HUMAN DNA MISMATCH REPAIR PROTEINS

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptides of the present invention are human homologs of the prokaryotic mutL4 gene and are hereinafter referred to as hMLHl, hMLH2 and hMLH3.

In both prolaryotes and eukaryotes, the DNA mismatch repair gene plays a prominent role in the correction of errors made during DNA replication and genetic recombination. The E. coli methyl-directed DNA mismatch repair system is the best understood DNA mismatch repair system to date. In E. coli , this repair pathway involves the products of the mutator genes mutS, mutL, mutH, and uvrD. Mutants of any one of these genes will reveal a mutator phenotype. MutS is a DNA mismatch-binding protein which initiates this repair process, uvrD is a DNA helicase and MutH is a latent

endonuclease that incises at the unmethylated strands of a hemi-methylated GATC sequence. MutL protein is believed to recognize and bind to the mis atch-DNA-MutS-MutH complex to enhance the endonuclease activity of MutH protein. After the unmethylated DNA strand is cut by the MutH, single-stranded DNA-binding protein, DNA polymerase III, exonuclease I and DNA ligase are required to complete this repair process (Modrich P., Annu. Rev. Genetics, 25:229-53 (1991)) .

Elements of the E. coli MutLHS system appears to be conserved during evolution in prokaryotes and eukaryotes. Genetic study analysis suggests that Saccharomyces cerevisiae has a mismatch repair system similar to the bacterial MutLHS system. In S . cerevisiae, at least two MutL homologs, PMSl and MLHl , have been reported. Mutation of either one of them leads to a mitotic mutator phenotype (Prolla et al, Mol. Cell. Biol. 14:407-415 (1994)). At least three MutS homologs have been found in S . cerevisiae, namely MSH1 , MSH2 , and MSH3 . Disruption of the MSH2 gene affects nuclear mutation rates. Mutants in S . cerevisae , MSH2 , PMSl , and MLHl have been found to exhibit increased rates of expansion and contraction of dinucleotide repeat sequences (Strand et al. , Nature, 365:274-276 (1993) ) .

It has been reported that a number of human tumors such as lung cancer, prostate cancer, ovarian cancer, breast cancer, colon cancer and stomach cancer show instability of repeated DNA sequences (Han et al. , Cancer, 53:5087-5089 (1993); Thibodeau et al. , Science 260:816-819 (1993); Risinger et al. , Cancer 53:5100-5103 (1993)). This phenomenon suggests that lack of the DNA mismatch repair is probably the cause of these tumors.

Little was known about the DNA mismatch repair system in humans until recently, the human homolog of the MutS gene was cloned and found to be responsible for hereditary nonpolyposis colon cancer (HNPCC) , (Fishel et al. , Cell, 75:1027-1038 (1993) and Leach et al., Cell, 75:1215-1225

(1993)) . HNPCC was first linked to a locus at chromosome 2pl6 which causes dinucleotide instability. It was then demonstrated that a DNA mismatch repair protein (MutS) homolog was located at this locus, and that C-->T transitional mutations at several conserved regions were specifically observed in HNPCC patients. Hereditary nonpolyposiε colorectal cancer is one of the most common hereditable diseases of man, affecting as many as one in two hundred individuals in the western world.

It has been demonstrated that hereditary colon cancer can result from mutations in several loci. Familial adenomatosis polyposis coli (APC) , linked to a gene on chromosome 5, is responsible for a small minority of hereditary colon cancer. Hereditary colon cancer is also associated with Gardner's syndrome, Turcot's syndrome, Peutz- Jaegherε syndrome and juvenile polyposis coli. In addition, hereditary nonpolyposis colon cancer may be involved in 5% of all human colon cancer. All of the different types of familial colon cancer have been shown to be transmitted by a dominant autosomal mode of inheritance.

In addition to localization of HNPCC, to the short arm of chromosome 2, a second locus has been linked to a pre¬ disposition to HNPCC (Lindholm, et al. , Nature Genetics, 5:279-282 (1993)) . A strong linkage was demonstrated between a polymorphic marker on the short arm of chromosome 3 and the disease locus.

This finding suggests that mutations on various DNA mismatch repair proteins probably play crucial roles in the development of human hereditary diseases and cancers.

HNPCC is characterized clinically by an apparent autosomal dominantly inherited predisposition to cancer of the colon, endometrium and other organs. (Lynch, H.T. et al., Gastroenterology, 104:1535-1549 (1993)). The identification of markers at 2pl6 and 3p2l-22 which were linked to disease in selected HNPCC kindred unequivocally

established its mendelian nature (Peltomaki, P. et al. , Science, 260:810-812 (1993)) . Tumors from HNPCC patients are characterized by widespread alterations of simple repeated sequences (microsatellites) (Aaltonen, L.A., et al. , Science, 260:812-816 (1993)). This type of genetic instability was originally observed in a subset (12 to 18% of sporadic colorectal cancers (Id. ) . Studies in bacteria and yeast indicated that a defect in DNA mismatch repair genes can result in a similar instability of microsatellites (Levinson, G. and Gutman, G.A. , Nuc. Acids Res. , 15:5325-5338 (1987)), and it was hypothesized that deficiency in mismatched repair was responsible for HNPCC (Strand, M. et al. , Nature, 365:274-276 (1993)) . Analysis of extracts from HNPCC tumor cell lines εhowed mismatch repair was indeed deficient, adding definitive support to this conjecture (Parsons, R.P., et al., Cell, 75:1227-1236 (1993)). As not all HNPCC kindred can be linked to the same loci, and as at least three genes can produce a similar phenotype in yeast, it seems likely that other mismatch repair genes could play a role in some cases of HNPCC. hMLHl is most homologous to the yeast mutL-homolog yMLHi while hMLH2 and hMLH3 have greater homology to the yeast mutL-homolog yPMSl (hMLH2 and hMLH3 due to their homology to yeast PMSl gene are sometimes referred to in the literature as hPMSl and hPMS2) . In addition to hMLHl, both the hMLH2 gene on chromosome 2q32 and the hMLH3 gene, on chromosome 7p22, were found to be mutated in the germ line of HNPCC patients. This doubles the number of genes implicated in HNPCC and may help explain the relatively high incidence of this disease.

In accordance with one aspect of the present invention, there are provided novel putative mature polypeptides which are hMLHl, hMLH2 and hMLH3, as well as biologically active and diagnostically or therapeutically useful fragments,

analogs and derivatives thereof. The polypeptides of the present invention are of human origin.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with still another aspect of the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to hMLHl, hMLH2 and hMLH3 sequences.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing an hMLHl, hMLH2 or hMLH3 nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said proteins.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptide, or polynucleotide encoding such polypeptide, for therapeutic purposes, for example, for the treatment of cancers.

In accordance with another aspect of the present invention there is provided a method of diagnosing a disease or a susceptibility to a disease related to a mutation in the hMLHl, hMLH2 or hMLH3 nucleic acid sequences and the proteins encoded by such nucleic acid sequences.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims .

Figure 1 illustrates the cDNA sequence and corresponding deduced amino acid sequence for the human DNA repair protein hMLHl. The amino acids are represented by their standard one-letter abbreviations. Sequencing was performed uεing a 373 Automated DNA sequencer (Applied Biosystems, Inc.) . Sequencing accuracy is predicted to be greater than 97% accurate.

Figure 2 illustrates the cDNA sequence and corresponding deduced amino acid sequence of hMLH2. The amino acids are represented by their standard one-letter abbreviations.

Figure 3 illustrates the cDNA sequence and corresponding deduced amino acid sequence of hMLH3. The amino acids are represented by their standard one-letter abbreviations.

Figure 4. Alignment of the predicted amino acid sequences of S. cerevisiae PMSl (yPMSl) , with the hMLH2 and hMLH3 amino acid sequences using MACAW (version 1.0) program. Amino acid in conserved blocks are capitalized and shaded on the mean of their pair-wise scores.

Figure 5. Mutational analysis of hMLH2. (A) IVSP analysis and mapping of the transcriptional stop mutation in HNPCC patient CW. Translation of codonε 1 to 369 (lane 1) , codons l to 290 (lane 2) , and codons l to 214 (lane 3) . CW is translated from the cDNA of patient CW, while NOR was translated from the cDNA of a normal individual. The arrowheadε indicate the truncated polypeptide due to the potential stop mutation. The arrows indicate molecular weight markers in kilodaltons. (B) Sequence analysis of CW indicates a C to T tranεition at codon 233 (indicated by the arrow) . Lanes l and 3 are sequence derived from control

patients; lane 2 is sequence derived from genomic DNA of CW. The ddA mixes from each sequencing mix were loaded in adjacent lanes to facilitate comparison aε were those for ddC, ddD, and ddT mixes.

Figure 6. Mutational analysis of hMLH3. (A) IVSP analysis of hMLH3 from patient GC. Lane GC is from fibroblasts of individual GC; lane GCx is from the tumor of patient GC; lanes NORi and 2 are from normal control individuals. FL indicates full-length protein, and the arrowheadε indicate the germ line truncated polypeptide. The arrowε indicate molecular weight markerε in kilodaltonε (B) PCR analyεiε of DNA from a patient GC shows that the lesion in present in both hMLH3 alleleε in tumor cells. Amplification was done using primers that amplify 5', 3', or within (MID) the region deleted in the cDNA. Lane 1, DNA derived from fibroblasts of patient GC; lane 2, DNA derived from tumor of patient GC; lane 3, DNA derived from a normal control patient; lane 4, reactions without DNA template. Arrows indicate molecular weight in base pairs.

In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode for the mature polypeptides having the deduced amino acid sequence of Figures l, 2 and 3 (SEQ ID No. 2, 4 and 6) or for the mature polypeptides encoded by the cDNA of the clone deposited aε ATCC Deposit No. 75649, 75651, 75650, deposited on January 25, 1994.

ATCC Deposit No. 75649 is a cDNA clone which contains the full length sequence encoding the human DNA repair protein referred to herein as hMLHl; ATCC Deposit No. 75651 is a cDNA clone containing the full length cDNA sequence encoding the human DNA repair protein referred to herein as hMLH2; ATCC Deposit No. 75650 is a cDNA clone containing the full length DNA εequence referred to herein aε hMLH3.

Polynucleotides encoding the polypeptides of the present invention may be obtained from one or more libraries prepared

from heart, lung, prostate, spleen, liver, gallbladder, fetal brain and testeε tissues . The polynucleotides of hMLHl were discovered from a human gallbladder cDNA library. In addition, six cDNA clones which are identical to the hMLHl at the N-terminal ends were obtained from human cerebellum, eight-week embryo, fetal heart, HSC172 cells and Jurket cell cDNA librarieε . The hMLHl gene containε an open reading frame of 756 amino acids encoding for an 85kD protein which exhibits homology to the bacterial and yeast mutL proteins. However, the 5' non-tranεlated region waε obtained from the cDNA clone obtained from the fetal heart for the purpoεe of extending the non-tranεlated region to deεign the oligonucleotideε .

The hMLH2 gene waε derived from a human T-cell lymphoma cDNA library. The hMLH2 cDNA clone identified an open reading frame of 2 , 796 base pairs flanked on both sides by in-frame termination codons. It is structurally related to the yeast PMSl family. It contains an open reading frame encoding a protein of 934 amino acid residues. The protein exhibits the highest degree of homology to yeast PMSl with 27% identity and 82 % similarity over the entire protein.

A second region of significant homology among the three PMS related proteins is in the carboxyl terminus, between codons 800 to 900. This region shares a 22% and 47% homology between yeast PMSl protein and hMLH2 and hMLH3 proteins, respectively, while very little homology of thiε region waε obεerved between these proteins, and the other yeast mutL homolog, yMLHl.

The hMLH3 gene waε derived from a human endometrial tumor cDNA library. The hMLH3 clone identified a 2,586 base pair open reading frame. It is εtructurally related to the yPMS2 protein family. It containε an open reading frame encoding a protein of 862 amino acid reεidueε. The protein exhibitε the highest degree of homology to yPMS2 with 32%

identity and 66% similarity over the entire amino acid sequence.

It is significant with respect to a putative identification of hMLHl, hMLH2 and hMLH3 that the GFRGEAL domain which is conserved in mutL homologs derived from E. coli is conserved in the amino acid sequences of , hMLHl, hMLH2 and hMLH3.

The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figures l, 2 and 3 (SEQ ID No. l) or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptides aε the DNA of Figureε 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or the deposited cDNA(ε) .

The polynucleotides which encode for the mature polypeptides of Figures l, 2 and 3 (SEQ ID No. 2, 4 and 6) or for the mature polypeptides encoded by the deposited cDNAs may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such aε introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivativeε of the polypeptideε having the deduced amino acid εequenceε of Figureε 1, 2 and 3 (SEQ

ID No. 2, 4 and 6) or the polypeptideε encoded by the cDNA of the depoεited cloneε . The variantε of the polynucleotides may be a naturally occurring allelic variant of the polynucleotideε or a non-naturally occurring variant of the polynucleotideε .

Thus, the present invention includes polynucleotideε encoding the same mature polypeptides as shown in Figureε l, 2 and 3 (SEQ ID No. 2, 4 and 6) or the same mature polypeptides encoded by the cDNA of the depoεited cloneε aε well aε variantε of such polynucleotides which variantε encode for a fragment, derivative or analog of the polypeptides of Figureε 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or the polypeptides encoded by the cDNA of the deposited clones. Such nucleotide variants include deletion variantε, subεtitution variantε and addition or inεertion variantε .

Aε hereinabove indicated, the polynucleotides may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figureε 1, 2 and 3 (SEQ ID No. 1, 3 and 5) or of the coding sequence of the depoεited clones. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a subεtitution, deletion or addition of one or more nucleotideε, which does not subεtantially alter the function of the encoded polypeptide.

The polynucleotideε of the preεent invention may alεo have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptides of the present invention. The marker sequence may be, for example, a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptides fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag correεpondε to an epitope derived from the influenza

hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984) ) .

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequenceε. The preεent invention particularly relateε to polynucleotideε which hybridize under εtringent conditionε to the hereinabove-deεcribed polynucleotideε. Aε herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptides encoded by the cDNA of Figures 1, 2 and 3 (SEQ ID No. 1, 3 and 5) or the deposited cDNA(s) .

The depoεit(s) referred to herein will be maintained under the terms of the Budapeεt Treaty on the International Recognition of the Depoεit of Micro-organiεmε for purposes of Patent Procedure. Theεe depoεitε are provided merely aε convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotideε contained in the deposited materials, as well as the amino acid sequence of the polypeptideε encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any deεcription of εequenceε herein. A license may be required to make, use or εell the depoεited materialε, and no εuch licenεe iε hereby granted.

The present invention further relateε to polypeptideε which have the deduced amino acid sequence of Figureε 1, 2 and 3 (SEQ ID No. 2, 4 and 6) or which have the amino acid sequence encoded by the deposited cDNA(s) , aε well aε fragmentε, analogε and derivativeε of such polypeptideε.

The terms "fragment," "derivative" and "analog" when referring to the polypeptideε of Figureε l, 2 and 3 (SEQ ID No. 2, 4 and 6) or that encoded by the deposited cDNA(s) , meanε polypeptideε which retain essentially the same biological function or activity as such polypeptideε. Thuε, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptides of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptideε of Figureε l, 2 and 3 (SEQ ID No. 2, 4 and 6) or that encoded by the depoεited cDNAε may be (i) one in which one or more of the amino acid reεidueε are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residueε includes a subεtituent group, or (iii) one in which the mature polypeptide iε fuεed with another compound, such aε a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) . Such fragments, derivatives and analogε are deemed to be within the εcope of thoεe skilled in the art from the teachings herein.

The polypeptideε and polynucleotideε of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it iε naturally occurring) . For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such

polynucleotides could be part of a vector and/or εuch polynucleotides or polypeptideε could be part of a compoεition, and εtill be isolated in that such vector or composition is not part of itε natural environment.

The present invention also relateε to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the hMLHl, hMLH2 and hMLH3 genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expresεion, and will be apparent to the ordinarily εkilled ar iεan.

The polynucleotideε of the present invention may be employed for producing polypeptides by recombinant techniques. Thuε, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromo omal, nonchromoεomal and εynthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculoviruε,- yeaεt plaεmidε; vectorε derived from combinations of plasmidε and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox viruε, and pseudorabieε. However, any other vector may be used as long as it iε replicable and viable in the hoεt.

The appropriate DNA sequence may be inserted into the vector by a variety of procedure . In general, the DNA

sequence iε inserted into an appropriate reεtriction endonuclease εite(ε) by procedureε known in the art. Such procedureε and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector iε operatively linked to an appropriate expression control sequence (ε) (promoter) to direct mRNA synthesiε . Aε repreεentative exampleε of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P _! promoter and other promoterε known to control expreεεion of geneε in prokaryotic or eukaryotic cellε or their viruεeε . The expression vector also containε a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed hoεt cellε such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such aε tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence aε hereinabove deεcribed, aε well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the proteins.

As representative examples of appropriate hostε, there may be mentioned: bacterial cellε, εuch aε E. coli, Streptomvceε. Salmonella typhimurium,• fungal cellε, εuch aε yeaεt; inεect cellε εuch aε Drosophila S2 and Spodoptera Sf9; animal cellε such aε CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host iε deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the

sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a εequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further compriseε regulatory εequenceε, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectorε and promoters are known to thoεe of εkill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen, Inc.), pbs, pDlO, phagescript, pεiXl74, pbluescript SK, pbsks, pNH8A, pNHi6a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223-3, pKK233-3, pDR540, pRlT5 (Pharmacia) . Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) . However, any other plasmid or vector may be used as long aε they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferaεe) vectorε or other vectorε with selectable markers. Two appropriate vectorε are pKK232-8 and pCM7. Particular named bacterial promoterε include lad, lacZ, T3, T7, gpt, lambda P _R , P, and TRP. Eukaryotic promoterε include CMV immediate early, HSV thymidine kinaεe, early and late SV40, LTRs from retrovirus, and mouse metallothionein-l. Selection of the appropriate vector and promoter iε well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-deεcribed constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such aε a yeaεt cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation (Davis, L.,

Dibner, M. , Battey, I., Baεic Methodε in Molecular Biology, (1986) ) .

The conεtructε in hoεt cellε can be uεed in a conventional manner to produce the gene product encoded by the recombinant εequence. Alternatively, the polypeptideε of the invention can be synthetically produced by conventional peptide syntheεizerε.

Mature proteins can be expressed in mammalian cellε, yeaεt, bacteria, or other cellε under the control of appropriate promoterε. Cell-free tranεlation syεtemε can alεo be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hoεtε are described by Sambrook, et al. , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

Tranεcription of the DNA encoding the polypeptideε of the present invention by higher eukaryoteε iε increased by inserting an enhancer sequence into the vector. Enhancerε are ciε-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its tranεcription. Exampleε including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenoviruε enhancerε.

Generally, recombinant expreεεion vectorε will include originε of replication and selectable markers permitting tranεformation of the hoεt cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expreεεed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes εuch aε 3-phoεphoglycerate kinaεe (PGK) , α-factor, acid phoεphataεe, or heat εhock proteinε, among others. The

heterologous structural sequence iε aεsembled in appropriate phase with translation initiation and termination sequences. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristicε, e.g., stabilization or simplified purification of expresεed recombinant product.

Uεeful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with εuitable tranεlation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonaε, Streptomyceε, and Staphylococcuε, although others may also be employed as a matter of choice.

Aε a repreεentative but nonlimiting example, useful expression vectorε for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmidε comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) . Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicalε, Uppεala, Sweden) and GEMl (Promega Biotec, Madiεon, Wl, USA) . Theεe pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter iε induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrif gation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

Microbial cellε employed in expreεsion of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art .

Various mammalian cell culture syεtemε can alεo be employed to expreεε recombinant protein. Exampleε of mammalian expreεεion systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expresεing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lineε . Mammalian expreεεion vectorε will compriεe an origin of replication, a suitable promoter and enhancer, and also any necesεary ribosome binding siteε, polyadenylation εite, splice donor and acceptor εiteε, transcriptional termination sequenceε, and 5' flanking nontranεcribed εequenceε . DNA εequenceε derived from the SV40 εplice, and polyadenylation εiteε may be uεed to provide the required nontranεcribed genetic elementε.

The polypeptideε can be recovered and purified from recombinant cell cultures by methodε including ammonium εulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocelluloεe chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding εtepε can be used, aε necesεary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps .

The polypeptideε of the present invention may be a naturally purified product, or a product of chemical synthetic procedureε, or produced by recombinant techniqueε

from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptideε of the preεent invention may be glycosylated or may be non-glycoεylated.

In accordance with a further aspect of the invention, there iε provided a process for determining susceptibility to cancer, in particular, a hereditary cancer. Thus, a mutation in a human repair protein, which is a human homolog of mutL, and in particular those described herein, indicates a susceptibility to cancer, and the nucleic acid sequenceε encoding such human homologs may be employed in an assay for ascertaining such susceptibility. Thus, for example, the assay may be employed to determine a mutation in a human DNA repair protein as herein described, such as a deletion, truncation, insertion, frame shift, etc., with such mutation being indicative of a suεceptibility to cancer.

A mutation may be ascertained for example, by a DNA sequencing asεay. Tiεεue εampleε, including but not limited to blood samples are obtained from a human patient . The samples are procesεed by methodε known in the art to capture the RNA. First strand cDNA is syntheεized from the RNA εampleε by adding an oligonucleotide primer conεiεting of polythymidine reεidueε which hybridize to the polyadenosine stretch present on the mRNA'ε. Reverεe transcriptase and deoxynucleotides are added to allow εyntheεis of the first strand cDNA. Primer sequenceε are synthesized baεed on the DNA sequence of the DNA repair protein of the invention. The primer sequence is generally comprised of 15 to 30 and preferably from 18 to 25 consecutive bases of the human DNA repair gene. Table 1 sets forth an illustrative example of oligonucleotide primer sequenceε based on hMLHl . The primers are used in pairs (one "sense" strand and one "anti-sense") to amplify the cDNA from the patients by the PCR method (Saiki et al . , Nature, 324:163-166 (1986)) such that three

overlapping fragments of the patient's cDNA's for such protein are generated. Table 1 also shows a list of preferred primer sequence pairs. The overlapping fragments are then subjected to dideoxynucleotide sequencing using a set of primer sequenceε εyntheεized to correεpond to the baεe pairs of the cDNA's at a point approximately every 200 base pairs throughout the gene.

TABLE 1

Primer Sequences used to amplify gene region using PCR

Start Site

Name and Arrangement Sequence

758 sense :-(-41) ^* GTTGAACATCTAGACGTCTC

1319 sense !-8 TCGTGGCAGGGGTTATTCG

1321 sensei-619 CTACCCAATGCCTCAACCG

1322 senεe :-677 GAGAACTGATAGAAATTGGATG

1314 εense -1548 GGGACATGAGGTTCTCCG

1323 sense -1593 GGGCTGTGTGAATCCTCAG

773 anti- 53 CGGTTCACCACTGTCTCGTC

1313 anti- 971 TCCAGGATGCTCTCCTCG

1320 anti- 1057 CAAGTCCTGGTAGCAAAGTC

1315 anti- 1760 ATGGCAAGGTCAAAGAGCG

1316 anti- 1837 CAACAATGTATTCAGXAAGTCC

1317 anti- 2340 TTGATACAACACTTTGTATCG

1318 anti- 2415 GGAATACTATCAGAAGGCAAG

* Numbers corresponding to location along nucleotide sequence of Figure 1 where ATG is number l. Preferred primer sequenceε pairs :

758, 1313

1319, 1320

660, 1909

725, 1995

1680, 2536

1727, 2610

The nucleotide sequenceε shown in Table l represent SEQ ID No. 7 through 19, reεpectively.

Table 2 lists representative examples of oligonucleotide primer sequenceε (sense and anti-senεe) which may be used, and preferably the entire set of primer sequences are used for sequencing to determine where a mutation in the patient DNA repair protein may be. The primer sequenceε may be from 15 to 30 bases in length and are preferably between 18 and 25 bases in length. The sequence information determined from the patient iε then compared to non-mutated εequences to determine if any mutations are present.

TABLE 2

Primer Sequences Uεed to Sequence the Amplified Fragmentε

Start Site Name Number and Arrangement Sequence

5282 εeqOl εenεe-377 ^* ACAGAGCAAGTTACTCAGATG

5283 εeq02 εenεe-552 GTACACAATGCAGGCATTAG

5284 εeq03 εense-904 AATGTGGATGTTAATGTGCAC

5285 seq04 εenεe-1096 CTGACCTCGTCTTCCTAC

5286 seq05 sense-1276 CAGCAAGATGAGGAGATGC

5287 seq06 senεe - 1437 GGAAATGGTGGAAGATGATTC

5288 εeq07 sense - 1645 CTTCTCAACACCAAGC

5289 seq08 senεe-1895 GAAATTGATGAGGAAGGGAAC 5295 εeq09 εenεe-1921 CrrC GATTGACAACTATGTGC 5294 εeqlO εenεe-2202 CACAGAAGATGGAAATATCCTG 5293 εeqll εense-2370 GTGTTGGTAGCACTTAAGAC

5291 seql2 anti-525 TTTCCCATA'i L'ri'CACTTG

5290 εeql3 anti-341 GTAACATGAGCCACATGGC

5292 εeql4 anti-46 CCACTGTCTCGTCCAGCCG

* Numberε corresponding to location along nucleotide sequence of Figure 1 where ATG is number 1.

The nucleotide sequenceε εhown in Table 2 repreεent SEQ ID No. 20 through 33, reεpectively.

In another embodiment, the primer sequences from Table

2 could be used in the PCR method to amplify a mutated region. The region could be sequenced and used aε a diagnostic to predict a predisposition to such mutated genes.

Alternatively, the assay to detect mutations in the genes of the present invention may be performed by genetic testing based on DNA sequence differences achieved by detection of alteration in electrophoretic mobility of DNA fragments in gelε with or without denaturing agentε. Small εequence deletionε and insertions can be visualized by high resolution gel electrophoresis. DNA fragmentε of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985) ) .

Sequence changes at specific locations may also be revealed by nuclease protection aεsays, εuch aε RNase and Si protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)). Perfectly matched sequences can be diεtinguiεhed from mismatched duplexeε by RNase A digestion or by differences in melting temperatureε.

Thuε, the detection of a specific DNA sequence may be achieved by methodε such as hybridization, RNase protection, chemical cleavage, Weεtern Blot analysiε,

direct DNA sequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphisms (RFLP) ) and Southern blotting of genomic DNA.

In addition to more conventional gel-electrophoresiε and DNA sequencing, mutations can also be detected by in si tu analysis.

The polypeptideε may also be employed to treat cancers or to prevent cancers, by expresεion of εuch polypeptideε in vivo, which is often referred to as "gene therapy."

Thus, for example, cellε from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cellε then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.

Similarly, cellε may be engineered in vivo for expression of a polypeptide in vivo by, for example, procedureε known in the art . Aε known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the preεent invention may be adminiεtered to a patient for engineering cellε in vivo and expreεεion of the polypeptide in vivo . These and other methodε for adminiεtering a polypeptide of the preεent invention by εuch method should be apparent to those skilled in the art from the teachings of the present

invention. For example, the expreεεion vehicle for engineering cellε may be other than a retroviruε, for example, an adenoviruε which may be used to engineer cellε in vivo after combination with a suitable delivery vehicle.

Each of the cDNA sequenceε identified herein or a portion thereof can be used in numerous ways aε polynucleotide reagents. The sequences can be used as diagnostic probes for the presence of a specific mRNA in a particular cell type. In addition, these sequenceε can be used aε diagnostic probes suitable for use in genetic linkage analysis (polymorphisms) .

The sequenceε of the preεent invention are alεo valuable for chromoεome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there iε a current need for identifying particular εiteε on the chromoεome. Few chromoεome marking reagentε baεed on actual εequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAs to chromoεomeε according to the present invention is an important firεt step in correlating those sequenceε with geneε associated with disease.

Briefly, sequenceε can be mapped to chromoεomeε by preparing PCR primerε (preferably 15-25 bp) from the cDNA. Computer analyεis of the 3' untranεlated region iε uεed to rapidly εelect primerε that do not span more than one exon

in the genomic DNA, thuε complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomeε. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids iε a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panelε of fragmentε from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping εtrategieε that can εimilarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomeε and preεelection by hybridization to conεtruct chromoεome-εpecific cDNA librarieε.

Fluoreεcence in si tu hybridization (FISH) of a cDNA clone to a metaphase chromoεomal εpread can be uεed to provide a preciεe chromoεomal location in one εtep. Thiε technique can be uεed with cDNA aε εhort aε 500 or 600 baεeε; however, cloneε larger than that have a higher likelihood of binding to a unique chromoεomal location with εufficient εignal intenεity for simple detection. FISH requires use of the cloneε from which the express sequence tag or EST waε derived, and the longer the better. For example, 2,000 bp iε good, 4,000 iε better, and more than

4,000 is probably not necessary to get good results a reasonable percentage of the time. For a review of thiε technique, see Verma et al., Human Chromoεomeε: a Manual of Baεic Techniques, Pergamon Presε, New York (1988) .

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) . The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes) .

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individualε. If a mutation iε obεerved in some or all of the affected individuals but not in any normal individuals, then the mutation is likely to be the causative agent of the disease.

With current resolution of physical mapping and genetic mapping techniqueε, a cDNA precisely localized to a chromosomal region associated with the diεease could be one of between 50 and 500 potential causative geneε. (Thiε assumeε 1 megabaεe mapping reεolution and one gene per 20 kb) . hMLH2 haε been localized uεing a genomic Pl clone (1670) which contained the 5' region of the hMLH2 gene.

Detailed analysiε of human metaphase chromoεome εpreadε, counterstained to reveal banding, indicated that the hMLH2 gene waε located within bands 2q32. Likewise, hMLH3 waε localized using a genomic Pl clone (2053) which contained the 3' region of the hMLH3 gene. Detailed analysiε of human metaphase chromosome spreads, counterstained to reveal banding, indicated that the hMLH3 gene was located within band 7p22, the most distal band on chromosome 7. Analyεiε with a variety of genomic cloneε showed that hMLH3 waε a member of a subfamily of related genes, all on chromosome 7.

The polypeptides, their fragments or other derivatives, or analogε thereof, or cellε expressing them can be used as an immunogen to produce antibodies thereto. These antibodies can be, for example, polyclonal or monoclonal antibodies. The preεent invention alεo includeε chimeric, εingle chain, and humanized antibodieε, aε well aε Fab fragmentε, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodieε and fragmentε.

Antibodieε generated againεt the polypeptideε correεponding to a εequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptideε to an animal, preferably a nonhuman. The antibody εo obtained will then bind the polypeptideε itεelf. In thiε manner, even a sequence encoding only a fragment of the polypeptides can

be uεed to generate antibodieε binding the whole native polypeptideε. Such antibodieε can then be used to isolate the polypeptide from tissue expresεing that polypeptide.

For preparation of monoclonal antibodieε, any technique which provideε antibodieε produced by continuouε cell line cultureε can be uεed. Exampleε include the hybridoma technique (Kohler and Milεtein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al. , 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodieε (Cole, et al., 1985, in Monoclonal Antibodieε and Cancer Therapy, Alan R. Liss, Inc., pp. 77- 96) .

Techniqueε described for the production of single chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide productε of thiε invention. Alεo, tranεgenic mice may be uεed to expreεε humanized antibodieε to immunogenic polypeptide productε of thiε invention.

The preεent invention will be further deεcribed with reference to the following exampleε; however, it iε to be underεtood that the preεent invention iε not limited to εuch exampleε. All partε or amountε, unleεε otherwise specified, are by weight.

In order to facilitate understanding of the following exampleε certain frequently occurring methodε and/or terms will be described.

"Plaεmidε" are deεignated by a lower caεe p preceded and/or followed by capital letterε and/or numbers . The starting plasmids herein are either commercially available, publicly available on an unrestricted baεiε, or can be conεtructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a reεtriction enzyme that actε only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactorε and other requirementε were uεed aε would be known to the ordinarily εkilled artisan. For analytical purposeε, typically 1 μg of plaεmid or DNA fragment iε used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid conεtruction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymeε are εpecified by the manufacturer. Incubation timeε of about 1 hour at 37'C are ordinarily uεed, but may vary in accordance with the εupplier'ε instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment .

Size separation of the cleaved fragmentε iε performed uεing 8 percent polyacrylamide gel deεcribed by Goeddel, D. et al. , Nucleic Acidε Reε. , 8:4057 (1980).

"Oligonucleotideε" referε to either a εingle stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thuε will not ligate to another oligonucleotide without adding a phosphate with an ATP in the preεence of a kinaεe. A εynthetic oligonucleotide will ligate to a fragment that haε not been dephosphorylated.

"Ligation" referε to the process of forming phosphodiester bondε between two double stranded nucleic acid fragments (Maniatiε, T. , et al. , Id., p. 146) . Unleεε otherwiεe provided, ligation may be accomplished using known bufferε and conditions with 10 unitε to T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragmentε to be ligated.

Unless otherwiεe εtated, transformation waε performed aε deεcribed in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973) .

Example 1 Bacterial Expreεsion of hMLHl

The full length DNA εequence encoding human DNA mismatch repair protein hMLHl, ATCC # 75649, iε initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3' ends of the DNA sequence to synthesize

insertion fragments. The 5' oligonucleotide primer haε the εequence 5' CGGGATCCATGTCGTTCGTGGCAGGG 3' (SEQ ID No. 34) , containε a BamHI restriction enzyme site followed by 18 nucleotides of hMLHl coding sequence following the initiation codon; the 3' sequence 5' GCTCTAGATTAACACCTCT CAAAGAC 3' (SEQ ID No. 35) contains complementary sequenceε to an Xbal εite and iε at the end of the gene. The reεtriction enzyme εiteε correspond to the restriction enzyme siteε on the bacterial expression vector pQE-9. (Qiagen, Inc., Chatsworth, CA) . The plasmid vector encodes antibiotic reεiεtance (Amp ^r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter/operator (P/O) , a ribosome binding site (RBS) , a 6-hiεtidine tag (6- Hiε) and reεtriction enzyme cloning εiteε. The pQE-9 vector iε digested with BamHI and Xbal and the insertion fragmentε are then ligated into the pQE-9 vector maintaining the reading frame initiated at the bacterial RBS. The ligation mixture iε then uεed to transform the E. coli strain Ml5/rep4 (Qiagen, Inc.) which containε multiple copieε of the plaεmid pREP4, which expresseε the lacl repreεεor and alεo conferε kanamycin reεiεtance (Kan ^r ) . Tranεformantε are identified by their ability to grow on LB plateε and ampicillin/kanamycin reεiεtant colonieε are εelected. Plaεmid DNA iε iεolated and confirmed by reεtriction analyεiε. Cloneε containing the desired constructs are grown overnight (O/N) in liquid culture in LB media supplemented with both .Amp (100 ug/ml) and Kan (25

ug/ml) . Tho O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cellε are grown to an optical density 600 (O.D. ^60ϋ ) of between 0.4 and 0.6. IPTG (isopropyl-B-D-thiogalacto pyranoside) iε then added to a final concentration of 1 mM. IPTG induceε by inactivating the lad repreεsor, clearing the P/O leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation (20 mins at 6000Xg) . The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HC1. After clarification, solubilized hMLHl is purified from thiε εolution by chromatography on a Nickel-Chelate column under conditionε that allow for tight binding by proteinε containing the 6-Hiε tag (Hochuli, E. et al . , Genetic Engineering, Principleε & Methodε, 12:87-98 (1990) . Protein renaturation out of GnHCl can be accomplished by several protocols (Jaenicke, R. and Rudolph, R. , Protein Structure - A Practical Approach, IRL Press, New York (1990) ) . Initially, εtep dialyεiε is utilized to remove the GnHCL. Alternatively, the purified protein isolated from the Ni-chelate column can be bound to a second column over which a decreasing linear GnHCL gradient is run. The protein is allowed to renature while bound to the column and is subsequently eluted with a buffer containing 250 mM Imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5 and 10% Glycerol. Finally, εoluble protein is dialyzed against a

storage buffer containing 5 mM Ammonium Bicarbonate. The purified protein waε analyzed by SDS-PAGE.

Example 2 Spontaneouε Mutation Aεsay for Detection of the Expression of hMLHl. hMLH2 and hMLH3 and Complementation to the E . coli mutl

The pQE9hMLHl, pQE9hMLH2 or pQE9hMLH3/GW3733, transformantε were subjected to the spontaneous mutation assay. The plasmid vector pQE9 was also transformed to AB1157 (k-12, argE3 hiεG4 , LeuB6 proA2 thr-1 ara-1 rpsL31 εupE44 tsx-33) and GW3733 to use as the positive and negative control respectively.

Fifteen 2 ml cultures, inoculated with approximately 100 to 1000 E. coli , were grown 2xl0 ⁸ cells per ml in LB ampicillin medium at 37°C. Ten microliters of each culture were diluted and plated on the LB ampicillin plateε to meaεure the number of viable cellε. The reεt of the cellε from each culture were then concentrated in εaline and plated on minimal plateε lacking of arginine to measure reversion of Argr ⁺ . In Table 3, the mean number of mutations per culture ( ) waε calculated from the median number {r) of mutantε per diεtribution, according to the equation (r/m) -In(m) = 1.24 (Lea et al. , J. Geneticε 49:264-285 (1949)) . Mutation rates per generation were recorded as m/N, with N representing the average number of cells per culture.

TABLE 3

Spontaneous Mutation Rates

Strain Mutation/generation

AB1157+vector (5.6+0.1) x 10-9a

GW3733+vector (1.1±0.2) x 10-6a

GW3733+pllMLHl (3.7±1.3 x 10-7a

GW3733+pllMLH2 (3.1±0.6) x 10-7b

GW3733+phMLH3 (2.1±0.8) X 10-7b

a: Average of three experiments, b: Average of four experiments.

The functional complementation result showed that the human mutL can partially rescue the E.coli mutL mutator phenotype, suggesting that the human mutL is not only εucceεsfully expreεεed in a bacterial expreεεion system, but also functionε in bacteria.

Example 3 Chromosomal Mapping of the hMLHl

An oligonucleotide primer set waε deεigned according to the sequence at the 5 ' end of the cDNA for HMLHl. This primer set would span a 94 bp segment. This primer set waε used in a polymerase chain reaction under the following set of conditions :

30 εecondε, 95 degreeε C

1 minute, 56 degreeε C

1 minute, 70 degreeε C Thiε cycle waε repeated 32 timeε followed by one 5 minute cycle at 70 degreeε C. Human, mouεe, and hamεter DNA were uεed aε template in addition to a somatic cell hybrid panel (Bios, Inc) . The reactions were analyzed on either 8% polyacrylamide gels or 3.5 % agarose gels. A 94 base pair band waε obεerved in the human genomic DNA sample and in the εomatic cell hybrid sample corresponding to chromosome 3. In addition, uεing variouε other somatic cell hybrid genomic DNA, the hMLHl gene was localized to chromosome 3p.

Example 4 Method for Determination of mutation of hMLHl gene in HNPCC kindred cDNA waε produced from RNA obtained from tiεsue sampleε from perεonε who are HNPCC kindred and the cDNA waε uεed aε a template for PCR, employing the primerε 5' GCATC TAGACGTTTCCTTGGC 3' (SEQ ID No. 36) and 5' CATCCAAGCTTCTGT TCCCG 3' (SEQ ID No. 37) , allowing amplification of codonε 1 to 394 of Figure 1; 5' GGGGTGCAGCAGCACATCG 3' (SEQ ID No. 38) and 5' GGAGGCAG.AATGTGTGAGCG 3' (SEQ ID No. 39) , allowing amplification of codonε 326 to 729 of Figure 1 (SEQ ID NO. 2) ; and 5' TCCCAAAGAAGGACTTGCT 3' (SEQ ID No. 40) and 5' AGTATAAGTCTTAAGTGCTACC 3' (SEQ ID No. 41) , allowing amplification of codonε 602 to 756 plus 128 nt of

3'- untranslated sequences of Figure 1 (SEQ ID No. 2) . The PCR conditions for all analyseε used consisted of 35 cycles at 95°C for 30 seconds, 52-58°C for 60 to 120 secondε, and 70°C for 60 to 120 εecondε, in the buffer solution deεcribed in San Sidransky, D. et al . , Science, 252:706 (1991) . PCR products were sequenced using primerε labeled at their 5' end with T4 polynucleotide kinase, employing SequiTherm Polymerase (Epicentre Technologies) . The intron-exon borders of selected exons were also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations were then cloned and sequenced to validate the results of the direct sequencing. PCR products were cloned into T-tailed vectors as deεcribed in Holton, T.A. and Graham, M.W. , Nucleic Acidε Research, 19:1156 (1991) and sequenced with T7 polymerase (United States Biochemical) . Affected individualε from seven kindreds all exhibited a heterozygous deletion of codonε 578 to 632 of the hMLHl gene. The derivation of five of these seven kindreds could be traced to a common ancestor. The genomic sequences surrounding codonε 578-632 were determined by cycle- sequencing of the Pl cloneε (a human genomic Pl library which containε the entire hMLHl gene (Genome Systems) ) using SequiTherm Polymerase, as described by the manufacturer, with the primers were labeled with T4 polynucleotide kinaεe, and by sequencing PCR products of genomic DNA. The primerε used to amplify the exon

containing codonε 578-632 were 5' TTTATGGTTTCTCACCTGCC 3' (SEQ ID No. 42) and 5' GTTATCTGCCCACCTCAGC 3' (SEQ ID No. 43) . The PCR product included 105 bp of intron C εequence upεtream of the exon and 117 bp downstream. No mutations in the PCR product were observed in the kindredε, εo the deletion in the RNA waε not due to a simple splice site mutation. Codonε 578 to 632 were found to conεtitute a εingle exon which waε deleted from the gene product in the kindredε deεcribed above. This exon containε several highly conserved amino acids.

In a second family (L7) , PCR was performed using the above primerε and a 4bp deletion waε obεerved beginning at the firεt nucleotide (nt) of codon 727. Thiε produced a frame shift with a new stop codon 166 nt downstream, resulting in a substitution of the carboxy-terminal 29 amino acids of hMLHl with 53 different amino acids, some encoded by nt normally in the 3' untranslated region.

A different mutation waε found in a different kindred (L2516) after PCR using the above primerε, the mutation conεiεting of a 4bp inεert between codonε 755 and 756. Thiε inεertion reεulted in a frame shift and extension of the ORF to include 102 nucleotides (34 amino acids) downstream of the normal termination codon. The mutationε in both kindredε L7 and L2516 were therefore predicted to alter the C-terminuε of hMLHl.

A possible mutation in the hMLHl gene was determined from alterations in size of the encoded protein, where

kindreds were too few for linkage studies. The primerε used for coupled tranεcription-translation of hMLHl were 5' GGATCCTi^TACGACrrClACTATAGGGAGACCACCATGGCATCT AGACGTTTCCCTTGGC 3' (SEQ ID No. 44) and 5' CATCCAAGCTTCTGTTCCCG 3' (SEQ ID No. 45) for codons 1 to 394 of Figure l and 5' GGATCCΓAATACGACTCACTATAGGGAGACCACCATGGG

GGTGCAGCAGCACATCG 3' (SEQ ID No. 46) and 5' GGAGGCAGAATGTG TGAGCG 3' (SEQ ID No. 47) for codonε 326 to 729 of Figure 1 (SEQ ID No. 2) . The resultant PCR products had signals for transcription by T7 RNA polymerase and for the initiation of translation at their 5' ends. RNA from lymphoblaεtoid cellε of patientε from 18 kindredε waε used to amplify two productε, extending from codon l to codon 394 or from codon 326 to codon 729, reεpectively. The PCR productε were then tranεcribed and tranεlated in vitro, making use of tranεcription-translation signalε incorporated into the PCR primerε. PCR productε were uεed aε templateε in coupled tranεcription-translation reactions performed as described by Powell, S.M. et al . , New England Journal of Medicine, 329:1982, (1993), using 40 micro CI of ³⁵ S labeled methionine. Samples were diluted in sample buffer, boiled for five minutes and analyzed by electropheresis on sodium dodecyl sulfate-polyacrylamide gels containing a gradient of 10% to 20% acrylamide. The gels were dried and subjected to radiography. All sampleε exhibited a polypeptide of the expected εize, but an abnormally migrating polypeptide waε additionally found in one case.

The sequence of the relevant PCR product was determined and found to include a 371 bp deletion beginning at the first nucleotide (nt) of codon 347. This alteration waε present in heterozygouε form, and resulted in a frame shift in a new stop codon 30 nt downstream of codon 346, thuε explaining the truncated polypeptide obεerved.

Four colorectal tumor cell lineε manifeεting microsatellite instability were examined. One of the four (cell line H6) showed no normal peptide in thiε assay and produced only a short product migrating at 27 kd. The εequence of the corresponding cDNA was determined and found to harbor a C to A transversion at codon 252, resulting in the substitution of a termination codon for serine. In accord with the translational analyses, no band at the normal C position was identified in the cDNA or genomic DNA from thiε tumor, indicating that it waε devoid of a functional hMLHl gene.

Table 4 εets forth the resultε of theεe εequencing aεεay . Deletionε were found in those people who were known to have a family history of the colorectal cancer. More particularly, 9 of 10 familieε showed an hMLHl mutation.

Table 4 - Summary of Mutations in hMLHl

cDNA Nucleotide Predicte

Sample Codon Change Coding Cha

Kindreds F2, F3 , F6, F8, 578-632 165 bp deletion In-frame F10, Fll, F52 deletion

Kindred L7 727/728 4 bp deletion Frarceshift (TCACACATTC to substrifattic TCATTCT) new amino a

Kindred L2516 755/756 4 bp insertion External cf (GTGTT.AA to terminuε GTGTTTGTTAA)

Kindred RA 347 371 bp deletion Frameshi Truncati

H6 Colorectal Tumor 252 Tr ans ver s i on Serine to S (TCA to TAA)

Example 5 Bacterial Expresεion and Purification of hMLH2

The DNA sequence encoding hMLH2, ATCC #75651, is initially amplified using PCR oligonucleotide primers corresponding to the 5' and 3 ' endε of the DNA sequence to synthesize insertion fragmentε. The 5' oligonucleotide primer has the sequence 5' CGGGATCCATGAAACAATTGCCTGCGGC 3' (SEQ ID No. 48) contains a BamHI restriction enzyme site

followed by 17 nucleotideε of hMLH2 following the initiation codon. The 3' εequence 5' GCTCTAGACCAGACTCAT GCTGTTTT 3' (SEQ ID No. 49) contains complementary sequenceε to an Xbal εite and iε followed by 18 nucleotideε of hMLH2. The restriction enzyme εiteε correspond to the reεtriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA) . pQE-9 encodes antibiotic resiεtance (Amp ^r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a riboεome binding site (RBS) , a 6-His tag and reεtriction enzyme εiteε. The amplified εequenceε and pQE- 9 are then digeεted with BamHI and Xbal. The amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture iε then used to transform E. coli strain Ml5/rep4 (Qiagen, Inc.) which containε multiple copies of the plasmid pREP4, which expreεεeε the lad repreεεor and alεo confers kanamycin resiεtance (Kan ^r ) . Tranεformantε are identified by their ability to grow on LB plateε and ampicillin/kanamycin reεiεtant colonieε are εelected. Plaεmid DNA is isolated and confirmed by restriction analysiε. Cloneε containing the deεired constructs are grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . Tho O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D. ⁶⁰⁰ ) of between 0.4 and 0.6.

IPTG (Isopropyl-B-D-thiogalacto pyranoside) is then added to a final concentration of 1 mM. IPTG induces by inactivating the lad repressor, clearing the P/0 leading to increased gene expression. Cellε are grown an extra 3 to 4 hourε . Cellε are then harvested by centrifugation (20 mins at 6000Xg) . The cell pellet iε solubilized in the chaotropic agent 6 Molar Guanidine HC1. After clarification, solubilized hMLH2 is purified from thiε εolution by chromatography on a Nickel-Chelate column under conditionε that allow for tight binding by proteinε containing the 6-Hiε tag (Hochuli, E. et al. , Genetic Engineering, Principleε &. Methodε, 12:87-98 (1990) . Protein renaturation out of GnHCl can be accomplished by several protocols (Jaenicke, R. and Rudolph, R. , Protein Structure - A Practical Approach, IRL Presε, New York (1990) ) . Initially, εtep dialyεiε iε utilized to remove the GnHCL. Alternatively, the purified protein isolated from the Ni-chelate column can be bound to a second column over which a decreasing linear GnHCL gradient iε run. The protein is allowed to renature while bound to the column and iε subsequently eluted with a buffer containing 250 mM Imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5 and 10% Glycerol. Finally, εoluble protein is dialyzed against a storage buffer containing 5 mM Ammonium Bicarbonate. The purified protein waε analyzed by SDS-PAGE.

Example 6 Bacterial Expression and Purification of hMLH3

The DNA sequence encoding hMLH3, ATCC #75650, iε initially amplified uεing PCR oligonucleotide primerε correεponding to the 5' and 3' ends of the DNA εequence to εynthesize insertion fragments. The 5' oligonucleotide primer has the εequence 5' CGGGATCCATGGAGCGAGCTGAGAGC 3' (SEQ ID No. 50) contains a BamHI restriction enzyme site followed by 18 nucleotides of hMLH3 coding εequence εtarting from the preεumed terminal amino acid of the proceεεed protein. The 3' εequence 5' GCTCTAGAGTGAAG ACTCTGTCT 3' (SEQ ID No. 51) containε complementary εequenceε to an Xbal εite and iε followed by 18 nucleotideε of hMLH3. The reεtriction enzyme sites correspond to the restriction enzyme εiteε on the bacterial expression vector pQE-9 (Qiagen, Inc. Chatsworth, CA) . pQE-9 encodes antibiotic resistance (Amp ^r ) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/O) , a ribosome binding εite (RBS) , a 6-Hiε tag and reεtriction enzyme εites. The amplified εequenceε and pQE- 9 are then digeεted with BamHI and Xbal. The amplified εequenceε are ligated into pQE-9 and are inεerted in frame with the εequence encoding for the hiεtidine tag and the RBS. The ligation mixture waε then uεed to tranεform E. coli εtrain M15/rep4 (Qiagen, Inc.) which containε multiple copies of the plasmid pREP4, which expresses the lad represεor and alεo conferε kanamycin reεiεtance (Kan ^r ) .

Transformantε are identified by their ability to grow on LB plateε and ampicillin/kanamycin reεiεtant colonieε are εelected. Plasmid DNA is isolated and confirmed by restriction analysiε. Clones containing the desired constructs are grown overnight (O/N) in liquid culture in LB media εupplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . Tho O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D. ⁶⁰⁰ ) of between 0.4 and 0.6. IPTG (Isopropyl-B-D-thiogalacto pyranoside) iε then added to a final concentration of 1 mM. IPTG induceε by inactivating the lad repreεεor, clearing the P/O leading to increased gene expresεion. Cellε are grown an extra 3 to 4 hourε. Cellε are then harveεted by centrifugation (20 minε at 6000Xg) . The cell pellet iε εolubilized in the chaotropic agent 6 Molar Guanidine HC1. After clarification, εolubilized εtanniocalcin iε purified from this solution by chromatography on a Nickel-Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al. , Genetic Engineering, Principles & Methods, 12:87-98 (1990). Protein renaturation out of GnHCl can be accomplished by several protocols (Jaenicke, R. and Rudolph, R. , Protein Structure - A Practical Approach, IRL Press, New York (1990)) . Initially, step dialyεiε iε utilized to remove the GnHCL. Alternatively, the purified protein iεolated from the Ni-chelate column can be bound to a second column

over which a decreaεing linear GnHCL gradient iε run. The protein is allowed to renature while bound to the column and iε subsequently eluted with a buffer containing 250 mM imidazole, 150 mM NaCl, 25 mM Tris-HCl pH 7.5 and 10% Glycerol. Finally, soluble protein is dialyzed against a storage buffer containing 5 mM Ammonium Bicarbonate. The purified protein was analyzed by SDS-PAGE.

Example 7 Method for determination of mutation of hMLH2 and hMLH3 in hereditary cancer Isolation of Genomic Clones

A human genomic Pl library (Genomic Systemε, Inc.) waε εcreened by PCR uεing primerε εelected for the cDNA sequence of hMLH2 and hMLH3. Two cloneε were isolated for hMLH2 using primerε 5' AAGCTGCTCTGTTAAAAGCG 3' (SEQ ID No. 52) and 5' GCACCAGCATCCAAGGAG 3' (SEQ ID No. 53) and reεulting in a 133 bp product. Three cloneε were isolated for hMLH3, using primerε 5' CAACCATGAGACACATCGC 3' (SEQ ID No. 54) and 5' AGGTTAGTGAAGACTCTGTC 3' (SEQ ID No. 55) reεulting in a 121 bp product. Genomic cloneε were nick- tranεlated with digoxigenindeoxy-uridine 5' -triphoεphate (Boehringer Manheim) , and FISH waε performed as described (Johnson, Cg. et al. , Methods Cell Biol. , 35:73-99 (1991)). Hybridization with the hMLH3 probe were carried out using a vast exceεε of human cot-l DNA for specific hybridization to the expressed hMLH3 locus. Chromoεomeε were counterεtained with 4,6-diamino-2-phenylidole andpropidium

iodide, producing a combination of C- and R-bandε. Aligned imageε for preciεe mapping were obtained uεing a triple- band filter set (Chroma Technology, Brattleboro, T) in combination with a cooled charge-coupled device camera (Photometries, Tucson, AZ) and variable excitation wavelength filterε (Johnson, Cv. et al. , Genet. Anal. Tech. Appl. , 8:75 (1991)). Image collection, analysiε and chromoεomal fractional length measurements were done suing the iSee Graphical Program System (Inovision Corporation, Durham, NC) .

Transcription coupled Translation Mutation Analysis

For purposes of IVSP analysiε the hMLH2 gene waε divided into three overlapping εegmentε. The firεt εegment included codonε 1 to 500, while the middle εegment included codonε 270 to 755, and the laεt εegment included codonε 485 to the tranεlational termination εite at codon 933. The primerε for the firεt εegment were 5' GGATCCTAATACGACTCACT ATAGGGAGACCACCATGGAACAATTGCCTGCGG 3' (SEQ ID No. 56) and 5' CCTGCTCCACTCATCTGC 3' (SEQ ID No. 57), for the middle εegment were 5' GGATCCΓΓAATACGACTCΆCTATAGGGAGACCACCATGGAAGA

TATCTTAAAGTTAATCCG 3' (SEQ ID No. 58) and 5' GGCTTCTTCTACTC TATATGG 3' (SEQ ID No. 59), and for the final εegment were 5' GGATCCT.AATACGACπ'C^CTATAGGGAGACCACCΑTGGCΑGGTCrrTGAAAACTC TTCG 3' (SEQ ID No. 60) and 5' AAAACAAGTCAGTGAATCCTC 3' (SEQ ID No. 61) . The primerε used for mapping the stop mutation in patient CW all used the same 5' primer as the

firεt segment. The 3' nested primerε were: 5' AAGCACΑTCTGTTTCTGCTG 3' (SEQ ID No. 62) COdonε 1 to 369; 5' ACGAGTAGATTCCTTTAGGC 3' (SEQ ID No. 63) COdonε 1 to 290; and 5' CAGAACTGACATGAGAGCC 3' (SEQ ID No. 64) codonε 1 to 214.

For analyεiε of hMLH3, the hMLH3 cDNA waε amplified aε a full-length product or aε two overlapping εegmentε. The primerε for full-length hMLH3 were 5'

GGATCCTAATACGACrrC^CrrATAGGGAGACCACCATGGAGCGAGCTGAGAGC 3 ' (SEQ ID No. 65) and 5' AGGTTAGTGAAGACTCTGTC 3' (SEQ ID No. 66) (codonε l to 863) . For segment 1, the sense primer waε the same as above and the antisense primer was 5' CTGAGGTCT CAGCAGGC 3' (SEQ ID No. 67) (codons 1 to 472) . Segment 2 primerε were 5' GGATCCTAATACGACTCACTATAGGGAGACCACCATGGTGTC CATTTCCAGACTGCG 3' (SEQ ID No. 68) and 5' AGGTTAGTGAAGACTCT GTC 3' (SEQ ID No. 69) (codonε 415 to 863) . Amplificationε were done as described below.

The PCR products contained recognition εignalε for tranεcription by T7 RNA polymeraεe and for the initiation of tranεlation at thei 5' endε. PCR productε were uεed aε templateε in coupled tranεcription-tranεlation reactionε containing 40 uCi of ³⁶ S-methionine (NEN, Dupont) . Sampleε were diluted in SDS εample buffer, and analyzed by electrophoreεiε on SDS-polyacrylamide gelε containing a gradient of 10 to 20% acrylamide. The gelε were fixed, treated with EnHance (Dupont) , dried and εubjected to autoradiography.

RT-PCR and Direct Sequencing of PCR Products cDNAs were generated from RNA of lymphoblastoid or tumor cellε with Superscript II (Life Technologies) . The cDNAs were then uεed aε templates for PCR. The conditions for all amplificationε were 35 cycleε at 95°C for 30ε, 52°C to 62°C for 60 to 120ε, and 70°C for 60 to 120ε, in buffer. The PCR productε were directly sequenced and cloned into the T-tailed cloning vector PCR2000 (Invitrogen) and sequenced with T7 polymerase (United States Biochemical) . For the direct sequencing of PCR productε, PCR reactionε were first phenolchloroform extracted and ethanol precipitated. Templates were directly sequenced using Sequitherm polymerase (Epicentre Technologies) and gamma- ³² P labelled primerε aε described by the manufacturer.

Intron/Exon Boundaries and Genomic Analysis of Mutations

Intron/exon borderε were determined by cycle- sequencing Pl cloneε uεing gamma- ³² P end labelled primerε and SequiTherm polymeraεe aε deεcribed by the manufacturer. The primerε used to amplify the hMLH2 exon containing codons 195 to 233 were 5' TTATTTGGCAGAAAAGCAGAG (SEQ ID No. 70) 3' and 5' TTAAAAGACTAACCTCTTGCC 3' (SEQ ID NO. 71), which produced a 215 bp product. The product waε cycle sequenced using the primer 5' CTGCTGTTATGAACAATATGG 3' (SEQ ID No. 72) . The primerε used to analyze the genomic deletion of hMLH3 in patient GC were: for the 5' region

amplification 5' CAGAAGCAGTTGCAAAGCC 3' (SEQ ID No. 73) and 5' AAACCGTACTCTTCACACAC 3' (SEQ ID No. 74) which produces a 74 bp product containing codons 233 to 257, primerε 5' GAGGAAAAGCTTTTGTTGGC 3' (SEQ ID No. 75) and 5' CAGTGGCTGCTGACTGAC 3' (SEQ ID No. 76) which produce a 93 bp product containing the codons 347 to 377, and primers 5' TCCAG.AACCAAGAAGGAGC 3' (SEQ ID No. 77) and 5' TGAGGTCTCAGCAGGC 3' (SEQ ID No. 78) which produce a 99 bp product containing the codons 439 to 472 of hMLH3.

TABLE 5

Summary of Mutations in HMLH2 and HMLH3 from patients affected with HNPCC

Genomic Predicted

Sample Codon Nucleotides cDNA Change Change Coding

Change

HMLH2

CW 233 Skipped CAG to TAG GLN to Sto Exon Codon

HMLH3

MM, NS, 20 CGG to CAG CGG to CAG ARG to GLN TF

GC 268 to 1,203 bp Deletion In-frame 669 Deletion deletion

GCx 268 to 1,203 bp Deletion Frameε ift

669 Deletion trucation

Numerouε modificationε and variationε of the preεent invention are poεsible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: HUMAN GENOME SCIENCES, INC.

(ii) TITLE OF INVENTION: Human DNA Mismatch Repair

Proteins

(iii) NUMBER OF SEQUENCES: 78

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART & OLSTEIN

(B) STREET: 6 BECKER F.ARM ROAD

(C) CITY: ROSE.LAND

(D) STATE: NEW JERSEY

(E) COUNTRY: USA

(F) ZIP: 07068

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 INCH DISKETTE

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTW.ARE: WORD PERFECT 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: PCT/US95/01035

(B) FILING DATE: 25 JAN 1995

(C) CLASSIFICATION: UNASSIGNED

(v) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/294,312

(B) FILING DATE: 23 AUG 1994

(C) CLASSIFICATION:

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/210,143

(B) FILING DATE: 16 MARCH 1994

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/187,757

(B) FILING DATE: 27 JAN 1994

(C) CLASSIFICATION:

(vi) ATTORNEY/AGENT INFORMATION:

(A) NAME: FERRARO, GREGORY D.

(B) REGISTRATION NUMBER: 36,134

(C) REFERENCE/DOCKET NUMBER: 325800-303

(viϋ) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 2525 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: CDNA

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

GTTGAACATC TAGACGTTTC CTTGGCTCTT CTGGCGCCAA AATGTCGTTC GTGGCAGGGG 60

TTATTCGGCG GCTGGACGAG ACAGTGGTGA ACCGCATCGC GGCGGGGGAA GTTATCCAGC 120

GGCCAGCTAA TGCTATCAAA GAGATGATTG AGAACTGTTT AGATGCAAAA TCCACAAGTA 180

TTCAAGTGAT TGTTAAAGAG GGAGGCCTGA AGTTGATTCA GATCCAAGAC AATGGCACCG 240

GGATCAGGAA AGAAGATCTG GATATTGTAT GTGAAAGTGT CACTACTAGT AAACTGCAGT 300

CCTTTGAGGA TTTAGCCAGT ATTTCTATCT ATGGCTTTCG AGGTGAGGCT TTGGCCAGCA 360

TAAGCCATGT GGCTCATGTT ACTATTACAA CGAAAACAGC TGATGGAAAG TGTGCATACA 420

GAGCAAGTTA CTCAGATGGA AAACTGAAAG CCCCTCCTAA ACCATGTGCT GGCAATCAAG 480

GGACCCAGAT CACGGTGGAG GACCTTTT T ACAACATAGC CACGAGGAGA AAAGCTTTAA 540

AAAATCCAAG TGAAGAATAT GGGAAAATTT TGGAAGTTGT TGGCAGGTAT TCAGTACACA 600

ATGCAGGCAT TAGTTTCTCA GTTAAAAAAC AAGGAGAGAC AGTAGCTGAT GTTAGGACAC 660

TACCCAATGC CTCAACCGTG GACAATATTC GCTCCGTCTT GGGAAATGCT GTTAGTCGAG 720

AACTGATAGA AATTGGATGT GAGGATAAAA CCCTAGCCTT CAAAATGAAT GGTTACATAT 780

CCAATGCAAA CTACTCAGTG AAGAAGTGCA TCTTCTTACT CTTCATCAAC CATCGTCTGG 840

TAGAATCAAC TTCCTTGAGA AAAGCCATAG AAACAGTGTA TGCAGCCTAT TTGCCAAAAA 900

ACACACACCC ATTCCTGTAC CTCAGTTTAG AAATCAGTCC CCAGAATGTG GATGTTAATG 960

TGAACCCCAC AAAGCATGAA GT CACTTCC TGCACGAGGA GAGCATCCTG GAGCGGGTGC 1020

AGCAGCACAT CGAGAGCAAG CTCCTGGGCT CCAATTCCTC CAGGATGTAC TTCACCCAGA 1080

CTTTGCTACC AGGACTTGCT GGCCCCTCTG GGGAGATGGT TAAATCCACA ACAAGTCTCA 1140

CCTCGTCTTC TACTTCTGGA AGTAGTGATA AGGTCTATGC CCACCAGATG GTTCGTACAG 1200

ATTCCCGGGA ACAGAAGCTT GATGCATTTC TGCAGCCTCT GAGCAAACCC CTGTCCAGTC 1260

AGCCCCAGGC CATTGTCACA GAGGATAAGA CAGATATTTC TAGTGGCAGG GCTAGGCAGC 1320

AAGATGAGGA GATGCTTGAA CTCCCAGCCC CTGCTGAAGT GGCTGCCAAA AATCAGAGCT 1380

TGGAGGGGGA TACAACAAAG GGGACTTCAG .AAATGTCAGA GAAGAGAGGA CCTACTTCCA 1440

GCAACCCCAG AAAGAGACAT CGGGAAGATT CTGATCTCCA AATCCTCGAA GATGATTCCC 1500

GAAAGGAAAT GACTGCAGCT TGTACCCCCC GGAGAAGGAT CATTAACCTC ACTAGTGTTT 1560

TGAGTCTCCA GGAAGAAATT AATGAGCAGG GACATGAGGT TCTCCGGGAG ATGTTGCATA 1620

ACCACTCCTT CGTGGGCTGT GTGAATCCTC AGTGGGCCTT GGCACAGCAT CAAACCAAGT 1680

TATACCTTCT CAACACCACC AAGCTTAGTG AAGAACTGTT CTACCAGATA CTCATTTATG 1740

ATTTTGCCAA TTTTGGTGTT CTCAGGTTAT CGGAGCCAGC ACCGCTCTTT GACCTTGCCA 1800

TGCTTCCCTT ACATAGTCCA GAGAGTGGCT GGACAGAGGA AGATGGTCCC AAAGAAGGAC 1860

TTGCTGAATA CATTGTTGAG TTTCTGAAGA AGAAGGCTGA GATGCTTGCA GACTATTTCT 1920

CTTTGGAAAT TGATGAGGAA GGGAACCTGA TTGGATTACC CCTTCTGATT GACAACTATG 1980

TGCCCCCTTT GGAGGGACTG CCTATCTTCA TTCTTCCACT AGCCACTGAG GTGAATTGGG 2040

ACGAAGAAAA GGAATGTTTT G-AAAGCCTCA GTAAAGAATG CGCTATGTTC TATTCCATCC 2100

GGAAGCAGTA CATATCTGAG GAGTCGACCC TCTCAGGCCA GCAGAGTGAA GTGCCTGGCT 2160

CCATTCCAAA CTCCTGGAAG TGGACTGTGG AACACATTGT CTATAAAGCC TTGCGCTCAC 2220

ACATTCTGCC TCCTAAACAT TCCACAGAAG ATGGAAATAT CCTGCAGCTT GCTAACCTGC 2280

CTGATCTATA CAAAGTCTTT GAGAGGTGTT AAATATGGTT ATTTATGCAC TGTGGGATGT 2340

GTTCTTCTTT CTCTGTATTC CGATACAAAG TGTTGTACTA AAGTGTGATA TACAAAGTGT 2400

ACCAACATAA GTGTTGGTAG CACTTAAGAC TTATACTTGC CTTCTGATAG TATTCCTTTA 2460

TACACAGTGG ATTGATTATA AATAAATAGA TGTGTCTTAA CATAAAAAAA AAAAAAAAAA 2520

AAAAA 2525

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

(A) LENGTH: 756 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS:

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

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

Met Ser Phe Val Ala Gly Val lie Arg Arg Leu Asp Glu Thr Val

5 10 15

Val Asn Arg lie Ala Ala Gly Glu Val lie Gin Arg Pro Ala Asn

20 25 30

Ala lie Lys Glu Met lie Glu Asn Cys Leu Asp Ala Lyε Ser Thr

35 40 45

Ser lie Gin Val lie Val Lyε Glu Gly Gly Leu Lys Leu lie Gin

50 55 60 lie Gin Asp Asn Gly Thr Gly lie Arg Lys Glu Asp Leu Asp lie

65 70 75

Val Cys Glu Arg Phe Thr Thr Ser Lyε Leu Gin Ser Phe Glu Asp

80 85 90

Leu Ala Ser lie Ser Thr Tyr Gly Phe Arg Gly Glu Ala Leu Ala

95 100 105

Ser lie Ser His Val Ala Hiε Val Thr lie Thr Thr Lyε Thr Ala

110 115 120

Asp Gly Lys Cys Ala Tyr Arg Ala Ser Tyr Ser Asp Gly Lyε Leu

125 130 135

Lyε Ala Pro Pro Lyε Pro Cyε Ala Gly Asn Gin Gly Thr Gin lie

140 145 150

Thr Val Glu Asp Leu Phe Tyr Asn lie Ala Thr Arg Arg Lys Ala

155 160 165

Leu Lyε Asn Pro Ser Glu Glu Tyr Gly Lys lie Leu Glu Val Val

170 175 180

Gly Arg Tyr Ser Val Hiε Aεn Ala Gly lie Ser Phe Ser Val Lyε

185 190 195

Lyε Gin Gly Glu Thr Val Ala Aεp Val Arg Thr Leu Pro Aεn Ala

200 205 210

Ser Thr Val Asp Asn lie Arg Ser Val Phe Gly Asn Ala Val Ser

215 220 225

Arg Glu Leu lie Glu lie Gly Cys Glu Aεp Lyε Thr Leu Ala Phe

230 235 240

Lyε Met Asn Gly Tyr lie Ser Asn Ala Asn Tyr Ser Val Lys Lyε

245 250 255

Cyε lie Phe Leu Leu Phe lie Aεn Hiε Arg Leu Val Glu Ser Thr

260 265 270

Ser Leu Arg Lyε Ala lie Glu Thr Val Tyr Ala Ala Tyr Leu Pro

275 280 285

Lys Asn Thr His Pro Phe Leu Tyr Leu Ser Leu Glu lie Ser Pro

290 295 300

Gin Asn Val Asp Val Asn Val His Pro Thr Lyε His Glu Val Hiε

305 310 315

Phe Leu Hiε Glu Glu Ser lie Leu Glu Arg Val Gin Gin Hiε lie

320 325 330

Glu Ser Lyε Leu Leu Gly Ser Asn Ser Ser Arg Met Tyr Phe Thr

335 340 345

Gin Thr Leu Leu Pro Gly Leu Ala Ala Pro Ser Gly Glu Met Val

350 355 360

Lys Ser Thr Thr Ser Leu Thr Ser Ser Ser Thr Ser Gly Ser Ser

365 370 375

Asp Lyε Val Tyr Ala Hiε Gin Met Val Arg Thr Aεp Ser Arg Glu

380 385 390

Gin Lyε Leu Asp Ala Phe Leu Gin Pro Leu Ser Lys Pro Leu Ser

395 400 405

Ser Gin Pro Gin Ala lie Val Thr Glu Asp Lyε Thr Asp lie Ser

410 415 420

Ser Gly Arg Ala Arg Gin Gin Asp Glu Glu Met Leu Glu Leu Pro

425 430 435

Ala Pro Ala Glu Val Ala Ala Lys Asn Gin Ser Leu Glu Gly Asp

440 445 450

Thr Thr Lyε Gly Thr Ser Glu Met Ser Glu Lyε Arg Gly Pro Thr

455 460 465

Ser Ser Aεn Pro Arg Lyε Arg Hiε Arg Glu Aεp Ser Aεp Val Glu

470 475 480

Met Val Glu Asp Asp Ser Arg Lys Glu Met Thr Ala Ala Cyε Thr

485 490 495

Pro Arg Arg Arg lie lie Aεn Leu Thr Ser Val Leu Ser Leu Gin

500 505 510

Glu Glu lie Asn Glu Gin Gly Hiε Glu Val Leu Arg Glu Met Leu

515 520 525

Hiε Aεn Hiε Ser Phe Val Gly Cyε Val Aεn Pro Gin Trp Ala Leu

530 535 540

Ala Gin Hiε Gin Thr Lyε Leu Tyr Leu Leu Asn Thr Thr Lyε Leu

545 550 555

Ser Glu Glu Leu Phe Tyr Gin lie Leu lie Tyr Aεp Phe Ala Asn

560 565 570

Phe Gly Val Leu Arg Leu Ser Glu Pro Ala Pro Leu Phe Asp Leu

575 580 585

Ala Met Leu Ala Leu Asp Ser Pro Glu Ser Gly Trp Thr Glu Glu

590 595 600

Asp Gly Pro Lys Glu Gly Leu Ala Glu Tyr lie Val Glu Phe Leu

605 610 615

Lys Lys Lyε Ala Glu Met Leu Ala Aεp Tyr Phe Ser Leu Glu lie

620 625 630

Asp Glu Glu Gly Asn Leu lie Gly Leu Pro Leu Leu Thr Asp Asn

635 640 645

Tyr Val Pro Pro Leu Glu Gly Leu Pro lie Phe lie Leu Arg Leu

650 655 660

Ala Thr Glu Val Asn Trp Asp Glu Glu Lyε Glu Cyε Phe Glu Ser

665 670 675

Leu Ser Lyε Glu Cyε Ala Met Phe Tyr Ser lie Arg Lyε Gin Tyr

680 685 690 lie Ser Glu Glu Ser Thr Leu Ser Gly Gin Gin Ser Glu Val Pro

695 700 705

Gly Ser lie Pro Asn Ser Trp Lyε Trp Thr Val Glu His lie Val

710 715 720

Tyr Lys Ala Leu Arg Ser Hiε lie Leu Pro Pro Lyε Hiε Phe Thr

725 730 735

Glu Aεp Gly Asn lie Leu Gin Leu Ala Asn Leu Pro Asp Leu Tyr

740 745 750

Lyε Val Phe Glu Arg Cyε

755

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

(A) LENGTH: 3063 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: CDNA

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3

GGCACGAGTG GCTGCTTGCG GCTAGTGGAT GGTAATTGCC TGCCTCGCGC TAGCAGCAAG 60

CTGCTCTGTT AAAAGCGAAA ATGAAACAAT TGCCTGCGGC AACAGTTCGA CTCCTTTCAA 120

GTTCTCAGAT CATCACTTCG GTGGTCAGTG TTGTAAAAGA GCTTATTGAA AACTCCTTGG 180

ATGCTGGTGC CACAAGCGTA GATGTTAAAC TGGAGAACTA TGGATTTGAT AAAATTGAGG 240

TGCGAGATAA CGGGGAGGGT ATCAAGGCTG TTGATGCACC TGTAATGGCA ATGAAGTACT 300

ACACCTCAAA AATAAATAGT CATGAAGATC TTGAAAATTT GACAACTTAC GGTTTTCGTG 360

GAGAAGCCTT GGGGTCAATT TGTTGTATAG CTGAGGTTTT AATTACAACA AGAACGGCTG 420

CTGATAATTT TAGCACCCAG TATGTTTTAG ATGGCAGTGG CCACATACTT TCTCAGAAAC 480

CTTCACATCT TGGTCAAGGT ACAACTGTAA CTGCTTTAAG ATTATTTAAG AATCTACCTG 540

TAAG.AAAGCA GTTTTACTCA ACTGCAAAAA AATGTAAAGA TGAAATAAAA AAGATCCAAG 600

ATCTCCTCAT GAGCTTTGGT ATCCTTAAAC CTGACTTAAG GATTGTCTTT GTACATAACA 660

AGGCAGTTAT TTGGCAGAAA AGCAGAGTAT CAGATCACAA GATGGCTCTC ATGTCAGTTC 720

TGGGGACTGC TGTTATGAAC AATATGGAAT CCTTTCAGTA CCACTCTGAA GAATCTCAGA 780

TTTATCTCAG TGGATTTCTT CCAAAGTGTG ATGCAGACCA CTCTTTCACT AGTCTTTCAA 840

CACCAGAAAG AAGTTTCATC TTCATAAACA GTCGACCAGT ACATCAAAAA GATATCTTAA 900

AGTTAATCCG ACATCATTAC AATCTGAAAT GCCTAAAGGA ATCTACTCGT TTGTATCCTG 960

TTTTCTTTCT GAAAATCGAT GTTCCTACAG CTGATGTTGA TGTAAATTTA ACACCAGATA 1020

AAAGCCAAGT ATTATTACAA AATAAGGAAT CTGTTTTAAT TGCTCTTGAA AATCTGATGA 1080

CGACTTGTTA TGGACCATTA CCTAGTACAA ATTCTTATGA AAATAATAAA ACAGATGTTT 1140

CCGCAGCTGA CATCGTTCTT AGTAAAACAG CAGAAACAGA TGTGCTTTTT AATAAAGTGG 1200

.AATCATCTGG AAAGAATTAT TCAAATGTTG ATACTTCAGT CATTCCATTC CAAAATGATA 1260

TGCATAATGA TGAATCTGGA AAAAACACTG ATGATTGTTT AAATCACCAG ATAAGTATTG 1320

GTGACTTTGG TTATGGTCAT TGTAGTAGTG AAATTTCTAA CATTGATAAA AACACTAAGA 1380

ATGCATTTCA GGACATTTCA ATGAGTAATG TATCATGGGA GAACTCTCAG ACGGAATATA 1440

GTAAAACTTG TTTTATAAGT TCCGTTAAGC ACACCCAGTC AGAAAATGGC AATAAAGACC 1500

ATATAGATGA GAGTGGGGAA AATGAGGAAG AAGCAGGTCT TGAAAACTCT TCGGAAATTT 1560

CTGCAGATGA GTGGAGCAGG GGAAATATAC TTAAAAATTC AGTGGGAGAG AATATTGAAC 1620

CTGTGAAAAT TTTAGTGCCT GAAAAAAGTT TACCATGTAA AGTAAGTAAT AATAATTATC 1680

CAATCCCTGA ACAAATGAAT CTTAATGAAG ATTCATGTAA CAAAAAATCA AATGTAATAG 1740

ATAATAAATC TGGAAAAGTT ACAGCTTATG ATTTACTTAG CAATCGAGTA ATCAAGAAAC 1800

CCATGTCAGC AAGTGCTCTT TTTGTTCAAG ATCATCGTCC TCAGTTTCTC ATAGAAAATC 1860

CTAAGACTAG TTTAGAGGAT GCAACACTAC AAATTGAAGA ACTGTGGAAG ACATTGAGTG 1920

AAGAGGAAAA ACTGAAATAT GAAGAGAAGG CTACTAAAGA CTTGGNACGA TACAATAGTC 1980

AAATGAAGAG AGCCATTGAA CAGGAGTCAC AAATGTCACT AAAAGATGGC AGAAAAAAGA 2040

TAAAACCCAC CAGCGCATGG AATTTGGCCC AGAAGCACAA GTTAAAAACC TCATTATCTA 2100

ATCAACCANA ACTTGATGAA CTCCTTCAGT CCCAAATTGA AAAAAGAAGG AGTCAAAATA 2160

TTAAAATGGT ACAGATCCCC TTTTCTATGA AAAACTTAAA AATAAATTTT AAGAAACAAA 2220

ACAAAGTTGA CTTAGAAGAG AAGGATGAAC CTTGCTTGAT CCACAATCTC AGGTTTCCTG 2280

ATGCATGGCT AATGACATCC AAAACAGAGG TAATGTTATT AAATCCATAT AGAGTAGAAG 2340

AAGCCCTGCT ATTTAAAAGA CTTCTTGAGA ATCATAAACT TCCTGCAGAG CCACTGGAAA 2400

AGCCAATTAT GTTAACAGAG AGTCTTTTTA ATGGATCTCA TTATTTAGAC GTTTTATATA 2460

AAATGACAGC AGATGACCAA AGATACAGTG GATCAACTTA CCTGTCTGAT CCTCGTCTTA 2520

CAGCGAATGG TTTCAAGATA AAATTGATAC CAGGAGTTTC AATTACTGAA AATTACTTGG 2580

AAATAGAAGG AATGGCTAAT TGTCTCCCAT TCTATGGAGT AGCAGATTTA AAAGAAATTC 2640

TTAATGCTAT ATTAAACAGA AATGCAAAGG AAGTTTATGA ATGTAGACCT CGCAAAGTGA 2700

TAAGTTATTT AGAGGGAGAA GCAGTGCGTC TATCCAGACA ATTACCCATG TACTTATCAA 2760

AAGAGGACAT CCAAGACATT ATCTACAGAA TGAAGCACCA GTTTGGAAAT GAAATTAAAG 2820

AGTGTGTTCA TGGTCGCCCA TTTTTTCATC ATTTAACCTA TCTTCCAGAA ACTACATGAT 2880

TAAATATGTT T.AAGAAGATT AGTTACCATT GAAATTGGTT CTGTCATAAA ACAGCATGAG 2940

TCTGGTTTTA AATTATCTTT GTATTATGTG TCACATGGTT ATTTTTTAAA TGAGGATTCA 3000

CTGACTTGTT TTTATATTGA AAAAAGTTCC ACGTATTGTA GAAAACGTAA ATAAACTAAT 3060

AAC 3063

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 931 BASE PAIRS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS:

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN (XI)

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

Met Lyε Gin Leu Pro Ala Ala Thr Val Arg Leu Leu Ser Ser Ser

5 10 15

Gin lie lie Thr Ser Val Val Ser Val Val Lyε Glu Leu lie Glu

20 25 30

Aεn Ser Leu Aεp Ala Gly Ala Thr Ser Val Aεp Val Lys Leu Glu

35 40 45

Aεn Tyr Gly Phe Asp Lyε lie Glu Val Arg Asp Asn Gly Glu Gly

50 55 60 lie Lys Ala Val Asp Ala Pro Val Met Ala Met Lys Tyr Tyr Thr

65 70 75

Ser Lyε lie Asn Ser Hiε Gly Aεp Leu Glu Asn Leu Thr Thr Tyr

80 85 90

Gly Phe Arg Gly Glu Ala Leu Gly Ser lie Cys Cyε lie Ala Glu

95 100 105

Val Leu lie Thr Thr Arg Thr Ala Ala Aεp Aεn Phe Ser Thr Gin

110 115 120

Tyr Val Leu Asp Gly Ser Gly His lie Leu Ser Gin Lys Pro Ser

125 130 135

Hiε Leu Gly Gin Gly Thr Thr Val Thr Ala Leu Arg Leu Phe Lyε

140 145 150

Asn Leu Pro Val Arg Lys Gin Phe Tyr Ser Thr Ala Lys Lyε Cyε

155 160 165

Lyε Asp Glu lie Lys Lyε lie Gin Aεp Leu Leu Met Ser Phe Gly

170 175 180 lie Leu Lyε Pro Asp Leu Arg lie Val Phe Val His Asn Lys Ala

185 190 195

Val lie Trp Gin Lys Ser Arg Val Ser Asp His Lys Met Ala Leu

200 205 210

Met Ser Val Leu Gly Thr Ala Val Met Asn Asn Met Glu Ser Phe

215 220 225

Gin Tyr Hiε Ser Glu Glu Ser Gin lie Tyr Leu Ser Gly Phe Leu

230 235 240

Pro Lyε Cyε Asp Ala Asp His Ser Phe Thr Ser Leu Ser Thr Pro

245 250 255

Glu Arg Ser Phe lie Phe lie Asn Ser Arg Pro Val Hiε Gin Lyε

260 265 270

Aεp lie Leu Lyε Leu lie Arg Hiε Hiε Tyr Aεn Leu Lyε Cyε Leu

275 280 285

Lyε Glu Ser Thr Arg Leu Tyr Pro Val Phe Phe Leu Lyε lie Asp

290 295 300

Val Pro Thr Ala Asp Val Aεp Val Aεn Leu Thr Pro Aεp Lyε Ser

305 310 315

Gin Val Leu Leu Gin Aεn Lyε Glu Ser Val Leu lie Ala Leu Glu

320 325 330

Aεn Leu Met Thr Thr Cyε Tyr Gly Pro Leu Pro Ser Thr Asn Ser

335 340 345

Tyr Glu Asn Asn Lyε Thr Asp Val Ser Ala Ala Asp lie Val Leu

350 355 360

Ser Lys Thr Ala Glu Thr Asp Val Leu Phe Asn Lyε Val Glu Ser

365 370 375

Ser Gly Lyε Aεn Tyr Ser Asn Val Asp Thr Ser Val lie Pro Phe

380 385 390

Gin Asn Asp Met Hiε Aεn Aεp Glu Ser Gly Lyε Aεn Thr Aεp Aεp

395 400 405

Cyε Leu Asn His Gin lie Ser lie Gly Aεp Phe Gly Tyr Gly Hiε

410 415 420

Cyε Ser Ser Glu lie Ser Asn lie Asp Lys Asn Thr Lys Asn Ala

425 430 435 Phe Gin Asp lie Ser Met Ser Aεn Val Ser Trp Glu Aεn Ser Gin

440 445 450

Thr Glu Tyr Ser Lyε Thr Cyε Phe lie Ser Ser Val Lyε Hiε Thr

455 460 465

Gin Ser Glu Aεn Gly Aεn Lyε Asp Hiε lie Asp Glu Ser Gly Glu

470 475 480

Asn Glu Glu Glu Ala Gly Leu Glu Asn Ser Ser Glu lie Ser Ala

485 490 495

Asp Glu Trp Ser Arg Gly Asn lie Leu Lyε Aεn Ser Val Gly Glu

500 505 510

Aεn lie Glu Pro Val Lyε lie Leu Val Pro Glu Lyε Ser Leu Pro

515 520 525

Cyε Lyε Val Ser Aεn Aεn Asn Tyr Pro lie Pro Glu Gin Met Asn

530 535 540

Leu Asn Glu Asp Ser Cyε Aεn Lyε Lyε Ser Asn Val lie Asp Asn

545 550 555

Lyε Ser Gly Lyε Val Thr Ala Tyr Aεp Leu Leu Ser Asn Arg Val

560 565 570 lie Lys Lys Pro Met Ser Ala Ser Ala Leu Phe Val Gin Asp Hiε

575 580 585

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

590 595 600 Ala Thr Leu Gin lie Glu Glu Leu Trp Lys Thr Leu Ser Glu Glu

605 610 615 Glu Lyε Leu Lyε Tyr Glu Glu Lyε Ala Thr Lyε Aεp Leu Xaa Arg

620 625 630 Tyr Asn Ser Gin Met Lys Arg Ala lie Glu Gin Glu Ser Gin Met

635 640 645 Ser Leu Lys Asp Gly Arg Lyε Lys lie Lyε Pro Thr Ser Ala Trp

650 655 660 Aεn Leu Ala Gin Lyε Hiε Lyε Leu Lyε Thr Ser Leu Ser Asn Gin

665 670 675

Pro Xaa Leu Asp Glu Leu Leu Gin Ser Gin lie Glu Lys Arg Arg

680 685 690

Ser Gin Asn lie Lys Met Val Gin lie Pro Phe Ser Met Lys Asn

695 700 705

Leu Lyε lie Aεn Phe Lyε Lyε Gin Asn Lys Val Asp Leu Glu Glu

710 715 720

Lys Asp Glu Pro Cys Leu lie His Asn Leu Arg Phe Pro Asp Ala

725 730 735

Trp Leu Met Thr Ser Lyε Thr Glu Val Met Leu Leu Asn Pro Tyr

740 745 750

Arg Val Glu Glu Ala Leu Leu Phe Lyε Arg Leu Leu Glu Aεn Hiε

755 760 765

Lyε Leu Pro Ala Glu Pro Leu Glu Lyε Pro lie Met Leu Thr Glu

770 775 780

Ser Leu Phe Asn Gly Ser Hiε Tyr Leu Aεp Val Leu Tyr Lyε Met

785 790 795

Thr Ala Asp Asp Gin Arg Tyr Ser Gly Ser Thr Tyr Leu Ser Asp

800 805 810

Pro Arg Leu Thr Ala Asn Gly Phe Lys lie Lys Leu lie Pro Gly

815 820 825

Val Ser lie Thr Glu Aεn Tyr Leu Glu lie Glu Gly Met Ala Asn

830 835 840

Cyε Leu Pro Phe Tyr Gly Val Ala Asp Leu Lyε Glu lie Leu Asn

845 850 855

Ala lie Leu Asn Arg Asn Ala Lyε Glu Val Tyr Glu Cyε Arg Pro

860 865 870

Arg Lyε Val lie Ser Tyr Leu Glu Gly Glu Ala Val Arg Leu Ser

875 880 885

Arg Gin Leu Pro Met Tyr Leu Ser Lyε Glu Aεp lie Gin Asp lie

890 895 900 lie Tyr Arg Met Lyε Hiε Gin Phe Gly Aεn Glu lie Lyε Glu Cyε

905 910 915

Val Hiε Gly Arg Pro Phe Phe Hiε Hiε Leu Thr Tyr Leu Pro Glu

920 925 930 Thr

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

(A) LENGTH: 2771 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS : SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

CGAGGCGGAT CGGGTGTTGC ATCCATGGAG CGAGCTGAGA GCTCGAGTAC AGAACCTGCT 60

AAGGCCATCA AACCTATTGA TCGGAAGTCA GTCCATCAGA TTTGCTCTGG GCAGGTGGTA 120

CTGAGTCTAA GCACTGCGGT AAAGGAGTTA GTAGAAAACA GTCTGGATGC TGGTGCCACT 180

AATATTGATC TAAAGCTTAA GGACTATGGA GTGGATCTTA TTGAAGTTTC AGACAATGGA 240

TGTGGGGTAG AAGAAGAAAA CTTCGAAGGC TTAACTCTGA AACATCACAC ATCTAAGATT 300

CAAGAGTTTG CCGACCTAAC TCAGGTTGAA ACTTTTGGCT TTCGGGGGGA AGCTCTGAGC 360

TCACTTTGTG CACTGAGCGA TGTCACCATT TCTACCTGCC ACGCATCGGC GAAGGTTGGA 420

ACTCGACTGA TGTTTGATCA CAATGGGAAA ATTATCCAGA AAACCCCCTA CCCCCGCCCC 480

AGAGGGACCA CAGTCAGCGT GCAGCAGTTA TTTTCCACAC TACCTGTGCG CCATAAGGAA 540

TTTCAAAGGA ATATTAAGAA GGAGTATGCC AAAATGGTCC AGGTCTTACA TGCATACTGT 600

ATCATTTCAG CAGGCATCCG TGTAAGTTGC ACCAATCAGC TTGGACAAGG AAAACGACAG 660

CCTGTGGTAT GCACAGGTGG AAGCCCCAGC ATAAAGGAAA ATATCGGCTC TGTGTTTGGG 720

CAGAAGCAGT TGCAAAGCCT CATTCCTTTT GTTCAGCTGC CCCCTAGTGA CTCCGTGTGT 780

GAAGAGTACG GTTTGAGCTG TTCGGATGCT CTGCATAATC TTTTTTACAT CTCAGGTTTC 840

ATTTCACAAT GCACGCATGG AGTTGGAAGG AGTTCAACAG ACAGACAGTT TTTCTTTATC 900

AACCGGCGGC CTTGTGACCC AGCAAAGGTC TGCAGACTCG TGAATGAGGT CTACCACATG 960

TATAATCGAC ACCAGTATCC ATTTGTTGTT CTTAACATTT CTGTTGATTC AGAATGCGTT 1020

GATATCAATG TTACTCCAGA TAAAAGGCAA ATTTTGCTAC AAGAGGAAAA GCTTTTGTTG 1080

GCAGTTTTAA AGACCTCTTT GATAGGAATG TTTGATAGTG ATGTCAACAA GCTAAATGTC 1140

AGTCAGCAGC CACTGCTGGA TGTTGAAGGT AACTTAATAA AAATGCATGC AGCGGATTTG 1200

GAAAAGCCCA TGGTAGAAAA GCAGGATCAA TCCCCTTCAT TAAGGACTGG AGAAGAAAAA 1260

AAAGACGTGT CCATTTCCAG ACTGCGAGAG GCCTTTTCTC TTCGTCACAC AACAGAGAAC 1320

AAGCCTCACA GCCCAAAGAC TCCAGAACCA AGAAGGAGCC CTCTAGGACA GAAAAGGGGT 1380

ATGCTGTCTT CTAGCACTTC AGGTGCCATC TCTGACAAAG GCGTCCTGAG ACCTCAGAAA 1440

GAGGCAGTGA GTTCCAGTCA CGGACCCAGT GACCCTACGG ACAGAGCGGA GGTGGAGAAG 1500

GACTCGGGGC ACGGCAGCAC TTCCGTGGAT TCTGAGGGGT TCAGCATCCC AGACACGGGC 1560

AGTCACTGCA GCAGCGAGTA TGCGGCCAGC TCCCCAGGGG ACAGGGGCTC GCAGGAACAT 1620

GTGGACTCTC AGGAGAAAGC GCCTGAAACT GACGACTCTT TTTCAGATGT GGACTGCCAT 1680

TCAAACCAGG AAGATACCGG ATGTAAATTT CGAGTTTTGC CTCAGCCAAC TAATCTCGCA 1740

ACCCCAAACA CAAAGCGTTT TAAAAAAGAA GAAATTCTTT CCAGTTCTGA CATTTGTCAA 1800

AAGTTAGTAA ATACTCAGGA CATGTCAGCC TCTCAGGTTG ATGTAGCTGT GAAAATTAAT 1860

AAGAAAGTTG TGCCCCTGGA CTTTTCTATG AGTTCTTTAG CTAAACGAAT AAAGCAGTTA 1920

CATCATGAAG CACAGCAAAG TGAAGGGGAA CAGAATTACA GGAAGTTTAG GGCAAAGATT 1980

TGTCCTGGAG AAAATCAAGC AGCCGAAGAT GAACTAAGAA AAGAGATAAG TAAAACGATG 2040

TTTGCAGAAA TGGAAATCAT TGGTCAGTTT AACCTGGGAT TTATAATAAC CACACTGAAT 2100

GAGGATATCT TCATAGTGGA CCAGCATGCC ACGGACGAGA AGTATAACTT CGAGATGCTG 2160

CAGCAGCACA CCGTGCTCCA GGGGCAGACG CTCATAGCAC CTCAGACTCT CAACTTAACT 2220

GCTGTTAATG AAGCTGTTCT GATAGAAAAT CTGGAAATAT TTAGAAAGAA TGGCTTTGAT 2280

TTTGTTATCG ATGAAAATGC TCCAGTCACT GAAAGGGCTA AACTGATTTC CTTGCCAACT 2340

AGTAAAAACT GGACCTTCGG ACCCCAGGAC GTCGATGAAC TGATCTTCAT GCTGAGCGAC 2400

AGCCCTGGGG TCATGTGCCG GCCTTCCCGA GTCAAGCAGA TGTTTGCCTC CAGAGCCTGC 2460

CGGAAGTCGG TGATGATTGG GACTGCTCTT AACACAAGCG AGATGAAGAA ACTGATCACC 2520

CACATGGGGG AGATGGACCA CCCCTGGAAC TGTCCCCATG GAAGGCCAAC CATGAGACAC 2580

ATCGCCAACC TGGGTGTCAT TTCTCAG.AAC TGACCGTAGT CACTGTATGG AATAATTGGT 2640

TTTATCGCAG ATTTTTATGT TTTGAAAGAC AGAGTCTTCA CTAACCTTTT TTGTTTTAAA 2700

ATGAAACCTG CTACTTAAAA AAAATACACA TCACACCCAT TTAAAAGTGA TCTTGAGAAC 2760

CTTTTCAAAC C 2771

(2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 862 AMINO ACIDS

(B) TYPE: AMINO ACID

( C) STRANDEDNESS :

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro Ala Lys Ala lie

5 10 15

Lys Pro lie Asp Arg Lys Ser Val His Gin lie Cyε Ser Gly Gin

20 25 30

Val Val Leu Ser Leu Ser Thr Ala Val Lyε Glu Leu Val Glu Asn

35 40 45

Ser Leu Asp Ala Gly Ala Thr Asn lie Asp Leu Lyε Leu Lyε Aεp

50 55 60

Tyr Gly Val Asp Leu lie Glu Val Ser Asp Asn Gly Cys Gly Val

65 70 75

Glu Glu Glu Asn Phe Glu Gly Leu Thr Leu Lyε Hiε Hiε Thr Ser

80 85 90

Lyε lie Gin Glu Phe Ala Aεp Leu Thr Gin Val Glu Thr Phe Gly

95 100 105

Phe Arg Gly Glu Ala Leu Ser Ser Leu Cys Ala Leu Ser Asp Val

110 115 120

Thr lie Ser Thr Cys His Ala Ser Ala Lys Val Gly Thr Arg Leu

125 130 135

Met Phe Asp His Asn Gly Lys lie lie Gin Lyε Thr Pro Tyr Pro

140 145 150

Arg Pro Arg Gly Thr Thr Val Ser Val Gin Gin Leu Phe Ser Thr

155 160 165

Leu Pro Val Arg Hiε Lyε Glu Phe Gin Arg Asn lie Lys Lyε Glu

170 175 180

Tyr Ala Lys Met Val Gin Val Leu His Ala Tyr Cys lie lie Ser

185 190 195

Ala Gly lie Arg Val Ser Cyε Thr Aεn Gin Leu Gly Gin Gly Lys

200 205 210

Arg Gin Leu Trp Tyr Ala Gin Val Glu Ala Pro Ala lie Lys Glu

215 220 225

Asn lie Gly Ser Val Phe Gly Gin Lys Gin Leu Gin Ser Leu lie

230 235 240

Pro Phe Val Gin Leu Pro Pro Ser Aεp Ser Val Cyε Glu Glu Tyr

245 250 255

Gly Leu Ser Cyε Ser Asp Ala Leu His Asn Leu Phe Tyr lie Ser

260 265 270

Gly Phe lie Ser Gin Cyε Thr Hiε Gly Val Gly Arg Ser Ser Thr

275 280 285

Asp Arg Gin Phe Phe Phe lie Asn Arg Arg Pro Cys Asp Pro Ala

290 295 300

Lys Val Cys Arg Leu Val Asn Glu Val Tyr Hiε Met Tyr Aεn Arg

305 310 315

Hiε Gin Tyr Pro Phe Val Val Leu Asn lie Ser Val Asp Ser Glu

320 325 330

Cyε Val Asp lie Asn Val Thr Pro Asp Lyε Arg Gin lie Leu Leu

335 340 345

Gin Glu Glu Lys Leu Leu Leu Ala Val Leu Lyε Thr Ser Leu lie

350 355 360

Gly Met Phe Aεp Ser Aεp Val Aεn Lyε Leu Aεn Val Ser Gin Gin

365 370 375

Pro Leu Leu Aεp Val Glu Gly Aεn Leu lie Lyε Met Hiε Ala Ala

380 385 390

Aεp Leu Glu Lyε Pro Met Val Glu Lys Gin Asp Gin Ser Pro Ser

395 400 405

Leu Arg Thr Gly Glu Glu Lyε Lyε Asp Val Ser lie Ser Arg Leu

410 415 420

Arg Glu Ala Phe Ser Leu Arg His Thr Thr Glu Asn Lyε Pro Hiε

425 430 435

Ser Pro Lys Thr Pro Glu Pro Arg Arg Ser Pro Leu Gly Gin Lyε

440 445 450

Arg Gly Met Leu Ser Ser Ser Thr Ser Gly Ala lie Ser Aεp Lyε

455 460 465

Gly Val Leu Arg Pro Gin Lyε Glu Ala Val Ser Ser Ser Hiε Gly

470 475 480

Pro Ser Aεp Pro Thr Asp Arg Ala Glu Val Glu Lys Asp Ser Gly

485 490 495

His Gly Ser Thr Ser Val Aεp Ser Glu Gly Phe Ser lie Pro Aεp

500 505 510

Thr Gly Ser Hiε Cyε Ser Ser Glu Tyr Ala Ala Ser Ser Pro Gly

515 520 525

Asp Arg Gly Ser Gin Glu Hiε Val Aεp Ser Gin Glu Lyε Ala Pro

530 535 540

Glu Thr Aεp Aεp Ser Phe Ser Aεp Val Aεp Cyε Hiε Ser Aεn Gin

545 550 555

Glu Aεp Thr Gly Cyε Lyε Phe Arg Val Leu Pro Gin Pro Thr Asn

560 565 570

Leu Ala Thr Pro Asn Thr Lys Arg Phe Lyε Lyε Glu Glu lie Leu

575 580 585

Ser Ser Ser Aεp lie Cyε Pro Gin Leu Val Asn Thr Gin Asp Met

590 595 600

Ser Ala Ser Gin Val Asp Val Ala Val Lys lie Aεn Lyε Lyε Val

605 610 615

Val Pro Leu Aεp Phe Ser Met Ser Ser Leu Ala Lyε Arg lie Lyε

620 625 630

Gin Leu Hiε Hiε Glu Ala Gin Gin Ser Glu Gly Glu Gin Aεn Tyr

635 640 645

Arg Lyε Phe Arg Ala Lyε lie Cyε Pro Gly Glu Aεn Gin Ala Ala

650 655 660

Glu Aεp Glu Leu Arg Lyε Glu lie Ser Lyε Thr Met Phe Ala Glu

665 670 675

Met Glu lie lie Gly Gin Phe Aεn Leu Gly Phe lie lie Thr Thr

680 685 690

Leu Asn Glu Asp lie Phe lie Val Asp Glu Hiε Ala Thr Asp Glu

695 700 705

Lyε Tyr Aεn Phe Glu Met Leu Gin Gin Hiε Thr Val Leu Gin Gly

710 715 720

Gin Arg Leu lie Ala Pro Glu Thr Leu Aεn Leu Thr Ala Val Aεn

725 730 735

Glu Ala Val Leu lie Glu Asn Leu Glu lie Phe Arg Lyε Asn Gly

740 745 750

Phe Asp Phe Val lie Asp Glu Asn Ala Pro Val Thr Glu Arg Ala

755 760 765

Lyε Leu lie Ser Leu Pro Thr Ser Lyε Asn Trp Thr Phe Gly Pro

770 775 780

Gin Asp Val Asp Glu Leu lie Phe Met Leu Ser Asp Ser Pro Gly

785 790 795

Val Met Cyε Arg Pro Ser Arg Val Lys Gin Met Phe Ala Ser Arg

800 805 810

Ala Cys Arg Lys Ser Val Met lie Gly Thr Ala Leu Asn Thr Ser

815 820 825

Glu Met Lys Lys Leu lie Thr His Met Gly Glu Met Asp Hiε Pro

830 835 840

Trp Aεn Cyε Pro Hiε Gly Arg Pro Thr Met Arg Hiε lie Ala Asn

845 850 855

Leu Gly Val lie Ser Gin Asn 860

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GTTGAACATC TAGACGTCTC 20 (2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID .(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

TCGTGGCAGG GGTTATTCG 19

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

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

CTACCCAATG CCTCAACCG 19

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 22 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10

GAGAACTGAT AGAAATTGGA TG 22

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 18 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GGGACATGAG GTTCTCCG 18

(2) INFORMATION FOR SEQ ID NO:12:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: GGGCTGTGTG AATCCTCAG 19

(2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CGGTTCACCA CTGTCTCGTC 20

(2) INFORMATION FOR SEQ ID NO: 14 :

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 18 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TCCAGGATGC TCTCCTCG 18

(2) INFORMATION FOR SEQ ID NO:15:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) ST.RANDEDNESS : SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: CAAGTCCTGG TAGCAAAGTC 20

(2) INFO.RMATION FOR SEQ ID NO:16:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: ATGGCAAGGT CAAAGAGCG 19

(2) INFORMATION FOR SEQ ID NO:17:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 22 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: CAACAATGTA TTCAGN.AAGT CC 22

(2) INFORMATION FOR SEQ ID NO:18:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

TTGATACAAC ACTTTGTATC G 21

(2) INFORMATION FOR SEQ ID NO:19:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GGAATACTAT CAGAAGGCAA G 21

(2) INFORMATION FOR SEQ ID NO:20:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: ACAGAGCAAG TTACTCAGAT G 21

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GTACACAATG CAGGCATTAG 20

(2) INFORMATION FOR SEQ ID NO:22:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: AATGTGGATG TTAATGTGCA C 21

(2) INFORMATION FOR SEQ ID NO:23:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CTGACCTCGT CTTCCTAC 19

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: CAGCAAGATG AGGAGATGC 19

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

GGAAATGGTG GAAGATGATT C 21

(2) INFORMATION FOR SEQ ID NO:26:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 16BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: CTTCTCAACA CCAAGC 16

(2) INFORMATION FOR SEQ ID NO:27:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GAAATTGATG AGGAAGGGAA C 21

(2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 22 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: CTTCTGATTG ACAACTATGT GC 22

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 22 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: CACAGAAGAT GGAAATATCC TG 22

(2) INFORMATION FOR SEQ ID NO:30:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: GTGTTGGTAG CACTTAAGAC 20

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TTTCCCATAT TCTTCACTTG 20

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: GTAACATGAG CCACATGGC 19

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CCACTGTCTC GTCCAGCCG 19

(2) INFORMATION FOR SEQ ID NO:34:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 26 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CGGGATCCAT GTCGTTCGTG GCAGGG 26

(2) INFORMATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 26 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: GCTCTAGATT AACACCTCTC AAAGAC 26

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: GCATCTAGAC GTTTCCTTGG C 21

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: CATCCAAGCT TCTGTTCCCG 20

(2) INFORMATION FOR SEQ ID NO:38:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: GGGGTGCAGC AGCACATCG 19

(2) INFORMATION FOR SEQ ID NO:39:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GGAGGCAGAA TGTGTGAGCG 20

(2) INFORMATION FOR SEQ ID NO:40:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40: TCCCAAAGAA GGACTTGCT 19

(2) INFORMATION FOR SEQ ID NO:41:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 22 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: AGTATAAGTC TTAAGTGCTA CC 22

(2) INFORMATION FOR SEQ ID NO:42:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi) SEQUENCE DESCRIPTION: SEQ ID N0:41: TTTATGGTTT CTCACCTGCC 20

(2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: GTTATCTGCC CACCTCAGC 19

(2) INFORMATION FOR SEQ ID N0:44:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 59 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:

GGATCCTAAT ACGACTCACT ATAGGGAGAC CACCATGGCA TCTAGACGTT TCCCTTGGC 59

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: CATCCAAGCT TCTGTTCCCG 20

(2) INFORMATION FOR SEQ ID NO:46:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 56 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:

GGATCCTAAT ACGACTCACT ATAGGGAGAC CACCATGGGG GTGCAGCAGC ACATCG 56

(2) INFORMATION FOR SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: GGAGGCAGAA TGTGTGAGCG 20

(2) INFORMATION FOR SEQ ID NO:48:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 28 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: CGGGATCCAT GAAACAATTG CCTGCGGC 28

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 26 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: GCTCTAGACC AGACTCATGC TGTTTT 26

(2) INFORMATION FOR SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 26 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:50: CGGGATCCAT GGAGCGAGCT GAGAGC 26

(2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 23 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

( i) SEQUENCE DESCRIPTION: SEQ ID NO:51:

GCTCTAGAGT GAAGACTCTG TCT 23

(2) INFORMATION FOR SEQ ID NO:52:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52: AAGCTGCTCT GTT.AAAAGCG 20

(2) INFORMATION FOR SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 18 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: GCACCAGCAT CCAAGGAG 18

(2) INFORMATION FOR SEQ ID NO: 54:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: CAACCATGAG ACACATCGC 19

(2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55: AGGTTAGTGA AGACTCTGTC 20

(2) INFORMATION FOR SEQ ID NO: 56:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 53 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

GGATCCTAAT ACGACTCACT ATAGGGAGAC CACCATGGAA CAATTGCCTG CGG 53

(2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 18 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57: CCTGCTCCAC TCATCTGC 18

(2) INFORMATION FOR SEQ ID NO: 58:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 60 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58: GGATCCTAAT ACGACTCACT ATAGGGAGAC CACCATGGAA GATATCTTAA AGTTAATCCG 60

(2) INFORMATION FOR SEQ ID NO: 59:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

( i) SEQUENCE DESCRIPTION: SEQ ID NO.-59: GGCTTCTTCT ACTCTATATG G 21

(2) INFORMATION FOR SEQ ID NO:60:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 58 BASE PAIRS

(B) TYPE: .NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 60 : GGATCCTAAT ACGACTCACT ATAGGGAGAC CACCATGGCA GGTCTTGAAA ACTCTTCG 58

( 2 ) INFORMATION FOR SEQ ID NO : 61 :

( i ) SEQUENCE CHARACTERISTICS

(A) LENGTH : 21 BASE PAIRS

(B ) TYPE : NUCLEIC ACID

( C) STRANDEDNESS : S INGLE

(D) TOPOLOGY : LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: AAAACAAGTC AGTGAATCCT C 21

(2) INFORMATION FOR SEQ ID NO: 62:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: AAGCACATCT GTTTCTGCTG 20

(2) INFORMATION FOR SEQ ID NO:63:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR (ii) MOLECULE TYPE: Oligonucleotide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: ACGAGTAGAT TCCTTTAGGC 20

(2) INFORMATION FOR SEQ ID NO: 64:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:

CAGAACTGAC ATGAGAGCC 19

(2) INFORMATION FOR SEQ ID NO: 65:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 52 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

( ii ) MOLECULE TYPE : Ol igonucleotide

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 65 : GGATCCTAAT ACGACTCACT ATAGGGAGAC CACCATGGAG CGAGCTGAGA GC 52

(2 ) INFORMATION FOR SEQ ID NO : 66 :

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEi^R

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66: AGGTTAGTGA AGACTCTGTC 20

(2) INFORMATION FOR SEQ ID NO:67:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 17 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67: CTGAGGTCTC AGCAGGC 17

(2) INFORMATION FOR SEQ ID NO:68:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 57 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

( ii ) MOLECULE TYPE : Oligonucleotide

(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 68 :

GGATCCTAAT ACGACTCACT ATAGGGAGAC CACCATGGTG TCCATTTCCA GACTGCG 57

( 2 ) INFORMATION FOR SEQ ID NO : 69 :

( i ) SEQUENCE CHAI?ACTERISTI CS

(A) LENGTH : 20 BASE PAIRS

(B) TYPE : NUCLEIC ACID

( C) STRANDEDNESS : SINGLE

(D) TOPOLOGY : LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69: AGGTTAGTGA AGACTCTGTC 20

(2) INFORMATION FOR SEQ ID NO: 70:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:

TTATTTGGCA GAAAAGCAGA G 21

(2) INFORMATION FOR SEQ ID NO: 71:

(i) SEQUENCE CHAIΪACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71: TTAAAAGACT AACCTCTTGC C 21

(2) INFORMATION FOR SEQ ID NO: 72:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 21 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72: CTGCTGTTAT GAACAATATG G 21

(2) INFORMATION FOR SEQ ID NO: 73:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: CAGAAGCAGT TGCAAAGCC 19

(2) INFORMATION FOR SEQ ID NO: 74:

(i) SEQUENCE CHAIΪACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS : SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: AAACCGTACT CTTCACACAC 20

(2) INFORMATION FOR SEQ ID NO: 75:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 20 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: GAGGAAAAGC TTTTGTTGGC 20

(2) INFORMATION FOR SEQ ID NO: 76:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 18 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76: CAGTGGCTGC TGACTGAC 18

(2) INFORMATION FOR SEQ ID NO: 77:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 19 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:

TCCAGAACCA AGAAGGAGC 19

(2) INFORMATION FOR SEQ ID NO: 78:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 16 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: Oligonucleotide ( i) SEQUENCE DESCRIPTION: SEQ ID NO: 78: TGAGGTCTCA GCAGGC 16