WO1995016694A1

WO1995016694A1 - Origin of replication complex genes, proteins and methods

Info

Publication number: WO1995016694A1
Application number: PCT/US1994/014563
Authority: WO
Inventors: Bruce W. Stillman; Stephen P. Bell; Ryuji Kobayashi; Jasper Rine; Margit Foss; Francis J. Mcnally; Patricia Laurenson; Ira Herskowitz; Joachim J. Li
Original assignee: Cold Spring Harbor Laboratory; The Regents Of The University Of California
Priority date: 1993-12-16
Filing date: 1994-12-16
Publication date: 1995-06-22
Also published as: EP0733057A4; AU1373495A; CA2178965A1; JPH09506768A; AU700405B2; EP0733057A1

Abstract

Origin of DNA Replication Complex (ORC) genes, recombinant ORC peptides and methods of identifying DNA binding proteins and using the subject compositions are provided. Vectors and cells comprising such ORC genes find use in the production of recombinant ORC peptides. The subject ORC peptides find particular use in screening for ORC selective agents useful in the diagnosis, prognosis or treatment of disease, particularly fungal infections and neoproliferative disease. Disclosed methods for identifying a gene encoding a protein which directly or indirectly associates with a selected DNA sequence involve: transforming an expression library of hybrid proteins into a reporter strain, wherein the library comprises protein-coding sequences fused to a constitutively expressed transcription activation domain and the reporter strain comprises a reporter gene with at least one copy of a selected DNA sequence in its promoter region. Clones expressing the transcription or translation product of the reporter gene are detected and recovered.

Description

ORIGIN OF REPLICATION COMPLEX GENES, PROTEINS AND METHODS

INTRODUCTION The research carried out in the subject application was supported in part by grants from the National Institutes of Health. The government may have rights in any patent issuing on this application.

Technical Field

The technical field of this invention concerns Origin of Replication Complex genes which are invovled with DNA transcription and replication.

Background

The elements involved in the early events of eukaryotic DNA replication have begun to emerge in the yeast Saccharomyces cerevisiae. A critical first step was the identification of ARS elements derived from yeast chromosomes, a subset of which were subsequently shown to act as chromosomal origins of DNA replication (reviewed in 11). Sequence comparison of a number of ARS elements resulted in the identification of the ARS consensus sequence (ACS, 12). This sequence is essential for the function of yeast origins of DNA replication (7, 12, 13). Three additional elements required for efficient ARSl function have been identified. When mutated individually, these elements, referred to as Bl, B2, and B3, result in a slight reduction of ARSl activity. When two or three of the B elements are simultaneously mutated, however, ARSl function is severely compromised (14). Proteins that recognize two elements of ARSl have been identified. The yeast transcription factor ABF1 binds to and mediates the function of the B3 element (11, 14). More recently we have identified a multi-protein complex that specifically recognizes the highly conserved ACS (15). This activity, referred to as the origin recognition complex (ORC), has several properties that make it an attractive candidate to act as an initiator protein at yeast origins of replication. Binding of this protein requires the ACS, and the effect of mutations in the consensus sequence on ARSl function parallels the effect of the same mutations on ORC DNA binding. ORC binds to more than 10 yeast ARS elements, several of which are known origins of DNA replication (15). Specific DNA binding by ORC requires ATP, suggesting that ORC binds ATP, a property of a number of known initiator proteins (17). ORC also interacts with other sequences outside of the ACS that are known to be important for ARS function (18, 19). Further support for the hypothesis that ORC mediates the function of the ACS is provided by in situ deoxyribonuclease I (DNase I) footprinting experiments that identify a protected region of ARSl remarkably similar to that observed with ORC in vitro (20).

Relevant Literature

A multi-protein complex that recognizes cellular origins of DNA replication was ^'reported in Bell and Stillman (1992) Nature 357, 128-134. Much of the present disclosure was published by Foss et al. (1993), Bell et al. (1993) and Li and Herskowicz (1993), in Science 262, 1838, 1843 and 1870, respectively, issue date December 17, 1993. Wang and Reed (1993) Nature 364, 121-126 report using a single-hybrid screen as disclosed herein.

SUMMARY OF THE INVENTION Origin of DNA Replication Complex (ORC) genes, recombinant ORC peptides and methods of identifying DNA binding proteins and using the subject compositions are provided. Provided are compositions comprising isolated nucleic acids encoding unique ORC gene portions, especially portions encoding biologically active unique portions of ORC1-ORC6 proteins. Vectors and cells comprising such DNA molecules find use in the production of recombinant ORC peptides. The subject compositions are used to isolate ORC genes from a wide variety of species, including human. The subject ORC peptides also find particular use in screening for ORC selective agents useful in the diagnosis, prognosis or treatment of disease, particulary fungal infections and neoproliferative disease. Particularly useful are agents capable of distinguishing an ORC protein of an infectious organism or transformed cell from the wild-type human homologue.

Also disclosed are methods for identifying a gene encoding a protein which directly or indirectly associates with a selected DNA sequence. Generally, the methods involve transforming an expression library of hybrid proteins into a reporter strain, wherein the library comprises protein-coding sequences fused to a constitutively expressed transcription activation domain and the reporter strain comprises a reporter gene with at least one copy of a selected DNA sequence in its promoter region. Clones expressing the transcription or translation product of the reporter gene are detected and recovered. A preferred method employs an activation domain from GAL4 and a lacZ reporter gene.

BREIF DESCRIPTION OF SEQUENCE ID NUMBERS SEQUENCE ID NO:l. DNA Sequence of ORC1. SEQUENCE ID NO:2. Amino Acid Sequence of ORC1. SEQUENCE ID NO: 3. DNA Sequence of ORC2.

SEQUENCE ID NO:4. Amino Acid Sequence of ORC2. SEQUENCE ID NO:5. DNA Sequence of ORC3. SEQUENCE ID NO:6. Amino Acid Sequence of ORC3. SEQUENCE ID NO:7. DNA Sequence of ORC4. SEQUENCE ID NO: 8. Amino Acid Sequence of ORC4. SEQUENCE ID NO:9. DNA Sequence of ORC5. SEQUENCE ID NO: 10. Amino Acid Sequence of ORC5. SEQUENCE ID NO: 11. DNA Sequence of ORC6. SEQUENCE ID NO: 12. Amino Acid Sequence of ORC6.

DESCRIPTION OF SPECIFIC EMBODIMENTS The recombinant polypeptides of the invention comprise unique portions of the disclosed ORC proteins which retain an binding affinity specific to the subject full-length ORC protein. A "unique portion" has an amino acid sequence unique to subject ORC in that it is not found in previously known protein and has a length at least long enough to define a peptide specific to that ORC. Unique portions are found to vary from about 5 to about 25 residues, usually from 5 to 10 residues in length, depending on the particular amino acid sequence and are readily identified by comparing the subject portion sequences with known peptide/protein sequence data bases. Hence, the term polypeptide as used herein defines an amino acid polymer with as few as five residues. ORCs used in the subject screening assays are frequently smaller deletion mutants of full-length ORC proteins. Typically, such deletion mutants are readily generated using conventional molecular techniques and screened for an ORC-specific binding affinity using the various assays described below, e.g. footprint analysis, coimmunoprecipitation, etc.

ORC-specific retained binding affinities include the ability to selectively bind a nucleic acid of a defined sequence, an ORC protein or an compound such as an antibody which is capable of selectively binding an ORC protein. As such, binding specificity may be provided by an ORC-specific immunological epitope, lectin binding site, etc. Selective binding is conveniently shown by competition with labeled ligand using recombinant ORC peptide either in vitro or in cell based systems as disclosed herein. Generally, selective binding requires a binding affinity of 10^"6M, preferably 10^"8M, more preferably 10^"10M, under in vitro conditions as exemplified below.

The subject recombinant polypeptides may be free or covalently coupled to other atoms or molecules. Frequently the polypeptides are present as a portion of a larger polypeptide comprising the subject polypeptide where the remainder of the larger polypeptide need not be ORC-derived. The subject polypeptides are typically "isolated", meaning unaccompanied by at least some of the material with which they are associated in their natural state. Generally, an isolated polypeptide constitutes at least about 1%, preferably at least about 10%, and more preferably at least about 50% by weight of the total poly/peptide in a given sample. By pure peptidepolypeptide is intended at least about 60% , preferably at least 80%, and more preferably at least about 90% by weight of total polypeptide. Included in the subject polypeptide weight are any atoms, molecules, groups, etc. covalently coupled to the subject polypeptides, such as detectable labels, glycosylations, phosphorylations, etc.

The subject polypeptides may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample and to what, if anything, the polypeptide is covalently linked. Purification methods include electrophoretic, molecular, immunological and chromatographic techniques, especially affinity chromatography and RP-HPLC in the case of peptides. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer- Verlag, NY (1982). The polypeptides may be modified or joined to other compounds using physical, chemical, and molecular techniques disclosed or cited herein or otherwise known to those skilled in the relevant art to affect their ORC/receptor binding specificity or other properties such as solubility, membrane transportability, stability, toxicity, bioavailability, localization, detectability, in vivo half-life, etc. as assayed by methods disclosed herein or otherwise known to those of ordinary skill in the art. Other modifications to further modulate binding specificity/affinity include chemical/enzymatic intervention (e.g. fatty acid-acylation, proteolysis, glycosylation) and especially where the poly /peptide is integrated into a larger polypeptide, selection of a particular expression host, etc. Amino and/or carboxyl termini may be functionalized e.g., for the amino group, acylation or alkylation, and for the carboxyl group, esterification or amidification, or the like.

Many of the disclosed poly/peptides contain glycosylation sites and patterns which may be disrupted or modified, e.g. by enzymes like glycosidases. For instance, N or O-linked glycosylation sites of the disclosed poly/peptides may be deleted or substituted for by another basic amino acid such as Lys or His for N- linked glycosylation alterations, or deletions or polar substitutions are introduced at Ser and Thr residues for modulating O-linked glycosylation. Glycosylation variants are also produced by selecting appropriate host cells, e.g. yeast, insect, or various mammalian cells, or by in vitro methods such as neuraminidase digestion. Other covalent modifications of the disclosed poly/peptides may be introduced by reacting the targeted amino acid residues with an organic derivatizing (e.g. ethyl¬ s' [(p-azido-phenyl)dithio] propioimidate) or crosslinking agent (e.g. 1, 1- bis(diazoacetyl)-2-phenylethane) capable of reacting with selected side chains or termini. For therapeutic and diagnostic localization, the subject poly/peptides thereof may be labeled directly (radioisotopes, fluorescers, etc.) or indirectly with an agent capable of providing a detectable signal, for example, a heart muscle kinase labeling site. 5 ORC poypeptides with ORC binding specificity are identified by a variety of ways including crosslinking, or preferably, by screening such polypeptides for binding to or disruption of ORC-ORC complexes. Additional ORC-specific agents include specific antibodies that can be modified to a monovalent form, such as Fab, Fab', or Fv, specifically binding oligopeptides or oligonucleotides and most

10 preferably, small molecular weight organic compounds. For example, the disclosed ORC peptides are used as immunogens to generate specific polyclonal or monoclonal antibodies. See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, for general methods.

Other prospective ORC specific agents are screened from large libraries of

15 synthetic or natural compounds. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily producible. Additionally, natural and synthetically produced libraries and compounds -are readily modified through conventional chemical, physical, and biochemical means. See, e.g. Houghten et al. and Lam et al (1991) Nature 354,

»

20 84 and 81, respectively and Blake and Litzi -Davis (1992), Bioconjugate Chem 3, 510.

Useful agents are identified with assays employing a compound comprising the subject polypeptides or encoding nucleic acids. A wide variety of in vitro, cell-free binding assays, especially assays for specific binding to immobilized

25 compounds comprising ORC polypeptide find convenient use. For example, immobilized ORC-ORC or ORC-nucleic acid complexes provide convenient targets for disruption, e.g. as measured by the disassociation of a labelled component of the complex. Such assays are amenable to scale-up, high throughput usage suitable for volume drug screening. While less preferred, cell-based assays may be used to

30 determine specific effects of prospective agents.

Preferred agents are ORC- and species-specific. Useful agents may be found within numerous chemical classes, though typically they are organic compounds; preferably small organic compounds. Small organic compounds have a molecular weight of more than 150 yet less than about 4,500, preferably less than about 1500, more preferably, less than about 500. Exemplary classes include steroids, heterocyclics, polycyclics, substituted aromatic compounds, and the like. Selected agents may be modified to enhance efficacy, stability, pharmaceutical compatibility, and the like. Structural identification of an agent may be used to identify, generate, or screen additional agents. For example, where peptide agents are identified, they may be modified in a variety of ways as described above, e.g. to enhance their proteolytic stability. Other methods of stabilization may include encapsulation, for example, in liposomes, etc. The subject binding agents are prepared in any convenient way known to those in the art.

For therapeutic uses, the compositions and agents disclosed herein may be administered by any convenient way. Small organics are preferably administered orally; other compositions and agents are preferably administered parenterally, conveniently in a pharmaceutically or physiologically acceptable carrier, e.g., phosphate buffered saline, or the like. Typically, the compositions are added to a retained physiological fluid. As examples, many of the disclosed therapeutics are amenable to direct injection or infusion, topical, in tratracheal/nasal- administration e.g. through aerosal, intraocularly, or within/on implants e.g. collagen, osmotic pumps, grafts comprising appropriately transformed cells, etc. Generally, the ' amount administered will be empirically determined, typically in the range of about 10 to 1000 μg/kg of the recipient. For peptide agents, the concentration will generally be in the range of about 50 to 500 μg/ml in the dose administered. Other additives may be included, such as stabilizers, bactericides, etc. These additives will be present in conventional amounts.

The invention provides isolated nucleic acids encoding ORC genes, their transcriptional regulatory regions and the disclosed unique ORC polypeptides which retain ORC-specific function. As used herein: an "isolated" nucleic acid is present as other than a naturally occurring chromosome or transcript in its natural state and is typically joined in sequence to at least one nucleotide with which it is not normally associated on a natural chromosome; nucleic acids with substantial sequence similarity hybridize under low stringency conditions, for example, at 50°C and SSC (0.9 M saline/0.09 M sodium citrate) and remain bound when subject to washing at 55 °C with SSC, wherein regions of non-identity of substantially similar nucleic acid sequences preferably encode redundant codons; a partially pure nucleotide sequence constitutes at least about 5 % , preferably at least about 30%, and more preferably at least about 90% by weight of total nucleic acid present in a given fraction; unique portions of the disclosed nucleic acids are of length sufficient to distinguish previously known nucleic acids, hence a unique portion has a nucleotide sequence at least long enough to define a novel oligonucleotide, usually at least about 18 bp in length, preferably at least about 36 nucleotides in length. Typically, the invention's ORC polypeptide encoding polynucleotides are associated with heterologous sequences. Examples of such heterologous sequences include regulatory sequences such as promoters, enhancers, response elements, signal sequences, polyadenylation sequences, etc., introns, 5' and 3' noncoding regions, etc. According to a particular embodiment of the invention, portions of the coding sequence are spliced with heterologous sequences to produce soluble, secreted fusion proteins, using appropriate signal sequences and optionally, a fusion partner such as β-Gal. For antisense applications where the inhibition of expression is indicated, especially useful oligonucleotides are between about 10 and 30 nucleotides in length and include sequences surrounding the disclosed ATG start site, especially the oligonucleotides defined by the disclosed sequence beginning about 5 nucleotides before the start site and ending about 10 nucleotides after the disclosed start site. The ORC encoding nucleic acids can be subject to alternative purification, synthesis, modification, sequencing, expression, transfection, administration or other use by methods disclosed in standard manuals such as Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc, Wiley-Interscience, NY, NY, 1992) or that are otherwise known in the art.

The invention also provides vectors comprising the described ORC nucleic acids. A large number of vectors, including plasmid and viral vectors, have been described for expression in a variety of eukaryotic and prokaryotic hosts.

Advantageously, vectors will often include a promotor operably linked to an ORC polypeptide-encoding portion, one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance. The inserted coding sequences may be synthesized, isolated from natural sources, prepared as hybrids, etc. Suitable host cells may be transformed/ transfected/infected by any suitable method including electroporation, CaCl₂ mediated DNA uptake, viral infection, microinjection, microprojectile, or other methods.

Appropriate host cells include bacteria, archebacteria, fungi, especially yeast, and plant and animal cells, especially mammalian cells. Of particular interest are E. coli. B. subtilis. Saccharomyces cerevisiae. SF9 cells, C129 cells, 293 cells, Neurospora, and CHO, COS, HeLa cells, immortalized mammalian myeloid and lymphoid cell lines, and pluripotent cells, especially mammalian ES cells and zygotes. Prefeπed expression systems include COS-7, 293, BHK, CHO, TM4, CV1, VERO-76, HELA, MDCK, BRL 3A, W138, Hep G2, MMT 060562, TRI cells, and baculovirus systems. Preferred replication systems include Ml 3, ColEl, SV40, baculovirus, lambda, adenovirus, AAV, BPV, etc. A large number of transcription initiation and termination regulatory regions have been isolated and shown to be effective in the transcription and translation of heterologous proteins in the various hosts. Examples of these regions, methods of isolation, manner oi^" manipulation, etc. are known in the art.

For the production of stably transformed cells and transgenic animals, the subject nucleic acids may be integrated into a host genome by recombination events. For example, such a nucleic acid can be electroporated into a cell, and thereby effect homologous recombination at the site of an endogenous gene, an analog or pseudogene thereof, or a sequence with substantial identity to an ORC- encoding gene. Other recombination-based methods such as nonhomologous recombinations, deletion of endogenous gene by homologous recombination, especially in pluripotent cells, etc. , provide additional applications. Preferred transgenics and stable transformants over-express or under-express (e.g. knock-out cells and animals) a disclosed ORC gene and find use in drug development and as a disease model. Methods for making transgenic animals, usually rodents, from ES cells or zygotes are known to those skilled in the art.

The compositions and methods disclosed herein may be used to effect gene therapy. See, e.g. Zhu et al. (1993) Science 261, 209-211; Gutierrez et al. (1992) Lancet 339, 715-721. For example, cells are transfected with ORC-encoding sequences operably linked to gene regulatory sequences capable of effecting altered ORC expression or regulation. To modulate ORC translation, target cells may be transfected with complementary antisense polynucleotides. For gene therapy involving the grafting/implanting/transfusion of transfected cells, administration will depend on a number of variables that are ascertained empirically. For example, the number of cells will vary depending on the stability of the transfered cells. Transfer media is typically a buffered saline solution or other pharmacologically acceptable solution. Similarly the amount of other administered compositions, e.g. transfected nucleic acid, protein, etc. , will depend on the manner of administration, purpose of the therapy, and the like.

The genes encoding six ORC subunits from S. cerevisiae are used to obtain the functional homologues of the ORC proteins from other species. For example, we have demonstrated that the ORCI gene is conserved in a related fungi klyuermyces lactis. The ORCI gene in both S. cerevisie and t lactis contain conserved primary protein sequence that are utilized to obtain the ORCI gene from other species including other fungi and from human. Using oligonucleotide primers based on the conserved sequences between S. cerevisiae and k lactis, PCR is used to identify the ORCI protein in any eukaryotic species. The cloned gene encoding ORCI polypeptide from any fungi or from human cells is used to express the protein in a bacterial expression system to make antibodies against the polypeptide. These antibodies are used to immunoprecipitate the ORC complex from the relevant species. Using the disclosed techniques for protein sequencing, the sequence the ORC polypeptides is obtained. Using the protein sequencing methodologies disclosed herein for cloning the S. cerevisiae protein, other genes or cDNAS encoding the ORC polypeptides from other fungi species and from human cells are obtained. As we demonstrate herein how to reconstitute the ORC complex by expressing each of the S. cerevisiae genes in a baculovirus expression vector and infecting Sf 9 insect cells with viruses expressing each of the ORC subunits, these genes are used to express the ORC polypeptides and reconstitute activity. In this way, large amounts of ORC protein from any fungi or mammalian species, including human cells, are obtained.

Inhibitors of ORC protein in fungi provide valuable reagents to selectively inhibit proliferation of fungal cell division by inhibiting the initiation of DNA replication. This offers a powerful, selective target for antifungal agents valuable in controlling fungal infections in human and other species. For example, as disclosed herein, inhibiting the ORC function by mutation in S. cerevisiae can actually cause the death of the mutant cells. In human proliferative disorders such as cancer, cells of the diseased tissue undergo uncontrolled cell proliferation. A key event in this cell proliferation is the initiation of DNA replication. Inhibiting the initiation of DNA replication through inhibition of ORC function provides a valuable target for inhibitors of cell growth. By expressing each of the cDNAS encoding the ORC proteins, either individually or together in an expression system, ORC function is reconstituted in vitro. Using this recombinant, expressed protein, inhibitors of ORC function are obtained that block the initiation of DNA replication in cell cycle. As described above, small molecular inhibitors of ORC DNA binding or other activities provide valuable reagents as anti-cancer and anti-proliferation drugs. The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES Example 1. Transcriptional silencing and ORC.

The binding of purified ORC to the ARS consensus sequence (ACS) at each of the mating type silencers was tested using a DNase I protection assay (22). ORC protected the match to the ACS at each of the four silencers in an ATP dependent manner. In addition, at each silencer characteristic hypersensitive sites of DNAse I cleavage were observed initiating 12-13 bp from the ACS and extending away from the consensus sequence at approximately 10 bp intervals. This pattern of DNase I protection and enhanced cleavage is nearly identical to that observed at non-silencer sequences and indicates that ORC binding to these elements is not fundamentally different from its binding at other ARS elements. At HML-E, HML-I, and HMR-E the only protection observed included the

ACS. At HMR-I, however, we observed a second unexpected footprint that did not overlap a strong match to the ACS. Moreover, unlike all previous sites bound by ORC, this protection showed little dependence upon the addition of ATP to the binding reaction. Although there are two partial matches to the ACS in this region, similar sequences in other ARS elements and silencers were not recognized by ORC, suggesting that these sequences did not direct this unusual ATP- independent binding of ORC to DNA. In combination with the protection observed at the ACS, the boundaries of the ORC footprint at HMR-I were very similar to the boundaries of HMR-I defined by deletion mutagenesis (23). These experiments demonstrate that ORC binds all four of the mating-type silencers, that ORC can bind sequences other than the ACS and that it plays an important role at HML and HMR. A clear link between ORC function and transcriptional silencing was provided by the finding that a mutation in a gene encoding a subunit of ORC was defective for repression at HMR (below). To clone the genes encoding the various ORC subunits, peptides derived from each of the ORC subunits were sequenced (24). A candidate gene, refeπed to as ORC2, was isolated by complementation of a temperature sensitive mutation that showed silencing defects at the permissive temperature. Genetic experiments suggested that ORC2 mediated the silencing function of the ACS at HMR-E, making it a good candidate to encode a subunit of ORC (below). Comparison of the predicted amino acid sequence of ORC2 showed that all of the peptides derived from the 72 kd subunit of ORC were within the open reading frame of the ORC2 gene indicating that it encoded the second largest subunit of ORC.

ORC2 mutations alter ORC function in vitro.

To address the effect of ORC2 mutations on ORC function in vitro, extracts were prepared from both orc2-I and ORC2 strains (25). Fractions derived from wild-type cells showed strong ORC DNAse I protection over the ACS and Bl elements of ARSl in DNAse I footprinting. In contrast, fractions derived from orc2-l cells showed a dramatic reduction in ORC DNA binding activity. The ACS and the Bl element were no longer protected from DNase I cleavage. Only the characteristic enhanced DNase I cleavages in the B domain of ARSl remained. Mutations that disrupt ORC DNA binding at ARSl prevented the residual DNA binding observed with the mutant fractions, indicating that this binding required the ACS. The DNA binding defects were also not due to a general inhibition of DNA binding as mixing of mutant and wild type fractions did not reduce binding of the wild type protein. Incubation of the mutant cells at the non-permissive temperature was not necessary to observe defects in ORC DNA binding, which explains the defect observed in mating-type regulation at the permissive temperature (below). To investigate the polypeptide composition of ORC derived from orc2-l and ORC2 cells, immuno-blots of these fractions were probed with polyclonal antibodies raised against ORC. 30 μg of partially purified ORC derived from either JRY3688 (ORC2) or JRY3687 (orc2-I) was separated on a 10% SDS- polyacrylamide gel and transfeπed to nitrocellulose. The resulting protein blot was incubated with polyclonal mouse sera raised against the entire ORC complex. This sera detects all but the 50 kd subunit of ORC. Antibody-antigen complexes were detected with horseradish peroxidase conjugated secondary antibodies followed by incubation with a chemiluminescent substrate.

Wild type fractions contained the 120, 72, 62, 56, and 53 kd subunits of ORC in roughly equal quantity. The mutant fractions, however, showed a distinctly different subunit composition. While the amount of the 120 and 56 kd subunits was only slightly reduced relative to the wild type fraction, the amount of the 72, 62, and 53 kd subunits was reduced dramatically. In UV cross-linking experiments the same three subunits are specifically cross-linked to DNA in an ACS and ATP dependent manner, suggesting an important role for one or more of these subunits in ORC DNA binding (15). Thus, the absence of these subunits explains the defects in DNA binding observed in vitro and indicates that the orc2-l mutation results in a reduction of ORC stability or a defect in Orc2p also results in reduced DNA binding of an intact ORC complex. orc2-I cells are defective for entry into S-phase. The point in the cell cycle the essential function of ORC2 is performed in vivo was investigated using alpha factor and hydroxyurea (HU) as cell cycle landmarks (26). Our results were consistent with the execution of the essential function of Orc2p between late Gl and the initiation of DNA synthesis. Arrest with HU followed by release into the non-permissive temperature resulted in 89% of the cells completing an additional cell cycle, indicating that the essential function for Orc2p was executed before the HU arrest point in the cell cycle. In contrast, blocking the cell cycle with alpha-factor followed by release at the non-permissive temperature resulted in the only 41 % of the cells completing an additional cell cycle. This phenotype indicates that the Orc2p function was performed at or near the Gl-S phase boundary.

To address the role of ORC in yeast DNA replication more directly, the DNA content of asynchronous cultures of either orc2-l or isogenic wild type cells was measured at various times after shifting from the permissive to the non- permissive temperature by fluorescent cytometric analysis (27). JRY3687 (orc2-l) or JRY3688 (ORC2) cells grown at 24 °C (0 minute time point) or at various times after shifting to the non-permissive temperature (37 °C) were fixed, stained with propidium iodide, and analyzed for DNA content using a Coulter Model Epics-C Flow Cytometer. In addition, a small number of cells (approximately 1000) from each time point were returned to the permissive temperature to determine the percentage of cells that remained viable at a given time point. Initially, the DNA content of both wild type and mutant cells was equally divided between 1C and 2C with approximately 10% of the cells in S phase. At early time points after the temperature shift (15-70 minutes) there was a dramatic loss of orc2-l cells in S- phase suggesting that entry into S-phase had been halted. Consistent with this hypothesis, as the time course continued the orc2-l mutant showed a rapid accumulation of cells with a 1C DNA content and a commensurate decrease in cells with a 2C DNA content (50-100 minutes). Between 100 and 120 minutes, a new population of orc2-l cells was observed that appeared to enter into a delayed S phase. By 150 minutes the bulk of the mutant cells were in this population and after 180 minutes only a few cells remained with a 1C DNA content.

Interestingly, we observed a strong coπelation between entry into the new round of DNA synthesis and a loss of orc2-l cell viability. Similar experiments with isogenic ORC2 cells showed that these effects were specific to the orc2-l mutation. These findings indicate that at the non-permissive temperature the orc2- 1 cells were initially unable to enter S phase, but later entered into an abortive round of DNA replication. Entry into this type of replication appears to be a lethal event. Overall, the analysis of the orc2-l mutation provides in vivo evidence showing that ORC acts early in S-phase in general, and as the initiator protein at yeast origins of replication in particular. Identification of the ORC6 gene. A second gene that represented a strong candidate to encode one of the subunits of ORC was the AAP1 gene. This gene was cloned using a novel screen fop proteins that bound to the ACS in vivo (below). When compared to the predicted amino acid sequence of this gene, we found that all of the peptides derived from the 50 kd subunit of ORC were encoded by the open reading frame of the AAP1 gene (28). For this reason we now refer to AAP1 as ORC6 , as it encodes the smallest of the six ORC subunits. The identification of this gene as a subunit of ORC provides direct evidence that ORC is bound to the ACS in vivo.

Numbered Citations for Introduction and Example 1 1. Callan, Cold Spring Harbor Symp. Quant. Biol. 38, 195-203 (1973).

2. Fangman and Brewer, Cell 71, 363-366 (1992).

3. P. Laurenson and J. Rine, Micro. Rev. 56, 543-560 (1992).

4. D. D. Dubey, et al., Mol. Cell. Biol. 11, 5346-5355 (1991).

5. D. H. Rivier and J. Rine, Science 256, 659-663 (1992). 6. A. M. Miller and K. A. Nasmyth, Nature 312, 247-251 (1984).

7. L. Pillus and J. Rine, Cell 59, 637-647 (1989).

8. A. Axlerod and J. Rine, Mol. Cell. Biol. 11, 1080-1091 (1991).

9. J. L. Campbell and C. S. Newlon, in The Molecular and Cellular Biology of the Yeast Saccharomyces J. R. Broach, J. R. Pringle, E. W. Jones, Eds. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1991) pp. 41-146.

10. J. Broach, et al., CSH Symp. Quant. Biol. Al, 1165-1173 (1983).

11. Deshpande and Newlon, Mol. & Cell. Bio 12, 4305-4313 (1992).

12. Y. Marahrens and B. Stillman, Science 255, 817-823 (1992).

13. S. P. Bell and B. Stillman, Nature 357, 128-134 (1992). 14. Kornberg & Baker, DNA Replicat'n (Freeman & Co, NY, 1991) v2.

15. C. S. Newlon, Microbiol. Rev. 52, 568-601 (1988).

16. Newlon and Theis, Current opinion in gen. and dev. 3, (1993).

17. J. F. X. Diffley and J. H. Cocker, Nature 357, 169-172 (1992).

18. Jacob, et al., CSH Symp. Quant. Biol , 28, 329-348 (1963). 19. DNAse I footprinting was performed as previously described (15).

20. J. B. Feldman,et al., J. Mol. Biol. 178, 815-834 (1984).

21. To obtain sufficient protein for peptide sequencing, a revised purification procedure for ORC was devised. Whole cell extract was prepared from 400g of frozen BJ926 cells using a bead beater (Biospec Products) until greater than 90% breakage was achieved. One twelfth volume of a saturated (at 4°C) solution of ammonium sulfate was added to the broken cells and stirred for 30 minutes. This solution was then spun at 13,000 x g for 20 minutes. The resulting supernatant was spun in a 45Ti rotor (Beckman) at 44,000 RPM for 1.5 hrs. 0.27g/ml of ammonium sulfate was added to the resulting supernatant, and the resulting precipitate was collected by spinning in the 45 Ti rotor at 40,000 RPM for 30 minutes. The resulting pellet was resuspended in buffer H/0.0 (15) and dialyzed versus H/0.15M KC1 (H with 0.15 M KC1 added). Preparation of ORC from this extract was similar to (15) with the following changes. The dsDNA cellulose column was omitted from the preparation and only a single glycerol gradient was performed. Sequencing of peptides derived from ORC subunits was performed using a modification of an "in gel" protocol described previously (40, 41). Purified ORC (— 10 μg per subunit) was separated by SDS-PAGE and stained with 0.1 % Coomassie Brilliant Blue G (Aldrich). After destaining the gel was soaked in water for one hour. The protein bands were excised, transferred to a microcentrifuge tube and treated with 200 ng of Achromobacteτ protease I (Lysylendopeptidase: Wako). The resulting peptides were separated by reverse- phase chromatography and sequenced by automated Edman degradation (Applied Biosystems model 470).

22. To isolate and assay ORC from ORC2 and orc2-I cells four liters of JRY3687 (orc2-l, MATa, hmrDAr. TRPl ade2 his3 leu2 trpl ura3) or the isogenic wild-type strain JRY3688 (ORC2 MATa., hmrDAr.TRPl ade2 his3 leu2 trpl ura3) were grown to a density of 2 x 10⁷ cells per ml. Extracts were prepared as described (24) and fractionated over the first two columns in the preparation of ORC. The peak fraction of ORC DNA binding activity eluted from the Q- Sepharose (Pharmacia) column of each preparation was used for subsequent analysis. Antibodies were raised against the entire ORC complex using a single mouse. The resulting sera was able to recognize all but the 50 kd subunit of ORC. Proteins were transfeπed to nitrocellulose and antigen-antibody complexes were detected with horse radish peroxidase conjugated secondary anitbodies and a chemiluminescent substrate. 23. Yeast cells were grown to a density of 1-4 x 10⁷ cells per ml at 24°C then diluted to a density of 2-4 x 10⁶ cells per ml into YPD containing 6 μM alpha- factor and incubated for 2-2.5 hours at 24°C (> 90% unbudded cells). For the hydroxyurea aπest experiments alpha factor was washed away and the cells were resuspended in YPD containing 100 mM hydroxyurea and incubated an additional 2.5 hours ( > 90% large budded cells). After incubation with the growth inhibitor, cells were briefly sonicated and plated on YPD plates pre-incubated at either 24°C or 37°C and observed at 0, 3, and 6 hours after plating.

24. Yeast cells were grown to a density of 1-4 x 10⁷ cells per ml at 24°C and diluted into fresh YPD at either 37°C or 24°C and a density of 2-4 x 10⁶ cells per ml. At times after dilution, 3 x 10⁶ cells were processed as described (42).

25. The position of the five peptides derived from the 50 kd subunit of ORC in the ORC6 gene were residues: 51-65; 91-102; 110-105; 207-226; 424-430.

26. K. M. Hennessy, et al, Genes Dev. 4, 2252-2263 (1990). 27. H. Renauld, et al., Genes Dev. 7, 1133-1145 (1993).

28. A. H. Brand, G. Micklem, and K. Nasmyth, Cell 51, 709-719 (1987).

29. McNally and Rine, Mol. and Cell. Biol. 11 , 5648-5659 (1991).

30. D. D. Brown, Cell 37, 359-365 (1984).

31. . A. P. Wolffe, J. Cell Sci. 99, 201-206 (1991). 32. D. Kitsberg, et al., Nature 364, 459-463 (1993).

33. K. S. Hatton, et al., Mol. Cell. Biol. 8, 2149-2158 (1988).

34. V. Dhar, et al., Mol. Cell. Biol. 9, 3524-3532 (1989).

35. L. G. Edgar and J. D. McGhee, Cell 53, 589-599 (1988).

36. L. P. Villaπeal, Micro. Rev. 55, 512-542 (1991). 37. H. Kawasaki, et al., Anal. Biochem. 191, 332-336 (1990).

38. H. Kawasaki and K. Suzuki, Anal. Biochem. 186, 264-268 (1990).

39. R. Nash, et al., EMBL Journal 7, 4335-4346 (1988).

40. J. Abraham, et al., J. Mol. Biol. 176, 307-331 (1984).

41. D. T. Stinchcomb, et al., Nature 282, 39-43 (1979).

Example 2.

ORC2, a gene required for viability and silencing In a mutant screen, a temperature-sensitive mutation called orc2-l was isolated that, at the permissive temperature, resulted in derepression of HMRa. flanked by the synthetic silencer and did not cause derepression of HMRa. flanked by the wild-type silencer (20). Because the orc2-l mutant was temperature- sensitive and silencing defective, it merited further analysis. The temperature resistance of a heterozygus orc2-l/ORC2 diploid (JRY2640) established that the mutation was recessive. The diploid was transformed with a plasmid containing HMRa flanked by a mutant silencer (pJR1212), to provide MATal function required for sporulation. The temperature-sensitive growth phenotype segregated 2 ts : 2 wild type in each of 23 tetrads, indicating that it was caused by a single nuclear mutation. An HMLOL mαtal HMR orc2-l segregant (JRY3683) was obtained from the diploid following sporulation.

Genetic crosses were used to determine which features in the wild-type silencer distinguished it from the synthetic silencer with respect to derepression by orc2-l. A mαtal HMRα strain (JRY3683) containing the orc2-l mutation was mated to a MATα strain containing a mutation in the RAPl binding site of HMR-E flanking HMRa (the HMRa-e-rαpl-10 allele; 5401-la) to determine whether orc2-l could derepress HMRa in the absence of a functional RAPl binding site. All 29 of the 96 MATα segregants that had little or no mating ability were temperature- sensitive for growth. Nineteen of the MATα temperature-sensitive segregants were mating competent, indicating that the orc2-l mutation per se was insufficient to disrupt mating ability, and suggesting that the HMRa-e-rαpl-10 allele was required in combination with orc2-l to block mating ability of α strains. A MATα temperature-sensitive segregant from this cross, which mated weakly as an α (JRY4133), was confirmed to have the genotype MATα HMRa-e-rαpl-10 orc2-l. As further evidence that orc2-l in combination with HMRa-e-rαpl-10 blocked the mating ability of MATα strains, a somewhat unusual cross was used to simplify the previous cross by having orc2-l as the only relevant heterozygous marker. Two MATα HMRa-e-rαpl-10 strains (JRY4133 and JRY4132) had complementary auxotrophic markers, allowing for the selection of the rare

MATα/ MATα diploid formed by a mating event between these two strains. This diploid was able to sporulate due to the low level of expression of HMRa in the diploid caused by the RAPl -site mutation in the HMR-E silencer (21). One of these strains had the orc2-l mutation (JRY4133) and the other did not. As expected, the temperature sensitivity segregated 2:2 in each of 34 tetrads. All of the temperature-resistant segregants (two per tetrad) exhibited the a mating phenotype, and all of the temperature-sensitive segregants were either very weak a -maters or were unable to mate at all. The absence of any recombinants between the temperature sensitivity and mating phenotype placed the gene(s) responsible for the temperature sensitivity and the mating defect less than 1.5 centimorgans apart, providing strong evidence that a lesion in a single gene was responsible for both phenotypes. This result was in agreement with the co-reversion of the ts and mating phenotypes described herein.

Isolation of multiple alleles of ORC2

Using the information from this analysis of orc2-l, a second screen was performed to identify additional mutations in essential genes with a role in silencer function. This second screen produced 50 mutants that were temperature sensitive for growth, and in which HMRa (flanked by a mutation in the RAPl -binding site) was derepressed at a semi-permissive temperature. Complementation tests for both growth at 37 °C and for mating phenotype were performed between orc2-l and the collection of temperature-sensitive mutants from the second screen. The collection of temperature sensitive mutants had the matal stel4 genotype, but were able to mate as α;'s due to the derepression of HMRα. These mutants were mated to a mαtal orc2-l strain (JRY3683) and the diploids were tested for growth at 37° C. All but three diploids were able to grow at the restrictive temperature. The three temperature-sensitive diploids were each presumed to be orc2/orc2 homozygotes due to the inability of the two mutations to complement one another. The mating type of the diploids was checked to determine whether the defect in repression of HMR was complemented. All three diploids mated as α's. Thus, the three mutants were unable to complement either the temperature sensitivity or the mating phenotype of the original orc2-l mutation. The new mutations (in strains JRY4136, 4137 and 4138) were designated orc2-2, orc2-3, and orc2-4. To investigate the possibility that the new mutations were in a gene other than ORC2 yet still failed to complement orc2-l, the allelism between orc2-l and orc2-3 was tested. The original mαtal orc2-3 stel4 mutant was cured of its HMRα plasmid, creating JRY 4137, and mated with a MATα HMRa-e-rαpl-10 orc2-l strain (JRY3685). In 24 tetrads from this diploid, all segregants were temperature sensitive for growth, indicating strong linkage between orc2-l and orc2-3 (<2 centimorgans). All further studies were performed using the orc2-l allele, which provided the stronger mutant phenotypes. Map position of ORC2

Linkage between ORC2 and LYS2, on chromosome II, was evident in crosses between two lys2 strains (JRY2640 and PSY152) and the original orc2-l isolate (JRY2903) that placed ORC2 approximately 24 centimorgans from LYS2.

A third cross (JRY4130 x JRY4134) tested the linkage between seel 8, which is centromere proximal to LYS2, and ORC2. Because both orc2-l and secl8 are temperature sensitive, an ORC2 allele marked by URA3 (from pJR1423) was used to determine that SEC18 and ORC2 were separated by 6.6 centimorgans (Table 1).

No previously-mapped genes involved in silencing map near SEC18. Table 1. Linkage of ORC2 to LYS2 and ORC2 to SEC18 Tetrad types Map distance

Cross PD 1 NPD (cM)

ORC2 vs LYS2 10 14 0 29

ORC2 vs LYS2 20 14 0 21 ORC2 vs LYS2 TOTAL 30 28 0 24

ORC2 vs SEC18 46 7 0 6.6

The ORC2 mutants aπested with a cell cycle terminal phenotype.

The effect of the orc2-l mutation on the cell division cycle was explored: mutant orc2-l strains were grown in liquid medium at 23 °C, the permissive temperature, and then shifted to 37 °C to test whether the cells aπested with a single terminal morphology. Specifically, orc2-l cells (JRY3683) were grown to log phase at the permissive temperature (23°C) and the culture was split. Half of the culture was grown an additional five hours at the permissive temperature and the other half was shifted to the nonpermissive temperature (37°C) and grown for an additional five hours. At that time, both cultures were fixed and stained with DAPI to allow visualization of the nucleus. In the culture maintained at the permissive temperature, cells at all phases of the cell cycle were observed. Cells later in the cell cycle, as evidenced by the presence of large buds, frequently exhibited nuclei in both the mother and the daughter cell. In contrast, in the culture shifted to the restrictive temperature, approximately 90% of the cells aπested as large budded cells. Nuclei were only present in the mother cell and not in the daughter cells. In addition, the cells were larger than those grown at the permissive temperature, indicating that protein synthesis and cell wall synthesis continued in the absence of ORC2 function. Similar results were obtained with two additional orc2-l strains (JRY3685 and JRY3687).

ORC2 cells harvested either after continuous growth at the permissive temperature or after a shift to the nonpermissive temperature were fixed and stained with DAPI allowing visualization of DNA with fluorescence microscopy. The cells grown permissively displayed a range of morphologies from small unbudded cells to cells with single buds of various sizes. The cells shifted to the nonpermissive temperature looked very different: the majority aπested as large budded cells, and for the most part, each mother-daughter pair contained only a single brightly-staining region, often at or near the neck. These data indicated that orc2-l mutants displayed cell cycle defects characteristic of mutants defective in DNA replication.

Cloning of the ORC2 gene:

The ORC2 gene was cloned by complementation of the orc2-l temperature sensitivity (22). One complementing clone (pJR1416) was chosen for further analysis. Subclones missing various fragments from the insert were retransformed into an orc2 strain to assay whether the deletion affected the clone's ability to complement orc2-F temperature sensitivity. The key observations were that the deletion of a 2.8-kb Sstl-SstI fragment destroyed complementation activity, whereas the deletions of flanking sequences (Xbal, and the larger_>_-tl fragment) had no effect. The 2.8-kb fragment was subcloned (pJR1263), and shown to possess complementing activity. To determine whether the gene on the clone was indeed allelic to the ORC2 mutation, a fragment of the original clone was subcloned into a yeast integrating vector. This plasmid (pJR1423) was cleaved within the insert to direct homologous integration and transformed into a wild-type strain (W303-1A). As a result, the site of integration was marked by the plasmid's URA3 gene. The resulting strain (JRY4134) was crossed to an orc2-l strain (JRY3685). In each of 59 tetrads, URA3 segregated opposite to the temperature sensitivity caused by orc2-l, indicating that ORC2 had indeed been cloned. ORC2 was essential for cell viability.

ORC2 was disrupted by URA3, (23), and integrated into a diploid homozygous for ura3 and ORC2, (JRY3444). Of the 41 tetrads dissected, 40 tetrads had two live and two dead segregants, and one tetrad had only one live segregant. The colonies that grew were, without exception, Ura-. By inference, the dead segregants contained the URA3 gene, and thus the ORC2 disruption, indicating that ORC2 function was essential for cell viability at all temperatures. The dead segregants were examined under a microscope to gain some insight into the true null phenotype. Most of the spores germinated into cells that were elongated or otherwise deformed and had not divided. In no case did the cell divide more than two times. Thus in many spores, the absence of ORC2 blocked cell division but not growth.

Role of ORC2 in Plasmid Replication

To test the role of ORC2 in plasmid stability, an isogenic pair of strains, one wild type (W303-1B) and one orc2-l (JRY4125), were transformed with a plasmid containing a centromere, a suppressor tRNA (SUPll-1), URA3, and

ARSl, a chromosomal origin of replication (YRP14/CEN4/ARS1/ARS1; (24, 25), selecting for uracil prototrophy. Transformants were grown on selective medium at 23°C, the permissive temperature for orc2-l. The colonies were picked from the selective plate, serially diluted, plated onto solid rich medium and grown to colonies at 23°C. The wild-type transformants grew into colonies most of which were white with a few exhibiting red sectors. The small fraction of red colonies were from cells in the selectively grown colony that had lost the plasmid. In contrast, the majority of colonies from the orc2-l mutant were red, reflecting a high degree of plasmid loss among the cells in the selectively grown colony. Moreover, in the orc2-l strain, red sectors were present in the majority of white colonies with some white colonies displaying multiple red sectors.

It is possible to quantitate the number of cell cycles in which a plasmid is lost from the number of colonies that are half red and half white. Only those colonies that lose the plasmid in the first cell division form half red, half white colonies. In the case of the wild-type strain, 0.9 % (10 / 1168) of the colonies were half red and half white, indicating that the plasmid was lost in 0.9 % of cell cycles. In contrast, the frequency of half red and half white colonies in the orc2-l strain grown at the permissive temperature was 11 % (58 / 512), indicating that the same plasmid was lost approximately 12 times as often in the strain with partially defective Orc2p. These data indicated a profound defect in plasmid stability specific to the orc2-l strain, and in combination with the cell-cycle phenotype of orc2-l, suggested that orc2-l strains were defective in DNA replication. These results were consistent with the flow cytometry studies of orc2-l strains herein. Sequence of ORC2

The sequence of the 2.8-kb Sstl-Sstl ørc2-complementing fragment was determined and deposited in Genbank (Accession # L23924). The only open reading frame of significant length was deduced to be ORC2, and predicted a 620 residue protein of approximately 68 kD. The Sstl fragment included 806-bp of upstream sequence and 140-bp of downstream sequence.

The deduced Orc2p protein was 15% basic residues and 16% serine/threonines. Fully 50% of the N-terminal residues (residues 15-280) were lysine, arginine, proline, serine, or threonine. The KeyBank motif program revealed several matches to peptide motifs within Orc2p. Orc2p contained many potential phosphorylation sites: 3 for cAMP- and cGMP-dependent protein kinase (starting at residues -57, 433 and 546), 12 for protein kinase C (24, 41, 42, 89, 101, 102, 176, 321, 335, 431, 521, and 549) and 14 for caseine kinase II (60, 148, 149, 182, 238, 270, 389, 481, 486, 491, 505, 552, 595, and 605), and match to the nuclear targeting sequence (residues 103-107). A perfect match to the RAPl binding site consensus (starting at nucleotide 595), and two near matches (12/15) to the ABF1 -binding consensus sequence (starting at 12 and 609). It was determined by sequence homology that a lysyl tRNA synthetase gene is located to the left of the Sstl fragment shown here (Mirande and Waller, 1988), and a kinase homolog to the right.

Another homolgy is with the region near the catalytic domain of human topoisomerase I proteins which has diverged among topoisomerase I proteins from other species except for the region suπounding the invariant active-site tyrosine. This region includes a consensus sequence consisting of a serine and lysine residue near the tyrosine (25). The Orc2p protein also contained such a consensus sequence near its C-terminus. However, mutation of this putative active-site tyrosine to phenylalanine had no detectable effect on the ability of ORC2 to complement the temperature-sensitivity or mating defect of an orc2-l strain. Table 2. Strain list. Strain Genotype ^(a)

DBY1034 MATa his4-539 lys2-801 ura3-52 SUC W303-1A MATa αde2-l cαnl-100 his3-ll,15 leu2-3,112 trpl-1 urα3-l

W303-1B MATα αde2-l cαnl-100 his3-ll,15 leu2-3,112 trpl-1 urα3-l PSY152 MATa his3D200 leu2-3,112 lys2-801 urα3-52

JRY4130 MATα his4 urα3 secl8 JRY438 MATα GaT his4-519 leu2-3,112 SUC2 urα3-52

JRY543 MATalMATα αde2-101/αde2-101 his3A200/his3A200

■ Iys2-801/lys2-801 met2/MET2 TYRl/tyrl urα3-52/urα3-52 JRY2640 mαtal αde2 leu2-3,112 lys2-801 urα3 JRY2698 MATα HMRα αde2-101 his3 leu2 trpl urα3-52

JRY2699 MATα HMRα αde2-101 his3 leu2 trpl urα3-52 sir4DN::HIS3 JRY2700 MATα HMRα αde2-101 his3 leu2 trpl urα3-52 + pJR924 JRY2903 MATα HMRα αde2-101 his3 leu2 orc2-l trpl urα3-52

JRY2904 MATα HMRα αde2-101 his3 leu2 orc2-l trpl urα3-52

+ pJR924 JRY3444 MATalMATα αde2-101/αde2-101 his3D200lhis3D200 lys2-801/lys2-801met2/MET2 TYRl/tyrl urα3-52/urα3-52 orc2::TnlOLUK/ORC2

JRY3683 mαtal {HMRα} αde2 his3 leu2 orc2-lurα3

JRY3685 MATα HMRa-e-rαpl-10 αde2 leu2 trpl orc2-l urα3 JRY3687 MATα hmrDAr. TRPl αde2 his3 leu2 trpl urα3 orc2-l JRY3690 MATa HMRa-e-rapl-10 αde2 his3-ll,15 leu2 orc2-l trpl urα3

JRY4125 MATα αde2-l cαnl-100 his3-ll,15 leu2-3,112 orc2-l trpl-1 urα3-l

JRY4132 MATα HMRa-e-rαpl-10 αde2 his3 urα3

JRY4133 MATα HMRa-e-rαpl-10 αde2 leu2 orc2-ltrpl urα3

JRY4134 MATa αde2-l cαnl-100 his3-ll,15 leu2-3,112 trpl-1 urα3-l ORC2::pJR1423

JRY4135 mαtal αde2 leu2-3,112 lys2-801 urα3 ste!4

JRY4136 mαtal αde2 leu2-3,112 lys2-801 orc2-2 urα3 stel4

JRY4137 mαtal αde2 leu2-3,112 lys2-801 orc2-3 urα3 stel4

JRY4138 mαtal αde2 leu2-3,112 lys2-801 orc2-4 urα3 stel4

(a) Unless otherwise noted, all strains were HMLα and HMRa. HMRa-e- rαpl-10 refers to the allele of HMR-E, originally described as PAS1-1, that contains a mutation in the RAPl binding site (21).

Numbered Citations for Example 2.

T. I. Herskowitz, et al Cold Spring Harbor Laboratory Press 583 (1992)._# 2. J. Abraham, J. Feldman, K.A. Nasmyth, J.N. Strathern, J.R. Broach, and J. Hicks, C.S.H. Symp. Quant. Biol. 47, 989 (1982). J.B. Feldman, J.B. Hicks, and J.R. Broach, J. Mol. Biol. 178, 815 (1984).

3. J. Rine, and I. Herskowitz, Genetics 116, 9 (1987).

4. Kurtz et al, Genes Dev. 5, 616 (1991); Sussel et al, PNAS 88, 7749 (1991). 5. J.R. Mullen, et al, PMBO J. 8, 2067 (1989).

6. P.S. Kayne, et al, Cell 55, 27 (1988). L.M. Johnson, et al, Proc. Natl. Acad. Sci. USA 87, 6286 (1990). P.D. Megee, et al, Science 247, 841 (1990). E. Park, and J. Szostak, Mol. Cell. Biol. 10, 4932 (1990).

7. P. Laurenson, and J. Rine, Microbiol. Rev. 56, 543 (1992). 8. Brand, et al., Cell 41, 41 (1985); Kimmerly, et al., EMBO J. 7, 2241 (1988). 9. D. Shore, and K. Nasmyth, Cell 51, 721 (1987). 10. M.S. Longtine, et al., Curr. Genet. 16, 225 (1989).

11. A.R. Buchman, et al, Mol. Cell. Biol. 8, 5086 (1988).

12. J.F.X. Diffley, and J.H. Cocker, Science 357, 169 (1992).

13. A.S. Buchman, and R.D. Kornberg, Mol. Cell. Biol. 10, 887 (1990).

14. J.A. Huberman, et al, Nucleic Acids Res.16, 6373 (1988). B.J. Brewer, and W.L. Fangman, Cell 51, 463 (1987).

15. S.P. Bell and B. Stillman, Nature 357, 128 (1992).

16. F.J. McNally, and J. Rine, Mol. Cell. Biol. 11, 5648 (1991).

17. A.M. Miller, and K.A. Nasmyth, Nature 312, 247 (1984). 18. D.H. Rivier, and J. Rine, J. Science 256, 659 (1992).

19. Two genetic screens were devised to identify temperature sensitive mutations in essential genes involved in silencing. The screen that led to isolation of orc2-l started with JRY2698 (HMLa, MATa, HMRa, ade2, his3, leu2, trpl, ura3-52), which had a mating-type cassettes at all three chromosomal mating-type loci and was transformed with a plasmid (pJR924) containing the a mating-type cassette at HMR (JRY2700). The plasmid-borne HMRa locus had two synthetic silencers substituted for the E silencer, and also had a deletion of the I element. The use of two silencers rather than one minimized the risk of being distracted by site mutations in the silencer. One hundred and sixty two thousand colonies of EMS-mutagenized colonies were grown on supplemented minimal media (without uracil) at 25°C and screened for derepression of the plasmid-borne a cassette at HMR. Mutagenized- colonies were replica-plated onto lawns of the mating tester strain DBY1034 {MATa, his4-539, lys2-801, urα3-52) on minimal media either with or without uracil supplementation. Replicas were incubated at 25°C for one hour, then overnight at 30°C. Only plasmid-containing JRY2700 cells were able to mate with the tester strain to yield diploids capable of growing on the unsupplemented plates because the only functional URA3 gene was on the plasmid.

Cells bearing mutations causing derepression of the plasmid-borne a cassette could be distinguished from the other classes of mutations by exploiting a feature of yeast plasmids. Approximately 10% of the cells in these colonies lacked the plasmid and thus could, in principle, mate with the tester strain and form Ura^" diploids capable of growth on the plates supplemented with uracil. If a colony had a mutation in the mating response pathway, the cells would be unable to mate even in the absence of the plasmid, and thus would be unable to form diploids capable of growth on medium supplemented with uracil. Twenty eight strains were identified that were temperature-sensitive for growth and that mated with the tester strain only on plates supplemented with uracil. Plasmid-free isolates of each strain were then retransformed with the plasmid bearing the synthetic silencer at the HMRa locus (pJR924) and with the plasmid bearing the wild-type HMRa locus (pJR919; McNally and Rine, 1991). Three strains were able to mate when carrying the wild-type HMR locus (pJR919) but not when carrying the synthetic silencer-containing HMR locus (pJR924). In order to determine if the ts growth phenotype and the mating phenotype were due to the same mutation, spontaneous revertants of the ts phenotype were selected. A spontaneous revertant of the ts growth of one strain, JRY2904, mated as well as the wild-type JRY2700, suggesting that the mating phenotype and temperature-sensitive growth were due to the same mutation which was named orc2-l. 20. Y. Kassir, et al, Genet. 109, 481 (1985). Foss and Rine, Genetics. (1993) 21. The ORC2 gene was cloned by complementation of the temperature sensitivity of orc2-l. An orc2-l strain (JRY3683) was transformed with a CEN EE/2-based Saccharomyces cerevisiae genomic library (32) Approximately 1000 to 1500 transformants formed colonies at 23 °C. Replica prints of these colonies were incubated at 37 °C to screen for the ability to grow at elevated temperatures. Plasmids were isolated from temperature-resistant strains and retested. Those plasmids that complemented the defect a second time were analyzed by restriction digestion. One plasmid from the CEN-LEU2 library (pJR1416) was chosen for further analysis. 22. ORC2 was disrupted with the TnlO LUK transposon (33), which inserted within the ORC2 coding sequence on the plasmid (pJR1146) carrying the Sstl orc2- 1 complementing fragment. Plasmid pJRl 147 had the Tnl QLUK insertion within the ORC2 coding region. The Q_-C2-containing Sstl fragment, disrupted by the transposon, was removed from pJR1147 by partial digestion with Sstl. The fragment was transformed into the wild-type diploid JRY543. The integration of this disruption allele at the ORC2 locus was confirmed by DNA blot hybridization analysis (Southern, 1975), and the diploid was named JRY3444. 23. P. Hieter, C. Mann, M. Snyder, and R.W. Davis, Cell 40, 381 (1985). 24. D. Koshland, J.C. Kent, and L.H. Hartwell, Cell 40, 393 (1985). R.M. Lynn, et al, Proc. Natl. Acad. Sci. USA 86, 3559 (1989). W.-K. Eng,S.D. Pandit, and R. Sternglanz, J. Biol. Chem. 264, 13373 (1989).

25, 26. A.H. Brand, G. Micklem, and K. Nasmyth, Cell 51, 709 (1987). 27. S. Shuman, et al, Proc. Natl. Acad. Sci. USA 86, 9793 (1989).

28, 29. J. Singh, and A.J.S. Klar, A. J. S. Genes and Dev. 6, 186 (1992).

30. D.D. Dubey, et al, Mol. Cell. Biol. 11, 5346 (1991).

31. CA. Hrycyna, et al, EMBO J. 10, 1699 (1991).

32. A mutation was introduced into the RAPl binding site at HMR-E adjacent to the HMRa locus by oligonucleotide-directed mutagenesis (35), and the change confirmed by sequencing. The RAPl site mutation was identical to the PAS1-1 mutation of HMR-E characterized previously that blocks RAPl protein binding in vitro (21), and is described here as HMRa-e-rapl-10. The plasmid consisting of the HMRa-e-rapl-10 Hinάlll fragment in pRS316 was named pJR1425. The wild- type HMRa version of the same plasmid was named pJR1426. Approximately 100,000 mutagenized cells from 12 independent cultures of the HMLa matal HMRa stel4 strain with the HMRα plasmid (pJR1425) were grown into colonies at 23 °C and replica-plated to a MATa urα3 mating-type tester lawn (PS Y 152) to identify mutants exhibiting the a mating phenotype. The mating plates were incubated at 30 °C in order to identify mutants defective enough to be derepressed at HMR yet not so defective as to be inviable. Of nine hundred haploid mating proficient colonies that were picked, fifty mutants were temperature sensitive for growth at 37°F to some degree. These mutants were subjected to further study and the remainder were discarded. All 50 mutants were recessive to wild-type. Only the subset of mutants relevant to ORC2 are presented here; the remainder will be discussed elsewhere.

33. The ORC2 gene was defined by the orc2-l mutation. An orc2- complementing plasmid (pJR1416) was obtained by complementation of the temperature sensitivity of orc2-l. In order to map the approximate position of the orc2 -complementing gene in the plasmid, six derivatives of pJR1416 were made and tested for complementation. The SαR-SαU. fragment was removed from the insert to yield pJR1418. Three adjacent Xbαl-Xbαl fragments were removed to yield pJR1422. Sphl cleaved once in the insert and once just inside the vector. Deleting this Sphl-Sphl fragment produced pJR1417. Cleavage by Sstl released two fragments from the insert. Deletion of both fragments created pJR1419. Isolates in which only the larger Sstl fragment (pJR1421) or only the smaller Sstl fragment (pJR1420) was deleted were also recovered. The 2.8-kb Sstl-Sstl orc2- complementing fragment was cloned into the Sstl site of the CEN URA3 vector pRS316 (36), to yield pJR1263. Two plasmids were made which allowed the chromosomal integration of part or all of ORC2. The first, pJR1423, contained an Xhol/Kpnl insert (from pJR1416) which extended from a few kb upstream of the ORC2 start codon to about 60-bp upstream of the stop codon inserted into Xhόl- Kpnl-cnt pRS306 (36), a yeast integrating vector marked by URA3. The second plasmid, pJR1424, contained the Sstl ørc2-complementing fragment inserted into the Sstl site of pRS306. 34. F. Spencer, et al Genetics 124, 237 (1990). 35. O. Huisman, et al, Genetics 116, 191 (1987).

36. E.M. Southern, J. Mol. Biol. 98, 503 (1975).

37. T.A. Kunkel, et al, Methods Enzymol. 154, 367 (1987).

38. R.S. Sikorski, and P. Hieter, Genetics 122, 19 (1989).

Example 3.

In order to identify potential yeast initiators, we developed a genetic strategy, the one-hybrid system, to find proteins that recognize a target sequence of interest. The one-hybrid system has two basic components: (i) a hybrid expression library, constructed by fusing a transcriptional activation domain to random protein segments, and (ii) a reporter gene containing a binding site of interest in its promoter region. Hybrid proteins that recognize this site are expected to induce expression of the reporter gene, because of their dual ability to bind the promoter region and activate transcription (8). This association may be indirect, since hybrids that interact with endogenous proteins already occupying the binding site will also activate transcription (7). Nevertheless, as long as the association is sequence-specific the protein incoφorated in the hybrid should be functionally relevant. We have used this method to look for proteins from the yeast Saccharomyces cerevisiae that recognize the ARS consensus sequence (ACS) of yeast origins of DNA replication. The protein component of this screen was provided by a set of three complementary yeast hybrid expression libraries, YL1-3, containing random yeast protein segments fused to the GAL4 transcriptional activation domain (GAL^*⁰) (9). The reporter gene for our screen contained four direct repeats of the ACS in its promoter region and was integrated into the yeast strain GGY1 to form JLY363(ACS^W) (10). To determine the dependence of lacZ induction on the ACS, we constructed in parallel JLY365(ACS^MUΓANT), which harbors a reporter gene carrying four copies of a nonfunctional multiply-mutated ACS (Fig. 4) (10).

We isolated nine plasmids that induced greater lacZ activity in JLY363(ACS^wr) than JLY365(ACS^MurA,,,τ) from a screen of 1.2 million YL1-3 transformants (11). Many of the plasmids that induced lacZ activity on initial screening of the library in JLY363(ACS^WT) failed to exhibit a dependence on the ACS when introduced into JLY365(ACS^MljTANT). Restriction analysis of these plasmids showed that the nine isolates represented five genomic clones, which we initially labeled AAP1-5 for ACS associated protein. AAPl was isolated four times, AAP5 twice, and the others only once. To examine the sequence specificity of lacZ induction with finer resolution, reporter constructs containing direct repeats of four ACS point mutants were each integrated into GGY1 to generate the set of reporter strains(lθ). The five AAP clones were individually examined in these strains for the ability to induce lacZ expression. AAPl displayed a coπespondence between the induction of this set of reporter genes and the ARS function (12) of their ACS. The AAP5 hybrid exhibited a slightly weaker coπelation, and the remaining clones showed poor coπelation. These findings suggest that AAPl, and possibly AAP5, encodes a protein that recognizes the ACS in a sequence-specific manner. Constructs with deletions in the AAPl coding sequence (14) were unable to induce lacZ expression, indicating that recognition of the ACS resided in the protein segment fused to GAL4.

The genomic segments fused to the GAL4^AD in AAP1-5 were sequenced (15) to determine the extent of the hybrid proteins that were made. AAPl and AAP5 had sizable protein coding sequences of 301 and 123 amino acids, respectively, fused in frame with the GAL4^AD. In principle, these segments are large enough to direct the hybrid protein to the promoter of the reporter gene. AAP2-4 encoded hybrid proteins with only short peptide extensions (10, 22, and 38 amino acids respectively) fused to the GAL4^AD, suggesting that these hybrids were not responsible for the transcriptional induction attributed to these clones. Because of this finding and the lack of proper sequence specificity for the ACS element, AAP2-4 were not studied further.

The full-length gene for AAPl was cloned from a yeast genomic library and sequenced (15) (Genbank accession no. L23323). AAPl contains an open reading frame for a protein 435 amino acids long with a predicted molecular weight of 50,302 daltons. The hybrid GAL4^AD-AAP1 protein obtained from the screen was a fusion of the GAL4^AD to the C-terminal two-thirds of the predicted full-length protein (residues 135-435) , indicating that this portion of the molecule is sufficient for association with the ACS. Comparison of peptide sequences from the 50kd subunit of ORC with the predicted protein sequence from AAPl demonstrated that our gene encodes this subunit and confirmed the association between the AAPl protein and the ACS. Because of this identity, we have renamed our gene ORC6. An overlapping ORF capable of encoding a protein 250 amino acids long exists on the complementary strand. The positions of the predicted start and stop codons for this ORF are at nt 1615-7 and nt 865-7, respectively. In pJL766 the C residue at 1471 was mutated to a T, preserving the amino acid sequence of ORC6 but introducing a stop codon in this overlapping ORF. The sequence of ORC6 indicates a connection with the regulatory machinery governing cell cycle progression. Orc6p contains four phosphorylation sites, (S/T)PXK, for cyclin- dependent protein kinases (20) clustered in the first half of the molecule. Using the more relaxed consensus site (S/T)P adds two more sites to this cluster. We have observed Orc6p phosphorylated in vivo on serine and threonine residues. However, since the initiation of yeast DNA replication commences promptly in response to the activation of this protein kinase in Gl, we believe that Orcόp and possibly other ORC subunits are regulated substrates of this kinase. Finally, as expected for a protein participating in nuclear events, Orcόp contains a potential nuclear localization signal (NLS) within the (S/T)PXK cluster and one in the C- terminal domain (amino acid residues 117-122 and 263-279). Orc6p can be seen in the nucleus by immunofluoresence.

A marked deletion of the ORC6 gene (pJL731) (21), removing all but 13 codons from its open reading frame, was introduced into diploids from three different strain backgrounds. The resulting heterozygous ORC6 deletion strains, JLY481, JLY475, and JLY469 were induced to undergo meiosis, and 20 tetrads of each strain were dissected (21). In all backgrounds the ORC6 disruption cosegregated with inviability, demonstrating that ORC6 is essential for cell growth. Microscopic examination revealed that mutant spores from JLY481 and JLY475 germinated, completed 1-2 rounds of cell division, and then aπested with a uniform large bud morphology reminiscent of cell division cycle mutants defective in DNA replication or nuclear division (22). The position of cell cycle aπest could not be pinpointed, however, since the DNA content of these cells could not be readily measured. Mutant spores derived from JLY469 germinated poorly.

The interpretation of these ORC6 deletion experiments was complicated by the presence of a second open reading frame (ORF2) of 250 amino acids on the antisense strand of the ORC6 gene. ORF2 spans nucleotides 1617 to 868 of the Genbank sequence and overlaps the C-terminal two-thirds of the ORC6 coding sequence. A marked deletion that removed the N-terminal third of the ORC6 coding sequence without affecting ORF2 (pJL733) was introduced into diploids (21). Tetrad analysis again showed the ORC6 deletion cosegregating with cell death. Finally, an ORC6 gene was constructed that contains a silent codon change for the ORC6 ORF but introduces a UGA stop codon in ORF2 (22). This gene was able to rescue a haploid strain containing a full deletion of the ORC6 ORF. We conclude that ORC6 is essential for cell viability.

Our results validate the one-hybrid system screen as a method to identify and clone genes for proteins that recognize a DNA sequence of interest. This screen has also been successful in identifying DNA-binding proteins (23), and a variation of this screen has been used to identify a binding site for a suspected DNA-binding protein (24). The one-hybrid approach is particularly useful for proteins that are difficult to detect biochemically or for which starting material in a purification is difficult to obtain. We identified genes that interact genetically with ORC6 using established cdc mutants because germinating spores bearing an ORC6 deletion appeared to exhibit a cell division cycle phenotype. pJL749 (28), a plasmid that overexpresses Orcόp several hundred-fold, was introduced into a virtually isogenic set of temperature-sensitive cdc mutants aπesting at various points in the cell cycle (29). Overexpression of ORC6 selectively affected cdcό and cdc46 mutants, lowering their restrictive temperature by 5-7° C; there was no significant effect on the other mutants examined or on the wild-type strain (Table 1).

viability with

Strain cdc mutant overexpression of ORC6

RDY488 wild-type +

RDY501 cdc28-l +

RDY510 cdc4-l +

RDY664 cdc34-2 +

RDY543 cdc7-4 +

JLY310 cdc6-l -

JLY179 cdc46-l -

JLY338 cdc2-l +

JLY353 cdcl7-l +

RDY619 cdcl5-2 +

Table 1. Viability of cdc Mutants in the Presence of High Levels of ORC6 Expression. JL749 (GALp-HA-ORC6), JL772 (GALp-HA), and RS425 were introduced into each cdc mutant, and examined for growth at various temperatures under conditions that induce expression of ORC6 (28, 29). + indicates mutants whose restrictive temperature remains unchanged in the presence of JL749 relative to JL772 and RS425. - indicates mutants whose restrictive temperature is lowered 5-7° C when JL749 is present. Numbered Citations for Example 3

1. Kelly, J. Biol. Chem. 263, 17889 (1988); Marians, Annu. Rev. Biochem.

61, 673 (1992); Kornberg, Baker, DNA Replication. (Freeman and Company, New York, 1992); B. Stillman, Annu. Rev. Cell Biol. 5, 197 (1989). 2. M. L. DePamphilis, Annu. Rev. Biochem. 62, 29 (1993).

3. Campbell and Newlon, in The Molecular and Cellular Biology of the Yeast Saccharomyces Broach, et al, Eds. (CSHL Press, 1991), vol. 1, pp. 41-146.

4. Fangman and Brewer, Annu. Rev. Cell Biol. 7, 375 (1991).

5. J.R. Broach et al., Cold Spring Harbor Symp. Quant. Biol. Al, 1165 (1983); Van Houton and C. S. Newlon, Mol. Cell. Biol. 10, 3917 (1990).

6. Y. Marahrens and B. Stillman, Science 255, 817 (1992).

7. S. Fields and O.-K. Song, Nature 340, 245 (1989); C.-T. Chien, P.T. Bartel, R. Sternglanz, S. Fields, Proc. Natl. Acad. Sci. USA 88, 9578 (1991).

8. R. Brent and M. Ptashne, Cell 43, 729 (1985). 9. The N-terminal portions of the hybrids from hree related hybrid expression libraries, YL1-3 (7), consist of the SV40 nuclear localization signal and amino acids 768-881 of the GAL4 activation domain (GAL4^AD). The C-terminal portions were derived from random yeast protein segments which have been fused to the end of the GAL4^AD. These segments are encoded by short (l-3kb) fragments from a Sau3a partial digest of yeast genomic DNA. Together, YL1-3 ensure that all three reading frames of these fragments can be expressed. 10. pLRlDl is described in R.W. West Jr., R.R. Rogers, M. Ptashne, Mol. Cell. Biol. A, 2467 (1984). We generated pBgl-lacZ from pLRlDl by (i) substituting an Xhol-Bglll-Xhol polylinker for the Xhol linker and (ii) precisely excising a Hind III fragment containing 2m sequences. The resulting vector has a unique Bgl II site approximately 100 bp upstream of the TATA box for insertion of DNA sequences in the promoter region and a unique Stul site for targeted integration of the plasmid at the URA3 locus. Multiple direct repeats of ARSl domain A and several of its mutant derivatives were inserted into the Bgl II site of pBgl-lacZ to generate all the reporter genes used in this work. The inserted repeat elements, derived from complementary oligonucleotides, were oriented with the TATA box to their right. Each reporter gene construct was integrated into the URA3 locus of GGY1 {MATa Dgαl4 Dgαl80 urα3 leu2 his3 αde2 tyr) [G. Gill and M. Ptashne, Cell 51, 121 (1987)] to create a reporter strain. Integration of pBgl- lacZ into GGY1 generated JLY387.

11. YEPD (rich complete) and SD (synthetic dropout) media are as described [J.B. Hicks and I. Herskowitz, Genetics 83, 245 (1976)]. Standard methods were used for manipulation of yeast cells [C. Guthrie and G.R. Fink, Ed., Guide to Yeαst Genetics and Moleculat Biology (Academic Press, San Diego 1991)] and DNA [F.M. Ausubel et al., Ed., Current Protocols in Molecular Biology (Wiley, New York 1989)]. Libraries YL1-3 were transformed [R.H. Schiestl and R.D. Geitz, Current Genetics 16, 339 (1989)] into JLY363 (10) and plated on SD-Leu at a density of 2-5000 colonies/ 10cm plate. 500,000 transformants were obtained for YL1 and YL2, and 200,000 for YL3. Transformants were assayed on filters for production of b-galactosidase [L. Breeden and K. Nasmyth, Cold Spring Harbor Symp. Quant. Biol. Al, 643 (1985)]. 49 isolates remained positive after colony purification (15 from YL-1; 22 from YL-2, 12 form YL-3), and library plasmids were extracted from them . These plasmids were each transformed into both JLY363 and its mutant counterpart JLY365 (10). Nine plasmids induced greater b-galactosidase activity in the wild type reporter strain than the control. These plasmids were classified into five clones, AAP1-5, based on their Hind III restriction pattern. Each clone was then retested in JLY360, JLY361, JLY387, JLY429, JLY431, JLY433, JLY435. The AAPl hybrid clone was called pJL720. The AAPl gene was later renamed ORC6.2

12. The ARS function of the mutant sequences was analyzed in the context of ARSl domain B (Bglll-Hinfl fragment, nt 853-734) in the following CEN-based URA3-containing plasmids: pJL347 (wt), pJL243 (multiple), pJL326 (A863T), pJL338 (T869A), pJL330 (T862C), and pJL316 (T867G). These plasmids were transformed into JLY106 {MATa urα3 leu2 his3 trpl lys2 αde2) and its homozygous diploid counterpart JLY162. pJL243, pJL326, and pJL338 did not yield a high frequency of transformation and could not be assayed quantitatively for ARS function. pJL347, pJL330, and pJL316 transformed cells with high efficiency and were assayed for mitotic stability [Stinchcomb, et al. Nature 282, 39 (1979)]. 13. pJL720, the ORC6 hybrid construct originally isolated from the YL3 library, has two BamHI sites. The 5' site created by the hybrid junction coπesponds to Sau3a site at nt. 843. Excision of the segment between the two sites generated pJL721, leaving amino acids 339-435 in frame with the GhlA . pGAD3R (11) the parent vector for the YL3 library, contains no ORC6 sequence. pRS425, Christianson, et al., Gene 110, 119 (1992), contains no components of the fusion protein.

14. All sequencing was performed with Sequenase (USB) on collapsed double- stranded templates. The protein coding segments of the AAP1-5 hybrid clones were sequenced from their junction with the GAL4^AD to their stop codon. Two of the ORC6 sequencing primers were used as colony hybridization probes to screen a high copy number yeast genomic library [M. Carlson and D. Botstein, Cell 28, 145 (1982)] for a clone of the full-length ORC6 gene (pJL724). The full-length gene was sequenced on both strands using oligonuclotide primers positioned approximately 200 nt apart.

15. S. P. Bell and B. Stillman, Nature 357, 128 (1992).

16. Hodgman, Nature 333, 22 (1988); Walker et al., EMBO J. 1, 945 (1982).

17. P. Linder, et al., Nature 337, 121 (1989).

18. E. A. Nigg, Seminars in Cell Biology 2, 261 (1991). 19. ORC6 deletions were constructed by replacing nucleotides 458-1721

(pJL731) or nucleotides 458-846 (pJL733) of the Genbank sequence with the URA3 Hindlll fragment oriented in the opposite direction to that of the ORC6 sequence. Each construct was used to generate heterozygous deletions of ORC6 in diploid strains by one-step gene replacement. ORC6 deletion analysis was performed in JLY461 {MATa/MATa urα3/urα3 Ieu2/leu2 his3/his3 trpl/trpl αde2/αde2 [cir°J), JLY462 {MATα/MATa urα3/urα3 Ieu2/leu2 trpl/trpl his4/his4 cαnl/cαnl), and JLY463 {MATα/MATa urα3/urα3 Ieu2/leu2 trpl/trpl his3/HIS3); their respective genetic backgrounds are S288c, EG123, and A364a. Disruption of JLY461, JLY462, and JLY463 by pJL731 (full deletion) created JLY481, JLY475, and JLY469, respectively. Disruption of JLY461, JLY462, and JLY463 by pJL733 (N-terminal deletion) created JLY485, JLY479, JLY473, respectively. These heterozygous marked deletion strains were sporulated, and twenty tetrads of each were dissected and grown on YEPD to assess viability.

20. Pringle and Hartwell, in The Molecular Biology of the Yeast Saccharomyces Strathern, et al, Eds. (CSHL Press, CSH, 1981), vol. 1, pp. 97-142. 21. A point mutant (pJL766) was made by replacing the BamHI-SphI fragment of the full-length clone with a BamHI/SphI fragment generated by PCR from pJL720 using primers. One mutation changes nucleotide 1471 of the Genbank sequence from C to T and was confirmed by sequence analysis. 22. M. M. Wang and R. R. Reed, Nature 364, 121 (1993). 23. T. E. Wilson, et alt, Science 252, 1296 (1991).

24. J. F. X. Diffley and J. H. Cocker, Nature 357, 169 (1992).

25. pJL749 contains the GAL1 promoter (nt 146-816) driving the expression of ORC6 (nt 443-2298) in the high-copy yeast shuttle vector RS425 [T. W. Christianson, et al., Gene 110, 119 (1992)]. 26. The cdc mutant strains have been backcrossed 4-5 times against two congenic strains derived from A364a , RDY487 {MATa leu2 urα3 trpl) and RDY488 {MATα leu2 urα3 trpl). All are urαS ' leu2 trpl. RDY510, RDY664, JLY310, and JLY179 are MATa; the rest are MATα. Additional markers can be found in JLY310(a_fe2), RDY543(Λw3), and RDY619 (pep4D::TRPl his3 αde2). pJL749, pJL772, and RS425 (28) were transformed into these strains and plated on SD-LEU at 22° C. Four colony-purified isolates from each transformation were patched onto SD-LEU plates and replica-plated to SGAL-LEU plates, all at 22° C. The patches on SGAL-LEU were replica-plated to a series of pre-warmed SGAL- LEU plates at 22°, 25°, 27°, 30°, 32.5°, 35°, 37°, and 38° C. The viability of cdc mutants containing pJL749 was compared to those containing pJL772 and pRS425.

27. Hartwell, JMB 104, 803 (1976); Hennessy, et al G&D A, 2252(1990).

28. Chen, et al., PNAS 89, 10459 (1992); Hogan, et al, ibid. 89, 3098.

29. B.J. Andrews and S.W. Mason, Science. 261, 1543 (1993).

Example 4. Ore protein purification and gene cloning

Protein Purification: To obtain sufficient protein for peptide sequencing, a revised purification procedure for ORC was devised, based on the procedure reported previously (Bell and Stillman, 1992). Whole cell extract was prepared from 400g of frozen BJ926 cells (frozen immediately after harvesting a 300 liter logarithmically growing culture, total of 1.6 kg per 300 liters). All buffers contained 0.5 mM PMSF, 1 mM benzamidine, 2 mM pepstatin A, 0.1 mg/ml bacitracin and 2mM DTT. 400 mis of 2X buffer H/0. l ^Np-* (100 mM Hepes-KOH, pH 7.5, 0.2 M KC1, 2 mM EDTA, 2 mM EGTA, 10 mM Mg

Acetate, and 20% glycerol) was added to the cells and after thawing the cells were broken using a bead beater (Biospec Products) until greater than 90% cell breakage was achieved (twenty 30 second pulses separated by 90 second pauses). After breakage is complete, the volume of the broken cells was measured and one twelfth volume of a saturated (at 4°C) solution of ammonium sulfate was added and stiπed for 30 minutes. This solution was then spun at 13,000 x g for 20 minutes. The resulting supernatant was transfeπed to 45Ti bottle assemblies (Beckman) and spun in a 45Ti rotor at 44,000 RPM for 1.5 hrs. The volume of the resulting supernatant was measured and 0.27g/ml of ammonium sulfate was added. After stirring for 30 minutes, the precipitate was collected by spinning in the 45 Ti rotor at 40,000 RPM or 30 minutes. The resulting pellet was resuspended using a B- pestle dounce in buffer H/0.0 (50 mM Hepes-KOH, pH 7.5, 1 mM EDTA, 1 mM EGTA, 5 mM Mg Acetate, 0.02% NP-40, 10% glycerol) and dialyzed versus H/0.15M KC1 (Buffer H with 0.15 M KC1 added). This preparation typically yielded 12-16 g soluble protein (determined by Bradford assay with a bovine serum albumin standard). Preparation of ORC from this extract was essentially as described (Bell and Stillman, 1992) with the following changes (column sizes used for preparation of ORC from 400g of cells are indicated in parenthesis). The S- Sepharose column was loaded at 20 mg protein per ml of resin ( — 300 ml). The Q-Sepharose (50 ml) and sequence specific affinity column (5ml) was run as described but the dsDNA cellulose column was omitted from the preparation. Only a single glycerol gradient was performed in an SW-41 rotor spun at 41,000 RPM for 20 hrs. We estimate a yield of 130 μg of ORC complex (all subunits combined) per 400 g of yeast cells. Protein Sequencing: Digestion of ORC subunits was performed using an

"in gel" protocol described by Kawasaki and Suzuki with some modification. Briefly, purified ORC (~ 10 μg per subunit) was first separated by 10% SDS- PAGE and stained with 0.1 % Coomassie Brilliant Blue G (Aldrich) for 15 min. After destaining (10% methanol, 10% acetic acid), the gel was soaked in water for one hour, then the protein bands were excised, transfeπed to a microcentrifuge tube and cut into 3-5 pieces to fit snugly into the bottom of the tube. A minimum volume of 0.1M Tris-HCl (pH=9.0) containing 0.1 % SDS was added to completely cover the gel pieces. Then 200 ng of Achromobacter protease I (Lysylendopeptidase: Wako) was added and incubated at 30 °C for 24 hrs. After digestion the samples were centrifuged and the supernatant was passed through an Ultrafree-MC filter (Millipore, 0.22μm). The gel slices were then washed twice in 0.1 % TFA for one hour and the washes were recovered and filtered as above. All filtrates were combined and reduced to a volume suitable for injection on the HPLC using a speed-vac. The digests were separated by reverse-phase HPLC (Hewlett-Packard 1090 system) using a Vydac C18 column (2.1x 250 mm, 5μm, 300 angstroms) with an ion exchange pre-column (Brownlee GAX-013, 3.2x 15mm). The peptides were eluted from the C-18 column by increasing acetonitrile concentration and monitored by their absorbance at 214, 280, 295, and 550 nm. Amino acid sequencing of the purified peptides was performed on an automated sequencer (Applied Biosystems model 470) with on-line HPLC (Applied Biosystems model 1020A) analysis of PTH-amino acids. ORC SUBUNIT CLONING: ORCI: To clone the gene for the largest (120 kd) subunit of ORC, the following degenerate oligonucleoide primers 1201 and 1202 were synthesized based on the sequence of the first ORCI peptide. These oligos were used to perform PCR reactions using total yeast genomic DNA from the strain W303 a as target. A 48 base pair fragment was specifically amplified. This fragment was subcloned and sequenced. The resulting sequence encoded the predicted peptide indicating that it was the coπect amplification product. A radioactively labeled form of the PCR product was then used to probe a genomic library of yeast DNA sequences resulting in the identification of two overlapping clones. Sequencing of these clones resulted in the identification of a large open reading frame that encoded a protein with a predicted molecular weight of 120 kd and that encoded all four of the ORCI peptide sequences.

ORC3: To clone the gene for the 62 kd subunit of ORC, the following degenerate oligonucleoide primers 621 and 624 were synthesized based on the sequence of the third peptide. These oligos were used to perform PCR reactions using total yeast genomic DNA from the strain W303 a as target. A 53 base pair fragment was specifically amplified. This fragment was subcloned and sequenced. The resulting sequence encoded the predicted peptide indicating that it was the coπect amplification product. A radioactively labeled form of the PCR product was then used to probe a genomic library of yeast DNA sequences resulting in the identification of two overlapping clones. Sequencing of these clones resulted in the identification of a large open reading frame that encoded a protein with a predicted molecular weight of 71 kd and encoded all three of the ORC3 peptide sequences. The inconsistency of the molecular weight is presumably due to anomalous migration of this protein during SDS-PAGE.

ORC4: By comparing the sequnce of the ORC4 peptides to that of the known potentially protein encoding sequnces in the genbank database we found that a portion of the ORC4 coding sequence had been previously cloned in the process of cloning the adjacent gene. Using the information from the database we were able to design a perfect match oligo and use this to immediately screen a yeast library. Using this oligo as a probe of the same yeast genomic DNA library a lambda clone was isolated that contained the entire ORC4 gene. This gene encoded a protein of predicted molecular weight 56 kd and also all of the peptides derived from the peptide sequencing of the 56 kd subunit.

ORC5: To clone the gene for the 53 kd subunit of ORC, the following degenerate oligonucleoide primers 535 and 536 were synthesized based on the sequence of the first ORC5 peptide. These oligos were used to perform PCR reactions using total yeast genomic DNA from the strain W303 a as target. A 47 base pair fragment was specifically amplified. This fragment was subcloned and sequenced. The resulting sequence encoded the predicted peptide indicating that it was the coπect amplification product. A radioactively labeled form of the PCR product was then used to probe a genomic library of yeast DNA sequences resulting in the identification of a single lambda clone. Sequencing of this clones resulted in the identification of a large open reading frame that encoded a several of the peptide sequences derived from the 53 kd subunit of ORC indicating that this was the coπect gene. However the sequence of the 5' end of the gene wasno present in this lambda clone. Fortuitoulsy, the mutations in the same gene had also been picked up in the same sreen that resulted in the identification of the ORC2 gene. A complementing clone to this mutation was found to overlap with the lambda clone and contain the entire 5' end of the gene. Sequencing of this complementing DNA fragment resulted in the identification of the entire sequence of the ORC5 gene.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: COLD SPRING HARBOR LABORATORY

THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

(ii) TITLE OF INVENTION: ORC GENES, RECOMBINANT ORC PEPTIDES AND METHODS OF IDENTIFYING DNA BINDING PROTEINS

(iii) NUMBER OF SEQUENCES: 12

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: FLEHR, HOHBACH, TEST, ALBRITTON & HERBERT (B) STREET: 4 Embarcadero Center, Suite 3400

(C) CITY: San Francisco

(D) STATE: California

(E) COUNTRY: USA

(F) ZIP: 94111-4187

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: (C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Osman, Richard A

(B) REGISTRATION NUMBER: 36,677 (C) REFERENCE/DOCKET NUMBER: FP-59032-PC/RAO

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (415) 781-1989 (B) TELEFAX: (415) 398-3249

(C) TELEX: 910 277299

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4940 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATAACATGCT CGCCCTTTTA TATTATGACA GAAAGAATAT ATATATTCAT ATATAAGATG 60 CTTCTATTTA TTAGTTTTAT CTTTTAATTG ATGATGTGTC CATAGAATTT AAGTAAGTGC 120

ATGGTATGGA GTGTATAATG GTTTATAATT TCCCCTAAGA TGACACAAAA AAATGTTCTC 180

CCAAAAATTT ACCAAGAAAA AAAATTAAGA ATACTACACA ATTGATGCTT GGGTTATTTT 240 AAATATCCGG TACATTCTAT TACAAATATG TTTGTACAAT GTAAGCCCCT TCATAATGGT 300

CAGTATTAAG ATAAGGACTG CTATGGGGCA TTTTTTGTCT TACTGGGTAT CACAGGATAA 360

TAACTTGGCG CCAAATTAGA AAAGATATAA ACCTCAAATA TTTGAAATTC TTTGGTGACC 420

TGTCTCATCG TTATATCAAC AAATATTGCA CCAACGAACA CCACTACATA TGTAACTACT 480

CTCTTCCTCG ACTTATTTTT TATTAACGTT GACACGGCCA GATCGAAAAT CATAGAAAAA 540 CAACAACATT GAGAAGAGAT GAAGTTGCGC AAAGGGAAAG AAAACTGCAT AGGCGGCAAA 600

TTCAGCCTAA AAGTTTCCAG AAGCAGGAAC TCATTCCCTA TTGATTAATA CTCATTACAA 660

AAACCACAAT AGAGTAGATA AGATGGCAAA AACGTTGAAG GATTTACAGG GTTGGGAGAT 720

AATAACAACT GATGAGCAGG GAAATATAAT CGATGGAGGT CAGAAGAGAT TACGCCGAAG 780 AGGTGCAAAA ACTGAACATT ACTTAAAGAG AAGTTCTGAT GGAATTAAAC TAGGTCGTGG 840 TGATAGTGTA GTCATGCACA ACGAAGCCGC TGGGACTTAC TCCGTTTATA TGATCCAGGA 900 GTTGAGACTT AATACATTAA ATAATGTTGT CGAACTCTGG GCTCTCACCT ATTTACGATG 960 GTTTGAAGTC AATCCTTTAG CTCATTATAG GCAGTTTAAT CCTGACGCTA ACATTTTGAA 1020

TCGTCCTTTA AATTATTACA ATAAACTGTT TTCTGAAACT GCAAATAAAA ATGAACTGTA 1080 TCTCACTGCA GAATTAGCCG AATTGCAGCT ATTTAACTTT ATCAGGGTTG CCAACGTAAT 1140 GGATGGAAGC AAATGGGAAG TATTGAAAGG AAATGTCGAT CCAGAAAGAG ACTTTACAGT 1200 TCGTTATATT TGTGAGCCGA CTGGGGAGAA ATTTGTGGAC ATTAATATTG AGGATGTCAA 1260 AGCTTACATA AAGAAAGTGG AGCCAAGGGA AGCCCAGGAA TATTTGAAAG ATTTAACACT 1320

TCCATCAAAG AAGAAAGAGA TCAAAAGAGG TCCTCAAAAG AAAGATAAGG CTACTCAAAC 1380

GGCACAAATT TCAGACGCAG AAACAAGAGC TACAGATATA ACGGATAATG AGGACGGTAA 1440 TGAAGATGAA TCATCTGATT ATGAAAGTCC GTCAGATATC GACGTTAGCG AGGATATGGA 1500

CAGCGGTGAA ATATCCGCAG ATGAGCTTGA GGAAGAAGAA GACGAAGAAG AAGACGAAGA 1560 CGAAGAAGAG AAAGAAGCTA GGCATACAAA TTCACCAAGG AAAAGAGGCC GTAAGATAAA 1620

ACTAGGTAAA GATGATATTG ACGCTTCTGT ACAACCTCCC CCCAAAAAAA GAGGTCGTAA 1680 ACCTAAAGAT CCTAGTAAAC CGCGTCAGAT GCTATTGATA TCTTCATGCC GTGCAAATAA 1740

TACTCCTGTG ATTAGGAAAT TTACAAAAAA GAATGTTGCT AGGGCGAAAA AGAAATATAC 1800

CCCGTTTTCG AAAAGATTTA AATCTATAGC TGCAATACCA GATTTAACTT CATTACCTGA 1860

ATTTTACGGA AATTCTTCGG AATTGATGGC ATCAAGGTTT GAAAACAAAT TAAAAACAAC 1920 CCAAAAGCAT CAGATTGTAG AAACAATTTT TTCTAAAGTC AAAAAACAGT TGAACTCTTC 1980 GTATGTCAAA GAAGAAATAT TGAAGTCTGC AAATTTCCAA GATTATTTAC CGGCTAGGGA 2040 GAATGAATTC GCCTCAATTT ATTTAAGTGC ATATAGTGCC ATTGAGTCCG ACTCCGCTAC 2100 TACTATATAC GTGGCTGGTA CGCCTGGTGT AGGGAAAACT TTAACCGTAA GGGAAGTCGT 2160

AAAGGAACTA CTATCGTCTT CTGCACAACG AGAAATACCA GACTTTCTTT ATGTGGAAAT 2220 AAATGGATTG AAAATGGTAA AACCCACAGA CTGTTACGAA ACTTTATGGA ACAAAGTGTC 2280 AGGAGAAAGG TTAACATGGG CAGCTTCAAT GGAGTCACTA GAGTTTTACT TTAAAAGAGT 2340 TCCAAAAAAT AAGAAGAAAA CCATTGTAGT CTTGTTGGAC GAACTCGATG CCATGGTAAC 2400 GAAATCTCAA GATATTATGT ACAATTTTTT CAATTGGACT ACTTACGAAA ATGCCAAACT 2460

TATTGTCATT GCAGTAGCCA ATACAATGGA CTTACCAGAA CGTCAGCTAG GCAATAAGAT 2520

TACTTCAAGA ATTGGGTTTA CCAGAATTAT GTTCACTGGG TATACGCACG AAGAGCTAAA 2580 AAATATCATT GATTTAAGAC TGAAGGGGTT GAACGACTCA TTTTTCTATG TTGATACAAA 2640

AACTGGCAAT GCTATTTTGA TTGATGCGGC TGGAAACGAC ACTACAGTTA AGCAAACGTT 2700.

GCCTGAAGAC GTGAGGAAAG TTCGCTTAAG AATGAGTGCT GATGCCATTG AAATAGCTTC 2760

GAGAAAAGTA GCAAGTGTTA GTGGTGATGC AAGAAGAGCA TTGAAGGTTT GTAAAAGAGC 2820 AGCTGAAATT GCTGAAAAAC ACTATATGGC TAAGCATGGT TATGGATATG ATGGAAAGAC 2880 GGTTATTGAA GATGAAAATG AGGAGCAAAT ATACGATGAT GAAGACAAGG ATCTTATTGA 2940 AAGTAACAAA GCCAAAGACG ATAATGATGA CGATGATGAC AATGATGGGG TACAAACAGT 3000 TCACATCACG CACGTTATGA AAGCCTTAAA CGAAACTTT AATTCTCATG TAATTACGTT 3060

TATGACGCGA CTTTCATTTA CAGCAAAACT GTTTATTTAT GCATTATTAA ACTTGATGAA 3120 AAAGAACGGA TCTCAAGAGC AAGAACTGGG CGATATTGTC GATGAAATCA AGTTACTTAT 3180 TGAAGTAAAT GGCAGTAATA AGTTTGTCAT GGAGATAGCC AAAACATTGT TCCAACAGGG 3240 AAGTGATAAT ATTTCTGAAC AATTGAGAAT TATATCATGG GATTTCGTTC TCAATCAGTT 3300 ACTTGACGCG GGAATATTGT TTAAACAAAC TATGAAGAAC GATAGAATAT GTTGTGTCAA 3360

GCTAAATATA TCAGTAGAAG AAGCCAAAAG AGCCATGAAT GAGGATGAGA CATTGAGAAA 3420

TTTATAGATT CGGTTTTTAT TATTCATGAC CTAGCATACA CA ACATATA CCTACATAGT 3480 AGCGCATTTA TCCAAAACAT ACGATATTGT GGATGTACAT ACCTTCTATA TCTCCTTAAA 3540

GCTATTGTGT AGCTTGATTT AAAATATGCT AACGCCAACT CTCACATGGT AGCAGGCGGG 3600 TATAGTTGTT TTCATGTATT AACGCCCGGC GATGGTGCCT TAGATGAGGG CGACGAGGAG 3660 GGCTTCCTGA TATTATGGCT CTTTCTATCC TGACTTTTGT TATGATGTCG ATGTTGCTGG 3720 CCACCTAGGT GCTTATATAT CAAAAGAGGA TCGCCGATTT CATTGATTTC TGGGATGGTT 3780 AATGTCAAAT TAAAGATCTT TGCCAGTGCA ATTTTGAAAA TTTTTTGAAT GTTTATAGAT 3840 TTGGCAGTAG AGCAGAATAT AAGAGGAGCA TTCATGACCT GTGCATACTT CATACTCGTT 3900

CTCGAGATTT GTTCCTGATA TTCCGGGTCT AAGTCTATTA GTAAATCGTA CTTTGTGCCC 3960 ACCAAAATAG GAATTGCCGA ATCATTTAGC CCGTACGCCT GCCTATACCA CTCCTTTATT 4020 GAACTCAACG TCTCTGGACG TGTCAGGTCA AACAGAAATA TGATCACTGA AGACCCTACC 4080 GTCGCAATTG GGAGCATGTT GATGAATTCT CTTTGTCCGC CTAAATCCAT TATAGAAAAT 4140 ATAATATCCG TGGAGCGTAT GCTTACTTTT CTTTTCAAAA AGTTCACTCC CAGCGTCTGT 4200

GTGTATTCCT TATCGTATAT GTTCTGTACG TACTTCACCA TCAGCGATGT TTTCCCTACT 4260

TGTGCATCCC CTACTAATCC AACCTGAACT TCAACCTGAT TTCGTACCGC AGGTATAGAA 4320 TTGTTTGCTC CCGTGCTTGG TGTAGCCATC TTAGCTTAAC TCAATTTAAT TTCTACAGCA 4380

AAATCCAAAC GTAATATCTA TATTTTTCTC GAAAAACTGA GGACAAGAGC CAATCAATCA 4440

TCTATAATCC AATTTATATT ATTTTTTCCC TTCTGGGTTC TTTTCTTCCT TTTCTTGTTT 4500

ACCTTTTTTG CTTTTTCATA AAATAATTTC TCTAGATTTG AAGACAGCAT TTTTGTACAT 4560 CCATACACCA TACACCATAC ACCATAGCAC CAGTACACTA TATTTTTATG AATTTTACTA 4620 AGAATTATTC CTGCAGGAGC TCCACTGAAA AAAAAAGAGC AGCATGGATG TCATGTCGGT 4680 AGAGTGCTAC TGAGTAAATG GGAGGACGCG GTAGATCCAG TGTGGAATCA AGGTGGTGCC 4740 GGTGTGAAGC CGCCTCGGCC GGCTGGACTC TCCAGGCCGG AGTGATGATT GCCACGCTGA 4800

AGCTAACACA GTTTCACAAT ACCAGTGTCC TCATTAGTGA GTTCCAATGT ATAGTTAGTA 4860

GTGGTATTTT GATATATGTG AGTGGTAGCA GATTTGAACT TAGTTAGTTG TATTCGCCTT 4920 TGAGGAAACC AAGCCAAAAA 4940

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 914 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

( i) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Ala Lys Thr Leu Lys Asp Leu Gin Gly Trp Glu lie lie Thr Thr 1 5 10 15

Asp Glu Gin Gly Asn lie lie Asp Gly Gly Gin Lys Arg Leu Arg Arg 20 25 30

Arg Gly Ala Lys Thr Glu His Tyr Leu Lys Arg Ser Ser Asp Gly lie 35 40 45 Lys Leu Gly Arg Gly Asp Ser Val Val Met His Asn Glu Ala Ala Gly 50 55 60

Thr Tyr Ser Val Tyr Met lie Gin Glu Leu Arg Leu Asn Thr Leu Asn 65 70 75 80

Asn Val Val Glu Leu Trp Ala Leu Thr Tyr Leu Arg Trp Phe Glu Val 85 90 95 Asn Pro Leu Ala His Tyr Arg Gin Phe Asn Pro Asp Ala Asn lie Leu

100 105 110

Asn Arg Pro Leu Asn Tyr Tyr Asn Lys Leu Phe Ser Glu Thr Ala Asn 115 120 125

Lys Asn Glu Leu Tyr Leu Thr Ala Glu Leu Ala Glu Leu Gin Leu Phe 130 135 140

Asn Phe lie Arg Val Ala Asn Val Met Asp Gly Ser Lys Trp Glu Val 145 150 155 160

Leu Lys Gly Asn Val Asp Pro Glu Arg Asp Phe Thr Val Arg Tyr lie

165 170 175 Cys Glu Pro Thr Gly Glu Lys Phe Val Asp lie Asn lie Glu Asp Val

180 185 190

Lys Ala Tyr lie Lys Lys Val Glu Pro Arg Glu Ala Gin Glu Tyr Leu 195 200 205

Lys Asp Leu Thr Leu Pro Ser Lys Lys Lys Glu lie Lys Arg Gly Pro 210 215 220

Gin Lys Lys Asp Lys Ala Thr Gin Thr Ala Gin He Ser Asp Ala Glu 225 230 235 240

Thr Arg Ala Thr Asp He Thr Asp Asn Glu Asp Gly Asn Glu Asp Glu 245 250 255 Ser Ser Asp Tyr Glu Ser Pro Ser Asp He Asp Val Ser Glu Asp Met

260 265 270

Asp Ser Gly Glu He Ser Ala Asp Glu Leu Glu Glu Glu Glu Asp Glu

275 280 285

Glu Glu Asp Glu Asp Glu Glu Glu Lys Glu Ala Arg His Thr Asn Ser

290 295 300

Pro Arg Lys Arg Gly Arg Lys He Lys Leu Gly Lys Asp Asp He Asp 305 310 315 320

Ala Ser Val Gin Pro Pro Pro Lys Lys Arg Gly Arg Lys Pro Lys Asp

325 330 335 Pro Ser Lys Pro Arg Gin Met Leu Leu He Ser Ser Cys Arg Ala Asn

340 345 350

Asn Thr Pro Val He Arg Lys Phe Thr Lys Lys Asn Val Ala Arg Ala 355 360 365

Lys Lys Lys Tyr Thr Pro Phe Ser Lys Arg Phe Lys Ser He Ala Ala 370 375 380

He Pro Asp Leu Thr Ser Leu Pro Glu Phe Tyr Gly Asn Ser Ser Glu 385 390 395 400

Leu Met Ala Ser Arg Phe Glu Asn Lys Leu Lys Thr Thr Gin Lys His 405 410 415 Gin He Val Glu Thr He Phe Ser Lys Val Lys Lys Gin Leu Asn Ser 420 425 430

Ser Tyr Val Lys Glu Glu He Leu Lys Ser Ala Asn Phe Gin Asp Tyr 435 440 445

Leu Pro Ala Arg Glu Asn Glu Phe Ala Ser He Tyr Leu Ser Ala Tyr 450 455 460 Ser Ala He Glu Ser Asp Ser Ala Thr Thr He Tyr Val Ala Gly Thr 465 470 475 480

Pro Gly Val Gly Lys Thr Leu Thr Val Arg Glu Val Val Lys Glu Leu 485 490 495

Leu Ser Ser Ser Ala Gin Arg Glu He Pro Asp Phe Leu Tyr Val Glu

500 505 510

He Asn Gly Leu Lys Met Val Lys Pro Thr Asp Cys Tyr Glu Thr Leu 515 520 525

Trp Asn Lys Val Ser Gly Glu Arg Leu Thr Trp Ala Ala Ser Met Glu 530 535 540

Ser Leu Glu Phe Tyr Phe Lys Arg Val Pro Lys Asn Lys Lys Lys Thr 545 550 555 560

He Val Val Leu Leu Asp Glu Leu Asp Ala Met Val Thr Lys Ser Gin 565 570 575

Asp He Met Tyr Asn Phe Phe Asn Trp Thr Thr Tyr Glu Asn Ala Lys 580 585 590

Leu He Val He Ala Val Ala Asn Thr Met Asp Leu Pro Glu Arg Gin 595 ^" 600 605

Leu Gly Asn Lys He Thr Ser Arg He Gly Phe Thr Arg He Met Phe

610 615 620 Thr Gly Tyr Thr His Glu Glu Leu Lys Asn He He Asp Leu Arg Leu

625 630 635 640

Lys Gly Leu Asn Asp Ser Phe Phe Tyr Val Asp Thr Lys Thr Gly Asn

645 650 655

Ala He Leu He Asp Ala Ala Gly Asn Asp Thr Thr Val Lys Gin Thr

660 665 670

Leu Pro Glu Asp Val Arg Lys Val Arg Leu Arg Met Ser Ala Asp Ala 675 680 685

He Glu He Ala Ser Arg Lys Val Ala Ser Val Ser Gly Asp Ala Arg 690 695 700 Arg Ala Leu Lys Val Cys Lys Arg Ala Ala Glu He Ala Glu Lys His 705 710 715 720

Tyr Met Ala Lys His Gly Tyr Gly Tyr Asp Gly Lys Thr Val He Glu 725 730 735

Asp Glu Asn Glu Glu Gin He Tyr Asp Asp Glu Asp Lys Asp Leu He 740 745 750

Glu Ser Asn Lys Ala Lys Asp Asp Asn Asp Asp Asp Asp Asp Asn Asp 755 760 765

Gly Val Gin Thr Val His He Thr His Val Met Lys Ala Leu Asn Glu 770 775 780 Thr Leu Asn Ser His Val He Thr Phe Met Thr Arg Leu Ser Phe Thr

785 790 795 800

Ala Lys Leu Phe He Tyr Ala Leu Leu Asn Leu Met Lys Lys Asn Gly 805 810 815

Ser Gin Glu Gin Glu Leu Gly Asp He Val Asp Glu He Lys Leu Leu 820 825 830 He Glu Val Asn Gly Ser Asn Lys Phe Val Met Glu He Ala Lys Thr 835 840 845

Leu Phe Gin Gin Gly Ser Asp Asn He Ser Glu Gin Leu Arg He He 850 855 860

Ser Trp Asp Phe Val Leu Asn Gin Leu Leu Asp Ala Gly He Leu Phe 865 870 875 880

Lys Gin Thr Met Lys Asn Asp Arg He Cys Cys Val Lys Leu Asn He 885 890 895

Ser Val Glu Glu Ala Lys Arg Ala Met Asn Glu Asp Glu Thr Leu Arg 900 905 910 Asn Leu

(2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2809 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS. (B) LOCATION: 807..2666

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

GAGCTCAACA CCACCATTGA GAACGTAGAA TTTCAATTTT TAAGCTGATT CTCTTTCTGC 60

ATGAACTCTC CTAGCAATGT GAAACTTCTC TTAAGGGAAA TTTTCGCCTT TTTGAATGGG 120

CATACTTGGC CAAAAATTCA GGATTGAATA TATATAATCG GAACTTGTAT GGATAAAAAT 180 TTATATCAAG AGTCTGTTTC TTAATTGGAT TTGCTGTGAT CTAGTATTGA GATGACTATA 240

AACCGGCCAG GAAATTAGTC TTTTCGAAGC TGGTTTTGGT TTCGCAAGAG TCTTTTTGAC 300

AGCTTTTTGG CCTCAATTTG TATTCCCTTA ATACGCTTCT TCAACTCTGT CTTAGAGACC 360

ATTTCTCCAG TGGCCTCATC TAGGTGTAAA CTAGCAATAG CGTCACTAGC TGCCGTGACA 420

TTAACTTGCT GTGGCACCTT TATATGTAAT ATGAACCATC TTTCAATGGA TCATAAGAAT 480 AAGTGTCGTA AAAGGCCAAA TATCCATGCA TAAATATCGA CTTATTCGCG TAAATGTGAT 540

ATGGATCAGC TAGTACCAAT TTCTAGTCTA GCAAAATCGG GAAAATTTTT CAGAACACCC 600

ACTCACCGCA TCATTGAGGT GGAAATGACA ATAGTAAGCA GAATTGTTAT TCTTCACAAT 660

GTGTAAAAGT TATAAAGAAA TAGGAACCAC CTTTAAATTA AGACAAAGTA GAATATATTA 720

GCTGAAATTG TATTTGATAA TTGATCATTG ATCTTATTTG CTATATCTTT AAAACAAGTT 700 TTTGTAGTAC TGCGAATTGC CATAAC ATG CTA AAT GGG GAA GAC TTT GTA GAG 833

Met Leu Asn Gly Glu Asp Phe Val Glu 1 5 CAT AAT GAT ATC CTA TCG TCT CCG GCA AAA AGC AGG AAT GTA ACC CCA 881 His Asn Asp He Leu Ser Ser Pro Ala Lys Ser Arg Asn Val Thr Pro 10 15 20 25

AAA AGG GTT GAC CCA CAT GGA GAA AGA CAA CTG AGA AGA ATT CAT TCA 929 Lys Arg Val Asp Pro His Gly Glu Arg Gin Leu Arg Arg He His Ser

30 35 40

TCA AAG AAG AAT TTG TTG GAA AGA ATC TCG CTT GTA GGC AAC GAA AGG 977 Ser Lys Lys Asn Leu Leu Glu Arg He Ser Leu Val Gly Asn Glu Arg 45 50 55

AAA AAT ACA TCT CCA GAT CCG GCA CTC AAA CCT AAA ACG CCA AGT AAA 1025 Lys Asn Thr Ser Pro Asp Pro Ala Leu Lys Pro Lys Thr Pro Ser Lys 60 65 70

GCT CCC CGT AAA CGT GGA AGA CCA AGA AAG ATA CAG GAA GAA TTA ACT 1073 Ala Pro Arg Lys Arg Gly Arg Pro Arg Lys He Gin Glu Glu Leu Thr 75 80 85 GAT AGG ATC AAG AAG GAT GAG AAA GAT ACA ATT TCC TCT AAG AAA AAG 1121 Asp Arg He Lys Lys Asp Glu Lys Asp Thr He Ser Ser Lys Lys Lys 90 95 100 105

AGG AAA TTG GAC AAA GAT ACA TCA GGT AAT GTC AAT GAG GAA AGC AAG 1169 Arg Lys Leu Asp Lys Asp Thr Ser Gly Asn Val Asn Glu Glu Ser Lys

110 115 120

ACT TCT AAC AAC AAG CAG GTG ATG GAA AAG ACG GGG ATA AAA GAG AAA 1217 Thr Ser Asn Asn Lys Gin Val Met Glu Lys Thr Gly He Lys Glu Lys 125 130 135

AGA GAA CGC GAA AAA ATA CAG GTA GCG ACC ACA ACA TAT GAA GAT AAT 1265 Arg Glu Arg Glu Lys He Gin Val Ala Thr Thr Thr Tyr Glu Asp Asn 140 145 150

GTG ACT CCA CAA ACT GAT GAT AAT TTT GTA TCA AAT TCA CCC GAG CCA 1313 Val Thr Pro Gin Thr Asp Asp Asn Phe Val Ser Asn Ser Pro Glu Pro 155 160 165 CCA GAA CCT GCA ACA CCA TCT AAG AAG TCT TTA ACC ACT AAT CAT GAT 1361 Pro Glu Pro Ala Thr Pro Ser Lys Lys Ser Leu Thr Thr Asn His Asp 170 175 180 185

TTT ACT TCG CCC CTA AAG CAA ATT ATA ATG AAT AAT TTA AAA GAA TAT 1409 Phe Thr Ser Pro Leu Lys Gin He He Met Asn Asn Leu Lys Glu Tyr

190 195 200

AAA GAC TCA ACC TCC CCA GGT AAA TTA ACC TTG AGT AGA AAT TTT ACT 1457 Lys Asp Ser Thr Ser Pro Gly Lys Leu Thr Leu Ser Arg Asn Phe Thr 205 210 215

CCA ACC CCT GTA CCG AAA AAT AAA AAG CTC TAC CAA ACT TCG GAA ACC 1505 Pro Thr Pro Val Pro Lys Asn Lys Lys Leu Tyr Gin Thr Ser Glu Thr 220 225 230

AAG TCA GCA AGC TCG TTT TTG GAT ACT TTT GAA GGA TAT TTC GAC CAA 1553 Lys Ser Ala Ser Ser Phe Leu Asp Thr Phe Glu Gly Tyr Phe Asp Gin 235 240 245 AGA AAA ATT GTC AGA ACT AAT GCG AAG TCA AGG CAC ACC ATG TCA ATG 1601 Arg Lys He Val Arg Thr Asn Ala Lys Ser Arg His Thr Met Ser Met 250 255 260 265 GCA CCT GAC GTT ACC AGA GAA GAG TTT TCC CTA GTA TCA AAC TTT TTC 1649 Ala Pro Asp Val Thr Arg Glu Glu Phe Ser Leu Val Ser Asn Phe Phe 270 275 280 AAC GAA AAT TTT CAA AAA CGT CCC AGG CAA AAG TTA TTT GAA ATT CAG 1697 Asn Glu Asn Phe Gin Lys Arg Pro Arg Gin Lys Leu Phe Glu He Gin 285 290 295

AAA AAA ATG TTT CCC CAG TAT TGG TTT GAA TTG ACT CAA GGA TTC TCC 1745 Lys Lys Met Phe Pro Gin Tyr Trp Phe Glu Leu Thr Gin Gly Phe Ser 300 305 310

TTA TTA TTT TAT GGT GTA GGT TCG AAA CGT AAT TTT TTG GAA GAG TTT 1793 Leu Leu Phe Tyr Gly Val Gly Ser Lys Arg Asn Phe Leu Glu Glu Phe 315 320 325

GCC ATT GAC TAC TTG TCT CCG AAA ATC GCG TAC TCG CAA CTG GCT TAT 1841 Ala He Asp Tyr Leu Ser Pro Lys He Ala Tyr Ser Gin Leu Ala Tyr 330 335 340 345

GAG AAT GAA TTA CAA CAA AAC AAA CCT GTA AAT TCC ATC CCA TGC CTT 1889 Glu Asn Glu Leu Gin Gin Asn Lys Pro Val Asn Ser He Pro Cys Leu 350 355 360 ATT TTA AAT GGT TAC AAC CCT AGC TGT AAC TAT CGT GAC GTC TTC AAA 1937 He Leu Asn Gly Tyr Asn Pro Ser Cys Asn Tyr Arg Asp Val Phe Lys 365 370 375

GAG ATT ACC GAT CTT TTG GTC CCC GCT GAG TTG ACA AGA AGC GAA ACT 1985 Glu He Thr Asp Leu Leu Val Pro Ala Glu Leu Thr Arg Ser Glu Thr 380 385 390

AAG TAC TGG GGC AAT CAT GTG ATT TTG CAG ATC CAA AAG ATG ATT GAT 2033 Lys Tyr Trp Gly Asn His Val He Leu Gin He Gin Lys Met He Asp 395 400 405

TTC TAC AAA AAT CAA CCT TTA GAT ATC AAA TTA ATA CTT GTA GTG CAT 2081 Phe Tyr Lys Asn Gin Pro Leu Asp He Lys Leu He Leu Val Val His 410 415 420 425

AAT CTG GAT GGT CCT AGC ATA AGG AAA AAC ACT TTT CAG ACG ATG CTA 2129 Asn Leu Asp Gly Pro Ser He Arg Lys Asn Thr Phe Gin Thr Met Leu 430 435 440 AGC TTC CTC TCC GTC ATC AGA CAA ATC GCC ATA GTC GCC TCT ACA GAC 2177 Ser Phe Leu Ser Val He Arg Gin He Ala He Val Ala Ser Thr Asp 445 450 455

CAC ATT TAC GCT CCG CTC CTC TGG GAC AAC ATG AAG GCC CAA AAC TAC 2225 His He Tyr Ala Pro Leu Leu Trp Asp Asn Met Lys Ala Gin Asn Tyr 460 465 470

AAC TTT GTC TTT CAT GAT ATT TCG AAT TTT GAA CCG TCG ACA GTC GAG 2273 Asn Phe Val Phe His Asp He Ser Asn Phe Glu Pro Ser Thr Val Glu 475 480 485

TCT ACG TTC CAA GAT GTG ATG AAG ATG GGT AAA AGC GAT ACC AGC AGT 2321 Ser Thr Phe Gin Asp Val Met Lys Met Gly Lys Ser Asp Thr Ser Ser 490 495 500 505

GGT GCT GAA GGT GCG AAA TAC GTC TTA CAA TCA CTT ACT GTG AAC TCC 2369 Gly Ala Glu Gly Ala Lys Tyr Val Leu Gin Ser Leu Thr Val Asn Ser 510 515 520 AAG AAG ATG TAT AAG TTG CTT ATT GAA ACA CAA ATG CAG AAT ATG GGG 2417 Lys Lys Met Tyr Lys Leu Leu He Glu Thr Gin Met Gin Asn Met Gly 525 530 535 AAT CTA TCC GCT AAC ACA GGT CCT AAG CGT GGT ACT CAA AGA ACT GGA 2465 Asn Leu Ser Ala Asn Thr Gly Pro Lys Arg Gly Thr Gin Arg Thr Gly 540 545 550 GTA GAA CTT AAA CTT TTC AAC CAT CTC TGT GCC GCT GAT TTT ATT GCT 2513 Val Glu Leu Lys Leu Phe Asn His Leu Cys Ala Ala Asp Phe He Ala 555 560 565

TCT AAT GAG ATA GCT CTA AGG TCG ATG CTT AGA GAA TTC ATA GAA CAT 2561 Ser Asn Glu He Ala Leu Arg Ser Met Leu Arg Glu Phe He Glu His 570 575 580 585

AAA ATG GCC AAC ATA ACT AAG AAC AAT TCT GGA ATG GAA ATT ATT TGG 2609 Lys Met Ala Asn He Thr Lys Asn Asn Ser Gly Met Glu He He Trp 590 595 600

GTA CCC TAC ACG TAT GCG GAA CTT GAA AAA CTT CTG AAA ACC GTT TTA 2657 Val Pro Tyr Thr Tyr Ala Glu Leu Glu Lys Leu Leu Lys Thr Val Leu 605 610 615

AAT ACT CTA TAAATGTATA CATATCACGA ACAATTGTAA TAGTACTAGG 2706

Asn Thr Leu 620 CTTGCTAGCT TTGCTTTCCC ATAACCAACA ATACTTAGTG ATGTATCTTA AAACGACTAA 2766

AAAACTTCTC ATATAACCCT ACTGAAAAAC GTCTGATGAG CTC 2809

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 620 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Met Leu Asn Gly Glu Asp Phe Val Glu His Asn Asp He Leu Ser Ser 1 5 10 15

Pro Ala Lys Ser Arg Asn Val Thr Pro Lys Arg Val Asp Pro His Gly * 20 25 30

Glu Arg Gin Leu Arg Arg He His Ser Ser Lys Lys Asn Leu Leu Glu 35^" 40 45 Arg He Ser Leu Val Gly Asn Glu Arg Lys Asn Thr Ser Pro Asp Pro 50 55 60

Ala Leu Lys Pro Lys Thr Pro Ser Lys Ala Pro Arg Lys Arg Gly Arg

65 70 75 80

Pro Arg Lys He Gin Glu Glu Leu Thr Asp Arg He Lys Lys Asp Glu

85 90 95

Lys Asp Thr He Ser Ser Lys Lys Lys Arg Lys Leu Asp Lys Asp Thr 100 105 110

Ser Gly Asn Val Asn Glu Glu Ser Lys Thr Ser Asn Asn Lys Gin Val 115 120 125 Met Glu Lys Thr Gly He Lys Glu Lys Arg Glu Arg Glu Lys He Gin 130 135 140 Val Ala Thr Thr Thr Tyr Glu Asp Asn Val Thr Pro Gin Thr Asp Asp 145 150 155 160

Asn Phe Val Ser Asn Ser Pro Glu Pro Pro Glu Pro Ala Thr Pro Ser 165 170 175

Lys Lys Ser Leu Thr Thr Asn His Asp Phe Thr Ser Pro Leu Lys Gin 180 185 190 He He Met Asn Asn Leu Lys Glu Tyr Lys Asp Ser Thr Ser Pro Gly 195 200 205

Lys Leu Thr Leu Ser Arg Asn Phe Thr Pro Thr Pro Val Pro Lys Asn 210 215 220

Lys Lys Leu Tyr Gin Thr Ser Glu Thr Lys Ser Ala Ser Ser Phe Leu 225 230 235 240

Asp Thr Phe Glu Gly Tyr Phe Asp Gin Arg Lys He Val Arg Thr Asn 245 250 255

Ala Lys Ser Arg His Thr Met Ser Met Ala Pro Asp Val Thr Arg Glu 260 265 270 Glu Phe Ser Leu Val Ser Asn Phe Phe Asn Glu Asn Phe Gin Lys Arg 275 280 285

Pro Arg Gin Lys Leu Phe Glu He Gin Lys Lys Met Phe Pro Gin Tyr 290 295 300

Trp Phe Glu Leu Thr Gin Gly Phe Ser Leu Leu Phe Tyr Gly Val Gly 305 310 315 320

Ser Lys Arg Asn Phe Leu Glu Glu Phe Ala He Asp Tyr Leu Ser Pro 325 330 335

Lys He Ala Tyr Ser Gin Leu Ala Tyr Glu Asn Glu Leu Gin Gin Asn 340 345 350 Lys Pro Val Asn Ser He Pro Cys Leu He Leu Asn Gly Tyr Asn Pro 355 360 365

Ser Cys Asn Tyr Arg Asp Val Phe Lys Glu He Thr Asp Leu Leu Val 370 375 380

Pro Ala Glu Leu Thr Arg Ser Glu Thr Lys Tyr Trp Gly Asn His Val 385 390 395 400

He Leu Gin He Gin Lys Met He Asp Phe Tyr Lys Asn Gin Pro Leu 405 410 415

Asp He Lys Leu He Leu Val Val His Asn Leu Asp Gly Pro Ser He 420 425 430 Arg Lys Asn Thr Phe Gin Thr Met Leu Ser Phe Leu Ser Val He Arg 435 440 445

Gin He Ala He Val Ala Ser Thr Asp His He Tyr Ala Pro Leu Leu 450 455 460

Trp Asp Asn Met Lys Ala Gin Asn Tyr Asn Phe Val Phe His Asp He 465 470 475 480

Ser Asn Phe Glu Pro Ser Thr Val Glu Ser Thr Phe Gin Asp Val Met 485 490 495

Lys Met Gly Lys Ser Asp Thr Ser Ser Gly Ala Glu Gly Ala Lys Tyr 500 505 510 Val Leu Gin Ser Leu Thr Val Asn Ser Lys Lys Met Tyr Lys Leu Leu 515 520 525

He Glu Thr Gin Met Gin Asn Met Gly Asn Leu Ser Ala Asn Thr Gly 530 535 540

Pro Lys Arg Gly Thr Gin Arg Thr Gly Val Glu Leu Lys Leu Phe Asn

545 550 555 560 His Leu Cys Ala Ala Asp Phe He Ala Ser Asn Glu He Ala Leu Arg

565 570 575

Ser Met Leu Arg Glu Phe He Glu His Lys Met Ala Asn He Thr Lys 580 585 590

Asn Asn Ser Gly Met Glu He He Trp Val Pro Tyr Thr Tyr Ala Glu 595 600 605

Leu Glu Lys Leu Leu Lys Thr Val Leu Asn Thr Leu 610 615 620

(2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2759 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TCTGAAATAA AAAGTACAAA AAAGAAAACA ATATACCAGA TATGAACCCT TTTAGTGAGA 60

TTCCAGCATG TCTTTGCGCA GATCCAAATC TTTCTTTGTC TTGAAATTTA TTCAGTAAAT 120

TAAAAGTCAG TTCTTTAGTA GCATTCATCT TCTTGGTAAG TCTTTTTCTT GTTTTTGAAA 180

AAGAGTTCCT GAAGTTTGTC TACTGTGAAT ATACTTTGCA CATTTGTTTA ATTTTTAAAC 240

ACGCTATAAT TTGTGTCATA AAGAATTTTT TGTAGAATAG CTTTTTTTTT AATAGGAAAA 300 AAAAATAAAA AAAGGTGGAA AAGACAATCT TTTCCAGAAA CTTGAAACTA TACTGGAGAT 360

GAAGGGTTGT CGTTGGTTGC GTTACGAGAC AGGCTTGACA ATTTCACAAG AGTAATGTTT 420

CATTACCTGC TGTTTTATTA TCTTTATATT TAGTAAGACC AGCAGAAACG CTACACGTGA 480

TGATAATGGA ACTAAGCATT CTGTTAGATG GTAAGAATTT TTTTTACCTT CCATTACCAC 540

TAACGCCTTT TTTAGTGTCT TTTTGATATT TACTGACGTA TTTTTCCGCA CCGTAATTTG 600 AAGAAAAAGA AAAGTGACAA AAGATGGCAT TGTTTACATA CAGAGTCGTA GTATCACAAG 660

AGTAGTCCAA CAGGATGAGC GACCTTAACC AATCCAAAAA GATGAACGTC AGCGAGTTTG 720

CTGACGCCCA AAGGAGCCAC TATACAGTAT ACCCCAGTTT GCCTCAAAGT AACAAAAATG 780

ATAAACACAT TCCCTTTGTC AAACTTCTAT CAGGCAAAGA ATCGGAAGTG AACGTGGAAA 840

AAAGATGGGA ATTGTATCAT CAGTTACATT CCCACTTTCA TGATCAAGTA GATCATATTA 900 TCGATAATAT TGAAGCAGAC TTGAAAGCAG AGATTTCAGA CCTTTTATAT AGTGAAACTA 960

CTCAGAAAAG GCGATGCTTT AACACTATTT TCCTATTAGG TTCAGATAGT ACGACAAAAA 1020 TTGAACTTAA AGACGAATCT TCTCGCTACA ACGTTTTGAT TGAATTGACT CCGAAAGAAT 1080 CTCCGAATGT AAGAATGATG CTTCGTAGGT CTATGTACAA ACTTTACAGC GCAGCTGATG 1140 CAGAAGAACA TCCAACTATC AAGTATGAAG ACATTAACGA TGAAGATGGC GATTTTACCG 1200 AGCAAAACAA TGATGTATCA TACGATCTGT CACTTGTGGA AAACTTCAAA AGGCTTTTTG 1260 GAAAAGACTT AGCAATGGTA TTTAATTTTA AAGATGTAGA TTCTATTAAC TTCAACACAT 1320

TGGATAACTT CATAATTCTA TTGAAAAGTG CCTTCAAGTA TGACCATGTT AAAATAAGTT 1380 TAATCTTTAA TATTAATACA AACTTGTCAA ATATTGAGAA AAATTTGAGA CAATCAACCA 1440 TACGACTTCT GAAGAGAAAT TATCATAAAC TAGACGTGTC GAGTAATAAA GGATTTAAGT 1500 ACGGAAACCA AATCTTTCAA AGCTTTTTGG ATACGGTTGA TGGCAAACTA AATCTTTCAG 1560 ATCGTTTTGT GGAATTCATT CTCAGCAAGA TGGCAAATAA TACTAATCAC AACTTACAAT 1620

TATTGACGAA GATGCTGGAT TATTCGTTGA TGTCGTACTT TTTCCAGAAT GCCTTTTCAG 1680 TATTCATTGA CCCTGTAAAT GTTGATTTTT TGAACGACGA CTACTTAAAA ATACTGAGCA 1740 GATGTCCTAC ATTCATGTTC TTTGTCGAAG GTCTTATAAA GCAGCATGCT CCTGCTGACG 1800 AAATTCTTTC ATTATTGACA AACAAAAACA GAGGCCTAGA AGAGTTTTTT GTTGAGTTTT 1860 TGGTAAGAGA GAACCCGATT AACGGGCATG CTAAGTTTGT TGCTCGATTC CTCGAAGAAG 1920

AATTGAATAT AACCAATTTT AATCTGATAG AATTATATCA TAATTTGCTT ATTGGCAAAC 1980 TAGACTCCTA TCTAGATCGT TGGTCAGCAT GTAAAGAGTA TAAGGATCGG CTTCATTTTG 2040 AACCCATTGA TACAATTTTT CAAGAGCTAT TTACTTTGGA CAACAGAAGT GGATTACTTA 2100 CCCAGTCGAT TTTCCCTTCT TACAAGTCAA ATATCGAAGA TAACTTACTA AGTTGGGAGC 2160 AGGTGCTGCC TTCGCTTGAT AAAGAAAATT ATGATACTCT TTCTGGAGAT TTGGATAAAA 2220

TAATGGCTCC GGTACTGGGT CAGCTATTCA AGCTTTATCG TGAGGCGAAT ATGACTATCA 2280

ACATTTACGA TTTCTACATT GCGTTCAGAG AAACATTACC AAAAGAGGAA ATATTAAATT*2340 TCATAAGAAA AGATCCCTCC AACACCAAAC TCTTAGAACT AGCAGAAACA CCGGACGCAT 2400

TTGACAAAGT AGCACTAATT TTATTCATGC AAGCAATCTT CGCCTTTGAA AACATGGGTC 2460

TCATTAAGTT TCAAAGCACC AAGAGTTACG ATCTGGTAGA AAAATGTGTC TGGAGAGGAA 2520

TTTAGATAAA GAATGCACGG ATAAATAAGT AAATAAATAA CCATACATAT ATAGAACCAT 2580

AGAACCACGT TTTTGTAATG AACAGTCTAC CTGTATCTCA TCATTTTTCT GTGTTAACTA 2640 TTATTATTAT TATTATCGAA TGGAGGGTAA TATTATGTAT AGGTAAAATA AATAGATAGT 2700

GCCATGATGC GCGAAGATTG GCAATGGGAA ACTCAAGAAG GCAGCAACAA AAAAATAAA 2759

(2) INFORMATION FOR SEQ ID NO:6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 615 amino acids

(B) TYPE: amino acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi ) SEQUENCE DESCRIPTION : SEQ ID NO: 6 :

Met Ser Asp Leu Asn Gin Ser Lys Lys Met Asn Val Ser Glu Phe Ala 1 5 10 15

Asp Ala Gin Arg Ser His Tyr Thr Val Tyr Pro Ser Leu Pro Gin Ser 20 25 30

Asn Lys Asn Asp Lys His He Pro Phe Val Lys Leu Leu Ser Gly Lys 35 40 45

Glu Ser Glu Val Asn Val Glu Lys Arg Trp Glu Leu Tyr His Gin Leu 50 55 60 His Ser His Phe His Asp Gin Val Asp His He He Asp Asn He Glu 65 70 75 80

Ala Asp Leu Lys Ala Glu He Ser Asp Leu Leu Tyr Ser Glu Thr Thr

85 90 95

Gin Lys Arg Arg Cys Phe Asn Thr He Phe Leu Leu Gly Ser Asp Ser

100 105 110

Thr Thr Lys He Glu Leu Lys Asp Glu Ser Ser Arg Tyr Asn Val Leu 115 120 125

He Glu Leu Thr Pro Lys Glu Ser Pro Asn Val Arg Met Met Leu Arg

130 135 140 Arg Ser Met Tyr Lys Leu Tyr Ser Ala Ala Asp Ala Glu Glu His Pro

145 150 155 160

Thr He Lys Tyr Glu Asp He Asn Asp Glu Asp Gly Asp Phe Thr Glu 165 170 175

Gin Asn Asn Asp Val Ser Tyr Asp Leu Ser Leu Val Glu Asn Phe Lys 180 185 190

Arg Leu Phe Gly Lys Asp Leu Ala Met Val Phe Asn Phe Lys Asp Val 195 200 205

Asp Ser He Asn Phe Asn Thr Leu Asp Asn Phe He He Leu Leu Lys 210 215 220 Ser Ala Phe Lys Tyr Asp His Val Lys He Ser Leu He Phe Asn He 225 ^* 230 235 240

Asn Thr Asn Leu Ser Asn He Glu Lys Asn Leu Arg Gin Ser Thr He 245 250 255

Arg Leu Leu Lys Arg Asn Tyr His Lys Leu Asp Val Ser Ser Asn Lys

260 265 270

Gly Phe Lys Tyr Gly Asn Gin He Phe Gin Ser Phe Leu Asp Thr Val 275 280 285

Asp Gly Lys Leu Asn Leu Ser Asp Arg Phe Val Glu Phe He Leu Ser 290 295 300 Lys Met Ala Asn Asn Thr Asn His Asn Leu Gin Leu Leu Thr Lys Met 305 310 315 320 Leu Asp Tyr Ser Leu Met Ser Tyr Phe Phe Gin Asn Ala Phe Ser Val

325 330 335

Phe He Asp Pro Val Asn Val Asp Phe Leu Asn Asp Asp Tyr Leu Lys 340 345 350 He Leu Ser Arg Cys Pro Thr Phe Met Phe Phe Val Glu Gly Leu He 355 360 365

Lys Gin His Ala Pro Ala Asp Glu He Leu Ser Leu Leu Thr Asn Lys 370 ^~_-3^*75 380

Asn Arg Gly Leu Glu Glu Phe Phe Val Glu Phe Leu Val Arg Glu Asn 385 390 395 400 Pro He Asn Gly His Ala Lys Phe Val Ala Arg Phe Leu Glu Glu Glu

405 410 415

Leu Asn He Thr Asn Phe Asn Leu He Glu Leu Tyr His Asn Leu Leu 420 425 430

He Gly Lys Leu Asp Ser Tyr Leu Asp Arg Trp Ser Ala Cys Lys Glu 435 440 445

Tyr Lys Asp Arg Leu His Phe Glu Pro He Asp Thr He Phe Gin Glu 450 455 460

Leu Phe Thr Leu Asp Asn Arg Ser Gly Leu Leu Thr Gin Ser He Phe

465 470 475 480 Pro Ser Tyr Lys Ser Asn He Glu Asp Asn Leu Leu Ser Trp Glu Gin

485 490 495

Val Leu Pro Ser Leu Asp Lys Glu Asn Tyr Asp Thr Leu Ser Gly Asp 500 505 510

Leu Asp Lys He Met Ala Pro Val Leu Gly Gin Leu Phe Lys Leu Tyr 515 520 525

Arg Glu Ala Asn Met Thr He Asn He Tyr Asp Phe Tyr He Ala Phe 530 535 540

Arg Glu Thr Leu Pro Lys Glu Glu He Leu Asn Phe He Arg Lys Asp

545 550 555 560 ^' Pro Ser Asn Thr Lys Leu Leu Glu Leu Ala Glu Thr Pro Asp Ala Phe

565 570 575

Asp Lys Val Ala Leu He Leu Phe Met Gin Ala He Phe Ala Phe Glu 580 585 590

Asn Met Gly Leu He Lys Phe Gin Ser Thr Lys Ser Tyr Asp Leu Val 595 600 605

Glu Lys Cys Val Trp Arg Gly 610 615

(2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2404 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: CTCGAGGCCA CCAAGAAGAG AAAGAGAAGA GCCAGATATT GACTGGAGTG CAGCCAGAGG 60 TTCCAACTTC CAAAGCTCCT CGGAGCCACC AAGAAGAGAA AGAGAAAAGG AAGAACCAGC 120 TTTGGATTGG GGTGCTGCCA GAGGTGCTCA GTTTGGTAAG CCTCAACAAA CCAAAAATAC 180

CTACAAGGAT AGGTCTCTAA CTAACAAAAA GACTACTGAT GAGCAACCAA AAATCCAGAA 240 GTCTGTTTAT GATGTTTTAC GTACTGAAGA TGATGATGAA GATGAAGAGG CTGAAAAGCA 300

AAATGGAGAC GCAAAAGAAA ACAAAGTTGA TGCGGCAGTT GAAAAGCTAC AGGATAAAAC 360

TGCTCAATTG ACTGTTGAAG ATGGTGACAA TTGGGAAGTT GTTGGTAAGA AATAGAGTGT 420

TGTATGATGA TAAAATGTAC ATTTGTATTT ACTGTTTGCT TTTTTTCTTT CTTGTTTTTC 480

TACTCTCCTT TCTACCAGGT ATTCTAACTC TATTATATAA TTAAAAAAAA AATAACCATA 540 TATTTTGTAT TAAGTTTCAT ACATGTGTTC AAGTGTATTT TTGGATTTAT CATTTTTCTA 600

TGTGAGGTAA GTTTTTGAAT GTCCCATTTT CCTTTCGTTT TTGGAAAGTT CTAAGAAAAA 660

GCATTAACAA TTAAAAAAAA AAAAAAAATC TAAATAATAC TGATAGAAAT ATCAAATATA 720

AACTACTAAT ATCGGTAATA TTCAAAAGAA GAAGCATGAC TATAAGCGAA GCTCGTCTAT 780 CACCGCAAGT CAATCTTCTC CCAATAAAGA GGCACTCAAA CGAAGAGGTA GAGGAGACTG 840 CAGCGATTCT AAAAAAGCGT ACTATAGATA ATGAAAAGTG TAAAGACAGC GACCCTGGTT 900 TTGGTTCCCT TCAAAGAAGG TTACTGCAGC AACTTTATGG CACACTTCCT ACGGACGAAA 960 AGATAATCTT CACATATTTA CAAGATTGTC AACAAGAGAT CGATAGAATC ATTAAACAAT 1020

CCATTATTCA GAAAGAGAGT CATTCAGTAA TTCTCGTGGG GCCCAGACAA AGTTACAAAA 1080 CATACTTATT AGACTATGAA CTGTCTTTGT TGCAACAATC TTATAAAGAG CAGTTTATAA 1140 CTATCAGGTT GAATGGGTTT ATTCACTCCG AACAAACAGC TATTAACGGT ATAGCAACTC 1200 AATTGGAACA GCAGTTGCAG AAAATTCATG GCAGTGAAGA AAAAATTGAC GATACTTCAT 1260 TAGAGACTAT TAGCAGTGGT TCTTTGACAG AAGTGTTTGA GAAAATTCTT TTACTCTTAG 1320

ATTCGACCAC GAAGACAAGA AATGAAGATA GTGGTGAGGT TGACAGAGAG AGTATAACAA 1380

AGATAACAGT TGTTTTTATA TTCGATGAAA TTGATACATT TGCTGGGCCT GTGAGGCAAA 1440 CTTTATTATA CAATCTTTTT GACATGGTAG AACATTCTCG GGTACCTGTT TGCATTTTTG 1500

GCTGCACAAC GAAATTAAAT ATCTTGGAAT ATTTAGAAAA GAGGGTAAAG AGTAGATTTT 1560

CTCAAAGAGT GATTTATATG CCGCAAATAC AGAATCTAGA CGATATGGTT GACGCCGTCA 1620

GAAATTTACT TACAGTTCGC TCTGAAATCT CCCCCTGGGT TTCACAATGG AATGAAACGT 1680 TGGAAAAAGA ACTATCCGAC CCTCGATCGA ATTTGAATAG ACATATTAGG ATGAATTTCG 1740 AAACCTTTAG GTCATTACCT ACATTGAAAA ATAGCATAAT TCCATTAGTA GCGACATCCA 1800 AAAATTTTGG TTCACTCTGC ACTGCCATAA AATCGTGTTC TTTTCTTGAC ATATACAATA 1860 AGAACCAACT ATCTAATAAT TTAACAGGAA GGCTCCAATC TTTATCCGAT TTAGAGTTAG 1920

CCATTTTGAT CTCAGCCGCT AGGGTTGCCT TAAGGGCGAA AGACGGATCT TTTAATTTTA 1980

ATTTAGCTTA TGCAGAGTAT GAAAAGATGA TTAAAGCTAT CAACTCCAGA ATTCCCACCG 2040 TGGCTCCTAC TACAAATGTG GGAACAGGTC AAAGTACTTT TTCTATCGAC AATACTATCA 2100

AACTATGGTT GAAAAAGGAC GTCAAGAACG TTTGGGAAAA TTTAGTGCAA CTGGATTTTT 2160 TTACCGAGAA ATCAGCCGTT GGTTTGAGAG ATAATGCGAC CGCAGCATTT TACGCTAGCA 2220

ATTATCAATT TCAGGGCACC ATGATCCCGT TTGACTTGAG AAGTTACCAG ATGCAGATCA 2280 TTCTTCAGGA ATTAAGAAGA ATTATCCCCA AATCTAATAT GTACTACTCC TGGACACAAC 2340

TGTGAATCTT GGGAACAATA TACAGACATT TTATTGGCGG TAGCAACTCT GATATTCCAC 2400

TGTT 2404

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 529 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Met Thr He Ser Glu Ala Arg Leu Ser Pro Gin Val Asn Leu Leu Pro 1 5 10 15

He Lys Arg His Ser Asn Glu Glu Val Glu Glu Thr Ala Ala He Leu 20 25 30

Lys Lys Arg Thr He Asp Asn Glu Lys Cys Lys Asp Ser Asp Pro Gly 35 40 45

Phe Gly Ser Leu Gin Arg Arg Leu Leu Gin Gin Leu Tyr Gly Thr Leu 50 55 60

Pro Thr Asp Glu Lys He He Phe Thr Tyr Leu Gin Asp Cys Gin Gin 65 70 75 80

Glu He Asp Arg He He Lys Gin Ser He He Gin Lys Glu Ser His 85 90 95

Ser Val He Leu Val Gly Pro Arg Gin Ser Tyr Lys Thr Tyr Leu Leu 100 105 110

Asp Tyr Glu Leu Ser Leu Leu Gin Gin Ser Tyr Lys Glu Gin Phe He

115 120 125

Thr He Arg Leu Asn Gly Phe He His Ser Glu Gin Thr Ala He Asn 130 135 140

Gly He Ala Thr Gin Leu Glu Gin Gin Leu Gin Lys He His Gly Ser 145 150 155 160 Glu Glu Lys He Asp Asp Thr Ser Leu Glu Thr He Ser Ser Gly Ser

165 170 175

Leu Thr Glu Val Phe Glu Lys He Leu Leu Leu eu Asp Ser Thr Thr 180 185 190

Lys Thr Arg Asn Glu Asp Ser Gly Glu Val Asp Arg Glu Ser He Thr 195 200 205

Lys He Thr Val Val Phe He Phe Asp Glu He Asp Thr Phe Ala Gly 210 215 220

Pro Val Arg Gin Thr Leu Leu Tyr Asn Leu Phe Asp Met Val Glu His 225 230 235 240 Ser Arg Val Pro Val Cys He Phe Gly Cys Thr Thr Lys Leu Asn He 245 250 255

Leu Glu Tyr Leu Glu Lys Arg Val Lys Ser Arg Phe Ser Gin Arg Val -^' 260 265 270

He Tyr Met Pro Gin He Gin Asn Leu Asp Asp Met Val Asp Ala Val 275 280 285 Arg Asn Leu Leu Thr Val Arg Ser Glu He Ser Pro Trp Val Ser Gin 290 295 300

Trp Asn Glu Thr Leu Glu Lys Glu Leu Ser Asp Pro Arg Ser Asn Leu 305 310 315 320

Asn Arg His He Arg Met Asn Phe Glu Thr Phe Arg Ser Leu Pro Thr 325 330 335

Leu Lys Asn Ser He He Pro Leu Val Ala Thr Ser Lys Asn Phe Gly 340 345 350

Ser Leu Cys Thr Ala He Lys Ser Cys Ser Phe Leu Asp He Tyr Asn 355 360 365 Lys Asn Gin Leu Ser Asn Asn Leu Thr Gly Arg Leu Gin Ser Leu Ser 370 375 380

Asp Leu Glu Leu Ala He Leu He Ser Ala Ala Arg Val Ala Leu Arg 385 390 395 400

Ala Lys Asp Gly Ser Phe Asn Phe Asn Leu Ala Tyr Ala Glu Tyr Glu 405 410 415

Lys Met He Lys Ala He Asn Ser Arg He Pro Thr Val Ala Pro Thr 420 . 425 430

Thr Asn Val Gly Thr Gly Gin Ser Thr Phe Ser He Asp Asn Thr He 435 440 445 ^' Lys Leu Trp Leu Lys Lys Asp Val Lys Asn Val Trp Glu Asn Leu Val 450 455 460

Gin Leu Asp Phe Phe Thr Glu Lys Ser Ala Val Gly Leu Arg Asp Asn

465 470 475 480

Ala Thr Ala Ala Phe Tyr Ala Ser Asn Tyr Gin Phe Gin Gly Thr Met

485 490 495

He Pro Phe Asp Leu Arg Ser Tyr Gin Met Gin He He Leu Gin Glu 500 505 510

Leu Arg Arg He He Pro Lys Ser Asn Met Tyr Tyr Ser Trp Thr Gin 515 520 525 Leu

(2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2306 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

GCTATTTTTT CATGCGTCAG ATGTCACAAA GCCTTTAATC AAGTATTGTT GCAAGAACAC 60 CTGATTCAAA AACTACGTTC TGATATCGAA TCCTATTTAA TTCAAGATTT GAGATGCTCC 120

AGATGTCATA AAGTGAAACG TGACTATATG AGTGCCCACT GTCCATGTGC CGGCGCGTGG 180

GAAGGAACTC TCCCCAGAGA AAGCATTGTT CAAAAGTTAA ATGTGTTTAA GCAAGTAGCC 240

AAGTATTACG GTTTTGATAT ATTATTGAGT TGTATTGCTG ATTTGACCAT ATGAGTAAGC 300

AGTATATAAC GCGAGGTTCA ATGGCCTCTT TACCATGAAA AAAAAAAAAA AAAAAAAAAA 360 AAGGTAAGGA AAAAGAGTAT TTTCAATTCG TTTCTGAACA TATAAATATA AATAACCGAA 420

AAATTAGCCC TTGAACATAA TTAACACTCT TCTTTGATAT TTAAATCACA AGTACTTTTC 480

TTTTATTTTC TTCTTAATAC TTTTGGAAAT AAAATGAATG TGACCACTCC GGAAGTTGCT 540

TTTAGGGAAT ATCAAACCAA CTGTCTCGCA TCGTATATTT CTGCTGATCC AGACATAACT 600

CCTTCAAATT TAATCTTGCA AGGTTATAGT GGAACAGGAA AAACCTACAC TTTGAAGAAG 660 TATTTTAATG CGAATCCAAA TTTGCATGCA GTATGGCTGG AACCTGTTGA GTTGGTTTCT 720

TGGAAGCCCT TACTGCAGGC GATAGCACGT ACTGTACAAT ATAAATTGAA AACCCTATAT 780

CCAAACATTC CCACCACAGA TTACGATCCT TTACAGGTTG AAGAGCCATT TCTTTTGGTA 840

AAGACGTTGC ACAATATTTT TGTCCAATAT GAATCTTTGC AAGAAAAGAC TTGCTTGTTC 900 TTGATATTGG ATGGTTTCGA TAGTTTACAA GATTTAGACG CCGCACTGTT TAACAAATAT 960 ATCAAACTAA ATGAATTACT TCCAAAAGAT TCTAAAATTA ATATAAAATT CATTTACACG 1020 ATGTTAGAGA CATCATTTTT GCAAAGATAT TCTACACATT GCATTCCAAC TGTTATGTTT 1080 CCGAGGTATA ATGTGGACGA AGTTTCTACT ATATTAGTGA TGTCTAGATG TGGCGAACTC 1140

ATGGAAGATT CTTGTCTACG TAAGCGTATC ATTGAAGAGC AGATAACGGA CTGTACAGAC 1200 GATCAATTTC AAAATGTAGC TGCGAACTTC ATTCACTTAA TTGTGCAGGC TTTTCATTCT 1260 TATACTGGAA ACGACATATT CGCATTGAAT GACTTGATAG ACTTCAAATG GCCCAAGTAT 1320 GTATCTCGCA TTACTAAGGA AAACATATTT GAACCACTGG CTCTTTACAA AAGTGCCATC 1380 AAACTATTTT TAAGCACAGA TGATAATTTA AGTGAAAATG GACAAGGTGA AAGCGCGATA 1440

ACCACAAATC GTGATGACCT TGAGAACAGT CAAACTTACG ACTTATCAAT AATTTCGAAG 1500 TATCTGCTCA TAGCCTCATA TATTTGTTCA TATCTGGAAC CTAGATACGA TGCGAGTATT 1560 TTCTCTAGGA AAACACGTAT CATACAAGGT AGAGCTGCTT ATGGACGAAG AAAGAAGAAA 1620 GAAGTTAACC CTAGATATTT ACAGCCTTCT TTATTTGCTA TTGAAAGACT TTTGGCTATT 1680 TTCCAAGCTA TATTCCCTAT TCAAGGTAAG GCGGAGAGTG GTTCCCTATC TGCACTTCGT 1740

GAGGAATCCT TAATGAAAGC GAATATCGAG GTTTTTCAAA ATTTATCCGA ATTGCATACA 1800

TTGAAATTAA TAGCTACAAC CATGAACAAG AATATCGACT ATTTGAGTCC TAAAGTCAGG 1860 TGGAAAGTAA ACGTTCCCTG GGAAATTATT AAAGAAATAT CAGAATCTGT TCATTTCAAT 1920

ATCAGCGATT ACTTCAGCGA TATTCACGAA TGATTATCTC CCTGGAAGGT ATCCAGAGGG 1980 CAGGATACGT TCGAAACAAC AACTACGTTA TATAAATATT TATACATAGT GGGATAGAAT 2040

GAACAATTAT CAAGTAAACC TTGTATTTTT TGTTCCCACG CTCTACGCTC TGTTTCTTGG 2100 ATATGGTAAT CAAAGATTAA TACGTATAAC CGTTATTAAT TCAGTCCACT AGAAACTATT 2160

AAAAGCGCCC TACTGTATGG AAAAACAATG AATGAGGAGA CTGAACGGCG CAAAATTGTT 2220

AGTTTAGTTG CTCTTTTTGG CGGCCGGCGA TAATGTTCTT CACTTGGTAT TCTTACCAGG 2280

ATTGAGCCTG ATTTTGTTTT GTCTTA 2306

(2) INFORMATION FOR SEQ ID NO:10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 479 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Met Asn Val Thr Thr Pro Glu Val Ala Phe Arg Glu Tyr Gin Thr Asn 1 5 10 15

Cys Leu Ala Ser Tyr He Ser Ala Asp Pro Asp He Thr Pro Ser Asn 20 25 30

Leu He Leu Gin Gly Tyr Ser Gly Thr Gly Lys Thr Tyr Thr Leu Lys 35 40 45

Lys Tyr Phe Asn Ala Asn Pro Asn Leu His Ala Val Trp Leu Glu Pro 50 55 60

Val Glu Leu Val Ser Trp Lys Pro Leu Leu Gin Ala He Ala Arg Thr 65 70 75 80

Val Gin Tyr Lys Leu Lys Thr Leu Tyr Pro Asn He Pro Thr Thr Asp 85 90 95

Tyr Asp Pro Leu Gin Val Glu Glu Pro Phe Leu Leu Val Lys Thr Leu 100 105 110

His Asn He Phe Val Gin Tyr Glu Ser Leu Gin Glu Lys Thr Cys Leu ^" 115 120 125 Phe Leu He Leu Asp Gly Phe Asp Ser Leu Gin Asp Leu Asp Ala Ala 130 135 140

Leu Phe Asn Lys Tyr He Lys Leu Asn Glu Leu Leu Pro Lys Asp Ser 145 150 155 160

Lys He Asn He Lys Phe He Tyr Thr Met Leu Glu Thr Ser Phe Leu 165 170 175

Gin Arg Tyr Ser Thr His Cys He Pro Thr Val Met Phe Pro Arg Tyr 180 185 190

Asn Val Asp .Glu Val Ser Thr He Leu Val Met Ser Arg Cys Gly Glu

195 200 205 Leu Met Glu Asp Ser Cys Leu Arg Lys Arg He He Glu Glu Gin He

210 215 220 Thr Asp Cys Thr Asp Asp Gin Phe Gin Asn Val Ala Ala Asn Phe He 225 230 235 240

His Leu He Val Gin Ala Phe His Ser Tyr Thr Gly Asn Asp He Phe 245 250 255

Ala Leu Asn Asp Leu He Asp Phe Lys Trp Pro Lys Tyr Val Ser Arg

260 265 270 He Thr Lys Glu Asn He Phe Glu Pro Leu Ala Leu Tyr Lys Ser Ala 275 280 285

He Lys Leu Phe Leu Ser Thr Asp Asp Asn Leu Ser Glu Asn Gly Gin

290 295 300

Gly Glu Ser Ala He Thr Thr Asn Arg Asp Asp Leu Glu Asn Ser Gin

305 310 315 320

Thr Tyr Asp Leu Ser He He Ser Lys Tyr Leu Leu He Ala Ser Tyr 325 330 335

He Cys Ser Tyr Leu Glu Pro Arg Tyr Asp Ala Ser He Phe Ser Arg 340 345 350 Lys Thr Arg He He Gin Gly Arg Ala Ala Tyr Gly Arg Arg Lys Lys 355 360 365

Lys Glu Val Asn Pro Arg Tyr Leu Gin Pro Ser Leu Phe Ala He Glu

370 375 380

Arg Leu Leu Ala He Phe Gin Ala He Phe Pro He Gin Gly Lys Ala

385 390 395 400

Glu Ser Gly Ser Leu Ser Ala Leu Arg Glu Glu Ser Leu Met Lys Ala ^' 405 410 415

Asn He Glu Val Phe Gin Asn Leu Ser Glu Leu His Thr Leu Lys Leu

420 425 430 He Ala Thr Thr Met Asn Lys Asn He Asp Tyr Leu Ser Pro Lys Val

435 440 445

Arg Trp Lys Val Asn Val Pro Trp Glu He He Lys Glu He Ser Glu 450 455 460

Ser Val His Phe Asn He Ser Asp Tyr Phe Ser Asp He His Glu 465 470 475

(2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1975 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: double

(D) TOPOLOGY: 1inear

(ii) MOLECULE TYPE: cDNA (ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 443..1747

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: CGTGTGCTCT TCTATAGTAA TTTGACATTC TCTAAACGCA GAGACCTCTT ATAAAGATTC 60 AACAAATAAG GAATGTTACC TATGCTAGTC GCAACTCTCT CGTAAGTTGA GGGTTGCTAA 120 CAGAAAAACG ATGAGAAGAA ACTTTTGAAA AATATTGTGT GAAAGCAGCA CGAAACAGAG 180

TATGAAAAAA GAATGCGGGC GTCCGTAAAG AGCTAGAATC GCAAGTGTCC AGAATATGCA 240 AGGCTTTCGA ATACACTCCT CACGCTTCTC TTCAGCAAAA ATCAACTCTT TGTGATAAAA 300

CTGTGTATTT CTTTGTTCTT TGCCGTTGTT TACGTTAGTA AGAAATCGGC ATTGAAAAAA 360

AAAATCTCAC ACTAAAATTG CAGAAAAAAG TGTACAATAT CAGTAAATAA AATTGGCCAA 420

AACAATACCA TTAAAACCAG TC ATG TCC ATG CAA CAA GTC CAA CAT TGT GTC 472

Met Ser Met Gin Gin Val Gin His Cys Val 1 5 10 GCA GAA GTA CTT CGA CTA GAT CCA CAA GAA AAA CCG GAC TGG TCG AGC 520 Ala Glu Val Leu Arg Leu Asp Pro Gin Glu Lys Pro Asp Trp Ser Ser 15 20 25

GGA TAT TTG AAG AAG TTG ACT AAT GCG ACA TCG ATT TTA TAT AAT ACT 568 Gly Tyr Leu Lys Lys Leu Thr Asn Ala Thr Ser He Leu Tyr Asn Thr 30 35 40

TCA CTG AAC AAG GTA ATG CTG AAA CAA GAT GAA GAG GTT GCT AGA TGT 616 Ser Leu Asn Lys Val Met Leu Lys Gin Asp Glu Glu Val Ala Arg Cys 45 50 55

CAC ATA TGT GCA TAC ATA GCG TCA CAG AAA ATG AAT GAA AAA CAC ATG 664 His He Cys Ala Tyr He Ala Ser Gin Lys Met Asn Glu Lys His Met 60 65 70

CCT GAC CTT TGC TAT TAT ATA GAC AGT ATT CCC TTG GAG CCG AAA AAA 712

Pro Asp Leu Cys Tyr Tyr He Asp Ser He Pro Leu Glu Pro Lys Lys 75 80 85 90

GCC AAG CAT TTA ATG AAC CTT TTC AGA CAA AGT TTA TCT AAT TCT TCA 760 Ala Lys His Leu Met Asn Leu Phe Arg Gin Ser Leu Ser Asn Ser Ser 95 100 105 CCT ATG AAA CAA TTT GCT TGG ACA CCG AGC CCC AAA AAG AAC AAA CGC 808 Pro Met Lys Gin Phe Ala Trp Thr Pro Ser Pro Lys Lys Asn Lys Arg 110 115 120

AGT CCA GTA AAG AAC GGT GGG AGG TTT ACT TCT TCT GAT CCG AAA GAG 856 Ser Pro Val Lys Asn Gly Gly Arg Phe Thr Ser Ser Asp Pro Lys Glu 125 130 135

TTG AGG AAT CAA CTG TTT GGT ACA CCA ACT AAA GTT AGG AAA AGC CAA 904 Leu Arg Asn Gin Leu Phe Gly Thr Pro Thr Lys Val Arg Lys Ser Gin 140 145 150

AAT AAT GAT TCG TTC GTA ATA CCA GAA CTA CCC CCC ATG CAA ACC AAT 952 Asn Asn Asp Ser Phe Val He Pro Glu Leu Pro Pro Met Gin Thr Asn 155 160 165 170

GAA TCG CCG TCT ATT ACT AGG AGA AAG TTA GCA TTT GAA GAG GAT GAG 1000 Glu Ser Pro Ser He Thr Arg Arg Lys Leu Ala Phe Glu Glu Asp Glu 175 180 185 GAT GAG GAT GAA GAG GAA CCA GGA AAC GAC GGT TTG TCT TTA AAA AGC 1048 Asp Glu Asp Glu Glu Glu Pro Gly Asn Asp Gly Leu Ser Leu Lys Ser 190 195 200

CAT AGT AAT AAG AGC ATT ACT GGA ACC AGA AAT GTA GAT TCT GAT GAG 1096 His Ser Asn Lys Ser He Thr Gly Thr Arg Asn Val Asp Ser Asp Glu 205 210 215

TAT GAA AAC CAT GAA AGT GAC CCT ACA AGT GAG GAA GAG CCA TTA GGT 1144 Tyr Glu Asn His Glu Ser Asp Pro Thr Ser Glu Glu Glu Pro Leu Gly 220 225 230

GTG CAA GAA AGC AGA AGC GGG AGA ACG AAA CAA AAT AAG GCA GTT GGA 1192 Val Gin Glu Ser Arg Ser Gly Arg Thr Lys Gin Asn Lys Ala Val Gly

235 240 245 250

AAA CCG CAA TCA GAA TTG AAG ACG GCA AAA GCC CTG AGG AAA AGG GGC 1240

Lys Pro Gin Ser Glu Leu Lys Thr Ala Lys Ala Leu Arg Lys Arg Gly 255 260 265

AGA ATA CCA AAT TCT TTG TTA GTA AAG AAG TAT TGC AAA ATG ACT ACT 1288

Arg He Pro Asn Ser Leu Leu Val Lys Lys Tyr Cys Lys Met Thr Thr 270 275 280

GAA GAA ATA ATA CGG CTT TGC AAC GAT TTT GAA TTA CCA AGA GAA GTA 1336 Glu Glu He He Arg Leu Cys Asn Asp Phe Glu Leu Pro Arg Glu Val 285 290 295 GCA TAT AAA ATT GTG GAT GAG TAC AAC ATA AAC GCG TCA AGA TTG GTT 1384 Ala Tyr Lys He Val Asp Glu Tyr Asn He Asn Ala Ser Arg Leu Val 300 305 310

TGC CCA TGG CAA TTA GTG TGT GGG TTA GTA TTA AAT TGT ACA TTC ATT 1432 Cys Pro Trp Gin Leu Val Cys Gly Leu Val Leu Asn Cys Thr Phe He 315 320 325 330

GTA TTT AAT GAA AGA AGA CGC AAG GAT CCA AGA ATT GAC CAT TTT ATA 1480 Val Phe Asn Glu Arg Arg Arg Lys Asp Pro Arg He Asp His Phe He 335 340 345

GTC AGT AAG ATG TGC AGC TTG ATG TTG ACG TCA AAA GTG GAT GAT GTT 1528 Val Ser Lys Met Cys Ser Leu Met Leu Thr Ser Lys Val Asp Asp Val 350 355 360

ATT GAA TGT GTA AAA TTA GTG AAG GAA TTA ATT ATC GGT GAA AAA TGG 1576 He Glu Cys Val Lys Leu Val Lys Glu Leu He He Gly Glu Lys Trp 365 370 375 TTC AGA GAT TTG CAA ATT AGG TAT GAT GAT TTT GAT GGC ATC AGA TAC 1624 Phe Arg Asp Leu Gin He Arg Tyr Asp Asp Phe Asp Gly He Arg Tyr 380 385 390

GAT GAA ATT ATA TTT AGG AAA CTG GGA TCG ATG TTA CAA ACC ACC AAT 1672 Asp Glu He He Phe Arg Lys Leu Gly Ser Met Leu Gin Thr Thr Asn 395 400 405 410

ATT TTG GTC ACA GAC GAC CAG TAC AAT ATT TGG AAG AAA AGA ATT GAA 1720 He Leu Val Thr Asp Asp Gin Tyr Asn He Trp Lys Lys Arg He Glu 415 420 425

ATG GAT TTG GCA TTA ACA GAA CCT TTA TAACATATCC AGTATTAACT 1767

Met Asp Leu Ala Leu Thr Glu Pro Leu 430 435

AAAAGTATAT ATTTGACCAA TACCTGACAT ATCTTCTAAA GCATGCCTTT AGCCCTATAA 1827

CGAGCTAATG TTAGCTCCAT CTTTGCACTT ATGATTGGAT CAGCCCTCAA ACGCTTTTGT 1887 ATCTTTGCAG CTTCCGCGAA GGTAGTAGCT TGAAGTTTTT CATCCATAGT TCTTGCTAAA 1947

ATTGCAGAAT CTTCAAACAA TTCTATGG 1975

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 435 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE T^PE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Met Ser Met Gin Gin Val Gin His Cys Val Ala Glu Val Leu Arg Leu 1 5 10 15

Asp Pro Gin Glu Lys Pro Asp Trp Ser Ser Gly Tyr Leu Lys Lys Leu 20 25 30

Thr Asn Ala Thr Ser He Leu Tyr Asn Thr Ser Leu Asn Lys Val Met 35 40 45

Leu Lys Gin Asp Glu Glu Val Ala Arg Cys His He Cys Ala Tyr He

50 55 60 Ala Ser Gin Lys Met Asn Glu Lys His Met Pro Asp Leu Cys Tyr Tyr

65 70 75 80

He Asp Ser He Pro Leu Glu Pro Lys Lys Ala Lys His Leu Met Asn 85 90 95

Leu Phe Arg Gin Ser Leu Ser Asn Ser Ser Pro Met Lys Gin Phe Ala 100 105 110

Trp Thr Pro Ser Pro Lys Lys Asn Lys Arg Ser Pro Val Lys Asn Gly 115 120 125

Gly Arg Phe Thr Ser Ser Asp Pro Lys Glu Leu Arg Asn Gin Leu Phe

130 135 140

Gly Thr Pro Thr Lys Val Arg Lys Ser Gin Asn Asn Asp Ser Phe Val

145 150 • 155 160

He Pro Glu Leu Pro Pro Met Gin Thr Asn Glu Ser Pro Ser He Thr 165 170 175

Arg Arg Lys Leu Ala Phe Glu Glu Asp Glu Asp Glu Asp Glu Glu Glu 180 185 190 Pro Gly Asn Asp Gly Leu Ser Leu Lys Ser His Ser Asn Lys Ser He 195 200 205

Thr Gly Thr Arg Asn Val Asp Ser Asp Glu Tyr Glu Asn His Glu Ser 210 215 220

Asp Pro Thr Ser Glu Glu Glu Pro Leu Gly Val Gin Glu Ser Arg Ser 225 230 235 240

Gly Arg Thr Lys Gin Asn Lys Ala Val Gly Lys Pro Gin Ser Glu Leu 245 250 255

Lys Thr Ala Lys Ala Leu Arg Lys Arg Gly Arg He Pro Asn Ser Leu 260 265 270 Leu Val Lys Lys Tyr Cys Lys Met Thr Thr Glu Glu He He Arg Leu 275 280 285

Cys Asn Asp Phe Glu Leu Pro Arg Glu Val Ala Tyr Lys He Val Asp 290 295 300

Glu Tyr Asn He Asn Ala Ser Arg Leu Val Cys Pro Trp Gin Leu Val 305 310 315 320 Cys Gly Leu Val Leu Asn Cys Thr Phe He Val Phe Asn Glu Arg Arg 325 330 335

Arg Lys Asp Pro Arg He Asp His Phe He Val Ser Lys Met Cys Ser 340 345 350

Leu Met Leu Thr Ser Lys Val Asp Asp Val He Glu Cys Val Lys Leu 355 360 365 Val Lys Glu Leu He He Gly Glu Lys Trp Phe Arg Asp Leu Gin He 370 375 380

Arg Tyr Asp Asp Phe Asp Gly He Arg Tyr Asp Glu He He Phe Arg 385 390 395 400

Lys Leu Gly Ser Met Leu Gin Thr Thr Asn He Leu Val Thr Asp Asp 405 410 415

Gin Tyr Asn He Trp Lys Lys Arg He Glu Met Asp Leu Ala Leu Thr 420 425 430

Glu Pro Leu 435

Claims

WHAT IS CLAIMED IS:

1. A composition comprising an isolated nucleic acid encoding a biologically active unique portion of an ORC polypeptide.

2. A composition according to claim 1, wherein said ORC gene is ORCI.

3. A composition according to claim 1, wherein said ORC gene is ORC2.

4. A composition according to claim 1, wherein said ORC gene is ORC3.

5. A composition according to claim 1, wherein said ORC gene is

ORC4.

6. A composition according to claim 1 , wherein said ORC gene is ORC5. «

7. A composition according to claim 1 , wherein said ORC gene is - ORC6.

8. A composition comprising a recombinant, biologically active unique portion of an ORC protein.

9. A method of identifying an ORC selective agent, said method comprising the steps of: contacting an agent with a composition according to claim 8; measuring in at least qualitative terms the binding affinity of said agent for said composition.

10. A method for identifying a gene encoding a protein which directly or indirectly associates with a selected DNA sequence, said method comprising the steps of: transforming an expression library of hybrid proteins into a reporter strain, wherein said library comprises protein-coding sequences fused to a constitutively expressed transcription activation domain and said reporter strain comprises a reporter gene with at least one copy of a selected DNA sequence in its promoter region; detecting the transcription or translation product of said reporter gene in a clone of said reporter strain; recovering said clone; whereby said clone comprises a gene encoding a protein which directly or indirectly associates with said selected DNA sequence.