 260 265 270 Ile Gln Phe Ile Glu Gly Lys Glu Gly Ile Gln Glu Glu Phe Phe His 275 280 285 Thr Phe Asn Thr Leu His Asn Ala Asn Lys Gln Ile Val Ile Ser Ser 290 295 300 Asp Arg Pro Pro Lys Gln Leu Ala Thr Leu Glu Asp Arg Leu Arg Thr 305 310 315 320 Arg Phe Glu Trp Gly Leu Ile Thr Asp Val Gln Pro Pro Glu Leu Glu 325 330 335 Thr Arg Ile Ala Ile Leu Arg Lys Lys Ala Gln Met Glu Arg Leu Ala 340 345 350 Val Pro Asp Asp Val Leu Glu Leu Ile Ala Ser Ser Ile Glu Arg Asn 355 360 365 Ile Arg Glu Leu Glu Gly Ala Leu Ile Arg Val Thr Ala Phe Ala Ser 370 375 380 Leu Asn Lys Thr Pro Ile Asp Lys Ala Leu Ala Glu Ile Val Leu Arg 385 390 395 400 Asp Leu Ile Ala Asp Ala Asn Thr Met Gln Ile Ser Ala Ala Thr Ile 405 410 415 Met Ala Ala Thr Ala Glu Tyr Phe Asp Thr Thr Val Glu Glu Leu Arg 420 425 430 Gly Pro Gly Lys Thr Arg Ala Leu Ala Gln Ser Arg Gln Ile Ala Met 435 440 445 Tyr Leu Cys Arg Glu Leu Thr Asp Leu Ser Leu Pro Lys Ile Gly Gln 450 455 460 Ala Phe Gly Arg Asp His Thr Thr Val Met Tyr Ala Gln Arg Lys Ile 465 470 475 480 Leu Ser Glu Met Ala Glu Arg Arg Glu Val Phe Asp His Val Lys Glu 485 490 495 Leu Thr Thr Arg Ile Arg Gln Arg Ser Lys Arg 500 505 100 446 PRT Thermus thermophilus 100 Met Ser His Glu Ala Val Trp Gln His Val Leu Glu His Ile Arg Arg 1 5 10 15 Ser Ile Thr Glu Val Glu Phe His Thr Trp Phe Glu Arg Ile Arg Pro 20 25 30 Leu Gly Ile Arg Asp Gly Val Leu Glu Leu Ala Val Pro Thr Ser Phe 35 40 45 Ala Leu Asp Trp Ile Arg Arg His Tyr Ala Gly Leu Ile Gln Glu Gly 50 55 60 Pro Arg Leu Leu Gly Ala Gln Ala Pro Arg Phe Glu Leu Arg Val Val 65 70 75 80 Pro Gly Val Val Val Gln Glu Asp Ile Phe Gln Pro Pro Pro Ser Pro 85 90 95 Pro Ala Gln Ala Gln Pro Glu Asp Thr Phe Lys Thr Ser Trp Trp Gly 100 105 110 Pro Thr Thr Pro Trp Pro His Gly Gly Ala Val Ala Val Ala Glu Ser 115 120 125 Pro Gly Arg Ala Tyr Asn Pro Leu Phe Ile Tyr Gly Gly Arg Gly Leu 130 135 140 Gly Lys Thr Tyr Leu Met His Ala Val Gly Pro Leu Arg Ala Lys Arg 145 150 155 160 Phe Pro His Met Arg Leu Glu Tyr Val Ser Thr Glu Thr Phe Thr Asn 165 170 175 Glu Leu Ile Asn Arg Pro Ser Ala Arg Asp Arg Met Thr Glu Phe Arg 180 185 190 Glu Arg Tyr Arg Ser Val Asp Leu Leu Leu Val Asp Asp Val Gln Phe 195 200 205 Ile Ala Gly Lys Glu Arg Thr Gln Glu Glu Phe Phe His Thr Phe Asn 210 215 220 Ala Leu Tyr Glu Ala His Lys Gln Ile Ile Leu Ser Ser Asp Arg Pro 225 230 235 240 Pro Lys Asp Ile Leu Thr Leu Glu Ala Arg Leu Arg Ser Arg Phe Glu 245 250 255 Trp Gly Leu Ile Thr Asp Asn Pro Ala Pro Asp Leu Glu Thr Arg Ile 260 265 270 Ala Ile Leu Lys Met Asn Ala Ser Ser Gly Pro Glu Asp Pro Glu Asp 275 280 285 Ala Leu Glu Tyr Ile Ala Arg Gln Val Thr Ser Asn Ile Arg Glu Trp 290 295 300 Glu Gly Ala Leu Met Arg Ala Ser Pro Phe Ala Ser Leu Asn Gly Val 305 310 315 320 Glu Leu Thr Arg Ala Val Ala Ala Lys Ala Leu Arg His Leu Arg Pro 325 330 335 Arg Glu Leu Glu Ala Asp Pro Leu Glu Ile Ile Arg Lys Ala Ala Gly 340 345 350 Pro Val Arg Pro Glu Thr Pro Gly Gly Ala His Gly Glu Arg Arg Lys 355 360 365 Lys Glu Val Val Leu Pro Arg Gln Leu Ala Met Tyr Leu Val Arg Glu 370 375 380 Leu Thr Pro Ala Ser Leu Pro Glu Ile Gly Gln Leu Phe Gly Gly Arg 385 390 395 400 Asp His Thr Thr Val Arg Tyr Ala Ile Gln Lys Val Gln Glu Leu Ala 405 410 415 Gly Lys Pro Asp Arg Glu Val Gln Gly Leu Leu Arg Thr Leu Arg Glu 420 425 430 Ala Cys Thr Asp Pro Val Asp Asn Leu Trp Ile Thr Cys Gly 435 440 445 101 467 PRT Escherichia coli 101 Met Ser Leu Ser Leu Trp Gln Gln Cys Leu Ala Arg Leu Gln Asp Glu 1 5 10 15 Leu Pro Ala Thr Glu Phe Ser Met Trp Ile Arg Pro Leu Gln Ala Glu 20 25 30 Leu Ser Asp Asn Thr Leu Ala Leu Tyr Ala Pro Asn Arg Phe Val Leu 35 40 45 Asp Trp Val Arg Asp Lys Tyr Leu Asn Asn Ile Asn Gly Leu Leu Thr 50 55 60 Ser Phe Cys Gly Ala Asp Ala Pro Gln Leu Arg Phe Glu Val Gly Thr 65 70 75 80 Lys Pro Val Thr Gln Thr Pro Gln Ala Ala Val Thr Ser Asn Val Ala 85 90 95 Ala Pro Ala Gln Val Ala Gln Thr Gln Pro Gln Arg Ala Ala Pro Ser 100 105 110 Thr Arg Ser Gly Trp Asp Asn Val Pro Ala Pro Ala Glu Pro Thr Tyr 115 120 125 Arg Ser Asn Val Asn Val Lys His Thr Phe Asp Asn Phe Val Glu Gly 130 135 140 Lys Ser Asn Gln Leu Ala Arg Ala Ala Ala Arg Gln Val Ala Asp Asn 145 150 155 160 Pro Gly Gly Ala Tyr Asn Pro Leu Phe Leu Tyr Gly Gly Thr Gly Leu 165 170 175 Gly Lys Thr His Leu Leu His Ala Val Gly Asn Gly Ile Met Ala Arg 180 185 190 Lys Pro Asn Ala Lys Val Val Tyr Met His Ser Glu Arg Phe Val Gln 195 200 205 Asp Met Val Lys Ala Leu Gln Asn Asn Ala Ile Glu Glu Phe Lys Arg 210 215 220 Tyr Tyr Arg Ser Val Asp Ala Leu Leu Ile Asp Asp Ile Gln Phe Phe 225 230 235 240 Ala Asn Lys Glu Arg Ser Gln Glu Glu Phe Phe His Thr Phe Asn Ala 245 250 255 Leu Leu Glu Gly Asn Gln Gln Ile Ile Leu Thr Ser Asp Arg Tyr Pro 260 265 270 Lys Glu Ile Asn Gly Val Glu Asp Arg Leu Lys Ser Arg Phe Gly Trp 275 280 285 Gly Leu Thr Val Ala Ile Glu Pro Pro Glu Leu Glu Thr Arg Val Ala 290 295 300 Ile Leu Met Lys Lys Ala Asp Glu Asn Asp Ile Arg Leu Pro Gly Glu 305 310 315 320 Val Ala Phe Phe Ile Ala Lys Arg Leu Arg Ser Asn Val Arg Glu Leu 325 330 335 Glu Gly Ala Leu Asn Arg Val Ile Ala Asn Ala Asn Phe Thr Gly Arg 340 345 350 Ala Ile Thr Ile Asp Phe Val Arg Glu Ala Leu Arg Asp Leu Leu Ala 355 360 365 Leu Gln Glu Lys Leu Val Thr Ile Asp Asn Ile Gln Lys Thr Val Ala 370 375 380 Glu Tyr Tyr Lys Ile Lys Val Ala Asp Leu Leu Ser Lys Arg Arg Ser 385 390 395 400 Arg Ser Val Ala Arg Pro Arg Gln Met Ala Met Ala Leu Ala Lys Glu 405 410 415 Leu Thr Asn His Ser Leu Pro Glu Ile Gly Asp Ala Phe Gly Gly Arg 420 425 430 Asp His Thr Thr Val Leu His Ala Cys Arg Lys Ile Glu Gln Leu Arg 435 440 445 Glu Glu Ser His Asp Ile Lys Glu Asp Phe Ser Asn Leu Ile Arg Thr 450 455 460 Leu Ser Ser 465 102 440 PRT Thermatoga maritima 102 Met Lys Glu Arg Ile Leu Gln Glu Ile Lys Thr Arg Val Asn Arg Lys 1 5 10 15 Ser Trp Glu Leu Trp Phe Ser Ser Phe Asp Val Lys Ser Ile Glu Gly 20 25 30 Asn Lys Val Val Phe Ser Val Gly Asn Leu Phe Ile Lys Glu Trp Leu 35 40 45 Glu Lys Lys Tyr Tyr Ser Val Leu Ser Lys Ala Val Lys Val Val Leu 50 55 60 Gly Asn Asp Ala Thr Phe Glu Ile Thr Tyr Glu Ala Phe Glu Pro His 65 70 75 80 Ser Ser Tyr Ser Glu Pro Leu Val Lys Lys Arg Ala Val Leu Leu Thr 85 90 95 Pro Leu Asn Pro Asp Tyr Thr Phe Glu Asn Phe Val Val Gly Pro Gly 100 105 110 Asn Ser Phe Ala Tyr His Ala Ala Leu Glu Val Ala Lys His Pro Gly 115 120 125 Arg Tyr Asn Pro Leu Phe Ile Tyr Gly Gly Val Gly Leu Gly Lys Thr 130 135 140 His Leu Leu Gln Ser Ile Gly Asn Tyr Val Val Gln Asn Glu Pro Asp 145 150 155 160 Leu Arg Val Met Tyr Ile Thr Ser Glu Lys Phe Leu Asn Asp Leu Val 165 170 175 Asp Ser Met Lys Glu Gly Lys Leu Asn Glu Phe Arg Glu Lys Tyr Arg 180 185 190 Lys Lys Val Asp Ile Leu Leu Ile Asp Asp Val Gln Phe Leu Ile Gly 195 200 205 Lys Thr Gly Val Gln Thr Glu Leu Phe His Thr Phe Asn Glu Leu His 210 215 220 Asp Ser Gly Lys Gln Ile Val Ile Cys Ser Asp Arg Glu Pro Gln Lys 225 230 235 240 Leu Ser Glu Phe Gln Asp Arg Leu Val Ser Arg Phe Gln Met Gly Leu 245 250 255 Val Ala Lys Leu Glu Pro Pro Asp Glu Glu Thr Arg Lys Ser Ile Ala 260 265 270 Arg Lys Met Leu Glu Ile Glu His Gly Glu Leu Pro Glu Glu Val Leu 275 280 285 Asn Phe Val Ala Glu Asn Val Asp Asp Asn Leu Arg Arg Leu Arg Gly 290 295 300 Ala Ile Ile Lys Leu Leu Val Tyr Lys Glu Thr Thr Gly Lys Glu Val 305 310 315 320 Asp Leu Lys Glu Ala Ile Leu Leu Leu Lys Asp Phe Ile Lys Pro Asn 325 330 335 Arg Val Lys Ala Met Asp Pro Ile Asp Glu Leu Ile Glu Ile Val Ala 340 345 350 Lys Val Thr Gly Val Pro Arg Glu Glu Ile Leu Ser Asn Ser Arg Asn 355 360 365 Val Lys Ala Leu Thr Ala Arg Arg Ile Gly Met Tyr Val Ala Lys Asn 370 375 380 Tyr Leu Lys Ser Ser Leu Arg Thr Ile Ala Glu Lys Phe Asn Arg Ser 385 390 395 400 His Pro Val Val Val Asp Ser Val Lys Lys Val Lys Asp Ser Leu Leu 405 410 415 Lys Gly Asn Lys Gln Leu Lys Ala Leu Ile Asp Glu Val Ile Gly Glu 420 425 430 Ile Ser Arg Arg Ala Leu Ser Gly 435 440 103 457 PRT Helicobacter pylori 103 Met Asp Thr Asn Asn Asn Ile Glu Lys Glu Ile Leu Ala Leu Val Lys 1 5 10 15 Gln Asn Pro Lys Val Ser Leu Ile Glu Tyr Glu Asn Tyr Phe Ser Gln 20 25 30 Leu Lys Tyr Asn Pro Asn Ala Ser Lys Ser Asp Ile Ala Phe Phe Tyr 35 40 45 Ala Pro Asn Gln Val Leu Cys Thr Thr Ile Thr Ala Lys Tyr Gly Ala 50 55 60 Leu Leu Lys Glu Ile Leu Ser Gln Asn Lys Val Gly Met His Leu Ala 65 70 75 80 His Ser Val Asp Val Arg Ile Glu Val Ala Pro Lys Ile Gln Ile Asn 85 90 95 Ala Gln Ser Asn Ile Asn Tyr Lys Ala Ile Lys Thr Ser Val Lys Asp 100 105 110 Ser Tyr Thr Phe Glu Asn Phe Val Val Gly Ser Cys Asn Asn Thr Val 115 120 125 Tyr Glu Ile Ala Lys Lys Val Ala Gln Ser Asp Thr Pro Pro Tyr Asn 130 135 140 Pro Val Leu Phe Tyr Gly Gly Thr Gly Leu Gly Lys Thr His Ile Leu 145 150 155 160 Asn Ala Ile Gly Asn His Ala Leu Glu Lys His Lys Lys Val Val Leu 165 170 175 Val Thr Ser Glu Asp Phe Leu Thr Asp Phe Leu Lys His Leu Asp Asn 180 185 190 Lys Thr Met Asp Ser Phe Lys Ala Lys Tyr Arg His Cys Asp Phe Phe 195 200 205 Leu Leu Asp Asp Ala Gln Phe Leu Gln Gly Lys Pro Lys Leu Glu Glu 210 215 220 Glu Phe Phe His Thr Phe Asn Glu Leu His Ala Asn Ser Lys Gln Ile 225 230 235 240 Val Leu Ile Ser Asp Arg Ser Pro Lys Asn Ile Ala Gly Leu Glu Asp 245 250 255 Arg Leu Lys Ser Arg Phe Glu Trp Gly Ile Thr Ala Lys Val Met Pro 260 265 270 Pro Asp Leu Glu Thr Lys Leu Ser Ile Val Lys Gln Lys Cys Gln Leu 275 280 285 Asn Gln Ile Thr Leu Pro Glu Glu Val Met Glu Tyr Ile Ala Gln His 290 295 300 Ile Ser Asp Asn Ile Arg Gln Met Glu Gly Ala Ile Ile Lys Ile Ser 305 310 315 320 Val Asn Ala Asn Leu Met Asn Ala Ser Ile Asp Leu Asn Leu Ala Lys 325 330 335 Thr Val Leu Glu Asp Leu Gln Lys Asp His Ala Glu Gly Ser Ser Leu 340 345 350 Glu Asn Ile Leu Leu Ala Val Ala Gln Ser Leu Asn Leu Lys Ser Ser 355 360 365 Glu Ile Lys Val Ser Ser Arg Gln Lys Asn Val Ala Leu Ala Arg Lys 370 375 380 Leu Val Val Tyr Phe Ala Arg Leu Tyr Thr Pro Asn Pro Thr Leu Ser 385 390 395 400 Leu Ala Gln Phe Leu Asp Leu Lys Asp His Ser Ser Ile Ser Lys Met 405 410 415 Tyr Ser Gly Val Lys Lys Met Leu Glu Glu Glu Lys Ser Pro Phe Val 420 425 430 Leu Ser Leu Arg Glu Glu Ile Lys Asn Arg Leu Asn Glu Leu Asn Asp 435 440 445 Lys Lys Thr Ala Phe Asn Ser Ser Glu 450 455 104 1305 DNA Thermus thermophilus 104 gtgtcgcacg aggccgtctg gcaacacgtt ctggagcaca tccgccgcag catcaccgag 60 gtggagttcc acacctggtt tgaaaggatc cgccccttgg ggatccggga cggggtgctg 120 gagctcgccg tgcccacctc ctttgccctg gactggatcc ggcgccacta cgccggcctc 180 atccaggagg gccctcggct cctcggggcc caggcgcccc ggtttgagct ccgggtggtg 240 cccggggtcg tagtccagga ggacatcttc cagcccccgc cgagcccccc ggcccaagct 300 caacccgaag atacctttaa aacttcgtgg tggggcccaa caactccatg gccccacggc 360 ggcgccgtgg ccgtggccga gtcccccggc cgggcctaca accccctctt catctacggg 420 ggccgtggcc tgggaaagac ctacctgatg cacgccgtgg gcccactccg tgcgaagcgc 480 ttcccccaca tgagattaga gtacgtttcc acggaaactt tcaccaacga gctcatcaac 540 cggccatccg cgagggaccg gatgacggag ttccgggagc ggtaccgctc cgtggacctc 600 ctgctggtgg acgacgtcca gttcatcgcc ggaaaggagc gcacccagga ggagtttttc 660 cacaccttca acgcccttta cgaggcccac aagcagatca tcctctcctc cgaccggccg 720 cccaaggaca tcctcaccct ggaggcgcgc ctgcggagcc gctttgagtg gggcctgatc 780 accgacaatc cagcccccga cctggaaacc cggatcgcca tcctgaagat gaacgccagc 840 agcgggcctg aggatcccga ggacgccctg gagtacatcg cccggcaggt cacctccaac 900 atccgggagt gggaaggggc cctcatgcgg gcatcgcctt tcgcctccct caacggcgtt 960 gagctgaccc gcgccgtggc ggccaaggct ctccgacatc ttcgccccag ggagctggag 1020 gcggacccct tggagatcat ccgcaaagcg gcgggaccag ttcggcctga aaccccggga 1080 ggagctcacg gggagcgccg caagaaggag gtggtcctcc cccggcagct cgccatgtac 1140 ctggtgcggg agctcacccc ggcctccctg cccgagatcg accagctcaa cgacgaccgg 1200 gaccacacca cggtcctcta cgccatccag aaggtccagg agctcgcgga aagcgaccgg 1260 gaggtgcagg gcctcctccg caccctccgg gaggcgtgca catga 1305 105 434 PRT Thermus thermophilus 105 Val Ser His Glu Ala Val Trp Gln His Val Leu Glu His Ile Arg Arg 1 5 10 15 Ser Ile Thr Glu Val Glu Phe His Thr Trp Phe Glu Arg Ile Arg Pro 20 25 30 Leu Gly Ile Arg Asp Gly Val Leu Glu Leu Ala Val Pro Thr Ser Phe 35 40 45 Ala Leu Asp Trp Ile Arg Arg His Tyr Ala Gly Leu Ile Gln Glu Gly 50 55 60 Pro Arg Leu Leu Gly Ala Gln Ala Pro Arg Phe Glu Leu Arg Val Val 65 70 75 80 Pro Gly Val Val Val Gln Glu Asp Ile Phe Gln Pro Pro Pro Ser Pro 85 90 95 Pro Ala Gln Ala Gln Pro Glu Asp Thr Phe Lys Thr Ser Trp Trp Gly 100 105 110 Pro Thr Thr Pro Trp Pro His Gly Gly Ala Val Ala Val Ala Glu Ser 115 120 125 Pro Gly Arg Ala Tyr Asn Pro Leu Phe Ile Tyr Gly Gly Arg Gly Leu 130 135 140 Gly Lys Thr Tyr Leu Met His Ala Val Gly Pro Leu Arg Ala Lys Arg 145 150 155 160 Phe Pro His Met Arg Leu Glu Tyr Val Ser Thr Glu Thr Phe Thr Asn 165 170 175 Glu Leu Ile Asn Arg Pro Ser Ala Arg Asp Arg Met Thr Glu Phe Arg 180 185 190 Glu Arg Tyr Arg Ser Val Asp Leu Leu Leu Val Asp Asp Val Gln Phe 195 200 205 Ile Ala Gly Lys Glu Arg Thr Gln Glu Glu Phe Phe His Thr Phe Asn 210 215 220 Ala Leu Tyr Glu Ala His Lys Gln Ile Ile Leu Ser Ser Asp Arg Pro 225 230 235 240 Pro Lys Asp Ile Leu Thr Leu Glu Ala Arg Leu Arg Ser Arg Phe Glu 245 250 255 Trp Gly Leu Ile Thr Asp Asn Pro Ala Pro Asp Leu Glu Thr Arg Ile 260 265 270 Ala Ile Leu Lys Met Asn Ala Ser Ser Gly Pro Glu Asp Pro Glu Asp 275 280 285 Ala Leu Glu Tyr Ile Ala Arg Gln Val Thr Ser Asn Ile Arg Glu Trp 290 295 300 Glu Gly Ala Leu Met Arg Ala Ser Pro Phe Ala Ser Leu Asn Gly Val 305 310 315 320 Glu Leu Thr Arg Ala Val Ala Ala Lys Ala Leu Arg His Leu Arg Pro 325 330 335 Arg Glu Leu Glu Ala Asp Pro Leu Glu Ile Ile Arg Lys Ala Ala Gly 340 345 350 Pro Val Arg Pro Glu Thr Pro Gly Gly Ala His Gly Glu Arg Arg Lys 355 360 365 Lys Glu Val Val Leu Pro Arg Gln Leu Ala Met Tyr Leu Val Arg Glu 370 375 380 Leu Thr Pro Ala Ser Leu Pro Glu Ile Asp Gln Leu Asn Asp Asp Arg 385 390 395 400 Asp His Thr Thr Val Leu Tyr Ala Ile Gln Lys Val Gln Glu Leu Ala 405 410 415 Glu Ser Asp Arg Glu Val Gln Gly Leu Leu Arg Thr Leu Arg Glu Ala 420 425 430 Cys Thr 106 1128 DNA Thermus thermophilus 106 atgaacataa cggttcccaa aaaactcctc tcggaccagc tttccctcct ggagcgcatc 60 gtcccctcta gaagcgccaa ccccctctac acctacctgg ggctttacgc cgaggaaggg 120 gccttgatcc tcttcgggac caacggggag gtggacctcg aggtccgcct ccccgccgag 180 gcccaaagcc ttccccgggt gctcgtcccc gcccagccct tcttccagct ggtgcggagc 240 cttcctgggg acctcgtggc cctcggcctc gcctcggagc cgggccaggg ggggcagctg 300 gagctctcct ccgggcgttt ccgcacccgg ctcagcctgg cccctgccga gggctacccc 360 gagcttctgg tgcccgaggg ggaggacaag ggggccttcc ccctccggac gcggatgccc 420 tccggggagc tcgtcaaggc cttgacccac gtgcgctacg ccgcgagcaa cgaggagtac 480 cgggccatct tccgcggggt gcagctggag ttctcccccc agggcttccg ggcggtggcc 540 tccgacgggt accgcctcgc cctctacgac ctgcccctgc cccaagggtt ccaggccaag 600 gccgtggtcc ccgcccggag cgtggacgag atggtgcggg tcctgaaggg ggcggacggg 660 gccgaggccg tcctcgccct gggcgagggg gtgttggccc tggccctcga gggcggaagc 720 ggggtccgga tggccctccg cctcatggaa ggggagttcc ccgactacca gagggtcatc 780 ccccaggagt tcgccctcaa ggtccaggtg gagggggagg ccctcaggga ggcggtgcgc 840 cgggtgagcg tcctctccga ccggcagaac caccgggtgg acctcctttt ggaggaaggc 900 cggatcctcc tctccgccga gggggactac ggcaaggggc aggaggaggt gcccgcccag 960 gtggaggggc cggacatggc cgtggcctac aacgcccgct acctcctcga ggccctcgcc 1020 cccgtggggg accgggccca cctgggcatc tccgggccca cgagcccgag cctcatctgg 1080 ggggacgggg aggggtaccg ggcggtggtg gtgcccctca gggtctag 1128 107 376 PRT Thermus thermophilus 107 Met Asn Ile Thr Val Pro Lys Lys Leu Leu Ser Asp Gln Leu Ser Leu 1 5 10 15 Leu Glu Arg Ile Val Pro Ser Arg Ser Ala Asn Pro Leu Tyr Thr Tyr 20 25 30 Leu Gly Leu Tyr Ala Glu Glu Gly Ala Leu Ile Leu Phe Gly Thr Asn 35 40 45 Gly Glu Val Asp Leu Glu Val Arg Leu Pro Ala Glu Ala Gln Ser Leu 50 55 60 Pro Arg Val Leu Val Pro Ala Gln Pro Phe Phe Gln Leu Val Arg Ser 65 70 75 80 Leu Pro Gly Asp Leu Val Ala Leu Gly Leu Ala Ser Glu Pro Gly Gln 85 90 95 Gly Gly Gln Leu Glu Leu Ser Ser Gly Arg Phe Arg Thr Arg Leu Ser 100 105 110 Leu Ala Pro Ala Glu Gly Tyr Pro Glu Leu Leu Val Pro Glu Gly Glu 115 120 125 Asp Lys Gly Ala Phe Pro Leu Arg Thr Arg Met Pro Ser Gly Glu Leu 130 135 140 Val Lys Ala Leu Thr His Val Arg Tyr Ala Ala Ser Asn Glu Glu Tyr 145 150 155 160 Arg Ala Ile Phe Arg Gly Val Gln Leu Glu Phe Ser Pro Gln Gly Phe 165 170 175 Arg Ala Val Ala Ser Asp Gly Tyr Arg Leu Ala Leu Tyr Asp Leu Pro 180 185 190 Leu Pro Gln Gly Phe Gln Ala Lys Ala Val Val Pro Ala Arg Ser Val 195 200 205 Asp Glu Met Val Arg Val Leu Lys Gly Ala Asp Gly Ala Glu Ala Val 210 215 220 Leu Ala Leu Gly Glu Gly Val Leu Ala Leu Ala Leu Glu Gly Gly Ser 225 230 235 240 Gly Val Arg Met Ala Leu Arg Leu Met Glu Gly Glu Phe Pro Asp Tyr 245 250 255 Gln Arg Val Ile Pro Gln Glu Phe Ala Leu Lys Val Gln Val Glu Gly 260 265 270 Glu Ala Leu Arg Glu Ala Val Arg Arg Val Ser Val Leu Ser Asp Arg 275 280 285 Gln Asn His Arg Val Asp Leu Leu Leu Glu Glu Gly Arg Ile Leu Leu 290 295 300 Ser Ala Glu Gly Asp Tyr Gly Lys Gly Gln Glu Glu Val Pro Ala Gln 305 310 315 320 Val Glu Gly Pro Asp Met Ala Val Ala Tyr Asn Ala Arg Tyr Leu Leu 325 330 335 Glu Ala Leu Ala Pro Val Gly Asp Arg Ala His Leu Gly Ile Ser Gly 340 345 350 Pro Thr Ser Pro Ser Leu Ile Trp Gly Asp Gly Glu Gly Tyr Arg Ala 355 360 365 Val Val Val Pro Leu Arg Val Glx 370 375 108 376 PRT Thermus thermophilus 108 Met Asn Ile Thr Val Pro Lys Lys Leu Leu Ser Asp Gln Leu Ser Leu 1 5 10 15 Leu Glu Arg Ile Val Pro Ser Arg Ser Ala Asn Pro Leu Tyr Thr Tyr 20 25 30 Leu Gly Leu Tyr Ala Glu Glu Gly Ala Leu Ile Leu Phe Gly Thr Asn 35 40 45 Gly Glu Val Asp Leu Glu Val Arg Leu Pro Ala Glu Ala Gln Ser Leu 50 55 60 Pro Arg Val Leu Val Pro Ala Gln Pro Phe Phe Gln Leu Val Arg Ser 65 70 75 80 Leu Pro Gly Asp Leu Val Ala Leu Gly Leu Ala Ser Glu Pro Gly Gln 85 90 95 Gly Gly Gln Leu Glu Leu Ser Ser Gly Arg Phe Arg Thr Arg Leu Ser 100 105 110 Leu Ala Pro Ala Glu Gly Tyr Pro Glu Leu Leu Val Pro Glu Gly Glu 115 120 125 Asp Lys Gly Ala Phe Pro Leu Arg Thr Arg Met Pro Ser Gly Glu Leu 130 135 140 Val Lys Ala Leu Thr His Val Arg Tyr Ala Ala Ser Asn Glu Glu Tyr 145 150 155 160 Arg Ala Ile Phe Arg Gly Val Gln Leu Glu Phe Ser Pro Gln Gly Phe 165 170 175 Arg Ala Val Ala Ser Asp Gly Tyr Arg Leu Ala Leu Tyr Asp Leu Pro 180 185 190 Leu Pro Gln Gly Phe Gln Ala Lys Ala Val Val Pro Ala Arg Ser Val 195 200 205 Asp Glu Met Val Arg Val Leu Lys Gly Ala Asp Gly Ala Glu Ala Val 210 215 220 Leu Ala Leu Gly Glu Gly Val Leu Ala Leu Ala Leu Glu Gly Gly Ser 225 230 235 240 Gly Val Arg Met Ala Leu Arg Leu Met Glu Gly Glu Phe Pro Asp Tyr 245 250 255 Gln Arg Val Ile Pro Gln Glu Phe Ala Leu Lys Val Gln Val Glu Gly 260 265 270 Glu Ala Leu Arg Glu Ala Val Arg Arg Val Ser Val Leu Ser Asp Arg 275 280 285 Gln Asn His Arg Val Asp Leu Leu Leu Glu Glu Gly Arg Ile Leu Leu 290 295 300 Ser Ala Glu Gly Asp Tyr Gly Lys Gly Gln Glu Glu Val Pro Ala Gln 305 310 315 320 Val Glu Gly Pro Asp Met Ala Val Ala Tyr Asn Ala Arg Tyr Leu Leu 325 330 335 Glu Ala Leu Ala Pro Val Gly Asp Arg Ala His Leu Gly Ile Ser Gly 340 345 350 Pro Thr Ser Pro Ser Leu Ile Trp Gly Asp Gly Glu Gly Tyr Arg Ala 355 360 365 Val Val Val Pro Leu Arg Val Glx 370 375 109 367 PRT Escherichia coli 109 Met Lys Phe Thr Val Glu Arg Glu His Leu Leu Lys Pro Leu Gln Gln 1 5 10 15 Val Ser Gly Pro Leu Gly Gly Arg Pro Thr Leu Pro Ile Leu Gly Asn 20 25 30 Leu Leu Leu Gln Val Ala Asp Gly Thr Leu Ser Leu Thr Gly Thr Asp 35 40 45 Leu Glu Met Glu Met Val Ala Arg Val Ala Leu Val Gln Pro His Glu 50 55 60 Pro Gly Ala Thr Thr Val Pro Ala Arg Lys Phe Phe Asp Ile Cys Arg 65 70 75 80 Gly Leu Pro Glu Gly Ala Glu Ile Ala Val Gln Leu Glu Gly Glu Arg 85 90 95 Met Leu Val Arg Ser Gly Arg Ser Arg Phe Ser Leu Ser Thr Leu Pro 100 105 110 Ala Ala Asp Phe Pro Asn Leu Asp Asp Trp Gln Ser Glu Val Glu Phe 115 120 125 Thr Leu Pro Gln Ala Thr Met Lys Arg Leu Ile Glu Ala Thr Gln Phe 130 135 140 Ser Met Ala His Gln Asp Val Arg Tyr Tyr Leu Asn Gly Met Leu Phe 145 150 155 160 Glu Thr Glu Gly Glu Glu Leu Arg Thr Val Ala Thr Asp Gly His Arg 165 170 175 Leu Ala Val Cys Ser Met Pro Ile Gly Gln Ser Leu Pro Ser His Ser 180 185 190 Val Ile Val Pro Arg Lys Gly Val Ile Glu Leu Met Arg Met Leu Asp 195 200 205 Gly Gly Asp Asn Pro Leu Arg Val Gln Ile Gly Ser Asn Asn Ile Arg 210 215 220 Ala His Val Gly Asp Phe Ile Phe Thr Ser Lys Leu Val Asp Gly Arg 225 230 235 240 Phe Pro Asp Tyr Arg Arg Val Leu Pro Lys Asn Pro Asp Lys His Leu 245 250 255 Glu Ala Gly Cys Asp Leu Leu Lys Gln Ala Phe Ala Arg Ala Ala Ile 260 265 270 Leu Ser Asn Glu Lys Phe Arg Gly Val Arg Leu Tyr Val Ser Glu Asn 275 280 285 Gln Leu Lys Ile Thr Ala Asn Asn Pro Glu Gln Glu Glu Ala Glu Glu 290 295 300 Ile Leu Asp Val Thr Tyr Ser Gly Ala Glu Met Glu Ile Gly Phe Asn 305 310 315 320 Val Ser Tyr Val Leu Asp Val Leu Asn Ala Leu Lys Cys Glu Asn Val 325 330 335 Arg Met Met Leu Thr Asp Ser Val Ser Ser Val Gln Ile Glu Asp Ala 340 345 350 Ala Ser Gln Ser Ala Ala Tyr Val Val Met Pro Met Arg Leu Glx 355 360 365 110 367 PRT Proteus mirabilis 110 Met Lys Phe Ile Ile Glu Arg Glu Gln Leu Leu Lys Pro Leu Gln Gln 1 5 10 15 Val Ser Gly Pro Leu Gly Gly Arg Pro Thr Leu Pro Ile Leu Gly Asn 20 25 30 Leu Leu Leu Lys Val Thr Glu Asn Thr Leu Ser Leu Thr Gly Thr Asp 35 40 45 Leu Glu Met Glu Met Met Ala Arg Val Ser Leu Ser Gln Ser His Glu 50 55 60 Ile Gly Ala Thr Thr Val Pro Ala Arg Lys Phe Phe Asp Ile Trp Arg 65 70 75 80 Gly Leu Pro Glu Gly Ala Glu Ile Ser Val Glu Leu Asp Gly Asp Arg 85 90 95 Leu Leu Val Arg Ser Gly Arg Ser Arg Phe Ser Leu Ser Thr Leu Pro 100 105 110 Ala Ser Asp Phe Pro Asn Leu Asp Asp Trp Gln Ser Glu Val Glu Phe 115 120 125 Thr Leu Pro Gln Ala Thr Leu Lys Arg Leu Ile Glu Ser Thr Gln Phe 130 135 140 Ser Met Ala His Gln Asp Val Arg Tyr Tyr Leu Asn Gly Met Leu Phe 145 150 155 160 Glu Thr Glu Asn Thr Glu Leu Arg Thr Val Ala Thr Asp Gly His Arg 165 170 175 Leu Ala Val Cys Ala Met Asp Ile Gly Gln Ser Leu Pro Gly His Ser 180 185 190 Val Ile Val Pro Arg Lys Gly Val Ile Glu Leu Met Arg Leu Leu Asp 195 200 205 Gly Ser Gly Glu Ser Leu Leu Gln Leu Gln Ile Gly Ser Asn Asn Leu 210 215 220 Arg Ala His Val Gly Asp Phe Ile Phe Thr Ser Lys Leu Val Asp Gly 225 230 235 240 Arg Phe Pro Asp Tyr Arg Arg Val Leu Pro Lys Asn Pro Thr Lys Thr 245 250 255 Val Ile Ala Gly Cys Asp Ile Leu Lys Gln Ala Phe Ser Arg Ala Ala 260 265 270 Ile Leu Ser Asn Glu Lys Phe Arg Gly Val Arg Ile Asn Leu Thr Asn 275 280 285 Gly Gln Leu Lys Ile Thr Ala Asn Asn Pro Glu Gln Glu Glu Ala Glu 290 295 300 Glu Ile Val Asp Val Gln Tyr Gln Gly Glu Glu Met Glu Ile Gly Phe 305 310 315 320 Asn Val Ser Tyr Leu Leu Asp Val Leu Asn Thr Leu Lys Cys Glu Glu 325 330 335 Val Lys Leu Leu Leu Thr Asp Ala Val Ser Ser Val Gln Val Glu Asn 340 345 350 Val Ala Ser Ala Ala Ala Ala Tyr Val Val Met Pro Met Arg Leu 355 360 365 111 366 PRT Haemophilus influenzae 111 Met Gln Phe Ser Ile Ser Arg Glu Asn Leu Leu Lys Pro Leu Gln Gln 1 5 10 15 Val Cys Gly Val Leu Ser Asn Arg Pro Asn Ile Pro Val Leu Asn Asn 20 25 30 Val Leu Leu Gln Ile Glu Asp Tyr Arg Leu Thr Ile Thr Gly Thr Asp 35 40 45 Leu Glu Val Glu Leu Ser Ser Gln Thr Gln Leu Ser Ser Ser Ser Glu 50 55 60 Asn Gly Thr Phe Thr Ile Pro Ala Lys Lys Phe Leu Asp Ile Cys Arg 65 70 75 80 Thr Leu Ser Asp Asp Ser Glu Ile Thr Val Thr Phe Glu Gln Asp Arg 85 90 95 Ala Leu Val Gln Ser Gly Arg Ser Arg Phe Thr Leu Ala Thr Gln Pro 100 105 110 Ala Glu Glu Tyr Pro Asn Leu Thr Asp Trp Gln Ser Glu Val Asp Phe 115 120 125 Glu Leu Pro Gln Asn Thr Leu Arg Arg Leu Ile Glu Ala Thr Gln Phe 130 135 140 Ser Met Ala Asn Gln Asp Ala Arg Tyr Phe Leu Asn Gly Met Lys Phe 145 150 155 160 Glu Thr Glu Gly Asn Leu Leu Arg Thr Val Ala Thr Asp Gly His Arg 165 170 175 Leu Ala Val Cys Thr Ile Ser Leu Glu Gln Glu Leu Gln Asn His Ser 180 185 190 Val Ile Leu Pro Arg Lys Gly Val Leu Glu Leu Val Arg Leu Leu Glu 195 200 205 Thr Asn Asp Glu Pro Ala Arg Leu Gln Ile Gly Thr Asn Asn Leu Arg 210 215 220 Val His Leu Lys Asn Thr Val Phe Thr Ser Lys Leu Ile Asp Gly Arg 225 230 235 240 Phe Pro Asp Tyr Arg Arg Val Leu Pro Arg Asn Ala Thr Lys Ile Val 245 250 255 Glu Gly Asn Trp Glu Met Leu Lys Gln Ala Phe Ala Arg Ala Ser Ile 260 265 270 Leu Ser Asn Glu Arg Ala Arg Ser Val Arg Leu Ser Leu Lys Glu Asn 275 280 285 Gln Leu Lys Ile Thr Ala Ser Asn Thr Glu His Glu Glu Ala Glu Glu 290 295 300 Ile Val Asp Val Asn Tyr Asn Gly Glu Glu Leu Glu Val Gly Phe Asn 305 310 315 320 Val Thr Tyr Ile Leu Asp Val Leu Asn Ala Leu Lys Cys Asn Gln Val 325 330 335 Arg Met Cys Leu Thr Asp Ala Phe Ser Ser Cys Leu Ile Glu Asn Cys 340 345 350 Glu Asp Ser Ser Cys Glu Tyr Val Ile Met Pro Met Arg Leu 355 360 365 112 367 PRT Pseudomonas putida 112 Met His Phe Thr Ile Gln Arg Glu Ala Leu Leu Lys Pro Leu Gln Leu 1 5 10 15 Val Ala Gly Val Val Glu Arg Arg Gln Thr Leu Pro Val Leu Ser Asn 20 25 30 Val Leu Leu Val Val Gln Gly Gln Gln Leu Ser Leu Thr Gly Thr Asp 35 40 45 Leu Glu Val Glu Leu Val Gly Arg Val Gln Leu Glu Glu Pro Ala Glu 50 55 60 Pro Gly Glu Ile Thr Val Pro Ala Arg Lys Leu Met Asp Ile Cys Lys 65 70 75 80 Ser Leu Pro Asn Asp Ala Leu Ile Asp Ile Lys Val Asp Glu Gln Lys 85 90 95 Leu Leu Val Lys Ala Gly Arg Ser Arg Phe Thr Leu Ser Thr Leu Pro 100 105 110 Ala Asn Asp Phe Pro Thr Val Glu Glu Gly Pro Gly Ser Leu Thr Cys 115 120 125 Asn Leu Glu Gln Ser Lys Leu Arg Arg Leu Ile Glu Arg Thr Ser Phe 130 135 140 Ala Met Ala Gln Gln Asp Val Arg Tyr Tyr Leu Asn Gly Met Leu Leu 145 150 155 160 Glu Val Ser Arg Asn Thr Leu Arg Ala Val Ser Thr Asp Gly His Arg 165 170 175 Leu Ala Leu Cys Ser Met Ser Ala Pro Ile Glu Gln Glu Asp Arg His 180 185 190 Gln Val Ile Val Pro Arg Lys Gly Ile Leu Glu Leu Ala Arg Leu Leu 195 200 205 Thr Asp Pro Glu Gly Met Val Ser Ile Val Leu Gly Gln His His Ile 210 215 220 Arg Ala Thr Thr Gly Glu Phe Thr Phe Thr Ser Lys Leu Val Asp Gly 225 230 235 240 Lys Phe Pro Asp Tyr Glu Arg Val Leu Pro Lys Gly Gly Asp Lys Leu 245 250 255 Val Val Gly Asp Arg Gln Ala Leu Arg Glu Ala Phe Ser Arg Thr Ala 260 265 270 Ile Leu Ser Asn Glu Lys Tyr Arg Gly Ile Arg Leu Gln Leu Ala Ala 275 280 285 Gly Gln Leu Lys Ile Gln Ala Asn Asn Pro Glu Gln Glu Glu Ala Glu 290 295 300 Glu Glu Ile Ser Val Asp Tyr Glu Gly Ser Ser Leu Glu Ile Gly Phe 305 310 315 320 Asn Val Ser Tyr Leu Leu Asp Val Leu Gly Val Met Thr Thr Glu Gln 325 330 335 Val Arg Leu Ile Leu Ser Asp Ser Asn Ser Ser Ala Leu Leu Gln Glu 340 345 350 Ala Gly Asn Asp Asp Ser Ser Tyr Val Val Met Pro Met Arg Leu 355 360 365 113 366 PRT Buchnera aphidicola 113 Met Lys Phe Thr Ile Gln Asn Asp Ile Leu Thr Lys Asn Leu Lys Lys 1 5 10 15 Ile Thr Arg Val Leu Val Lys Asn Ile Ser Phe Pro Ile Leu Glu Asn 20 25 30 Ile Leu Ile Gln Val Glu Asp Gly Thr Leu Ser Leu Thr Thr Thr Asn 35 40 45 Leu Glu Ile Glu Leu Ile Ser Lys Ile Glu Ile Ile Thr Lys Tyr Ile 50 55 60 Pro Gly Lys Thr Thr Ile Ser Gly Arg Lys Ile Leu Asn Ile Cys Arg 65 70 75 80 Thr Leu Ser Glu Lys Ser Lys Ile Lys Met Gln Leu Lys Asn Lys Lys 85 90 95 Met Tyr Ile Ser Ser Glu Asn Ser Asn Tyr Ile Leu Ser Thr Leu Ser 100 105 110 Ala Asp Thr Phe Pro Asn His Gln Asn Phe Asp Tyr Ile Ser Lys Phe 115 120 125 Asp Ile Ser Ser Asn Ile Leu Lys Glu Met Ile Glu Lys Thr Glu Phe 130 135 140 Ser Met Gly Lys Gln Asp Val Arg Tyr Tyr Leu Asn Gly Met Leu Leu 145 150 155 160 Glu Lys Lys Asp Lys Phe Leu Arg Ser Val Ala Thr Asp Gly Tyr Arg 165 170 175 Leu Ala Ile Ser Tyr Thr Gln Leu Lys Lys Asp Ile Asn Phe Phe Ser 180 185 190 Ile Ile Ile Pro Asn Lys Ala Val Met Glu Leu Leu Lys Leu Leu Asn 195 200 205 Thr Gln Pro Gln Leu Leu Asn Ile Leu Ile Gly Ser Asn Ser Ile Arg 210 215 220 Ile Tyr Thr Lys Asn Leu Ile Phe Thr Thr Gln Leu Ile Glu Gly Glu 225 230 235 240 Tyr Pro Asp Tyr Lys Ser Val Leu Phe Lys Glu Lys Lys Asn Pro Ile 245 250 255 Ile Thr Asn Ser Ile Leu Leu Lys Lys Ser Leu Leu Arg Val Ala Ile 260 265 270 Leu Ala His Glu Lys Phe Cys Gly Ile Glu Ile Lys Ile Glu Asn Gly 275 280 285 Lys Phe Lys Val Leu Ser Asp Asn Gln Glu Glu Glu Thr Ala Glu Asp 290 295 300 Leu Phe Glu Ile Asp Tyr Phe Gly Glu Lys Ile Glu Ile Ser Ile Asn 305 310 315 320 Val Tyr Tyr Leu Leu Asp Val Ile Asn Asn Ile Lys Ser Glu Asn Ile 325 330 335 Ala Leu Phe Leu Asn Lys Ser Lys Ser Ser Ile Gln Ile Glu Ala Glu 340 345 350 Asn Asn Ser Ser Asn Ala Tyr Val Val Met Leu Leu Lys Arg 355 360 365 114 39 DNA Artificial Sequence Description of Artificial Sequence primer 114 gtgtggatcc tcgtccccct catgcgcgac caggaaggg 39 115 27 DNA Artificial Sequence Description of Artificial Sequence primer 115 gtgtggatcc gtggtgacct tagccac 27 116 30 DNA Artificial Sequence Description of Artificial Sequence primer 116 ttcgtgtccg aggaccttgt ggtccacaac 30 117 3514 DNA Aquifex aeolicus 117 atgagtaagg atttcgtcca ccttcacctg cacacccagt tctcactcct ggacggggct 60 ataaagatag acgagctcgt gaaaaaggca aaggagtatg gatacaaagc tgtcggaatg 120 tcagaccacg gaaacctctt cggttcgtat aaattctaca aagccctgaa ggcggaagga 180 attaagccca taatcggcat ggaagcctac tttaccacgg gttcgaggtt tgacagaaag 240 actaaaacga gcgaggacaa cataaccgac aagtacaacc accacctcat acttatagca 300 aaggacgaaa aggtctaaag aacttaatga agctctcaac cctcgcctac aaagaaggtt 360 tttactacaa acccagaatt gattacgaac tccttgaaaa gtacggggag ggcctaatag 420 cccttaccgc atgcctgaaa ggtgttccca cctactacgc ttctataaac gaagtgaaaa 480 aggcggagga atgggtaaag aagttcaagg atatattcgg agatgacctt tatttagaac 540 ttcaagcgaa caacattcca gaacaggaag tggcaaacag gaacttaata gagatagcca 600 aaaagtacga tgtgaaactc atagcgacgc aggacgccca ctacctcaat cccgaagaca 660 ggtacgccca cacggttctt atggcacttc aaatgaaaaa gaccattcac gaactgagtt 720 cgggaaactt caagtgttca aacgaagacc ttcactttgc tccacccgag tacatgtgga 780 aaaagtttga aggtaagttc gaaggctggg aaaaggcact cctgaacact ctcgaggtaa 840 tggaaaagac agcggacagc tttgagatat ttgaaaactc cacctacctc cttcccaagt 900 acgacgttcc gcccgacaaa acccttgagg aatacctcag agaactcgcg tacaaaggtt 960 taagacagag gatagaaagg ggacaagcta aggatactaa agagtactgg gagaggctcg 1020 agtacgaact ggaagttata aacaaaatgg gctttgcggg atacttcttg atagttcagg 1080 acttcataaa ctgggctaag aaaaacgaca tacctgttgg acccggaagg ggaagtgctg 1140 gaggttccct cgtcgcatac gccatcggaa taacggacgt tgaccctata aagcacggat 1200 tcctttttga gaggttctta aaccccgaaa gggtttccat gccggatata gacgtggatt 1260 tctgtcagga caacagggaa aaggtcatag agtacgtaag gaacaagtac ggacacgaca 1320 acgtagctca gataatcacc tacaacgtaa tgaaggcgaa gcaaacactg agagacgtcg 1380 caagggccat gggactcccc tactccaccg cggacaaact cgcaaaactc attcctcagg 1440 gggacgttca gggaacgtgg ctcagtctgg aagagatgta caaaacgcct gtggaggaac 1500 tccttcagaa gtacggagaa cacagaacgg acatagagga caacgtaaag aagttcagac 1560 agatatgcga agaaagtccg gagataaaac agctcgttga gacggccctg aagcttgaag 1620 gtctcacgag acacacctcc ctccacgccg cgggagtggt tatagcacca aagcccttga 1680 gcgagctcgt tcccctctac tacgataaag agggcgaagt cgcaacccag tacgacatgg 1740 ttcagctcga agaactcggt ctcctgaaga tggacttcct cggactcaaa accctcacag 1800 aactgaaact catgaaagaa ctcataaagg aaagacacgg agtggatata aacttccttg 1860 aacttcccct tgacgacccg aaagtttaca aactccttca ggaaggaaaa accacgggag 1920 tgttccagct cgaaagcagg ggaatgaaag aactcctgaa gaaactaaag cccgacagct 1980 ttgacgacat cgttgcggtc ctcgcactct acagacccgg acctctaaag agcggactcg 2040 ttgacacata cattaagaga aagcacggaa aagaacccgt tgagtacccc ttcccggagc 2100 ttgaacccgt ccttaaggaa acctacggag taatcgttta tcaggaacag gtgatgaaga 2160 tgtctcagat actttccggc tttactcccg gagaggcgga taccctcaga aaggcgatag 2220 gtaagaagaa agcggattta atggctcaga tgaaagacaa gttcatacag ggagcggtgg 2280 aaaggggata ccctgaagaa aagataagga agctctggga agacatagag aagttcgctt 2340 cctactcctt caacaagtct cactcggtag cttacgggta catctcctac tggaccgcct 2400 acgttaaagc ccactatccc gcggagttct tcgcggtaaa actcacaact gaaaagaacg 2460 acaacaagtt cctcaacctc ataaaagacg ctaaactctt cggatttgag atacttcccc 2520 ccgacataaa caagagtgat gtaggattta cgatagaagg tgaaaacagg ataaggttcg 2580 ggcttgcgag gataaaggga gtgggagagg aaactgctaa gataatcgtt gaagctagaa 2640 agaagtataa gcagttcaaa gggcttgcgg acttcataaa caaaaccaag aacaggaaga 2700 taaacaagaa agtcgtggaa gcactcgtaa aggcaggggc ttttgacttt actaagaaaa 2760 agaggaaaga actactcgct aaagtggcaa actctgaaaa agcattaatg gctacacaaa 2820 actccctttt cggtgcaccg aaagaagaag tggaagaact cgacccctta aagcttgaaa 2880 aggaagttct cggtttttac atttcagggc acccccttga caactacgaa aagctcctca 2940 agaaccgcta cacacccatt gaagatttag aagagtggga caaggaaagc gaagcggtgc 3000 ttacaggagt tatcacggaa ctcaaagtaa aaaagacgaa aaacggagat tacatggcgg 3060 tcttcaacct cgttgacaag acgggactaa tagagtgtgt cgtcttcccg ggagtttacg 3120 aagaggcaaa ggaactgata gaagaggaca gagtagtggt agtcaaaggt tttctggacg 3180 aggaccttga aacggaaaat gtcaagttcg tggtgaaaga ggttttctcc cctgaggagt 3240 tcgcaaagga gatgaggaat accctttata tattcttaaa aagagagcaa gccctaaacg 3300 gcgttgccga aaaactaaag ggaattattg aaaacaacag gacggaggac ggatacaact 3360 tggttctcac ggttgatctg ggagactact tcgttgattt agcactccca caagatatga 3420 aactaaaggc tgacagaaag gttgtagagg agatagaaaa actgggagtg aaggtcataa 3480 tttagtaaat aacccttact tccgagtagt cccc 3514 118 1161 PRT Aquifex aeolicus 118 Met Ser Lys Asp Phe Val His Leu His Leu His Thr Gln Phe Ser Leu 1 5 10 15 Leu Asp Gly Ala Ile Lys Ile Asp Glu Leu Val Lys Lys Ala Lys Glu 20 25 30 Tyr Gly Tyr Lys Ala Val Gly Met Ser Asp His Gly Asn Leu Phe Gly 35 40 45 Ser Tyr Lys Phe Tyr Lys Ala Leu Lys Ala Glu Gly Ile Lys Pro Ile 50 55 60 Ile Gly Met Glu Ala Tyr Phe Thr Thr Gly Ser Arg Phe Asp Arg Lys 65 70 75 80 Thr Lys Thr Ser Glu Asp Asn Ile Thr Asp Lys Tyr Asn His His Leu 85 90 95 Ile Leu Ile Ala Lys Asp Asp Lys Gly Leu Lys Asn Leu Met Lys Leu 100 105 110 Ser Thr Leu Ala Tyr Lys Glu Gly Phe Tyr Tyr Lys Pro Arg Ile Asp 115 120 125 Tyr Glu Leu Leu Glu Lys Tyr Gly Glu Gly Leu Ile Ala Leu Thr Ala 130 135 140 Cys Leu Lys Gly Val Pro Thr Tyr Tyr Ala Ser Ile Asn Glu Val Lys 145 150 155 160 Lys Ala Glu Glu Trp Val Lys Lys Phe Lys Asp Ile Phe Gly Asp Asp 165 170 175 Leu Tyr Leu Glu Leu Gln Ala Asn Asn Ile Pro Glu Gln Glu Val Ala 180 185 190 Asn Arg Asn Leu Ile Glu Ile Ala Lys Lys Tyr Asp Val Lys Leu Ile 195 200 205 Ala Thr Gln Asp Ala His Tyr Leu Asn Pro Glu Asp Arg Tyr Ala His 210 215 220 Thr Val Leu Met Ala Leu Gln Met Lys Lys Thr Ile His Glu Leu Ser 225 230 235 240 Ser Gly Asn Phe Lys Cys Ser Asn Glu Asp Leu His Phe Ala Pro Pro 245 250 255 Glu Tyr Met Trp Lys Lys Phe Glu Gly Lys Phe Glu Gly Trp Glu Lys 260 265 270 Ala Leu Leu Asn Thr Leu Glu Val Met Glu Lys Thr Ala Asp Ser Phe 275 280 285 Glu Ile Phe Glu Asn Ser Thr Tyr Leu Leu Pro Lys Tyr Asp Val Pro 290 295 300 Pro Asp Lys Thr Leu Glu Glu Tyr Leu Arg Glu Leu Ala Tyr Lys Gly 305 310 315 320 Leu Arg Gln Arg Ile Glu Arg Gly Gln Ala Lys Asp Thr Lys Glu Tyr 325 330 335 Trp Glu Arg Leu Glu Tyr Glu Leu Glu Val Ile Asn Lys Met Gly Phe 340 345 350 Ala Gly Tyr Phe Leu Ile Val Gln Asp Phe Ile Asn Trp Ala Lys Lys 355 360 365 Asn Asp Ile Pro Val Gly Pro Gly Arg Gly Ser Ala Gly Gly Ser Leu 370 375 380 Val Ala Tyr Ala Ile Gly Ile Thr Asp Val Asp Pro Ile Lys His Gly 385 390 395 400 Phe Leu Phe Glu Arg Phe Leu Asn Pro Glu Arg Val Ser Met Pro Asp 405 410 415 Ile Asp Val Asp Phe Cys Gln Asp Asn Arg Glu Lys Val Ile Glu Tyr 420 425 430 Val Arg Asn Lys Tyr Gly His Asp Asn Val Ala Gln Ile Ile Thr Tyr 435 440 445 Asn Val Met Lys Ala Lys Gln Thr Leu Arg Asp Val Ala Arg Ala Met 450 455 460 Gly Leu Pro Tyr Ser Thr Ala Asp Lys Leu Ala Lys Leu Ile Pro Gln 465 470 475 480 Gly Asp Val Gln Gly Thr Trp Leu Ser Leu Glu Glu Met Tyr Lys Thr 485 490 495 Pro Val Glu Glu Leu Leu Gln Lys Tyr Gly Glu His Arg Thr Asp Ile 500 505 510 Glu Asp Asn Val Lys Lys Phe Arg Gln Ile Cys Glu Glu Ser Pro Glu 515 520 525 Ile Lys Gln Leu Val Glu Thr Ala Leu Lys Leu Glu Gly Leu Thr Arg 530 535 540 His Thr Ser Leu His Ala Ala Gly Val Val Ile Ala Pro Lys Pro Leu 545 550 555 560 Ser Glu Leu Val Pro Leu Tyr Tyr Asp Lys Glu Gly Glu Val Ala Thr 565 570 575 Gln Tyr Asp Met Val Gln Leu Glu Glu Leu Gly Leu Leu Lys Met Asp 580 585 590 Phe Leu Gly Leu Lys Thr Leu Thr Glu Leu Lys Leu Met Lys Glu Leu 595 600 605 Ile Lys Glu Arg His Gly Val Asp Ile Asn Phe Leu Glu Leu Pro Leu 610 615 620 Asp Asp Pro Lys Val Tyr Lys Leu Leu Gln Glu Gly Lys Thr Thr Gly 625 630 635 640 Val Phe Gln Leu Glu Ser Arg Gly Met Lys Glu Leu Leu Lys Lys Leu 645 650 655 Lys Pro Asp Ser Phe Asp Asp Ile Val Ala Val Leu Ala Leu Tyr Arg 660 665 670 Pro Gly Pro Leu Lys Ser Gly Leu Val Asp Thr Tyr Ile Lys Arg Lys 675 680 685 His Gly Lys Glu Pro Val Glu Tyr Pro Phe Pro Glu Leu Glu Pro Val 690 695 700 Leu Lys Glu Thr Tyr Gly Val Ile Val Tyr Gln Glu Gln Val Met Lys 705 710 715 720 Met Ser Gln Ile Leu Ser Gly Phe Thr Pro Gly Glu Ala Asp Thr Leu 725 730 735 Arg Lys Ala Ile Gly Lys Lys Lys Ala Asp Leu Met Ala Gln Met Lys 740 745 750 Asp Lys Phe Ile Gln Gly Ala Val Glu Arg Gly Tyr Pro Glu Glu Lys 755 760 765 Ile Arg Lys Leu Trp Glu Asp Ile Glu Lys Phe Ala Ser Tyr Ser Phe 770 775 780 Asn Lys Ser His Ser Val Ala Tyr Gly Tyr Ile Ser Tyr Trp Thr Ala 785 790 795 800 Tyr Val Lys Ala His Tyr Pro Ala Glu Phe Phe Ala Val Lys Leu Thr 805 810 815 Thr Glu Lys Asn Asp Asn Lys Phe Leu Asn Leu Ile Lys Asp Ala Lys 820 825 830 Leu Phe Gly Phe Glu Ile Leu Pro Pro Asp Ile Asn Lys Ser Asp Val 835 840 845 Gly Phe Thr Ile Glu Gly Glu Asn Arg Ile Arg Phe Gly Leu Ala Arg 850 855 860 Ile Lys Gly Val Gly Glu Glu Thr Ala Lys Ile Ile Val Glu Ala Arg 865 870 875 880 Lys Lys Tyr Lys Gln Phe Lys Gly Leu Ala Asp Phe Ile Asn Lys Thr 885 890 895 Lys Asn Arg Lys Ile Asn Lys Lys Val Val Glu Ala Leu Val Lys Ala 900 905 910 Gly Ala Phe Asp Phe Thr Lys Lys Lys Arg Lys Glu Leu Leu Ala Lys 915 920 925 Val Ala Asn Ser Glu Lys Ala Leu Met Ala Thr Gln Asn Ser Leu Phe 930 935 940 Gly Ala Pro Lys Glu Glu Val Glu Glu Leu Asp Pro Leu Lys Leu Glu 945 950 955 960 Lys Glu Val Leu Gly Phe Tyr Ile Ser Gly His Pro Leu Asp Asn Tyr 965 970 975 Glu Lys Leu Leu Lys Asn Arg Tyr Thr Pro Ile Glu Asp Leu Glu Glu 980 985 990 Trp Asp Lys Glu Ser Glu Ala Val Leu Thr Gly Val Ile Thr Glu Leu 995 1000 1005 Lys Val Lys Lys Thr Lys Asn Gly Asp Tyr Met Ala Val Phe Asn Leu 1010 1015 1020 Val Asp Lys Thr Gly Leu Ile Glu Cys Val Val Phe Pro Gly Val Tyr 1025 1030 1035 1040 Glu Glu Ala Lys Glu Leu Ile Glu Glu Asp Arg Val Val Val Val Lys 1045 1050 1055 Gly Phe Leu Asp Glu Asp Leu Glu Thr Glu Asn Val Lys Phe Val Val 1060 1065 1070 Lys Glu Val Phe Ser Pro Glu Glu Phe Ala Lys Glu Met Arg Asn Thr 1075 1080 1085 Leu Tyr Ile Phe Leu Lys Arg Glu Gln Ala Leu Asn Gly Val Ala Glu 1090 1095 1100 Lys Leu Lys Gly Ile Ile Glu Asn Asn Arg Thr Glu Asp Gly Tyr Asn 1105 1110 1115 1120 Leu Val Leu Thr Val Asp Leu Gly Asp Tyr Phe Val Asp Leu Ala Leu 1125 1130 1135 Pro Gln Asp Met Lys Leu Lys Ala Asp Arg Lys Val Val Glu Glu Ile 1140 1145 1150 Glu Lys Leu Gly Val Lys Val Ile Ile 1155 1160 119 2408 DNA Aquifex aeolicus 119 atgaactacg ttcccttcgc gagaaagtac agaccgaaat tcttcaggga agtaatagga 60 caggaagctc ccgtaaggat actcaaaaac gctataaaaa acgacagagt ggctcacgcc 120 tacctctttg ccggaccgag gggggttggg aagacgacta ttgcaagaat tctcgcaaaa 180 gctttgaact gtaaaaatcc ctccaaaggt gagccctgcg gtgagtgcga aaactgcagg 240 gagatagaca ggggtgtgtt ccctgactta attgaaatgg atgccgcctc aaacaggggt 300 atagacgacg taagggcatt aaaagaagcg gtcaattaca aacctataaa aggaaagtac 360 aaggtttaca taatagacga agctcacatg ctcacgaaag aagctttcaa cgctctctta 420 aaaaccctcg aagagccccc tcccagaact gttttcgtcc tttgtaccac ggagtacgac 480 aaaattcttc ccacgatact ctcaaggtgt cagaggataa tcttctcaaa ggtaagaaag 540 gaaaaagtaa tagagtatct aaaaaagata tgtgaaaagg aagggattga gtgcgaagag 600 ggagcccttg aggttctggc tcatgcctct gaagggtgca tgagggatgc agcctctctc 660 ctggaccagg cgagcgttta cggggaaggc agggtaacaa aagaagtagt ggagaacttc 720 ctcggaattc tcagtcagga aagcgttagg agttttctga aattgcttct gaactcagaa 780 gtggacgaag ctataaagtt cctcagagaa ctctcagaaa agggctacaa cctgaccaag 840 ttttgggaga tgttagaaga ggaagtgaga aacgcaattt tagtaaagag cctgaaaaat 900 cccgaaagcg tggttcagaa ctggcaggat tacgaagact tcaaagacta ccctctggaa 960 gccctcctct acgttgagaa cctgataaac aggggtaaag ttgaagcgag aacgagagaa 1020 cccttaagag cctttgaact cgcggtaata aagagcctta tagtcaaaga cataattccc 1080 gtatcccagc tcggaagtgt ggtaaaggaa accaaaaagg aagaaaagaa agttgaagta 1140 aaagaagagc caaaagtaaa agaagaaaaa ccaaaggagc aggaagagga caggttccag 1200 aaagttttaa acgctgtgga cggcaaaatc cttaaaagaa tacttgaagg ggcaaaaagg 1260 gaagaaagag acggaaaaat cgtcctaaag atagaagcct cttatctgag aaccatgaaa 1320 aaggaatttg actcactaaa ggagactttt ccttttttag agtttgaacc cgtggaggat 1380 aaaaaaaaac ctcagaagtc cagcgggacg aggctgtttt aaaggtaaag gagctcttca 1440 atgcaaaaat actcaaagta cgaagtaaaa gctaaggtca taaaggtgag aatgcccgtg 1500 gaagagatag ggctgtttaa cgcactaata gacggcttgc ccaggtacgc actcacgagg 1560 acgaaggaaa agggaaaggg agaagttttc gttttagcga ctccttataa agtcaaggaa 1620 ttgatggaag ctatggaggg tatgaaaaaa cacataaagg atttagaaat cctcggagag 1680 acggatgagg atttaacttt ttaaagtatg ggtgtatctg agcaaaggtt taagctaaaa 1740 acaaacctga aacccgcagg ggaccagccg aaagccataa aaaaactcct tgaaaaccta 1800 aggaaaggcg taaaagaaca aacacttctc ggagtcacgg gaagcggaaa gacttttact 1860 ctagcaaacg taatagcgaa gtacaacaaa ccaactcttg tggtagttca caacaaaatt 1920 ctcgcggcac agctatacag ggagtttaaa gaactattcc ctgaaaacgc tgtagagtac 1980 tttgtctctt actacgacta ttaccaacct gaagcctaca ttcccgaaaa agatttatac 2040 atagaaaagg acgcgagtat aaacgaaagc tggaacgttt cagacactcc gccacgatat 2100 ccgttctaga aaggagggac gttatagtag ttgcttcagt ttcttgcata tacggactcg 2160 ggaaacctga gcactacgaa aacctgagga taaaactcca aaggggaata agactgaact 2220 tgagtaagct cctgaggaaa ctcgttgagc taggatatca gagaaatgac tttgccataa 2280 agagggctac cttctcggtt aggggagacg tggttgagat agtcccttct cacacggaag 2340 attacctcgt gagggtagag ttctgggacg acgaagttga aagaatagtc ctcatggacg 2400 ctctgaac 2408 120 473 PRT Aquifex aeolicus 120 Met Asn Tyr Val Pro Phe Ala Arg Lys Tyr Arg Pro Lys Phe Phe Arg 1 5 10 15 Glu Val Ile Gly Gln Glu Ala Pro Val Arg Ile Leu Lys Asn Ala Ile 20 25 30 Lys Asn Asp Arg Val Ala His Ala Tyr Leu Phe Ala Gly Pro Arg Gly 35 40 45 Val Gly Lys Thr Thr Ile Ala Arg Ile Leu Ala Lys Ala Leu Asn Cys 50 55 60 Lys Asn Pro Ser Lys Gly Glu Pro Cys Gly Glu Cys Glu Asn Cys Arg 65 70 75 80 Glu Ile Asp Arg Gly Val Phe Pro Asp Leu Ile Glu Met Asp Ala Ala 85 90 95 Ser Asn Arg Gly Ile Asp Asp Val Arg Ala Leu Lys Glu Ala Val Asn 100 105 110 Tyr Lys Pro Ile Lys Gly Lys Tyr Lys Val Tyr Ile Ile Asp Glu Ala 115 120 125 His Met Leu Thr Lys Glu Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu 130 135 140 Glu Pro Pro Pro Arg Thr Val Phe Val Leu Cys Thr Thr Glu Tyr Asp 145 150 155 160 Lys Ile Leu Pro Thr Ile Leu Ser Arg Cys Gln Arg Ile Ile Phe Ser 165 170 175 Lys Val Arg Lys Glu Lys Val Ile Glu Tyr Leu Lys Lys Ile Cys Glu 180 185 190 Lys Glu Gly Ile Glu Cys Glu Glu Gly Ala Leu Glu Val Leu Ala His 195 200 205 Ala Ser Glu Gly Cys Met Arg Asp Ala Ala Ser Leu Leu Asp Gln Ala 210 215 220 Ser Val Tyr Gly Glu Gly Arg Val Thr Lys Glu Val Val Glu Asn Phe 225 230 235 240 Leu Gly Ile Leu Ser Gln Glu Ser Val Arg Ser Phe Leu Lys Leu Leu 245 250 255 Leu Asn Ser Glu Val Asp Glu Ala Ile Lys Phe Leu Arg Glu Leu Ser 260 265 270 Glu Lys Gly Tyr Asn Leu Thr Lys Phe Trp Glu Met Leu Glu Glu Glu 275 280 285 Val Arg Asn Ala Ile Leu Val Lys Ser Leu Lys Asn Pro Glu Ser Val 290 295 300 Val Gln Asn Trp Gln Asp Tyr Glu Asp Phe Lys Asp Tyr Pro Leu Glu 305 310 315 320 Ala Leu Leu Tyr Val Glu Asn Leu Ile Asn Arg Gly Lys Val Glu Ala 325 330 335 Arg Thr Arg Glu Pro Leu Arg Ala Phe Glu Leu Ala Val Ile Lys Ser 340 345 350 Leu Ile Val Lys Asp Ile Ile Pro Val Ser Gln Leu Gly Ser Val Val 355 360 365 Lys Glu Thr Lys Lys Glu Glu Lys Lys Val Glu Val Lys Glu Glu Pro 370 375 380 Lys Val Lys Glu Glu Lys Pro Lys Glu Gln Glu Glu Asp Arg Phe Gln 385 390 395 400 Lys Val Leu Asn Ala Val Asp Gly Lys Ile Leu Lys Arg Ile Leu Glu 405 410 415 Gly Ala Lys Arg Glu Glu Arg Asp Gly Lys Ile Val Leu Lys Ile Glu 420 425 430 Ala Ser Tyr Leu Arg Thr Met Lys Lys Glu Phe Asp Ser Leu Lys Glu 435 440 445 Thr Phe Pro Phe Leu Glu Phe Glu Pro Val Glu Asp Lys Lys Lys Pro 450 455 460 Gln Lys Ser Ser Gly Thr Arg Leu Phe 465 470 121 1090 DNA Aquifex aeolicus 121 atgcgcgtta aggtggacag ggaggagctt gaagaggttc ttaaaaaagc aagagaaagc 60 acggaaaaaa aagccgcact cccgatactc gcgaacttct tactctccgc aaaagaggaa 120 aacttaatcg taagggcaac ggacttggaa aactaccttg tagtctccgt aaagggggag 180 gttgaagagg aaggagaggt ttgcgtccac tctcaaaaac tctacgatat agtcaagaac 240 ttaaattccg cttacgttta ccttcatacg gaaggtgaaa aactcgtcat aacgggagga 300 aagagtacgt acaaacttcc gacagctccc gcggaggact ttcccgaatt tccagaaatc 360 gtagaaggag gagaaacact ttcgggaaac cttctcgtta acggaataga aaaggtagag 420 tacgccatag cgaaggaaga agcgaacata gcccttcagg gaatgtatct gagaggatac 480 gaggacagaa ttcactttgt gttcggacgg tcacaggctt gcactttatg aacctctacg 540 taaacattga aaagagtgaa gacgagtctt ttgcttactt ctccactccc gagtggaaac 600 tcgccgttag ctcctggaag gagaattccc ggactacatg agtgtcatcc ctgaggagtt 660 ttcggcggaa gtcttgtttg agacagagga agtcttaaag gttttaaaga ggttgaaggc 720 tttaagcgaa ggaaaagttt ttcccgtgaa gattacctta agcgaaaacc ttgccatctt 780 tgagttcgcg gatccggagt tcggagaagc gagagaggaa attgaagtgg agtacacggg 840 agagcccttt gagataggat tcaacggaaa taccttatgg aggcgcttga cgcctacgac 900 agcgaaagag tgtggttcaa gttcacaacc cccgacacgg ccactttatt ggaggctgaa 960 gattacgaaa aggaacctta caagtgcata ataatgccga tgagggtgta gccatgaaaa 1020 aagctttaat ctttttattg agcttgagcc ttttaattcc tgcgtttagc gaagccaaac 1080 ccaagtcttc 1090 122 363 PRT Aquifex aeolicus 122 Met Arg Val Lys Val Asp Arg Glu Glu Leu Glu Glu Val Leu Lys Lys 1 5 10 15 Ala Arg Glu Ser Thr Glu Lys Lys Ala Ala Leu Pro Ile Leu Ala Asn 20 25 30 Phe Leu Leu Ser Ala Lys Glu Glu Asn Leu Ile Val Arg Ala Thr Asp 35 40 45 Leu Glu Asn Tyr Leu Val Val Ser Val Lys Gly Glu Val Glu Glu Glu 50 55 60 Gly Glu Val Cys Val His Ser Gln Lys Leu Tyr Asp Ile Val Lys Asn 65 70 75 80 Leu Asn Ser Ala Tyr Val Tyr Leu His Thr Glu Gly Glu Lys Leu Val 85 90 95 Ile Thr Gly Gly Lys Ser Thr Tyr Lys Leu Pro Thr Ala Pro Ala Glu 100 105 110 Asp Phe Pro Glu Phe Pro Glu Ile Val Glu Gly Gly Glu Thr Leu Ser 115 120 125 Gly Asn Leu Leu Val Asn Gly Ile Glu Lys Val Glu Tyr Ala Ile Ala 130 135 140 Lys Glu Glu Ala Asn Ile Ala Leu Gln Gly Met Tyr Leu Arg Gly Tyr 145 150 155 160 Glu Asp Arg Ile His Phe Val Gly Ser Asp Gly His Arg Leu Ala Leu 165 170 175 Tyr Glu Pro Leu Gly Glu Phe Ser Lys Glu Leu Leu Ile Pro Arg Lys 180 185 190 Ser Leu Lys Val Leu Lys Lys Leu Ile Thr Gly Ile Glu Asp Val Asn 195 200 205 Ile Glu Lys Ser Glu Asp Glu Ser Phe Ala Tyr Phe Ser Thr Pro Glu 210 215 220 Trp Lys Leu Ala Val Arg Leu Leu Glu Gly Glu Phe Pro Asp Tyr Met 225 230 235 240 Ser Val Ile Pro Glu Glu Phe Ser Ala Glu Val Leu Phe Glu Thr Glu 245 250 255 Glu Val Leu Lys Val Leu Lys Arg Leu Lys Ala Leu Ser Glu Gly Lys 260 265 270 Val Phe Pro Val Lys Ile Thr Leu Ser Glu Asn Leu Ala Ile Phe Glu 275 280 285 Phe Ala Asp Pro Glu Phe Gly Glu Ala Arg Glu Glu Ile Glu Val Glu 290 295 300 Tyr Thr Gly Glu Pro Phe Glu Ile Gly Phe Asn Gly Lys Tyr Leu Met 305 310 315 320 Glu Ala Leu Asp Ala Tyr Asp Ser Glu Arg Val Trp Phe Lys Phe Thr 325 330 335 Thr Pro Asp Thr Ala Thr Leu Leu Glu Ala Glu Asp Tyr Glu Lys Glu 340 345 350 Pro Tyr Lys Cys Ile Ile Met Pro Met Arg Val 355 360 123 1093 DNA Aquifex aeolicus 123 gtggaaacca caatattcca gttccagaaa acttttttca caaaacctcc gaaggagagg 60 gtcttcgtcc ttcatggaga agagcagtat ctcataagaa cctttttgtc taagctgaag 120 gaaaagtacg gggagaatta cacggttctg tggggggatg agataagcga ggaggaattc 180 tacactgccc tttccgagac cagtatattc ggcggttcaa aggaaaaagc ggtggtcatt 240 tacaacttcg gggatttcct gaagaagctc ggaaggaaga aaaaggaaaa agaaaggctt 300 ataaaagtcc tcagaaacgt aaagagtaac tacgtattta tagtgtacga tgcgaaactc 360 cagaaacagg aactttcttc ggaacctctg aaatccgtag cgtctttcgg cggtatagtg 420 gtagcaaaca ggctgagcaa ggagaggata aaacagctcg tccttaagaa gttcaaagaa 480 aaagggataa acgtagaaaa cgatgccctt gaataccttc tccagctcac gggttacaac 540 ttgatggagc tcaaacttga ggttgaaaaa ctgatagatt acgcaagtga aaagaaaatt 600 ttaacactcg atgaggtaaa gagagtagcc ttctcagtct cagaaaacgt aaacgtattt 660 gagttcgttg atttactcct cttaaaagat tacgaaaagg ctcttaaagt tttggactcc 720 ctcatttcct tcggaataca ccccctccag attatgaaaa tcctgtcctc ctatgctcta 780 aaactttaca ccctcaagag gcttgaagag aagggagagg acctgaataa ggcgatggaa 840 agcgtgggaa taaagaacaa ctttctcaag atgaagttca aatcttactt aaaggcaaac 900 tctaaagagg acttgaagaa cctaatcctc tccctccaga ggatagacgc tttttctaaa 960 ctttactttc aggacacagt gcagttgctg gggatttctt gacctcaaga ctggagaggg 1020 aagttgtgaa aaatacttct catggtggat aatctttttt atgaagtttg cggtttgcgt 1080 ttttcccggt tct 1093 124 350 PRT Aquifex aeolicus 124 Val Glu Thr Thr Ile Phe Gln Phe Gln Lys Thr Phe Phe Thr Lys Pro 1 5 10 15 Pro Lys Glu Arg Val Phe Val Leu His Gly Glu Glu Gln Tyr Leu Ile 20 25 30 Arg Thr Phe Leu Ser Lys Leu Lys Glu Lys Tyr Gly Glu Asn Tyr Thr 35 40 45 Val Leu Trp Gly Asp Glu Ile Ser Glu Glu Glu Phe Tyr Thr Ala Leu 50 55 60 Ser Glu Thr Ser Ile Phe Gly Gly Ser Lys Glu Lys Ala Val Val Ile 65 70 75 80 Tyr Asn Phe Gly Asp Phe Leu Lys Lys Leu Gly Arg Lys Lys Lys Glu 85 90 95 Lys Glu Arg Leu Ile Lys Val Leu Arg Asn Val Lys Ser Asn Tyr Val 100 105 110 Phe Ile Val Tyr Asp Ala Lys Leu Gln Lys Gln Glu Leu Ser Ser Glu 115 120 125 Pro Leu Lys Ser Val Ala Ser Phe Gly Gly Ile Val Val Ala Asn Arg 130 135 140 Leu Ser Lys Glu Arg Ile Lys Gln Leu Val Leu Lys Lys Phe Lys Glu 145 150 155 160 Lys Gly Ile Asn Val Glu Asn Asp Ala Leu Glu Tyr Leu Leu Gln Leu 165 170 175 Thr Gly Tyr Asn Leu Met Glu Leu Lys Leu Glu Val Glu Lys Leu Ile 180 185 190 Asp Tyr Ala Ser Glu Lys Lys Ile Leu Thr Leu Asp Glu Val Lys Arg 195 200 205 Val Ala Phe Ser Val Ser Glu Asn Val Asn Val Phe Glu Phe Val Asp 210 215 220 Leu Leu Leu Leu Lys Asp Tyr Glu Lys Ala Leu Lys Val Leu Asp Ser 225 230 235 240 Leu Ile Ser Phe Gly Ile His Pro Leu Gln Ile Met Lys Ile Leu Ser 245 250 255 Ser Tyr Ala Leu Lys Leu Tyr Thr Leu Lys Arg Leu Glu Glu Lys Gly 260 265 270 Glu Asp Leu Asn Lys Ala Met Glu Ser Val Gly Ile Lys Asn Asn Phe 275 280 285 Leu Lys Met Lys Phe Lys Ser Tyr Leu Lys Ala Asn Ser Lys Glu Asp 290 295 300 Leu Lys Asn Leu Ile Leu Ser Leu Gln Arg Ile Asp Ala Phe Ser Lys 305 310 315 320 Leu Tyr Phe Gln Asp Thr Val Gln Leu Leu Arg Asp Phe Leu Thr Ser 325 330 335 Arg Leu Glu Arg Glu Val Val Lys Asn Thr Ser His Gly Gly 340 345 350 125 1051 DNA Aquifex aeolicus 125 atggaaaaag tttttttgga aaaactccag aaaaccttgc acatacccgg aggactcctt 60 ttttacggca aagaaggaag cggaaagacg aaaacagctt ttgaatttgc aaaaggtatt 120 ttatgtaagg aaaacgtacc tggggatgcg gaagttgtcc ctcctgcaaa cacgtaaacg 180 agctggagga agccttcttt aaaggagaaa tagaagactt taaagtttat aagacaagga 240 cggtaaaaag cacttcgttt accttatggg cgaacatccc gactttgtgg taataatccc 300 gagcggacat tacataaaga tagaacagat aagggaagtt aagaactttg cctatgtgaa 360 gcccgcacta agcaggagaa aagtaattat aatagacgac gcccacgcga tgacctctca 420 ggcggcaaac gctcttttaa aggtattgga agagccacct gcggacacca cctttatctt 480 gaccacgaac aggcgttctg caatcctgcc gactatcctc tccagaactt ttcaagtgga 540 gttcaagggc ttttcagtaa aagaggttat ggaaatagcg aaagtagacg aggaaatagc 600 gaaactctct ggaggcagtc taaaaagggc tatcttacta aaggaaaaca aagatatcct 660 aaacaaagta aaggaattct tggaaaacga gccgttaaaa gtttacaagc ttgcaagtga 720 attcgaaaag tgggaacctg aaaagcaaaa actcttcctt gaaattatgg aagaattggt 780 atctcaaaaa ttgaccgaag agaaaaaaga caattacacc taccttcttg atacgatcag 840 actctttaaa gacggactcg caaggggtgt aaacgaacct ctgtggctgt ttacgttagc 900 cgttcaggcg gattaataaa ccgttattga ttccgtaaca tttaaacctt aatctaaatt 960 atgagagcct ttgaaggagg tctggtatgg aaaatttgaa gattagatat atagatacga 1020 ggaagatagg aaccgtgagc ggtgtaaaag t 1051 126 305 PRT Aquifex aeolicus 126 Met Glu Lys Val Phe Leu Glu Lys Leu Gln Lys Thr Leu His Ile Pro 1 5 10 15 Gly Gly Leu Leu Phe Tyr Gly Lys Glu Gly Ser Gly Lys Thr Lys Thr 20 25 30 Ala Phe Glu Phe Ala Lys Gly Ile Leu Cys Lys Glu Asn Val Pro Trp 35 40 45 Gly Cys Gly Ser Cys Pro Ser Cys Lys His Val Asn Glu Leu Glu Glu 50 55 60 Ala Phe Phe Lys Gly Glu Ile Glu Asp Phe Lys Val Tyr Lys Asp Lys 65 70 75 80 Asp Gly Lys Lys His Phe Val Tyr Leu Met Gly Glu His Pro Asp Phe 85 90 95 Val Val Ile Ile Pro Ser Gly His Tyr Ile Lys Ile Glu Gln Ile Arg 100 105 110 Glu Val Lys Asn Phe Ala Tyr Val Lys Pro Ala Leu Ser Arg Arg Lys 115 120 125 Val Ile Ile Ile Asp Asp Ala His Ala Met Thr Ser Gln Ala Ala Asn 130 135 140 Ala Leu Leu Lys Val Leu Glu Glu Pro Pro Ala Asp Thr Thr Phe Ile 145 150 155 160 Leu Thr Thr Asn Arg Arg Ser Ala Ile Leu Pro Thr Ile Leu Ser Arg 165 170 175 Thr Phe Gln Val Glu Phe Lys Gly Phe Ser Val Lys Glu Val Met Glu 180 185 190 Ile Ala Lys Val Asp Glu Glu Ile Ala Lys Leu Ser Gly Gly Ser Leu 195 200 205 Lys Arg Ala Ile Leu Leu Lys Glu Asn Lys Asp Ile Leu Asn Lys Val 210 215 220 Lys Glu Phe Leu Glu Asn Glu Pro Leu Lys Val Tyr Lys Leu Ala Ser 225 230 235 240 Glu Phe Glu Lys Trp Glu Pro Glu Lys Gln Lys Leu Phe Leu Glu Ile 245 250 255 Met Glu Glu Leu Val Ser Gln Lys Leu Thr Glu Glu Lys Lys Asp Asn 260 265 270 Tyr Thr Tyr Leu Leu Asp Thr Ile Arg Leu Phe Lys Asp Gly Leu Ala 275 280 285 Arg Gly Val Asn Glu Pro Leu Trp Leu Phe Thr Leu Ala Val Gln Ala 290 295 300 Asp 305 127 630 DNA Aquifex aeolicus 127 atgaacttcc tgaaaaagtt ccttttactg agaaaagctc aaaagtctcc ttacttcgaa 60 gagttctacg aagaaatcga tttgaaccag aaggtgaaag atgcaaggtt tgtagttttt 120 gactgcgaag ccacagaact cgacgtaaag aaggcaaaac tcctttcaat aggtgcggtt 180 gaggttaaaa acctggaaat agacctctct aaatcttttt acgagatact caaaagtgac 240 gagataaagg cggcggagat acatggaata accagggaag acgttgaaaa gtacggaaag 300 gaaccaaagg aagtaatata cgactttctg aagtacataa agggaagcgt tctcgttggc 360 tactacgtga agtttgacgt ctcactcgtt gagaagtact ccataaagta cttccagtat 420 ccaatcatca actacaagtt agacctgttt agtttcgtga agagagagta ccagagtggc 480 aggagtcttg acgaccttat gaaggaactc ggtgtagaaa taagggcaag gcacaacgcc 540 cttgaagatg cctacataac cgctcttctt ttcctaaagt acgtttaccc gaacagggag 600 tacagactaa aggatctccc gattttcctt 630 128 210 PRT Aquifex aeolicus 128 Met Asn Phe Leu Lys Lys Phe Leu Leu Leu Arg Lys Ala Gln Lys Ser 1 5 10 15 Pro Tyr Phe Glu Glu Phe Tyr Glu Glu Ile Asp Leu Asn Gln Lys Val 20 25 30 Lys Asp Ala Arg Phe Val Val Phe Asp Cys Glu Ala Thr Glu Leu Asp 35 40 45 Val Lys Lys Ala Lys Leu Leu Ser Ile Gly Ala Val Glu Val Lys Asn 50 55 60 Leu Glu Ile Asp Leu Ser Lys Ser Phe Tyr Glu Ile Leu Lys Ser Asp 65 70 75 80 Glu Ile Lys Ala Ala Glu Ile His Gly Ile Thr Arg Glu Asp Val Glu 85 90 95 Lys Tyr Gly Lys Glu Pro Lys Glu Val Ile Tyr Asp Phe Leu Lys Tyr 100 105 110 Ile Lys Gly Ser Val Leu Val Gly Tyr Tyr Val Lys Phe Asp Val Ser 115 120 125 Leu Val Glu Lys Tyr Ser Ile Lys Tyr Phe Gln Tyr Pro Ile Ile Asn 130 135 140 Tyr Lys Leu Asp Leu Phe Ser Phe Val Lys Arg Glu Tyr Gln Ser Gly 145 150 155 160 Arg Ser Leu Asp Asp Leu Met Lys Glu Leu Gly Val Glu Ile Arg Ala 165 170 175 Arg His Asn Ala Leu Glu Asp Ala Tyr Ile Thr Ala Leu Leu Phe Leu 180 185 190 Lys Tyr Val Tyr Pro Asn Arg Glu Tyr Arg Leu Lys Asp Leu Pro Ile 195 200 205 Phe Leu 210 129 526 DNA Aquifex aeolicus 129 atgctcaata aggtttttat aataggaaga cttacgggtg accccgttat aacttatcta 60 ccgagcggaa cgcccgtagt agagtttact ctggcttaca acagaaggta taaaaaccag 120 aacggtgaat ttcaggagga aagtcacttc tttgacgtaa aggcgtacgg aaaaatggct 180 gaagactggg ctacacgctt ctcgaaagga tacctcgtac tcgtagaggg aagactctcc 240 caggaaaagt gggagaaaga aggaaagaag ttctcaaagg tcaggataat agcggaaaac 300 gtaagattaa taaacaggcc gaaaggtgct gaacttcaag cagaagaaga ggaggaagtt 360 cctcccattg aggaggaaat tgaaaaactc ggtaaagagg aagagaagcc ttttaccgat 420 gaagaggacg aaataccttt ttaattttga ggaggttaaa gtatggtagt gagagctcct 480 aagaagaaag tttgtatgta ctgtgaacaa aagagagagc cagatt 526 130 147 PRT Aquifex aeolicus 130 Met Leu Asn Lys Val Phe Ile Ile Gly Arg Leu Thr Gly Asp Pro Val 1 5 10 15 Ile Thr Tyr Leu Pro Ser Gly Thr Pro Val Val Glu Phe Thr Leu Ala 20 25 30 Tyr Asn Arg Arg Tyr Lys Asn Gln Asn Gly Glu Phe Gln Glu Glu Ser 35 40 45 His Phe Phe Asp Val Lys Ala Tyr Gly Lys Met Ala Glu Asp Trp Ala 50 55 60 Thr Arg Phe Ser Lys Gly Tyr Leu Val Leu Val Glu Gly Arg Leu Ser 65 70 75 80 Gln Glu Lys Trp Glu Lys Glu Gly Lys Lys Phe Ser Lys Val Arg Ile 85 90 95 Ile Ala Glu Asn Val Arg Leu Ile Asn Arg Pro Lys Gly Ala Glu Leu 100 105 110 Gln Ala Glu Glu Glu Glu Glu Val Pro Pro Ile Glu Glu Glu Ile Glu 115 120 125 Lys Leu Gly Lys Glu Glu Glu Lys Pro Phe Thr Asp Glu Glu Asp Glu 130 135 140 Ile Pro Phe 145 131 1472 DNA Aquifex aeolicus 131 atgcaatttg tggataaact tccctgtgac gaatccgccg agagggcggt tcttggcagt 60 atgcttgaag accccgaaaa catacctctg gtacttgaat accttaaaga agaagacttc 120 tgcatagacg agcacaagct acttttcagg gttcttacaa acctctggtc cgagtacggc 180 aataagctcg atttcgtatt aataaaggat caccttgaaa agaaaaactt actccagaaa 240 atacctatag actggctcga agaactctac gaggaggcgg tatcccctga cacgcttgag 300 gaagtctgca aaatagtaaa acaacgttcc gcacagaggg cgataattca actcggtata 360 gaactcattc acaaaggaaa ggaaaacaaa gactttcaca cattaatcga ggaagcccag 420 agcaggatat tttccatagc ggaaagtgct acatctacgc agttttacca tgtgaaagac 480 gttgcggaag aagttataga actcatttat aaattcaaaa gctctgacag gctagtcacg 540 ggactcccaa gcggtttcac ggaactcgat ctaaagacga cgggattcca ccctggagac 600 ttaataatac tcgccgcaag acccggtatg gggaaaaccg cctttatgct ctccataatc 660 tacaatctcg caaaagacga gggaaaaccc tcagctgtat tttccttgga aatgagcaag 720 gaacagctcg ttatgagact cctctctatg atgtcggagg tcccactttt caagataagg 780 tctggaagta tatcgaatga agatttaaag aagcttgaag caagcgcaat agaactcgca 840 aagtacgaca tatacctcga cgacacaccc gctctcacta caacggattt aaggataagg 900 gcaagaaagc tcagaaagga aaaggaagtt gagttcgtgg cggtggacta cttgcaactt 960 ctgagaccgc cagtccgaaa gagttcaaga caggaggaag tggcagaggt ttcaagaaac 1020 ttaaaagccc ttgcaaagga acttcacatt cccgttatgg cacttgcgca gctctcccgt 1080 gaggtggaaa agaggagtga taaaagaccc cagcttgcgg acctcagaga atccggacag 1140 atagaacagg acgcagacct aatccttttc ctccacagac ccgagtacta caagaaaaag 1200 ccaaatcccg aagagcaggg tatagcggaa gtgataatag ccaagcaaag gcaaggaccc 1260 acggacattg tgaagctcgc atttattaag gagtacacta agtttgcaaa cctagaagcc 1320 cttcctgaac aacctcctga agaagaggaa ctttccgaaa ttattgaaac acaggaggat 1380 gaaggattcg aagatattga cttctgaaaa ttaaggtttt ataattttat cttggctatc 1440 cggggtagct caatcggcag agcgggtggc tg 1472 132 438 PRT Aquifex aeolicus 132 Met Gln Phe Val Asp Lys Leu Pro Cys Asp Glu Ser Ala Glu Arg Ala 1 5 10 15 Val Leu Gly Ser Met Leu Glu Asp Pro Glu Asn Ile Pro Leu Val Leu 20 25 30 Glu Tyr Leu Lys Glu Glu Asp Phe Cys Ile Asp Glu His Lys Leu Leu 35 40 45 Phe Arg Val Leu Thr Asn Leu Trp Ser Glu Tyr Gly Asn Lys Leu Asp 50 55 60 Phe Val Leu Ile Lys Asp His Leu Glu Lys Lys Asn Leu Leu Gln Lys 65 70 75 80 Ile Pro Ile Asp Trp Leu Glu Glu Leu Tyr Glu Glu Ala Val Ser Pro 85 90 95 Asp Thr Leu Glu Glu Val Cys Lys Ile Val Lys Gln Arg Ser Ala Gln 100 105 110 Arg Ala Ile Ile Gln Leu Gly Ile Thr Ser Thr Gln Phe Tyr His Val 115 120 125 Lys Asp Val Ala Glu Glu Val Ile Glu Leu Ile Tyr Lys Phe Lys Ser 130 135 140 Ser Asp Arg Leu Val Thr Gly Leu Pro Ser Gly Phe Thr Glu Leu Asp 145 150 155 160 Leu Lys Thr Thr Gly Phe His Pro Gly Asp Leu Ile Ile Leu Ala Ala 165 170 175 Arg Pro Gly Met Gly Lys Thr Ala Phe Met Leu Ser Ile Ile Tyr Asn 180 185 190 Leu Ala Lys Asp Glu Gly Lys Pro Ser Ala Val Phe Ser Leu Glu Met 195 200 205 Ser Lys Glu Gln Leu Val Met Arg Leu Leu Ser Met Met Ser Glu Val 210 215 220 Pro Leu Phe Lys Ile Arg Ser Gly Ser Ile Ser Asn Glu Asp Leu Lys 225 230 235 240 Lys Leu Glu Ala Ser Ala Ile Glu Leu Ala Lys Tyr Asp Ile Tyr Leu 245 250 255 Asp Asp Thr Pro Ala Leu Thr Thr Thr Asp Leu Arg Ile Arg Ala Arg 260 265 270 Lys Leu Arg Lys Glu Lys Glu Val Glu Phe Val Ala Val Asp Tyr Leu 275 280 285 Gln Leu Leu Arg Pro Pro Val Arg Lys Ser Ser Arg Gln Glu Glu Val 290 295 300 Ala Glu Val Ser Arg Asn Leu Lys Ala Leu Ala Lys Glu Leu His Ile 305 310 315 320 Pro Val Met Ala Leu Ala Gln Leu Ser Arg Glu Val Glu Lys Arg Ser 325 330 335 Asp Lys Arg Pro Gln Leu Ala Asp Leu Arg Glu Ser Gly Gln Ile Glu 340 345 350 Gln Asp Ala Asp Leu Ile Leu Phe Leu His Arg Pro Glu Tyr Tyr Lys 355 360 365 Lys Lys Pro Asn Pro Glu Glu Gln Gly Ile Ala Glu Val Ile Ile Ala 370 375 380 Lys Gln Arg Gln Gly Pro Thr Asp Ile Val Lys Leu Ala Phe Ile Lys 385 390 395 400 Glu Tyr Thr Lys Phe Ala Asn Leu Glu Ala Leu Pro Glu Gln Pro Pro 405 410 415 Glu Glu Glu Glu Leu Ser Glu Ile Ile Glu Thr Gln Glu Asp Glu Gly 420 425 430 Phe Glu Asp Ile Asp Phe 435 133 1526 DNA Aquifex aeolicus 133 atgtcctcgg acatagacga acttagacgg gaaatagata tagtagacgt catttccgaa 60 tacttaaact tagagaaggt aggttccaat tacagaacga actgtccctt tcaccctgac 120 gatacaccct ccttttacgt gtctccaagt aaacaaatat tcaagtgttt cggttgcggg 180 gtagggggag acgcgataaa gttcgtttcc ctttacgagg acatctccta ttttgaagcc 240 gcccttgaac tcgcaaaacg ctacggaaag aaattagacc ttgaaaagat atcaaaagac 300 gaaaaggtat acgtggctct tgacagggtt tgtgatttct acagggaaag ccttctcaaa 360 aacagagagg caagtgagta cgtaaagagt aggggaatag accctaaagt agcgaggaag 420 tttgatcttg ggtacgcacc ttccagtgaa gcactcgtaa aagtcttaaa agagaacgat 480 cttttagagg cttaccttga aactaaaaac ctcctttctc ctacgaaggg tgtttacagg 540 gatctctttc ttcggcgtgt cgtgatcccg ataaaggatc cgaggggaag agttataggt 600 ttcggtggaa ggaggatagt agaggacaaa tctcccaagt acataaactc tccagacagc 660 agggtattta aaaaggggga gaacttattc ggtctttacg aggcaaagga gtatataaag 720 gaagaaggat ttgcgatact tgtggaaggg tactttgacc ttttgagact tttttccgag 780 ggaataagga acgttgttgc acccctcggt acagccctga cccaaaatca ggcaaacctc 840 ctttccaagt tcacaaaaaa ggtctacatc ctttacgacg gagatgatgc gggaagaaag 900 gctatgaaaa gtgccattcc cctactcctc agtgcaggag tggaagttta tcccgtttac 960 ctccccgaag gatacgatcc cgacgagttt ataaaggaat tcgggaaaga ggaattaaga 1020 agactgataa acagctcagg ggagctcttt gaaacgctca taaaaaccgc aagggaaaac 1080 ttagaggaga aaacgcgtga gttcaggtat tatctgggct ttatttccga tggagtaagg 1140 cgctttgctc tggcttcgga gtttcacacc aagtacaaag ttcctatgga aattttatta 1200 atgaaaattg aaaaaaattc tcaagaaaaa gaaattaaac tctcctttaa ggaaaaaatc 1260 ttcctgaaag gactgataga attaaaacca aaaatagacc ttgaagtcct gaacttaagt 1320 cctgagttaa aggaactcgc agttaacgcc ttaaacggag aggagcattt acttccaaaa 1380 gaagttctcg agtaccaggt ggataacttg gagaaacttt ttaacaacat ccttagggat 1440 ttacaaaaat ctgggaaaaa gaggaagaaa agagggttga aaaatgtaaa tacttaatta 1500 actttaataa atttttagag ttagga 1526 134 498 PRT Aquifex aeolicus 134 Met Ser Ser Asp Ile Asp Glu Leu Arg Arg Glu Ile Asp Ile Val Asp 1 5 10 15 Val Ile Ser Glu Tyr Leu Asn Leu Glu Lys Val Gly Ser Asn Tyr Arg 20 25 30 Thr Asn Cys Pro Phe His Pro Asp Asp Thr Pro Ser Phe Tyr Val Ser 35 40 45 Pro Ser Lys Gln Ile Phe Lys Cys Phe Gly Cys Gly Val Gly Gly Asp 50 55 60 Ala Ile Lys Phe Val Ser Leu Tyr Glu Asp Ile Ser Tyr Phe Glu Ala 65 70 75 80 Ala Leu Glu Leu Ala Lys Arg Tyr Gly Lys Lys Leu Asp Leu Glu Lys 85 90 95 Ile Ser Lys Asp Glu Lys Val Tyr Val Ala Leu Asp Arg Val Cys Asp 100 105 110 Phe Tyr Arg Glu Ser Leu Leu Lys Asn Arg Glu Ala Ser Glu Tyr Val 115 120 125 Lys Ser Arg Gly Ile Asp Pro Lys Val Ala Arg Lys Phe Asp Leu Gly 130 135 140 Tyr Ala Pro Ser Ser Glu Ala Leu Val Lys Val Leu Lys Glu Asn Asp 145 150 155 160 Leu Leu Glu Ala Tyr Leu Glu Thr Lys Asn Leu Leu Ser Pro Thr Lys 165 170 175 Gly Val Tyr Arg Asp Leu Phe Leu Arg Arg Val Val Ile Pro Ile Lys 180 185 190 Asp Pro Arg Gly Arg Val Ile Gly Phe Gly Gly Arg Arg Ile Val Glu 195 200 205 Asp Lys Ser Pro Lys Tyr Ile Asn Ser Pro Asp Ser Arg Val Phe Lys 210 215 220 Lys Gly Glu Asn Leu Phe Gly Leu Tyr Glu Ala Lys Glu Tyr Ile Lys 225 230 235 240 Glu Glu Gly Phe Ala Ile Leu Val Glu Gly Tyr Phe Asp Leu Leu Arg 245 250 255 Leu Phe Ser Glu Gly Ile Arg Asn Val Val Ala Pro Leu Gly Thr Ala 260 265 270 Leu Thr Gln Asn Gln Ala Asn Leu Leu Ser Lys Phe Thr Lys Lys Val 275 280 285 Tyr Ile Leu Tyr Asp Gly Asp Asp Ala Gly Arg Lys Ala Met Lys Ser 290 295 300 Ala Ile Pro Leu Leu Leu Ser Ala Gly Val Glu Val Tyr Pro Val Tyr 305 310 315 320 Leu Pro Glu Gly Tyr Asp Pro Asp Glu Phe Ile Lys Glu Phe Gly Lys 325 330 335 Glu Glu Leu Arg Arg Leu Ile Asn Ser Ser Gly Glu Leu Phe Glu Thr 340 345 350 Leu Ile Lys Thr Ala Arg Glu Asn Leu Glu Glu Lys Thr Arg Glu Phe 355 360 365 Arg Tyr Tyr Leu Gly Phe Ile Ser Asp Gly Val Arg Arg Phe Ala Leu 370 375 380 Ala Ser Glu Phe His Thr Lys Tyr Lys Val Pro Met Glu Ile Leu Leu 385 390 395 400 Met Lys Ile Glu Lys Asn Ser Gln Glu Lys Glu Ile Lys Leu Ser Phe 405 410 415 Lys Glu Lys Ile Phe Leu Lys Gly Leu Ile Glu Leu Lys Pro Lys Ile 420 425 430 Asp Leu Glu Val Leu Asn Leu Ser Pro Glu Leu Lys Glu Leu Ala Val 435 440 445 Asn Ala Leu Asn Gly Glu Glu His Leu Leu Pro Lys Glu Val Leu Glu 450 455 460 Tyr Gln Val Asp Asn Leu Glu Lys Leu Phe Asn Asn Ile Leu Arg Asp 465 470 475 480 Leu Gln Lys Ser Gly Lys Lys Arg Lys Lys Arg Gly Leu Lys Asn Val 485 490 495 Asn Thr 135 705 DNA Aquifex aeolicus 135 atgcaagata ccgctacctg cagtatttgt caggggacgg gattcgtaaa gaccgaagac 60 aacaaggtaa ggctctgcga atgcaggttc aagaaaaggg atgtaaacag ggaactaaac 120 atcccaaaga ggtactggaa cgccaactta gacacttacc accccaagaa cgtatcccag 180 aacagggcac ttttgacgat aagggtcttc gtccacaact tcaatcccga ggaagggaaa 240 gggcttacct ttgtaggatc tcctggagtc ggcaaaactc accttgcggt tgcaacatta 300 aaagcgattt atgagaagaa gggaatcaga ggatacttct tcgatacgaa ggatctaata 360 ttcaggttaa aacacttaat ggacgaggga aaggatacaa agtttttaaa aactgtctta 420 aactcaccgg ttttggttct cgacgacctc ggttctgaga ggctcagtga ctggcagagg 480 gaactcatct cttacataat cacttacagg tataacaacc ttaagagcac gataataacc 540 acgaattact cactccagag ggaagaagag agtagcgtga ggataagtgc ggatcttgca 600 agcagactcg gagaaaacgt agtttcaaaa atttacgaga tgaacgagtt gctcgttata 660 aagggttccg acctcaggaa gtctaaaaag ctatcaaccc catct 705 136 235 PRT Aquifex aeolicus 136 Met Gln Asp Thr Ala Thr Cys Ser Ile Cys Gln Gly Thr Gly Phe Val 1 5 10 15 Lys Thr Glu Asp Asn Lys Val Arg Leu Cys Glu Cys Arg Phe Lys Lys 20 25 30 Arg Asp Val Asn Arg Glu Leu Asn Ile Pro Lys Arg Tyr Trp Asn Ala 35 40 45 Asn Leu Asp Thr Tyr His Pro Lys Asn Val Ser Gln Asn Arg Ala Leu 50 55 60 Leu Thr Ile Arg Val Phe Val His Asn Phe Asn Pro Glu Glu Gly Lys 65 70 75 80 Gly Leu Thr Phe Val Gly Ser Pro Gly Val Gly Lys Thr His Leu Ala 85 90 95 Val Ala Thr Leu Lys Ala Ile Tyr Glu Lys Lys Gly Ile Arg Gly Tyr 100 105 110 Phe Phe Asp Thr Lys Asp Leu Ile Phe Arg Leu Lys His Leu Met Asp 115 120 125 Glu Gly Lys Asp Thr Lys Phe Leu Lys Thr Val Leu Asn Ser Pro Val 130 135 140 Leu Val Leu Asp Asp Leu Gly Ser Glu Arg Leu Ser Asp Trp Gln Arg 145 150 155 160 Glu Leu Ile Ser Tyr Ile Ile Thr Tyr Arg Tyr Asn Asn Leu Lys Ser 165 170 175 Thr Ile Ile Thr Thr Asn Tyr Ser Leu Gln Arg Glu Glu Glu Ser Ser 180 185 190 Val Arg Ile Ser Ala Asp Leu Ala Ser Arg Leu Gly Glu Asn Val Val 195 200 205 Ser Lys Ile Tyr Glu Met Asn Glu Leu Leu Val Ile Lys Gly Ser Asp 210 215 220 Leu Arg Lys Ser Lys Lys Leu Ser Thr Pro Ser 225 230 235 137 4101 DNA Thermatoga maritima 137 atgaaaaaga ttgaaaattt gaagtggaaa aatgtctcgt ttaaaagcct ggaaatagat 60 cccgatgcag gtgtggttct cgtttccgtg gaaaaattct ccgaagagat agaagacctt 120 gtgcgtttac tggagaagaa gacgcggttt cgagtcatcg tgaacggtgt tcaaaaaagt 180 aacggggatc taaggggaaa gatactttcc cttctcaacg gtaatgtgcc ttacataaaa 240 gatgttgttt tcgaaggaaa caggctgatt ctgaaagtgc ttggagattt cgcgcgggac 300 aggatcgcct ccaaactcag aagcacgaaa aaacagctcg atgaactgct gcctcccgga 360 acagagatca tgctggaggt tgtggagcct ccggaagatc ttttgaaaaa ggaagtacca 420 caaccagaaa agagagaaga accaaagggt gaagaattga agatcgagga tgaaaaccac 480 atctttggac agaaacccag aaagatcgtc ttcaccccct caaaaatctt tgagtacaac 540 aaaaagacat cggtgaaggg caagatcttc aaaatagaga agatcgaggg gaaaagaacg 600 gtccttctga tttacctgac agacggagaa gattctctga tctgcaaagt cttcaacgac 660 gttgaaaagg tcgaagggaa agtatcggtg ggagacgtga tcgttgccac aggagacctc 720 cttctcgaaa acggggagcc caccctttac gtgaagggaa tcacaaaact tcccgaagcg 780 aaaaggatgg acaaatctcc ggttaagagg gtggagctcc acgcccatac caagttcagc 840 gatcaggacg caataacaga tgtgaacgaa tatgtgaaac gagccaagga atggggcttt 900 cccgcgatag ccctcacgga tcatgggaac gttcaggcca taccttactt ctacgacgcg 960 gcgaaagaag ctggaataaa gcccattttc ggtatcgaag cgtatctggt gagtgacgtg 1020 gagcccgtca taaggaatct ctccgacgat tcgacgtttg gagatgccac gttcgtcgtc 1080 ctcgacttcg agacgacggg tctcgacccg caggtggatg agatcatcga gataggagcg 1140 gtgaagatac agggtggcca gatagtggac gagtaccaca ctctcataaa gccttccagg 1200 gagatctcaa gaaaaagttc ggagatcacc ggaatcactc aagagatgct ggaaaacaag 1260 agaagcatcg aggaagttct gccggagttc ctcggttttc tggaagattc catcatcgta 1320 gcacacaacg ccaacttcga ctacagattt ctgaggctgt ggatcaaaaa agtgatggga 1380 ttggactggg aaagacccta catagatacg ctcgccctcg caaagtccct tctcaaactg 1440 agaagctact ctctggattc cgttgtggaa aagctcggat tgggtccctt ccggcaccac 1500 agggccctgg atgacgcgag ggtcaccgct caggttttcc tcaggttcgt tgagatgatg 1560 aagaagatcg gtatcacgaa gctttcagaa atggagaagt tgaaggatac gatagactac 1620 accgcgttga aacccttcca ctgcacgatc ctcgttcaga acaaaaaggg attgaaaaac 1680 ctatacaaac tggtttctga ttcctatata aagtacttct acggtgttcc gaggatcctc 1740 aaaagtgagc tcatcgagaa cagagaagga ctgctcgtgg gtagcgcgtg tatctccggt 1800 gagctcggac gtgccgccct cgaaggagcg agtgattcag aactcgaaga gatcgcgaag 1860 ttctacgact acatagaagt catgccgctc gacgttatag ccgaagatga agaagaccta 1920 gacagagaaa gactgaaaga agtgtaccga aaactctaca gaatagcgaa aaaattgaac 1980 aagttcgtcg tcatgaccgg tgatgttcat ttcctcgatc ccgaagatgc caggggcaga 2040 gctgcacttc tggcacctca gggaaacaga aacttcgaga atcagcccgc actctacctc 2100 agaacgaccg aagaaatgct cgagaaggcg atagagatat tcgaagatga agagatcgcg 2160 agggaagtcg tgatagagaa tcccaacaga atagccgata tgatcgagga agtgcagccg 2220 ctcgagaaaa aacttcaccc gccgatcata gagaacgccg atgaaatagt gagaaacctc 2280 accatgaagc gggcgtacga gatctacggt gatccgcttc ccgaaatcgt ccagaagcgt 2340 gtggaaaagg aactgaacgc catcataaat catggatacg ccgttctcta tctcatcgct 2400 caggagctcg ttcagaaatc tatgagcgat ggttacgtgg ttggatccag aggatccgtc 2460 gggtcttcac tcgtggccaa tctcctcgga ataacagagg tgaatcccct accaccacat 2520 tacaggtgtc cagagtgcaa atactttgaa gttgtcgaag acgacagata cggagcgggt 2580 tacgaccttc ccaacaagaa ctgtccaaga tgtggggctc ctctcagaaa agacggccac 2640 ggcataccgt ttgaaacgtt catggggttc gagggtgaca aggtccccga catagatctc 2700 aacttctcag gagagtatca ggaacgtgct catcgttttg tggaagaact cttcggtaaa 2760 gaccacgtct atagggcggg aaccataaac accatcgcgg aaagaagtgc ggtgggttac 2820 gtgagaagct acgaagagaa aaccggaaag aagctcagaa aggcggaaat ggaaagactc 2880 gtttccatga tcacgggagt gaagagaacg acgggtcagc acccaggggg gctcatgatc 2940 ataccgaaag acaaagaagt ctacgatttc actcccatac agtatccagc caacgataga 3000 aacgcaggtg tgttcaccac gcacttcgca tacgagacga tccatgatga cctggtgaag 3060 atagatgcgc tcggccacga tgatcccact ttcatcaaga tgctcaagga cctcaccgga 3120 atcgatccca tgacgattcc catggatgac cccgatacgc tcgccatatt cagttctgtg 3180 aagcctcttg gtgtggatcc cgttgagctg gaaagcgatg tgggaacgta cggaattccg 3240 gagttcggaa ccgagtttgt gaggggaatg ctcgttgaaa cgagaccaaa gagtttcgcc 3300 gagcttgtga gaatctcagg actgtcacac ggtacggacg tctggttgaa caacgcacgt 3360 gattggataa acctcggcta cgccaagctc tccgaggtta tctcgtgtag ggacgacatc 3420 atgaacttcc tcatacacaa aggaatggaa ccgtcacttg ccttcaagat catggaaaac 3480 gtcaggaagg gaaagggtat cacagaagag atggagagcg agatgagaag gctgaaggtt 3540 ccagaatggt tcatcgaatc ctgtaaaagg atcaaatatc tcttcccgaa agctcacgct 3600 gtggcttacg tgagtatggc cttcagaatt gcttacttca aggttcacta tcctcttcag 3660 ttttacgcgg cgtacttcac gataaaaggt gatcagttcg atccggttct cgtactcagg 3720 ggaaaagaag ccataaagag gcgcttgaga gaactcaaag cgatgcctgc caaagacgcc 3780 cagaagaaaa acgaagtgag tgttctggag gttgccctgg aaatgatact gagaggtttt 3840 tccttcctac cgcccgacat cttcaaatcc gacgcgaaga aatttctgat agaaggaaac 3900 tcgctgagaa ttccgttcaa caaacttcca ggactgggtg acagcgttgc cgagtcgata 3960 atcagagcca gggaagaaaa gccgttcact tcggtggaag atctcatgaa gaggaccaag 4020 gtcaacaaaa atcacataga gctgatgaaa agcctgggtg ttctcgggga ccttccagag 4080 acggaacagt tcacgctttt c 4101 138 1367 PRT Thermatoga maritima 138 Met Lys Lys Ile Glu Asn Leu Lys Trp Lys Asn Val Ser Phe Lys Ser 1 5 10 15 Leu Glu Ile Asp Pro Asp Ala Gly Val Val Leu Val Ser Val Glu Lys 20 25 30 Phe Ser Glu Glu Ile Glu Asp Leu Val Arg Leu Leu Glu Lys Lys Thr 35 40 45 Arg Phe Arg Val Ile Val Asn Gly Val Gln Lys Ser Asn Gly Asp Leu 50 55 60 Arg Gly Lys Ile Leu Ser Leu Leu Asn Gly Asn Val Pro Tyr Ile Lys 65 70 75 80 Asp Val Val Phe Glu Gly Asn Arg Leu Ile Leu Lys Val Leu Gly Asp 85 90 95 Phe Ala Arg Asp Arg Ile Ala Ser Lys Leu Arg Ser Thr Lys Lys Gln 100 105 110 Leu Asp Glu Leu Leu Pro Pro Gly Thr Glu Ile Met Leu Glu Val Val 115 120 125 Glu Pro Pro Glu Asp Leu Leu Lys Lys Glu Val Pro Gln Pro Glu Lys 130 135 140 Arg Glu Glu Pro Lys Gly Glu Glu Leu Lys Ile Glu Asp Glu Asn His 145 150 155 160 Ile Phe Gly Gln Lys Pro Arg Lys Ile Val Phe Thr Pro Ser Lys Ile 165 170 175 Phe Glu Tyr Asn Lys Lys Thr Ser Val Lys Gly Lys Ile Phe Lys Ile 180 185 190 Glu Lys Ile Glu Gly Lys Arg Thr Val Leu Leu Ile Tyr Leu Thr Asp 195 200 205 Gly Glu Asp Ser Leu Ile Cys Lys Val Phe Asn Asp Val Glu Lys Val 210 215 220 Glu Gly Lys Val Ser Val Gly Asp Val Ile Val Ala Thr Gly Asp Leu 225 230 235 240 Leu Leu Glu Asn Gly Glu Pro Thr Leu Tyr Val Lys Gly Ile Thr Lys 245 250 255 Leu Pro Glu Ala Lys Arg Met Asp Lys Ser Pro Val Lys Arg Val Glu 260 265 270 Leu His Ala His Thr Lys Phe Ser Asp Gln Asp Ala Ile Thr Asp Val 275 280 285 Asn Glu Tyr Val Lys Arg Ala Lys Glu Trp Gly Phe Pro Ala Ile Ala 290 295 300 Leu Thr Asp His Gly Asn Val Gln Ala Ile Pro Tyr Phe Tyr Asp Ala 305 310 315 320 Ala Lys Glu Ala Gly Ile Lys Pro Ile Phe Gly Ile Glu Ala Tyr Leu 325 330 335 Val Ser Asp Val Glu Pro Val Ile Arg Asn Leu Ser Asp Asp Ser Thr 340 345 350 Phe Gly Asp Ala Thr Phe Val Val Leu Asp Phe Glu Thr Thr Gly Leu 355 360 365 Asp Pro Gln Val Asp Glu Ile Ile Glu Ile Gly Ala Val Lys Ile Gln 370 375 380 Gly Gly Gln Ile Val Asp Glu Tyr His Thr Leu Ile Lys Pro Ser Arg 385 390 395 400 Glu Ile Ser Arg Lys Ser Ser Glu Ile Thr Gly Ile Thr Gln Glu Met 405 410 415 Leu Glu Asn Lys Arg Ser Ile Glu Glu Val Leu Pro Glu Phe Leu Gly 420 425 430 Phe Leu Glu Asp Ser Ile Ile Val Ala His Asn Ala Asn Phe Asp Tyr 435 440 445 Arg Phe Leu Arg Leu Trp Ile Lys Lys Val Met Gly Leu Asp Trp Glu 450 455 460 Arg Pro Tyr Ile Asp Thr Leu Ala Leu Ala Lys Ser Leu Leu Lys Leu 465 470 475 480 Arg Ser Tyr Ser Leu Asp Ser Val Val Glu Lys Leu Gly Leu Gly Pro 485 490 495 Phe Arg His His Arg Ala Leu Asp Asp Ala Arg Val Thr Ala Gln Val 500 505 510 Phe Leu Arg Phe Val Glu Met Met Lys Lys Ile Gly Ile Thr Lys Leu 515 520 525 Ser Glu Met Glu Lys Leu Lys Asp Thr Ile Asp Tyr Thr Ala Leu Lys 530 535 540 Pro Phe His Cys Thr Ile Leu Val Gln Asn Lys Lys Gly Leu Lys Asn 545 550 555 560 Leu Tyr Lys Leu Val Ser Asp Ser Tyr Ile Lys Tyr Phe Tyr Gly Val 565 570 575 Pro Arg Ile Leu Lys Ser Glu Leu Ile Glu Asn Arg Glu Gly Leu Leu 580 585 590 Val Gly Ser Ala Cys Ile Ser Gly Glu Leu Gly Arg Ala Ala Leu Glu 595 600 605 Gly Ala Ser Asp Ser Glu Leu Glu Glu Ile Ala Lys Phe Tyr Asp Tyr 610 615 620 Ile Glu Val Met Pro Leu Asp Val Ile Ala Glu Asp Glu Glu Asp Leu 625 630 635 640 Asp Arg Glu Arg Leu Lys Glu Val Tyr Arg Lys Leu Tyr Arg Ile Ala 645 650 655 Lys Lys Leu Asn Lys Phe Val Val Met Thr Gly Asp Val His Phe Leu 660 665 670 Asp Pro Glu Asp Ala Arg Gly Arg Ala Ala Leu Leu Ala Pro Gln Gly 675 680 685 Asn Arg Asn Phe Glu Asn Gln Pro Ala Leu Tyr Leu Arg Thr Thr Glu 690 695 700 Glu Met Leu Glu Lys Ala Ile Glu Ile Phe Glu Asp Glu Glu Ile Ala 705 710 715 720 Arg Glu Val Val Ile Glu Asn Pro Asn Arg Ile Ala Asp Met Ile Glu 725 730 735 Glu Val Gln Pro Leu Glu Lys Lys Leu His Pro Pro Ile Ile Glu Asn 740 745 750 Ala Asp Glu Ile Val Arg Asn Leu Thr Met Lys Arg Ala Tyr Glu Ile 755 760 765 Tyr Gly Asp Pro Leu Pro Glu Ile Val Gln Lys Arg Val Glu Lys Glu 770 775 780 Leu Asn Ala Ile Ile Asn His Gly Tyr Ala Val Leu Tyr Leu Ile Ala 785 790 795 800 Gln Glu Leu Val Gln Lys Ser Met Ser Asp Gly Tyr Val Val Gly Ser 805 810 815 Arg Gly Ser Val Gly Ser Ser Leu Val Ala Asn Leu Leu Gly Ile Thr 820 825 830 Glu Val Asn Pro Leu Pro Pro His Tyr Arg Cys Pro Glu Cys Lys Tyr 835 840 845 Phe Glu Val Val Glu Asp Asp Arg Tyr Gly Ala Gly Tyr Asp Leu Pro 850 855 860 Asn Lys Asn Cys Pro Arg Cys Gly Ala Pro Leu Arg Lys Asp Gly His 865 870 875 880 Gly Ile Pro Phe Glu Thr Phe Met Gly Phe Glu Gly Asp Lys Val Pro 885 890 895 Asp Ile Asp Leu Asn Phe Ser Gly Glu Tyr Gln Glu Arg Ala His Arg 900 905 910 Phe Val Glu Glu Leu Phe Gly Lys Asp His Val Tyr Arg Ala Gly Thr 915 920 925 Ile Asn Thr Ile Ala Glu Arg Ser Ala Val Gly Tyr Val Arg Ser Tyr 930 935 940 Glu Glu Lys Thr Gly Lys Lys Leu Arg Lys Ala Glu Met Glu Arg Leu 945 950 955 960 Val Ser Met Ile Thr Gly Val Lys Arg Thr Thr Gly Gln His Pro Gly 965 970 975 Gly Leu Met Ile Ile Pro Lys Asp Lys Glu Val Tyr Asp Phe Thr Pro 980 985 990 Ile Gln Tyr Pro Ala Asn Asp Arg Asn Ala Gly Val Phe Thr Thr His 995 1000 1005 Phe Ala Tyr Glu Thr Ile His Asp Asp Leu Val Lys Ile Asp Ala Leu 1010 1015 1020 Gly His Asp Asp Pro Thr Phe Ile Lys Met Leu Lys Asp Leu Thr Gly 1025 1030 1035 1040 Ile Asp Pro Met Thr Ile Pro Met Asp Asp Pro Asp Thr Leu Ala Ile 1045 1050 1055 Phe Ser Ser Val Lys Pro Leu Gly Val Asp Pro Val Glu Leu Glu Ser 1060 1065 1070 Asp Val Gly Thr Tyr Gly Ile Pro Glu Phe Gly Thr Glu Phe Val Arg 1075 1080 1085 Gly Met Leu Val Glu Thr Arg Pro Lys Ser Phe Ala Glu Leu Val Arg 1090 1095 1100 Ile Ser Gly Leu Ser His Gly Thr Asp Val Trp Leu Asn Asn Ala Arg 1105 1110 1115 1120 Asp Trp Ile Asn Leu Gly Tyr Ala Lys Leu Ser Glu Val Ile Ser Cys 1125 1130 1135 Arg Asp Asp Ile Met Asn Phe Leu Ile His Lys Gly Met Glu Pro Ser 1140 1145 1150 Leu Ala Phe Lys Ile Met Glu Asn Val Arg Lys Gly Lys Gly Ile Thr 1155 1160 1165 Glu Glu Met Glu Ser Glu Met Arg Arg Leu Lys Val Pro Glu Trp Phe 1170 1175 1180 Ile Glu Ser Cys Lys Arg Ile Lys Tyr Leu Phe Pro Lys Ala His Ala 1185 1190 1195 1200 Val Ala Tyr Val Ser Met Ala Phe Arg Ile Ala Tyr Phe Lys Val His 1205 1210 1215 Tyr Pro Leu Gln Phe Tyr Ala Ala Tyr Phe Thr Ile Lys Gly Asp Gln 1220 1225 1230 Phe Asp Pro Val Leu Val Leu Arg Gly Lys Glu Ala Ile Lys Arg Arg 1235 1240 1245 Leu Arg Glu Leu Lys Ala Met Pro Ala Lys Asp Ala Gln Lys Lys Asn 1250 1255 1260 Glu Val Ser Val Leu Glu Val Ala Leu Glu Met Ile Leu Arg Gly Phe 1265 1270 1275 1280 Ser Phe Leu Pro Pro Asp Ile Phe Lys Ser Asp Ala Lys Lys Phe Leu 1285 1290 1295 Ile Glu Gly Asn Ser Leu Arg Ile Pro Phe Asn Lys Leu Pro Gly Leu 1300 1305 1310 Gly Asp Ser Val Ala Glu Ser Ile Ile Arg Ala Arg Glu Glu Lys Pro 1315 1320 1325 Phe Thr Ser Val Glu Asp Leu Met Lys Arg Thr Lys Val Asn Lys Asn 1330 1335 1340 His Ile Glu Leu Met Lys Ser Leu Gly Val Leu Gly Asp Leu Pro Glu 1345 1350 1355 1360 Thr Glu Gln Phe Thr Leu Phe 1365 139 567 DNA Thermatoga maritima 139 gtgctcgcca tgatatggaa cgacaccgtt ttttgcgtcg tagacacaga aaccacggga 60 accgatccct ttgccggaga ccggatagtt gaaatagccg ctgttcctgt cttcaagggg 120 aagatctaca gaaacaaagc gtttcactct ctcgtgaatc ccagaataag aatccctgcg 180 ctgattcaga aagttcacgg tatcagcaac atggacatcg tggaagcgcc agacatggac 240 acagtttacg atcttttcag ggattacgtg aagggaacgg tgctcgtgtt tcacaacgcc 300 aacttcgacc tcacttttct ggatatgatg gcaaaggaaa cgggaaactt tccaataacg 360 aatccctaca tcgacacact cgatctttca gaagagatct ttggaaggcc tcattctctc 420 aaatggctct ccgaaagact tggaataaaa accacgatac ggcaccgtgc tcttccagat 480 gccctggtga ccgcaagagt ttttgtgaag cttgttgaat ttcttggtga aaacagggtc 540 aacgaattca tacgtggaaa acggggg 567 140 189 PRT Thermatoga maritima 140 Met Leu Ala Met Ile Trp Asn Asp Thr Val Phe Cys Val Val Asp Thr 1 5 10 15 Glu Thr Thr Gly Thr Asp Pro Phe Ala Gly Asp Arg Ile Val Glu Ile 20 25 30 Ala Ala Val Pro Val Phe Lys Gly Lys Ile Tyr Arg Asn Lys Ala Phe 35 40 45 His Ser Leu Val Asn Pro Arg Ile Arg Ile Pro Ala Leu Ile Gln Lys 50 55 60 Val His Gly Ile Ser Asn Met Asp Ile Val Glu Ala Pro Asp Met Asp 65 70 75 80 Thr Val Tyr Asp Leu Phe Arg Asp Tyr Val Lys Gly Thr Val Leu Val 85 90 95 Phe His Asn Ala Asn Phe Asp Leu Thr Phe Leu Asp Met Met Ala Lys 100 105 110 Glu Thr Gly Asn Phe Pro Ile Thr Asn Pro Tyr Ile Asp Thr Leu Asp 115 120 125 Leu Ser Glu Glu Ile Phe Gly Arg Pro His Ser Leu Lys Trp Leu Ser 130 135 140 Glu Arg Leu Gly Ile Lys Thr Thr Ile Arg His Arg Ala Leu Pro Asp 145 150 155 160 Ala Leu Val Thr Ala Arg Val Phe Val Lys Leu Val Glu Phe Leu Gly 165 170 175 Glu Asn Arg Val Asn Glu Phe Ile Arg Gly Lys Arg Gly 180 185 141 1434 DNA Thermatoga maritima 141 gtggaagttc tttacaggaa gtacaggcca aagacttttt ctgaggttgt caatcaggat 60 catgtgaaga aggcaataat cggtgctatt cagaagaaca gcgtggccca cggatacata 120 ttcgccggtc cgaggggaac ggggaagact actcttgcca gaattctcgc aaaatccctg 180 aactgtgaga acagaaaggg agttgaaccc tgcaattcct gcagagcctg cagagagata 240 gacgagggaa ccttcatgga cgtgatagag ctcgacgcgg cctccaacag aggaatagac 300 gagatcagaa gaatcagaga cgccgttgga tacaggccga tggaaggtaa atacaaagtc 360 tacataatag acgaagttca catgctcacg aaagaagcct tcaacgcgct cctcaaaaca 420 ctcgaagaac ctccttccca cgtcgtgttc gtgctggcaa cgacaaacct tgagaaggtt 480 cctcccacga ttatctcgag atgtcaggtt ttcgagttca gaaacattcc cgacgagctc 540 atcgaaaaga ggctccagga agttgcggag gctgaaggaa tagagataga cagggaagct 600 ctgagcttca tcgcaaaaag agcctctgga ggcttgagag acgcgctcac catgctcgag 660 caggtgtgga agttctcgga aggaaagata gatctcgaga cggtacacag ggcgctcggg 720 ttgataccga tacaggttgt tcgcgattac gtgaacgcta tcttttctgg tgatgtgaaa 780 agggtcttca ccgttctcga cgacgtctat tacagcggga aggactacga ggtgctcatt 840 caggaagcag tcgaggatct ggtcgaagac ctggaaaggg agagaggggt ttaccaggtt 900 tcagcgaacg atatagttca ggtttcgaga caacttctga atcttctgag agagataaag 960 ttcgccgaag aaaaacgact cgtctgtaaa gtgggttcgg cttacatagc gacgaggttc 1020 tccaccacaa acgttcagga aaacgatgtc agagaaaaaa acgataattc aaatgtacag 1080 cagaaagaag agaagaaaga aacggtgaag gcaaaagaag aaaaacagga agacagcgag 1140 ttcgagaaac gcttcaaaga actcatggaa gaactgaaag aaaagggcga tctctctatc 1200 tttgtcgctc tcagcctctc agaggtgcag tttgacggag aaaaggtgat tatttctttt 1260 gattcatcga aagctatgca ttacgagttg atgaagaaaa aactgcctga gctggaaaac 1320 attttttcta gaaaactcgg gaaaaaagta gaagttgaac ttcgactgat gggaaaagaa 1380 gaaacaatcg agaaggtttc tcagaagatc ctgagattgt ttgaacagga ggga 1434 142 478 PRT Thermatoga maritima 142 Met Glu Val Leu Tyr Arg Lys Tyr Arg Pro Lys Thr Phe Ser Glu Val 1 5 10 15 Val Asn Gln Asp His Val Lys Lys Ala Ile Ile Gly Ala Ile Gln Lys 20 25 30 Asn Ser Val Ala His Gly Tyr Ile Phe Ala Gly Pro Arg Gly Thr Gly 35 40 45 Lys Thr Thr Leu Ala Arg Ile Leu Ala Lys Ser Leu Asn Cys Glu Asn 50 55 60 Arg Lys Gly Val Glu Pro Cys Asn Ser Cys Arg Ala Cys Arg Glu Ile 65 70 75 80 Asp Glu Gly Thr Phe Met Asp Val Ile Glu Leu Asp Ala Ala Ser Asn 85 90 95 Arg Gly Ile Asp Glu Ile Arg Arg Ile Arg Asp Ala Val Gly Tyr Arg 100 105 110 Pro Met Glu Gly Lys Tyr Lys Val Tyr Ile Ile Asp Glu Val His Met 115 120 125 Leu Thr Lys Glu Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu Glu Pro 130 135 140 Pro Ser His Val Val Phe Val Leu Ala Thr Thr Asn Leu Glu Lys Val 145 150 155 160 Pro Pro Thr Ile Ile Ser Arg Cys Gln Val Phe Glu Phe Arg Asn Ile 165 170 175 Pro Asp Glu Leu Ile Glu Lys Arg Leu Gln Glu Val Ala Glu Ala Glu 180 185 190 Gly Ile Glu Ile Asp Arg Glu Ala Leu Ser Phe Ile Ala Lys Arg Ala 195 200 205 Ser Gly Gly Leu Arg Asp Ala Leu Thr Met Leu Glu Gln Val Trp Lys 210 215 220 Phe Ser Glu Gly Lys Ile Asp Leu Glu Thr Val His Arg Ala Leu Gly 225 230 235 240 Leu Ile Pro Ile Gln Val Val Arg Asp Tyr Val Asn Ala Ile Phe Ser 245 250 255 Gly Asp Val Lys Arg Val Phe Thr Val Leu Asp Asp Val Tyr Tyr Ser 260 265 270 Gly Lys Asp Tyr Glu Val Leu Ile Gln Glu Ala Val Glu Asp Leu Val 275 280 285 Glu Asp Leu Glu Arg Glu Arg Gly Val Tyr Gln Val Ser Ala Asn Asp 290 295 300 Ile Val Gln Val Ser Arg Gln Leu Leu Asn Leu Leu Arg Glu Ile Lys 305 310 315 320 Phe Ala Glu Glu Lys Arg Leu Val Cys Lys Val Gly Ser Ala Tyr Ile 325 330 335 Ala Thr Arg Phe Ser Thr Thr Asn Val Gln Glu Asn Asp Val Arg Glu 340 345 350 Lys Asn Asp Asn Ser Asn Val Gln Gln Lys Glu Glu Lys Lys Glu Thr 355 360 365 Val Lys Ala Lys Glu Glu Lys Gln Glu Asp Ser Glu Phe Glu Lys Arg 370 375 380 Phe Lys Glu Leu Met Glu Glu Leu Lys Glu Lys Gly Asp Leu Ser Ile 385 390 395 400 Phe Val Ala Leu Ser Leu Ser Glu Val Gln Phe Asp Gly Glu Lys Val 405 410 415 Ile Ile Ser Phe Asp Ser Ser Lys Ala Met His Tyr Glu Leu Met Lys 420 425 430 Lys Lys Leu Pro Glu Leu Glu Asn Ile Phe Ser Arg Lys Leu Gly Lys 435 440 445 Lys Val Glu Val Glu Leu Arg Leu Met Gly Lys Glu Glu Thr Ile Glu 450 455 460 Lys Val Ser Gln Lys Ile Leu Arg Leu Phe Glu Gln Glu Gly 465 470 475 143 1098 DNA Thermatoga maritima 143 atgaaagtaa ccgtcacgac tcttgaattg aaagacaaaa taaccatcgc ctcaaaagcg 60 ctcgcaaaga aatccgtgaa acccattctt gctggatttc ttttcgaagt gaaagatgga 120 aatttctaca tctgcgcgac cgatctcgag accggagtca aagcaaccgt gaatgccgct 180 gaaatctccg gtgaggcacg ttttgtggta ccaggagatg tcattcagaa gatggtcaag 240 gttctcccag atgagataac ggaactttct ttagaggggg atgctcttgt tataagttct 300 ggaagcaccg ttttcaggat caccaccatg cccgcggacg aatttccaga gataacgcct 360 gccgagtctg gaataacctt cgaagttgac acttcgctcc tcgaggaaat ggttgaaaag 420 gtcatcttcg ccgctgccaa agacgagttc atgcgaaatc tgaatggagt tttctgggaa 480 ctccacaaga atcttctcag gctggttgca agtgatggtt tcagacttgc acttgctgaa 540 gagcagatag aaaacgagga agaggcgagt ttcttgctct ctttgaagag catgaaagaa 600 gttcaaaacg tgctggacaa cacaacggag ccgactataa cggtgaggta cgatggaaga 660 agggtttctc tgtcgacaaa tgatgtagaa acggtgatga gagtggtcga cgctgaattt 720 cccgattaca aaagggtgat ccccgaaact ttcaaaacga aagtggtggt ttccagaaaa 780 gaactcaggg aatctttgaa gagggtgatg gtgattgcca gcaagggaag cgagtccgtg 840 aagttcgaaa tagaagaaaa cgttatgaga cttgtgagca agagcccgga ttatggagaa 900 gtggtcgatg aagttgaagt tcaaaaagaa ggggaagatc tcgtgatcgc tttcaacccg 960 aagttcatcg aggacgtttt gaagcacatt gagactgaag aaatcgaaat gaacttcgtt 1020 gattctacca gtccatgtca gataaatcca ctcgatattt ctggatacct ttacatagtg 1080 atgcccatca gactggca 1098 144 366 PRT Thermatoga maritima 144 Met Lys Val Thr Val Thr Thr Leu Glu Leu Lys Asp Lys Ile Thr Ile 1 5 10 15 Ala Ser Lys Ala Leu Ala Lys Lys Ser Val Lys Pro Ile Leu Ala Gly 20 25 30 Phe Leu Phe Glu Val Lys Asp Gly Asn Phe Tyr Ile Cys Ala Thr Asp 35 40 45 Leu Glu Thr Gly Val Lys Ala Thr Val Asn Ala Ala Glu Ile Ser Gly 50 55 60 Glu Ala Arg Phe Val Val Pro Gly Asp Val Ile Gln Lys Met Val Lys 65 70 75 80 Val Leu Pro Asp Glu Ile Thr Glu Leu Ser Leu Glu Gly Asp Ala Leu 85 90 95 Val Ile Ser Ser Gly Ser Thr Val Phe Arg Ile Thr Thr Met Pro Ala 100 105 110 Asp Glu Phe Pro Glu Ile Thr Pro Ala Glu Ser Gly Ile Thr Phe Glu 115 120 125 Val Asp Thr Ser Leu Leu Glu Glu Met Val Glu Lys Val Ile Phe Ala 130 135 140 Ala Ala Lys Asp Glu Phe Met Arg Asn Leu Asn Gly Val Phe Trp Glu 145 150 155 160 Leu His Lys Asn Leu Leu Arg Leu Val Ala Ser Asp Gly Phe Arg Leu 165 170 175 Ala Leu Ala Glu Glu Gln Ile Glu Asn Glu Glu Glu Ala Ser Phe Leu 180 185 190 Leu Ser Leu Lys Ser Met Lys Glu Val Gln Asn Val Leu Asp Asn Thr 195 200 205 Thr Glu Pro Thr Ile Thr Val Arg Tyr Asp Gly Arg Arg Val Ser Leu 210 215 220 Ser Thr Asn Asp Val Glu Thr Val Met Arg Val Val Asp Ala Glu Phe 225 230 235 240 Pro Asp Tyr Lys Arg Val Ile Pro Glu Thr Phe Lys Thr Lys Val Val 245 250 255 Val Ser Arg Lys Glu Leu Arg Glu Ser Leu Lys Arg Val Met Val Ile 260 265 270 Ala Ser Lys Gly Ser Glu Ser Val Lys Phe Glu Ile Glu Glu Asn Val 275 280 285 Met Arg Leu Val Ser Lys Ser Pro Asp Tyr Gly Glu Val Val Asp Glu 290 295 300 Val Glu Val Gln Lys Glu Gly Glu Asp Leu Val Ile Ala Phe Asn Pro 305 310 315 320 Lys Phe Ile Glu Asp Val Leu Lys His Ile Glu Thr Glu Glu Ile Glu 325 330 335 Met Asn Phe Val Asp Ser Thr Ser Pro Cys Gln Ile Asn Pro Leu Asp 340 345 350 Ile Ser Gly Tyr Leu Tyr Ile Val Met Pro Ile Arg Leu Ala 355 360 365 145 972 DNA Thermatoga maritima 145 atgccagtca cgtttctcac aggtactgca gaaactcaga aggaagaatt gataaagaaa 60 ctcctgaagg atggtaacgt ggagtacata aggatccatc cggaggatcc cgacaagatc 120 gatttcataa ggtctttact caggacaaag acgatctttt ccaacaagac gatcattgac 180 atcgtcaatt tcgatgagtg gaaagcacag gagcagaagc gtctcgttga acttttgaaa 240 aacgtaccgg aagacgttca tatcttcatc cgttctcaaa aaacaggtgg aaagggagta 300 gcgctggagc ttccgaagcc atgggaaacg gacaagtggc ttgagtggat agaaaagcgc 360 ttcagggaga atggtttgct catcgataaa gatgcccttc agctgttttt ctccaaggtt 420 ggaacgaacg acctgatcat agaaagggag attgaaaaac tgaaagctta ttccgaggac 480 agaaagataa cggtagaaga cgtggaagag gtcgttttta cctatcagac tccgggatac 540 gatgattttt gctttgctgt ttccgaagga aaaaggaagc tcgctcactc tcttctgtcg 600 cagctgtgga aaaccacaga gtccgtggtg attgccactg tccttgcgaa tcacttcttg 660 gatctcttca aaatcctcgt tcttgtgaca aagaaaagat actacacctg gcctgatgtg 720 tccagggtgt ccaaagagct gggaattccc gttcctcgtg tggctcgttt cctcggtttc 780 tcctttaaga cctggaaatt caaggtgatg aaccacctcc tctactacga tgtgaagaag 840 gttagaaaga tactgaggga tctctacgat ctggacagag ccgtgaaaag cgaagaagat 900 ccaaaaccgt tcttccacga gttcatagaa gaggtggcac tggatgtata ttctcttcag 960 agagatgaag aa 972 146 324 PRT Thermatoga maritima 146 Met Pro Val Thr Phe Leu Thr Gly Thr Ala Glu Thr Gln Lys Glu Glu 1 5 10 15 Leu Ile Lys Lys Leu Leu Lys Asp Gly Asn Val Glu Tyr Ile Arg Ile 20 25 30 His Pro Glu Asp Pro Asp Lys Ile Asp Phe Ile Arg Ser Leu Leu Arg 35 40 45 Thr Lys Thr Ile Phe Ser Asn Lys Thr Ile Ile Asp Ile Val Asn Phe 50 55 60 Asp Glu Trp Lys Ala Gln Glu Gln Lys Arg Leu Val Glu Leu Leu Lys 65 70 75 80 Asn Val Pro Glu Asp Val His Ile Phe Ile Arg Ser Gln Lys Thr Gly 85 90 95 Gly Lys Gly Val Ala Leu Glu Leu Pro Lys Pro Trp Glu Thr Asp Lys 100 105 110 Trp Leu Glu Trp Ile Glu Lys Arg Phe Arg Glu Asn Gly Leu Leu Ile 115 120 125 Asp Lys Asp Ala Leu Gln Leu Phe Phe Ser Lys Val Gly Thr Asn Asp 130 135 140 Leu Ile Ile Glu Arg Glu Ile Glu Lys Leu Lys Ala Tyr Ser Glu Asp 145 150 155 160 Arg Lys Ile Thr Val Glu Asp Val Glu Glu Val Val Phe Thr Tyr Gln 165 170 175 Thr Pro Gly Tyr Asp Asp Phe Cys Phe Ala Val Ser Glu Gly Lys Arg 180 185 190 Lys Leu Ala His Ser Leu Leu Ser Gln Leu Trp Lys Thr Thr Glu Ser 195 200 205 Val Val Ile Ala Thr Val Leu Ala Asn His Phe Leu Asp Leu Phe Lys 210 215 220 Ile Leu Val Leu Val Thr Lys Lys Arg Tyr Tyr Thr Trp Pro Asp Val 225 230 235 240 Ser Arg Val Ser Lys Glu Leu Gly Ile Pro Val Pro Arg Val Ala Arg 245 250 255 Phe Leu Gly Phe Ser Phe Lys Thr Trp Lys Phe Lys Val Met Asn His 260 265 270 Leu Leu Tyr Tyr Asp Val Lys Lys Val Arg Lys Ile Leu Arg Asp Leu 275 280 285 Tyr Asp Leu Asp Arg Ala Val Lys Ser Glu Glu Asp Pro Lys Pro Phe 290 295 300 Phe His Glu Phe Ile Glu Glu Val Ala Leu Asp Val Tyr Ser Leu Gln 305 310 315 320 Arg Asp Glu Glu 147 936 DNA Thermatoga maritima 147 atgaacgatt tgatcagaaa gtacgctaaa gatcaactgg aaactttgaa aaggatcata 60 gaaaagtctg aaggaatatc catcctcata aatggagaag atctctcgta tccgagagaa 120 gtatcccttg aacttcccga gtacgtggag aaatttcccc cgaaggcctc ggatgttctg 180 gagatagatc ccgaggggga gaacataggc atagacgaca tcagaacgat aaaggacttc 240 ctgaactaca gccccgagct ctacacgaga aagtacgtga tagtccacga ctgtgaaaga 300 atgacccagc aggcggcgaa cgcgtttctg aaggcccttg aagaaccacc agaatacgct 360 gtgatcgttc tgaacactcg ccgctggcat tatctactgc cgacgataaa gagccgagtg 420 ttcagagtgg ttgtgaacgt tccaaaggag ttcagagatc tcgtgaaaga gaaaatagga 480 gatctctggg aggaacttcc acttcttgag agagacttca aaacggctct cgaagcctac 540 aaacttggtg cggaaaaact ttctggattg atggaaagtc tcaaagtttt ggagacggaa 600 aaactcttga aaaaggtcct ttcaaaaggc ctcgaaggtt atctcgcatg tagggagctc 660 ctggagagat tttcaaaggt ggaatcgaag gaattctttg cgctttttga tcaggtgact 720 aacacgataa caggaaaaga cgcgtttctt ttgatccaga gactgacaag aatcattctc 780 cacgaaaaca catgggaaag cgttgaagat caaaaaagcg tgtctttcct cgattcaatt 840 ctcagggtga agatagcgaa tctgaacaac aaactcactc tgatgaacat cctcgcgata 900 cacagagaga gaaagagagg tgtcaacgct tggagc 936 148 311 PRT Thermatoga maritima 148 Met Asn Asp Leu Ile Arg Lys Tyr Ala Lys Asp Gln Leu Glu Thr Leu 1 5 10 15 Lys Arg Ile Ile Glu Lys Ser Glu Gly Ile Ser Ile Leu Ile Asn Gly 20 25 30 Glu Asp Leu Ser Tyr Pro Arg Glu Val Ser Leu Glu Leu Pro Glu Tyr 35 40 45 Val Glu Lys Phe Pro Pro Lys Ala Ser Asp Val Leu Glu Ile Asp Pro 50 55 60 Glu Gly Glu Asn Ile Gly Ile Asp Asp Ile Arg Thr Ile Lys Asp Phe 65 70 75 80 Leu Asn Tyr Ser Pro Glu Leu Tyr Thr Arg Lys Tyr Val Ile Val His 85 90 95 Asp Cys Glu Arg Met Thr Gln Gln Ala Ala Asn Ala Phe Leu Lys Ala 100 105 110 Leu Glu Glu Pro Pro Glu Tyr Ala Val Ile Val Leu Asn Thr Arg Arg 115 120 125 Trp His Tyr Leu Leu Pro Thr Ile Lys Ser Arg Val Phe Arg Val Val 130 135 140 Val Asn Val Pro Lys Glu Phe Arg Asp Leu Val Lys Glu Lys Ile Gly 145 150 155 160 Asp Leu Trp Glu Glu Leu Pro Leu Leu Glu Arg Asp Phe Lys Thr Ala 165 170 175 Leu Glu Ala Tyr Lys Leu Gly Ala Glu Lys Leu Ser Gly Leu Met Glu 180 185 190 Ser Leu Lys Val Leu Glu Thr Glu Lys Leu Leu Lys Lys Val Leu Ser 195 200 205 Lys Gly Leu Glu Gly Tyr Leu Ala Cys Arg Glu Leu Leu Glu Arg Phe 210 215 220 Ser Lys Val Glu Ser Lys Glu Phe Phe Ala Leu Phe Asp Gln Val Thr 225 230 235 240 Asn Thr Ile Thr Gly Lys Asp Ala Phe Leu Leu Ile Gln Arg Leu Thr 245 250 255 Arg Ile Ile Leu His Glu Asn Thr Trp Glu Ser Val Glu Asp Lys Ser 260 265 270 Val Ser Phe Leu Asp Ser Ile Leu Arg Val Lys Ile Ala Asn Leu Asn 275 280 285 Asn Lys Leu Thr Leu Met Asn Ile Leu Ala Ile His Arg Glu Arg Lys 290 295 300 Arg Gly Val Asn Ala Trp Ser 305 310 149 423 DNA Thermatoga maritima 149 atgtctttct tcaacaagat catactcata ggaagactcg tgagagatcc cgaagagaga 60 tacacgctca gcggaactcc agtcaccacc ttcaccatag cggtggacag ggttcccaga 120 aagaacgcgc cggacgacgc tcaaacgact gatttcttca ggatcgtcac ctttggaaga 180 ctggcagagt tcgctagaac ctatctcacc aaaggaaggc tcgttctcgt cgaaggtgaa 240 atgagaatga gaagatggga aacacccact ggagaaaaga gggtatctcc ggaggttgtc 300 gcaaacgttg ttagattcat ggacagaaaa cctgctgaaa cagttagcga gactgaagag 360 gagctggaaa taccggaaga agacttttcc agcgatacct tcagtgaaga tgaaccacca 420 ttt 423 150 141 PRT Thermatoga maritima 150 Met Ser Phe Phe Asn Lys Ile Ile Leu Ile Gly Arg Leu Val Arg Asp 1 5 10 15 Pro Glu Glu Arg Tyr Thr Leu Ser Gly Thr Pro Val Thr Thr Phe Thr 20 25 30 Ile Ala Val Asp Arg Val Pro Arg Lys Asn Ala Pro Asp Asp Ala Gln 35 40 45 Thr Thr Asp Phe Phe Arg Ile Val Thr Phe Gly Arg Leu Ala Glu Phe 50 55 60 Ala Arg Thr Tyr Leu Thr Lys Gly Arg Leu Val Leu Val Glu Gly Glu 65 70 75 80 Met Arg Met Arg Arg Trp Glu Thr Pro Thr Gly Glu Lys Arg Val Ser 85 90 95 Pro Glu Val Val Ala Asn Val Val Arg Phe Met Asp Arg Lys Pro Ala 100 105 110 Glu Thr Val Ser Glu Thr Glu Glu Glu Leu Glu Ile Pro Glu Glu Asp 115 120 125 Phe Ser Ser Asp Thr Phe Ser Glu Asp Glu Pro Pro Phe 130 135 140 151 1353 DNA Thermatoga maritima 151 atgcgtgttc ccccgcacaa cttagaggcc gaagttgctg tgctcggaag catattgata 60 gatccgtcgg taataaacga cgttcttgaa attttgagcc acgaagattt ctatctgaaa 120 aaacaccaac acatcttcag agcgatggaa gagctttacg acgaaggaaa accggtggac 180 gtggtttccg tctgtgacaa gcttcaaagc atgggaaaac tcgaggaagt aggtggagat 240 ctggaagtgg cccagctcgc tgaggctgtg cccagttctg cacacgcact tcactacgcg 300 gagatcgtca aggaaaaatc cattctgagg aaactcattg agatctccag aaaaatctca 360 gaaagtgcct acatggaaga agatgtggag atcctgctcg acaacgcaga aaagatgatc 420 ttcgagatct cagagatgaa aacgacaaaa tcctacgatc atctgagagg catcatgcac 480 cgggtgtttg aaaacctgga gaacttcagg gaaagagcca accttataga acccggtgtg 540 ctcataacgg gactaccaac gggattcaaa agtctggaca aacagaccac agggttccac 600 agctccgatc tggtgataat agcagcgaga ccctccatgg gaaaaacctc cttcgcactc 660 tcaatagcga ggaacatggc tgtcaatttc gaaatccccg tcggaatatt cagtctcgag 720 atgtccaagg aacagctcgc tcaaagacta ctcagcatgg agtccggtgt ggatctttac 780 agcatcagaa caggatacct ggatcaggag aagtgggaaa gactcacaat agcggcttct 840 aaactctaca aagcacccat agttgtggac gatgagtcac tcctcgatcc gcgatcgttg 900 agggcaaaag cgagaaggat gaaaaaagaa tacgatgtaa aagccatttt tgtcgactat 960 ctccagctca tgcacctgaa aggaagaaaa gaaagcagac agcaggagat atccgagatc 1020 tcgagatctc tgaagctcct tgcgagggaa ctcgacatag tggtgatagc gctttcacag 1080 ctttcgaggg ccgtagaaca gagagaagac aaaagaccga ggctgagtga cctcagggaa 1140 tccggtgcga tagaacagga cgcagacaca gtcatcttca tctacaggga ggaatattac 1200 aggagcaaaa aatccaaaga ggaaagcaag cttcacgaac ctcacgaagc tgaaatcata 1260 ataggtaaac agagaaacgg tcccgttgga acgatcactc tgatcttcga ccccagaacg 1320 gttacgttcc atgaagtcga tgtggtgcat tca 1353 152 451 PRT Thermatoga maritima 152 Met Arg Val Pro Pro His Asn Leu Glu Ala Glu Val Ala Val Leu Gly 1 5 10 15 Ser Ile Leu Ile Asp Pro Ser Val Ile Asn Asp Val Leu Glu Ile Leu 20 25 30 Ser His Glu Asp Phe Tyr Leu Lys Lys His Gln His Ile Phe Arg Ala 35 40 45 Met Glu Glu Leu Tyr Asp Glu Gly Lys Pro Val Asp Val Val Ser Val 50 55 60 Cys Asp Lys Leu Gln Ser Met Gly Lys Leu Glu Glu Val Gly Gly Asp 65 70 75 80 Leu Glu Val Ala Gln Leu Ala Glu Ala Val Pro Ser Ser Ala His Ala 85 90 95 Leu His Tyr Ala Glu Ile Val Lys Glu Lys Ser Ile Leu Arg Lys Leu 100 105 110 Ile Glu Ile Ser Arg Lys Ile Ser Glu Ser Ala Tyr Met Glu Glu Asp 115 120 125 Val Glu Ile Leu Leu Asp Asn Ala Glu Lys Met Ile Phe Glu Ile Ser 130 135 140 Glu Met Lys Thr Thr Lys Ser Tyr Asp His Leu Arg Gly Ile Met His 145 150 155 160 Arg Val Phe Glu Asn Leu Glu Asn Phe Arg Glu Arg Ala Asn Leu Ile 165 170 175 Glu Pro Gly Val Leu Ile Thr Gly Leu Pro Thr Gly Phe Lys Ser Leu 180 185 190 Asp Lys Gln Thr Thr Gly Phe His Ser Ser Asp Leu Val Ile Ile Ala 195 200 205 Ala Arg Pro Ser Met Gly Lys Thr Ser Phe Ala Leu Ser Ile Ala Arg 210 215 220 Asn Met Ala Val Asn Phe Glu Ile Pro Val Gly Ile Phe Ser Leu Glu 225 230 235 240 Met Ser Lys Glu Gln Leu Ala Gln Arg Leu Leu Ser Met Glu Ser Gly 245 250 255 Val Asp Leu Tyr Ser Ile Arg Thr Gly Tyr Leu Asp Gln Glu Lys Trp 260 265 270 Glu Arg Leu Thr Ile Ala Ala Ser Lys Leu Tyr Lys Ala Pro Ile Val 275 280 285 Val Asp Asp Glu Ser Leu Leu Asp Pro Arg Ser Leu Arg Ala Lys Ala 290 295 300 Arg Arg Met Lys Lys Glu Tyr Asp Val Lys Ala Ile Phe Val Asp Tyr 305 310 315 320 Leu Gln Leu Met His Leu Lys Gly Arg Lys Glu Ser Arg Gln Gln Glu 325 330 335 Ile Ser Glu Ile Ser Arg Ser Leu Lys Leu Leu Ala Arg Glu Leu Asp 340 345 350 Ile Val Val Ile Ala Leu Ser Gln Leu Ser Arg Ala Val Glu Gln Arg 355 360 365 Glu Asp Lys Arg Pro Arg Leu Ser Asp Leu Arg Glu Ser Gly Ala Ile 370 375 380 Glu Gln Asp Ala Asp Thr Val Ile Phe Ile Tyr Arg Glu Glu Tyr Tyr 385 390 395 400 Arg Ser Lys Lys Ser Lys Glu Glu Ser Lys Leu His Glu Pro His Glu 405 410 415 Ala Glu Ile Ile Ile Gly Lys Gln Arg Asn Gly Pro Val Gly Thr Ile 420 425 430 Thr Leu Ile Phe Asp Pro Arg Thr Val Thr Phe His Glu Val Asp Val 435 440 445 Val His Ser 450 153 1695 DNA Thermatoga maritima 153 gtgattcctc gagaggtcat cgaggaaata aaagaaaagg ttgacatcgt agaggtcatt 60 tccgagtacg tgaatcttac ccgggtaggt tcctcctaca gggctctctg tccctttcat 120 tcagaaacca atccttcttt ctacgttcat ccgggtttga agatatacca ttgtttcggc 180 tgcggtgcga gtggagacgt catcaaattt cttcaagaaa tggaagggat cagtttccag 240 gaagcgctgg aaagacttgc caaaagagct gggattgatc tttctctcta cagaacagaa 300 gggacttctg aatacggaaa atacattcgt ttgtacgaag aaacgtggaa aaggtacgtc 360 aaagagctgg agaaatcgaa agaggcaaaa gactatttaa aaagcagagg cttctctgaa 420 gaagatatag caaagttcgg ctttgggtac gtccccaaga gatccagcat ctctatagaa 480 gttgcagaag gcatgaacat aacactggaa gaacttgtca gatacggtat cgcgctgaaa 540 aagggtgatc gattcgttga tagattcgaa ggaagaatcg ttgttccaat aaagaacgac 600 agtggtcata ttgtggcttt tggtgggcgt gctctcggca acgaagaacc gaagtatttg 660 aactctccag agaccaggta tttttcgaag aagaagaccc tttttctctt cgatgaggcg 720 aaaaaagtgg caaaagaggt tggttttttc gtcatcaccg aaggctactt cgacgcgctc 780 gcattcagaa aggatggaat accaacggcg gtcgctgttc ttggggcgag tctttcaaga 840 gaggcgattc taaaactttc ggcgtattcg aaaaacgtca tactgtgttt cgataatgac 900 aaagcaggct tcagagccac tctcaaatcc ctcgaggatc tcctagacta cgaattcaac 960 gtgcttgtgg caaccccctc tccttacaaa gacccagatg aactctttca gaaagaagga 1020 gaaggttcat tgaaaaagat gctgaaaaac tcgcgttcgt tcgaatattt tctggtgacg 1080 gctggtgagg tcttctttga caggaacagc cccgcgggtg tgagatccta cctttctttc 1140 ctcaaaggtt gggtccaaaa gatgagaagg aaaggatatt tgaaacacat agaaaatctc 1200 gtgaatgagg tttcatcttc tctccagata ccagaaaacc agattttgaa cttttttgaa 1260 agcgacaggt ctaacactat gcctgttcat gagaccaagt cgtcaaaggt ttacgatgag 1320 gggagaggac tggcttattt gtttttgaac tacgaggatt tgagggaaaa gattctggaa 1380 ctggacttag aggtactgga agataaaaac gcgagggagt ttttcaagag agtctcactg 1440 ggagaagatt tgaacaaagt catagaaaac ttcccaaaag agctgaaaga ctggattttt 1500 gagacaatag aaagcattcc tcctccaaag gatcccgaga aattcctcgg tgacctctcc 1560 gaaaagttga aaatccgacg gatagagaga cgtatcgcag aaatagatga tatgataaag 1620 aaagcttcaa acgatgaaga aaggcgtctt cttctctcta tgaaagtgga tctcctcaga 1680 aaaataaaga ggagg 1695 154 565 PRT Thermatoga maritima 154 Met Ile Pro Arg Glu Val Ile Glu Glu Ile Lys Glu Lys Val Asp Ile 1 5 10 15 Val Glu Val Ile Ser Glu Tyr Val Asn Leu Thr Arg Val Gly Ser Ser 20 25 30 Tyr Arg Ala Leu Cys Pro Phe His Ser Glu Thr Asn Pro Ser Phe Tyr 35 40 45 Val His Pro Gly Leu Lys Ile Tyr His Cys Phe Gly Cys Gly Ala Ser 50 55 60 Gly Asp Val Ile Lys Phe Leu Gln Glu Met Glu Gly Ile Ser Phe Gln 65 70 75 80 Glu Ala Leu Glu Arg Leu Ala Lys Arg Ala Gly Ile Asp Leu Ser Leu 85 90 95 Tyr Arg Thr Glu Gly Thr Ser Glu Tyr Gly Lys Tyr Ile Arg Leu Tyr 100 105 110 Glu Glu Thr Trp Lys Arg Tyr Val Lys Glu Leu Glu Lys Ser Lys Glu 115 120 125 Ala Lys Asp Tyr Leu Lys Ser Arg Gly Phe Ser Glu Glu Asp Ile Ala 130 135 140 Lys Phe Gly Phe Gly Tyr Val Pro Lys Arg Ser Ser Ile Ser Ile Glu 145 150 155 160 Val Ala Glu Gly Met Asn Ile Thr Leu Glu Glu Leu Val Arg Tyr Gly 165 170 175 Ile Ala Leu Lys Lys Gly Asp Arg Phe Val Asp Arg Phe Glu Gly Arg 180 185 190 Ile Val Val Pro Ile Lys Asn Asp Ser Gly His Ile Val Ala Phe Gly 195 200 205 Gly Arg Ala Leu Gly Asn Glu Glu Pro Lys Tyr Leu Asn Ser Pro Glu 210 215 220 Thr Arg Tyr Phe Ser Lys Lys Lys Thr Leu Phe Leu Phe Asp Glu Ala 225 230 235 240 Lys Lys Val Ala Lys Glu Val Gly Phe Phe Val Ile Thr Glu Gly Tyr 245 250 255 Phe Asp Ala Leu Ala Phe Arg Lys Asp Gly Ile Pro Thr Ala Val Ala 260 265 270 Val Leu Gly Ala Ser Leu Ser Arg Glu Ala Ile Leu Lys Leu Ser Ala 275 280 285 Tyr Ser Lys Asn Val Ile Leu Cys Phe Asp Asn Asp Lys Ala Gly Phe 290 295 300 Arg Ala Thr Leu Lys Ser Leu Glu Asp Leu Leu Asp Tyr Glu Phe Asn 305 310 315 320 Val Leu Val Ala Thr Pro Ser Pro Tyr Lys Asp Pro Asp Glu Leu Phe 325 330 335 Gln Lys Glu Gly Glu Gly Ser Leu Lys Lys Met Leu Lys Asn Ser Arg 340 345 350 Ser Phe Glu Tyr Phe Leu Val Thr Ala Gly Glu Val Phe Phe Asp Arg 355 360 365 Asn Ser Pro Ala Gly Val Arg Ser Tyr Leu Ser Phe Leu Lys Gly Trp 370 375 380 Val Gln Lys Met Arg Arg Lys Gly Tyr Leu Lys His Ile Glu Asn Leu 385 390 395 400 Val Asn Glu Val Ser Ser Ser Leu Gln Ile Pro Glu Asn Gln Ile Leu 405 410 415 Asn Phe Phe Glu Ser Asp Arg Ser Asn Thr Met Pro Val His Glu Thr 420 425 430 Lys Ser Ser Lys Val Tyr Asp Glu Gly Arg Gly Leu Ala Tyr Leu Phe 435 440 445 Leu Asn Tyr Glu Asp Leu Arg Glu Lys Ile Leu Glu Leu Asp Leu Glu 450 455 460 Val Leu Glu Asp Lys Asn Ala Arg Glu Phe Phe Lys Arg Val Ser Leu 465 470 475 480 Gly Glu Asp Leu Asn Lys Val Ile Glu Asn Phe Pro Lys Glu Leu Lys 485 490 495 Asp Trp Ile Phe Glu Thr Ile Glu Ser Ile Pro Pro Pro Lys Asp Pro 500 505 510 Glu Lys Phe Leu Gly Asp Leu Ser Glu Lys Leu Lys Ile Arg Arg Ile 515 520 525 Glu Arg Arg Ile Ala Glu Ile Asp Asp Met Ile Lys Lys Ala Ser Asn 530 535 540 Asp Glu Glu Arg Arg Leu Leu Leu Ser Met Lys Val Asp Leu Leu Arg 545 550 555 560 Lys Ile Lys Arg Arg 565 155 804 DNA Thermus thermophilus 155 atggctctac acccggctca ccctggggca ataatcgggc acgaggccgt tctcgccctc 60 cttccccgcc tcaccgccca gaccctgctc ttctccggcc ccgagggggt ggggcggcgc 120 accgtggccc gctggtacgc ctgggggctc aaccgcggct tccccccgcc ctccctgggg 180 gagcacccgg acgtcctcga ggtggggccc aaggcccggg acctccgggg ccgggccgag 240 gtgcggctgg aggaggtggc gcccctcttg gagtggtgct ccagccaccc ccgggagcgg 300 gtgaaggtgg ccatcctgga ctcggcccac ctcctcaccg aggccgccgc caacgccctc 360 ctcaagctcc tggaggagcc cccttcctac gcccgcatcg tcctcatcgc cccaagccgc 420 gccaccctcc tccccaccct ggcctcccgg gccacggagg tggcattcgc ccccgtgccc 480 gaggaggccc tgcgcgccct cacccaggac ccggagctcc tccgctacgc cgccggggcc 540 ccgggccgcc tccttagggc cctccaggac ccggaggggt accgggcccg catggccagg 600 gcgcaaaggg tcctgaaagc cccgcccctg gagcgcctcg ctttgcttcg ggagcttttg 660 gccgaggagg agggggtcca cgccctccac gccgtcctaa agcgcccgga gcacctcctt 720 gccctggagc gggcgcggga ggccctggag gggtacgtga gccccgagct ggtcctcgcc 780 cggctggcct tagacttaga gaca 804 156 268 PRT Thermus thermophilus 156 Met Ala Leu His Pro Ala His Pro Gly Ala Ile Ile Gly His Glu Ala 1 5 10 15 Val Leu Ala Leu Leu Pro Arg Leu Thr Ala Gln Thr Leu Leu Phe Ser 20 25 30 Gly Pro Glu Gly Val Gly Arg Arg Thr Val Ala Arg Trp Tyr Ala Trp 35 40 45 Gly Leu Asn Arg Gly Phe Pro Pro Pro Ser Leu Gly Glu His Pro Asp 50 55 60 Val Leu Glu Val Gly Pro Lys Ala Arg Asp Leu Arg Gly Arg Ala Glu 65 70 75 80 Val Arg Leu Glu Glu Val Ala Pro Leu Leu Glu Trp Cys Ser Ser His 85 90 95 Pro Arg Glu Arg Val Lys Val Ala Ile Leu Asp Ser Ala His Leu Leu 100 105 110 Thr Glu Ala Ala Ala Asn Ala Leu Leu Lys Leu Leu Glu Glu Pro Pro 115 120 125 Ser Tyr Ala Arg Ile Val Leu Ile Ala Pro Ser Arg Ala Thr Leu Leu 130 135 140 Pro Thr Leu Ala Ser Arg Ala Thr Glu Val Ala Phe Ala Pro Val Pro 145 150 155 160 Glu Glu Ala Leu Arg Ala Leu Thr Gln Asp Pro Glu Leu Leu Arg Tyr 165 170 175 Ala Ala Gly Ala Pro Gly Arg Leu Leu Arg Ala Leu Gln Asp Pro Glu 180 185 190 Gly Tyr Arg Ala Arg Met Ala Arg Ala Gln Arg Val Leu Lys Ala Pro 195 200 205 Pro Leu Glu Arg Leu Ala Leu Leu Arg Glu Leu Leu Ala Glu Glu Glu 210 215 220 Gly Val His Ala Leu His Ala Val Leu Lys Arg Pro Glu His Leu Leu 225 230 235 240 Ala Leu Glu Arg Ala Arg Glu Ala Leu Glu Gly Tyr Val Ser Pro Glu 245 250 255 Leu Val Leu Ala Arg Leu Ala Leu Asp Leu Glu Thr 260 265 157 729 DNA Thermus thermophilus 157 atgctggacc tgagggaggt gggggaggcg gagtggaagg ccctaaagcc ccttttggaa 60 agcgtgcccg agggcgtccc cgtcctcctc ctggacccta agccaagccc ctcccgggcg 120 gccttctacc ggaaccggga aaggcgggac ttccccaccc ccaaggggaa ggacctggtg 180 cggcacctgg aaaaccgggc caagcgcctg gggctcaggc tcccgggcgg ggtggcccag 240 tacctggcct ccctggaggg ggacctcgag gccctggagc gggagctgga gaagcttgcc 300 ctcctctccc cacccctcac cctggagaag gtggagaagg tggtggccct gaggcccccc 360 ctcacgggct ttgacctggt gcgctccgtc ctggagaagg accccaagga ggccctcctg 420 cgcctaggcg gcctcaagga ggagggggag gagcccctca ggctcctcgg ggccctctcc 480 tggcagttcg ccctcctcgc ccgggccttc ttcctcctcc gggaaaaccc caggcccaag 540 gaggaggacc tcgcccgcct cgaggcccac ccctacgccg cccgccgcgc cctggaggcg 600 gcgaagcgcc tcacggaaga ggccctcaag gaggccctgg acgccctcat ggaggcggaa 660 aagagggcca agggggggaa agacccgtgg ctcgccctgg aggcggcggt cctccgcctc 720 gcccgttga 729 158 292 PRT Thermus thermophilus 158 Met Val Ile Ala Phe Thr Gly Asp Pro Phe Leu Ala Arg Glu Ala Leu 1 5 10 15 Leu Glu Glu Ala Arg Leu Arg Gly Leu Ser Arg Phe Thr Glu Pro Thr 20 25 30 Pro Glu Ala Leu Ala Gln Ala Leu Ala Pro Gly Leu Phe Gly Gly Gly 35 40 45 Gly Ala Met Leu Asp Leu Arg Glu Val Gly Glu Ala Glu Trp Lys Ala 50 55 60 Leu Lys Pro Leu Leu Glu Ser Val Pro Glu Gly Val Pro Val Leu Leu 65 70 75 80 Leu Asp Pro Lys Pro Ser Pro Ser Arg Ala Ala Phe Tyr Arg Asn Arg 85 90 95 Glu Arg Arg Asp Phe Pro Thr Pro Lys Gly Lys Asp Leu Val Arg His 100 105 110 Leu Glu Asn Arg Ala Lys Arg Leu Gly Leu Arg Leu Pro Gly Gly Val 115 120 125 Ala Gln Tyr Leu Ala Ser Leu Glu Gly Asp Leu Glu Ala Leu Glu Arg 130 135 140 Glu Leu Glu Lys Leu Ala Leu Leu Ser Pro Pro Leu Thr Leu Glu Lys 145 150 155 160 Val Glu Lys Val Val Ala Leu Arg Pro Pro Leu Thr Gly Phe Asp Leu 165 170 175 Val Arg Ser Val Leu Glu Lys Asp Pro Lys Glu Ala Leu Leu Arg Leu 180 185 190 Gly Gly Leu Lys Glu Glu Gly Glu Glu Pro Leu Arg Leu Leu Gly Ala 195 200 205 Leu Ser Trp Gln Phe Ala Leu Leu Ala Arg Ala Phe Phe Leu Leu Arg 210 215 220 Glu Asn Pro Arg Pro Lys Glu Glu Asp Leu Ala Arg Leu Glu Ala His 225 230 235 240 Pro Tyr Ala Ala Arg Arg Ala Leu Glu Ala Ala Lys Arg Leu Thr Glu 245 250 255 Glu Ala Leu Lys Glu Ala Leu Asp Ala Leu Met Glu Ala Glu Lys Arg 260 265 270 Ala Lys Gly Gly Lys Asp Pro Trp Leu Ala Leu Glu Ala Ala Val Leu 275 280 285 Arg Leu Ala Arg 290 159 37 DNA Artificial Sequence Description of Artificial Sequence primer 159 gtgtgtcata tgagtaagga tttcgtccac cttcacc 37 160 34 DNA Artificial Sequence Description of Artificial Sequence primer 160 gtgtgtggat ccggggacta ctcggaagta aggg 34 161 36 DNA Artificial Sequence Description of Artificial Sequence primer 161 gtgtgtcata tggaaaccac aatattccag ttccag 36 162 39 DNA Artificial Sequence Description of Artificial Sequence primer 162 gtgtgtggat ccttatccac catgagaagt atttttcac 39 163 41 DNA Artificial Sequence Description of Artificial Sequence primer 163 gtgtgtcata tggaaaaagt tttttttgga aaaaactcca g 41 164 35 DNA Artificial Sequence Description of Artificial Sequence primer 164 gtgtgtggat ccttaatccg cctgaacggc taacg 35 165 41 DNA Artificial Sequence Description of Artificial Sequence primer 165 gtgtgtcata tgaactacgt tcccttcgcg agaaagtaca g 41 166 36 DNA Artificial Sequence Description of Artificial Sequence primer 166 gtgtgtggat ccttaaaaca gcctcgtccc gctgga 36 167 33 DNA Artificial Sequence Description of Artificial Sequence primer 167 gtgtgtcata tgcgcgttaa ggtggacagg gag 33 168 35 DNA Artificial Sequence Description of Artificial Sequence primer 168 tgtgtctcga gtcatggcta caccctcatc ggcat 35 169 47 DNA Artificial Sequence Description of Artificial Sequence primer 169 gtgtgtcata tgctcaataa ggtttttata ataggaagac ttacggg 47 170 39 DNA Artificial Sequence Description of Artificial Sequence primer 170 gtgtggatcc ttaaaaaggt atttcgtcct cttcatcgg 39 171 807 DNA Thermus thermophilus 171 atggctcgag gcctgaaccg cgttttcctc atcggcgccc tcgccacccg gccggacatg 60 cgctacaccc cggcggggct cgccattttg gacctgaccc tcgccggtca ggacctgctt 120 ctttccgata acggggggga accggaggtg tcctggtacc accgggtgag gctcttaggc 180 cgccaggcgg agatgtgggg cgacctcttg gaccaagggc agctcgtctt cgtggagggc 240 cgcctggagt accgccagtg ggaaagggag ggggagaagc ggagcgagct ccagatccgg 300 gccgacttcc ggaccccctg gacgaccggg ggaagaagcg ggcggaggac agccggggcc 360 agcccaggct ccgcgccgcc ctgaaccagg tcttcctcat gggcaacctg acccgggacc 420 cggaactccg ctacaccccc cagggcaccg cggtggcccg gctgggcctg gcggtgaacg 480 agcgccgcca gggggcggag gagcgcaccc acttcgtgga ggttcaggcc tggcgcgacc 540 tggcggagtg ggccgccgag ctgaggaagg gcgacggcct tttcgtgatc ggcaggttgg 600 tgaacgactc ctggaccagc tccagcggcg agcggcgctt ccagacccgt gtggaggccc 660 tcaggctgga gcgccccacc cgtggacctg cccaggcctg cccaggccgg cggaacaggt 720 cccgcgaagt ccagacgggt ggggtggaca ttgacgaagg cttggaagac tttccgccgg 780 aggaggattt gccgttttga gcacgaa 807 172 266 PRT Thermus thermophilus 172 Met Ala Arg Gly Leu Asn Arg Val Phe Leu Ile Gly Ala Leu Ala Thr 1 5 10 15 Arg Pro Asp Met Arg Tyr Thr Pro Ala Gly Leu Ala Ile Leu Asp Leu 20 25 30 Thr Leu Ala Gly Gln Asp Leu Leu Leu Ser Asp Asn Gly Gly Glu Pro 35 40 45 Glu Val Ser Trp Tyr His Arg Val Arg Leu Leu Gly Arg Gln Ala Glu 50 55 60 Met Trp Gly Asp Leu Leu Asp Gln Gly Gln Leu Val Phe Val Glu Gly 65 70 75 80 Arg Leu Glu Tyr Arg Gln Trp Glu Arg Glu Gly Glu Lys Arg Ser Glu 85 90 95 Leu Gln Ile Arg Ala Asp Phe Leu Asp Pro Leu Asp Asp Arg Gly Lys 100 105 110 Lys Arg Ala Glu Asp Ser Arg Gly Gln Pro Arg Leu Arg Ala Ala Leu 115 120 125 Asn Gln Val Phe Leu Met Gly Asn Leu Thr Arg Asp Pro Glu Leu Arg 130 135 140 Tyr Thr Pro Gln Gly Thr Ala Val Ala Arg Leu Gly Leu Ala Val Asn 145 150 155 160 Glu Arg Arg Gln Gly Ala Glu Glu Arg Thr His Phe Val Glu Val Gln 165 170 175 Ala Trp Arg Asp Leu Ala Glu Trp Ala Ala Glu Leu Arg Lys Gly Asp 180 185 190 Gly Leu Phe Val Ile Gly Arg Leu Val Asn Asp Ser Trp Thr Ser Ser 195 200 205 Ser Gly Glu Arg Arg Phe Gln Thr Arg Val Glu Ala Leu Arg Leu Glu 210 215 220 Arg Pro Thr Arg Gly Pro Ala Gln Ala Cys Pro Gly Arg Arg Asn Arg 225 230 235 240 Ser Arg Glu Val Gln Thr Gly Gly Val Asp Ile Asp Glu Gly Leu Glu 245 250 255 Asp Phe Pro Pro Glu Glu Asp Leu Pro Phe 260 265 173 992 DNA Bacillus stearothermophilus 173 aattccgaca tttcaattga atcgtttatt ccgcttgaaa aagaaggcaa gttgctcgtt 60 gatgtgaaaa gaccggggag catcgtactg caggcgcgct ttttctctga aatcgtgaaa 120 aaactgccgc aacaaacggt ggaaatcgaa acggaagaca actttttgac gatcatccgc 180 tcggggcact cagaattccg cctcaatggg ctaaacgccg acgaatatcc gcgcctgccg 240 caaattgaag aagaaaacgt gtttcaaatc ccggctgatt tattgaaaac cgtgattcgg 300 caaacggtgt tcgccgtttc tacatcggaa acgcgcccaa tcttgacagg tgtcaactgg 360 aaagttgaac atggcgagct tgtctgcaca gcgaccgaca gtcatcgctt agccatgcgc 420 aaagtgaaaa ttgagtcgga aaatgaagta tcatacaacg tcgtcatccc tggaaaaagt 480 cttaatgagc tcagcaaaat tttggatgac ggcaaccacc cggtggacat cgtcatgaca 540 gccaatcaag tgctatttaa ggccgagcac cttctcttct tttcccggct gcttgacggc 600 aactatccgg agacggcccg cttgattcca acagaaagca aaacgaccat gatcgtcaat 660 gcaaaagagt ttctgcaggc aatcgaccga gcgtccttgc ttgctcgaga aggaaggaac 720 aacgttgtga aactgacgac gcttcctgga ggaatgctcg aaatttcttc gatttctccg 780 agatcgggaa agtgacggag cagctgcaaa cggagtctct tgaaggggaa gagttgaaca 840 tttcgttcag cgcgaaatat atgatggacg cgttgcgggc gcttgatgga acagacattt 900 caaatcagct tcactggggc catgcggccg ttcctgttgc gcccgcttca accgattcga 960 tgcttcagct cattttgccg gtgagaacat at 992 174 334 PRT Bacillus stearothermophilus 174 Asn Ser Asp Ile Ser Ile Ile Glu Ser Phe Ile Pro Leu Glu Lys Glu 1 5 10 15 Gly Lys Leu Leu Val Asp Val Lys Arg Pro Gly Ser Ile Val Leu Gln 20 25 30 Ala Arg Phe Phe Ser Glu Ile Val Lys Lys Leu Pro Gln Gln Thr Val 35 40 45 Glu Ile Glu Thr Glu Asp Asn Phe Leu Thr Ile Ile Arg Ser Gly His 50 55 60 Ser Glu Phe Arg Leu Asn Gly Leu Asn Ala Asp Glu Tyr Pro Arg Leu 65 70 75 80 Pro Gln Ile Glu Glu Glu Asn Val Phe Gln Ile Pro Ala Asp Leu Leu 85 90 95 Lys Thr Val Ile Arg Gln Thr Val Phe Ala Val Ser Thr Ser Glu Thr 100 105 110 Arg Pro Ile Leu Thr Gly Val Asn Trp Lys Val Glu His Gly Glu Leu 115 120 125 Val Cys Thr Ala Thr Asp Ser His Arg Leu Ala Met Arg Lys Val Lys 130 135 140 Ile Ile Glu Ser Glu Asn Glu Val Ser Tyr Asn Val Val Ile Pro Gly 145 150 155 160 Lys Ser Leu Asn Glu Leu Ser Lys Ile Ile Leu Asp Asp Gly Asn His 165 170 175 Pro Val Asp Ile Val Met Thr Ala Asn Gln Val Leu Phe Lys Ala Glu 180 185 190 His Leu Leu Phe Phe Ser Arg Leu Leu Asp Gly Asn Tyr Pro Glu Thr 195 200 205 Ala Arg Leu Ile Pro Thr Glu Ser Lys Thr Thr Met Ile Val Asn Ala 210 215 220 Lys Glu Phe Leu Gln Ala Ile Asp Arg Ala Ser Leu Leu Ala Arg Glu 225 230 235 240 Gly Arg Asn Asn Val Val Lys Leu Thr Thr Leu Pro Gly Gly Met Leu 245 250 255 Glu Ile Ser Ser Ile Ser Pro Glu Ile Gly Lys Val Thr Glu Gln Leu 260 265 270 Gln Thr Glu Ser Leu Glu Gly Glu Glu Leu Asn Ile Ser Phe Ser Ala 275 280 285 Lys Tyr Met Met Asp Ala Leu Arg Ala Leu Asp Gly Thr Asp Ile Gln 290 295 300 Ile Ser Phe Thr Gly Ala Met Arg Pro Phe Leu Leu Arg Pro Leu His 305 310 315 320 Thr Asp Ser Met Leu Gln Leu Ile Leu Pro Val Arg Thr Tyr 325 330 175 492 DNA Bacillus stearothermophilus 175 atgattaacc gcgtcatttt ggtcggcagg ttaacgagag atccggagtt gcgttacact 60 ccaagcggag tggctgttgc cacgtttacg ctcgcggtca accgtccgtt tacaaatcag 120 cagggcgagc gggaaacgga ttttattcaa tgtgtcgttt ggcgccgcca ggcggaaaac 180 gtcgccaact ttttgaaaaa ggggagcttg gctggtgtcg atggccgact gcaaacccgc 240 agctatgaaa atcaagaagg tcggcgtgtg tacgtgacgg aagtggtggc tgatagcgtc 300 caatttcttg agccgaaagg aacgagcgag cagcgagggg cgacagcagg cggctactat 360 ggggatccat tcccattcgg gcaagatcag aaccaccaat atccgaacga aaaagggttt 420 ggccgcatcg atgacgatcc tttcgccaat gacggccagc cgatcgatat ttctgatgat 480 gatttgccgt tt 492 176 164 PRT Bacillus stearothermophilus 176 Met Ile Asn Arg Val Ile Leu Val Gly Arg Leu Thr Arg Asp Pro Glu 1 5 10 15 Leu Arg Tyr Thr Pro Ser Gly Val Ala Val Ala Thr Phe Thr Leu Ala 20 25 30 Val Asn Arg Pro Phe Thr Asn Gln Ser Tyr Glu Asn Gln Glu Gly Arg 35 40 45 Arg Val Tyr Val Thr Glu Val Val Ala Asp Ser Val Gln Phe Leu Glu 50 55 60 Pro Lys Gly Thr Ser Glu Gln Arg Gly Ala Thr Ala Gly Gly Tyr Tyr 65 70 75 80 Gln Gly Glu Arg Glu Thr Asp Phe Ile Gln Cys Val Val Trp Arg Arg 85 90 95 Gln Ala Glu Asn Val Ala Asn Phe Leu Lys Lys Gly Ser Leu Ala Gly 100 105 110 Val Asp Gly Arg Leu Gln Thr Arg Gly Asp Pro Phe Pro Phe Gly Gln 115 120 125 Asp Gln Asn His Gln Tyr Pro Asn Glu Lys Gly Phe Gly Arg Ile Asp 130 135 140 Asp Asp Pro Phe Ala Asn Asp Gly Gln Pro Ile Asp Ile Ser Asp Asp 145 150 155 160 Asp Leu Pro Phe 177 1044 DNA Bacillus stearothermophilus 177 atgctggaac gcgtatgggg aaacattgaa aaacggcgtt tttctcccct ttatttatta 60 tacggcaatg agccgttttt attaacggaa acgtatgagc gattggtgaa cgcagcgctt 120 ggccccgagg agcgggagtg gaacttggct gtgtacgact gcgaggaaac gccgatcgag 180 gcggcgcttg aggaggccga gacggtgccg tttttcggcg agcggcgtgt cattctcatc 240 aagcatccat atttttttac gtctgaaaaa gagaaggaga tcgaacatga tttggcgaag 300 ctggaggcgt acttgaaggc gccgtcgccg ttttcgatcg tcgtcttttt cgcgccgtac 360 gagaagcttg atgagcgaaa aaaaattacg aagctcgcca aagagcaaag cgaagtcgtc 420 atcgccgccc cgctcgccga agcggagctg cgtgcctggg tgcggcgccg catcgagagc 480 caaggggcgc aagcaagcga cgaggcgatt gatgtcctgt tgcggcgggc cgggacgcag 540 ctttccgcct tggcgaatga aatcgataaa ttggccctgt ttgccggatc gggcggaacc 600 atcgaggcgg cggcggttga gcggcttgtc gcccgcacgc cggaagaaaa cgtatttgtg 660 cttgtcgagc aagtggcgaa gcgcgacatt ccagcagcgt tgcagacgtt ttatgatctg 720 cttgaaaaca atgaagagcc gatcaaaatt ttggcgttgc tcgccgccca tttccgcttg 780 ctttcgcaag tgaaatggct tgcctcctta ggctacggac aggcgcaaat tgctgcggcg 840 ctcaaggtgc acccgttccg cgtcaagctc gctcttgctc aagcggcccg cttcgctgac 900 ggagagcttg ctgaggcgat caacgagctc gctgacgccg attacgaagt gaaaagcggg 960 gcggtcgatc gccggttggc cgttgagctg cttctgatgc gctggggcgc ccgcccggcg 1020 caagcggggc gccacggccg gcgg 1044 178 348 PRT Bacillus stearothermophilus 178 Met Leu Glu Arg Val Trp Gly Asn Ile Glu Lys Arg Arg Phe Ser Pro 1 5 10 15 Leu Tyr Leu Leu Tyr Gly Asn Glu Pro Phe Leu Leu Thr Glu Thr Tyr 20 25 30 Glu Arg Leu Val Asn Ala Ala Leu Gly Pro Glu Glu Arg Glu Trp Asn 35 40 45 Leu Ala Val Tyr Asp Cys Glu Glu Thr Pro Ile Glu Ala Ala Leu Glu 50 55 60 Glu Ala Glu Thr Val Pro Phe Phe Gly Glu Arg Arg Val Ile Leu Ile 65 70 75 80 Lys His Pro Tyr Phe Phe Thr Ser Glu Lys Glu Lys Glu Ile Glu His 85 90 95 Asp Leu Ala Lys Leu Glu Ala Tyr Leu Lys Ala Pro Ser Pro Phe Ser 100 105 110 Ile Val Val Phe Phe Ala Pro Tyr Glu Lys Leu Asp Glu Arg Lys Lys 115 120 125 Ile Thr Lys Leu Ala Lys Glu Gln Ser Glu Val Val Ile Ala Ala Pro 130 135 140 Leu Ala Glu Ala Glu Leu Arg Ala Trp Val Arg Arg Arg Ile Glu Ser 145 150 155 160 Gln Gly Ala Gln Ala Ser Asp Glu Ala Ile Asp Val Leu Leu Arg Arg 165 170 175 Ala Gly Thr Gln Leu Ser Ala Leu Ala Asn Glu Ile Asp Lys Leu Ala 180 185 190 Leu Phe Ala Gly Ser Gly Gly Thr Ile Glu Ala Ala Ala Val Glu Arg 195 200 205 Leu Val Ala Arg Thr Pro Glu Glu Asn Val Phe Val Leu Val Glu Gln 210 215 220 Val Ala Lys Arg Asp Ile Pro Ala Ala Leu Gln Thr Phe Tyr Asp Leu 225 230 235 240 Leu Glu Asn Asn Glu Glu Pro Ile Lys Ile Leu Ala Leu Leu Ala Ala 245 250 255 His Phe Arg Leu Leu Ser Gln Val Lys Trp Leu Ala Ser Leu Gly Tyr 260 265 270 Gly Gln Ala Gln Ile Ala Ala Ala Leu Lys Val His Pro Phe Arg Val 275 280 285 Lys Leu Ala Leu Ala Gln Ala Ala Arg Phe Ala Asp Gly Glu Leu Ala 290 295 300 Glu Ala Ile Asn Glu Leu Ala Asp Ala Asp Tyr Glu Val Lys Ser Gly 305 310 315 320 Ala Val Asp Arg Arg Leu Ala Val Glu Leu Leu Leu Met Arg Trp Gly 325 330 335 Ala Arg Pro Ala Gln Ala Gly Arg His Gly Arg Arg 340 345 179 757 DNA Bacillus stearothermophilus 179 atgcgatggg aacagctagc gaaacgccag ccggtggtgg cgaaaatgct gcaaagcggc 60 ttggaaaaag ggcggatttc tcatgcgtac ttgtttgagg ggcagcgggg gacgggcaaa 120 aaagcggcca gtttgttgtt ggcgaaacgt ttgttttgtc tgtccccaat cggagtttcc 180 ccgtgtctag agtgccgcaa ctgccggcgc atcgactccg gcaaccaccc tgacgtccgg 240 gtgatcggcc cagatggagg atcaatcaaa aaggaacaaa tcgaatggct gcagcaagag 300 ttctcgaaaa cagcggtcga gtcggataaa aaaatgtaca tcgttgagca cgccgatcaa 360 atgacgacaa gcgctgccaa cagccttctg aaatttttgg aagagccgca tccggggacg 420 gtggcggtat tgctgactga gcaataccac cgcctgctag ggacgatcgt ttcccgctgt 480 caagtgcttt cgttccggcc gttgccgccg gcagagctcg cccagggact tgtcgaggag 540 cacgtgccgt tgccgttggc gctgttggct gcccatttga caaacagctt cgaggaagca 600 ctggcgcttg ccaaagatag ttggtttgcc gaggcgcgaa cattagtgct acaatggtat 660 gagatgctgg gcaagccgga gctgcagctt ttgtttttca tccacgaccg cttgtttccg 720 cattttttgg aaagccatca gcttgacctt ggacttg 757 180 252 PRT Bacillus stearothermophilus 180 Met Arg Trp Glu Gln Leu Ala Lys Arg Gln Pro Val Val Ala Lys Met 1 5 10 15 Leu Gln Ser Gly Leu Glu Lys Gly Arg Ile Ser His Ala Tyr Leu Phe 20 25 30 Glu Gly Gln Arg Gly Thr Gly Lys Lys Ala Ala Ser Leu Leu Leu Ala 35 40 45 Lys Arg Leu Phe Cys Leu Ser Pro Ile Gly Val Ser Pro Cys Leu Glu 50 55 60 Cys Arg Asn Cys Arg Arg Ile Asp Ser Gly Asn His Pro Asp Val Arg 65 70 75 80 Val Ile Gly Pro Asp Gly Gly Ser Ile Lys Lys Glu Gln Ile Glu Trp 85 90 95 Leu Gln Gln Glu Phe Ser Lys Thr Ala Val Glu Ser Asp Lys Lys Met 100 105 110 Tyr Ile Val Glu His Ala Asp Gln Met Thr Thr Ser Ala Ala Asn Ser 115 120 125 Leu Leu Lys Phe Leu Glu Glu Pro His Pro Gly Thr Val Ala Val Leu 130 135 140 Leu Thr Glu Gln Tyr His Arg Leu Leu Gly Thr Ile Val Ser Arg Cys 145 150 155 160 Gln Val Leu Ser Phe Arg Pro Leu Pro Pro Ala Glu Leu Ala Gln Gly 165 170 175 Leu Val Glu Glu His Val Pro Leu Pro Leu Ala Leu Leu Ala Ala His 180 185 190 Leu Thr Asn Ser Phe Glu Glu Ala Leu Ala Leu Ala Lys Asp Ser Trp 195 200 205 Phe Ala Glu Ala Arg Thr Leu Val Leu Gln Trp Tyr Glu Met Leu Gly 210 215 220 Lys Pro Glu Leu Gln Leu Leu Phe Phe Ile His Asp Arg Leu Phe Pro 225 230 235 240 His Phe Leu Glu Ser His Gln Leu Asp Leu Gly Leu 245 250 181 1677 DNA Bacillus stearothermophilus 181 gtggcatacc aagcgttata tcgcgtgttt cggccgcagc gctttgcgga catggtcggc 60 caagaacacg tgaccaagac gttgcaaagc gccctgcttc aacataaaat atcgcacgct 120 tacttatttt ccggcccgcg cggtacagga aaaacgagcg cagcgaaaat tttcgccaag 180 gcggtcaact gtgaacaggc gccagcggcg gagccatgca atgagtgtcc agcttgcctc 240 ggcattacga atggaacggt tcccgatgtg ctggaaattg acgctgcttc caacaaccgc 300 gtcgatgaaa ttcgtgatat ccgtgagaag gtgaaatttg cgccaacgtc ggcccgctac 360 aaagtgtata tcatcgacga ggtgcatatg ctgtcgatcg gtgcgtttaa cgcgctgttg 420 aaaacgttgg aggagccgcc gaaacacgtc attttcattt tggccacgac cgagccgcac 480 aaaattccgg cgacgatcat ttcccgctgc caacggttcg attttcgccg catcccgctt 540 caggcgatcg tttcacggct aaagtacgtc gcaagcgccc aaggtgtcga ggcgtcagat 600 gaggcattgt ccgccatcgc ccgtgctgca gacgggggga tgcgcgatgc gctcagcttg 660 cttgatcaag ccatttcgtt cagcgacggg aaacttcggc tcgacgacgt gctggcgatg 720 accggggctg catcatttgc cgccttatcg agcttcatcg aagccatcca ccgcaaagat 780 acagcggcgg ttcttcagca cttggaaacg atgatggcgc aagggaaaga tccgcatcgt 840 ttggttgaag acttgatttt gtactatcgc gatttattgc tgtacaaaac cgctccctat 900 gtggagggag cgattcaaat tgctgtcgtt gacgaagcgt tcacttcact gtcggaaatg 960 attccggttt ccaatttata cgaggccatc gagttgctga acaaaagcca gcaagagatg 1020 aagtggacaa accacccgcg ccttctgttg gaagtggcgc ttgtgaaact ttgccatcca 1080 tcagccgccg ccccgtcgct gtcggcttcc gagttggaac cgttgataaa gcggattgaa 1140 acgctggagg cggaattgcg gcgcctgaag gaacaaccgc ctgcccctcc gtcgaccgcc 1200 gcgccggtga aaaaactgtc caaaccgatg aaaacggggg gatataaagc cccggttggc 1260 cgcatttacg agctgttgaa acaggcgacg catgaagatt tagctttggt gaaaggatgc 1320 tgggcggatg tgctcgacac gttgaaacgg cagcataaag tgtcgcacgc tgccttgctg 1380 caagagagcg agccggttgc agcgagcgcc tcagcgtttg tattaaaatt caaatacgaa 1440 atccactgca aaatggcgac cgatcccaca agttcggtca aagaaaacgt cgaagcgatt 1500 ttgtttgagc tgacaaaccg ccgctttgaa atggtagcca ttccggaggg agaatgggga 1560 aaaataagag aagagttcat ccgcaataag gacgccatgg tggaaaaaag cgaagaagat 1620 ccgttaatcg ccgaagcgaa gcggctgttt ggcgaagagc tgatcgaaat taaagaa 1677 182 559 PRT Bacillus stearothermophilus 182 Val Ala Tyr Gln Ala Leu Tyr Arg Val Phe Arg Pro Gln Arg Phe Ala 1 5 10 15 Asp Met Val Gly Gln Glu His Val Thr Lys Thr Leu Gln Ser Ala Leu 20 25 30 Leu Gln His Lys Ile Ser His Ala Tyr Leu Phe Ser Gly Pro Arg Gly 35 40 45 Thr Gly Lys Thr Ser Ala Ala Lys Ile Phe Ala Lys Ala Val Asn Cys 50 55 60 Glu Gln Ala Pro Ala Ala Glu Pro Cys Asn Glu Cys Pro Ala Cys Leu 65 70 75 80 Gly Ile Thr Asn Gly Thr Val Pro Asp Val Leu Glu Ile Asp Ala Ala 85 90 95 Ser Asn Asn Arg Val Asp Glu Ile Arg Asp Ile Arg Glu Lys Val Lys 100 105 110 Phe Ala Pro Thr Ser Ala Arg Tyr Lys Val Tyr Ile Ile Asp Glu Val 115 120 125 His Met Leu Ser Ile Gly Ala Phe Asn Ala Leu Leu Lys Thr Leu Glu 130 135 140 Glu Pro Pro Lys His Val Ile Phe Ile Leu Ala Thr Thr Glu Pro His 145 150 155 160 Lys Ile Pro Ala Thr Ile Ile Ser Arg Cys Gln Arg Phe Asp Phe Arg 165 170 175 Arg Ile Pro Leu Gln Ala Ile Val Ser Arg Leu Lys Tyr Val Ala Ser 180 185 190 Ala Gln Gly Val Glu Ala Ser Asp Glu Ala Leu Ser Ala Ile Ala Arg 195 200 205 Ala Ala Asp Gly Gly Met Arg Asp Ala Leu Ser Leu Leu Asp Gln Ala 210 215 220 Ile Ser Phe Ser Asp Gly Lys Leu Arg Leu Asp Asp Val Leu Ala Met 225 230 235 240 Thr Gly Ala Ala Ser Phe Ala Ala Leu Ser Ser Phe Ile Glu Ala Ile 245 250 255 His Arg Lys Asp Thr Ala Ala Val Leu Gln His Leu Glu Thr Met Met 260 265 270 Ala Gln Gly Lys Asp Pro His Arg Leu Val Glu Asp Leu Ile Leu Tyr 275 280 285 Tyr Arg Asp Leu Leu Leu Tyr Lys Thr Ala Pro Tyr Val Glu Gly Ala 290 295 300 Ile Gln Ile Ala Val Val Asp Glu Ala Phe Thr Ser Leu Ser Glu Met 305 310 315 320 Ile Pro Val Ser Asn Leu Tyr Glu Ala Ile Glu Leu Leu Asn Lys Ser 325 330 335 Gln Gln Glu Met Lys Trp Thr Asn His Pro Arg Leu Leu Leu Glu Val 340 345 350 Ala Leu Val Lys Leu Cys His Pro Ser Ala Ala Ala Pro Ser Leu Ser 355 360 365 Ala Ser Glu Leu Glu Pro Leu Ile Lys Arg Ile Glu Thr Leu Glu Ala 370 375 380 Glu Leu Arg Arg Leu Lys Glu Gln Pro Pro Ala Pro Pro Ser Thr Ala 385 390 395 400 Ala Pro Val Lys Lys Leu Ser Lys Pro Met Lys Thr Gly Gly Tyr Lys 405 410 415 Ala Pro Val Gly Arg Ile Tyr Glu Leu Leu Lys Gln Ala Thr His Glu 420 425 430 Asp Leu Ala Leu Val Lys Gly Cys Trp Ala Asp Val Leu Asp Thr Leu 435 440 445 Lys Arg Gln His Lys Val Ser His Ala Ala Leu Leu Gln Glu Ser Glu 450 455 460 Pro Val Ala Ala Ser Ala Ser Ala Phe Val Leu Lys Phe Lys Tyr Glu 465 470 475 480 Ile His Cys Lys Met Ala Thr Asp Pro Thr Ser Ser Val Lys Glu Asn 485 490 495 Val Glu Ala Ile Leu Phe Glu Leu Thr Asn Arg Arg Phe Glu Met Val 500 505 510 Ala Ile Pro Glu Gly Glu Trp Gly Lys Ile Arg Glu Glu Phe Ile Arg 515 520 525 Asn Lys Asp Ala Met Val Glu Lys Ser Glu Glu Asp Pro Leu Ile Ala 530 535 540 Glu Ala Lys Arg Leu Phe Gly Glu Glu Leu Ile Glu Ile Lys Glu 545 550 555 183 4301 DNA Bacillus stearothermophilus 183 atggtgacaa aagagcaaaa agagcggttt ctcatcctgc ttgagcagct gaagatgacg 60 tcggacgaat ggatgccgca ttttcgtgag gcagccattc gcaaagtcgt gatcgataaa 120 gaggagaaaa gctggcattt ttattttcag ttcgacaacg tgctgccggt tcatgtatac 180 aaaacgtttg ccgatcggct gcagacggcg ttccgccata tcgccgccgt ccgccatacg 240 atggaggtcg aagcgccgcg cgtaactgag gcggatgtgc aggcgtattg gccgctttgc 300 cttgccgagc tgcaagaagg catgtcgccg cttgtcgatt ggctcagccg gcagacgcct 360 gagctgaaag gaaacaagct gcttgtcgtt gcccgccatg aagcggaagc gctggcgatc 420 aaacggcggt tcgccaaaaa aatcgctgat gtgtacgctt cgtttgggtt tccccccctt 480 cagcttgacg tcagcgtcga gccgtccaag caagaaatgg aacagttttt ggcgcaaaaa 540 cagcaagagg acgaagagcg agcgcttgct gtactgaccg atttagcgag ggaagaagaa 600 aaggccgcgt ctgcgccgcc gtccggtccg cttgtcatcg gctatccgat ccgcgacgag 660 gagccggtgc ggcggcttga aacgatcgtc gaagaagagc ggcgcgtcgt tgtgcaaggc 720 tatgtatttg acgccgaagt gagcgaatta aaaagcggcc gcacgctgtt gaccatgaaa 780 atcacagatt acacgaactc gattttagtc aaaatgttct cgcgcgacaa agaggacgcc 840 gagcttatga gcggcgtcaa aaaaggcatg tgggtgaaag tgcgcggcag cgtgcaaaac 900 gatacgttcg tccgtgattt ggtcatcatc gccaacgatt tgaacgaaat cgccgcaaac 960 gaacggcaag atacggcgcc ggaaggggaa aagagggtcg agctccattt gcataccccg 1020 atgagccaaa tggacgcggt cacctcggtg acaaaactca ttgagcaagc gaaaaaatgg 1080 gggcatccgg cgatcgccgt caccgaccat gccgttgttc agtcgtttcc ggaggcctac 1140 agcgcggcga aaaaacacgg catgaaggtc atttacggcc ttgaggcgaa catcgtcgac 1200 gatggcgtgc cgatcgccta caatgagacg caccgccgtc tttcggagga aacgtacgtc 1260 gtctttgacg tcgagacgac gggcctgtcg gctgtgtaca atacgatcat tgagctggcg 1320 gcggtgaaag tgaaagacgg cgagatcatc gaccgattca tgtcgtttgc caaccctgga 1380 catccgttgt cggtgacaac gatggagctg actgggatca ccgatgagat ggtgaaagac 1440 gccccgaagc cggacgaggt gctagcccgt tttgttgact gggccggcga tgcgacgctt 1500 gttgcccaca acgccagctt tgacatcggt tttttaaacg cgggcctcgc tcgcatgggg 1560 cgcggcaaaa tcgcgaatcc agtcatcgat acgctcgagc tggcccgttt tttatacccg 1620 gatttgaaaa accatcggct caatacattg tgcaaaaaat ttgacattga attgacgcag 1680 catcaccgcg ccatctacga cgcggaggcg accgggcatt tgcttatgcg gctgttgaag 1740 gaagcggaag agcgcggcat actgtttcat gacgaattaa acagccgcac gcacagcgaa 1800 gcgtcctatc ggcttgcgcg cccgttccat gtgacgctgt tggcgcaaaa cgagactgga 1860 ttgaaaaatt tgttcaagct tgtgtcattg tcgcacattc aatattttca ccgtgtgccg 1920 cgcatcccgc gctccgtgct cgtcaagcac cgcgacggcc tgcttgtcgg ctcgggctgc 1980 gacaaaggag agctgtttga caacttgatc caaaaggcgc cggaagaagt cgaagacatc 2040 gcccgttttt acgattttct tgaagtgcat ccgccggacg tgtacaagcc gctcatcgag 2100 atggattatg tgaaagacga agagatgatc aaaaacatca tccgcagcat cgtcgccctt 2160 ggtgagaagc ttgacatccc ggttgtcgcc actggcaacg tccattactt gaacccagaa 2220 gataaaattt accggaaaat cttaatccat tcgcaaggcg gggcgaatcc gctcaaccgc 2280 catgaactgc cggatgtata tttccgtacg acgaatgaaa tgcttgactg cttctcgttt 2340 ttagggccgg aaaaagcgaa ggaaatcgtc gttgacaaca cgcaaaaaat cgcttcgtta 2400 atcggcgatg tcaagccgat caaagatgag ctgtatacgc cgcgcattga aggggcggac 2460 gaggaaatca gggaaatgag ctaccggcgg gcgaaggaaa tttacggcga cccgttgccg 2520 aaacttgttg aagagcggct tgagaaggag ctaaaaagca tcatcggcca tggctttgcc 2580 gtcatttatt tgatctcgca caagcttgtg aaaaaatcgc tcgatgacgg ctaccttgtc 2640 gggtcgcgcg gatcggtcgg ctcgtcgttt gtcgcgacga tgacggaaat caccgaggtc 2700 aatccgctgc cgccgcatta cgtttgcccg aactgcaagc attcggagtt ctttaacgac 2760 ggttcagtcg gctcagggtt tgatttgccg gataaaaact gcccgcgatg tgggacgaaa 2820 tacaagaaag acgggcacga catcccgttt gagacgtttc tcggctttaa aggcgacaaa 2880 gtgccggata tcgacttgaa cttttccggc gaataccagc cgcgcgccca caactatacg 2940 aaagtgctgt ttggcgaaga caacgtctac cgcgccggga cgattggcac ggtcgctgac 3000 aaaacggcgt acggatttgt caaagcgtat gcgagcgacc ataacttaga gctgcgcggc 3060 gcggaaatcg acggctcgcg gctggctgca ccggggtgaa gcggacgacc gggcagcatc 3120 cgggcggcat catcgtcgtc ccggattata tggaaattta cgattttacg ccgattcaat 3180 atccggccga tgacacgtcc tctgaatggc ggacgaccca tttcgacttc cattcgatcc 3240 acgacaattt gttgaagctc gatattctcg ggcacgacga tccgacggtc attcgcatgc 3300 tgcaagattt aagcggcatc gatccgaaaa cgatcccgac cgacgacccg gatgtgatgg 3360 gcattttcag cagcaccgag ccgcttggcg ttacgccgga gcaaatcatg tgcaatgtcg 3420 gcacgatcgg cattccggag tttggcacgc gcttcgttcg gcaaatgttg gaagagacaa 3480 ggccaaaaac gttttccgaa ctcgtgcaaa tttccggctt gtcgcacggc accgatgtgt 3540 ggctcggcaa cgcgcaagag ctcattcaaa acggcacgtg tacgttatcg gaagtcatcg 3600 gctgccgcga cgacattatg gtctatttga tttaccgcgg gctcgagccg tcgctcgctt 3660 ttaaaatcat ggaatccgtg cgcaaaggaa aaggcttaac gccggagttt gaagcagaaa 3720 tgcgcaaaca tgacgtgccg gagtggtaca tcgattcatg caaaaaaatc aagtacatgt 3780 tcccgaaagc gcacgccgcc gcctacgtgt taatggcggt gcgcatcgcc tactttaagg 3840 tgcaccatcc gcttttgtat tacgcgtcgt actttacggt gcgggcggag gactttgacc 3900 ttgacgccat gatcaaagga tcacccgcca ttcgcaagcg gattgaggaa atcaacgcca 3960 aaggcattca ggcgacggcg aaagaaaaaa gcttgctcac ggttcttgag gtggccttag 4020 agatgtgcga gcgcggcttt tcctttaaaa atatcgattt gtaccgctcg caggcgacgg 4080 aattcgtcat tgacggcaat tctctcattc cgccgttcaa cgccattccg gggcttggga 4140 cgaacgtggc gcaggcgatc gtgcgcgccc gcgaggaagg cgagtttttg tcgaaggagg 4200 atttgcaaca gcgcggcaaa ttgtcgaaaa cgctgctcga gtatctagaa agccgcggct 4260 gccttgactc gcttccagac cataaccagc tgtcgctgtt t 4301 184 1433 PRT Bacillus stearothermophilus 184 Met Val Thr Lys Glu Gln Lys Glu Arg Phe Leu Ile Leu Leu Glu Gln 1 5 10 15 Leu Lys Met Thr Ser Asp Glu Trp Met Pro His Phe Arg Glu Ala Ala 20 25 30 Ile Arg Lys Val Val Ile Asp Lys Glu Glu Lys Ser Trp His Phe Tyr 35 40 45 Phe Gln Phe Asp Asn Val Leu Pro Val His Val Tyr Lys Thr Phe Ala 50 55 60 Asp Arg Leu Gln Thr Ala Phe Arg His Ile Ala Ala Val Arg His Thr 65 70 75 80 Met Glu Val Glu Ala Pro Arg Val Thr Glu Ala Asp Val Gln Ala Tyr 85 90 95 Trp Pro Leu Cys Leu Ala Glu Leu Gln Glu Gly Met Ser Pro Leu Val 100 105 110 Asp Trp Leu Ser Arg Gln Thr Pro Glu Leu Lys Gly Asn Lys Leu Leu 115 120 125 Val Val Ala Arg His Glu Ala Glu Ala Leu Ala Ile Lys Arg Arg Phe 130 135 140 Ala Lys Lys Ile Ala Asp Val Tyr Ala Ser Phe Gly Phe Pro Pro Leu 145 150 155 160 Gln Leu Asp Val Ser Val Glu Pro Ser Lys Gln Glu Met Glu Gln Phe 165 170 175 Leu Ala Gln Lys Gln Gln Glu Asp Glu Glu Arg Ala Leu Ala Val Leu 180 185 190 Thr Asp Leu Ala Arg Glu Glu Glu Lys Ala Ala Ser Ala Pro Pro Ser 195 200 205 Gly Pro Leu Val Ile Gly Tyr Pro Ile Arg Asp Glu Glu Pro Val Arg 210 215 220 Arg Leu Glu Thr Ile Val Glu Glu Glu Arg Arg Val Val Val Gln Gly 225 230 235 240 Tyr Val Phe Asp Ala Glu Val Ser Glu Leu Lys Ser Gly Arg Thr Leu 245 250 255 Leu Thr Met Lys Ile Thr Asp Tyr Thr Asn Ser Ile Leu Val Lys Met 260 265 270 Phe Ser Arg Asp Lys Glu Asp Ala Glu Leu Met Ser Gly Val Lys Lys 275 280 285 Gly Met Trp Val Lys Val Arg Gly Ser Val Gln Asn Asp Thr Phe Val 290 295 300 Arg Asp Leu Val Ile Ile Ala Asn Asp Leu Asn Glu Ile Ala Ala Asn 305 310 315 320 Glu Arg Gln Asp Thr Ala Pro Glu Gly Glu Lys Arg Val Glu Leu His 325 330 335 Leu His Thr Pro Met Ser Gln Met Asp Ala Val Thr Ser Val Thr Lys 340 345 350 Leu Ile Glu Gln Ala Lys Lys Trp Gly His Pro Ala Ile Ala Val Thr 355 360 365 Asp His Ala Val Val Gln Ser Phe Pro Glu Ala Tyr Ser Ala Ala Lys 370 375 380 Lys His Gly Met Lys Val Ile Tyr Gly Leu Glu Ala Asn Ile Val Asp 385 390 395 400 Asp Gly Val Pro Ile Ala Tyr Asn Glu Thr His Arg Arg Leu Ser Glu 405 410 415 Glu Thr Tyr Val Val Phe Asp Val Glu Thr Thr Gly Leu Ser Ala Val 420 425 430 Tyr Asn Thr Ile Ile Glu Leu Ala Ala Val Lys Val Lys Asp Gly Glu 435 440 445 Ile Ile Asp Arg Phe Met Ser Phe Ala Asn Pro Gly His Pro Leu Ser 450 455 460 Val Thr Thr Met Glu Leu Thr Gly Ile Thr Asp Glu Met Val Lys Asp 465 470 475 480 Ala Pro Lys Pro Asp Glu Val Leu Ala Arg Phe Val Asp Trp Ala Gly 485 490 495 Asp Ala Thr Leu Val Ala His Asn Ala Ser Phe Asp Ile Gly Phe Leu 500 505 510 Asn Ala Gly Leu Ala Arg Met Gly Arg Gly Lys Ile Ala Asn Pro Val 515 520 525 Ile Asp Thr Leu Glu Leu Ala Arg Phe Leu Tyr Pro Asp Leu Lys Asn 530 535 540 His Arg Leu Asn Thr Leu Cys Lys Lys Phe Asp Ile Glu Leu Thr Gln 545 550 555 560 His His Arg Ala Ile Tyr Asp Ala Glu Ala Thr Gly His Leu Leu Met 565 570 575 Arg Leu Leu Lys Glu Ala Glu Glu Arg Gly Ile Leu Phe His Asp Glu 580 585 590 Leu Asn Ser Arg Thr His Ser Glu Ala Ser Tyr Arg Leu Ala Arg Pro 595 600 605 Phe His Val Thr Leu Leu Ala Gln Asn Glu Thr Gly Leu Lys Asn Leu 610 615 620 Phe Lys Leu Val Ser Leu Ser His Ile Gln Tyr Phe His Arg Val Pro 625 630 635 640 Arg Ile Pro Arg Ser Val Leu Val Lys His Arg Asp Gly Leu Leu Val 645 650 655 Gly Ser Gly Cys Asp Lys Gly Glu Leu Phe Asp Asn Leu Ile Gln Lys 660 665 670 Ala Pro Glu Glu Val Glu Asp Ile Ala Arg Phe Tyr Asp Phe Leu Glu 675 680 685 Val His Pro Pro Asp Val Tyr Lys Pro Leu Ile Glu Met Asp Tyr Val 690 695 700 Lys Asp Glu Glu Met Ile Lys Asn Ile Ile Arg Ser Ile Val Ala Leu 705 710 715 720 Gly Glu Lys Leu Asp Ile Pro Val Val Ala Thr Gly Asn Val His Tyr 725 730 735 Leu Asn Pro Glu Asp Lys Ile Tyr Arg Lys Ile Leu Ile His Ser Gln 740 745 750 Gly Gly Ala Asn Pro Leu Asn Arg His Glu Leu Pro Asp Val Tyr Phe 755 760 765 Arg Thr Thr Asn Glu Met Leu Asp Cys Phe Ser Phe Leu Gly Pro Glu 770 775 780 Lys Ala Lys Glu Ile Val Val Asp Asn Thr Gln Lys Ile Ala Ser Leu 785 790 795 800 Ile Gly Asp Val Lys Pro Ile Lys Asp Glu Leu Tyr Thr Pro Arg Ile 805 810 815 Glu Gly Ala Asp Glu Glu Ile Arg Glu Met Ser Tyr Arg Arg Ala Lys 820 825 830 Glu Ile Tyr Gly Asp Pro Leu Pro Lys Leu Val Glu Glu Arg Leu Glu 835 840 845 Lys Glu Leu Lys Ser Ile Ile Gly His Gly Phe Ala Val Ile Tyr Leu 850 855 860 Ile Ser His Lys Leu Val Lys Lys Ser Leu Asp Asp Gly Tyr Leu Val 865 870 875 880 Gly Ser Arg Gly Ser Val Gly Ser Ser Phe Val Ala Thr Met Thr Glu 885 890 895 Ile Thr Glu Val Asn Pro Leu Pro Pro His Tyr Val Cys Pro Asn Cys 900 905 910 Lys His Ser Glu Phe Phe Asn Asp Gly Ser Val Gly Ser Gly Phe Asp 915 920 925 Leu Pro Asp Lys Asn Cys Pro Arg Cys Gly Thr Lys Tyr Lys Lys Asp 930 935 940 Gly His Asp Ile Pro Phe Glu Thr Phe Leu Gly Phe Lys Gly Asp Lys 945 950 955 960 Val Pro Asp Ile Asp Leu Asn Phe Ser Gly Glu Tyr Gln Pro Arg Ala 965 970 975 His Asn Tyr Thr Lys Val Leu Phe Gly Glu Asp Asn Val Tyr Arg Ala 980 985 990 Gly Thr Ile Gly Thr Val Ala Asp Lys Thr Ala Tyr Gly Phe Val Lys 995 1000 1005 Ala Tyr Ala Ser Asp His Asn Leu Glu Leu Arg Gly Ala Glu Ile Asp 1010 1015 1020 Leu Ala Ala Gly Cys Thr Gly Val Lys Arg Thr Thr Gly Gln His Pro 1025 1030 1035 1040 Gly Gly Ile Ile Val Val Pro Asp Tyr Met Glu Ile Tyr Asp Phe Thr 1045 1050 1055 Pro Ile Gln Tyr Pro Ala Asp Asp Thr Ser Ser Glu Trp Arg Thr Thr 1060 1065 1070 His Phe Asp Phe His Ser Ile His Asp Asn Leu Leu Lys Leu Asp Ile 1075 1080 1085 Leu Gly His Asp Asp Pro Thr Val Ile Arg Met Leu Gln Asp Leu Ser 1090 1095 1100 Gly Ile Asp Pro Lys Thr Ile Pro Thr Asp Asp Pro Asp Val Met Gly 1105 1110 1115 1120 Ile Phe Ser Ser Thr Glu Pro Leu Gly Val Thr Pro Glu Gln Ile Met 1125 1130 1135 Cys Asn Val Gly Thr Ile Gly Ile Pro Glu Phe Gly Thr Arg Phe Val 1140 1145 1150 Arg Gln Met Leu Glu Glu Thr Arg Pro Lys Thr Phe Ser Glu Leu Val 1155 1160 1165 Gln Ile Ser Gly Leu Ser His Gly Thr Asp Val Trp Leu Gly Asn Ala 1170 1175 1180 Gln Glu Leu Ile Gln Asn Gly Thr Cys Thr Leu Ser Glu Val Ile Gly 1185 1190 1195 1200 Cys Arg Asp Asp Ile Met Val Tyr Leu Ile Tyr Arg Gly Leu Glu Pro 1205 1210 1215 Ser Leu Ala Phe Lys Ile Met Glu Ser Val Arg Lys Gly Lys Gly Leu 1220 1225 1230 Thr Pro Glu Phe Glu Ala Glu Met Arg Lys His Asp Val Pro Glu Trp 1235 1240 1245 Tyr Ile Asp Ser Cys Lys Lys Ile Lys Tyr Met Phe Pro Lys Ala His 1250 1255 1260 Ala Ala Ala Tyr Val Leu Met Ala Val Arg Ile Ala Tyr Phe Lys Val 1265 1270 1275 1280 His His Pro Leu Leu Tyr Tyr Ala Ser Tyr Phe Thr Val Arg Ala Glu 1285 1290 1295 Asp Phe Asp Leu Asp Ala Met Ile Lys Gly Ser Pro Ala Ile Arg Lys 1300 1305 1310 Arg Ile Glu Glu Ile Asn Ala Lys Gly Ile Gln Ala Thr Ala Lys Glu 1315 1320 1325 Lys Ser Leu Leu Thr Val Leu Glu Val Ala Leu Glu Met Cys Glu Arg 1330 1335 1340 Gly Phe Ser Phe Lys Asn Ile Asp Leu Tyr Arg Ser Gln Ala Thr Glu 1345 1350 1355 1360 Phe Val Ile Asp Gly Asn Ser Leu Ile Pro Pro Phe Asn Ala Ile Pro 1365 1370 1375 Gly Leu Gly Thr Asn Val Ala Gln Ala Ile Val Arg Ala Arg Glu Glu 1380 1385 1390 Gly Glu Phe Leu Ser Lys Glu Asp Leu Gln Gln Arg Gly Lys Leu Ser 1395 1400 1405 Lys Thr Leu Leu Glu Tyr Leu Glu Ser Arg Gly Cys Leu Asp Ser Leu 1410 1415 1420 Pro Asp His Asn Gln Leu Ser Leu Phe 1425 1430 185 199 PRT Thermus thermophilus 185 Thr Pro Lys Gly Lys Asp Leu Val Arg His Leu Glu Asn Arg Ala Lys 1 5 10 15 Arg Leu Gly Leu Arg Leu Pro Gly Gly Val Ala Gln Tyr Leu Ala Ser 20 25 30 Leu Glu Gly Asp Leu Glu Ala Leu Glu Arg Glu Leu Glu Lys Leu Ala 35 40 45 Leu Leu Ser Pro Pro Leu Thr Leu Glu Lys Val Glu Lys Val Val Ala 50 55 60 Leu Arg Pro Pro Leu Thr Gly Phe Asp Leu Val Arg Ser Val Leu Glu 65 70 75 80 Lys Asp Pro Lys Glu Ala Leu Leu Arg Leu Gly Arg Leu Lys Glu Glu 85 90 95 Gly Glu Glu Pro Leu Arg Leu Leu Gly Ala Leu Ser Trp Gln Phe Ala 100 105 110 Leu Leu Ala Arg Ala Phe Phe Leu Leu Arg Glu Met Pro Arg Pro Lys 115 120 125 Glu Glu Asp Leu Ala Arg Leu Glu Ala His Pro Tyr Ala Ala Lys Lys 130 135 140 Ala Leu Leu Glu Ala Ala Arg Arg Leu Thr Glu Glu Ala Leu Lys Glu 145 150 155 160 Ala Leu Asp Ala Leu Met Glu Ala Glu Lys Arg Ala Lys Gly Gly Lys 165 170 175 Asp Pro Trp Leu Ala Leu Glu Ala Ala Val Leu Arg Leu Ala Arg Pro 180 185 190 Ala Gly Gln Pro Arg Val Asp 195 186 27 DNA Artificial Sequence Description of Artificial Sequence PCR primer 186 gcccagtacc tcgcctccct cgagggg 27 187 27 DNA Artificial Sequence Description of Artificial Sequence PCR primer 187 ggcccccttg gccttctcgg cctccat 27 188 331 DNA Thermus thermophilus 188 agactcgagg ccctggagcg ggagctggag aagcttgccc tcctctcccc acccctcacc 60 ctggagaagg tggagaaggt ggtggccctg aggccccccc tcacgggctt tgacctggtg 120 cgctccgtcc tggagaagga ccccaaggag gccctcctgc gcctcaggcg cctcagggag 180 gagggggagg agcccctcag gctcctcggg gccctctcct ggcagttcgc cctcctcgcc 240 cgggccttct tcctcctccg ggaaaacccc aggcccaagg aggaggacct cgcccgcctc 300 gaggcccacc cctacgccgc caagaaggcc a 331 189 110 PRT Thermus thermophilus 189 Arg Leu Glu Ala Leu Glu Arg Glu Leu Glu Lys Leu Ala Leu Leu Ser 1 5 10 15 Pro Pro Leu Thr Leu Glu Lys Val Glu Lys Val Val Ala Leu Arg Pro 20 25 30 Pro Leu Thr Gly Phe Asp Leu Val Arg Ser Val Leu Glu Lys Asp Pro 35 40 45 Lys Glu Ala Leu Leu Arg Leu Arg Arg Leu Arg Glu Glu Gly Glu Glu 50 55 60 Pro Leu Arg Leu Leu Gly Ala Leu Ser Trp Gln Phe Ala Leu Leu Ala 65 70 75 80 Arg Ala Phe Phe Leu Leu Arg Glu Asn Pro Arg Pro Lys Glu Glu Asp 85 90 95 Leu Ala Arg Leu Glu Ala His Pro Tyr Ala Ala Lys Lys Ala 100 105 110 190 31 DNA Artificial Sequence Description of Artificial Sequence PCR primer 190 gtggtgtcta gacatcataa cggttctggc a 31 191 27 DNA Artificial Sequence Description of Artificial Sequence PCR Primer 191 gagggccacc accttctcca ccttctc 27 192 25 DNA Artificial Sequence Description of Artificial Sequence PCR Primer 192 ctccgtcctg gagaaggacc ccaag 25 193 29 DNA Artificial Sequence Description of Artificial Sequence PCR primer 193 cgcgaattca acgcsctcct caagacsct 29 194 31 DNA Artificial Sequence Description of Artificial Sequence PCR primer 194 gacacttaac atatggtcat cgccttcacc g 31 195 38 DNA Artificial Sequence Description of Artificial Sequence PCR primer 195 gtgtgtgaat tcgggtcaac gggcgaggcg gaggaccg 38 196 10 PRT Deinococcus radiodurans 196 Val Ile Leu Asn Pro Gly Ser Val Gly Gln 1 5 10 197 10 PRT Methanococcus jannaschii 197 Tyr Leu Ile Asn Pro Gly Ser Val Gly Gln 1 5 10 198 10 PRT Thermotoga maritima 198 Leu Val Leu Asn Pro Gly Ser Ala Gly Arg 1 5 10 199 28 DNA Artificial Sequence Description of Artificial Sequence PCR primer 199 ctggtgaacc cgggctccgt gggccagc 28 200 10 PRT Artificial Sequence Description of Artificial Sequence polypeptide 200 Leu Leu Val Asn Pro Gly Ser Val Gly Gln 1 5 10 201 27 DNA Artificial Sequence Description of Artificial Sequence PCR primer 201 ctcgaggagc ttgaggaggg tgttggc 27 202 9 PRT Artificial Sequence Description of Artificial Sequence polypeptide 202 Ala Asn Thr Leu Leu Lys Leu Leu Glu 1 5 203 32 PRT Deinococcus radiodurans 203 Gly Phe Gly Gly Val Gln Leu His Ala Ala His Gly Tyr Leu Leu Ser 1 5 10 15 Gln Phe Leu Ser Pro Arg His Asn Val Arg Glu Asp Glu Tyr Gly Gly 20 25 30 204 32 PRT Caenorhabditis elegans 204 Gly Phe Asp Gly Ile Gln Leu His Gly Ala His Gly Tyr Leu Leu Ser 1 5 10 15 Gln Phe Thr Ser Pro Thr Thr Asn Lys Arg Val Asp Lys Tyr Gly Gly 20 25 30 205 32 PRT Pseudomonas aeruginosa 205 Gly Phe Ser Gly Val Glu Ile His Ala Ala His Gly Tyr Leu Leu Ser 1 5 10 15 Gln Phe Leu Ser Pro Leu Ser Asn Arg Arg Ser Asp Ala Trp Gly Gly 20 25 30 206 32 PRT Archaeoglobus fulgidus 206 Gly Phe Asp Ala Val Gln Leu His Ala Ala His Gly Tyr Leu Leu Ser 1 5 10 15 Glu Phe Ile Ser Pro His Val Asn Arg Arg Lys Asp Glu Tyr Gly Gly 20 25 30 207 30 DNA Artificial Sequence Description of Artificial Sequence PCR primer 207 catcctggac tcggcccacc tcctcaccga 30 208 9 PRT Artificial Sequence Description of Artificial Sequence polypeptide 208 Ile Leu Asp Ser Ala His Leu Leu Thr 1 5 209 33 DNA Artificial Sequence Description of Artificial Sequence PCR primer 209 gaggaggtag ccgtgggccg cgtggagctc cac 33 210 11 PRT Artificial Sequence Description of Artificial Sequence polypeptide 210 Val Glu Leu His Ala Ala His Gly Tyr Leu Leu 1 5 10 211 32 DNA Artificial Sequence Description of Artificial Sequence PCR primer 211 ggctttccca tatggctcta cacccggctc ac 32 212 29 DNA Artificial Sequence Description of Artificial Sequence PCR primer 212 gcgtggatcc acggtcatgt ctctaagtc 29

Claims

What is claimed:

1. An isolated Bacillus single-strand binding protein:

(i) comprising the amino acid sequence of SEQ ID NO: 176; or

(ii) being encoded by a nucleic acid molecule hybridizing to the complement of SEQ ID NO: 175 under hybridization conditions comprising at most about 0.9M sodium citrate buffer at a temperature of at least about 37° C.

2. The isolated Bacillus single-strand binding protein according to claim 1 wherein the Bacillus species is Bacillus stearothermophilus.

3. The isolated Bacillus single-strand binding protein according to claim 1 wherein the single-strand binding protein comprises the amino acid sequence of SEQ ID NO: 176.

4. The isolated Bacillus single-strand binding protein according to claim 1 wherein the single-strand binding protein is encoded by a nucleic acid molecule that hybridizes to the complement of SEQ ID NO: 175 under hybridization conditions comprising at most about 0.9M sodium citrate buffer at a temperature of at least about 37° C.

5. The isolated Bacillus single-strand binding protein according to claim 1 wherein the single-strand binding protein is purified.

6. A DNA-protein complex comprising:

a DNA molecule comprising a single-stranded region and a single-strand binding protein according to claim 1 that is bound to the single-stranded region of the DNA molecule.

7. A kit comprising:

a container that contains therein either a deoxynucleoside triphosphate or a dideoxynucleoside triphosphate;

a container that contains therein a DNA polymerase III-type enzyme complex; and

a container that contains therein a single-strand binding protein according to claim 1.