CA2310854C - Isolation and identification of novel polymerases - Google Patents

Abstract

The invention provides purified thermostable enzymes derived from various extremophilic prokaryotic organisms. The enzymes have polymerase activity and can be used to catalyze DNA synthesis by addition of deoxynucleotides to the 3' end of a polynucleotide chain, using complementary polynucleotide strands as a template.

Description

ISOLAT10N AND IDE11'TIFICATION OF 1~'OVEL POLYMERASES
s Field of the Invention This invention relates to newly identified polynucleotides, polypeptides encoded by such polvnucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention have been identified as polymerises ~o Back rg ound of the Invention Thermophilic bacteria have received considerable attention as sources of highly active and thermostable enz5nrzes. Recently, the most extremely thermophilic organotrophic eubacteria presently known have been isolated and characterized. These bacteria, which belong to the genus ~s thermotoga, are fermentative microorganisms metabolizing a variety of carbohydrates (Huber, R.
and Stetter, K.O., in Billows, et al., (Ed.), The Procaryotes. 2nd Ed., Springer-Verlaz, New York, pgs. 3809-3819 (1992)).
In Huber et al., 1986, Arch. Microbiol. 144:324-333, the isolation of the bacterium Thermotoga maritima is described. T. maritima is a eubacterium that is strictly anaerobic, rod-zo .shaped, fermentative, hyperthermophilic, and grows between »°C and 90°C, with an optimum growth temperature of about 80°C. This eubacterium has been isolated from geothermally heated sea floors in Italy and the Azores. T. maritima cells have a sheath-like structure and monotrichous flagellation. T. maritima is classified in the eubacterium kingdom by virtue of having murein and fatty acid-containing lipids, diphtheria-toxin-resistant elongation factor 2.
an RNA polymerise zs subunit pattern, and sensitivity to antibiotics.
Since, to date, most organisms identified from the archaeal domain are thermophiles or hyperthermophiles, archaea are also considered a fertile source of thermophilic enzymes.

Summary of the Invention The present im~ention provides polynucleotides and polypeptides encoded thereby which have been identified as polyerase ertzynes. In accordance with one aspect of the present s invention. there is provided novel enzymes. as well as active fragments.
analogs and derivatives thereof.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encodins enzymes of the present invention including mRNAs. DNAs.
cDNAs. genomic DST ~s as well as active analogs and fragments of such enzynes.
~o In accordance W th another aspect of the present invention there are provided isolated nucleic acid molecules encoding mature polspeptides expressed by the DNA in SEQ
ID'~os:l. 3. s, 7. 9.
11.
In accordance with yet a further aspect of the present invention. there is provided a process for producing such polypeptide by recombinant techniques comprising culturing recombinant is prokar,~otic andior eukaryotic host cells, containing a nucleic acid sequence encodin_ an enzyme of the present invention, under conditions promoting expression of said enzyme and subsequent recovew of said enzwrte.
In accordance with yet a further aspect of the present invention. there is provided a process for utilizing such enzymes, or polvnucleotide encoding such enzymes for polymerizing DNA.
Zo In accordance with yet a further aspect ofthe present invention. there is also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
In accordance with yet a further aspect of the present invention. there is provided a process for utilizing such enzymes, or polynucleotides encoding such enzymes. for in riwo purposes related 2s to scientific research. for example. to generate probes for identifying similar sequences »~hich might encode similar enzymes from other organisms.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
_ Brief Description of the Drawin; s The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims. Sequencing was performed using s a 378 automated DNA sequencer (Applied Bios~stems. Inc.).
FIGURE 1 shows the nucleotide and deduced amino acid sequence of D:~'A
polymerise (3py 1 ) from Ammonifer degensii.
1o FIGURE 2 shows the nucleotide and deduced amino acid sequence of DNA
polymerise ( 1 P1'2) from Pyrolobus fumarius.
FIGURE 3 shows the nucleotide and deduced amino acid sequence of DNA
polymerise (SPY 1 ) from Archaeoglobus lithotrophicus.
FIGURE 4 shows the nucleotide and deduced amino acid sequence of DNA
polymerise (23PY 1 ) from Metallosphaera prunae.
FIGURE ~ shows the nucleotide and deduced amino acid sequence of DNA po-lvmerase (29PY 1 ) from Desulfurococcus.
FIGURE 6 shows the nucleotide and deduced amino acid sequence of DNA
polymerise (34PY1 ) from Aquifex VF-5.
~3-WO 99!07837 PCT/US98/17152 Description of the Preferred Emhudimentc The term "gene" means the segment of Dpi A involved in producing a poivpeptide chain: it includes regions preceding and following the coding region (leader and trailers as well as inten~enin~
s sequences (introns~ betwjeen individual coding se;ments (exons).
-~ coding sequence is "operably linked to" another codin~,~ sequence when R.'~A polvmerase will transcribe the m~o coding sequences into a single mRNA. which is then translated into a single polvpeptide having amino acids derived from both coding sequences. The coding sequences need not he contiguous to one another so long as the expressed sequences are ultimately processed to io produce the desired protein.
"Recombinant" enzymes refer to enzymes produced by recombinant D\'.~
techniques: i.e..
produced from cells transformed by an exogenous D'~:A construct encoding the desired enzyme.
"Synthetic" enzymes are those prepared by chemical sythesis.
.A DNA "coding sequence of or a "nucleotide sequence encoding" a particular enzyme. is ~s a DN A sequence which is transcribed and translated into an enzyme when placed under the control of appropriate regulaton~ sequences. A "promotor sequence" is a DNA. regulatoy region capable of binding RNA poh~merase in a cell and initiating transcription of a dov~mstream (3' direction) coding sequence. The promoter is part of the DNA sequence. This sequence region has a start codon at its 3' terminus. The promoter sequence does include the minimum number of bases 2o where elements necessary to initiate transcription at levels detectable above back_round.
However. after the R.'~'.4 poiymerase binds the sequence and transcription is initiated at the start codon (~' terminus with a promoter). transcription proceeds downstream in the 3' direction.
Within the promotor sequence will be found a transcription initiation site (com°enientlv defined by mapping with nuclease Sl) as well as protein binding domains (consensus sequences) zs responsible for the binding of RNA polvmerase.
The present invention provides purified tltermostable enzymes that catalyze D1T.A synthesis by addition of deo~wnucleotides to the 3' end of a polvnucleotide chain. using a complementary polynucleotide strand as a template. An exemplay purified enzyme is a polymerase derived from an organism referred herein as ".Ammonifex de~ensii KC:~" is a gram negative.
so chemolithoautotrophic eubacteria and has a vey high temperature optimum.
Ammonifex degensii KC4 was discovered in a deep sea isolate from the Middle Atlantic Ride.
Ammonifex de~ensii KCB grovys optimally at 70°C and pl-I 7.0 in a low salt medium. This exemplary enzyme is shown in Fi~~ure 1.
The polvnucleotide encoding SEQ ID NO:I was originally recovered from a genomic gene s libraw derived from Ammonifex degensii 1CC4 as described below. It contains an open reading frame encoding a protein of 867 amino acid residues.
In one embodiment, the representative polymerase of SEQ 1D NO:1 of the present invention has a molecular weight of about 9~.6 kilodaltons as measured by SDS-PAGE ael electrophoresis and an inferred molecular weight from the nucleotide sequence of the gene. This ~o purified enzyme may be used to polymerize DNA where desired. The polymerase enzyme of the present invention has a very high thermostability and has the closest homology to polvmerase from Bacillus steatotherntopltilus with ~6% identity and 7~°~o similariy at the amino acid level.
In accordance with an aspect of the present invention, there are provided isolated nucleic acid molecules (polynucleotides) which encode for the mature enzymes having the deduced amino ~s acid sequence of Figures 1-6 and SEQ ID NOs:l, 3. ~. 7. 9, 11.
This invention. in addition to the isolated nucleic acid molecule encoding an polymerase enzyme disclosed in Figures '1-6 (SEQ ID NOs:I, 3. ~. 7. 9. 11 ). also provides substantially similar sequences. Isolated nucleic acid sequences are substantially similar if: (i) they are capable of hybridizing under stringent conditions, hereinafter described. to SEQ ID NO:1: or (ii j 2o they encode DNA sequences which are degenerate to SEQ ID N0:1. De_enerate DNA sequences encode the amino acid sequence of SEQ ID NO:?. but hare variations in the nucleotide coding sequences. As used herein. "substantially similar" refers to the sequences haying similar identity to the sequences of the instant invention. The nucleotide sequences that are substantially similar can be identified by hybridization or by sequence comparison. Enzyme sequences that are zs substantially similar can be identified by one or more of the following:
proteolyic digestion. Qel electrophoresis and~'or microsequencing. One means for isolating a nucleic acid molecule encoding a polymerise enzyme is to probe a genomic gene library with a natural or artificially designed probe using art recognized procedures (see, for example: Current Protocols in Molecular Biology. Ausubel F h4. et al. (EDS.) Green Publishing Company Assoc.
and Tohn so Wiley Interscience. I\ew fork. 1989, 1992). It is appreciated to one skilled in the art that SEQ
ID NO:1, or fra_ments thereof (comprising at least 10 contiguous nucleotides and at least 70%
-s-complementan~ to a target sequence). is a particularly useful probe. Other particular useful probes f or this purpose are hybridizable fra~:ments to the sequences of SEQ
ID ~ O:1 ( i. e..
comprising at least 10 conti~~uous nucleotides and at least 70°~o complementary to a target sequence).
~~~ith respect to nucleic acid sequences which hybridize to specific nucleic acid sequences disclosed herein. hybridization may be carried out under conditions of reduced stringency.
medium stringency or even stringent conditions. As an example of oligonucieotide hybridization.
a polymer membrane containing immobilized denatured nucleic acid is first prehybridized for 30 ~o minutes at 4s°C in a solution consisting of 0.9 M NaCI. ~0 mM
NaH,PO,. pH 7Ø ~.0 mM
Na,EDT.~. 0.~°/o SDS. IOX Denhardt's: and 0.~ mQ/mL polvriboadenvlic acid. Approximatel~~
? X 10' cpm (specinc activity 4-9 X 16 cpm/~,e) oi'= P end-labeled oli~onucleotide probe are then added to the solution. After 1?-16 hours of incubation, the membrane is washed for 30 minutes at room temperature in 1 X SET ( 1 ~0 mM NaCI, ? 0 mM Tris hydrochloride, pH 7.8. 1 is mM Na,EDTA) containing 0.~% SDS, followed by a 30 minute wash in fresh 1X
SET at Tm-10°C for the oligo-nucleotide probe. The membrane is then exposed to auto-radiographic film for detection of hybridization signals.
In nucleic acid hybridization reactions. the conditions used to achieve a particular level of stringency will vary, depending on the nature of the nucleic acids being hybridized. For example, zo the length. degree of complementariy. nucleotide sequence composition (e.g.. GC u. AT contend.
and nucleic acid type (e.g.. RNA v. DNA) of the hybridizing regions of the nucleic acids can be considered in selecting hybridization conditions. An additional consideration is whether one of the nucleic acids is immobilized. for example. on a filter.
An example of progressively higher stringency conditions is as follow°s: ? x SSC/0.1 °ro SDS
25 at about room temperature (hybridization conditions): 0.2 x SSC/0.1 °ro SDS at about room temperature (low stringency conditions): 0.? x SSC/0.1 % SDS at about 4?
°C (moderate strin,encv conditional: and 0.1 x SSC at about 68°C (high stringency conditional.
Washing can be carried out using only one of these conditions, e.g.. high stringency conditions. or each of the conditions can be used, e.g.. for 10-1 ~ minutes each. in the order listed above, repeating any or all of the steps 30 listed. However. as mentioned above, optimal conditions v~~il1 van', depending on the particular hybridization reaction involved. and can be determined empirically.

"Identiy" as the term is used herein. refers to a polyucleotide sequence which comprises a percentage of the same bases as a reference polynucleotide (SEQ ID NO: l ?.
For example. a s polynucleotide which is at least 90% identical to a reference polwucleotide.
has Folwucleotide bases which are identical in 90% of the bases which make up the reference polvnucleotide and may have different bases in 10% of the bases which comprise that polynucleotide sequence.
The present invention also relates to polynucleotides which differ from the reference pohnucleotide such that the changes are silent changes, for example the changes do not alter the ~o amino acid sequence encoded by the polvnucleotide. The present invention also relates to nucleotide changes which result in amino acid substitutions. additions.
deletions. fusions and truncations in the enzyme encoded by the reference polynucleotide (SEQ ID NO:1 ). In a preferred aspect of the invention these enzymes retain the same biological action as the enzyme encoded by the reference polynucleotide.
~s It is also appreciated that such probes can be and are preferably labeled with an anah~ticallv detectable reagent to facilitate identification of the probe.
Useful reagents include but are not limited to radioactivity. fluorescent dyes or enzymes capable of catalyzing the formation of a detectable product. The probes are thus useful to isolate complementary copies of DNA from other animal sources or to screen such sources for related sequences.
2o The present invention provides substantially pure polymerase enzymes. The term "substantially pure" is used herein to describe a molecule. such as a poly~peptide (e.g.. a poh-tnerase polypeptide. or a fragment thereof) that is substantially free of other proteins. lipids.
carbohydrates. nucleic acids, and other biological materials with which it is naturally associated.
For example, a substantially pure molecule. such as a polypeptide. can be at least 60%, by dry 2s weight, the molecule of interest. The parity of the polypeptides can be determined using standard methods including. e. g.. polyacrylamide gel electrophoresis (e. g.. SDS-P4GE). column chromatography (e. g.. high performance liquid chromatography (HPLC)). and amino- terminal amino acid sequence analysis.
Polymerase polypeptides included in the invention can have one of the amino acid 3o sequences of polymerases shown in Figures 1 through 6 (SEQ ID Nos:2. 4. 6.
8. 10. 1?), for example. the amino acid sequence of Ammonifex degensii KC4 (SEQ ID NO:? 1.
Polymerase WO 99/07837 PCT/(JS98/17152 polvpeptides. such as those isolated from Ammonifex degensii KC4. can be characterized by polymerizin~~ DNA.
Also included in the invention are polypeptides having sequences that are "substantially s identical' to the sequence of a polymerise polypeptide. such as one of SEQ
ID I\'O:'_, e. ,.. SEQ
ID NO::1. A "substantially identical" amino acid sequence is a sequence that differs from a reference sequence only by conservative amino acid substitutions. for example.
substitutions of one amino acid for another of the same class (e.g.. substitution of one hydrophobic amino acid.
such as isoleucine. valine. leucine; or methionine; for another. or substitution of one polar amino ~o acid for another, such as substitution of arginine for lysine. glutamic acid for aspartic acid. or elutamine for aspara~ine). or by one or more non-conservative substitutions.
deletions. or insertions. provided that the polypeptide retains at least one polymerise-specific activim or a polymerise-specific epitope. For example. one or more amino acids can be deleted from a polymerise polypeptide; resulting in modification of the structure of the polypeptide. without ~s si~nificantlv altering its biological activity. For example, amino- or carboxyl-terminal amino acids that are not required for polymerise biological activity; can be removed. Such modifications can result in the development of smaller active polymerise polypeptides.
Other polymerise polypeptides included in the invention are polypeptides having amino acid sequences that are at least 50% identical to the amino acid sequence of a polymerise zo polypeptide. such as any of polymerises in SEQ ID Nos:2. 4. 6. 8, 10. I2, e.g.. SEQ ID NO:1?.
The length of comparison in determining amino acid sequence homology can be.
for example.
at least 1 ~ amino acids. for example. it least 20. 2~. or 35 amino acids.
Homolo~~~ can be measured using standard sequence analysis software (e.g.; Sequence Analysis Sofmare Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center.
1710 University 2s Avenue. Madison. ~T~'I ~370~: also see Ausubel; et al.. supra). Such procedures and algorithms include, for example. a BLAST program (Basic Local Alignment Search Tool at the \ational Center for Biological Information). ALIGN. AMAS (Analysis of Multiply Ali_ned Seauencesl.
AMPS (Protein Multiple Sequence Alignment); ASSET ( Aligned Segment Statistical Evaluation Tool). BANDS. BESTSCOR, BIOSCAN (Biological Sequence Comparative Analysis 'vode);
so BLIA~IPS (BLocks IMProved Searcher). FASTA; Intervals 8: Points. BMB.
CLLTST.~L t:
CLL'STAL V4CONSENSUS, LCONSENSUS; Vs~CONSENSUS, Smith-Waterman algorithm.
_g_ DARV'I~'. Las Ve_tas algorithm, FN AT (Forced Nucleotide .~li~nment Tooll.
Framcali_~n.
Framesewch. DS'NAI\~IIC. FILTER FSAP (Fristensky Sequence Analysis Packa<_e~.
GAP (Global Alignment Programo. GENAL. GIBBS. GenQuest. ISSC (Sensitive Sequence Comparison), s LALIGN (Local Sequence Alignment). LCP (Local Content Program 1. MACAW
(Multiple Alignment Construction &, Analysis Workbench). MAP (Multiple .41i=nment Proeram). MBLhP
I~~IBLhN: PIMA (Pattern-Induced Multi-sequence Alignment). SAGA (Sequence Alignmnet by Genetic .'-lLgorithm) and WHAT IF.
The invention also includes fragments of polymerise polypeptides that retain at least one ~o polymerise-specific activity or epitope. Polymerise activity can be assayed by examining the polymerizing of DIVA. For example. a polymerise polypeptide fragment containing. e. g.. at least s-I U amino acids can be used as an imrnunogen in the production of polymerise-specific antibodies. The fragment can contain, for example. an amino acid sequence that is consen~ed in polymerises. and this amino acid sequence can contain amino acids that are conserved in ~s polymerises. Such fragments can easily be identified by comparing the sequences of polymerises found in Figures 1-X. In addition to their use as peptide immunogens, the above-described polymerise fragments can be used in immunoassays. such as ELISAs. to detect the presence of polymerise-specific antibodies in samples.
The polymerise polypeptides of the invention can be obtained using any of several 2o standard methods. For example, polymerise polypeptides can be produced in a standard recombinant expression systems (see beloy). chemically synthesized (this approach may be limited to small polymerise peptide fragments), or purified from organisms in which they are naturally expressed.
The invention also provides isolated nucleic acid molecules that encode the polymerise 2s polvpeptides described above. as well as fragments thereof. For example.
nucleic acids that encode am~ of SEQ ID NOs:l, 3. ~, 7, 9. 11 are included in the invention.
These nucleic acids can contain naturally occurring nucleotide sequences. or sequences that differ from those of the naturally occurring nucleic acids that encode polymerises. but encode the same amino acids, due to the degeneracy of the genetic code. The nucleic acids of the invention can contain DN A or so RNA nucleotides. or combinations or modifications thereof. Exemplary nucleic acids of the invention are shown in SEQ ID NO:1.
.g.

WO 99107837 PCT/US98l17152 Bv "isolated nucleic acid" is meant a nucleic acid. e.o., a D'M'A or R'~:~
molecule. that is not immediately contiguous with the ~' and 3' f3anlin!, sequences with which it normally is immediately contiguous w.~hen present in the naturally occurring genome of the organism from s which it is derived. The term thus describes. for example, a nucleic acid that is incorporated into a vector. such as a piasmid or viral vector: a nucleic acid that is incorporated into the genome of a heterologous cell (or the genome of a homologous cell. but at a site different from that at which it naturally occursj: and a nucleic acid that exists as a separate molecule. e.g., a DNA
fragment produced by PCR amplification or restriction enzyme digestion. or an R~'A molecule ~o produced by in aitro transcription. The term also describes a recombinant nucleic acid that forms part of a hybrid Gene encoding additional polypeptide sequences that can be used. for example.
in the production of a fusion protein.
The nucleic acid molecules of the invention can be used as templates in standard methods for production of polymerise gene products (e. g. , polymerise RNAs and polvmerase is poiy~peptides). In addition. the nucleic acid molecules that encode polymerise polypeptides (and fragments thereof) and related nucleic acids. such as { 1 ) nucleic acids containing sequences that are complementary to. or that hybridize to. nucleic acids encodin' polymerise polvpeptides, or fragments thereof (e.~.. fragments containing at least 10. 12. 1~, 20. or 25 nucleotidesj: and (2) nucleic acids containing sequences that hybridize to sequences that are complementary to nucleic 2o acids encoding polymerise polypeptides. or fragments thereof (e. g..
fragments containing at least 10. 12. I ~. '_'0. or '_'~ nucleotides): can be used in methods focused on their hybridization properties. For example. as is described in further detail below: such nucleic acid molecules can be used in the following methods: PCR methods for synthesizing polymerise nucleic acids.
methods for detecting the presence of an polymerise nucleic acid in a sample.
screening methods 2s for identiying nucleic acids encoding new polymerise family members.
OIigonucleotide probes useful for screening methods are from 10 to about 1 ~0 nucleotides in lenttth.
Further. such probes are preferably 10 to about 100 nucleotides in length and more preferably from 10 to about ~0 nucleotides in ien~th.
The invention also includes methods for identifying nucleic acid molecules that encode 3o members of the polymerise polypeptide family in addition to SEQ ID NOs:I.
=. =. i. 9. 11. In these methods. a sample, e. g., a nucleic acid library, such as a cDNA
library: that contains a -~ o-nucleic acid encodin'_ a polymerise pol~~peptide is screened with a polymerise-specific probe:
e.g., a polymerise-specific nucleic acid probe. Polvmerase-specific nucleic acid probes are nucleic acid molecules (e.b., molecules containing DIVA or R1v'A nucleotides.
or combinations s or modincations thereof) that specifically hybridize to nucleic acids eneodin~ polymerise polypeptides. or to complementaw sequences thereof. The term "polymerise-specinc probe", in the context of this method of invention. refers to probes that bind to nucleic acids encoding polvmerase polypeptides. or to complementary sequences thereof,. to a detectable greater extent than to nucleic acids encoding other enzymes. or to complementary sequences thereof.
The invention facilitates production of poh~merase-specific nucleic acid probes. Methods for obtaining such probes can be designed based on the amino acid sequences show in Figure 1. The probes. which can contain at least 10. e.g., 15. ?~, 3s. 50. 100. or 150 nucleotides. can be produced using any of several standard methods (see. e.g.. Ausubel, et al., supra). For example. preferabh: the probes are generated using PCR amplification methods.
In these ~s methods, primers are designed that correspond to polymerise-conserved sequences (see Figure 1), v~=hich can include pol~~merase-specific amino acids. and the resulting PCR product is used as a probe to screen a nucleic acid librar~r, such as a eDI~'A librar:
The coding sequences for the polymerise enzymes of the present invention were identified by preparins an Ammonifex degensii KC4 genomic DNA library; for example, and screening the 20 library for the clones having polymerise activity: Such methods for constructing a ~enomic gene library are well-know in the art. One means. for example. comprises shearin_ DNA isolated from Ammonifex degensii KC4 by physical disruption. A small amount of the sheared DNA is checked on an a~arose gel to verify that the majority of the DNA is in the desired size range (appro.~imately 3-6 kb). The DNA is then blunt ended using Mung Bean Nuclease.
incubated at 2s 3TC and phenol/chloroform extracted. The DTiA is then methylated using Eco RI Methylase.
Eco RI linkers are then ligated to the blunt ends through the use of T~ DNA
lipase and incubation at 4'C. The ligation reaction is then terminated and the DNA is cut-back W th Eco R.I restriction enzyme. The DNA is then size fractionated on a sucrose gradient follow~in~
procedures known in the art, for example. Maniatis. T.. et al., Molecular Cloning. Cold Spring so Harbor Press. New fork. 1982.

.~ plate assay is then performed to 'et an approximate concentration of the D1~IA.
Ligation reactions are then performed and 1 pl of the ligation reaction is packs=ed to construct a librar~~. Packagins, for example. may occur throuch the use of purified (i.gtl l phase arms cut s with EcoFl and DNA cut v~ith EcoRI after attaching EcoRI linkers. The D'~A
and (i._tl l arms are ligated with DN A ligase. The Ii~ated DNA is then packaged into infectious phase panicles.
The packased phaYes are used to infect E. coli cultures and the infected cells are spread on agar plates to yield plates carn~ing thousands of individual phage plaques. The library is then amplified.
~o Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA or a ~enomic library to isolate the full length D'~.-~ and to isolate other DN As which have a high sequence similarity to the gene or similar biological activim. Probes of this type have at least 10. preferably at least 15. and even more preferably at least 30 bases and may contain. for example. at least ~0 or more bases. The probe may also be used to identify a DNA
is clone corresponding to a full length transcript and a ~enomic clone or clones that contain the complete gene including regulatory and promotor regions. exons. and introns.
The isolated nucleic acid sequences and other enzymes may then be measured for retention of biological activiy characteristic to the enzyme of the present im~ention, for example. in an assay for detecting enzymatic polymerase activity. Such enzymes include truncated forms of 2o polvmerase. and variants such as deletion and insertion variants.
The polynucleotide of the present invention may be in the form of D:~A which DNA
includes cD?~ta. ~enomic DNA, and synthetic DNA. The DI\.~ may be double-stranded or single-stranded. and if single stranded may be the coding strand or non-coding (anti-sense) strand.
The coding sequence which encodes the mature enzyme may be identical to the coding sequences 2s show in Figures 1-6. or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the 'enetic code. encodes the same mature enzyme as the DNA of Figures I-6 (e.g., SEQ ID NO:1).
The polyucleotide which encodes the mature enzyme of Figure 1 (e.g.. SEQ ID
NO:1) may include. but is not limited to: only the coding sequence for the mature enzyme: the coding so sequence for the mature enzyme and additional coding sequence such as a leader sequence or a WO 99/0783? . PCT/US98/17152 proprotcin sequence: the coding sequence for the mature cnzymc (and optionally additional coding sequence) and non-coding sequence. such as introns or non-coding sequence ~' and%or ~' of the codin<_ sequence for the mature enzyrrte.
Thus. the term "polynucleotide encoding an enzyme (protein)" encompasses a poh~ttucleotide which includes only coding sequence for the enzyme as well as a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention further relates to variants of the hereinabove described polvnucleotides which encode for fragments. analogs and derivatives of the enzyme having the ~o deduced amino acid sequence of Figure 1 (e.g.. SEQ ID N0:? 1. The variant of the polwucieotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurrin; variant of the pol~mucleotide.
Thus. the present invention includes polynucleotides encoding the same mature enzyme as shown in Figure 1 as well as variants of such polynucleotides which variants encode for a ~s fragment. derivative or analog of the enzyme of Figtue 1. Such nucleotide variants include deletion variants. substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shov~~n in Figure 1. ~s know in the art. an allelic variant is an alternate form of a polynucleotide sequence which may have a 20 .substitution. deletion or addition of one or more nucleotides. which does not substantially alter the function of the encoded enzyme.
The present invention also includes polynucleotides. wherein the coding sequence for the mature enzyme may be fused in the same reading frame to a polynucleotide sequence which aids in expression and secretion of an ettzytrte from a host cell. for example. a leader sequence which 2s functions to control transport of an enzyme from the cell. The enzyme having a leader sequence is a preprotein and may have the leader sequence cleaved by the host cell to forth the mature form of the enzyme. The polynucleotides may also encode for a proprotein which is the mature protein plus additional s' amino acid residues. A mature protein having a prosequence is a proprotein and is an inactive form of the protein. Once the prosequence is cleaved an active 3o mature protein remains.
_t;.

Thus. for example, the polynucleotide of the present invention may encode for a mature enzyme. or for an enn me having a prosequence or for an enzyme having both a prosequence and a presequence !leader sequence).
The present im~ention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 70%, preferably at least 90°r'o, and more preferably at least 9~% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polyucleotides. As herein used, the term "stringent conditions" means hybridization will occur ~o only if there is at least 9~% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode enzymes which either retain substantially the same biological function or activity as the mature enzyme encoded by the DNA of Figure 1.
Alternativeh: the pohmucleotide may have at least I ~ bases. preferably at least 30 bases, and more preferably at least 50 bases which hybridize to a poiynucleotide of the present invention and which has an identity thereto, as hereinabove described. and which may or may not retain activity: For example. such polynucleotides may be employed as probes for the polynucleotide of SEQ ID NO:1. for example. for recovery of the polynucleotide or as a PCR
primer.
Thus. the present invention is directed to polynucleotides having at least a 70% identity.
zo preferably at least 90% identity and more preferably at least a 9~%
identity to a polynucleotide which encodes the enzyme of SEQ ID NO:1 as well as fragments thereof. which fragments have at least 30 bases and preferably at least ~0 bases to enzy°mes encoded by such polynucleotides.
The present invention further relates to an enzyme which has the deduced amino acid sequence of Figures 1-6. as well as fragments. analogs and derivatives of such enzyme.
zs The terms "fra~tnent." "derivative" and "analog" when referring to the enzyme of Figure 1 means a enzyme which retains essentially the same biological function or acti~rity as such enzyme. Thus. an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature enzyme.
The enzyme of the present invention may be a recombinant enzyme. a natural enzyme or so a synthetic enzyme. preferably a recombinant enzyme.

WO 99!07837 PCT/US98/17152 The fragment. derivative or analog of the enzyme of Fi_ure 1 may be ( il one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue s may or may not be one encoded by the genetic code. or (iil one in which one or more of the amino acid residues includes a substituent group. or (iii) one in which the mature enzyme is fused v,~ith another compound, such as a compound to increase the half life of the enzyme (for example.
polyethylene glycol). or (iv) one in which the additional amino acids are fused to the mature enzyme. such as a leader or secreton~ sequence or a sequence which is employed for purification to of the mature enzyme or a proprotein sequence. Such fragments. derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The enzymes and polynucleotides of the present invention are preferably provided in an isolated form. and preferably are purified to homogeneity.
The term "isolated" means that the material is removed from its original environment (e.g..
~s the natural environment if it is naturally occurring). For example. a naturally-occurring polynucleotide or enzyme present in a living animal is not isolated. but the same polynucleotide or enzyme. separated from some or all of the coexisting materials in the natural system. is isolated. Such polyucleotides could be part of a vector and/or such polynucleotides or enzymes could be part of a composition, and still be isolated in that such vector or composition is not part 20 of its natural environment.
The enzymes of the present invention include an enzyme of Figures 1-6 (in particular the mature enzyme) as well as enzymes wjhich have at least 70% similarity (preferably at least 70%
identity) to an enzyme of Figures 1-6 and more preferably at least 90°o similarity (more preferably at least 90% identity) to an enzymes of Figures 1-6 and still more preferably at least 2s 9~% similariy (still more preferably at least 9~% identity) to an enzyme of Figures 1-6 and also include portions of such enzymes with such portion of the enzyme eenerally containing at least 30 amino acids and more preferably at least ~0 amino acids.
As know in the art "similarin~" between two enzymes is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one enzyme to the sequence of 3o a second enzyme. Similarity in nucleic acid and amino acid sequences may be determined by procedures and algorithms which are well-known in the art. Such procedures and algorithms -~ s-include. for example. a BL AST program (Basic Local Ali_nment Search Tool at the ?sational Center for Biological Information), ALIGiv. AMAS (Analysis of Multiply Ali«ned Sequences).
AMPS (Protein Multiple Sequence Alignment). ASSET (Aliened Segment Statistical Evaluation Tool 1. BANDS. BESTSCOR. BIOSCA;\' (Biological Sequence Comparative Analysis Node).
BLIMPS (BLocks IMProved Searched. F4STA. Intervals 8: Points. BMB. CLUSTAL ~:
CLL'STAL W.. CONSENSUS. LCONSEIvSUS. Vv'CO?~SENSUS. Smith-Vv'aterman algorithm.
DARVv'II\T. Las degas algorithm, FNAT (Forced Nucleotide Alignment Tool ).
FrameaIign.
Framesearch, DYNAMIC, FILTER, FSAP (Fristensl.~~ Sequence Analysis Packager.
GAP (Global ~o Alignment Program), GENAL, GIBBS. GenQuest. ISSC (Sensitive Sequence Comparison).
LALIGN (Local Sequence Alignment). LCP (Local Content Programl. MACAW
(Multiple Alignment Construction & Analysis Vl~brkbench). MAP (Multiple Alignment Program). MBLKP
MBLKN. PIMA (Pattern-Induced Multi-sequence Alignment). SAGA (Sequence Alignmnet b~~
Genetic ALgorithm) and WHAT IF.
s A variant. i. e. a "fragment". "analo;" or "derivative" enzyme. and reference enzyme may dii~'er in amino acid sequence by one or more substitutions. additions.
deletions. fusions and truncations. which may be present in am' combination.
:4mong preferred variants are those that vary from a reference by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid in a polypeptide by 2o another amino acid of like characteristics. Typically seen as consen~ative substitutions are the replacements. one for another. among the aliphatic amino acids Ala. Val. Leu and Iie: irnerchange of the hvdroa~~l residues Ser and Thr, exchange of the acidic residues Asp and Glu. substitution between the amide residues Asn and Gln. exchange of the basic residues Lys and Arg~ and replacements among the aromatic residues Phe, Tyr.
2s Most highly preferred are variants which retain the same biological function and activity as the reference polypeptide from which it varies.
Fragments or portions of the enzymes of the present invention may be employed for producing the corresponding full-length enzyme by peptide synthesis:
therefore. the fragments may be employed as intermediates for producing the full-length enzymes.
Fragments or portions 30 of the polynucleotides of the present invention may be used to synthesize full-length polvnucleotides of the present invention.
-m The present invention also relates to vectors which include polynucleotides of the present invention. host cells which are ~eneticallv engineered with vectors of the invention and the production of enzymes of the invention by recombinant techniques.
s Host cells are genetically engineered (transduced or transformed or transfected'i with the vectors containing the polynucleotides of this invention. Such vectors may be.
for example. a cloning vector or an expression vector. The vector may be. for example, in the form of a plasmid. a viral particle. a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or ~o ampliyin~ the Genes of the present invention. The culture conditions. such as temperature, pH
and the like. are those previously used with the host cell selected for expression. and will he apparent to the ordinarily skilled artisan.
The polyucleotides of the present invention may be employed for producing enzymes by recombinant techniques. Thus. for example. the polynucleotide may be included in any one of ~s a variety of expression vectors for expressing an enzyme. Such vectors include chromosomal.
nonchromosomal and synthetic DNA sequences, e.g.. derivatives of SV40:
bacterial plasmids:
phage DNA: baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DN A. viral DNA such as vaccinia. adenovirus. fowl pox virus. and pseudorabies.
However. any other vector may be used as long as it is replicable and viable in the host.
2o The appropriate DNA sequence may be inserted into the vector by a variet~~
of procedures.
In seneral. the DNA sequence is inserted into an appropriate restriction endonuclease sites) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
The DNA sequence in the expression vector is operatively linked to an appropriate zs expression control sequences) (promoter) to direct mRNA synthesis. As representative examples of such promoters. there may be mentioned: LTR or SV.~O promoter. the E. coli.
lac or trp. the phage lambda PL promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include so appropriate sequences for amplifying expression.
_o.

WO 99/07837 PCT/US98l17152 In addition. the expression vectors preferably contain one or more selectable marker genes to proviae a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture. or such as tetracycline or ampicillin s resistance in L~. coli.
The vector containing the appropriate DI\A sequence as hereinabove described.
as well as an appropriate promoter or convol sequence. may be employed to vansform an appropriate host to permit the host to express the protein.
As representative examples of appropriate hosts. there may be mentioned:
bacterial cells.
~o such as E. coli. Sveptomyces, Bacillus subtilis; fungal cells. such as yeast: insect cells such as Drosophila S2 and SDOdoptera Sf~: animal cells such as CHO. COS or Bowes melanoma:
adenoviruses; plant cells. etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly the present invention also includes recombinant consvucts comprising ~s one or more of the sequences as broadly described above. The consvucts comprise a vector. such as a plasmid or viral vector. into which a sequence of the invention has been inserted. in a forward or reverse orientation. In a preferred aspect of this embodiment. the consvuct further comprises regulatory sequences, including, for example. a promoter. operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the 2o art. and are commercially available. The following vectors are provided by way of example;
Bacterial: pQE70. pQE60. pQE-9 (Qiagenj, pBluescript Ii (Stratagene): pTRC99a.
phh2?3-s.
pDR~40. pRIT?T (Phatmacia); Eukaryotic: pXTl, pSG~ (Strataeene) pSVh3. pBPV
pMSG, pSVLSV.~O (Pharmaciaj. However, any other plasmid or vector may be used as long as they are replicable and viable in the host.
2s Promoter regions can be selected from any desired gene using CAT
(chloramphenicol vansferase) vectors or other vectors with selectable markers. Two appropriate vectors are pKK333-8 and pCM7. Particular named bacterial promoters include laci. lacZ.
T3. T7. gpt.
lambda PR. P~ and ttp. Eukaryotic promoters include CMV immediate early: HSV
thymidine kinase. early and late SV40, LTRs from revovirus, and mouse metallothionein-I.
Selection of 3o the appropriate vector and promoter is well within the level of ordinary skill in the art.
_t s.

In a further embodiment. the present im~ention relates to host cells containing the above-described conswets. The host cell can be a higher eukaryotic cell, such as a mtunmalian cell, or a lower eukaryotie cell. such as a yeast cell. or the host cell can be a prokaryotic cell, s such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection. DEAF-Dextran mediated uansfection. or electroporation (Davis, L..
Dibner. ~~i.. Batted; L, Basic Methods in Molecular Biology, (1986)).
The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the enzymes of the invention can ~o be synthetically produced by conventional peptide synthesizers.
!~4ature proteins can be e.~;pressed in mammalian cells, yeast. bacteria. or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are is described by Sambrook et al., Molecular Cloning: A Laborato»~ Manual, Second Edition. Cold Sprin' Harbor, N.Y, ( 1989).
Transcription of the DNA encoding the enzymes of the present invention by higher eukar~~otes is increased by inserting an enhancer sequence into the vector.
Enhancers are cis-cretins elements of DNA, usually about from 10 to 300 by that act on a promoter to increase Zo its transcription. Examples include the SV40 enhancer on the late side of the replication origin by 100 to '_70. a cytomegalovirus early promoter enhancer. the pnh~oma enhancer on the late side of the replication origin, and adenovirus enhancers.
Generalh: recombinant expression vectors W 11 include ori~~ins of replication and selectable markers p~rrnitting transformation of the host cell, e.g., the ampicillin resistance gene of E. colt 25 and S. cerevisiae TRPI gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream smictu~ral sequence. Such promoters can be derived from operons encoding glycolvtic enzymes such as 3-phospho~lycerate kinase (PGh'.). a-factor. acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences. and 3o preferabh: a leader sequence capable of direcLn~ secretion of translated enzyme. Optionally; the heterolo=ous sequence can encode a fusion enzyme including an N-terminal identification peptide impartin~_ desired characteristics. e.~=., stabilization or simplified purification of expressed recombinant product.
Useful expression vectors for bacterial use are constructed by inserting a structural DNA
sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase W th a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to. if desirable, provide amplification within the host. Suitable prokan~otic hosts for ~o transformation include E. coli, Bacillus subtilis, Salmonella typlzimurium and various species within the genera Pseudomonas. Streptomyces. and Staphylococcus. although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially is available plasmids comprising genetic elements of the well known cloning vector pBR3?? {.4TCC
37017). Such commercial vectors include, for example. phIC2,23-3 (Pharmacia Fine Chemicals, Uppsala. Sweden) and GEMI (Pmmega Biotec. Madison. V~%I. USA). These pBR3'_'_' "backbone"
sections are combined with an appropriate promoter and the structural sequence to be expressed.
Following transformation of a suitable host strain and growth of the host strain to an 2o appropriate cell density; the selected promoter is induced by appropriate means (e.~.. temperature shift or chemical induction) and cells are cultured for an additional period.
Cells are n~pically harvested by centrifugation. disrupted by physical or chemical means.
and the resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any convenient 23 method. including freeze-thaw cycling, sonication, mechanical disruption.
or use of cell lysing agents. such methods are well known to those skilled in the art.
t%arious mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian ea-pression systems include the COS-7 lines of monkey kidney fibroblans. described by Gluzman, Cell. ?3:17 (1981), and other cell lines capable of expressing so a compatible vector; for example, the C127, 3T3. CHO. HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer. and -20.

also am~ necessay ribosome binding sites, polyadenylation site, splice donor and acceptor sites.
transcriptional termination sequences, and ~' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice. and polyadenylation sites may be used to provide the required s nontranscribed genetic elements.
The enzyme can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction. anion or canon exchan_e chromatography, phosphocellulose chromatography: hydrophobic interaction chromatography, affinit,~ chromatography. hydroxylapatite chromatography and lectin chromatography. Protein ~o refolding steps can be used, as necessary; in completing configuration of the mature protein.
Finalh: high performance liquid chromatography (HPLC) can be employed for final purification steps.
The enzymes of the present invention may be a natwally purified product. or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or ~s eukaryotic host (for example, by bacterial, yeast. higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedwe, the enzymes of the present invention may be glycosylated or may be non-glycosylated.
Enzymes of the invention may or may not also include an initial methionine amino acid residue.
The enzyme of this invention may be employed for any purpose in which such enzyme zo activity is necessary or desired. In a preferred embodiment the enzyme is employed for catalyzin_ D1~:4 synthesis by addition of deoxvnucleotides to the 3' end of a polynucleotide chain. using a complementary polynucleotide strand as a template.
In a preferred embodiment, the enzyme of the present invention is a thermostable enzyme which is stable to heat and is heat resistant and polymerizes DNA. i.e., the enzyme is able to Zs renatwe and regain activity after a brief (i. e., ~ to 30 seconds), or longer period. for example, minutes or hows. exposure to temperatwes of up to 70'C and has a temperature optimum above 60'C.
The enzymes. their fragments or other derivatives, or analogs thereof. or cells expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be. for 3o example. polyclonal or monoclonal antibodies. The present invention also includes chimeric.
single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression libraw. \~arious procedures know in the art may be used for the production of such antibodies and fras~ments.
antibodies generated against the enzymes correspondinL to a sequence of the present s invention ca~~ be obtained byr direct injection of the enzymes into an animal or by administerint the enr~mes to an animal. preferably a nonhuman. The antibody so obtained wilt then bind the enzymes itself. In this manner. even a sequence encoding only a fragment of the enr~mes can be used to generate antibodies binding the whole native enzymes. Such antibodies can then be used to isolate the enz~nne from cells expressing that enzyme. .
For preparation of monoclonal antibodies. any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hvbridoma technique ~l~ohler and Milstein. 197. Nature, ?~6:49~-497), the trioma technique. the human B-cell hybridoma technique (hozbor et al.. 1983, Immunoloav Today 4:72). and the EBV hyridoma technique to produce human monoclonal antibodies (Cole, et al..
1985, in ~s Monoclonal Antibodies and Cancer Therapy, Alan R. Liss. Inc.. pp. 77-96).
T:chniques described for the production of single chain antibodies (U.S.
Patent 4.946.778) can be adapted to produce single chain antibodies to immunogenic enzyme products of this invention. Also. transgenic mice may be used to express humanized antibodies to immunogenic enzyme products of this invention.
2o Antibodies =enerated against the enzyme of the present invention may be used in screenins for similar enzymes from other organisms and samples. Such screening techniques are Imow in the art. for example. one such screening assay is described in "Methods for Measuring Cellulose Activities". MetJ:ods in F.ynwlogy, bbl 160, pp. 87-1I6.
Antibodies may also be employed as a probe to screen gene libraries 2s generated from this or other organisms to identify this or cross reactive activities.
Isolation and purification of polypeptides produced in the systems described above can be carried out using conventional methods., appropriate for the particular system. For example, preparative chromatography and immunological separations employin_ antibodies.
sych as .
monoclonal or pol~~clonal antibodies. can be used.
3o Tne term "antibody:" as used herein. refers to intact immunoglobulin molecules. as well as fragments of immunoglobulin molecules, such as Fab. Fab'. (Fob' ),. F~: and SC A frasmenu.

that are capable of bindin' to an epitope of a polymerise polypeptide. These antibody fragments, which retain some abiliy to selectively: bind to the antigen (e.g.. a polymerise antigen) of the antibody from which they are derived, can be made using well known methods in the art (see, e. g.. Harlow and Lane, supra). and are described further. as follows.
( 1 ) A Fab fragment consists of a monovalent antigen-binding fragment of an antibody molecule. and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain.

(2) A Fab' fragment of an antibody molecule can be obtained by treating a whole antibody molecule W th pepsin, followed by reduction. to yield a molecule consisting of an intact light chain and a portion of a heave chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.

(3) A (Fib'), fragment of an antibody can be obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. A (Fib'), fragment is a dimer of two Fab' fragments, held together by two disulfide bonds.

(4) An Fv fragment is defined as a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.
20 (5) A single chain antibody ("SCA") is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain. linked by a suitable, flexible polypeptide linker.
As used in this invention, the term "epitope" refers to an antigenic determinant on an antigen. such as a polymerise polypeptide, to which the paratope of an antibody; such as a 2s poh~rrterase-specific antibody binds. Antigenic determinants usually consist of chemically active surface groupings of molecules, such as amino acids or sugar side chains. and can have specific three-dimensional structural characteristics. as well as specific charge characteristics.
As is mentioned above, antigens that can be used in producing polymerise-specific antibodies include polymerise polypeptides, e.g., any of the polymerises shown in Figures 1-X
3o polypeptide fragments. The polypeptide or peptide used to immunize an animal can be obtained by standard recombinant, chemical synthetic, or purification methods. As is well known in the *rB

WO 99/07837 PCTlUS98/17152 art, in order to increase immuno~_eniciy. an antigen can be conjugated to a carrier protein.
Commonly used carriers include keyhole limpet hemocyanin (KLH). thyroglobulin.
bovine serum albtunin (BSA). and tetanus toxoid. The coupled peptide is then used to immunize the animal s (e. g.. a mouse. a rat. or a rabbit). In addition to such carriers. well knoum adjuvants can be administered W th the antigen to facilitate induction of a strong immune response.
Polvlnerase-specific polvclonal and monoclonal antibodies can be purified. for example.
by binding to. and elution from, a matrix containing a polyrrterase polvpeptide. e.g.. the pohmlerase polypeptide (or fragment thereof) to which the antibodies were raised. Additional io methods for antibody purification and concentration are well known in the art and can be practiced with the polwtterase-specific antibodies of the invention (see. for example. Coligan. et al.. Unit 9. Current Protocols in Immunologa. Wiley Interscience. 1994).
Anti-idiotype antibodies corresponding to polymerise-specific antigens are also included in the invention, and can be produced using standard methods. These antibodies are raised to ~s polymerise-specific antibodies. and thus mimic polymerise-specific epitopes.
The members of a pair of molecules (e.g.. an antibody-antigen pair or a nucleic acid pair) are said to "specifically bind" to each other if they bind to each other with greater affiniy~ than to other, non-specific molecules. For example. an antibody raised against an antigen to which it binds more efficiently than to a non-specific protein can be described as specificall~~ binding 20 .to the antigen. (Similarly, a nucleic acid probe can be described as specifically binding to a nucleic acid target if it forms a specific duplex with the target by base pairing interactions tsee above).) The present invention is further described W th reference to the following examples:
however, it is to be understood that the present invention is not limited to such examples. All 2s parts or amounts, unless otherwise specified. are by weight.
In one aspect of the invention. a method for producing a polymerise enzyme.
such as those show in Figures 1-6. is provided. The method includes crow ng a host cell which comains a polynucleotide encoding the enzyme (e.g., SEQ ID Nos:2. 4, 6. 8. 10. 12).
under conditions which allow the expression of the nucleic acid. and isolating the enzyme encoded by the nucleic ao acid. A~ethods of culturing the host cell are described in the Examples and are know by those of skill in the art.

In order to facilitate understanding of the follow ng examples certain frequently occurring methods andior terms will be described.
"Plasmids" are designated by a lower case p preceded andior followed by capital letters s andior numbers. The starting plasmids herein are either commercially available. publicly a~~ailable on an unrestricted basis. or can be constructed from available plasmids in accord wide published procedures. In addition, equivalent plasmids to those described are l:nov~~n in the art and will be apparent to the ordinarily skilled artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA W th a restriction enzyme that ~o acts only at certain sequences in the DN A. The various restriction enzymes used herein are commercially available and their reaction conditions. cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For anah~tical purposes.
mpicaIlv 1 ug of plasmid or DrA fragment is used with about ? units of enzyme in about 20 ~l of buffer solution.
For the purpose of isolating DNA fragments for plasmid construction. wpicallv 5 to ~0 ug of ~s Dr'.~ are digested W th 20 to 2~0 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular restriction enrymes are specified by the manufacturer. lncubation times of about 1 hour at 37°C are ordinarily used, but may vary in accordance with the supplier's instructions. After digestion the reaction is electrophoresed directly on a polyacrvlamide gel to isolate the desired fragment.
zo Size separation of the cleaved fragments is generally performed using 8 percent polyacrylamide gel described by Goeddel, D. et al., Nucleic Acids Res.. 8;407 (1980), for example.
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized.
Such 2s s<nthetic oligonucleotides may or may not have a ~' phosphate. Those that do not will not ligate to another oligonucleotide writhout adding a phosphate with an ATP in the presence of a kinase.
A synthetic oligonucleotide will ligate to a fragment that has not been dephosphoy fated.
"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. 146). Unless otherwise provided.
30 ligation may be accomplished using known buffers and conditions with 10 units of T=1 DNA
_2 ~.

ligase ("lipase") per ~.~ p.a of approximately equimolar amounts of the DNA
fras:ments to be lieated.
Unless otherwise stated. transformation was performed as described in the method of s Sambrook. Fritsch and Maniatis. 1989. The following examples are intended to illustrate. but not to limit. the invention. VVltile the procedures described in the eaatnples are ypical of those that can be used to cam' out certain aspects of the invention, other procedures known to those skilled in the art can also be used. The following materials and methods were used in carrying out the experiments described in the examples.

Example l DNA Isolation and Library Construction The following outlines the procedures used to generate a gene library from a sample.
Isolate DNA.
IsoQuick Procedure as per manufacturer's instructions (Orca. Research Inc..
Botheil. WA).
Sbear DNA
~ngorously push and pull DNA through a 2SG double-hub needle and 1-cc syringes about X00 times.
Check a small amount (0.5 fig) on a 0.8% agarose gel to make sure the majorit,~ of the DNA is in the desired size range (about 3-6 kb).
~5 Blunt DNA
Add:
I-hO to a final volume of 40~ pl 4S ~l lOX Mung Bean Buffer 3.0 ~l Mung Bean Nuclease ( 1 SO u/~el) 2o Incubate 37°C, 1 ~ minutes.
Phenol/chloroform extract once.
Chloroform extract once.
Add 1 ml ice cold ethanol- to precipitate.
Place on ice for 10 minutes.
25 Spin in microfuge, high speed. 30 minutes.
Wash with 1 ml 70% ethanol.
Spin in microfuge, high speed. 10 minutes and dn~.

Mcthvlate DNA
Gently resuspend DIVA in ?6 ~l TE.
Add:
s 4.0 ~tl I OX EcoR I Methylase Buffer 0.~ ~tl SAM (33 mM) ~.0 ~tl EcoR I Methylase (40 u/~l) Incubate 37°. 1 hour.
Insure Blunt Ends Add to the methylation reaction:
5.0 ~tl 100 mM MQCh 8.0 ~tl dNTP min (2.~ mM of each dGTP, d.ATE dTTP dCTP) 4.0 ~tl Klenow (~ u/pl) incubate 12°C, 30 minutes.
Add 4~0 ~l 1X STE.
Phenol/chloroform extract once.
Chloroform extract once.
Add 1 ml ice cold ethanol to precipitate and place on ice for 10 minutes.
Spin in microfuge, high speed. 30 minutes.
2o Wash with 1 ml 70% ethanol.
Spin in microfu~e. high speed. 10 minutes and dry.
Adaptor Ligation Gently resuspend DNA in 8 ~.1 EcoR I adaptors (from Stratagene's cDN A
Synthesis Kit).
Add:
1.0 p,l l OX Ligation Buffer 1.0 ~l 10 mM rATP
1.0 ~.l T4 DNA Lipase (4 ul~.l) Incubate 4°C. ~ days.
so *rB

Phosphor~~latc Adaptors Heat kill li<,ation reaction 70°C. 30 minutes.
Add:
1.0 pl lOX Libation Buffer ?.0 ~.1 lOmM rATP

6.0 p.l H,O
1.0 pl Polynucleotide kinase (PNK) Incubate 37°C. 30 minutes.
Add 31 ~tl H,O and ~ ~ I l OX STE.
Size fractionate on a Sephacryl S-X00 spin column (pool fractions 1-3).
Phenollchloroform extract once.
Chloroform ea~tract once.
Add ice cold ethanol to precipitate. ' Place on ice, 10 minutes.
Spin in microfuge, high speed. 30 minutes.
Wash with 1 m! 70% ethanol.
Spin in microfuge, high speed. 10 minutes and dry Resuspend in 10.5 p,t TE buffer.
2o Do not plate assay Instead_ ligate directly to arms as above except use 3.~
~1 of DNA and no water.
Sucrose Gradient (2.2 ml) Size Fractionation Heat sample to 65°C, 10 minutes.
23 Gently load on 2.2 ml sucrose gradient.
Spin in mini-ultracentrifuge, 45h. 20°C. 4 hours (no brake).
Collect fractions by puncturing the bottom of the gradient tube v~zth a 30G
needle and allowin, the sucrose to flow through the needle. Collect the first 20 drops in a FalconTM
309 tube then collect 10 1-drop fractions (labelled i-10). Each drop is about 60 ~1 in so volume.
Run ~ ~tl of each fraction on a 0:8% agarose gel to check the size.

Pool fractions 1-4 (about 10-1.5 kb) and, in a separate tube. pool fractions s-7 (about ~-0.5 kb).
Add 1 ml ice cold ethanol to precipitate and place on ice for 10 minutes.
s Spin in microfuge. high speed, 30 minutes.
~~~ash with 1 ml 70% ethanol.
Spin in microfuge, high speed. 10 minutes and dr~~.
Resuspend each in 10 ~l TE buffer.
~o Test Ligation to Lambda Arms Plate assa~~ to get an approximate concentration. Spot 0.~ pl of the sample on anarose containing ethidium bromide along with standards (DNA samples of know concentration). View in UV light and estimate concentration compared to the standards. Fraction 1-4 = >I.0 pg/p,l.
Fraction 5-7 = 500 ng/ltl.
~s Prepare the following Iigation reactions (~ p,l reactions) and incubate 4°C. overnight:
Sample H=O lOX lOmM Lambd Insert T4 DNA

Ligase rATP a arms DNA Ligase (4 Buffer (ZAP) u/~l) Fraction 1-4 O.Sp,I 0.51 O.Sltl 1.0~t1 2.O~tl O.~p,l Fraction ~-7 O.Sp.I O.S~tI O.S~tI 1.01 2.Owl O.SpI

Test Package and Plate Package the Iigation reactions following manufacturer's protocol.
Stop packaging reactions with 500 p.l SM buffer and pool packaging that came from the same Iisation.
2s Titer 1.0 ~1 of each on appropriate host (ODboo = 1.0) [XLI-Blue MRFj Add 200 p,l host (in mM MgS04) to Falcon 2059 tubes Inoculate with 1 ~1 packaged phage Incubate 37°C. 1 ~ minutes Add about 3 ml 48°C top agar so [SOmI stock containing 150 ~l IPTG (O.SM) and 300 ~1~ X-GAL (350 mg/ml)]
Plate on 100mm plates and incubate 37°C, overnight.

Amplification of Libraries (5.0 a 105 recombinants from each library) Add 3.0 ml host cells (ODbou I.0) to two ~0 ml conical tube.
Inoculate with 2.5 X 105 pfu per conical tube.
s Incubate 37°C. 20 minutes.
Add top agar to each tube to a final volume of 4~ ml.
Plate the tube across five 1 ~0 mm plates.
Incubate 37°C, 6-8 hours or until plaques are about pin-head in size.
Overlay with 8-10 ml SM Buffer and place at 4°C overnight (with gentle rocking if ~o possible).
Har~~est Phage Recover phage suspension by pouring the SM buffer off each plate into a ~0 ml conical tube.
~s Add 3 ml chloroform, shake vigorously and incubate at room temperature, 1 ~
minutes.
Centrifuge at 2K rpm, l0 minutes to remove cell debris.
Pour supernatant into a sterile flask, add X00 ~l chloroform.
Store at 4°C.
2o Titer Amplified Library Make serial dilutions:
10'5= I ~1 amplified phage in 1 ml SM Buffer 10'6= I ~,l of the 10' dilution in I ml SM Buffer Add 200 ~1 host (in 10 mM MeSO;) to two tubes.
zs Inoculate one with 10 ~1 I 0'6 dilution ( I ~5 ).
Inoculate the other with I ~1 10'6 dilution (IU6).
Incubate 37°C, 1 ~ minutes.
Add about 3 ml 48°C top agar.
[~Oml stock containing I~0 ~.I IPTG (O.~M) and 37~ ~1 X-GAL (3s0 mg/ml)]
so Plate on 100 mm plates and incubate 37°C, overnight.

Excise the ZAP II librar;~ to create the pBluescript library according to manufacturers protocols (Stratagene).
s Example 2 .4ctivated Calf Thymus DNA Polvmerase Assay Streak out the clone to isolation:
1. Inoculate ~ml LB/Amp/Meth/Kan culture with isolated clone 2. Grow to turbidity ~0 3. Inoculate a ~Oml culture of LB/Amp/Meth/Kan Grow to OD600 of 0.7 to 0.9: induce culture with IPTG at a final concentration of 1 mM for 3 hours Centrifuge at 400 RPM for 20 minutes and discard supernate Resuspend pellet in 3mls of 20mM Tris pH 8.0 and sonicate twice for 1 minute each ~s Microcentrifu~e lml of sonicate for 30 minutes at 4°C
Remove 1 ul of the sonicate supernatent and add to 101 of the following Activated Calf Thymus Reaction Cocktail in a O.~ml eppendorf:
~ units/ml activated calf thymus DNA (Pharmacia 27-457-O1 ) 1 mMDTT
20 40mg/ml BSA
~OuM dATP ~OuM dCTP, SOuM dGTP, SuM dTTP
~OmM Tris pH 7.6 ~mM MaC1?
~O~Ci/ml H'-dTTP
2s bring to volume with H20 Incubate at 70°C for 10-30 minutes Stop reaction by cooling the tube Spot sample onto Whatrnan DE-81 filter paper (catalog#368-323) Dry completely 3o Vvash filters in 2X SSC five times for 2 minutes each Final wash in 100% ethanol to remove most of remaining water *rB

Allow the filters to dry to completion Count incorporation of H'-dTTP using a scintillation counter s The incorporation of nucleotides by the polymerase is proportional to counts, by at least five fold over background. (Maki,H, et al. .T.Biol.Chem. (1988) 263:670-6578 and Tabor. et al, US Patent 4791699).
Example 3 PCR Screening Polymerase sequences from Therntococcus litoralis, Pvrococcus GB-D (Deep Vent j.
and Pvrococcus furiosus were scanned to determine conserved regions. The following nucleic acid sequences were identified and corresponding amino acid sequences were utilized to derive degenerate oligonucleotide primers to be used in downstream screening:
~s Thermococcus litoralis: 37-45, 1045-1051 Pvrococcus GB-D (Deep Vent): 37-45, 1042-1049 P~.~rococcus furiosus: ~ 37-45, 505-512 The following corresponding amino acid sequences were used to produce degenerate 20 oligonucleotide primers:
YIl'.ALL''/RDD
WY~!SKECAE
2s The primers have been labeled Poldgenl forward and Poldgen2 reverse:
Poldgenl forward (26mer): 5'-TAc/TAT"/TTAc/TGCTCTc/TCTCA"/GAGATGA-3' Poldgen? reverse (23mer): 5'-TC"ITGC"/GCAc/TTCc/TTT:4CA~~/GTACCA-3' These primers were used to amplify potential polymerase genes directly from genomic 3o DNA (Template DN.4).

l0U~t1 PCR conditions:
1 ~tl Poldgen 1 forward (~OOns~/p.l) 1 ~1 Poldgen2 reverse (SOOnel~,l j s 1 ~.l ?~mM dNTP mix .
1 pl Template DNA (~l OOn~/~.11 1 ~.1 TaqPlus Polymerase (Strata?ene j l0y~l lOX low salt reaction buffer (Stratagene) 8~~.1 H:O
Number of Temperature Time Cycles 9~C 30 seconds 42C 30 seconds 72C 2 minutes, 30 seconds 30 95C 30 seconds 50C 30 seconds 72C '? minutes, 30 seconds 1 72C 10 minutes PCR products (l.4kb bands from both organisms) were phenol chloroform extracted (ref. Maniatis) and cloned using the TA cloning system into the pGemT PCR
Cloning Vector (Promega) using the following ligation reaction:
2o O.~p.l pGemT Cloning Vector (~OngJ~l) 2pl PCR Product (~1000n~,~.1~.1) 2ltl rATP (lOmM) ?~l lOX T4 Liease Buffer 1 ~.l T4 Liaase 13.5 ~1 H,O
Incubate 4°C overnight.

?.~~.1 of the above reaction was transformed into XL1-Blue MRF' competent cells (Strata~ene) l.4kb PCR products were also restriction analyzed using the appropriate restriction enzymes:
Potential clones were verified by restriction analysis and sequenced.
BLASTX and BLASTN database comparisons of the sequences indicated whether the sequences were homologous to the nucleic acid sequence of a knov~~n polymerase from another organism. Amplification primers were then generated to both ends of the known polymerase ~o gene. and were used in an amplification reaction on the genomic DNA in an attempt to pull out a full length polymerase gene from this organism. These primers include restriction sites and a new Ribosome Binding Site for downstream processing of the gene:
PCR Conditions:
~s 1 ~.l forward primer (250ng/ltl) l ltl reverse primer (250ng/~,l) 1 ~.l 2~mM dNTP
1 p.l template DNA ( 1 OOng/~tl) 1 ~l Taq polymerase 20 l0~tl lOX Taq Buffer 8~~1 H,O

WO 99!07837 PCTIUS98/17152 Number of Temperature Time Cycles s 2 9~C 30 seconds 42C 30 seconds 72C 2 minutes. 30 seconds 30 9~C 30 seconds ~0C 30 seconds 72C 2 minutes, 30 seconds GENE LIBRARY SCREE:~'ING
PCR products generated in the above reactions (using degenerate primers) were used to 1o make long "run off' single stranded DNA probes using (P'~ as a label).
The genomic library was screened using the single stranded (P''- labeled probe.
Hybridization conditions for these screenings were as per Maniatis (maximum stringency for aqueous solutions: 68°C in rolling hybridization chamber).
All positive clones were then excised into pBluescript SK and seduenced.
Example 4 Expression Positive clones were identified and isolated from the genomic library by the above methods. DNA from the clones were then used as templates in a 100 ul PCR
reaction. DI\TA
2o encoding the enzymes of the present invention were initially amplified from a pBluescript vector containing the DNA by the PCR technique. The amplified sequences were then inserted into a PQE vector and the enzyme was expressed according to the protocols set forth herein.
The pQE vector (Qiagen, Inc. Chatsworth. CA) encodes antibiotic resistance (Amp'). a bacterial origin of replication (ori), an IPTG-regulatable promoter operator (P/O), a ribosome 2s binding site (RBS). a 6-His tag and restriction enzyme sites.
The pQE vector was digested with the appropriate restriction enzymes. The amplified sequences were ligated into the respective pQE vector and inserted in frame with the sequence encoding for the PvBS. The ligation mixture was then used to transform the E.
cali strain M 1 ~IpREP4 (Qia~en. lne.) by electroporation. M 1 ~/pREP4 contains multiple copies of the plasmid pREP~. wrhich expresses the lacl repressor and also confers kanamycin resistance (Kan').
s Transformants were identified by their ability to ~row~ on LB plates and ampicillin~kanamvcin resistant colonies were selected. Plasmid DNA was isolated and confirmed by restriction analysis. Clones containing the desired constructs were grown overnight (O/Iv) in liquid culture in LB media supplemented with both Amp (100 ltg/ml) and Kan (2~ ~tJml). The O/N culture was used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells were grown to an ~o optical density 600 (O.D 6°°) of between 0.4 and 0.6. IPTG
("Isopropyl-B-D-thiocalacto pyanoside") was then added to a final concentration of 1 mM. IPTG induces by inactiyatin, the lacl repressor, clearing the P/O leading to increased gene expression. Cells were grown an extra 3 to ~ hours. Cells were then harvested by centrifugation.
Numerous modifications and variations of the present invention are possible in light of ~s the above teachings and, therefore, within the scope of the appended claims. the invention may be practiced otherwise than as particularly described. It is to be understood that, while the in~~ention has been described with reference to the above detailed description. the foregoing description is intended to illustrate, but not to limit. the scope of the invention. Other aspects.
advantages, and modifications of the invention are within the scope of the following claims.

SEQUENCE LISTING
<110> Diversa Corporation <120> ISOLATION AND IDENTIFICATION OF NOVEL POLYMERASES
<130> 581-179 <140> 2,310,854 <141> 1998-08-06 <150> US 08j907,166 <151> 1997-08-06 <160> 12 <170> FastSEQ'" for Windows'" Version 3.0 <210> 1 <211> 2607 <212> DNA
<213> Ammonifex degensii <220>
<221> CDS
<222> (1)...(2604) <400> 1 gtg aag gga aaa acc ttg ctc ctt ttg gac ggc tcg agc ata gcc tac 48 Val Lys Gly Lys Thr Leu Leu Leu Leu Asp Gly Ser Ser Ile Ala Tyr cgg gcc ttt ttc gcc ctt ccc tcc ctc cgc acc cgt acc ggc ctg ccc 96 Arg Ala Phe Phe Ala Leu Pro Ser Leu Arg Thr Arg Thr Gly Leu Pro acc ggt gcc gtg tac ggc ttt acc tcc atg ctc ttc aaa gtg ctg gaa 144 Thr Gly Ala Val Tyr Gly Phe Thr Ser Met Leu Phe Lys Val Leu Glu gaa agg cgt ccc acg gcc ata gtg gcg get ttc gat aaa agc aag acc 192 Glu Arg Arg Pro Thr Ala Ile Val Ala Ala Phe Asp Lys Ser Lys Thr acc ttc cgg cac gcc ctg gcg gag acc tac aag gcc cac cgc ccc gcc 240 Thr Phe Arg His Ala Leu Ala Glu Thr Tyr Lys Ala His Arg Pro Ala act ccg gat gaa ctg cgc cag cag ttc aac ctc atc aag gaa gtg ctg 288 Thr Pro Asp Glu Leu Arg Gln Gln Phe Asn Leu Ile Lys Glu Val Leu -37.1-act gcc ctc aac gtt ccg gta gtg gaa aag gag ggt ttt gag gcc gac 336 Thr Ala Leu Asn Val Pro Val Val Glu Lys Glu Gly Phe Glu Ala Asp gac ctc atc ggc act ctg gta gac cgg gcg gaa aaa gag ggt tgg cag 384 Asp Leu Ile Gly Thr Leu Val Asp Arg Ala Glu Lys Glu Gly Trp Gln tgc ctt atc gtc acc ggc gac ctc gac gcc ctg cag ctg gtt tcc ccc 432 Cys Leu Ile Val Thr Gly Asp Leu Asp Ala Leu Gln Leu Val Ser Pro ctc acc acc gtc gtc ctc atg cgc aag ggg ata agc gaa ata gcg gtc 480 Leu Thr Thr Val Val Leu Met Arg Lys Gly Ile Ser Glu Ile Ala Val ttt aac gag gcg gag gtg aaa cgc cgc ttc ggc gtc aca ccc cgc caa 528 Phe Asn Glu Ala Glu Val Lys Arg Arg Phe Gly Val Thr Pro Arg Gln ctc ccc gac ttc aaa gcc ttg gcc gga gat gcc tcg gac aac atc ccc 576 Leu Pro Asp Phe Lys Ala Leu Ala Gly Asp Ala Ser Asp Asn Ile Pro ggg ctt ccg ggc ata ggg ccc aaa act gcc tcc cgt ctg cta cag tcc 624 Gly Leu Pro Gly Ile Gly Pro Lys Thr Ala Ser Arg Leu Leu Gln Ser cac cag agc ctg gag aaa ttg ctg gag agc aag gaa ttt ttt ccg gcc 672 His Gln Ser Leu Glu Lys Leu Leu Glu Ser Lys Glu Phe Phe Pro Ala aag ctg cgc gaa acc tta gaa agg cac aag gaa gaa gcg gtt ttg gcc 720 Lys Leu Arg Glu Thr Leu Glu Arg His Lys Glu Glu Ala Val Leu Ala aag aaa ctg gcc ctc atc cgc cgc gat gtg ccg ctg gaa gag gag atc 768 Lys Lys Leu Ala Leu Ile Arg Arg Asp Val Pro Leu Glu Glu Glu Ile atc cgg ccc tgg ccg gga ccc aac att tta gcc acg ctg gag gtc ttc 816 Ile Arg Pro Trp Pro Gly Pro Asn Ile Leu Ala Thr Leu Glu Val Phe tcg cgc ctg gaa ttc cgc acc ttg gcc aag aga ttc ctc gag ctt ttc 864 Ser Arg Leu Glu Phe Arg Thr Leu Ala Lys Arg Phe Leu Glu Leu Phe ccc gag gca cgc ctc ctg tcc gcc agt ggc ctt acc ccc tcc get gtc 912 Pro Glu Ala Arg Leu Leu Ser Ala Ser Gly Leu Thr Pro Ser Ala Val -37.2-cgc gta aag gta gaa aga ccc gaa gaa cta gaa aga ctg ggg gaa gag 960 Arg Val Lys Val Glu Arg Pro Glu Glu Leu Glu Arg Leu Gly Glu Glu ctc gga agg caa gaa ttt gcg gcc ctg get tac ccc ccc gtt ctt cgg 1008 Leu Gly Arg Gln Glu Phe Ala Ala Leu Ala Tyr Pro Pro Val Leu Arg cgc aaa gcc act tct tct ttc ttg get ctc tgt ctg gga ggg gaa aag 1056 Arg Lys Ala Thr Ser Ser Phe Leu Ala Leu Cys Leu Gly Gly Glu Lys gtc ttc ctg ctg gaa ggg ccg gag gtg ctc aag agc ttc ttc cgg ctg 1104 Val Phe Leu Leu Glu Gly Pro Glu Val Leu Lys Ser Phe Phe Arg Leu ctc gaa gaa aag gga ggt ctt gtc agt acc tac gac get aaa tcc tgc 1152 Leu Glu Glu Lys Gly Gly Leu Val Ser Thr Tyr Asp Ala Lys Ser Cys ctt cac gcc ctg gaa cct tac ggc ttc aag ccc gaa atg atc ggg ttt 1200 Leu His Ala Leu Glu Pro Tyr Gly Phe Lys Pro Glu Met Ile Gly Phe gac gtc ctg ctg gca gcc tac ctg gtg aac ccc gcc gcc aac aac gaa 1248 Asp Val Leu Leu Ala Ala Tyr Leu Val Asn Pro Ala Ala Asn Asn Glu ctg ggg gcg atc gcc ttc gag cac gcg ggc ttt atg ctc tcc ccg gga 1296 Leu Gly Ala Ile Ala Phe Glu His Ala Gly Phe Met Leu Ser Pro Gly gca gag ctc ccg gaa aaa gcc cag gcg atc tac cag ctc acc ccc atc 1344 Ala Glu Leu Pro Glu Lys Ala Gln Ala Ile Tyr Gln Leu Thr Pro Ile cta aaa agt aag att aag ctt cag gaa cag gag tac ctt tat tac tcc 1392 Leu Lys Ser Lys Ile Lys Leu Gln Glu Gln Glu Tyr Leu Tyr Tyr Ser gtg gag ctt ccc tta gcc gcc gtc ttg gcc gac atg gag aaa gtc ggg 1440 Val Glu Leu Pro Leu Ala Ala Val Leu Ala Asp Met Glu Lys Val Gly gtg aaa gtt tcg gag gaa agg ctg cgt tct ctc tcc aag gag ctg gga 1488 Val Lys Val Ser Glu Glu Arg Leu Arg Ser Leu Ser Lys Glu Leu Gly gag cag ctg get cag ctt tcc gag gaa atc tat aag ctc gcc ggc gag 1536 Glu Gln Leu Ala Gln Leu Ser Glu Glu Ile Tyr Lys Leu Ala Gly Glu -37.3-cgc ttc aac ctg aat tcc ccc cgc cag ctc ggc tac atc ctg ttc gag 1584 Arg Phe Asn Leu Asn Ser Pro Arg Gln Leu Gly Tyr Ile Leu Phe Glu aag ttg gga ctc aaa ccg gtc aag aag acc aaa acc ggc tac tcc acc 1632 Lys Leu Gly Leu Lys Pro Val Lys Lys Thr Lys Thr Gly Tyr Ser Thr gac get tcg gtc cta gaa aag cta gcc gag cac gag atc gtg get aag 1680 Asp Ala Ser Val Leu Glu Lys Leu Ala Glu His Glu Ile Val Ala Lys gtg ctc gtc tac cgg cag ctg gcc aaa cta aag agc act tac acc gac 1728 Val Leu Val Tyr Arg Gln Leu Ala Lys Leu Lys Ser Thr Tyr Thr Asp gca ctt cca gag ctc atc gac ccg gcc acc ggg cgc ctg cac acc acc 1776 Ala Leu Pro Glu Leu Ile Asp Pro Ala Thr Gly Arg Leu His Thr Thr ttc ttg cag gca ggg acg gca acg gga aga ctg gcc tcc gcc gag ccc 1824 Phe Leu Gln Ala Gly Thr Ala Thr Gly Arg Leu Ala Ser Ala Glu Pro aac ctg cag aac att ccc gta cgc gat tct ctg gga agg cgc atc cgg 1872 Asn Leu Gln Asn Ile Pro Val Arg Asp Ser Leu Gly Arg Arg Ile Arg cag gcc ttc gtg get gag ggc ccc gac tac gtg cta cta agc gcc gac 1920 Gln Ala Phe Val Ala Glu Gly Pro Asp Tyr Val Leu Leu Ser Ala Asp tac tcc cag ata gag ctt cgg gtc ttg gcc cac ctt tcc gaa gat ccg 1968 Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Glu Asp Pro ggg ctg tgt gag gcc ttt gtt aaa gga gaa gac att cac gcc cgc acg 2016 Gly Leu Cys Glu Ala Phe Val Lys Gly Glu Asp Ile His Ala Arg Thr gcg gcc gag atc ttc ggc gtt tct cct cag gaa gtg acg ccg gag atg 2064 Ala Ala Glu Ile Phe Gly Val Ser Pro Gln Glu Val Thr Pro Glu Met cgg gcc aag gcc aag gtg gta aac ttc ggg atc gtt tac ggc atg agc 2112 Arg Ala Lys Ala Lys Val Val Asn Phe Gly Ile Val Tyr Gly Met Ser gat tac ggc ctt tcc cag gag ctc aag atc gag ccc ggc gag gcg cac 2160 Asp Tyr Gly Leu Ser Gln Glu Leu Lys Ile Glu Pro Gly Glu Ala His -3 7.4-gag tat ata gaa cgg tac ttc cgg cgc tat ccg cgc gtg aag cag ttc 2208 Glu Tyr Ile Glu Arg Tyr Phe Arg Arg Tyr Pro Arg Val Lys Gln Phe atc gag cgg gtg atc gcc cag gcc cga gag aag ggc tac gtg acc act 2256 Ile Glu Arg Val Ile Ala Gln Ala Arg Glu Lys Gly Tyr Val Thr Thr att ctc aac cgc cgc cgc tac atc cct gaa ata ctg agc agc aac cgc 2304 Ile Leu Asn Arg Arg Arg Tyr Ile Pro Glu Ile Leu Ser Ser Asn Arg aac cag cgt cag ctg ggg gag cgc ctg gcc atc aac acc acc att caa 2352 Asn Gln Arg Gln Leu Gly Glu Arg Leu Ala Ile Asn Thr Thr Ile Gln gga agt gcg gcc gat ctt ata aaa aag gcc atg gtg gac atc cac cgg 2400 Gly Ser Ala Ala Asp Leu Ile Lys Lys Ala Met Val Asp Ile His Arg caa ctg aaa ggg caa gga ttt aaa tgc cgg atg atc ctc cag gtg cac 2448 Gln Leu Lys Gly Gln Gly Phe Lys Cys Arg Met Ile Leu Gln Val His gac gaa ctc ctc ttc gag gtg cct aaa gaa gaa ctg gaa aag gtg gca 2496 Asp Glu Leu Leu Phe Glu Val Pro Lys Glu Glu Leu Glu Lys Val Ala cct ata ata aaa agc acc atg gag caa gcc tta cct ttt aag gtt ccc 2544 Pro Ile Ile Lys Ser Thr Met Glu Gln Ala Leu Pro Phe Lys Val Pro ata aag gcc aac ctc aag gta ggg cct aac tgg caa gac atg gaa gag 2592 Ile Lys Ala Asn Leu Lys Val Gly Pro Asn Trp Gln Asp Met Glu Glu tac gag gtg gaa tga 2607 Tyr Glu Val Glu <210> 2 <211> 868 <212> PRT
<213> Ammonifex degensii <400> 2 Val Lys Gly Lys Thr Leu Leu Leu Leu Asp Gly Ser Ser Ile Ala Tyr Arg Ala Phe Phe Ala Leu Pro Ser Leu Arg Thr Arg Thr Gly Leu Pro -37.5-Thr Gly Ala Val Tyr Gly Phe Thr Ser Met Leu Phe Lys Val Leu Glu Glu Arg Arg Pro Thr Ala Ile Val Ala Ala Phe Asp Lys Ser Lys Thr Thr Phe Arg His Ala Leu Ala Glu Thr Tyr Lys Ala His Arg Pro Ala Thr Pro Asp Glu Leu Arg Gln Gln Phe Asn Leu Ile Lys Glu Val Leu Thr Ala Leu Asn Val Pro Val Val Glu Lys Glu Gly Phe Glu Ala Asp Asp Leu Ile Gly Thr Leu Val Asp Arg Ala Glu Lys Glu Gly Trp Gln Cys Leu Ile Val Thr Gly Asp Leu Asp Ala Leu Gln Leu Val Ser Pro Leu Thr Thr Val Val Leu Met Arg Lys Gly Ile Ser Glu Ile Ala Val Phe Asn Glu Ala Glu Val Lys Arg Arg Phe Gly Val Thr Pro Arg Gln Leu Pro Asp Phe Lys Ala Leu Ala Gly Asp Ala Ser Asp Asn Ile Pro Gly Leu Pro Gly Ile Gly Pro Lys Thr Ala Ser Arg Leu Leu Gln Ser His Gln Ser Leu Glu Lys Leu Leu Glu Ser Lys Glu Phe Phe Pro Ala Lys Leu Arg Glu Thr Leu Glu Arg His Lys Glu Glu Ala Val Leu Ala Lys Lys Leu Ala Leu Ile Arg Arg Asp Val Pro Leu Glu Glu Glu Ile Ile Arg Pro Trp Pro Gly Pro Asn Ile Leu Ala Thr Leu Glu Val Phe Ser Arg Leu Glu Phe Arg Thr Leu Ala Lys Arg Phe Leu Glu Leu Phe Pro Glu Ala Arg Leu Leu Ser Ala Ser Gly Leu Thr Pro Ser Ala Val Arg Val Lys Val Glu Arg Pro Glu Glu Leu Glu Arg Leu Gly Glu Glu Leu Gly Arg Gln Glu Phe Ala Ala Leu Ala Tyr Pro Pro Val Leu Arg Arg Lys Ala Thr Ser Ser Phe Leu Ala Leu Cys Leu Gly Gly Glu Lys Val Phe Leu Leu Glu Gly Pro Glu Val Leu Lys Ser Phe Phe Arg Leu Leu Glu Glu Lys Gly Gly Leu Val Ser Thr Tyr Asp Ala Lys Ser Cys Leu His Ala Leu Glu Pro Tyr Gly Phe Lys Pro Glu Met Ile Gly Phe Asp Val Leu Leu Ala Ala Tyr Leu Val Asn Pro Ala Ala Asn Asn Glu Leu Gly Ala Ile Ala Phe Glu His Ala Gly Phe Met Leu Ser Pro Gly Ala Glu Leu Pro Glu Lys Ala Gln Ala Ile Tyr Gln Leu Thr Pro Ile -37.6-Leu Lys Ser Lys Ile Lys Leu Gln Glu Gln Glu Tyr Leu Tyr Tyr Ser Val Glu Leu Pro Leu Ala Ala Val Leu Ala Asp Met Glu Lys Val Gly Val Lys Val Ser Glu Glu Arg Leu Arg Ser Leu Ser Lys Glu Leu Gly Glu Gln Leu Ala Gln Leu Ser Glu Glu Ile Tyr Lys Leu Ala Gly Glu Arg Phe Asn Leu Asn Ser Pro Arg Gln Leu Gly Tyr Ile Leu Phe Glu Lys Leu Gly Leu Lys Pro Val Lys Lys Thr Lys Thr Gly Tyr Ser Thr Asp Ala Ser Val Leu Glu Lys Leu Ala Glu His Glu Ile Val Ala Lys Val Leu Val Tyr Arg Gln Leu Ala Lys Leu Lys Ser Thr Tyr Thr Asp Ala Leu Pro Glu Leu Ile Asp Pro Ala Thr Gly Arg Leu His Thr Thr Phe Leu Gln Ala Gly Thr Ala Thr Gly Arg Leu Ala Ser Ala Glu Pro Asn Leu Gln Asn Ile Pro Val Arg Asp Ser Leu Gly Arg Arg Ile Arg Gln Ala Phe Val Ala Glu Gly Pro Asp Tyr Val Leu Leu Ser Ala Asp Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Leu Ser Glu Asp Pro Gly Leu Cys Glu Ala Phe Val Lys Gly Glu Asp Ile His Ala Arg Thr Ala Ala Glu Ile Phe Gly Val Ser Pro Gln Glu Val Thr Pro Glu Met Arg Ala Lys Ala Lys Val Val Asn Phe Gly Ile Val Tyr Gly Met Ser Asp Tyr Gly Leu Ser Gln Glu Leu Lys Ile Glu Pro Gly Glu Ala His Glu Tyr Ile Glu Arg Tyr Phe Arg Arg Tyr Pro Arg Val Lys Gln Phe Ile Glu Arg Val I1e Ala Gln Ala Arg Glu Lys Gly Tyr Val Thr Thr Ile Leu Asn Arg Arg Arg Tyr Ile Pro Glu Ile Leu Ser Ser Asn Arg Asn Gln Arg Gln Leu Gly Glu Arg Leu Ala Ile Asn Thr Thr Ile Gln Gly Ser Ala Ala Asp Leu Ile Lys Lys Ala Met Val Asp Ile His Arg Gln Leu Lys Gly Gln Gly Phe Lys Cys Arg Met Ile Leu Gln Val His Asp Glu Leu Leu Phe Glu Val P:ro Lys Glu Glu Leu Glu Lys Val Ala Pro Ile Ile Lys Ser Thr Met Glu Gln Ala Leu Pro Phe Lys Val Pro Ile Lys Ala Asn Leu Lys Val Gly Pro Asn Trp Gln Asp Met Glu Glu -37.7-Tyr Glu Val Glu <210> 3 <211> 2412 <212> DNA
<213> Pyrolobus fumarius <220>
<221> CDS
<222> (1)...(2410) <400> 3 atg act gaa gtt gta ttc acg gtt tta gac tct agc tac gag gtt gtt 48 Met Thr Glu Val Val Phe Thr Val Leu Asp Ser Ser Tyr Glu Val Val ggt aaa gag cct cag gta atc ata tgg ggt att get gag aac ggc gag 96 Gly Lys Glu Pro Gln Val Ile Ile Trp Gly Ile Ala Glu Asn Gly Glu agg gta gtc ctc att gac agg tct ttt cgc cca tac ttc tat gcg ctg 144 Arg Val Val Leu Ile Asp Arg Ser Phe Arg Pro Tyr Phe Tyr Ala Leu ctt gca ccg ggc gcc gat cct aag cag gta gca caa cgt att cgt gca 192 Leu Ala Pro Gly Ala Asp Pro Lys Gln Val Ala Gln Arg Ile Arg Ala ttg agt agg cca aag agc ccg att ata ggt gta gag gat gac aag agg 240 Leu Ser Arg Pro Lys Ser Pro Ile Ile Gly Val Glu Asp Asp Lys Arg aag tac ttc ggg agg cct cgt agg gtc tta cgt att cgc acc gtg cta 288 Lys Tyr Phe Gly Arg Pro Arg Arg Val Leu Arg Ile Arg Thr Val Leu ccc gag get gtt agg gag tat cgc gaa ctc gta aag aac gtt gat ggt 336 Pro Glu Ala Val Arg Glu Tyr Arg Glu Leu Val Lys Asn Val Asp Gly gtt gag gat gtt cta gag gcg gat ata cgc ttc get atg cgc tat ctc 384 Val Glu Asp Val Leu Glu Ala Asp Ile Arg Phe Ala Met Arg Tyr Leu ata gat cac gat cta ttt cct ttc acc tgg tac cgt gta gag get gag 432 Ile Asp His Asp Leu Phe Pro Phe Thr Trp Tyr Arg Val Glu Ala Glu ccc ctc gag aac aag atg ggc ttc cgt gtc gac aag gta tac ctg gtt 480 Pro Leu Glu Asn Lys Met Gly Phe Arg Val Asp Lys Val Tyr Leu Val -37.8-aag agc agg ccg gag cca ctt tat ggt gag get ctc gca cca acc aag 528 Lys Ser Arg Pro Glu Pro Leu Tyr Gly Glu Ala Leu Ala Pro Thr Lys ctt ccc gat ctt agg ata ctc gcg ttc gat att gaa gtt tat agc aag 576 Leu Pro Asp Leu Arg Ile Leu Ala Phe Asp Ile Glu Val Tyr Ser Lys caa ggg tcg ccg cgt cca gag cgc gat cct gta ata gtg ata get gtg 624 Gln Gly Ser Pro Arg Pro Glu Arg Asp Pro Val Ile Val Ile Ala Val aag act gac gat ggc gat gag gtg cta ttc att gca gag ggc aaa gac 672 Lys Thr Asp Asp Gly Asp Glu Val Leu Phe Ile Ala Glu Gly Lys Asp gat cga aaa ccg ata cgc gag ttt gta gag tac gtg aag agg tat gac 720 Asp Arg Lys Pro Ile Arg Glu Phe Val Glu Tyr Val Lys Arg Tyr Asp ccc gac ata ata gtc ggt tat aac aac aat cat ttc gat tgg cct tat 768 Pro Asp Ile Ile Val Gly Tyr Asn Asn Asn His Phe Asp Trp Pro Tyr ctt ttg agg cgc gcc cgc atc cta ggc ata aag ctt gat gtg act aga 816 Leu Leu Arg Arg Ala Arg Ile Leu Gly Ile Lys Leu Asp Val Thr Arg aga gtt ggc gcc gag ccc acc act agc gta cat ggg cac gtc tct gtc 864 Arg Val Gly Ala Glu Pro Thr Thr Ser Val His Gly His Val Ser Val cct ggc agg ctt aac gta gat ctg tac gac tat gcc gaa gag atg cca 912 Pro Gly Arg Leu Asn Val Asp Leu Tyr Asp Tyr Ala Glu Glu Met Pro gag atc aag ata aag agt ctc gag gag gtc gca gag tat cta ggc gtg 960 Glu Ile Lys Ile Lys Ser Leu Glu Glu Val Ala Glu Tyr Leu Gly Val atg aag aag agt gaa cgc gtt atc atc aat tgg tgg gag att cca gac 1008 Met Lys Lys Ser Glu Arg Val Ile Ile Asn Trp Trp Glu Ile Pro Asp tat tgg gac gac ccg aag aag aga cca cta tta ctg caa tac gcg cgc 1056 Tyr Trp Asp Asp Pro Lys Lys A.rg Pro Leu Leu Leu Gln Tyr Ala Arg gac gat gtc cgc get act tac ggc tta gcc gag aag ata ttg ccg ttt 1104 Asp Asp Val Arg Ala Thr Tyr Gly Leu Ala Glu Lys Ile Leu Pro Phe -37.9-get atc cag ttg tcg tac gta aca ggt ctc cca cta gac cag gta ggt 1152 Ala Ile Gln Leu Ser Tyr Val Thr Gly Leu Pro Leu Asp Gln Val Gly gcg atg agt gtt ggc ttt cga ctt gaa tgg tac ctg ata cgc gcg gcg 1200 Ala Met Ser Val Gly Phe Arg Leu Glu Trp Tyr Leu Ile Arg Ala Ala ttt aag atg aaa gag ctt gtg ccg aac cgc gtt gag cgc cca gaa gag 1248 Phe Lys Met Lys Glu Leu Val Pro Asn Arg Val Glu Arg Pro Glu Glu act tac cgt ggc get ata gtt ctt gag ccg ttg aga ggc gtg cac gag 1296 Thr Tyr Arg Gly Ala Ile Val Leu Glu Pro Leu Arg Gly Val His Glu aat ata gcc gta ctc gac ttt agc tcg atg tac cca aac atc atg ata 1344 Asn Ile Ala Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Ile Met Ile aag tac aat gtt ggt cct gac acg ctt gtg agg cct ggt gaa aag tgt 1392 Lys Tyr Asn Val Gly Pro Asp Thr Leu Val Arg Pro Gly Glu Lys Cys ggc gag tgt ggt tgc tgg gag gcc ccg gag gtc aag cac agg ttc cgt 1440 Gly Glu Cys Gly Cys Trp Glu Ala Pro Glu Val Lys His Arg Phe Arg agg tgt ccg ccc ggc ttc ttc aag aca gtt ctt gag agg ctg tta gag 1488 Arg Cys Pro Pro Gly Phe Phe Lys Thr Val Leu Glu Arg Leu Leu Glu ctt cgt aag cgt gtg cgt get gaa atg aag aag tat cct ccg gat agc 1536 Leu Arg Lys Arg Val Arg Ala Glu Met Lys Lys Tyr Pro Pro Asp Ser cca gaa tat cga ctg ttg gat gaa agg cag aag gcg ttg aag gtt ctt 1584 Pro Glu Tyr Arg Leu Leu Asp Glu Arg Gln Lys Ala Leu Lys Val Leu gca aac get agt tac ggc tac atg ggt tgg agc ggc get agg tgg tat 1632 Ala Asn Ala Ser Tyr Gly Tyr Met Gly Trp Ser Gly Ala Arg Trp Tyr tgc agg gag tgc gca aag get gtc acg get tgg ggt agg cac ctc ata 1680 Cys Arg Glu Cys Ala Lys Ala Val Thr Ala Trp Gly Arg His Leu Ile cgc acc gcc atc aac ata get cgt aaa cta ggc ctc aag gtg atc tac 1728 Arg Thr Ala Ile Asn Ile Ala Arg Lys Leu Gly Leu Lys Val Ile Tyr -37.10-ggt gac aca gat tcg ctc ttc gtg acc tat gat ccg gag aag gtg gaa 1776 Gly Asp Thr Asp Ser Leu Phe Val Thr Tyr Asp Pro Glu Lys Val Glu aat ttc atc aaa att ata aag gag gag ctg ggg ttc gaa atc aag cta 1824 Asn Phe Ile Lys Ile Ile Lys Glu Glu Leu Gly Phe Glu Ile Lys Leu gag aag gtg tac aaa cgc tta ttc ttt aca gag get aag aag agg tac 1872 Glu Lys Val Tyr Lys Arg Leu Phe Phe Thr Glu Ala Lys Lys Arg Tyr get ggc ctt ctc gag gac gga cgt ata gat att gtc ggt ttc gag get 1920 Ala Gly Leu Leu Glu Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Ala gta cgt ggc gat tgg tgt gaa ctc gcc aag gag gtt cag act aag gtt 1968 Val Arg Gly Asp Trp Cys Glu Leu Ala Lys Glu Val Gln Thr Lys Val gtc gaa ata gta ttg aag acg agt gag gtg aac aag get gta gag tac 2016 Val Glu Ile Val Leu Lys Thr Ser Glu Val Asn Lys Ala Val Glu Tyr gtc agg aag att gtg aaa gag ttg gag gag ggc aag gtt ccc ata gag 2064 Val Arg Lys Ile Val Lys Glu Leu Glu Glu Gly Lys Val Pro Ile Glu aag ctt gta atc tgg aag acc ctt agt aag cgt ctt gag gag tac aca 2112 Lys Leu Val Ile Trp Lys Thr Leu Ser Lys Arg Leu Glu Glu Tyr Thr acg gag gca cca cac gtc gtt gca gcg aag agg atg ctg tca gca ggc 2160 Thr Glu~Ala Pro His Val Val Ala Ala Lys Arg Met Leu Ser Ala Gly tac cgg gta agc cca ggc gac aag ata ggg tat gta ata gtg aag ggt 2208 Tyr Arg Val Ser Pro Gly Asp Lys Ile Gly Tyr Val Ile Val Lys Gly ggt ggc cgt atc agt caa aga gca tgg cca tac ttc atg gtc aag gat 2256 Gly Gly Arg Ile Ser Gln Arg Ala Trp Pro Tyr Phe Met Val Lys Asp cct agc cag ata gac gtg acc tac tat gtt gac cac caa atc atc ccg 2304 Pro Ser Gln Ile Asp Val Thr Tyr Tyr Val Asp His Gln Ile Ile Pro get gca ttg aga ata ctg ggc tac ttt ggc atc acc gag aag aag ctg 2352 Ala Ala Leu Arg Ile Leu Gly Tyr Phe Gly Ile Thr Glu Lys Lys Leu -37.11-aaa gca agt gca act ggg cag aag act ctc ttc gac ttt cta gcc aag 2400 Lys Ala Ser Ala Thr Gly Gln Lys Thr Leu Phe Asp Phe Leu Ala Lys aag agc aag t as 2412 Lys Ser Lys <210> 4 <211> 803 <212> PRT
<213> Pyrolobus fumarius <400> 4 Met Thr Glu Val Val Phe Thr Val Leu Asp Ser Ser Tyr Glu Val Val Gly Lys Glu Pro Gln Val Ile Ile Trp Gly Ile Ala Glu Asn Gly Glu Arg Val Val Leu Ile Asp Arg Ser Phe Arg Pro Tyr Phe Tyr Ala Leu Leu Ala Pro Gly Ala Asp Pro Lys Gln Val Ala Gln Arg Ile Arg Ala Leu Ser Arg Pro Lys Ser Pro Ile Ile Gly Val Glu Asp Asp Lys Arg Lys Tyr Phe Gly Arg Pro Arg Arg Val Leu Arg Ile Arg Thr Val Leu Pro Glu Ala Val Arg Glu Tyr Arg Glu Leu Val Lys Asn Val Asp Gly Val Glu Asp Val Leu Glu Ala Asp Ile Arg Phe Ala Met Arg Tyr Leu Ile Asp His Asp Leu Phe Pro Phe Thr Trp Tyr Arg Val Glu Ala Glu Pro Leu Glu Asn Lys Met Gly Phe Arg Val Asp Lys Val Tyr Leu Val Lys Ser Arg Pro Glu Pro Leu Tyr Gly Glu Ala Leu Ala Pro Thr Lys Leu Pro Asp Leu Arg Ile Leu Ala Phe Asp Ile Glu Val Tyr Ser Lys Gln Gly Ser Pro Arg Pro Glu Arg Asp Pro Val Ile Val Ile Ala Val Lys Thr Asp Asp Gly Asp Glu Val Leu Phe Ile Ala Glu Gly Lys Asp Asp Arg Lys Pro Ile Arg Glu Phe Val Glu Tyr Val Lys Arg Tyr Asp Pro Asp Ile Ile Val Gly Tyr Asn Asn Asn His Phe Asp Trp Pro Tyr Leu Leu Arg Arg Ala Arg Ile Leu Gly Ile Lys Leu Asp Val Thr Arg Arg Val Gly Ala Glu Pro Thr Thr Ser Val His Gly His Val Ser Val Pro Gly Arg Leu Asn Val Asp Leu Tyr Asp Tyr Ala Glu Glu Met Pro -37.12-Glu Ile Lys Ile Lys Ser Leu Glu Glu Val Ala Glu Tyr Leu Gly Val Met Lys Lys Ser Glu Arg Val I.le Ile Asn Trp Trp Glu Ile Pro Asp Tyr Trp Asp Asp Pro Lys Lys Arg Pro Leu Leu Leu Gln Tyr Ala Arg Asp Asp Val Arg Ala Thr Tyr Gly Leu Ala Glu Lys Ile Leu Pro Phe Ala Ile Gln Leu Ser Tyr Val Thr Gly Leu Pro Leu Asp Gln Val Gly Ala Met Ser Val Gly Phe Arg Leu Glu Trp Tyr Leu Ile Arg Ala Ala Phe Lys Met Lys Glu Leu Val Pro Asn Arg Val Glu Arg Pro Glu Glu Thr Tyr Arg Gly Ala Ile Val Leu Glu Pro Leu Arg Gly Val His Glu Asn Ile Ala Val Leu Asp Phe Ser Ser Met Tyr Pro Asn Ile Met Ile Lys Tyr Asn Val Gly Pro Asp Thr Leu Val Arg Pro Gly Glu Lys Cys Gly Glu Cys Gly Cys Trp Glu Ala Pro Glu Val Lys His Arg Phe Arg Arg Cys Pro Pro Gly Phe Phe Lys Thr Val Leu Glu Arg Leu Leu Glu Leu Arg Lys Arg Val Arg Ala Glu Met Lys Lys Tyr Pro Pro Asp Ser Pro Glu Tyr Arg Leu Leu Asp Glu Arg Gln Lys Ala Leu Lys Val Leu Ala Asn Ala Ser Tyr Gly Tyr Met Gly Trp Ser Gly Ala Arg Trp Tyr Cys Arg Glu Cys Ala Lys Ala Val Thr Ala Trp Gly Arg His Leu Ile Arg Thr Ala Ile Asn Ile Ala Arg Lys Leu Gly Leu Lys Val Ile Tyr Gly Asp Thr Asp Ser Leu Phe Val Thr Tyr Asp Pro Glu Lys Val Glu Asn Phe Ile Lys Ile Ile Lys Glu Glu Leu Gly Phe Glu Ile Lys Leu Glu Lys Val Tyr Lys Arg Leu Phe Phe Thr Glu Ala Lys Lys Arg Tyr Ala Gly Leu Leu Glu Asp Gly Arg Ile Asp Ile Val Gly Phe Glu Ala Val Arg Gly Asp Trp Cys Glu Leu Ala Lys Glu Val Gln Thr Lys Val Val Glu Ile Val Leu Lys Thr Ser Glu Val Asn Lys Ala Val Glu Tyr Val Arg Lys Ile Val Lys Glu Leu Glu Glu Gly Lys Val Pro Ile Glu Lys Leu Val Ile Trp Lys Thr Leu Ser Lys Arg Leu Glu Glu Tyr Thr Thr Glu Ala Pro His Val Val Ala Ala Lys Arg Met Leu Ser Ala Gly -37.13-Tyr Arg Val Ser Pro Gly Asp Lys Ile Gly Tyr Val Ile Val Lys Gly Gly Gly Arg Ile Ser Gln Arg Ala Trp Pro Tyr Phe Met Val Lys Asp Pro Ser Gln Ile Asp Val Thr Tyr Tyr Val Asp His Gln Ile Ile Pro Ala Ala Leu Arg Ile Leu Gly Tyr Phe Gly Ile Thr Glu Lys Lys Leu Lys Ala Ser Ala Thr Gly Gln Lys Thr Leu Phe Asp Phe Leu Ala Lys Lys Ser Lys <210> 5 <211> 2367 <212> DNA
<213> Archaeoglobus lithotrophicus <220>
<221> CDS
<222> (1)...(2364) <400> 5 atg ata aag gtc aag ggc tgg ctg ctc gat gca gat tat atc acc gaa 48 Met Ile Lys Val Lys Gly Trp Leu Leu Asp Ala Asp Tyr Ile Thr Glu aac gat cga gcc gtt ata agg cta tgg tgt aag gat gag gaa gga ata 96 Asn Asp Arg Ala Val Ile Arg Leu Trp Cys Lys Asp Glu Glu Gly Ile ttt atc gca tac gat cac tca ttc cag ccc tac ttt tac gca ctc aaa 144 Phe Ile Ala Tyr Asp His Ser Phe Gln Pro Tyr Phe Tyr Ala Leu Lys gaa gag ggt atc act gcc gaa gat ata gtg aaa ata aag gtt caa acg 192 Glu Glu Gly Ile Thr Ala Glu Asp Ile Val Lys Ile Lys Val Gln Thr aaa aaa gaa gta att acg ccg tta aaa gtt gag gaa acc aca gcc aaa 240 Lys Lys Glu Val Ile Thr Pro Leu Lys Val Glu Glu Thr Thr Ala Lys aat ctt ggt agg gag gtt gaa gtt ttc aag ata tat gca aga cac cct 288 Asn Leu Gly Arg Glu Val Glu Val Phe Lys Ile Tyr Ala Arg His Pro cag cac gtc ccc aaa ctt cgt gag gtt gtt tcg cag tat ctg gag att 336 Gln His Val Pro Lys Leu Arg Glu Val Val Ser Gln Tyr Leu Glu Ile -37.14-agg gag gca gac ata cct ttt gcc tat cga tac ctc ata gat aaa aat 384 Arg Glu Ala Asp Ile Pro Phe Ala Tyr Arg Tyr Leu Ile Asp Lys Asn ctt gcg tgt atg gat gga gtt gta att gaa ggc gtt gaa aga cgt gag 432 Leu Ala Cys Met Asp Gly Val Val Ile Glu Gly Val Glu Arg Arg Glu aag ggg ttg aga tgt tac gaa atc aag aga ata gaa aga gat tcc aga 480 Lys Gly Leu Arg Cys Tyr Glu Ile Lys Arg Ile Glu Arg Asp Ser Arg cag gat ttt ccc gaa ctc aag gtt atg gcg ttt gat tgc gaa atg ctc 528 Gln Asp Phe Pro Glu Leu Lys Val Met Ala Phe Asp Cys Glu Met Leu tca gag gtt ggt atg ccc gat cca gag aaa gat cct atc ata gtc ata 576 Ser Glu Val Gly Met Pro Asp Pro Glu Lys Asp Pro Ile Ile Val Ile tca att aaa tcg ggt gaa tac gag gaa atc ctc aac ggt gat aac gag 624 Ser Ile Lys Ser Gly Glu Tyr Glu Glu Ile Leu Asn Gly Asp Asn Glu aga gaa ttg ctt acc aga ttt gtc aag ata att cgc gat att gat ccc 672 Arg Glu Leu Leu Thr Arg Phe Val Lys Ile Ile Arg Asp Ile Asp Pro gac att ata gtt gga tac aat cag gac agc ttt gac tgg ccc tat atc 720 Asp Ile Ile Val Gly Tyr Asn Gln Asp Ser Phe Asp Trp Pro Tyr Ile aag aag aga get gag aaa ctg agg gtt aag ctt gac atc gga aga gat 768 Lys Lys Arg Ala Glu Lys Leu Arg Val Lys Leu Asp Ile Gly Arg Asp aga agc gaa ctg get atc agg gga gga aga cca aag att get ggc agg 816 Arg Ser Glu Leu Ala Ile Arg Gly Gly Arg Pro Lys Ile Ala Gly Arg ttg aac gtg gat ctc tat gat att gca atg agg agt ctc gat gta aag 864 Leu Asn Val Asp Leu Tyr Asp Ile Ala Met Arg Ser Leu Asp Val Lys gtg aag aag ctc gaa aac gtt gca gag ttt ctg ggt aag aaa ata gag 912 Val Lys Lys Leu Glu Asn Val Ala Glu Phe Leu Gly Lys Lys Ile Glu ctt gca gat att gaa gcg aag gat atc tac aag cac tgg aca tcg ggc 960 Leu Ala Asp Ile Glu Ala Lys Asp Ile Tyr Lys His Trp Thr Ser Gly -37.15-gac agg gaa agc gta atc aaa tac tcc cgg cag gac atc ctg cac acg 1008 Asp Arg Glu Ser Val Ile Lys Tyr Ser Arg Gln Asp Ile Leu His Thr tac ttc ata get gaa gaa ttg ctg cca atg cat tac gaa ctt tcc aga 1056 Tyr Phe Ile Ala Glu Glu Leu Leu Pro Met His Tyr Glu Leu Ser Arg atg ata cgc ata cct ctc gat gat gtg aca agg agc ggg aga ggt aag 1104 Met Ile Arg Ile Pro Leu Asp Asp Val Thr Arg Ser Gly Arg Gly Lys cag gtt gag tgg ctg ctg tta agc gaa gca cac aaa ctt ggc gaa ctt 1152 Gln Val Glu Trp Leu Leu Leu Ser Glu Ala His Lys Leu Gly Glu Leu gca ccc aac ccc aga gag atg gcc gac agc tat gaa gga gca ttc gtg 1200 Ala Pro Asn Pro Arg Glu Met Ala Asp Ser Tyr Glu Gly Ala Phe Val ctc gag ccc gca aga gga ttg cat gag aac gta atc tgc ctg gac ttt 1248 Leu Glu Pro Ala Arg Gly Leu His Glu Asn Val Ile Cys Leu Asp Phe gcg tcc atg tat ccc tca ata atg att tca tac aac atc agc ccc gac 1296 Ala Ser Met Tyr Pro Ser Ile Met Ile Ser Tyr Asn Ile Ser Pro Asp acg ctt gta ata ggc aaa tgc gac gat tgc aat gta gcg ccg gag gtg 1344 Thr Leu Val Ile Gly Lys Cys Asp Asp Cys Asn Val Ala Pro Glu Val ggg cac aaa ttc agg aaa cat cct gat ggt ttt ttc aaa aga ata ctc 1392 Gly His Lys Phe Arg Lys His Pro Asp Gly Phe Phe Lys Arg Ile Leu aaa atg ctg att gag aaa aga aga gaa ata aag aag gtt atg aaa aca 1440 Lys Met Leu Ile Glu Lys Arg Arg Glu Ile Lys Lys Val Met Lys Thr ctt gac tac aac tcg cca gaa tac aag ctg ctc gat ata aag cag gca 1488 Leu Asp Tyr Asn Ser Pro Glu Tyr Lys Leu Leu Asp Ile Lys Gln Ala acg ctg aaa gtt ctt aca aac tcg ttt tac ggt tat act ggg tgg agt 1536 Thr Leu Lys Val Leu Thr Asn Ser Phe Tyr Gly Tyr Thr Gly Trp Ser ctt gcg aga tgg tac tgc aag gag tgc get gaa get aca acg gca tgg 1584 Leu Ala Arg Trp Tyr Cys Lys G.lu Cys Ala Glu Ala Thr Thr Ala Trp -37.16-ggc aga cac ttt atc aaa aca tct gca aga att gcg aaa gag ctt gga 1632 Gly Arg His Phe Ile Lys Thr Ser Ala Arg Ile Ala Lys Glu Leu Gly ttt gaa gtg cta tat ggg gat aca gat agc atc ttt gtt aaa aaa gat 1680 Phe Glu Val Leu Tyr Gly Asp Thr Asp Ser Ile Phe Val Lys Lys Asp gga ttg agc ctg gaa gag ctc aaa aaa gaa gtt aaa aag ctc ata ggt 1728 Gly Leu Ser Leu Glu Glu Leu Lys Lys Glu Val Lys Lys Leu Ile Gly aaa ctt tcg gaa gag atg cca ata caa ata gag ata gat gaa tac tac 1776 Lys Leu Ser Glu Glu Met Pro Ile Gln Ile Glu Ile Asp Glu Tyr Tyr gag aca ata ttc ttc gtt gaa aag aaa agg tat get gga ttg aca cag 1824 Glu Thr Ile Phe Phe Val Glu Lys Lys Arg Tyr Ala Gly Leu Thr Gln gat gga aga ata att gta aag ggt ctt gaa gtc aga aga ggc gac tgg 1872 Asp Gly Arg Ile Ile Val Lys Gly Leu Glu Val Arg Arg Gly Asp Trp tgc gag ctt gca aag aag ata cag aaa ggt gta ata gaa atc att ctg 1920 Cys Glu Leu Ala Lys Lys Ile Gln Lys Gly Val Ile Glu Ile Ile Leu aag gaa aag aat cct gaa aaa get get gag tat gtg aaa gga gtc ata 1968 Lys Glu Lys Asn Pro Glu Lys Ala Ala Glu Tyr Val Lys Gly Val Ile gag gag ata aag gca ggc aaa att ccg ctt gaa gat tat atc atc tac 2016 Glu Glu Ile Lys Ala Gly Lys Ile Pro Leu Glu Asp Tyr Ile Ile Tyr aag gga ttg acg aga aaa cca tca aag tac gag agt atg cag get cac 2064 Lys Gly Leu Thr Arg Lys Pro Ser Lys Tyr Glu Ser Met Gln Ala His gta aaa get gcc atg aag gcg gca aag aga gga ata gta tac aca atc 2112 Val Lys Ala Ala Met Lys Ala Ala Lys Arg Gly Ile Val Tyr Thr Ile ggc tca aag gtt ggt ttt gtc gtt aca aaa ggt gtg ggg aac ata ggt 2160 Gly Ser Lys Val Gly Phe Val Val Thr Lys Gly Val Gly Asn Ile Gly gat agg get ttt cca tct gat ctg ata gag gac ttt gac ggt gaa gtg 2208 Asp Arg Ala Phe Pro Ser Asp Leu Ile Glu Asp Phe Asp Gly Glu Val -37.17-atc aca gat ctt gac gga aac aag tac aag atc gac aag gaa tac tat 2256 Ile Thr Asp Leu Asp Gly Asn Lys Tyr Lys Ile Asp Lys Glu Tyr Tyr ata gac cat cag gta ctg cca tcg gtt ctt cga att ctc gag agg ttc 2304 Ile Asp His Gln Val Leu Pro Ser Val Leu Arg Ile Leu Glu Arg Phe gga tac acc gag gca cag cta aaa ggt get gcg gag cag caa acg cta 2352 Gly Tyr Thr Glu Ala Gln Leu Lys Gly Ala Ala Glu Gln Gln Thr Leu gat get ttc tgg taa 2367 Asp Ala Phe Trp <210> 6 <211> 788 <212> PRT
<213> Archaeoglobus lithotrophicus <400> 6 Met Ile Lys Val Lys Gly Trp Leu Leu Asp Ala Asp Tyr Ile Thr Glu Asn Asp Arg Ala Val Ile Arg Leu Trp Cys Lys Asp Glu Glu Gly Ile Phe Ile Ala Tyr Asp His Ser Phe Gln Pro Tyr Phe Tyr Ala Leu Lys Glu Glu Gly Ile Thr Ala Glu Asp Ile Val Lys Ile Lys Val Gln Thr Lys Lys Glu Val Ile Thr Pro Leu Lys Val Glu Glu Thr Thr Ala Lys Asn Leu Gly Arg Glu Val Glu Val Phe Lys Ile Tyr Ala Arg His Pro Gln His Val Pro Lys Leu Arg Glu Val Val Ser Gln Tyr Leu Glu Ile Arg Glu Ala Asp Ile Pro Phe Ala Tyr Arg Tyr Leu Ile Asp Lys Asn Leu Ala Cys Met Asp Gly Val Val Ile Glu Gly Val Glu Arg Arg Glu Lys Gly Leu Arg Cys Tyr Glu Ile Lys Arg Ile Glu Arg Asp Ser Arg Gln Asp Phe Pro Glu Leu Lys Val Met Ala Phe Asp Cys Glu Met Leu Ser Glu Val Gly Met Pro Asp P.ro Glu Lys Asp Pro Ile Ile Val Ile Ser Ile Lys Ser Gly G1u Tyr Glu Glu Ile Leu Asn Gly Asp Asn Glu Arg Glu Leu Leu Thr Arg Phe Val Lys Ile Ile Arg Asp Ile Asp Pro Asp Ile Ile Val Gly Tyr Asn Gln Asp Ser Phe Asp Trp Pro Tyr Ile -37.18-Lys Lys Arg Ala Glu Lys Leu Arg Val Lys Leu Asp Ile Gly Arg Asp Arg Ser Glu Leu Ala Ile Arg Gly Gly Arg Pro Lys Ile Ala Gly Arg Leu Asn Val Asp Leu Tyr Asp Ile Ala Met Arg Ser Leu Asp Val Lys Val Lys Lys Leu Glu Asn Val Ala Glu Phe Leu Gly Lys Lys Ile Glu Leu Ala Asp Ile Glu Ala Lys Asp Ile Tyr Lys His Trp Thr Ser Gly Asp Arg Glu Ser Val Ile Lys Tyr Ser Arg Gln Asp Ile Leu His Thr Tyr Phe Ile Ala Glu Glu Leu Leu Pro Met His Tyr Glu Leu Ser Arg Met Ile Arg Ile Pro Leu Asp Asp Val Thr Arg Ser Gly Arg Gly Lys Gln Val Glu Trp Leu Leu Leu Ser Glu Ala His Lys Leu Gly Glu Leu Ala Pro Asn Pro Arg Glu Met Ala Asp Ser Tyr Glu Gly Ala Phe Val Leu Glu Pro Ala Arg Gly Leu His Glu Asn Val Ile Cys Leu Asp Phe Ala Ser Met Tyr Pro Ser Ile Met Ile Ser Tyr Asn Ile Ser Pro Asp Thr Leu Val Ile Gly Lys Cys Asp Asp Cys Asn Val Ala Pro Glu Val Gly His Lys Phe Arg Lys His Pro Asp Gly Phe Phe Lys Arg Ile Leu Lys Met Leu Ile Glu Lys Arg Arg Glu Ile Lys Lys Val Met Lys Thr Leu Asp Tyr Asn Ser Pro Glu Tyr Lys Leu Leu Asp Ile Lys Gln Ala Thr Leu Lys Val Leu Thr Asn Ser Phe Tyr Gly Tyr Thr Gly Trp Ser Leu Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ala Thr Thr Ala Trp Gly Arg His Phe Ile Lys Thr Ser Ala Arg Ile Ala Lys Glu Leu Gly Phe Glu Val Leu Tyr Gly Asp Thr Asp Ser Ile Phe Val Lys Lys Asp Gly Leu Ser Leu Glu G1u Leu Lys Lys Glu Val Lys Lys Leu Ile Gly Lys Leu Ser Glu Glu Met Pro Ile Gln Ile Glu Ile Asp Glu Tyr Tyr Glu Thr Ile Phe Phe Val Glu Lys Lys Arg Tyr Ala Gly Leu Thr Gln Asp Gly Arg Ile Ile Val Lys Gly Leu Glu Val Arg Arg Gly Asp Trp Cys Glu Leu Ala Lys Lys Ile Gln Lys Gly Val Ile Glu Ile Ile Leu Lys Glu Lys Asn Pro Glu Lys Ala Ala Glu Tyr Val Lys Gly Val Ile -37.19-Glu Glu Ile Lys Ala Gly Lys Ile Pro Leu Glu Asp Tyr Ile Ile Tyr Lys Gly Leu Thr Arg Lys Pro Ser Lys Tyr Glu Ser Met Gln Ala His Val Lys Ala Ala Met Lys Ala Ala Lys Arg Gly Ile Val Tyr Thr Ile Gly Ser Lys Val Gly Phe Val Val Thr Lys Gly Val Gly Asn Ile Gly Asp Arg Ala Phe Pro Ser Asp Leu Ile Glu Asp Phe Asp Gly Glu Val Ile Thr Asp Leu Asp Gly Asn Lys Tyr Lys Ile Asp Lys Glu Tyr Tyr Ile Asp His Gln Val Leu Pro Ser Val Leu Arg Ile Leu Glu Arg Phe Gly Tyr Thr Glu Ala Gln Leu Lys Gly Ala Ala Glu Gln Gln Thr Leu Asp Ala Phe Trp <210> 7 <211> 2634 <212> DNA
<213> Metallosphaera prunae <220>
<221> CDS
<222> (1)...(2631) <400> 7 atg agt ata atg gcc aga cag ctt acc ctt get gac ttc tct ggg atc 48 Met Ser Ile Met Ala Arg Gln Leu Thr Leu Ala Asp Phe Ser Gly Ile aag aga gag gaa cca gtt aaa cag gaa gag aag acg cag gag gaa gag 96 Lys Arg Glu Glu Pro Val Lys Gln Glu Glu Lys Thr Gln Glu Glu Glu agg cct ctg gaa agg cca gcg agg cta aga aag gac aca gtt aaa cag 144 Arg Pro Leu Glu Arg Pro Ala Arg Leu Arg Lys Asp Thr Val Lys Gln gcg cag gag gag aga aag tac ttt ctt ctc tcc gta gac tat gat ggt 192 Ala Gln Glu Glu Arg Lys Tyr Phe Leu Leu Ser Val Asp Tyr Asp Gly aaa atg ggg aag get gtc tgc aag ctt tat gat cct gaa acg ggt gag 240 Lys Met Gly Lys Ala Val Cys Lys Leu Tyr Asp Pro Glu Thr Gly Glu cta cac gtc ctt tac gac agc acg ggt cac aag tca tac ttc ctt gtg 288 Leu His Val Leu Tyr Asp Ser Thr Gly His Lys Ser Tyr Phe Leu Val -37.20-gat tta gag cca gat cag atc caa aaa att cca aag att gtt aag gat 336 Asp Leu Glu Pro Asp Gln Ile Gln Lys Ile Pro Lys Ile Val Lys Asp gag tcc ttt gtt agg ctt gag aag acc act aaa ata gac ccc tac act 384 Glu Ser Phe Val Arg Leu Glu Lys Thr Thr Lys Ile Asp Pro Tyr Thr tgg aaa cct att aac cta acc aag att gtg gtg aat gac ccc ctc get 432 Trp Lys Pro Ile Asn Leu Thr Lys Ile Val Val Asn Asp Pro Leu Ala gtg aga cgc cta aga gaa tat gtc cca agg gcc tat gaa get cat ata 480 Val Arg Arg Leu Arg Glu Tyr Val Pro Arg Ala Tyr Glu Ala His Ile aaa tat ttt aac aat tat att tac gat ttc agc ctc ata cca ggg atg 528 Lys Tyr Phe Asn Asn Tyr Ile Tyr Asp Phe Ser Leu Ile Pro Gly Met ccc tac gtg gta aag aag ggg aag cta gtc ccc ctt aag ccg gag gtt 576 Pro Tyr Val Val Lys Lys Gly Lys Leu Val Pro Leu Lys Pro Glu Val gac gtc aaa gag gta aag gaa gcg ttc aag gat get gac cag ata get 624 Asp Val Lys Glu Val Lys Glu Ala Phe Lys Asp Ala Asp Gln Ile Ala caa gag atg gcg cta gac tgg get ccc ctc ttt gag tcc gag att ccg 672 Gln Glu Met Ala Leu Asp Trp Ala Pro Leu Phe Glu Ser Glu Ile Pro tcg gtg aag agg gtc gca ata gat ata gag gtt tat act ccc atg atg 720 Ser Val Lys Arg Val Ala Ile Asp Ile Glu Val Tyr Thr Pro Met Met ggt agg gta ccg gat cca gta aag gcc gag tac ccc gtg ata agc gta 768 Gly Arg Val Pro Asp Pro Val Lys Ala Glu Tyr Pro Val Ile Ser Val gcc cta gca ggg agc gat ggc ctg aaa ctg gtc cta gtc ctt gat agg 816 Ala Leu Ala Gly Ser Asp Gly Leu Lys Leu Val Leu Val Leu Asp Arg gga gat agt ccg att caa agt aag gat atc aag gtt gag gtc ttc cgc 864 Gly Asp Ser Pro Ile Gln Ser Lys Asp Ile Lys Val Glu Val Phe Arg aca gag agg gag ctt ctc tcc agg ttg ttt gac att ctt aag gaa tat 912 Thr Glu Arg Glu Leu Leu Ser Arg Leu Phe Asp Ile Leu Lys Glu Tyr -37.21-ccc atg gtt ctg acc ttt aac gga gac gac ttc gat atc cca tac ctg 960 Pro Met Val Leu Thr Phe Asn G.ly Asp Asp Phe Asp Ile Pro Tyr Leu atc ttc aga ggt ttc aag ctc ggg tta cta cag gat gag ata ccc ttc 1008 Ile Phe Arg Gly Phe Lys Leu Gly Leu Leu Gln Asp Glu Ile Pro Phe gag atc tct agt ttt ggc agg aaa cct gac gcg aag ttc aga tat gga 1056 Glu Ile Ser Ser Phe Gly Arg Lys Pro Asp Ala Lys Phe Arg Tyr Gly ttt cac ata gat ttg tac agg ttc ttc ttc aac aag gcg gtc agg aac 1104 Phe His Ile Asp Leu Tyr Arg Phe Phe Phe Asn Lys Ala Val Arg Asn tat gca ttt gag ggg aag tac tca gag tac aac ctt gac acc gta gcc 1152 Tyr Ala Phe Glu Gly Lys Tyr Ser Glu Tyr Asn Leu Asp Thr Val Ala cag gca ctc ttg ggt ctc tcc aag gtc aag ttg gac gag tcc att agc 1200 Gln Ala Leu Leu Gly Leu Ser Lys Val Lys Leu Asp Glu Ser Ile Ser gac cta aac atg tct aaa ctc gtg gag tac aac tac agg gac tcg gag 1248 Asp Leu Asn Met Ser Lys Leu Val Glu Tyr Asn Tyr Arg Asp Ser Glu atc acg ctg aag ttg acc acg ttc aac aac gaa cta gta tgg aag ttg 1296 Ile Thr Leu Lys Leu Thr Thr Phe Asn Asn Glu Leu Val Trp Lys Leu att gta ctc ttc tcc aga att tcc aag ctt ggt ata gag gag cta act 1344 Ile Val Leu Phe Ser Arg Ile Ser Lys Leu Gly Ile Glu Glu Leu Thr agg aca gag ata tca gcc tgg gta aag aac ctg tac tac tgg gaa cat 1392 Arg Thr Glu Ile Ser Ala Trp Val Lys Asn Leu Tyr Tyr Trp Glu His agg aaa agg aac tgg tta atc ccc ctc aag gag gaa atc ctt gaa cgc 1440 Arg Lys Arg Asn Trp Leu Ile Pro Leu Lys Glu Glu Ile Leu Glu Arg tcc tct ggg ttg aag aca get gcc att atc aag gga aag gga tac aag 1488 Ser Ser Gly Leu Lys Thr Ala Ala Ile Ile Lys Gly Lys Gly Tyr Lys ggc gca gtg gtc ata gac cca cct gtg ggg gtt tac ttt gac gta gtt 1536 Gly Ala Val Val Ile Asp Pro Pro Val Gly Val Tyr Phe Asp Val Val -37.22-gttctg gacttc gcc tcactg tat ccc tccatc atc agg aactgg aac 1584 ValLeu AspPhe Ala SerLeu Tyr Pro SerIle Ile Arg AsnTrp Asn ctcagt tatgaa acc gttgat gtg aag gaatgt aac aag aaaagg gat 1632 LeuSer TyrGlu Thr ValAsp Val Lys GluCys Asn Lys LysArg Asp ataagg gatgag agt ggggcg aaa atc catgag gtg tgc gtggac agg 1680 IleArg AspGlu Ser GlyAla Lys Ile HisGlu Val Cys ValAsp Arg cccggg attact gca gtggta act ggc ttactt agg gac ttcagg gtc~ 1728 ProGly IleThr Ala ValVal Thr Gly LeuLeu Arg Asp PheArg Val aaa att tac aag aag aaa ggg aaa cag agc aac ata gac gag gag aga 1776 Lys Ile Tyr Lys Lys Lys Gly Lys Gln Ser Asn Ile Asp Glu Glu Arg aag atg ttg tac gac gtg gta cag agg ggc atg aag gtg ttc att aat 1824 Lys Met Leu Tyr Asp Val Val Gln Arg Gly Met Lys Val Phe Ile Asn gcg acc tat ggc gtc ttc ggt gcg gag acc ttc ccc ttg tac gcc cca 1872 Ala Thr Tyr Gly Val Phe Gly Ala Glu Thr Phe Pro Leu Tyr Ala Pro gca gtt gca gag agc gtt aca gcc cta ggt agg tac gta atc acg tcc 1920 Ala Val Ala Glu Ser Val Thr Ala Leu Gly Arg Tyr Val Ile Thr Ser acc aag gaa atg get aac aag ctt ggg ctg aag gtt gtg tac ggg gat 1968 Thr Lys Glu Met Ala Asn Lys Leu Gly Leu Lys Val Val Tyr Gly Asp acg gac tcg ctc ttc att cac cag cct gat aag aag aag ctg gag gaa 2016 Thr Asp Ser Leu Phe Ile His Gln Pro Asp Lys Lys Lys Leu Glu Glu ctg gtg gag tgg acc agg cag aac ttc ggg ctt gat cta gag gtg gac 2064 Leu Val Glu Trp Thr Arg Gln Asn Phe Gly Leu Asp Leu Glu Val Asp aaa act tac agg ttc att gcc ttc tcc ggt ctt aag aag aac tac ttc 2112 Lys Thr Tyr Arg Phe Ile Ala Phe Ser Gly Leu Lys Lys Asn Tyr Phe ggt gtg ttc aag gat tcc aag gtt gac ata aag ggc atg ttg gca aag 2160 Gly Val Phe Lys Asp Ser Lys Val Asp Ile Lys Gly Met Leu Ala Lys -37.23-aag agg aac acc cca gag ttt ctg aag cag gcc ttc aat gag get aag 2208 Lys Arg Asn Thr Pro Glu Phe Leu Lys Gln Ala Phe Asn Glu Ala Lys gag agg cta gcg aag gtt cag aac cag gag gag ctc gaa aag gca att 2256 Glu Arg Leu Ala Lys Val Gln Asn Gln Glu Glu Leu Glu Lys Ala Ile caa gac tta acg gcg cag gtt aag gag gtg tac agg aag ctt aag atg 2304 Gln Asp Leu Thr Ala Gln Val Lys Glu Val Tyr Arg Lys Leu Lys Met aag gaa tat aac ttg gat gag ctc gcc ttc agg gtc atg tta tcc agg 2352 Lys Glu Tyr Asn Leu Asp Glu Leu Ala Phe Arg Val Met Leu Ser Arg gac gtg aag tcc tat gag aag aac acc cca cag cac gtt aag get gcg 2400 Asp Val Lys Ser Tyr Glu Lys Asn Thr Pro Gln His Val Lys Ala Ala gca cag ctg gcg gag atg aac gta caa gtg atg tca agg gat ata att 2448 Ala Gln Leu Ala Glu Met Asn Val Gln Val Met Ser Arg Asp Ile Ile agc ttc gta aag gta aag act aag gag gga gtt aaa cct gtc cag cta 2496 Ser Phe Val Lys Val Lys Thr Lys Glu Gly Val Lys Pro Val Gln Leu get aag ctt tca gag att gat gtg gat aaa tac tat gag agc gtg aga 2544 Ala Lys Leu Ser Glu Ile Asp Val Asp Lys Tyr Tyr Glu Ser Val Arg agt acc ttc gaa cag tta ttg aaa agc ttc aat gtg agc tgg gat aga 2592 Ser Thr Phe Glu Gln Leu Leu Lys Ser Phe Asn Val Ser Trp Asp Arg ata gag tcc acg aca tca atc gac tcg ttc ttc aag act tag 2634 Ile Glu Ser Thr Thr Ser Ile Asp Ser Phe Phe Lys Thr <210> 8 <211> 877 <212> PRT
<213> Metallosphaera prunae <400> 8 Met Ser Ile Met Ala Arg Gln Leu Thr Leu Ala Asp Phe Ser Gly Ile Lys Arg Glu Glu Pro Val Lys Gln Glu Glu Lys Thr Gln Glu Glu Glu -37.24-Arg Pro Leu Glu Arg Pro Ala Arg Leu Arg Lys Asp Thr Val Lys Gln Ala Gln Glu Glu Arg Lys Tyr Phe Leu Leu Ser Val Asp Tyr Asp Gly Lys Met Gly Lys Ala Val Cys Lys Leu Tyr Asp Pro Glu Thr Gly Glu Leu His Val Leu Tyr Asp Ser Thr Gly His Lys Ser Tyr Phe Leu Val Asp Leu Glu Pro Asp Gln Ile Gln Lys Ile Pro Lys Ile Val Lys Asp Glu Ser Phe Val Arg Leu Glu Lys Thr Thr Lys Ile Asp Pro Tyr Thr Trp Lys Pro Ile Asn Leu Thr Lys Ile Val Val Asn Asp Pro Leu Ala Val Arg Arg Leu Arg Glu Tyr Va1 Pro Arg Ala Tyr Glu Ala His Ile Lys Tyr Phe Asn Asn Tyr Ile Tyr Asp Phe Ser Leu Ile Pro Gly Met Pro Tyr Val Val Lys Lys Gly Lys Leu Val Pro Leu Lys Pro Glu Val Asp Val Lys Glu Val Lys Glu Ala Phe Lys Asp Ala Asp Gln Ile Ala Gln Glu Met Ala Leu Asp Trp Ala Pro Leu Phe Glu Ser Glu Ile Pro Ser Val Lys Arg Val Ala Ile Asp Ile Glu Val Tyr Thr Pro Met Met Gly Arg Val Pro Asp Pro Val Lys Ala Glu Tyr Pro Val Ile Ser Val Ala Leu Ala Gly Ser Asp Gly Leu Lys Leu Val Leu Val Leu Asp Arg Gly Asp Ser Pro Ile Gln Ser Lys Asp Ile Lys Val Glu Val Phe Arg Thr Glu Arg Glu Leu Leu Ser Arg Leu Phe Asp Ile Leu Lys Glu Tyr Pro Met Val Leu Thr Phe Asn Gly Asp Asp Phe Asp Ile Pro Tyr Leu Ile Phe Arg Gly Phe Lys Leu Gly Leu Leu Gln Asp Glu Ile Pro Phe Glu Ile Ser Ser Phe Gly Arg Lys Pro Asp Ala Lys Phe Arg Tyr Gly Phe His Ile Asp Leu Tyr Arg Phe Phe Phe Asn Lys Ala Val Arg Asn Tyr Ala Phe Glu Gly Lys Tyr Ser Glu Tyr Asn Leu Asp Thr Val Ala Gln Ala Leu Leu Gly Leu Ser Lys Val Lys Leu Asp Glu Ser Ile Ser Asp Leu Asn Met Ser Lys Leu Val Glu Tyr Asn Tyr Arg Asp Ser Glu Ile Thr Leu Lys Leu Thr Thr Phe Asn Asn Glu Leu Val Trp Lys Leu Ile Val Leu Phe Ser Arg Ile Ser Lys Leu Gly Ile Glu Glu Leu Thr -37.25-Arg Thr Glu Ile Ser Ala Trp Val Lys Asn Leu Tyr Tyr Trp Glu His Arg Lys Arg Asn Trp Leu Ile Pro Leu Lys Glu Glu Ile Leu Glu Arg Ser Ser Gly Leu Lys Thr Ala Ala Ile Ile Lys Gly Lys Gly Tyr Lys Gly Ala Val Val Ile Asp Pro Pro Val Gly Val Tyr Phe Asp Val Val Val Leu Asp Phe Ala Ser Leu Tyr Pro Ser Ile Ile Arg Asn Trp Asn Leu Ser Tyr Glu Thr Val Asp Val Lys Glu Cys Asn Lys Lys Arg Asp Ile Arg Asp Glu Ser Gly Ala Lys Ile His Glu Val Cys Val Asp Arg Pro Gly Ile Thr Ala Val Val Thr Gly Leu Leu Arg Asp Phe Arg Val Lys Ile Tyr Lys Lys Lys Gly Lys Gln Ser Asn Ile Asp Glu Glu Arg Lys Met Leu Tyr Asp Val Val Gln Arg Gly Met Lys Val Phe Ile Asn Ala Thr Tyr Gly Val Phe Gly Ala Glu Thr Phe Pro Leu Tyr Ala Pro Ala Val Ala Glu Ser Val Thr Ala Leu Gly Arg Tyr Val Ile Thr Ser Thr Lys Glu Met Ala Asn Lys Leu Gly Leu Lys Val Val Tyr Gly Asp Thr Asp Ser Leu Phe Ile His Gln Pro Asp Lys Lys Lys Leu Glu Glu Leu Val Glu Trp Thr Arg Gln Asn Phe Gly Leu Asp Leu Glu Val Asp Lys Thr Tyr Arg Phe Ile Ala Phe Ser Gly Leu Lys Lys Asn Tyr Phe Gly Val Phe Lys Asp Ser Lys Val Asp Ile Lys Gly Met Leu Ala Lys Lys Arg Asn Thr Pro Glu Phe Leu Lys Gln Ala Phe Asn Glu Ala Lys Glu Arg Leu Ala Lys Val Gln Asn Gln Glu Glu Leu Glu Lys Ala Ile Gln Asp Leu Thr Ala Gln Val Lys Glu Val Tyr Arg Lys Leu Lys Met Lys Glu Tyr Asn Leu Asp Glu Leu Ala Phe Arg Val Met Leu Ser Arg Asp Val Lys Ser Tyr Glu Lys Asn Thr Pro Gln His Val Lys Ala Ala Ala Gln Leu Ala Glu Met Asn Val Gln Val Met Ser Arg Asp Ile Ile Ser Phe Val Lys Val Lys Thr Lys Glu Gly Val Lys Pro Val Gln Leu Ala Lys Leu Ser Glu Ile Asp Val Asp Lys Tyr Tyr Glu Ser Val Arg Ser Thr Phe Glu Gln Leu Leu Lys Ser Phe Asn Val Ser Trp Asp Arg -37.26-Ile Glu Ser Thr Thr Ser Ile Asp Ser Phe Phe Lys Thr <210> 9 <211> 2289 <212> DNA
<213> Desulfurococcus sp.
<220>
<221> CDS
<222> (1)...(2286) <220>
<221> variation <222> (1801)..(1801) <223> s at position 1801 is either c or g <400> 9 atg gag agg gtt cgc cta gtg aag gtg gtt acc aag gat cct cta atc 48 Met Glu Arg Val Arg Leu Val Lys Val Val Thr Lys Asp Pro Leu Ile gtg agg aag att agg agc aag ttt aac act gcg tgg gag get aag ata 96 Val Arg Lys Ile Arg Ser Lys Phe Asn Thr Ala Trp Glu Ala Lys Ile aag tat cat gca aac tac atc tac gat aat agg ctg ata cct gga atg 144 Lys Tyr His Ala Asn Tyr Ile Tyr Asp Asn Arg Leu Ile Pro Gly Met agg tat gtt aca gac ttc tcc aac ggt gcg caa aag ctt gta atg gtt 192 Arg Tyr Val Thr Asp Phe Ser Asn Gly Ala Gln Lys Leu Val Met Val aag cca gag ata ccc caa tcc ctt gtt gag aaa gta agg gag ttg ttc 240 Lys Pro Glu Ile Pro Gln Ser Leu Val Glu Lys Val Arg Glu Leu Phe agg aat gag cct cct gaa aca gtg aag ctg get gag gaa ctc ctc ctc 288 Arg Asn Glu Pro Pro Glu Thr Val Lys Leu Ala Glu Glu Leu Leu Leu ttg ttc gag gag tca ccg ccc aag gtg aag cgc gta gca gtc gac ata 336 Leu Phe Glu Glu Ser Pro Pro Lys Val Lys Arg Val Ala Val Asp Ile gag gtt ttc acc cca ttc aaa ggg cgt atc ccc agc ccg aag ctc gcc 384 Glu Val Phe Thr Pro Phe Lys Gly Arg Ile Pro Ser Pro Lys Leu Ala -37.27-gaa tac cct gtg att agc ata gca ttg gcc ggt agc gac ggc ttg aag 432 Glu Tyr Pro Val Ile Ser Ile Ala Leu Ala Gly Ser Asp Gly Leu Lys aaa atc ctc ctg ctg gcc agg gaa tac aag cat gat ttc gac tac atg 480 Lys Ile Leu Leu Leu Ala Arg Glu Tyr Lys His Asp Phe Asp Tyr Met atg gag gat tac cct gtt gaa gcc gag gtg gag gtg ttc gac tcc gag 528 Met Glu Asp Tyr Pro Val Glu Ala Glu Val Glu Val Phe Asp Ser Glu aaa gac atg ttg ctg gaa gcc ttc aga ata atg ggg agc tat ccc gtc 576 Lys Asp Met Leu Leu Glu Ala Phe Arg Ile Met Gly Ser Tyr Pro Val gtc ctc act tac aac ggt gat aat ttc gac ctt caa tac ctg tac gtg 624 Val Leu Thr Tyr Asn Gly Asp Asn Phe Asp Leu Gln Tyr Leu Tyr Val aga gcc ttc aag ctg ggg att ctg aga agc cat atc ccg ttg aag ata 672 Arg Ala Phe Lys Leu Gly Ile Leu Arg Ser His Ile Pro Leu Lys Ile ggg gag gat atg att aga att gac aca agc ata cac cta gat cta tac 720 Gly Glu Asp Met Ile Arg Ile Asp Thr Ser Ile His Leu Asp Leu Tyr aag ttc ttc tcg aac agg get gtt aaa aac tat get ttc ggg ggg aaa 768 Lys Phe Phe Ser Asn Arg Ala Val Lys Asn Tyr Ala Phe Gly Gly Lys tac cag gag gag aag ctt gac get gtt tca ggg gca ctg cta gga gtg 816 Tyr Gln Glu Glu Lys Leu Asp Ala Val Ser Gly Ala Leu Leu Gly Val tcg aaa ata ggt ttc gag gaa aca atc ggc ggc ata tcg gcc tca cta 864 Ser Lys Ile Gly Phe Glu Glu Thr Ile Gly Gly Ile Ser Ala Ser Leu tta gcc gcc tac aac tac agg gat gcc gag atc acg tta aac cta acc 912 Leu Ala Ala Tyr Asn Tyr Arg Asp Ala Glu Ile Thr Leu Asn Leu Thr atg ttc agt aat gaa ctc gtt tgg aaa ctc att att ctt cta get agg 960 Met Phe Ser Asn Glu Leu Val Trp Lys Leu Ile Ile Leu Leu Ala Arg gtt tcc aag aca agc att gaa gac ctg tgt agg agg cag att tcc tac 1008 Val Ser Lys Thr Ser Ile Glu Asp Leu Cys Arg Arg Gln Ile Ser Tyr -37.28-tgg att caa aat ctg ttc ttc tgg gag cgc agg aag ctc ggc tac ctc 1056 Trp Ile Gln Asn Leu Phe Phe Trp Glu Arg Arg Lys Leu Gly Tyr Leu ata cct aac aag gag gac att ctg agg cat gta agg ggg acg ggg acg 1104 Ile Pro Asn Lys Glu Asp Ile Leu Arg His Val Arg Gly Thr Gly Thr aag gcg att att gag ggt aag aag tac get gga gcc tta gtg gtt gag 1152 Lys Ala Ile Ile Glu Gly Lys Lys Tyr Ala Gly Ala Leu Val Val Glu cct ccg aaa ggg get ttc ttc aac gtg gtc gtc ctc gac ata gcc tcc 1200 Pro Pro Lys Gly Ala Phe Phe Asn Val Val Val Leu Asp Ile Ala Ser cta tac cct agc att atc aaa aaa tac aat ctg agc tat gag acc gtt 1248 Leu Tyr Pro Ser Ile Ile Lys Lys Tyr Asn Leu Ser Tyr Glu Thr Val gac atg aag tgg tgt agc aag aca ata gat att gtc gat gaa acc ggg 1296 Asp Met Lys Trp Cys Ser Lys Thr Ile Asp Ile Val Asp Glu Thr Gly aga agg ctt cac gaa gtc tgc gtt gac aag ccc ggg ttg acc gcg caa 1344 Arg Arg Leu His Glu Val Cys Val Asp Lys Pro Gly Leu Thr Ala Gln cta acc ggt att cta agg gat tac agg gtt gga ata tat aag aag agg 1392 Leu Thr Gly Ile Leu Arg Asp Tyr Arg Val Gly Ile Tyr Lys Lys Arg tct aag gat aag agc ctt ccc cct gaa acc ctg gcc tgg tac gag gtg 1440 Ser Lys Asp Lys Ser Leu Pro Pro Glu Thr Leu Ala Trp Tyr Glu Val gtt cag aga get att aag gtg ttc ata aac get agc tac ggg gtc ttc 1488 Val Gln Arg Ala Ile Lys Val Phe Ile Asn Ala Ser Tyr Gly Val Phe ggg gat gag aag ttc tct ctg tac tcc cca gca gtg get gaa agc gtt 1536 Gly Asp Glu Lys Phe Ser Leu Tyr Ser Pro Ala Val Ala Glu Ser Val acc gcg atg ggt agg aag tcc ttc tac act att gtg aga aag gcc gcg 1584 Thr Ala Met Gly Arg Lys Ser Phe Tyr Thr Ile Val Arg Lys Ala Ala gat ctc ggg gtt aaa aca ctg tat ggc gac acg gac tcg ata ttc gtc 1632 Asp Leu Gly Val Lys Thr Leu Tyr Gly Asp Thr Asp Ser Ile Phe Val -37.29-tgg gcc cca acc cag gag cag ttg agg aag cta cag tca tgg atc ctt 1680 Trp Ala Pro Thr Gln Glu Gln Leu Arg Lys Leu Gln Ser Trp Ile Leu gag aag cta ggc ctg gag atc gag att gac aag tct ttt aca tac gtg 1728 Glu Lys Leu Gly Leu Glu Ile G.lu Ile Asp Lys Ser Phe Thr Tyr Val gtt ttc aca ggg ctt aag aag aac tac ctg ggc aga acg gtt gac ggc 1776 Val Phe Thr Gly Leu Lys Lys Asn Tyr Leu Gly Arg Thr Val Asp Gly ggc ata gag atc aag ggg ctt gtc scg aag aag agg aat act ccg gag 1824 Gly Ile Glu Ile Lys Gly Leu Val Xaa Lys Lys Arg Asn Thr Pro Glu ttc ctg aaa gac ttg ttc gag aat gtt atc gaa aag ctt aaa agc gtt 1872 Phe Leu Lys Asp Leu Phe Glu Asn Val Ile Glu Lys Leu Lys Ser Val gaa aac ccc gcg ggt ttc ata gag ttc gtc aag tgg ttg gag cat cag 1920 Glu Asn Pro Ala Gly Phe Ile Glu Phe Val Lys Trp Leu Glu His Gln gtg aag aca ata cat aac gat att agg agg aag gag ata acg ctc gac 1968 Val Lys Thr Ile His Asn Asp Ile Arg Arg Lys Glu Ile Thr Leu Asp cgg ctc gcc ata agg gtg gcc tta acc aag acg cca tcc ctc tac act 2016 Arg Leu Ala Ile Arg Val Ala Leu Thr Lys Thr Pro Ser Leu Tyr Thr aag act aag ccg ccg cat gtt aag gca gcc ctc caa tta atg aac tac 2064 Lys Thr Lys Pro Pro His Val Lys Ala Ala Leu Gln Leu Met Asn Tyr ggg tac agc gtg gag gag ggg gat att ata acg ttt gtc aag gtg aag 2112 Gly Tyr Ser Val Glu Glu Gly Asp Ile Ile Thr Phe Val Lys Val Lys agc aag gag ggc tat aag get ata cag tta acg agg ctt cac gaa gta 2160 Ser Lys Glu Gly Tyr Lys Ala Ile Gln Leu Thr Arg Leu His Glu Val gac cct gat aag tac att gag ctt gtt aaa agc ggt ctt gaa caa ttc 2208 Asp Pro Asp Lys Tyr Ile Glu Leu Val Lys Ser Gly Leu Glu Gln Phe ctc tca gcc ttc gga ata agg tgg gag gat atc ata ggc tcc ggc ggg 2256 Leu Ser Ala Phe Gly Ile Arg T.rp Glu Asp Ile Ile Gly Ser Gly Gly -3 7.3 0-tta acc gag ctt ttg aga aac aat agg gcg tag 2289 Leu Thr Glu Leu Leu Arg Asn Asn Arg Ala <210> 10 <211> 762 <212> PRT
<213> Desulfurococcus sp.
<220>
<221> variation <222> (601)..(601) <223> Xaa at position 601 is alanine or proline <400> 10 Met Glu Arg Val Arg Leu Val Lys Val Val Thr Lys Asp Pro Leu Ile Val Arg Lys Ile Arg Ser Lys Phe Asn Thr Ala Trp Glu Ala Lys Ile Lys Tyr His Ala Asn Tyr Ile Tyr Asp Asn Arg Leu Ile Pro Gly Met Arg Tyr Val Thr Asp Phe Ser Asn Gly Ala Gln Lys Leu Val Met Val Lys Pro Glu Ile Pro Gln Ser Leu Val Glu Lys Val Arg Glu Leu Phe Arg Asn Glu Pro Pro Glu Thr Val Lys Leu Ala Glu Glu Leu Leu Leu Leu Phe Glu Glu Ser Pro Pro Lys Val Lys Arg Val Ala Val Asp Ile Glu Val Phe Thr Pro Phe Lys Gly Arg Ile Pro Ser Pro Lys Leu Ala Glu Tyr Pro Val Ile Ser Ile Ala Leu Ala Gly Ser Asp Gly Leu Lys Lys Ile Leu Leu Leu Ala Arg Glu Tyr Lys His Asp Phe Asp Tyr Met Met Glu Asp Tyr Pro Val Glu Ala Glu Val Glu Val Phe Asp Ser Glu Lys Asp Met Leu Leu Glu Ala Phe Arg Ile Met Gly Ser Tyr Pro Val Val Leu Thr Tyr Asn Gly Asp Asn Phe Asp Leu Gln Tyr Leu Tyr Val Arg Ala Phe Lys Leu Gly Ile Leu Arg Ser His Ile Pro Leu Lys Ile Gly Glu Asp Met Ile Arg Ile Asp Thr Ser Ile His Leu Asp Leu Tyr Lys Phe Phe Ser Asn Arg Ala Val Lys Asn Tyr Ala Phe Gly Gly Lys -37.31-Tyr Gln Glu Glu Lys Leu Asp Ala Val Ser Gly Ala Leu Leu Gly Val Ser Lys Ile Gly Phe Glu Glu Thr Ile Gly Gly Ile Ser Ala Ser Leu Leu Ala Ala Tyr Asn Tyr Arg Asp Ala Glu Ile Thr Leu Asn Leu Thr Met Phe Ser Asn Glu Leu Val Trp Lys Leu Ile Ile Leu Leu Ala Arg Val Ser Lys Thr Ser Ile Glu Asp Leu Cys Arg Arg Gln Ile 5er Tyr Trp Ile Gln Asn Leu Phe Phe Trp Glu Arg Arg Lys Leu Gly Tyr Leu Ile Pro Asn Lys Glu Asp Ile Leu Arg His Val Arg Gly Thr Gly Thr Lys Ala Ile Ile Glu Gly Lys Lys Tyr Ala Gly Ala Leu Val Val Glu Pro Pro Lys Gly Ala Phe Phe Asn Val Val Val Leu Asp Ile Ala Ser Leu Tyr Pro Ser Ile Ile Lys Lys Tyr Asn Leu Ser Tyr Glu Thr Val Asp Met Lys Trp Cys Ser Lys Thr Ile Asp Ile Val Asp Glu Thr Gly Arg Arg Leu His Glu Val Cys Val Asp Lys Pro Gly Leu Thr Ala Gln Leu Thr Gly Ile Leu Arg Asp Tyr Arg Val Gly Ile Tyr Lys Lys Arg Ser Lys Asp Lys Ser Leu Pro Pro Glu Thr Leu Ala Trp Tyr Glu Val Val Gln Arg Ala Ile Lys Val Phe Ile Asn Ala Ser Tyr Gly Val Phe Gly Asp Glu Lys Phe Ser Leu Tyr Ser Pro Ala Val Ala Glu Ser Val Thr Ala Met Gly Arg Lys Ser Phe Tyr Thr Ile Val Arg Lys Ala Ala Asp Leu Gly Val Lys Thr Leu Tyr Gly Asp Thr Asp Ser Ile Phe Val Trp Ala Pro Thr Gln Glu Gln Leu Arg Lys Leu Gln Ser Trp Ile Leu Glu Lys Leu Gly Leu Glu Ile Glu Ile Asp Lys Ser Phe Thr Tyr Val Val Phe Thr Gly Leu Lys Lys Asn Tyr Leu Gly Arg Thr Val Asp Gly Gly Ile Glu Ile Lys Gly Leu Val Xaa Lys Lys Arg Asn Thr Pro Glu Phe Leu Lys Asp Leu Phe Glu Asn Val Ile Glu Lys Leu Lys Ser Val Glu Asn Pro Ala Gly Phe Ile Glu Phe Val Lys Trp Leu Glu His Gln Val Lys Thr Ile His Asn Asp Ile Arg Arg Lys Glu Ile Thr Leu Asp Arg Leu Ala Ile Arg Val Ala Leu Thr Lys Thr Pro Ser Leu Tyr Thr -37.32-Lys Thr Lys Pro Pro His Val Lys Ala Ala Leu Gln Leu Met Asn Tyr Gly Tyr Ser Val Glu Glu Gly Asp Ile Ile Thr Phe Val Lys Val Lys Ser Lys Glu Gly Tyr Lys Ala Ile Gln Leu Thr Arg Leu His Glu Val Asp Pro Asp Lys Tyr Ile Glu Leu Val Lys Ser Gly Leu Glu Gln Phe Leu Ser Ala Phe Gly Ile Arg Trp Glu Asp Ile Ile Gly Ser Gly Gly Leu Thr Glu Leu Leu Arg Asn Asn Arg Ala <210> 11 <211> 1725 <212> DNA
<213> Aquifex pyrophilus <220>
<221> CDS
<222> (1)...(1722) <400> 11 atg gat ttt gaa tac gta acg gga gaa gag gga tta aaa aag gca ata 48 Met Asp Phe Glu Tyr Val Thr Gly Glu Glu Gly Leu Lys Lys Ala Ile aaa agg ctc gaa aat tct cca tac ctt tac ctg gat acg gaa acc aca 96 Lys Arg Leu Glu Asn Ser Pro Tyr Leu Tyr Leu Asp Thr Glu Thr Thr gga gac agg ata agg ctc gta caa atc gga gac gaa gaa aac acc tac 144 Gly Asp Arg Ile Arg Leu Val Gln Ile Gly Asp Glu Glu Asn Thr Tyr gtt att gac ctc tac gaa att cag gat ata gaa cct ctg agg aaa tta 192 Val Ile Asp Leu Tyr Glu Ile Gln Asp Ile Glu Pro Leu Arg Lys Leu ata aac gaa agg ggg ata gta ggg cac aac ctt aag ttc gat ctt aag 240 Ile Asn Glu Arg Gly Ile Val Gly His Asn Leu Lys Phe Asp Leu Lys tac ctc tac agg tac ggg ata ttt ccc tcg gca acg ttt gac act atg 288 Tyr Leu Tyr Arg Tyr Gly Ile Phe Pro Ser Ala Thr Phe Asp Thr Met ata gcg agc tac ctc ctc gga tac gag aga cac tcc ctc aat cac ata 336 Ile Ala Ser Tyr Leu Leu Gly Tyr Glu Arg His Ser Leu Asn His Ile gtt tca aac cta ctc gga tat tcc atg gac aag agt tat cag act tcc 384 -37.33-Val Ser Asn Leu Leu Gly Tyr Ser Met Asp Lys Ser Tyr Gln Thr Ser gac tgg gga gcg agc gtt ctg agc gac get cag ctc aag tac get gca 432 Asp Trp Gly Ala Ser Val Leu Ser Asp Ala Gln Leu Lys Tyr Ala Ala aac gac gtt ata gtc ctc aga gaa ctc ttc cct aag atg agg gac atg 480 Asn Asp Val I1e Val Leu Arg Glu Leu Phe Pro Lys Met Arg Asp Met tta aac gag cta gac get gag agg gga gag gaa ctg ctc aag act aga 528 Leu Asn Glu Leu Asp Ala Glu Arg Gly Glu Glu Leu Leu Lys Thr Arg acg gca aag att ttc gat ctg aag agt ccc gta gca ata gtg gaa atg 576 Thr Ala Lys Ile Phe Asp Leu Lys Ser Pro Val Ala Ile Val Glu Met get ttc gta agg gaa gtt gca aaa ctc gag ata aac ggc ttt ccc gtg 624 Ala Phe Val Arg Glu Val Ala Lys Leu Glu Ile Asn Gly Phe Pro Val gac gta gaa gag cta acc aac aag tta aaa get gtg gaa agg gaa acc 672 Asp Val Glu Glu Leu Thr Asn Lys Leu Lys Ala Val Glu Arg Glu Thr cag aag agg ata cag gag ttt tac ata aag tac aga gtt gac cct ctc 720 Gln Lys Arg Ile Gln Glu Phe Tyr Ile Lys Tyr Arg Val Asp Pro Leu tct ccg aaa cag ctc gcc tca ctc ctg acg aag aag ttt aaa ctg aac 768 Ser Pro Lys Gln Leu Ala Ser Leu Leu Thr Lys Lys Phe Lys Leu Asn ctt ccc aag act cct aaa ggg aac gta tct aca gac gac aag get ctt 816 Leu Pro Lys Thr Pro Lys Gly Asn Val Ser Thr Asp Asp Lys Ala Leu act tcc tat cag gac gta gaa ccc gta aaa ctc gtt ctg gaa ata aga 864 Thr Ser Tyr Gln Asp Val Glu Pro Val Lys Leu Val Leu Glu Ile Arg aag ctt aag aag atc gcg gac aag tta aag gag tta aaa gaa cac ttg 912 Lys Leu Lys Lys Ile Ala Asp Lys Leu Lys Glu Leu Lys Glu His Leu aag aac ggg aga gtt tac ccg gag ttc aag caa ata gga get gta acg 960 Lys Asn Gly Arg Val Tyr Pro Glu Phe Lys Gln Ile Gly Ala Val Thr gga agg atg tcc tcc gca cac cca aat atc cag aac ata cac agg gat 1008 -37.34-Gly Arg Met Ser Ser Ala His Pro Asn Ile Gln Asn Ile His Arg Asp atg aga gga att ttc aag gcg gag gag gga aat act ttc gtc att tcg 1056 Met Arg Gly Ile Phe Lys Ala Glu Glu Gly Asn Thr Phe Val Ile Ser gac ttt tct cag ata gag ctc agg att gcg gcc gaa tac gta aag gac 1104 Asp Phe Ser Gln Ile Glu Leu Arg Ile Ala Ala Glu Tyr Val Lys Asp ccg ctt atg ctg gac gcc ttc aaa aag gga aag gac atg cac agg tac 1152 Pro Leu Met Leu Asp Ala Phe Lys Lys Gly Lys Asp Met His Arg Tyr acc get tca gtg gta ctc gga aag aaa gag gaa gaa ata aca aaa gag 1200 Thr Ala Ser Val Val Leu Gly Lys Lys Glu Glu Glu Ile Thr Lys Glu gag aga cag ctc gca aaa get ata aac ttc ggt ctc ata tac ggc att 1248 Glu Arg Gln Leu Ala Lys Ala Ile Asn Phe Gly Leu Ile Tyr Gly Ile tcc get aaa ggg ctt gca gaa tac gca aag ctt ggt tac ggc gtt gaa 1296 Ser Ala Lys Gly Leu Ala Glu Tyr Ala Lys Leu Gly Tyr Gly Val Glu att tct tta gaa gaa get cag gtt ttg aga gag agg ttt ttc aag aac 1344 Ile Ser Leu Glu Glu Ala Gln Val Leu Arg Glu Arg Phe Phe Lys Asn ttc aaa get ttc aaa gag tgg cac gac aga gtt aag aaa gaa cta aag 1392 Phe Lys Ala Phe Lys Glu Trp His Asp Arg Val Lys Lys Glu Leu Lys gaa aag gga gag gta aaa ggt cat acg ctt ctt gga agg aga ttt tcc 1440 Glu Lys Gly Glu Val Lys Gly His Thr Leu Leu Gly Arg Arg Phe Ser gca aat acc ttt aac gac get gta aat tac ccc ata cag gga acg ggt 1488 Ala Asn Thr Phe Asn Asp Ala Val Asn Tyr Pro Ile Gln Gly Thr Gly gcg gac cta cta aaa ctg gca gtt cta ctt ttt gac gca aac ctc cag 1536 Ala Asp Leu Leu Lys Leu Ala Val Leu Leu Phe Asp Ala Asn Leu Gln aaa aag gga ata gat gca aag ctc gtg aac ctc gtg cac gac gag ata 1584 Lys Lys Gly Ile Asp Ala Lys Leu Val Asn Leu Val His Asp Glu Ile gtc gta gag tgc gaa aag gaa aaa gcg gaa gaa gta aaa gaa ata ctc 1632 -37.35-Val Val Glu Cys Glu Lys Glu Lys Ala Glu Glu Val Lys Glu Ile Leu gaa aaa tcc atg aaa acg gcg gga aag ata ata ctg aaa gag gtt ccc 1680 Glu Lys Ser Met Lys Thr Ala Gly Lys Ile Ile Leu Lys Glu Val Pro gtg gaa gta gaa agc gtt ata aac gaa agg tgg acg aaa gat 1722 Val Glu Val Glu Ser Val Ile Asn Glu Arg Trp Thr Lys Asp taa 1725 <210> 12 <211> 574 <212> PRT
<213> Aquifex pyrophilus <400> 12 Met Asp Phe Glu Tyr Val Thr Gly Glu Glu Gly Leu Lys Lys Ala Ile Lys Arg Leu Glu Asn Ser Pro Tyr Leu Tyr Leu Asp Thr Glu Thr Thr Gly Asp Arg Ile Arg Leu Val Gln Ile Gly Asp Glu Glu Asn Thr Tyr Val Ile Asp Leu Tyr Glu Ile Gln Asp Ile Glu Pro Leu Arg Lys Leu Ile Asn Glu Arg Gly Ile Val Gly His Asn Leu Lys Phe Asp Leu Lys Tyr Leu Tyr Arg Tyr Gly Ile Phe Pro Ser Ala Thr Phe Asp Thr Met Ile Ala Ser Tyr Leu Leu Gly Tyr Glu Arg His Ser Leu Asn His Ile Val Ser Asn Leu Leu Gly Tyr Ser Met Asp Lys Ser Tyr Gln Thr Ser Asp Trp Gly Ala Ser Val Leu Ser Asp Ala Gln Leu Lys Tyr Ala Ala Asn Asp Val Ile Val Leu Arg Glu Leu Phe Pro Lys Met Arg Asp Met Leu Asn Glu Leu Asp Ala Glu Arg Gly Glu Glu Leu Leu Lys Thr Arg Thr Ala Lys Ile Phe Asp Leu Lys Ser Pro Val Ala Ile Val Glu Met Ala Phe Val Arg Glu Val Ala Lys Leu Glu Ile Asn Gly Phe Pro Val Asp Val Glu Glu Leu Thr Asn Lys Leu Lys Ala Val Glu Arg Glu Thr Gln Lys Arg Ile Gln Glu Phe Tyr Ile Lys Tyr Arg Val Asp Pro Leu Ser Pro Lys Gln Leu Ala Ser Leu Leu Thr Lys Lys Phe Lys Leu Asn -37.36-Leu Pro Lys Thr Pro Lys Gly Asn Val Ser Thr Asp Asp Lys Ala Leu Thr Ser Tyr Gln Asp Val Glu Pro Val Lys Leu Val Leu Glu Ile Arg Lys Leu Lys Lys Ile Ala Asp Lys Leu Lys Glu Leu Lys Glu His Leu Lys Asn Gly Arg Val Tyr Pro Glu Phe Lys Gln Ile Gly Ala Val Thr Gly Arg Met Ser Ser Ala His Pro Asn Ile Gln Asn Ile His Arg Asp Met Arg Gly Ile Phe Lys Ala Glu Glu Gly Asn Thr Phe Val Ile Ser Asp Phe Ser Gln Ile Glu Leu Arg Ile Ala Ala Glu Tyr Val Lys Asp Pro Leu Met Leu Asp Ala Phe Lys Lys Gly Lys Asp Met His Arg Tyr Thr Ala Ser Val Val Leu Gly Lys Lys Glu Glu Glu Ile Thr Lys Glu Glu Arg Gln Leu Ala Lys Ala Ile Asn Phe Gly Leu Ile Tyr Gly Ile Ser Ala Lys Gly Leu Ala Glu Tyr Ala Lys Leu Gly Tyr Gly Val Glu Ile Ser Leu Glu Glu Ala Gln Val Leu Arg Glu Arg Phe Phe Lys Asn Phe Lys Ala Phe Lys Glu Trp His Asp Arg Val Lys Lys Glu Leu Lys Glu Lys Gly Glu Val Lys Gly His Thr Leu Leu Gly Arg Arg Phe Ser Ala Asn Thr Phe Asn Asp Ala Val Asn Tyr Pro Ile Gln Gly Thr Gly Ala Asp Leu Leu Lys Leu Ala Val Leu Leu Phe Asp Ala Asn Leu Gln Lys Lys Gly Ile Asp Ala Lys Leu Val Asn Leu Val His Asp Glu Ile Val Val Glu Cys Glu Lys Glu Lys Ala Glu Glu Val Lys Glu Ile Leu Glu Lys Ser Met Lys Thr Ala Gly Lys Ile Ile Leu Lys Glu Val Pro Val Glu Val Glu Ser Val Ile Asn Glu Arg Trp Thr Lys Asp -37.37-

Claims (32)

THE EMBODIMENTS OF THE INVENTION FOR WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. Substantially pure polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10 and 12.

2. An isolated polynucleotide sequence encoding a polypeptide of claim 1.

3. An isolated polynucleotide selected from the group consisting of:
a) SEQ m NO:1, 3, 5, 7, 9, 11;
b) SEQ ID NO:1, 3, 5, 7, 9, 11 wherein T can be replaced by U;
c) nucleic acid sequences complementary to a) and b); and d) fragments of a), b), or c) that are at least 15 bases in length and that will hybridize to DNA which encodes the amino acid sequences of SEQ ID NO:2, 4, 6, 8, 10 or 12 under moderate to highly stringent conditions.

4. The polynucleotide of claim 2, wherein the polynucleotide is isolated from a prokaryote.

5. An expression vector including the polynucleotide of claim 2.

6. The vector of claim 5, wherein the vector is a plasmid.

7. The vector of claim 5, wherein the vector is derived from a virus.

8. A host cell stably transformed with the vector of claim 5.

9. The host cell of claim 8, wherein the cell is prokaryotic.

10. The host cell of claim 8, wherein the cell is eukaryotic.

11. Antibodies that specifically bind to the polypeptide of claim 1 or to a fragment thereof, wherein said fragment is at least 8 amino acids in length.

12. The antibodies according to claim 11, wherein said fragment is at least 10 amino acids in length.

13. The antibodies according to claim 11, wherein said fragment is at least 30 amino acids in length.

14. The antibodies according to claim 11, wherein said fragment is at least 50 amino acids in length.

15. The antibodies of any one of claims 11 to 14, wherein the antibodies are polyclonal.

16. An enzyme selected from the group consisting of:
a) an enzyme comprising an amino acid sequence which is at least 70% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 6, 8, 10, and 12; and b) an enzyme which comprises at least 30 consecutive amino acid residues having at least 70% identity to the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 5, 6, 8, 10 and 12, wherein the enzyme is a polymerase.

17. A method for producing a polypeptide comprising:
a) culturing the host cell of claim 8;
b) expressing from the host cell of claim 8 a polypeptide encoded by said DNA;
and c) isolating the polypeptide.

18. A process for producing a transformed cell comprising transforming or transfecting the cell with the vector of claim 5 such that the cell expresses the polypeptide encoded by the DNA contained in the vector.

19. An isolated polynucleotide selected from the group consisting of:
a) a polynucleotide having at least 70% identity to a nucleic acid encoding an enzyme comprising an amino acid sequence selected from the group of amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, and 12, wherein the polynucleotide encodes a polymerase;
b) a polynucleotide which is complementary to the polynucleotide of a); and c) a polynucleotide comprising at least 15 bases of the polynucleotide of a) or b).

20. An oligonucleotide probe that hybridizes to a nucleic acid target region corresponding to a region selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9 and 11, under hybridization conditions comprising hybridization in a solution comprising 0.9M NaCl, 50 mM NaH2PO4, pH 7.0, 5.0 mM Na2EDTA, 0.5% SDS, 10X Denhardt's, and 0.5 mg/mL polyriboadenylic acid at 45°C.

21. The oligonucleotide probe of claim 20, wherein the hybridization conditions further comprise: a wash for 30 minutes at room temperature in a buffer comprising 150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1 mM Na2EDTA, 0.5% SDS, followed by a 30 minute wash in fresh buffer at Tm-10°C.

22. The oligonucleotide probe of claim 20, wherein the probe is from 10 to about 150 nucleotides in length.

23. The oligonucleotide probe of claim 20, wherein the probe is from 10 to about 100 nucleotides in length.

24. The oligonucleotide probe of claim 20, wherein the probe is from 10 to about 50 nucleotides in length.

25. The oligonucleotide probe of claim 20, wherein the probe is from 10 to about 30 nucleotides in length.

26. The oligonucleotide probe of claim 20, wherein the probe is from 10 to about 15 nucleotides in length.

27. The oligonucleotide probe of claim 20, wherein the probe comprises a segment of contiguous bases which is at least 70% complementary to a target sequence of 10 contiguous nucleotides present in the target region.

28. The oligonucleotide probe of claim 20, wherein the probe is detestably labeled.

29. The oligonucleotide probe of claim 28, wherein the detectable label is selected from the group consisting of a radioisotope, a fluorescent compound, a bioluminescent compound, a chemiluminescent compound, a metal chelator and an enzyme.

30. An antibody that specifically binds to a polypeptide as set forth in any one of SEQ
ID NOs: 2, 4, 6, 8, 10 and 12.

31. The antibody of claim 30, wherein the antibody is polyclonal.

32. The antibody of claim 30, wherein the antibody is detectably labeled.