CA2178965A1 - Origin of replication complex genes, proteins and methods - Google Patents

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

WO g5/16694 PCT/US94/14563

2 1 78965 ORIGIN OF REPLICATION COMPLEX GENES, PROIEINS AND METHODS

INTRODUCTION
The research carried out in the subject application was ~uppolLed in part by grants from the National Institutes of Health. The government may have rights inany 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.

10 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
15 replication (reviewed in 11). Sequence co..-pafison of a number of ARS elements resulted in the identification of the ARS consellc~c 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 ARSI function have been identified. When mutated individually, these el~rnentC, referred to as Bl, B2, and 20 B3, result in a slight reduction of ARS1 activity. When two or three of the B elements are simultaneously mutated, however, ARS1 function is severely co~ ol.,ised (14) .

wo 95/16694 2 1 7 8 9 6 5 PcrlUS94/14s63 Proteins that recognize two elements of ARS1 have been identified. The yeast transcription factor ABFl binds to and me~i~tes the function of the B3 element (11, 14). More recently we have identified a multi-protein complex that spe~ ific~lly recognizes the highly conserved ACS (15). This activity, referred to as the origin recognition complex (ORC), has several propellies that make it an attractive candidate to act as an initiator protein at yeast origins of replication.
Binding of this protein ~ uires the ACS, and the effect of mutations in the con~n~-ls 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 plupelly 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 mYli~t~s the function of the ACS is provided by in situ deoxyribonuclease I (DNase I) footprinting experiments that identify a protectedregion of ARSl relll~l~bly 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 co"~posilions are provided.
Provided are compositions comprising isolated nucleic acids encoding unique ORC gene portions, especially portions encoding biologically active unique portions of ORCl-ORC6 proteins. Vectors and cells comprising such DNA
molecules find use in the production of recombinant ORC peptides.

wo95/16694 ~ ~ 2 1 78 ~65 p~US94/14563 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.
S 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 ~csoCi~t~s with a selected DNA sequence. Generally, the methods involve tran~ro.",ing an eA~lession library of hybrid proteins into a 10 re~,ler strain, wherein the library comprises protein-coding sequences fused to a constitutively eApl~ssed transcription activation domain and the reporter straincomprises a reporter gene with at least one copy of a selected DNA sequence in its promoter region. Clones e~-p-cssillg the transcription or translation product of the c;po-lel gene are detected and recovered. A p-~ d method employs an 15 activation domain from GAL4 and a lacZ reporter gene.

BREIF DESCRIPTION OF SEQUENCE ID NUMBERS
SEQUENCE ID NO: 1. DNA Sequence of ORCl .
SEQUENCE ID NO:2. Amino Acid Sequence of ORCl.
20 SEQUENCE ID NO:3. DNA Sequence of ORC2.
SEQUENCE ID NO:4. Amino Acid Sequence of ORC2.
SEQUENCE ID NO:S. DNA Sequence of ORC3.
SEQUENCE ID NO:6. Amino Acid Sequence of ORC3.
SEQUENCE ID NO:7. DNA Sequence of ORC4.
25 SEQUENCE ID NO:8. Amino Acid Sequence of ORC4.
SEQUENCE ID NO:9. DNA Sequence of ORCS.
SEQUENCE ID NO:10. Amino Acid Sequence of ORCS.
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 ~lis~lose~ ORC proteins which retain an binding affinity specific to the subject W0 95/16694 2 ~ ~ ~ 9 6 5 PCT/US94114563 full-length ORC protein. A "unique portion" has an amino acid sequence unique tcsubject 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 5 length, depending on the particular amino acid sequence and are readily idçntified by co",p~ing 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, 10 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 as15 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, seiective binding requires a binding 20 affinity of 10~M, preferably 10-8M, more preferably 10-'M, 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 25 larger polypeptide need not be ORC-derived. The subject polypeptides are typically "isolated", m~ning unaccompanied by at least some of the m~teri~l withwhich they are ~soci~t~ 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 30 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 WO95/16694 2 1 7 8 ~ 6 5 Pcrluss4ll4563 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 colllponents are present 5 in the sample and to what, if anything, the polypeptide is covalently linked.
Purification methods include electrophoretic, molecular, immunological and - chromatog,dphic techniques, especi~lly affinity chr~--latog,dphy 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 prope,lies such as solubility, membrane transportability, stability, toxicity, bioavailability, loc~li7~tion, detect~bility, in vivo half-life, etc.
15 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 inteNention (e.g. fatty acid-acylation, proteolysis, glycosylation) and especially where the poly/peptide is integrated into a largerpolypeptide, se~ection of a particular eAl)~ession host, etc. Amino and/or carboxyl 20 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 25 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 modul~ing O-linked glycosylation. Glycosylation variants are also produced by selecting approp,iate host cells, e.g. yeast, insect, or various m~mm~ n ~lls, or by in vitro methods such as neuraminidase digestion.
30 Other covalent modifications of the disclosed poly/peptides may be introduced by reacting the targeted amino acid residues with an organic derivatizing (e.g. methyl-

3-[(p azido-phenyl)dithio] propioimidate) or crosclinking agent (e.g. 1,1-bis(di~70~retyl)-2-phenylethane) capable of reacting with selected side chains or W095116694 . ~ 1 78965 PCT/US94/14563 termini. For theld~ulic and diagnostic loc~li7~tion, the subject poly/peptides thereof may be labeled directly (radioisotopes, fluorescers, etc.) or indirectly with an agent capable of providing a dete~t~hle signal, for example, a heart muscle kinase labeling site.
ORC poypeptides with ORC binding specificity are identified by a variety of ways including crosclinking, or prefeMbly, by screening such polypeptides forbinding 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 preferably, small molecular weight organic compounds. For example, the disclosed ORC peptides are used as immunogens to generate specific polyclonal ormonoclonal antibodies. See, Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, for geneMl methods.
Other pros~ecli~e ORC specific agents are screened from large libMries of synthetic or natural co.,-pounds. 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 co"lpounds are readily modified through conventional chemical, physical, and biochPmi~l means. See, e.g. Houghten et al. and Lam et al (1991) Nature 354, 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, espe~i~lly assays for specific binding to immobilized col--pounds 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 ~lic~csoci~tion of a labelled component of the complex. Such assays are amenable to scale-up, high throughput usage suitable for volume drug screening. While less plefelled, cell-based assays may be used to determine specific effects of prospective agents.
~efelled agents are ORC- and species-specific. Useful agents may be found within numerous chemical classes, though typically they are organic co",pounds; prefeMbly small organic compounds. Small organic compounds have WOgS/16694 ~; ~ ~ 2 1 /8965 PCT/USs4/14563 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 includesteroids, heterocyclics, polycyclics, substituted aromatic compounds, and the like.
Sele~t~d agents may be modified to enhance efficacy, stability, S pharm~ceutical colllpalibility, 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 enh~nce their proteolytic stability. Other methods of stabilization may include encapsulation, for example, in liposo-lles, etc. The 10 subject binding agents are ~repar~d in any convenient way known to those in the art.
For the~ eulic uses, the compositions and agents disclosed herein may be ~rlministçred by any convenient way. Small organics are preferably ~iminictered orally; other compositions and agents are preferably administered parenterally, 15 conveniently in a pharmaceutically or physiologically acceptable carrier, e.g., phosphate buffered saline, or the like. Typically, the compositions are added to a retained phyciological.fluid. As examples, many of the disclosed thel~peulics are ~m~n~hle to direct injection or infusion, topical, intratracheal/nasal ~rlminictration e.g. through aerosal, intraocularly, or within/on implants e.g. collagen, osmotic 20 pumps, grafts comprising applupliately transformed cells, etc. Generally, theamount ~dminict~red will be empirically determined, typically in the range of about 10 to 1000 ~lg/kg of the recipient. For peptide agents, the concentration will generally be in the range of about 50 to 500 ~gtml in the dose adminict~red.
Other additives may be included, such as stabilizers, bactericides, etc. These 25 additives will be present in conventional amounts.
The invention provides icol~ted 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 30 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 simil~rity hybridize under low stringency conditions, for example, at 50C and SSC (0.9 M saline/0.09 M sodium citrate) and remain bound when

4 2 1 7 8 9 6 5 PCT/US94/14563 subject to washing at 55C with SSC, wherein regions of non-identity of subst~nti~lly similar nucleic acid sequences preferably encode redundant codons; a partially pure nucleotide sequence con~tituteC at least about 5%, preferably at least about 30%, and more preferably at least about 90% by weight of total nucleic acid

5 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 of15 the coding sequence are spliced with heterologous sequences to produce soluble, secreted fusion proteins, using appropliate signal sequences and optionally, a fusion partner such as ~-Gal. For antisense applications where the inhibition ofeAples~ion is indicated, especially useful oligonucleotides are between about 10 and 30 nucleotides in length and include sequences surrounding the disclosed ATG start 20 site, espe~i~lly the oligonucleotides defined by the disclosed sequence beginnin~
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, ~ministration or other use by methods disclosed in standard manuals such as 25 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 hnown 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 been30 described for eApr~ssion in a variety of euharyotic 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 t;~ression, one or more l--alhel~ for selection in the host, e.g. antibiotic wo 95/16694 2 1 7 8 9 6 5 Pcr,Us94/l4563 recict~nce. The inserted coding sequences may be synthPsi7PA, isolated from natural sources, p~ aled as hybrids, etc. Suitable host cells may be transformed/transfected/infected by any suitable method including electroporation, CaCl2 mPAi~tPA DNA uptake, viral infection, microinjection, microprojectile, or 5 other methods.
Appropriate host cells include bacteria, archeb~teria, fungi, espe~i~lly yeast, and plant and animal cells, espe~i~lly m~mm~ n cells. Of particular interest are E. coli, B. subtilis, Saccharomyces cerevisiae, SF9 cells, C129 cells, 293 cells, Neurospora, and CHO, COS, HeLa cells, immortalized m~mm~ n 10 myeloid and lymphoid cell lines, and pluripotent cells, esperi~lly m~mm~ n EScells and zygotes. Preferred eA~l~ession systems include COS-7, 293, BHK, CHO, TM4, CVl, VERO-76, HELA, MDCK, BRL 3A, W138, Hep G2, MMT 060562, TRI cells, and baculovirus systems. Preferred replication systems include M13, ColEl, SV40, baculovirus, lambda, adenovirus, AAV, BPV, etc. A large number 15 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 of manipulation, etc. are known in the art.
For the production of stably transformed cells and transgenic ~nim~l.c, the - 20 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 25 recombinations, deletion of endogenous gene by homologous recombination, especi~lly in plu~i~lent cells, etc., provide additional applications. Preferredtransgenics and stable transrol,--ants over-express or under-express (e.g. knock-out cells and ~nim~l.c) a disclosed ORC gene and find use in drug development and asa disease model. Methods for making transgenic ~nim~lc, usually rodents, from 30 ES cells or zygotes are known to those skilled in the art.
The compositions and methods disclosed herein may be used to effect ~erle therapy. See, e.g. Zhu et al. (1993) Science 261, 209-211; Gutierrez et al. (.992) Lancet 339, 715-721. For example, cells are transfected with ORC-encoding WO95tl6694 2 1 7 8 ~ 6 5 PCT/USg4/l4s63 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, a~lmini~tration 5 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 ~mini~ttored compositions, e.g. transfected nucleic acid, protein, etc., will depend on the 10 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 ORCl gene in both S. cerevisie and k lactis contain lS conserved primary protein sequence that are utliized 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 ORCl protein in any eukaryotic species. The cloned gene - encoding ORCI polypeptide from any fungi or from human cells is used to express 20 the protein in a bacterial eAI)ression system to make antibodies against the polypeptide. These antibodies are used to immunoprecipi~te 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 25 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 eApres~ing each of the S. cerevisiae genes in a baculovirus e~pr~ssion 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 30 activity. In this way, large amounts of ORC protein from any fungi or m~mm~ n 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

- -wo 95tl6694 ~ ~ 2 1 7 8 9 6 5 Pcrluss4ll4563 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.
S In human proliferative disorders such as cancer, cells of the dice~ced 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 e,~p,essing each of the cDNAS encoding the ORC proteins, either individually or together in an c;Apre~ion 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, smallmolecular 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
Fy~rnple 1.
Transcriptional silencing and ORC.
The binding of purified ORC to the ARS consenc~ls sequence (ACS) at each of the mating type silencers was tested using a DNase I protection assay (22).
ORC p,ulecled 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 conc~ncuc sequence at approximately 10 bp intervals.
This pattern of DNase I l,iol~;Lion and enhanced cleavage is nearly identical to that observed at non-silencer sequences and indicates that ORC binding to these elements is not fund~mentally 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 boundby ORC, this p~oleclion showed little dependence upon the addition of ATP to the WOgS/16694 2 1 7896~ PcT/uS94/14563 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 proteclion observed 5 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 andHMR.
A clear link between ORC function and transcriptional ~ilen~in~ was provided by the finding that a mutation in a gene encoding a subunit of ORC was defective for lepression at HMR (below). To clone the genes encoding the variousORC subunits, peptides derived from each of the ORC subunits were sequenced (24). A candidate gene, referred to as ORC2, was isolated by complementation of 15 a ~"")el~ture sensitive mutation that showed silencing defects at the permissive le-"~ lu~e. Genetic experiments suggested that ORC2 me li~ted 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 20 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 p~epaled from both orc2-1 and ORC2 strains (25). Fractions derived from 25 wild-type cells showed strong ORC DNAse I protection over the ACS and B1 elementc of ARSl in DNAse I footprinting. In contrast, fractions derived from orc2-1 cells showed a dramatic reduction in ORC DNA binding activity. The ACS
and the B1 element were no longer protected from DNase I cleavage. Only the char~t~ri~tic enhanced DNase I cleavages in the B domain of ARS1 rem~in~d.
30 Mutations that disrupt ORC DNA binding at ARS1 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 wo gsl16694 2 1 7 8 9 6 5 P~ ,5 ~rl4s63 wild type protein. Incubation of the mutant cells at the non-permissive te"~peldlure was not necesc~ry to observe defects in ORC DNA binding, which explains the defect observed in mating-type regulation at the permissive te",pt;ldture (below).
To investig~te the polypeptide composition of ORC derived from orc2-1 5 and ORC2 cells, immuno-blots of these fractions were probed with polyclonal antibodies raised against ORC. 30 ~g of partially purified ORC derived from either JRY3688 (ORC2) or JRY3687 (orc2-1) was separated on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. The resllltin~ protein blot was incub~ted with polyclonal mouse sera raised against the entire ORC complex. This10 sera detects all but the 50 kd subunit of ORC. Antibody-antigen complexes were detected with horseradish peroxidase conjugated secondary antibodies followed byincubation 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 15 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 20 these ~ubunils in ORC DNA binding (15). Thus, the absence of these subunits explains the defects in DNA binding observed in vitro and indicates that the orc2-1 mutation results in a reduction of ORC stability or a defect in Orc2p also results in reduced DNA binding of an intact ORC complex.
orc2-1 cells are defective for entry into S-phase.
The point in the cell cycle the essential function of ORC2 is pelrolllled in vivo was investig~t~d using alpha factor and hydroxyurea (HU) as cell cycle l~n-lm~rkc (26). Our results were consistent with the execution of the essentialfunction of Orc2p between late Gl and the initiation of DNA synthesis. Arrest with HU followed by release into the non-permissive lelllpeldlul~ resulted in 89%
of the cells completing an addition~.' cell cycle, indicating that the essential function for Orc2p was executed before the J arrest point in the cell cycle. In contrast,blocking the cell cycle with alpha-factor followed by release at the non-permissive le",peldture resulted in the only 41 % of the cells completing an additional cell WO 95/16694 2 1 7 8 '~ 6 5 PCT/US94/14563 cycle. This phenotype indicates that the Orc2p function was ~l~ll,led at or nearthe Gl-S phase boundary.
To address the role of ORC in yeast DNA replication more directly, the DNA content of asynchronous cultures of either orc2-1 or isogenic wild type cells 5 was measured at various times after shifting from the permissive to the non-permissive tellll)e,dture by fluorescent cytometric analysis (27). JRY3687 (orc2-1) or JRY3688 (ORC2) cells grown at 24C (0 minute time point) or at various times after shifting to the non-permissive tel-.pel~ re (37C) were fixed, stained with propidium iodide, and analyzed for DNA content using a Coulter Model Epics-C
10 Flow Cytometer. In addition, a small number of cells (app~o~imately 1000) from each time point were returned to the permissive le-..peldture to determine the pe~ent~ge of cells that rem~inecl viable at a given time point. Initially, the DNA
content of both wild type and mutant cells was equally divided between lC and 2Cwith approximately 10% of the cells in S phase. At early time points after the 15 telllpeldlure shift (15-70 minutes) there was a dramatic loss of orc2-1 cells in S-phase suggesting that entry into S-phase had been halted. Consistent with this hypothesis, as the time course continued the orc2-1 mutant showed a rapid accumulation of cells with a lC DNA content and a commencurate decrease in cells with a 2C DNA content (50-100 minutes). Between l00 and 120 minutes, a 20 new population of orc2-1 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 Qnly a few cells rem~ine~ with a lC DNA content.
Interestingly, we observed a strong correlation between entry into the new round of DNA synthesis and a loss of orc2-1 cell viability. Similar experiments 25 with isogenic ORC2 cells showed that these effects were specific to the orc2-1 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 orc2-1 mutation provides in vivo evidence 30 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.

wo 95/16694 2 1 7 8 9 6 5 PCTtUS94/14563 A second gene that represen~ed a strong candidate to encode one of the subunits of ORC was the AAPI gene. This gene was cloned using a novel screen for proteins that bound to the ACS in vivo (below). When col"pa~t;d to the predicted amino acid sequence of this gene, we found that all of the peptides 5 derived from the 50 kd subunit of ORC were encoded by the open reading frame of the AAPI gene (28). For this reason we now refer to AAPI as ORC6, as it encodes the sm~ st of the six ORC subunits. The identification of this gene as asubunit 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. F~gm~n 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 Cel1ul(7r Biology - of the Yeast Saccharomyces J. R. Broach,~J. R. Pringle, E. W. Jones, Eds. (Cold 20 Spring Harbor Labol~toly Press, Gold Spring Harbor, NY, 1991) pp. 41-146.

10. J. Broach, et al., CSH Symp. Quant. Biol. 47, 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 pelro""ed 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 pllnfic~tionprocedure for ORC was devised. Whole cell extract was p~epal~d from 400g of wo gS/16694 2 1 7 8 q 6 5 PCT/US94/14563 frozen BJ926 cells using a bead beater (Biospec Products) until greater than 90%breakage was achieved. One twelfth volume of a saturated (at 4C) 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 5 was spun in a 45Ti rotor (Reckm~n) 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 res~-lting pellet was resuspended in buffer H/0.0 (15) and dialyzedversus H/0.lSM KCI (H with 0.15 M KCl added). Preparation of ORC from this 10 extract was similar to (15) with the following changes. The dsDNA cellulose column was omitted from the prepaMtion and only a single glycerol gradient was pelrol---ed. Sequencing of peptides derived from ORC subunits was performed using a modification of an "in gel" protocol described previously (40, 41).
Purified ORC (~ 10 ~g per subunit) was separated by SDS-PAGE and stained with 15 0.1 % Coomassie Brilliant Blue G (Aldrich). After dest~ining 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 Achromobacter protease I
(Lysylendopeptidase: Wako). The resulting peptides were separated by reverse-phase chromatography and sequenced by automated Edman degradation (Applied 20 Biosystems model 470).

22. To isolate and assay ORC from ORC2 and orc2-1 cells four liters of JRY3687 (orc2-1, MATa, hmrDA::TRPI ade2 his3 leu2 trpl ura3) or the isogenic wild-type strain JRY3688 (ORC2 MATa, hmrDA::TRPI ade2 his3 leu2 trpl ura3) were grown to a density of 2 x 107 cells per ml. Extracts were prepared as 25 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.
30 Proteins were transferred to nitrocellulose and antigen-antibody complexes were detected with horse radish peroxidase conjugated secondary anitbodies and a chemiluminescent substrate.

wo 95/16694 2 1 7 8 9 6 5 PCT/US94114563

23. Yeast cells were grown to a density of 1-4 x 107 cells per ml at 24C then diluted to a density of 2-4 x 106 cells per ml into YPD containing 6 ~M alpha-factor and incub~ted for 2-2.5 hours at 24C (> 90% unbudded cells). For the hydroxyurea arrest experiments alpha factor was washed away and the cells were S res~-~pen-iecl in YPD conl;~ining 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 24C or 37C 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 24C and 10 diluted into fresh YPD at either 37C or 24C and a density of 2-4 x 106 cells per ml. At times after dilution, 3 x 106cells 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. an~ 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. Villarreal, 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 wo 95/16694 2 1 ~ 8 9 6 5 p~US94114563 In a mutant screen, a ~I~peldture-sensitive mutation called orc2-1 was isolated that, at the permissive temperature, resulted in derepression of HMRa flanked by the synthetic ~il.oncer and did not cause dercples~ion of HMRa flanked by the wild-type silencer (20). Rer~usP the orc2-1 mutant was temperature-S sensitive and silencing defective, it mPrit~d further analysis. The telll~ldturere~ict~nce of a heterozygus orc2-1/ORC2 diploid (JRY2640) established that themutation was recessive. The diploid was transformed with a plasmid cont~ining HMRa flanked by a mutant silencer (pJR1212), to provide MATal function required for sporulation. The telll~ldture-sensitive growth phenotype segregated 2 10 ts: 2 wild type in each of 23 tetrads, indicating that it was caused by a single nuclear mutation. An HMLo~ matal HMRo~ orc2-1 segregant aRY3683) 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 15 orc2-1. A matal HMRo~ strain (JRY3683) containing the orc2-1 mutation was mated to a MATo~ strain cont~ining a mutation in the RAPl binding site of HMR-E
fl~nking HMRa (the HMRa-e-rapl-10 allele; 5401-la) to determine whether orc2-1 could dclcl~lcss HMRa in the absence of a functional RAPl binding site. All 29 of the 96 MATo~ segregallts that had little or no mating ability were ~elllpeldture-20 sensitive for growth. Nineteen of the MATo~ lel"~ldture-sensitive seg~cgant~ were mating colllpetent, indicating that the orc2-1 mutation per se was in~ufficient to disrupt mating ability, and suggesting that the HMRa-e-rapl-10 allele was required in combination with orc2-1 to block mating ability of ~ strains. A MATo~
telllpeld~ure-sensitive segregant from this cross, which mated weakly as an cY
25 (JRY4133), was confirmed to have the genotype MATo~ HMRa-e-rapl-10 orc2-1.
As further evidence that orc2-1 in combination with HMRa-e-rapl-10 blocked the mating ability of MATa strains, a somewhat unusual cross was used tosimplify the previous cross by having orc2-1 as the only relevant heterozygous marker. Two MATo~ HMRa-e-rapl-10 strains (JRY4133 and JRY4132) had 30 complementary auxotrophic markers, allowing for the selection of the rare MAT~IMATo~ diploid formed by a mating event between these two strains. This diploid was able to sporulate due to the low level of e~l)lession of HMRa in thediploid caused by the RAPl-site mutation in the HMR-E silencer (21). One of wo 95/16694 2 1 7 8 9 6 5 PCT~ s94~l4s63 these strains had the orc2-1 mutation (JRY4133) and the other did not. As expected, the le~ dture sensitivity segregated 2:2 in each of 34 tetrads. All ofthe ~ll~ tule-resistant segle~ts (two per tetrad) exhibited the ~ mating phenotype, and all of the t~ ture-sensitive segregants were either very weak 5 ~-maters or were unable to mate at all. The absence of any recombinants between the l~"~ldture sensitivity and mating phenotype placed the gene(s) responsible for the lelll~ldlu~ 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 10 mating phenotypes described herein.
Isolation of multiple alleles of ORC2 Using the information from this analysis of orc2-1, a second screen was o-llled to identify additional mutations in essential genes with a role in silencer function. This second screen produced 50 mutants that were temperature sensitive15 for growth, and in which HMRo~ (flanked by a mutation in the RAP1-binding site) was derepressed at a semi-permissive temperature. Complementation tests for bothgrowth at 37C and for mating phenotype were performed between orc2-1 and the collection of temperature-sensitive mutants from the second screen. The collection of te"-~lature sensitive mutants had the matal stel4 genotype, but were able to 20 mate as ~'s due to the derepl~;ssion of HMRo~. These mutants were mated to a matal orc2-1 strain (JRY3683) and the diploids were tested for growth at 37C.
All but three diploids were able to grow at the restrictive te...~ldture. 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 che~k~l to determine whether the defect in repression of HMR was complemented. All three diploids mated as ~'s. Thus, the three mutants were unable to complement either the ten.~ldture sensitivity or the mating phenotype of the original orc2-1 mutation. The new mutations (in strains JRY4136, 4137 and 4138) were decign~t~d orc2-2, orc2-3, and orc2-4.
To investigate the possibility that the new mutations were in a gene other than ORC2 ~e still failed to complement orc2-1, the allelism between orc2-1 and orc2-3 was tested. The original matal orc2-3 stel4 mutant was cured of its HMRo~ plasmid, creating JRY 4137, and mated with a MATo~ HMRa-e-rapl-10 wo 95/16694 - ~ l 7 ~ q 6 5 Pcr~US94/14563 orc2-1 strain aRY3685). In 24 tetrads from this diploid, all segr~ants were le",peldture sensitive for growth, intli~.~ting strong linkage between orc2-1 and orc2-3 (~2 centimorgans). All further studies were pelrol,--ed using the orc2-1 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 Iys2 strains (JRY2640 and PSY152) and the original orc2-1 isolate aRY2903) that placed ORC2 approximately 24 centimorgans from LYS2.
A third cross (JRY4130 x JRY4134) tested the linkage between secl8, which is centromere proximal to LYS2, and ORC2. Re~ se both orc2-1 and secl8 are ~e..,peldture 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 distadnce Cross PD T NPD (cM
ORC2 vs LYS2 10 14 0 29 ORC2 vsLYS2 20 14 0 21 ORC2 vs LYS2 TOTAL 30 28 0 24 ORC2 vs SEC18 46 7 0 6.6 The ORC2 mutants arrested with a cell cycle terminal phenotype.
The effect of the orc2-1 mutation on the cell division cycle was explored:
25 mutant orc2-1 strains were grown in liquid medium at 23C, the permissive t~lllpelature~ and then shifted to 37C to test whether the cells arrested with a single lel",inal morphology. Specifically, orc2-1 cells (JRY3683) were grown to log phase at the permissive lelllpela~re (23C) and the culture was split. Half of the culture was grown an additional five hours at the permissive l~--l~;ldture and 30 the other half was shifted to the nonpell,lissive telllpeldture (37C) and grown for an additional five hours. At that time, both cultures were fixed and stained with DAPI to allow visll~li7~tion of the nucleus. In the culture m~int~ined at the permissive lelllpeldture, cells at all phases of the cell cycle were observed. Cells wo 95/16694 2 ~ 7 ~ 9 6 5 PCT/US94/14563 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 ~e-,-peldture, approximately 90% of the cellsarrested as large budded cells. Nuclei were only present in the mother cell and not - 5 in the daughter cells. In addition, the cells were larger than those grown at the permissive le"~peldture, indicating that protein synthesis and cell wall synthesis continued in the absence of ORC2 function. Similar results were obtained with two additional orc2-1 strains aRY3685 and JRY36~7).
ORC2 cells harvested either after continuous growth at the permissive l~;-.-~:ldtule or after a shift to the nonpelmissive len.pe~dlule were fixed andstained with DAPI allowing vi~ li7~tion 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 nonpel--,issive ~e"~peldture looked very different: the majority arrested 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 in-iic~tP~ that orc2-1 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 orc2-1 temperature sensitivity (22). One complementing clone (pJR1416) was chosen for further analysis. Subclones mi~cing various fragments from the insert were retransformedinto an orc2 strain to assay whether the deletion affected the clone's ability to complement orc2-l 's te"~ ture sensitivity. The key observations were that the deletion of a 2.8-kb SstI-SstI fragment destroyed complementation activity, whereas the deletions of fl~nking sequences (XbaI, and the largerSstI fragment) had no effect. The 2.8-kb fragment was subcloned (pJR1263), and shown to possess complemP-nting 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 inlegl~lion and transformed into a wild-type strain (W303-lA). As a result, the wo 95/16694 ~ 2 1 7 8 9 6 5 P,~lus94ll4s63 site of integration was marked by the plasmid's URA3 gene. The resulting strain (JRY4134) was crossed to an orc2-1 strain (JRY3685). In each of 59 tetrads, URA3 seg~cgated opposile to the te,."~ldt~lre sensitivity caused by orc2-1, 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 ~i~cect~, 40 tetrads had two live and two dead segr~ants, and one tetrad had only one live segregant. The colonies that grew were, without exception, Ura-. By inference, 10 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 se~l~ants were .o~mined 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 15 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-lB) and one orc2-1 (JRY4125), were transformed with a 2.0 plasmid cont~ining a centromere, a supp~essor tRNA (SUPII-I), URA3, and ARS1, a chromosomal origin of replication (YRP14/CEN4/ARS1/ARS1; (24, 25), s~l~ting for uracil pr~totlophy. Transrol-,-ants were grown on selective medium at 23C, the permissive te~peldture for orc2-1. The colonies were picked from the selective plate, serially diluted, plated onto solid rich medium and grown to 25 colonies at 23C. The wild-type tran~ro~l"ants grew into colonies most of which were white with a few exhibiting red sectors. The small fraction of red colonieswere from cells in the selectively grown colony that had lost the plasmid. In contrast, the majority of colonies from the orc2-1 mutant were red, reflecting ahigh degree of plasmid loss among the cells in the selectively grown colony.
30 Moreover, in the orc2-1 strain, red sectors were present in the majority of white colonies with some white colonies displaying multiple red sectors.
It is possible to quanlilate 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 colonieswere 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 orc2-1 - S strain grown at the permissive le~,dture was 11% (58 / 512), in~ic~ting that the same plasmid was lost appro~cimately 12 times as often in the strain with partially defective Orc2p. These data indicated a profound defect in plasmid stability specific to the orc2-1 strain, and in combination with the cell-cycle phenotype of orc2-1, suggested that orc2-1 strains were defective in DNA replication. These results were consistent with the flow cytometry studies of orc2-1 strains herein.
Sequence of ORC2 The sequence of the 2.8-kb SstI-SstI orc2-complementing fragment was determined and deposited in Gçnb~nk (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 SstI 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 - 20 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 25 the nuclear ~r~,~ling sequence (residues 103-107). A perfect match to the RAP1 binding site concenC~s (starting at nucleotide 595), and two near ."~t~hPs (12/15) to the ABF1-binding concensus sequence (starting at 12 and 609). It was determined by sequence homology that a lysyl tRNA synthet~ce gene is located to the left ofthe Sstl fragment shown here (MiMnde and Waller, 1988), and a kinase homolog 30 to the right.
Another homolgy is with the region near the catalytic domain of human topoisomeMse I proteins which has diverged among topoisomeMse I proteins from other species except for the region surrounding the invariant active-site tyrosine.

WO 95/16694 2 1 1 8 9 6 5 PcTlus94ll4563 This region includes a conC~-nCl~c sequence consicting of a serine and lysine residue near the tyrosine (25). The Orc2p protein also contained such a con~Pn~us 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 5 complement the te~lpe,dture-sensitivity or mating defect of an orc2-1 strain.
Table 2. Strain list.
Strain Genotype ( ~
DBY1034 MATa his4-539 Iys2-801 ura3-52 SUC
W303-lA MATa ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-l ura3-1 W303-lB MATa ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-l ura3-1 PSY152 MATa his3D200 leu2-3,112 Iys2-801 ura3-52 JRY4130 MATa his4 ura3 secl8 JRY438 MATa Gal+ his4519 leu2-3,112 SVC2 ura3-52 JRY543 MATalMATa ade2-101/ade2-101 his3~200/his3~200 Iys2-801/lys2-801 met2/MET2 TYRI/tyrl ura3-52/ura3-52 - JRY2640 matal ade2 leu2-3,112 Iys2-801 ura3 JRY2698 MATa HMRa ade2-101 his3 leu2 trpl ura3-52 JRY2699 MATa HMRa ade2-101 his3 leu2 trpl ura3-52 sir4DN::H153 JRY2700 MATa HMRa ade2-101 his3 leu2 trpl ura3-52 + pJR924 JRY2903 MATa HMRa ade2-101 his3 leu2 orc2-1 trpl ura3-52 JRY2904 MATa HMRa ade2-101 his3 leu2 orc2-1 trpl ura3-52 + pJR924 JRY3444 MATalMATa ade2-101/ade2-101 his3D200mis3D200 Iys2-801/lys2-801met2/MET2 TYRI/tyrl ura3-52/ura3-52 orc2::Tnl OLUK/ORC2 JRY3683 matal {HMRa} ade2 his3 leu2 orc2-lura3 JRY3685 MATa HMRa-e-rapl -10 ade2 leu2 trpl orc2-1 ura3 JRY3687 MATa hmrDA::TRPI ade2 his3 leu2 trpl ura3 orc2-1 wo 9S/16694 2 ~ 7 8 9 6 5 Pcrluss4ll4563 JRY3690 MATa HMRa-e-rapl-10 ade2 his3-11,15 leu2 orc2-1 trpl ura3 JRY4125 MATa ade2-1 canl-100 his3-11,15 leu2-3,112 orc2-1 trpl-1 ura3-1 JRY4132 MATol HMRa-e-rapl-10 ade2 his3 ura3 JRY4133 MATo~ HMRa-e-rapl-10 ade2 leu2 orc2-ltrpl ura3 JRY4134 MATa ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3-1 ORC2:.~JR1423 JRY4135 matal ade2 leu2-3,112 Iys2-801 ura3 stel4 JRY4136 matal ade2 leu2-3,112 Iys2-801 orc2-2 ura3 stel4 JRY4137 matal ade2 leu2-3,112 Iys2-801 orc2-3 ura3 stel4 JRY4138 matal ade2 leu2-3,112 Iys2-801 orc2-4 ura3 stel4 (a) Unless otherwise noted, all strains were HMLo~ and HMRa. HMRa-e-15 rapl-10 refers to the allele of HMR-E, originally described as PAS1-1, that contains a mutatlon in the RAPl binding site (21).

Numbered Citations for Example 2.
1. . I. Herskowitz, et al Cold Spring Harbor Laboratory Press 583 (1992).
20 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).25 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. .Szost~k Mol. Cell. Biol. 10, 4932 (1990).
7. P. Laurenson, and J. Rine, Microbiol. Rev. 56, 543 (1992).
30 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).

21 7~i~65 WO 95/16694 : PCTtUS94/14563 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. Buc~m~n, 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. F~ngm~n, 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 orc2-1 started with JRY2698 (HMLo~, MATo~, HMRo~, ade2, his3, leu2, trpl, ura3-52), which had a mating-type c~settes at all three chromosomal mating-type 15 loci and was transformed with a plasmid (pJR924) containing the a mating-typec~ccette 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 minimi7ed the risk of being distracted by site mutations in the silencer. One hundred and sixty two thousand colonies of 20 EMS-mutagenized colonies were grown on supplemented minimal media (without uracil) at 25C and screened for del~pres~ion of the plasmid-borne a c~sette at HMR. Mutagenized colonies were replica-plated onto lawns of the mating tester strain DBY1034 (MATa, his4539, Iys2-801, ura3-52) on minimal media either - with or without uracil supplementation. Replicas were incubated at 25C for one 25 hour, then overnight at 30C. 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 de~epr~ssion of the plasmid-borne a c~sette could be distinguished from the other classes of mutations by exploiting a 30 feature of yeast pl~cmi-ls. 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 WO95/16694 2 1 78965 PCT~US94/14563 in the absence of the plasmid, and thus would be unable to form diploids capableof growth on medium supplemented with uracil. Twenty eight strains were identified that were te",~ld~ule-sensitive for growth and that mated with the tester strain only on plates supplemented with uracil. Plasmid-free isolates of each strain S were then retransformed with the plasmid bearing the synthetic ~ nc~r 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-con~ining HMR locus (pJR924). In order to determine if the ts growth 10 phenotype and the mating phenotype were due to the same mutation, spontaneousrevertants 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 orc2-1.
15 20. Y. Kassir, et al, Genet. 109, 481 (1985). Foss and Rine, Genetics. (1993) 21. The ORC2 gene was cloned by complementation of the tempelature sensitivity of orc2-1. An orc2-1 strain (JRY3683) was transformed with a CEN
LEU2-based Saccharomyces cerevisiae genomic library (32) Approximately 1000 to 1500 tran~rorlllanls formed colonies at 23C. Replica prints of these colonies 2.0 were incub~t~ at 37C to screen for the ability to grow at elevated te---~l~tures.
Plasmids were isolated from ~e",pel~ture-resistant strains and retested. Those plasmids that complemented the defect a second time were analyzed by restrictiondigestion. One plasmid from the CEN-LEU2 library (pJR1416) was chosen for further analysis.
25 22. ORC2 was disrupted with the TnlO LUK transposon (33), which inserted within the ORC2 coding sequence on the plasmid (pJR1146) carrying the SstI orc2-I complern~nting fragment. Plasmid pJR1147 had the TnlOLUK insertion within the ORC2 coding region. The ORC2-cont~ining SstI fragment, disrupted by the transposon, was removed from pJR1147 by partial digestion with SstI. The 30 fragment was transformed into the wild-type diploid JRY543. The integration of this disruption allele at the ORC2 locus was confirmed by DNA blot hybridizationanalysis (Southern, 1975), and the diploid was named JRY3444.
23. P. Hieter, C. Mann, M. Snyder, and R.W. Davis, Cell 40, 381 (1985).

WO g5/16694 2 1 7 8 9 6 5 Pcr,l,S94,l4563 24. D. Koshl~nd, 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. Sterngl~n7, J. Biol. Chem. 264, 13373 (1989).
25, 26. A.H. Brand, G. Micklem, and K. Nasmyth, Cell 51, 709 (1987).
5 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. C.A. Hrycyna, et al, EMBO J. 10, 1699 (I991).
32. A mutation was introduced into the RAPl binding site at HMR-E ~dj~cçnt 10 to the HMRol locus by oligonucleotide-directed mutagenesis (35), and the change confirmed by sequencing. The RAPl site mutation was identical to the PASI-l mutation of HMR-E characterized previously that blocks RAPl protein binding in vitro (21), and is described here as HMRo~-e-rapl-10. The plasmid con~i~ting of the HMRo~-e-rapl-10 HindIII fragment in pRS316 was named pJR1425. The wild-15 type HMRa version of the same plasmid was named pJR1426. Approximately100,000 mutagenized cells from 12 independent cultures of the HMLo~ matal HMRa stel4 strain with the HMRo~ plasmid (pJR1425) were grown into colonies at 23C and replica-plated to a MATa ura3 mating-type tester lawn (PSY152) to identify mutants exhibiting the a mating phenotype. The mating plates were 20 incubatçd at 30C 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 ~elllpeldture sensitive for growth at 37F to some degree. These mutants were subjected to further study andthe remainder were discarded. All 50 mutants were recessive to wild-type. Only 25 the subset of mutants relevant to ORC2 are presented here; the remainder will be ~i~cucse~ elsewhere.
33. The ORC2 gene was defined by the orc2-1 mutation. An orc2-complerne~ting plasmid (pJR1416) was obtained by complementation of the te-"l)eldtulc sensitivity of orc2-1. In order to map the approximate position of the 30 orc2 -complementing gene in the plasmid, six derivatives of pJR1416 were madeand tested for complementation. The SalI-SalI fragment was removed from the insert to yield pJR1418. Three adjacent XbaI-XbaI fragments were removed to wo 95/16694 2 1 7 8 ~ 6 5 PcTrUSs4/14563 yield pJR1422. SphI cleaved once in the insert and once just inside the vector.
Deleting this SphI-SphI fragment produced pJR1417. Cleavage by SstI released two fr~gmPnt~ from the insert. Deletion of both fragments created pJR1419.
Isolates in which only the larger SstI fragment (pJR1421) or only the smaller SstI
5 fragment (pJR1420) was deleted were also recovered. The 2.8-kb SstI-SstI orc2-compl~nting fragment was cloned into the SstI site of the CEN URA3 vector pRS316 (36), to yield pJR1263. Two plasmids were made which allowed the ch,o",oso",al integration of part or all of ORC2. The first, pJR1423, contained an XhoIlKpnI insert (from pJR1416) which extended from a few kb upstream of the 10 ORC2 start codon to about 60-bp upstream of the stop codon inserted into X71oI-KpnI-cut pRS306 (36), a yeast integrating vector marked by URA3. The second plasmid, pJR1424, contained the SstI orc2-complementing fragment inserted into the SstI 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 Enymol. 154, 367 (1987).
38. R.S. Sikorski, and P. Hieter, Genetics 122, 19 (1989).

20 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 eA~ression library, constructed by fusing a transcriptional activation domain to random protein 25 segments, and (ii) a lc~x~ltel gene conti~ining a binding site of interest in its promoter region. Hybrid proteins that recognize this site are expected to induce,ression of the repoller 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 30 will also activate transcription (7). Nevertheless, as long as the ~Csoci~tion is sequence-specific the protein incorporated in the hybrid should be functionally relevant.

wo 95/16694 2 1 7 8 q 6 ~ pCT/US94/14563 We have used this method to look for proteins from the yeast Saccharomyces cerevisiae that recognize the ARS conCPn.cuc sequence (ACS) of yeast origins of DNA replication. The protein co-,-ponent of this screen was provided by a set of three complementary yeast hybrid eAplession libraries, YLl-3, containing random yeast protein segmentc fused to the GAL4 transcriptional activation domain (GAL4AD) (9). The ,e~,ler gene for our screen contained four direct repeats of the ACS in its promoter region and was integrated into the yeast strain GGYl to form JLY363(ACSW') (10). To determine the dependence of lacZ
induction on the ACS, we constructed in parallel JLY365(ACSMUrA~), which harbors a l~ller gene carrying four copies of a nonfunctional multiply-mutated ACS (Fig. 4) (10).
We isolated nine plasmids that induced greater lacZ activity in JLY363(ACSWr) than JLY365(ACSMUr^NT) from a screen of 1.2 million YLl-3 transformants (11). Many of the plasmids that induced lacZ activity on initial screening of the library in JLY363(ACSWT) failed to exhibit a dependence on the ACS when introduced into JLY365(ACSMUrA~). Restriction analysis of these pl~cmi~c sho~wed that the nine isolates repl~;sented five genomic clones, which we initially labeled AAPI-5 for ACS associated protein. AAPI was isolated four times, AAP5 twice, and the others only once.
To eY~mine the sequence specificity of lacZ induction with finer resolution, repo"er constructs containing direct repeats of four ACS point mutants were eachinteg,~ted into GGYl to generate the set of reporter strains(10). The five AAP
clones were individually ex~min~d in these strains for the ability to induce lacZ
eA~,ession. AAPI displayed a collespondence between the induction of this set of~ r genes and the ARS function (12) of their ACS. The AAPS hybrid exhibited a slightly weaker correlation, and the rem~ining clones showed poor correlation. These findings suggest that AAPI, 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 GAL4AD in AAPI-S were sequenced (15) to determine the extent of the hybrid proteins that were made. AAPl and AAPS

had sizable protein coding sequences of 301 and 123 amino acids, respectively, fused in frame with the GAL4AD. In principle, these segments are large enough todirect the hybrid protein to the promoter of the ,~ er gene. AAP2-4 encoded hybrid proteins with only short peptide extensions (10, 22, and 38 amino acids respectively) fused to the GAL4^D, suggesting that these hybrids were not responsible for the transcriptional induction attributed to these clones. Rec~use 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 AAPI was cloned from a yeast genomic library and sequenced (15) (Genb~nk accession no. L23323). AAPI contains an open reading frame for a protein 435 amino acids long with a predicted molecular weight of 50,302 daltons. The hybrid GAL4^D-AAPl protein obtained from the screen was a fusion of the GAL4^D 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 ~Csoci~tion with the ACS. Comparison of peptide sequences fromthe 50kd subunit of ORC with the predicted protein sequence from MPI
demonstrated that our gene encodes this subunit and confirmed the association between the AAPl protein and the ACS. Rec~llse of this identity, we have rPn~mPd 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 Cresidue 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 indic~tes a connection with the regulatory machinery goveming cell cycle ~rogression. 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 conC~ncus site (S/T)P adds two more sites to this cluster. We have observed Orc6p phosphorylated in vivo on serine and threonine residues.
30 However, since the initiation of yeast DNA replication commences pro~llplly in response to the activatio!l of this protein kinase in Gl, we believe that Orc6p and possibly other ORC subunits are regulated substrates of this kinase. Finally, asexpected for a protein participating in nuclear events, Orc6p contains a potential 2 1 7 ~ q 6~

nuclear loc~li7~tion signal (NLS) within the (S/T)PXK cluster and one in the C-terminal domain (amino acld 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 5 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 di~ected (21). In all backgrounds the ORC6 disruption coseglegated with inviability, demonstrating that ORC6 is essential for cell growth.
10 Microscopic ex~min~tion revealed that mutant spores from JLY481 and JLY475 g~l",in~t~d, completed 1-2 rounds of cell division, and then arrested with a uniform large bud morphology rernini~cent of cell division cycle mutants defective in DNA replication or nuclear division (22). The position of cell cycle arrest could not be pinpointed, however, since the DNA content of these cells could not be 15 readily measured. Mutant spores derived from JLY469 germin~t~ poorly.
The inle,~"t;~tion 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 Genb~nk sequence and overlaps the C-terminal two-thirds of the ORC6 coding - 20 sequence. A marked deletion that removed the N-terminal third of the ORC6coding 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 changefor the ORC6 ORF but introduces a UGA stop codon in ORF2 (22). This gene 25 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 30 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.

wo 95/16694 2 1 7 8 9 6 5 PCT/US94/14563 We identified genes that interact gçneti~lly with ORC6 using established cdc mutants because gel..~in~ g spores bearing an ORC6 deletion al)peared to exhibit a cell division cycle phenotype. pJL749 (28), a plasmid that overe~,esses Orc6p several hundred-fold, was introduced into a virtually isogenic set of 5 te.l-pc~dture-sensitive cdc mutants arresting at various points in the cell cycle (29).
Ove.eAI,lc;ssion of ORC6 selectively affected cdc6 and cdc46 mutants, lowering their restrictive telllpeldtu~e by 5-7 C; there was no significant effect on the other mutants e~mine~ or on the wild-type strain (Table l).

viability with Strain cdc mutantovereA~ression of ORC6 RDY488 wild-type +
RDY501 cdc28-1 +
RDY510 cdc4-1 +
RDY664 cdc34-2 +
RDY543 cdc7-4 +
JLY310 cdc6-1 -JLY179 cdc46-1 JLY338 cdc2- 1 +
JLY353 cdc17-1 +
RDY619 cdc15-2 +

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 eY~mine~ for growth at various telll~;~dtu~es 25 under conditions that induce ~A~,ession of ORC6 (28, 29). + in~ tes mutants whose restrictive tell~ldture remains unchanged in the presence of JL749 relative to JL772 and RS425. - indicates ll-ul~ls whose restrictive tel~lpeldtul~ is lowered 5-7 C when JL749 is present.

wo 95/16694 2 1 7 ~ 9 6 5 pCT/US94/14563 Numbered Citations for Fx~mple 3 1. Kelly, J. Biol. Chem. 263, 17889 (1988); Marians, Annu. Kev. Biochem.
61, 673 (1992); Kornberg, Baker, DNA Replication. (Freeman and Company, New York, 1992); B. Stillman, Annu. Rev. Cell Biol. 5, 197 (1989).
5 2. M. L. DePamphilis, Annu. Rev. Biochem. 62, 29 (1993).
3. Campbell and Newlon, in The Molecular an~l Cellular Biology of the Yeast Sa~charomyces Broach, et al, Eds. (CSHL Press, 1991), vol. 1, pp. 41-146.
4. F~ngm~n and Brewer, Annu. Rev. Cell Biol. 7, 375 (1991).
5. J.R. Broach et al., Cold Spring Harbor Symp. Quant. Biol. 47, 1165 10 (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. Stemglanz, S. Fields, Proc. Natl. Acad. Sci. USA 88, 9578 (1991).
8. R. Brent and M. Ptdshne, Cell 43, 729 (1985).
15 9. The N-terminal portions of the hybrids from hree related hybrid expressionlibraries, YLl-3 (7), consist of the SV40 nuclear localization signal and amino acids 768-881 of the GAL4 activation domain (GAL4AD). The C-terminal portions were derived from random yeast protein segments which have been fused to the end of the GAL4AD. These segments are encoded by short (1-3kb) fragments from 20 a Sau3a partial digest of yeast genomic DNA. Together, YLl-3 ensure that all three reading frames of these fr~gmentc can be c~p,cssed.
10. pLRlDl is described in R.W. West Jr., R.R. Rogers, M. Ptashne, Mol.
Cell. Biol. 4, 2467 (1984). We generated pBgl-lacZ from pLRlDl by (i) subsl;4~ g dn XhoI-BglII-XhoI polylinker for the XhoI linker and (ii) precisely 25 excising a Hind III fragment cont~ining 2m sequences. The resulting vector has a unique Bgl II site appro~Limately 100 bp upstream of the TATA box for insertion of DNA sequences in the promoter region and a unique Stul site for targeted integld~ion 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 30 pBgl-lacZ to generate all the reporter genes used in this work. The inserted repeat elernP-ntc, derived from complementary oligonucleotides, were oriented with the TATA box to their right. Each ~epo.ler gene construct was integrated into the wo 95/16694 ' ~ 2 ! 7 8 ~ 6 5 PCT/US94/14563 URA3 locus of GGYl (MATa Dgal4 Dgal80 ura3 leu2 his3 ade2 tyr) [G. Gill and M. Ptashne, Cell 51, 121 (1987)] to create a reporter strain. Integration of pBgl-lacZ into GGYl generated JLY387.
11. YEPD (rich complete) and SD (synthetic dropout) media are as described 5 tJ.B. Hicks and I. Herskowitz, Genetics 83, 245 (1976)]. Standard methods wereused for manipulation of yeast cells ~C. Guthrie and G.R. Fink, Ed., Guide to Yeast 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 YLl-3 were transformed [R.H. Schiestl and R.D.
10 Geitz, Current Genetics 16, 339 (1989)] into JLY363 (10) and plated on SD-Leu at a density of 2-5000 colonies/lOcm plate. 500,000 tran~ro~ ants were obtained forYLl and YL2, and 200,000 for YL3. Transformants were assayed on filters for production of b-galactositl~ce [L. Breeden and K. Nasmyth, Cold Spring Harbor Symp. Quant. Biol. 47, 643 (1985)]. 49 isolates remained positive after colony 15 purification (15 from YL-l; 22 from YL-2, 12 form YL-3), and library plasmidswere extracted from them . These plasmids were each transformed into both JLY363 and its mutant counterpart JLY365 (10). Nine plasmids induced greater b-g~ tocidase activity in the wild type reporter strain than the control. These pl~cmi~is were cl~csified into five clones, AAPI-5, based on their Hind III
20 restriction pattern. Each clone was then retested in JLY360, JLY361, JLY387, JLY429, JLY431, JLY433, JLY435. The AAPI hybrid clone was called pJL720.
The AAPI gene was later ren~med ORC6.2 12. The ARS function of the mutant sequences was analyzed in the context of ARS1 domain B (BglII-Hinf~ fragment, nt 853-734) in the following CEN-based 25 URA3-containing plasmids: pJL347 (wt), pJL243 (multiple), pJL326 (A863T), pJL338 (T869A), pJL330 (T862C), and pJL316 (T867G). These plasmids were transformed into JLY106 (MATa ura3 leu2 his3 trpl Iys2 ade2) and its homozygous diploid counte~ JLY162. pJL243, pJL326, and pJL338 did not yield a high frequency of transformation and could not be assayed quantitatively30 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)]-wo 95/16694 2 1 7 8 9 6 5 PCT/USs4/14563 13. pJL720, the ORC6 hybrid construct originally isolated from the YL3library, has two BamHI sites. The 5' site created by the hybrid junction collesponds 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 GAL4^D.
5 pGAD3R (11) the parent vector for the YL3 library, contains no ORC6 sequence.
pRS425, Christi~n~on, et al., Gene 110, 119 (1992), contains no co...ponents of the fusion protein.
14. All sequçnçing was pe,l~ led with Sequenase (USB) on collapsed double-stranded templates. The protein coding segments of the AAPI-S hybrid clones were10 sequenced from their junction with the GAL4^D to their stop codon. Two of theORC6 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 15 a~plo~imately 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).
lg. ORC6 deletions were constructed by replacing nuc!eotides 458-1721 (pJI,731) or nucleotides 458-846 (pJL733) of the Genbank sequence with the URA3 HindIII 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 repl~cçrn~nt. ORC6 deletion analysis was pel~l"led in 25 JLY461 (MATa/MATa ura3/ura3 leu2/leu2 his3/his3 trpl/trpl ade2/ade2 lcirl), JLY462 (MATa/MATa ura3/ura3 leu2/leu2 trpl/trpl his4/his4 canl/canl), and JLY463 (MATa/MATa ura3/ura3 leu2/leu2 trpl/trpl his3/HI53); their respective genetic backgrounds are S288c, EG123, and A364a. Disruption of JLY461, JLY462, and JLY463 by pJL731 (full deletion) created JLY481, JLY475, and 30 JLY469, l~eclively. Disruption of JLY461, JLY462, and JLY463 by pJL733 (N-terrninal deletion) created JLY485, JLY479, JLY473, respectively. These WO 95/16694 . 2 1 7 8 9 6 5 PCT/US94/14563 helefozygous marked deletion strains were sporulated, and twenty tetrads of eachwere tli~c~te l 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 R~mH~/SphI fragment generated by PCR from pJL720 using primers. One mutation changes nucleotide 1471 of the C~çnb~nk 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 GALI promoter (nt 146-816) driving the t;A~ression 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 ura3 trpl) and RDY488 (MATa leu2 ura3 trpl). All are ura3 leu2 trpl. RDY510, RDY664, JLY310, and JLY179 are MATa; the rest are MATa. Additional markers can be found in JLY310(ade2), RDY543(his3), and RDY619 (pep4D::TRPI his3 ade2).
20 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 p~tc~ed 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 lllu~nts con~ining pJL749 was col,lpal~d to those con~ining pJL772 and pRS425.
27. Hartwell, JMB 104, 803 (1976); Hennessy, et al G&D 4, 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).

30 Example 4. Orc protein purification and gene cloning Protein Purification: To obtain sufficient protein for peptide sequençing~ a revised purification procedure for ORC was devised, based on the procedure reported previously (Bell and Stillman, 1992). Whole cell extract was wo 95/16694 2 1 7 8 9 6 5 PcrlUSs4/l4s63 t d 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 ben7~mi~ine, 2 mM pep~Lin A, 0.1 mg/ml bacitracin and 2mM DTT. 400 mls of 2X buffer H/0.1-NP~ (100 mM
5 Hepes-KOH, pH 7.5, 0.2 M KCl, 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 10 volume of a saturated (at 4C) solution of ammonium sulfate was added and stirred for 30 minutes. This solution was then spun at 13,000 x g for 20 minutes. The res--lting supernatant was transferred to 45Ti bottle assemblies (Re~m~n) and spun in a 45Ti rotor at 44,000 RPM for 1.5 hrs. The volume of the resulting su~",atant was measured and 0.27g/ml of ammonium sulfate was added. After 15 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.lSM KCl (Buffer H with 0.15 M KCl added). This preparation typically 20 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-Sel)har~se column was loaded at 20 mg protein per ml of resin ( ~ 300 ml). The Q-Sepharose (50 ml) and sequence specific affinity column (Sml) was run as described but the dsDNA cellulose column was omitted from the preparation.
Only a single glycerol gradient was pelro",led in an SW-41 rotor spun at 41,000 RPM for 20 hrs. We estim~t~ a yield of 130 ~g of ORC complex (all subunits combined) per 400 g of yeast cells.
Protein Sequencing: Digestion of ORC subunits was pelrol."ed using an "in gel" protocol described by Kawasaki and Suzuki with some modification.
Briefly, purified ORC (~ 10 ~g per subunit) was first separated by 10% SDS-PAGE and stained with 0.1 % Coomassie Brilliant Blue G (Aldrich) for 15 min.

wo 95/16694 ;~ 1 7 8 9 6 5 P~/US94/~4s63 After dest~ining (10% meth~nol, 10% acetic acid), the gel was soaked in water for one hour, then the protein bands were excised, transferred to a microcentrifuge tube and cut into 3-5 pieces to fit snugly into the bottom of the tube. A minimum volume of 0. lM 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 incub~ted at 30C for 24 hrs. After digestion the samples were centrifuged and the su~lna~ was passed through an Ultrafree-MC filter (Millipore, 0.22~m). 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, S~m, 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 pelrolnled 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 syntheci7çd basedon the sequence of the first ORCl peptide. These oligos were used to ~~
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 sequencecl. The resulting sequence encoded the predicted peptide indicating that it was the correct amplification product. A radioactively labeled form of the PCR product was then used to probe a genomic library of yeast DNA sequences r~s~-lting in the identification of two overlapping clones. Sequencing of these clones resulted in the identification of a large open reading frame that encoded a 30 protein with a predicted molecular weight of 120 kd and that encoded all four of the ORCl peptide sequences.
ORC3: To clone the gene for the 62 kd subunit of ORC, the following degen~.dte oligonucleoide primers 621 and 624 were synthe~i7~A based on the WOg5/16694 2 1 78965 PCTIUS94/14563 sequence of the third peptide. These oligos were used to pe~ 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 in~ic~ting that it was the 5 correct amplification product. A radioactively labeled form of the PCR productwas then used to probe a genomic library of yeast DNA sequences reslllting in the i~lPntific~tion 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 10 sequences. The inconsistency of the molecular weight is presumably due to anomalous migration of this protein during SDS-PAGE.
ORC4: By co",l)aling the sequnce of the ORC4 peptides to that of the known potentially protein encoding sequnces in the genbank ~l~t~h~ce we found that a portion of the ORC4 coding sequence had been previously cloned in the process 15 of cloning the adjacent gene. Using the information from the l~t~h~e we wereable 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 20 derived from the peptide sequencing of the 56 kd subunit.
ORCS: 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 25 base pair fragment was specifically amplified. This fragment was subcloned and sequenced. The reslllting sequence encoded the predicted peptide indicating that it was the correct amplification product. A radioactively labeled form of the PCR
product was then used to probe a genomic library of yeast DNA sequences resl-lting in the identification of a single lambda clone. Sequencing of this clones 30 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 correct 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 wo 95/16694 2 1 7 8 9 6 5 PCTIUS94tl4563 been picked up in the same sreen that resulted in the identification of the ORC2gene. A complementing clone to this mutation was found to overlap with the l~mb(l~ clone and contain the entire 5' end of the gene. Sequencing of this complementing DNA fragment resulted in the identification of the entire sequence5 of the ORCS gene.
All publications and patent applications cited in this specification are herein inc~ ed 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 10 illustration and example for purposes of clarity of understanding, it will be readily a~alent 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.

Sr;Q~N~r; 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) COuh-~: 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) ~u~R~ 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) *~rr;k~N~r;/DOCRET NUMBER: FP-59032-PC/RAO
(ix) TFT-FCOMMUNICATION INFORMATION: ..
(A) TELEPHONE: (415) 781-1989 21 7896~
W O95/16694 - PCT~us94/14563 ~B) TELEFAX: (415) 398-3249 (C) TELEX: 910 277299 5 ( 2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4940 base pair~
(B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ATAArATGcT CGCC~.-~-A TATTATGACA GAAAGAATAT ATATATTCAT ATATAArA-TG 60 ATGGTATGGA GTGTATAATG GTTTATAATT lCCC~lAAGA TGACACAAAA AAA1~Ll~lC 180 CAGTATTAAG ATAAGGACTG CTATGGGGCA l~ l~l~i TACTGGGTAT CACAGGATAA 360 TAACTTGGCG CCAAATTAGA APArATATAA ACCTCAAATA TTTGAAATTC TTTGGTGACC 420 ,~, ~ATCG TTATATCAAC AAATATTGCA CCAACGAACA CCACTACATA TGTAACTACT 480 ~ C~CG ACTTATTTTT TATTAACGTT GACACGGCCA GATCGAAAAT CATAGAAAAA 540 35 rAACAACATT GAGAAGAGAT GAAGTTGCGC AAAGGGAAAG AAAACTGCAT AGGCGGCAAA 600 TTCAGCCTAA AAG~C~AG AAGCAGGAAC TCATTCCCTA TTGATTAATA CTCATTACAA 660 ApA-ccAcAAT AGAGTAGATA AGATGGCAAA AACGTTGAAG GATTTACAGG GTTGGGAGAT 720 pATAArAACT GATGAGCAGG GAAATATAAT CGATGGAGGT rArAAGAGAT TACGCCGAAG 780 45 TGATAGTGTA GTCATGCACA ACGAAGCCGC TGGGACTTAC ~CCG1~ATA TGATCCAGGA 900 GTTTGAAGTC AA~C~AG CTCATTATAG GCAGTTTAAT CCTGACGCTA ACATTTTGAA 1020 ~CG~C~lA AATTATTACA ATAAACTGTT TTCTGAAACT GCAAATAAAA ATGAACTGTA 1080 55 GGATGGAAGC AAATGGGAAG TATTGAAAGG AAATGTCGAT CrAr-AAAr7AG ACTTTACAGT 1200 GGCACAAATT TCAGACGCAG AAACAAGAGC TArAGATATA ACGGATAATG AGGACGGTAA 1440 CAGCGGTGAA ATATCCGCAG ATGAGCTTGA GGAAGAAGAA GACGAAGAAr, AArA~r,AAr.A 1560

42 WO 95/16694 2 ~ 7 ~ 9 6 5 PCT/US94/14563 CrAACAAr~G AAAGAAGCTA GGCATACAAA TTrAC~AGG AAAAGAGGCC GTAA~-ATAAA 1620 ACTAGGTAAA GATGATATTG ACG~l-~-~ ACAACCTCCC CCCAAAAAAA GAGGTCGTAA 1680 S ACCTAAA~-AT CCTAGTAAAC CGCGTCAGAT GCTATTGATA TCTTCATGCC GTGrPAATAA 1740 TA~.C~.~.G ATTAGGAAAT TTArAAAAPA GAATGTTGCT AGGGCGAAAA AGAAATATAC 1800 CCCG.~..CG AAAAGATTTA AATCTATAGC TGCAATACCA GATTTAACTT CATTACCTGA 1860 ATTTTACGGA AA-~..CGG AATTGATGGC ATCAAGGTTT GAAAACAAAT TAAAAACAAC 1920 CCAPAAGCAT CAGATTGTAG AAArAATTTT TTCTAAAGTC AAAAAACAGT TGAACTCTTC 1980 lS GTATGTCAAA GPAGAAATAT TGAAGTCTGC AAATTTCCAA GATTATTTAC CGGCTAGGGA 2040 AAAGGAACTA CTA~C~.~.. CTGr~CAACG A~AAATACCA GA~ ATGTGGAAAT 2220 25 AG~-A~-AAAGG TTAACATGGG CAGCTTCAAT GGAGTCACTA GAGTTTTACT TTAAAAGAGT 2340 TCCAAAAAAT AAGAAGAAAA CCATTGTAGT ~.l~.~GGAC GAACTCGATG CCATGGTAAC 2400 TATTGTCATT GCAGTAGCCA ATA~AATGGA cTTAcrA~AA CGTCAGCTAG GCAATAAGAT 2520 35 AAATATCATT GATTTAAGAC TGAAGGGGTT GAACGACTCA ..~..~.ATG TTGATACAAA 2640 GCCTGAAGAC GT~-AGr-AAAG TTCGCTTAAG AAT-GAGTGCT GATGCCATTG AAATAGCTTC 2760 ~ AGPAAAGTA GCAAGTGTTA GTGGTGATGC AAGAA~AGCA TTGAAGGTTT GTAAAAGAGC 2820 AAGTAA~AA GCCAAAGACG ATAATGATGA CGATGATGAC AATGATGGGG TAcAAAcAGT 3000 pAAGAACGGA TCTCAAGAGC AAGAACTGGG CGATATTGTC GATGAAATCA AGTTACTTAT 3180 SS TGAAGTAAAT GGCAGTAATA AG~..~.~AT GGAGATAGCC AAAACATTGT TCCAACAGGG 3240 AAGTGATAAT ATTTCTGAAC AATTGAGAAT TATATCATGG GA...CGl.C TCAATCAGTT 3300 ACTTGACGCG GGAATATTGT TTAAA~AAAC TATGAAGAAC GATA~-AATAT G--~.~.~AA 3360 TTTPTArATT CG~..~..AT TATTCATGAC CTAGCATACA CATACATATA CCTACATAGT 3480 65 AGCGCATTTA TCCAAAACAT ACGATATTGT GGATGTACAT AC~ ATA ~-C~.lAAA 3540

43 GG~l.C~.GA TATTATGGCT ~l~ ATCC TGAC~ TATGATGTCG ATGTTGCTGG 3720 AATGTCAAAT TAAAGATCTT TGCCAGTGCA ATTTTGAAAA l~ lGAAT GTTTATAGAT 3840 TTGGCAGTAG AG~AGAATAT AAGAGGAGCA TTCATGACCT GTGCATACTT CATACTCGTT 3900 CTCGAGATTT G-~C~GATA TTCCGGGTCT AAGTCTATTA GTAAATCGTA ~ GCCC 3960 A~APAATAG GAATTGCCGA ATCATTTAGC CCGTACGCCT GCCTATACrA ~C~ATT 4020 15 GAACTCAACG ~ GGACG TGTCAGGTCA AACAGAAATA TGATCACTGA A-r~AcccTAcc 4080 GTCGCAATTG GGAGCATGTT GATGAATTCT ~..~G~CCGC CTAAATCCAT TATAGAAAAT 4140 ATAATATCCG TGGAGCGTAT GCTTACTTTT CTTTTCAAAA AGTTCACTCC CAGCC~Gl 4200 GTGTATTCCT TATCGTATAT Gl~G~ACG TACTTCACCA TCAGCGATGT l~cc~AcT 4260 25 ,~ ,GCTC CCGTGCTTGG TGTAGCCATC TTAGCTTAAC TCAATTTAAT TTCTACAGCA 4380 AAATCCAAAC GTAATATCTA TA,,,ll~lC GAAAAACTGA GGACAAGAGC CAATCAATCA 4440 TCTATAATCC AATTTATATT A..~...CCC TTCTGGGTTC l,ll~llC~l l~llG1~ 4500 AC~...l~lG ~l~ ~ATA AAATAATTTC TCTAGATTTG AAGACAGCAT llll~lACAT 4560 CrATAr~CCA TACACCATAC ACCATAGCAC CAGTACACTA TATTTTTATG AATTTTACTA 4620 35 AGAATTATTC CTGCAGGAGC TCCACTGAAA AAAAAAGAGc AGCATGGATG TCATGTCGGT 4680 GTGGTATTTT ~ATATATGTG AGTGGTAGCA GATTTGAACT TAGTTAGTTG TATTCGCCTT 4920 (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 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 Met Ala Lys Thr Leu Lys Asp Leu Gln Gly Trp Glu Ile Ile Thr Thr Asp Glu Gln Gly Asn Ile Ile Asp Gly Gly Gln Lys Arg Leu Arg Arg Arg Gly Ala Lys Thr Glu His Tyr Leu Lys Arg Ser Ser Asp Gly Ile

44 W 095/16694 ~ ! 7 ~ 9 6 5 PCTrUS94/14563 Ly6 Leu Gly Arg Gly Asp Ser Val Val Met His Asn Glu Ala Ala Gly Thr Tyr Ser Val Tyr Met Ile Gln Glu Leu Arg Leu Asn Thr Leu Asn Asn Val Val Glu Leu Trp Ala Leu Thr Tyr Leu Arg Trp Phe Glu Val 0 Asn Pro Leu Ala Hi8 Tyr Arg Gln Phe Asn Pro Asp Ala Asn Ile Leu Asn Arg Pro Leu Asn Tyr Tyr Asn Lys Leu Phe Ser Glu Thr Ala Asn Lys Asn Glu Leu Tyr Leu Thr Ala Glu Leu Ala Glu Leu Gln Leu Phe Asn Phe Ile Arg Val Ala Asn Val Met A~p Gly Ser Lys Trp Glu Val Leu Lys Gly Asn Val Asp Pro Glu Arg Asp Phe Thr Val Arg Tyr Ile cys Glu Pro Thr Gly Glu Lys Phe Val Asp Ile Asn Ile Glu Asp Val Lys Ala Tyr Ile Lys Lys Val Glu Pro Arg Glu Ala Gln Glu Tyr Leu Lys Asp Leu Thr Leu Pro Ser Lys Lys Lys Glu Ile Lys Arg Gly Pro Gln Lys Lys Asp Lys Ala Thr Gln Thr Ala Gln Ile Ser Asp Ala Glu Thr Arg Ala Thr Asp Ile Thr Asp Asn Glu Asp Gly Asn Glu Asp Glu - 40 Ser Ser Asp Tyr Glu Ser Pro Ser Asp Ile Asp Val Ser Glu Asp Met Asp Ser Gly Glu Ile Ser Ala Asp Glu Leu Glu Glu Glu Glu Asp Glu Glu Glu Asp Glu Asp Glu Glu Glu Lys Glu Ala Arg His Thr Asn Ser Pro Arg Lys Arg Gly Arg Lys Ile Lys Leu Gly Lys Asp Asp Ile Asp Ala Ser Val Gln Pro Pro Pro Lys Lys Arg Gly Arg Lys Pro Lys Asp Pro Ser Lys Pro Arg Gln Met Leu Leu Ile Ser Ser Cys Arg Ala Asn Asn Thr Pro Val Ile Arg Lys Phe Thr Lys Lys Asn Val Ala Arg Ala Lys Lys Lys Tyr Thr Pro Phe Ser Lys Arg Phe Lys Ser Ile Ala Ala Ile Pro Asp Leu Thr Ser Leu Pro Glu Phe Tyr Gly Asn Ser Ser Glu Leu Met Ala Ser Arg Phe Glu Asn Lys Leu Lys Thr Thr Gln Lys His Gln Ile Val Glu Thr Ile Phe Ser Lys Val Lys Lys Gln Leu Asn Ser Ser Tyr Val Lys Glu Glu Ile Leu Lys Ser Ala Asn Phe Gln Asp Tyr Leu Pro Ala Arg Glu Asn Glu Phe Ala Ser Ile Tyr Leu Ser Ala Tyr 0 Ser Ala I le Glu Ser Asp Ser Ala Thr Thr I le Tyr Val Ala Gly Thr Pro Gly Val Gly Lys Thr Leu Thr Val Arg Glu Val Val Lys Glu Leu Leu Ser Ser Ser Ala Gln Arg Glu I le Pro Asp Phe Leu Tyr Val Glu Ile Asn Gly Leu Lys Met Val Lys Pro Thr Asp Cys Tyr Glu Thr Leu Trp Asn Lys Val Ser Gly Glu Arg Leu Thr Trp Ala Ala Ser Met Glu Ser Leu Glu Phe Tyr Phe Lys Arg Val Pro Lys Asn Lys Lys Lys Thr Ile Val Val Leu Leu Asp Glu Leu Asp Ala Met Val Thr Lys Ser Gln Asp Ile Met Tyr Asn Phe Phe Asn Trp Thr Thr Tyr Glu Asn Ala Lys Leu Ile Val Ile Ala Val Ala Asn Thr Met Asp Leu Pro Glu Arg Gln Leu Gly Asn Lys Ile Thr Ser Arg . Ile Gly Phe Thr Arg Ile Met Phe Thr Gly Tyr Thr His Glu Glu Leu Lys Asn Ile Ile Asp Leu Arg Leu Lys Gly Leu Asn Asp Ser Phe Phe Tyr Val Asp Thr Lys Thr Gly Asn Ala Ile Leu Ile Asp Ala Ala Gly Asn Asp Thr Thr Val Lys Gln Thr Leu Pro Glu Asp Val Arg Lys Val Arg Leu Arg Met Ser Ala Asp Ala Ile Glu Ile Ala Ser Arg Lys Val Ala Ser Val Ser Gly Asp Ala Arg Arg Ala Leu Lys Val Cys Lys Arg Ala Ala Glu Ile Ala Glu Lys His Tyr Met Ala Lys His Gly Tyr Gly Tyr Asp Gly Lys Thr Val Ile Glu Asp Glu Asn Glu Glu Gln Ile Tyr Asp Asp Glu Asp Lys Asp Leu Ile Glu Ser Asn Lys Ala Lys Asp Asp Asn Asp Asp Asp Asp Asp Asn Asp Gly Val Gln Thr Val His Ile Thr His Val Met Lys Ala Leu Asn Glu Thr Leu Asn Ser HiR Val Ile Thr Phe Met Thr Arg Leu Ser Phe Thr Ala Lys Leu Phe Ile Tyr Ala Leu Leu Asn Leu Met Lys Lys Asn Gly Ser Gln Glu Gln Glu Leu Gly Asp Ile Val Asp Glu Ile Lys Leu Leu Ile Glu Val Asn Gly Ser Asn Lys Phe Val Met Glu Ile Ala Lys Thr Leu Phe Gln Gln Gly Ser Asp Asn Ile Ser Glu Gln Leu Arg Ile Ile Ser Trp Asp Phe Val Leu Asn Gln Leu Leu Asp Ala Gly Ile Leu Phe Lys Gln Thr Met Lys Asn Asp Arg Ile Cys Cy8 Val Lys Leu Asn Ile Ser Val Glu Glu Ala Lys Arg Ala Met Asn Glu Asp Glu Thr Leu Arg Asn Leu (2) INFORMATION FOR SEQ ID NO:3 ( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2809 base pair~
(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 ~.C~ GC 60 CATACTTGGC rAAAAATTCA GGATTGAATA TATATAATCG GAACTTGTAT GrATAAAAAT 180 TTATATCAAG AGl~.~.-C TTAATTGGAT TTGCTGTGAT CTAGTATTGA GATGACTATA 240 AACCGGCCAG GAAATTAGTC TTTTCGAAGC TGGTTTTGGT TTCGCAAGAG l~lll.~GAC 300 AG~.l..lGG CCTCAATTTG TAl.CCC.lA ATACGCTTCT TCAACTCTGT CTTAGAr-ACC 360 A..~.CCAG TGGCCTCATC TAGGTGTAAA CTAGCAATAG CGTCACTAGC TGCCGTGACA 420 TTAACTTGCT GTGGCACCTT TATATGTAAT ATr-AAr,r~TC TTTCAATGGA Tr~TAAG~AT 480 60 AA~..,.C~,.A AAAGGCCAAA TATCCATGCA TAAATATCGA CTTATTCGCG TAAATGTGAT 540 ATGGATCAGC TAGTACr~AT TTCTAGTCTA GCA~AATCGG GAAAATTTTT CAGAACACCC 600 GCTGAAATTG TATTTGATAA TTGATCATTG ATCTTATTTG CTATATCTTT AAAAcAArGTT 780 TTTGTAGTAC TGCGAATTGC CATAAC ATG CTA AAT GGG GAA GAC TTT GTA GAG 8:
Met Leu Asn Gly Glu Asp Phe Val Glu l 5 His Asn Asp Ile Leu Ser Ser Pro Ala Lys Ser Arg Asn Val Thr Pro 0 Lys Arg Val Asp Pro His Gly Glu Arg Gln Leu Arg Arg Ile His Ser Ser Ly~ Lys Asn Leu Leu Glu Arg Ile Ser Leu Val Gly Asn Glu Arg

45 50 55 Lys Asn Thr Ser Pro Asp Pro Ala Leu Lys Pro Lys Thr Pro Ser Lys Ala Pro Arg Lys Arg Gly Arg Pro Arg Lys Ile Gln Glu Glu Leu Thr Asp Arg Ile Lys Lys Asp Glu Lys Asp Thr Ile Ser Ser Lys Lys Lys 30 Arg Lys Leu Asp Lys Asp Thr Ser Gly Asn Val Asn Glu Glu Ser Lys Thr Ser Asn Asn Lys Gln Val Met Glu Lys Thr Gly Ile Lys Glu Lys 125 130. 135 Arg Glu Arg Glu Lys Ile Gln Val Ala Thr Thr Thr Tyr Glu Asp Asn Val Thr Pro Gln Thr Asp Asp Asn Phe Val Ser Asn Ser Pro Glu Pro Pro Glu Pro Ala Thr Pro Ser Lys Lys Ser Leu Thr Thr Asn His Asp Phe Thr Ser Pro Leu Lys Gln Ile Ile Met Asn Asn Leu Lys Glu Tyr l90 195 200 Lys Asp Ser Thr Ser Pro Gly Lys Leu Thr Leu Ser Arg Asn Phe Thr Pro Thr Pro Val Pro Lys Asn Lys Lys Leu Tyr Gln Thr Ser Glu Thr Lys Ser Ala Ser ser Phe Leu Asp Thr Phe Glu Gly Tyr Phe Asp Gln Arg Lys Ile Val Arg Thr Asn Ala Lys Ser Arg His Thr Met Ser Met W 095/16694 2 1 7 8 9 65 pcTnus94/14563 Ala Pro Asp Val Thr Arg Glu Glu Phe Ser Leu Val Ser Asn Phe Phe Asn Glu Asn Phe Gln Lys Arg Pro Arg Gln Lys Leu Phe Glu Ile Gln 0 Lys Lys Met Phe Pro Gln Tyr Trp Phe Glu Leu Thr Gln Gly Phe Ser Leu Leu Phe Tyr Gly Val Gly Ser Lys Arg Asn Phe Leu Glu Glu Phe Ala Ile Asp Tyr Leu Ser Pro Lys Ile Ala Tyr Ser Gln Leu Ala Tyr Glu Asn Glu Leu Gln Gln Asn Lys Pro Val Asn Ser Ile Pro Cys Leu Ile Leu Asn Gly Tyr Asn Pro Ser Cys Asn Tyr Arg Asp Val Phe Lys 30 Glu Ile Thr Asp Leu Leu Val Pro Ala Glu Leu Thr Arg Ser Glu Thr Lys Tyr Trp Gly Asn. His Val Ile Leu Gln Ile Gln Lys Met Ile Asp TTC TAC AAA AAT CAA CCT TTA GAT ATC AAA TTA ATA CTT GTA GTG CAT 2081.
Phe Tyr Lys Asn Gln Pro Leu Asp Ile Lys Leu Ile Leu Val Val His Asn Leu Asp Gly Pro Ser Ile Arg Lys Asn Thr Phe Gln Thr Met Leu Ser Phe Leu Ser Val Ile Arg Gln Ile Ala Ile Val Ala Ser Thr Asp S0 His Ile Tyr Ala Pro Leu Leu Trp Asp Asn Met Lys Ala Gln Asn Tyr Asn Phe Val Phe His Asp Ile Ser Asn Phe Glu Pro Ser Thr Val Glu Ser Thr Phe Gln Asp Val Met Lys Met Gly Lys Ser Asp Thr Ser Ser Gly Ala Glu Gly Ala Lys Tyr Val Leu Gln Ser Leu Thr Val Asn Ser Lys Lys Met Tyr Lys Leu Leu Ile Glu Thr Gln Met Gln Asn Met Gly WO 95/16694 2 1 7 8 ~ 6 5 PCT/US94/14563 A~n Leu Ser Ala Asn Thr Gly Pro Ly~ Arg Gly Thr Gln Arg Thr Gly GTA GAA CTT A~A CTT TTC AAC CAT CTC TGT GCC GCT GAT TTT ATT GCT 2513 Val Glu Leu Lys Leu Phe Asn Hi~ Leu Cys Ala Ala Asp Phe Ile Ala Ser Asn Glu Ile Ala Leu Arg Ser Met Leu Arg Glu Phe Ile Glu His Ly~ Met Ala Asn Ile Thr Lys Asn Asn Ser Gly Met Glu Ile Ile Trp GTA CCC TAC ACG TAT GCG GAA CTT GAA A~A CTT CTG A~A ACC GTT TTA 2657 Val Pro Tyr Thr Tyr Ala Glu Leu Glu Lys Leu Leu Lys Thr Val Leu AAT ACT CTA TA~ATGTATA CATATCACGA ACAATTGTAA TAGTACTAGG 2706 Asn Thr Leu AAAACTTCTC ATATAACCCT ACTGA~AAAC GTCTGATGAG CTC 2809 30 ( 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 Ile Leu Ser Ser Pro Ala Lys Ser Arg Asn Val Thr Pro Lys Arg Val Asp Pro His Gly Glu Arg Gln Leu Arg Arg Ile His Ser Ser Lys Lys Asn Leu Leu Glu 35~ 40 45 50 Arg Ile Ser Leu Val Gly Asn Glu Arg Lys Asn Thr Ser Pro Asp Pro Ala Leu Lys Pro Ly~ Thr Pro Ser Lys Ala Pro Arg Lys Arg Gly Arg Pro Arg Lys Ile Gln Glu Glu Leu Thr ARP Arg Ile Lys Lys Asp Glu Lys A~p Thr Ile Ser Ser Lys Lys Lys Arg Lys Leu Asp Lys Asp Thr Ser Gly Asn Val Asn Glu Glu Ser Lys Thr Ser Asn Asn Lys Gln Val 65 Met Glu Lys Thr Gly Ile Lys Glu Lys Arg Glu Arg Glu Lys Ile Gln Val Ala Thr Thr Thr Tyr Glu Asp Asn Val Thr Pro Gln Thr Asp Asp Asn Phe Val Ser Asn Ser Pro Glu Pro Pro Glu Pro Ala Thr Pro Ser Lys Lyn Ser Leu Thr Thr Asn His Asp Phe Thr Ser Pro Leu Ly~ Gln 0 Ile Ile Met Asn Asn Leu Lys Glu Tyr Lys Asp Ser Thr Ser Pro Gly Lys Leu Thr Leu Ser Arg Asn Phe Thr Pro Thr Pro Val Pro LYB Asn Lys Lys Leu Tyr Gln Thr Ser Glu Thr Lys Ser Ala Ser Ser Phe Leu Asp Thr Phe Glu Gly Tyr Phe Asp Gln Arg Lys Ile Val Arg Thr Asn Ala Lys Ser Arg His Thr Met Ser Met Ala Pro Asp Val Thr Arg Glu Glu Phe Ser Leu Val Ser Asn Phe Phe Asn Glu Asn Phe Gln Lys Arg Pro Arg Gln Lys Leu Phe Glu Ile Gln Lys Lys Met Phe Pro Gln Tyr Trp Phe Glu Leu Thr Gln Gly Phe Ser Leu Leu Phe Tyr Gly Val Gly Ser Lys Arg Asn Phe Leu Glu Glu Phe Ala Ile Asp Tyr Leu Ser Pro Lys Ile Ala Tyr Ser Gln Leu Ala Tyr Glu Asn Glu Leu Gln Gln Asn Lys Pro Val Asn Ser Ile Pro Cys Leu Ile Leu Asn Gly Tyr Asn Pro Ser Cys Asn Tyr Arg Asp Val Phe Lys Glu Ile Thr Asp Leu Leu Val Pro Ala Glu Leu Thr Arg Ser Glu Thr Lys Tyr Trp Gly Asn His Val Ile Leu Gln Ile Gln Lys Met Ile Asp Phe Tyr Lys Asn Gln Pro Leu Asp Ile Lys Leu Ile Leu Val Val His Asn Leu Asp Gly Pro Ser Ile Arg Lys Asn Thr Phe Gln Thr Met Leu Ser Phe Leu Ser Val Ile Arg Gln Ile Ala Ile Val Ala Ser Thr Asp His Ile Tyr Ala Pro Leu Leu Trp Asp Asn Met Lys Ala Gln A~n Tyr Asn Phe Val Phe His Asp Ile Ser Asn Phe Glu Pro Ser Thr Val Glu Ser Thr Phe Gln Asp Val Met Lys Met Gly Lys Ser Asp Thr Ser Ser Gly Ala Glu Gly Ala Lys Tyr WO 95/16694 2 1 7 8 9 ~ 5 PCT/US94/14563 Val Leu Gln Ser Leu Thr Val Asn Ser Lys Lys Met Tyr Lys Leu Leu Ile Glu Thr Gln Met Gln A~n Met Gly Asn Leu Ser Ala Asn Thr Gly Pro Lys Arg Gly Thr Gln Arg Thr Gly Val Glu Leu Lys Leu Phe Asn 0 His Leu Cys Ala Ala ABP Phe Ile Ala Ser Asn Glu Ile Ala Leu Arg Ser Met Leu Arg Glu Phe Ile Glu His Lys Met Ala Asn Ile Thr Lys A~n A~n Ser Gly Met Glu Ile Ile Trp Val Pro Tyr Thr Tyr Ala Glu Leu Glu Lys Leu Leu Lys Thr Val Leu Asn Thr Leu (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:

TTCCAGCATG TCTTTGCGCA GATCCAAATC lll~lll~lC TTGAAATTTA TTCAGTAAAT 120 TAAAAGTCAG l.~ll~AGTA GCATTCATCT TCTTGGTAAG 1~lllll~l L G 1 ~lllGAAA 180 AAGAGTTCCT GAAGlli~lC TACTGTGAAT ATACTTTGCA CAIll~lllA ATTTTTAAAC 240 ACGCTATAAT ll~lCATA AAGAATTTTT TGTAGAATAG ~1.ll L ~1l AATAGGAAAA 300 CATTACCTGC l~ll~lATTA TCTTTATATT TAGTAAGACC AG~r-~AACG CTACACGTGA 480 TAACGCCTTT TTTAGTGTCT TTTTGATATT TACTGACGTA lllllCCGCA CCGTAATTTG 600 AGTAGTCCAA CAGGATGAGC GACCTTAACC AATCr~AAAA GATGAACGTC AGCGAGTTTG 720 ATAAACACAT lCC~lll~lC AAACTTCTAT CAGGCAAAGA ATCGGAAGTG AACGTGGAAA 840 65 TCGATAATAT TGAAGCAGAC TTGAAAGCAG AGATTTCAGA C~llllATAT AGTGAAACTA 960 CT~A~-~AAAG GCGATGCTTT AACACTATTT TCCTATTAGG TT~-A~-ATAGT ACGAcAAAAA 1020 WO95/16694 : 2 1 78965 PCTJUS94/14563 TTGAACTTAA A~-ACr-~ATCT TCTCGCTACA ACC1l~lGAT TGAATTGACT CC~-AAA~-AAT 1080 CTCCGAATGT AAGAATGATG ~lC6~AGGT CTATGTACAA ACTTTACAGC GCAGCTGATG 1140 AGCAAAACAA TGATGTATCA TACGATCTGT CACTTGTGGA AAACTTCAAA AGG~llll~G 1260 ~AAAA~-ACTT AGCAATGGTA TTTAATTTTA AAGATGTAGA TTCTATTAAC TTCAACACAT 1320 TGGATAACTT CATAATTCTA TTGAAAAGTG CCTTCAAGTA TGACCATGTT AAAATpA~TT 1380 TAATCTTTAA TATTAATACA AA~l~AA ATATTGAGAA AAATTTGAGA CAATCAACCA 1440 TACGACTTCT GAA~-AGAAAT TAT~ATAAPC TAGACGTGTC GAGTAATAAA GGATTTAAGT 1500 ACGGAAACCA AA~ ~AA AG~ GG ATACGGTTGA TGGCAAACTA AA~ CAG 1560 A,C6,,,,~1 GGAATTCATT CTCAGCAAGA TGGCAAATAA TACTAATCAC AACTTACAAT 1620 TATTGACGAA GATGCTGGAT TA,,C6,1GA l61C6lACTT TTTCCAGAAT GCCl.ll`CAG 1680 TATTCATTGA CC~l6TAAAT GTTGATTTTT TGAACGACGA CTACTTAAAA ATACTGAGCA 1740 25 GAl~,~C~AC ATTCATGTTC l~ CGAAG GTCTTATAAA GCAGCATGCT CCTGCTGACG 1800 AAAl~ .C ATTATTGACA AACAAAAACA GAGGCCTAGA AGAC,~ l GTTGAGTTTT 1860 TGGTAA~-A~-A GAACCCGATT AACGGGCATG CTAAGTTTGT TGCTCGATTC CTCr-AA~-AAG 1920 35 AACCCATTGA TACAATTTTT CAAGAGCTAT TTACTTTGGA ~AACAGAAGT GGATTACTTA 2100 CCCAGTCGAT lllCC~l TACAAGTCAA ATATCGAAGA TAACTTACTA AGTTGGGAGC 2160 AGG~GCTGCC TTCGCTTGAT AAAC-AAAATT ATGATACTCT TTCTGGAGAT TTGGATAAAA 2220 45 T~ATAAr.AAA AGAlCC~lCC AACACCAAAC TCTTAGAACT AGCAGAAACA CCGGACGCAT 2400 TTGArAAAGT AGCACTAATT TTATTCATGC AAGCAATCTT CGCCTTTGAA AACATGGGTC 2460 A~A-c~rGT l~ lAATG AACAGTCTAC CTGTATCTCA TCAll~ l GTGTTAACTA 2640 TTATTATTAT TATTATCGAA TGGAGGGTAA TATTATGTAT AGGTAAAATA AATAr.~TAGT 2700 60 ( 2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 615 amino acids (B TYPE: amino acid (C STRANDEDNESS: ~ingle (D TOPOLOGY: 1inear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Met Ser Asp Leu Asn Gln Ser Lys Lys Met Asn Val Ser Glu Phe Ala Asp Ala Gln Arg Ser His Tyr Thr Val Tyr Pro Ser Leu Pro Gln Ser Asn Lys Asn Asp Lys His Ile Pro Phe Val Lys Leu Leu Ser Gly Lys Glu Ser Glu Val AEn Val Glu Lys Arg Trp Glu Leu Tyr His Gln Leu His Ser His Phe His Asp Gln Val Asp His Ile Ile Asp Asn Ile Glu Ala Asp Leu Lys Ala Glu Ile Ser Asp Leu Leu Tyr Ser Glu Thr Thr Gln Lys Arg Arg Cys Phe Asn Thr Ile Phe Leu Leu Gly Ser Asp Ser Thr Thr Lys Ile Glu Leu Lys Asp Glu Ser Ser Arg Tyr Asn Val Leu Ile Glu Leu Thr Pro Lys Glu Ser Pro Asn Val Arg Met Met Leu Arg Arg Ser Met Tyr Lys Leu Tyr Ser Ala Ala Asp Ala Glu Glu His Pro Thr Ile Lys Tyr Glu Asp Ile Asn Asp Glu Asp Gly Asp Phe Thr Glu Gln Asn Asn Asp Val Ser Tyr Asp Leu Ser Leu Val Glu Asn Phe Lys Arg Leu Phe Gly Lys Asp Leu Ala Met Val Phe Asn Phe Lys Asp Val Asp Ser Ile Asn Phe Asn Thr Leu Asp Asn Phe Ile Ile Leu Leu Lys Ser Ala Phe Lys Tyr Asp His Val Lys Ile Ser Leu Ile Phe Asn Ile Asn Thr Asn Leu Ser Asn Ile Glu Lys Asn Leu Arg Gln Ser Thr Ile Arg Leu Leu Lys Arg Asn Tyr His Lys Leu Asp Val Ser Ser Asn Lys Gly Phe Lys Tyr Gly Asn Gln Ile Phe Gln Ser Phe Leu Asp Thr Val Asp Gly Lys Leu Asn Leu Ser Asp Arg Phe Val Glu Phe Ile Leu Ser Lys Met Ala Asn Asn Thr Asn His Asn Leu Gln Leu Leu Thr Lys Met Leu Asp Tyr Ser Leu Met Ser Tyr Phe Phe Gln Asn Ala Phe Ser Val Phe Ile Asp Pro Val Asn Val Asp Phe Leu Asn Asp Asp Tyr Leu Lys WO 95/16694 ' 2 1 7 8 9 6 5 PCT/US94/14563 Ile Leu Ser Arg Cys Pro Thr Phe Met Phe Phe Val Glu Gly Leu Ile Lys Gln His Ala Pro Ala Asp Glu Ile Leu Ser Leu Leu Thr Asn Lys Asn Arg Gly Leu Glu Glu Phe Phe Val Glu Phe Leu Val Arg Glu Asn Pro Ile Asn Gly His Ala Lys Phe Val Ala Arg Phe Leu Glu Glu Glu Leu Asn Ile Thr Asn Phe Asn Leu Ile Glu Leu Tyr His Asn Leu Leu Ile Gly Lys Leu Asp Ser Tyr Leu Asp Arg Trp Ser Ala Cys Lys Glu Tyr Lys Asp Arg Leu His Phe Glu Pro Ile Asp Thr Ile Phe Gln Glu Leu Phe Thr Leu Asp Asn Arg Ser Gly Leu Leu Thr Gln Ser Ile Phe Pro Ser Tyr Lys Ser Asn Ile Glu Asp Asn Leu Leu Ser Trp Glu Gln Val Leu Pro Ser Leu Asp LYB Glu Asn Tyr Asp Thr Leu Ser Gly Asp Leu Asp Lys Ile Met Ala Pro Val Leu Gly Gln Leu Phe Lys Leu Tyr Arg Glu Ala Asn Met Thr Ile Asn Ile Tyr Asp Phe Tyr Ile Ala Phe Arg Glu Thr Leu Pro Lys Glu Glu Ile Leu Asn Phe Ile Arg Lys Asp Pro Ser Asn Thr Lys Leu Leu Glu Leu Ala Glu Thr Pro Asp Ala Phe Asp Lys Val Ala Leu Ile Leu Phe Met Gln Ala Ile Phe Ala Phe Glu Asn Met Gly Leu Ile Lys Phe Gln Ser Thr Lys Ser Tyr Asp Leu Val Glu Lys Cys Val Trp Arg Gly ~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 ) MnT FCUT-~ TYPE: cDNA

(xi) ~Q~N~: DESCRIPTION: SEQ ID NO:7:
65 CTCGAGGCCA C~AA~-AAGAG AAA~-AGA~AGA GC~-ATATT GACTGGAGTG CAGCCAGAGG 60 2 1 7~3 i65 CTACAAGGAT AGG~-~.AA CTAACAAAAA GACTACTGAT GAGCAACCAA AAATCCAGAA 240 5 ~.~..,...AT GA.~,-...AC GTACTGAAGA TGATGATGAA QATGAAGAGG CTGAAAAGCA 300 AAATGGAGAC GCAAAA~-AAA ACAAAGTTGA TGCGGCAGTT GAAAAGCTAC AG~.ATAAAAC 360 TGCTCAATTG ACTGTTGAAG ATGGTGACAA TTGGGAAGTT GTTGGTAAGA AATA~AGTGT 420 TGTATGATGA TAAAATGTAC ATTTGTATTT A~-.-GCT .1....~--- ~..G.l.--C 480 TA~.`.C~.- TCTACCAGGT ATTCTAACTC TATTATATAA TTAAAAAAAA AATAAc~ATA 540 15 TA..--~-AT TAAGTTTCAT ACAl~l~llC AAGTGTATT~ TTGGATTTAT CAll.llClA 600 TGTGAGGTAA Gl..l.GAAT GTCCCATTTT C~l..C6111 TTGGAAAGTT CTAAGAAAAA 660 AACTACTAAT ATCGGTAATA TTCAAAAGAA GAAGCATGAC TATAAGCGAA G~lCGl~lAT 780 CACCGCAAGT CAA-~l.~-C CCAATAAAGA GGCACTCAAA CGAAGAGGTA GAGGAGACTG 840 TTGGllCC~l TCAAAGAAGG TTACTGCAGC AACTTTATGG CACACTTCCT ACGGACGAAA 960 CCATTATTCA G~A~GAGAGT CATTCAGTAA ll~lCClGGG GCCrA~-A~-AA AGTTACAAAA 1080 CATACTTATT AGACTATGAA ~1~1~11~1 TGCAACAATC TTATAAAGAG CAGTTTATAA 1140 TAGAGACTAT TAGCAGTGGT TCTTTGACAG AACl-,11.GA GAAAATTCTT TTACTCTTAG 1320 ATTCr-AC~AC GAAGACAAGA AATrAAr-ATA GTGGTGAGGT TGACAGAGAG AGT~TAACAA 1380 Ar-ATAA~A-GT .~...l.ATA TTCGATGAAA TTGATACATT TGCTGGGCCT GTGAGGCAAA 1440 45 CTTTATTATA CAAl~l lll GACATGGTAG AACATTCTCG GGTACCTGTT TGCATTTTTG 1500 AAAATTTTGG TTCACTCTGC ACTGCCATAA AAlCGl~l.C lll~'.lGAC ATATACAATA 1860 Ar~AA~AAcT ATCTAATAAT TTAACAGGAA GGCTCCAATC TTTATCCGAT TTAGAGTTAG 1920 ~ 217~3965 TTA~CGA~-AA ATCAGCCGTT GGTTTGAGAG ATAATGCGAC CGCAGCATTT TACGCTAGCA 2220 ~ AGGA ATTAAGAAGA ATTATCCCCA AATCTAATAT GTACTACTCC TGGACACAAC 2340 TGTGAATCTT GG~AA~AATA TAr~ AC~TT TTATTGGCGG TAGCAACTCT GATATTCCAC 2400 (2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A LENGTH: 529 amino acids B TYPE: amino acid C STRANDEDNESS: single Dl TOPOLOGY: linear ( ii ) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Met Thr Ile Ser Glu Ala Arg Leu Ser Pro Gln Val Asn Leu Leu Pro Ile Lys Arg His Ser Asn Glu Glu Val Glu Glu Thr Ala Ala Ile Leu Lys Lys Arg Thr Ile Asp Asn Glu Lys Cys LYB Asp Ser Asp Pro Gly Phe Gly Ser Leu Gln Arg Arg Leu Leu Gln Gln Leu Tyr Gly Thr Leu Pro Thr Asp Glu Lys Ile Ile Phe Thr Tyr Leu Gln Asp Cys Gln Gln 40 Glu Ile Asp Arg Ile Ile Lys Gln Ser Ile Ile Gln Lys Glu Ser His Ser Val Ile Leu Val Gly Pro Arg Gln Ser Tyr Lys Thr Tyr Leu Leu Asp Tyr Glu Leu Ser Leu Leu Gln Gln Ser Tyr Lys Glu Gln Phe Ile Thr Ile Arg Leu Asn Gly Phe Ile His Ser Glu Gln Thr Ala Ile Asn Gly Ile Ala Thr Gln Leu Glu Gln Gln Leu Gln Lys Ile His Gly Ser 55 Glu Glu Lys Ile Asp Asp Thr Ser Leu Glu Thr Ile Ser Ser Gly Ser Leu Thr Glu Val Phe Glu Lys Ile Leu Leu Leu Leu Asp Ser Thr Thr Lys Thr Arg Asn Glu Asp Ser Gly Glu Val Asp Arg Glu Ser Ile Thr Lys Ile Thr Val Val Phe Ile Phe Asp Glu Ile Asp Thr Phe Ala Gly Pro Val Arg Gln Thr Leu Leu Tyr Asn Leu Phe Asp Met Val Glu His WOg5/16694 ; 2 1 78965 PCT~S94~l4s63 Ser Arg Val Pro Val Cys Ile Phe Gly CYR Thr Thr Lys Leu Asn Ile Leu Glu Tyr Leu Glu Lys Arg Val Lys Ser Arg Phe Ser Gln Arg Val Ile Tyr Met Pro Gln Ile Gln Asn Leu Asp Asp Met Val Asp Ala Val Arg Asn Leu Leu Thr Val Arg Ser Glu Ile Ser Pro Trp Val Ser Gln Trp Asn Glu Thr Leu Glu Lys Glu Leu Ser Asp Pro Arg Ser Asn Leu Asn Arg His Ile Arg Met Asn Phe Glu Thr Phe Arg Ser Leu Pro Thr Leu Lys Asn Ser Ile Ile Pro Leu Val Ala Thr Ser Lys Asn Phe Gly Ser Leu Cys Thr Ala Ile Lys Ser Cys Ser Phe Leu Asp Ile Tyr Asn Lys Asn Gln Leu Ser Asn Asn Leu Thr Gly Arg Leu Gln Ser Leu Ser Asp Leu Glu Leu Ala Ile Leu Ile Ser Ala Ala Arg Val Ala Leu Arg Ala Lys Asp Gly Ser Phe Asn Phe Asn Leu Ala Tyr Ala Glu Tyr Glu Lys Me~t Ile Lys Ala Ile Asn Ser Arg Ile Pro Thr Val Ala Pro Thr Thr Asn Val Gly Thr Gly Gln Ser Thr Phe Ser Ile Asp Asn Thr Ile Ly6 Leu Trp Leu Lys Lys Asp Val Lys Asn Val Trp Glu Asn Leu Val Gln Leu Asp Phe Phe Thr Glu Lys Ser Ala Val Gly Leu Arg Asp Asn Ala Thr Ala Ala Phe Tyr Ala Ser Asn Tyr Gln Phe Gln Gly Thr Met Ile Pro Phe Asp Leu Arg Ser Tyr Gln Met Gln Ile Ile Leu Gln Glu Leu Arg Arg Ile Ile Pro Lys Ser Asn Met Tyr Tyr Ser Trp Thr Gln 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

WO 9S/16694 2 1 7 8 9 6 5 Pcr/Us94~l4563 (xi) S~QD~ DESCRIPTION: SEQ ID NO:9:

GAAGGAACTC TCCCCAGAGA AAGCATTGTT CAAAAGTTAA AlGl~l.lAA GCAAGTAGCC 240 AGTATATAAC GCGAGGTTCA ATGGCCTCTT TACCATGAAA AAJUiUU~AAA AAAAAAAAAA 360 AAATTAGCCC TT~ArATAA TTAACACTCT TCTTTGATAT TTAAATCACA AGTACTTTTC 480 TTTAGGGAAT ATCAAACCAA ~.G.~.CGCA TCGTATATTT CTGCTGATCC AGACATAACT 600 25 TATTTTAATG CGAATCCAAA TTTGCATGCA GTATGGCTGG AAC~lGl-GA GTTGGTTTCT 720 CrPAAr-ATTC CCACCACAGA TTACGATCCT TTACAGGTTG AAGAGCCATT ~ GGTA 840 AAGACGTTGC ACAATATTTT TGTCCAATAT GAATCTTTGC AAGAAAAGAC TTG~ll~l.C 900 CCGAGGTATA ATGTGGACGA A~--l~lACT ATATTAGTGA TGTCTAGATG TGGCGAACTC 1140 ATGGAAGATT ~l.`-.~.ACG TAAGCGTATC ATTGAAGAGC AGATAACGGA CTGTACAGAC 1200 GTATCTCGCA TTACTAAGGA AAAcATATTT GAACCACTGG ~.~l~lACAA AAGTGCCATC 1380 AC~AAATC GTGATGACCT TGAGAACAGT CAAACTTACG ACTTATCAAT AATTTCGAAG 1500 TATCTGCTCA TAGCCTCATA TAl..Gl.`A TATCTGGAAC CTAGATACGA TGCGAGTATT 1560 55 ..C.~.AGGA AAACACGTAT ~ATA~AGGT AGAGCTGCTT ATGGACGAAG AAA~AAr~AAA 1620 TTCCAAGCTA TA..CC~lAT TCAAGGTAAG GCGGAGAGTG G..CC~lATC TGCACTTCGT 1740 GAGGAATCCT TAATGAAAGC GAATATCGAG Gl...l~AAA ATTTATCCGA ATTGCATACA 1800 W O95/16694 2 i 7 8 9 6 5 pCTrUS94/14S63 CAGGATACGT TCGAAACAAC AACTACGTTA TATAAATATT TATACATAGT GG~-ATAGAAT 204 GAACAATTAT CAAGTAAACC TTGTATTTTT .~CC~ACG CTCTACGCTC iGl-l~llGG 2100 ATATGGTAAT CAAArATTAA TACGTATAAC CGTTATTAAT TCAGTCCACT A~AA~CTATT 2160 AGTTTAGTTG ~C~l~llGG CGGCCGGCGA TAA`~ l CACTTGGTAT TCTTACCAGG 2280 ATTGAGCCTG Al,.. ~..... 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 Gln Thr Asn cys Leu Ala Ser Tyr Ile Ser Ala Asp Pro Asp Ile Thr Pro Ser Asn Leu Ile Leu Gln Gly Tyr Ser Gly Thr Gly Lys Thr Tyr Thr Leu Lys Lys Tyr Phe Asn Ala Asn Pro Asn Leu His Ala Val Trp Leu Glu Pro Val Glu Leu Val Ser Trp Lys Pro Leu Leu Gln Ala Ile Ala Arg Thr 65 70 75 8,0 Val Gln Tyr Lys Leu Lys Thr Leu Tyr Pro Asn Ile Pro Thr Thr Asp Tyr Asp Pro Leu Gln Val Glu Glu Pro Phe Leu Leu Val Lys Thr Leu His Asn Ile Phe Val Gln Tyr Glu Ser Leu Gln Glu Lys Thr Cys Leu Phe Leu Ile Leu Asp Gly Phe Asp Ser Leu Gln Asp Leu Asp Ala Ala Leu Phe Asn Lys Tyr Ile Lys Leu Asn Glu Leu Leu Pro Lys Asp Ser Lys Ile Asn Ile Lys Phe Ile Tyr Thr Met Leu Glu Thr Ser Phe Leu Gln Arg Tyr Ser Thr His Cys Ile Pro Thr Val Met Phe Pro Arg Tyr Asn Val Asp Glu Val Ser Thr Ile Leu Val Met Ser Arg Cys Gly Glu Leu Met Glu Asp Ser Cys Leu Arg Lys Arg Ile Ile Glu Glu Gln Ile 21 7~965 W 095/16694 PCTrUS94/14563 Thr Asp Cy8 Thr Asp Asp Gln Phe Gln Asn Val Ala Ala Asn Phe Ile His Leu Ile Val Gln Ala Phe His Ser Tyr Thr Gly Asn Asp Ile Phe Ala Leu Asn Asp Leu Ile Acp Phe Lys Trp Pro Lys Tyr Val Ser Arg Ile Thr Lys Glu Asn Ile Phe Glu Pro Leu Ala Leu Tyr Lys Ser Ala Ile Lys Leu Phe Leu Ser Thr Asp Asp Asn Leu Ser Glu Asn Gly Gln Gly Glu Ser Ala Ile Thr Thr Asn Arg Asp Asp Leu Glu Asn Ser Gln Thr Tyr Asp Leu Ser Ile Ile Ser Lys Tyr Leu Leu Ile Ala Ser Tyr Ile Cys Ser Tyr Leu Glu Pro Arg Tyr Asp Ala Ser Ile Phe Ser Arg Lys Thr Arg Ile Ile Gln Gly Arg Ala Ala Tyr Gly Arg Arg Lys Lys Lys Glu Val Asn Pro Arg Tyr Leu Gln Pro Ser Leu Phe Ala Ile Glu Arg Leu Leu Ala Ile Phe Gln Ala Ile Phe Pro Ile Gln Gly Lys Ala Glu Ser Gly Ser Leu Ser Ala Leu Arg Glu Glu Ser Leu Met Lys Ala Asn Ile Çlu Val Phe Gln Asn Leu S~r Glu Leu His Thr Leu Lys Leu 0 . Ile Ala Thr Thr Met Asn Lys Asn Ile Asp Tyr Leu Ser Pro Lys Val Arg Trp Lys Val Asn Val Pro Trp Glu Ile Ile Lys Glu Ile Ser Glu Ser Val His Phe Asn Ile Ser Asp Tyr Phe Ser Asp Ile His Glu (2) INFORMATION FOR SEQ ID NO:ll:
( i ) s~:Qu~l._~ CHARACTERISTICS:
A) LENGTH: 1975 base pairs B) TYPE: nucleic acid C) STRANDEDNESS: double ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
( ix ) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 443..1747 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll:

AA~AATAAG GAATGTTACC TATGCTAGTC GCAACTCTCT CGTAAGTTGA GGGTTGCTAA 120 W O 95/16694 2 1 7 8 9 6 5 PcTrus94ll4563 CAGAAAAACG ATGAGAAGAA ACTTTTGAAA AATATTGTGT GAAAGCAGCA cGA-AA~Ar-AG 18( TATGAAAAAA GAATGCGGGC ~CCG~AAAG AGCTAGAATC GCAAGTGTCC AGAATATGCA 240 AGGCTTTCGA ATACACTCCT CACGCTTCTC TTCAGCAAAA ATCAACTCTT TGT~-ATAAAA 300 ~ ATTT ~ TGCCG--~.. TACGTTAGTA AGAAATCGGC ATTr~AAAAAA- 360 AAAATCTCAC ACTAAAATTG ~AGAAAAAAG TGTACAATAT CAGTAAATAA AATTGGCCAA 420 AArAATAc~A TTAAAACCAG TC ATG TCC ATG CAA CAA GTC CAA CAT TGT GTC 472 Met Ser Met Gln Gln Val Gln His Cys Val l 5 10 Ala Glu Val Leu Arg Leu Asp Pro Gln Glu Ly~ Pro Asp Trp Ser Ser 20 Gly Tyr Leu Lys Lys Leu Thr Asn Ala Thr Ser Ile Leu Tyr Asn Thr Ser Leu Asn Lys Val Met Leu Lys Gln Asp Glu Glu Val Ala Arg Cys His Ile Cys Ala Tyr Ile Ala Ser Gln Lys Met Asn Glu Lys His Met Pro Asp Leu Cys Tyr Tyr Ile Asp Ser Ile Pro Leu Glu Pro Lys Lys Ala Lys His Leu Met Asn Leu Phe Arg Gln Ser Leu Ser Asn Ser Ser lO0 105 Pro Met Lys Gln Phe Ala Trp Thr Pro Ser Pro Lys Lys Asn Lys Arg Ser Pro Val Lys Asn Gly Gly Arg Phe Thr Ser Ser Asp Pro Lys Glu Leu Arg Asn Gln Leu Phe Gly Thr Pro Thr Lys Val Arg Lys Ser Gln Asn Asn Asp Ser Phe Val Ile Pro Glu Leu Pro Pro Met Gln Thr Asn Glu Ser Pro Ser Ile Thr Arg Arg Lys Leu Ala Phe Glu Glu Asp Glu Asp Glu Asp Glu Glu Glu Pro Gly Asn Asp Gly Leu Ser Leu Lys Ser 65 His Ser Asn Lys Ser Ile Thr Gly Thr Arg Asn Val Asp Ser Asp Glu WO95/16694 ; :. 2 1 78965 PCT/US94/14563 Tyr Glu Asn His Glu Ser Asp Pro Thr Ser Glu Glu Glu Pro Leu Gly Val Gln Glu Ser Arg Ser Gly Arg Thr Lys Gln Asn Lys Ala Val Gly Lys Pro Gln Ser Glu Leu Lys Thr Ala Lys Ala Leu Arg Lys Arg Gly Arg Ile Pro Asn Ser Leu Leu Val Lys Lys Tyr Cys Lys Met Thr Thr Glu Glu Ile Ile Arg Leu Cys Asn Asp Phe Glu Leu Pro Arg Glu Val Ala Tyr Ly~ Ile Val Asp Glu Tyr Asn Ile Asn Ala Ser Arg Leu Val Cy~ Pro Trp Gln Leu Val Cys Gly Leu Val Leu Asn Cys Thr Phe Ile Val Phe Asn Glu Arg Arg Arg Lys Asp Pro Arg Ile Asp His Phe Ile Val Ser Lys Met Cys Ser Leu Met Leu Thr Ser Lys Val Asp Asp Val ~350 355 360 Ile Glu Cys Val Lys Leu Val Lys Glu Leu Ile Ile Gly Glu Lys Trp Phe Arg Asp Leu Gln Ile Arg Tyr Asp Asp Phe Asp Gly Ile Arg Tyr Asp Glu Ile Ile Phe Arg Lys Leu Gly Ser Met Leu Gln Thr Thr Asn Ile Leu Val Thr Asp Asp Gln Tyr Asn Ile Trp Lys Lys Arg Ile Glu Met Asp Leu Ala Leu Thr Glu Pro Leu AAAAGTATAT ATTTGACCAA TACCTGACAT A~ ~.AAA GCATGCCTTT AGCCCTATAA 1827 CGAGCTAATG TTAGCTCCAT CTTTGCACTT ATGATTGGAT CAGCCCTCAA ACG~L~..~. 1887 65 ~ 2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 435 amino acids WO 95/16694 ~ 2 ~ 7 8 9 6 5 PCT/US94/]4563 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Met Ser Met Gln Gln Val Gln His Cys Val Ala Glu Val Leu Arg Leu Asp Pro Gln Glu Lys Pro Asp Trp Ser Ser Gly Tyr Leu Lys Lys Leu Thr Asn Ala Thr Ser Ile Leu Tyr Asn Thr Ser Leu Asn Lys Val Met Leu Lys Gln Asp Glu Glu Val Ala Arg Cys His Ile Cys Ala Tyr Ile 20 Ala Ser Gln Lys Met A~n Glu Lys His Met Pro Asp Leu Cys Tyr Tyr Ile A~p Ser Ile Pro Leu Glu Pro Lys Lys Ala Lys His Leu Met Asn Leu Phe Arg Gln Ser Leu Ser Asn Ser Ser Pro Met Lys Gln Phe Ala Trp Thr Pro Ser Pro Lys Lys Asn Lys Arg Ser Pro Val Lys Asn Gly Gly Arg Phe Thr Ser Ser Asp Pro Lys Glu Leu Arg Asn Gln Leu Phe Gly Thr Pro Thr Lys Val Arg Lys Ser Gln Asn Asn Asp Ser Phe Val ; Ile Pro Glu Leu Pro Pro Met.Gln Thr Asn Glu Ser Pro Ser Ile Thr . 165 170 . 175 Arg Arg Lys Leu Ala Phe Glu Glu Asp Glu.Asp Glu Asp Glu Glu Glu 45 Pro Gly Asn Asp Gly Leu Ser Leu Lys Ser His Ser Asn Lys Ser Ile Thr Gly Thr Arg Asn Val Asp Ser Asp Glu Tyr Glu Asn His Glu Ser Asp Pro Thr Ser Glu Glu Glu Pro Leu Gly Val Gln Glu Ser Arg Ser Gly Arg Thr Lys Gln Asn Lys Ala Val Gly Lys Pro Gln Ser Glu Leu Lys Thr Ala Lys Ala Leu Arg Lys Arg Gly Arg Ile Pro Asn Ser Leu 60 Leu Val Lys Lys Tyr Cys Lys Met Thr Thr Glu Glu Ile Ile Arg Leu Cys Asn Asp Phe Glu Leu Pro Arg Glu Val Ala Tyr Lys Ile Val Asp Glu Tyr Asn Ile Asn Ala Ser Arg Leu Val Cys Pro Trp Gln Leu Val 2 t 78965 WO 95/16694 ~ . . PCTtUS94tl4563 Cys Gly Leu Val Leu Asn Cys Thr Phe Ile Val Phe Asn Glu Arg Arg Arg Lys Asp Pro Arg Ile Asp His Phe Ile Val Ser Lys Met Cys Ser Leu Met Leu Thr Ser Lys Val Asp Asp Val I le Glu Cys Val Lys Leu 0 Val LYB Glu Leu Ile Ile Gly Glu Lys Trp Phe Arg Asp Leu Gln Ile Arg Tyr ABP Asp Phe ABP Gly Ile Arg Tyr ABP Glu Ile Ile Phe Arg LYB Leu Gly Ser Met Leu Gln Thr Thr Asn Ile Leu Val Thr Asp Asp Gln Tyr Asn I le Trp LYB LYB Arg I le Glu Met Asp Leu Ala Leu Thr Glu Pro Leu

Claims (10)

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 ORC1.

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.