METHODS FOR THE DIAGNOSIS AND PROGNOSIS OF CANCER

Reference to Government Grant

The invention described herein was supported in part by National Institutes of Health grant ROl CA60999-01A1. The U.S. government has certain rights in the invention.

Cross-Reference to Related Applications

This application claims priority from U.S. provisional patent application No. 60/039,532 filed March 3, 1997, U.S. Provisional Application

No. 60/020, 196 filed June 21, 1996, U.S. Provisional Application No. 60/019,372 filed June 5, 1996 and U.S. Provisional Application No. 60/014,943 filed April 5, 1996.

Field of the Invention

The invention relates to methods for the identification of individuals at risk for cancer, and for the detection and evaluation of cancers.

Background of the Invention

A. The Rb Family of Tumor Suppressors

Many types of human cancer are believed to be caused by an imbalance of growth regulators within a cell. A decrease in negative control growth regulators and/or their deactivation can cause a cancerous condition. Alternatively, an increase in positive control growth regulators can also cause a cancerous condition.

Since the identification of the first tumor suppressor gene, much effort in cancer research has been focused on the identification of new tumor suppressor genes and their involvement in human cancer. Many types of human cancers are thought to develop by a loss of heterozygosity of putative tumor suppressor genes not yet identified (Lasko et al. , Annu. Rev. Genetics, 25. 281- 296 (1991)) according to Knudson's "two-hit" hypothesis (Knudson, Proc. Natl. Acad. Sci. USA, 68, 820-823 (1971)).

One of the most studied tumor suppressor genes is the retinoblastoma susceptibility gene (Rb), whose gene product (pRb, pl05, or pRb/pl05) has been shown to play a key role in the regulation of cell division. In interphasic cells, pRb contributes to maintaining the quiescent state of the cell by repressing transcription of genes required for the cell cycle through interaction with transcription factors, such as E2F (Wagner et al.. Nature, 352, 189-190 (1991); Nevins, Science, 258, 424-429 (1992); and Hiebert et al. , Genes Develop., 6, 177-185 (1992)). The loss of this activity can induce cell transformation as evidenced by the reversion of the transformed phenotype in pRb cells after replacement of a functional pRb (Huang et al , Science 242 1563-1565 (1988); Bookstein et al , Science, 247: 712-715 (1990); and Sumegi et al., Cell Growth Differ., 1 247-250 (1990)). Upon entrance into the cell cycle, pRb seems to be phosphorylated by cell cycle-dependent kinases (Lees et al, EMBO J. 10:4219- 4290 (1991); Hu et al. , Mol Cell. Biol , 12:971-980 (1992); Hinds et al. , Cell, 70:993-1006 (1992); and Matsushime et al . Nature, 35:295-300)) which is thought to permit its dissociation from transcription factors and, hence, the expression of genes required for progression through the cell cycle.

It has been found that the retinoblastoma protein family includes at least three members. Two other proteins, pi 07, and the recently cloned pRb2/pl30, share regions of homology with pRb/pl05, especially in two discontinuous domains which make up the "pocket region" . Ewen et al , Cell 66: 1155-1 164 (1993); Mayol et al , Oncogene 8: 1561-2566 (1993); Li et al .

Genes Dev. 7: 2366-2377 (1993); and Harmon et al. . Genes Dev. 7: 2378-2391

(1993). The pocket domain is required for binding with several viral transforming oncoproteins (Moran, Curr. Opin. Genet. Dev. 3: 63-70 (1993)).

The pRb2/pl30 cDNA and putative amino acid sequence are set forth by Li et al. The pl07 cDNA and putative amino acid sequence are set forth by Ewen et al. The entire disclosures of Li et al. and Ewen et al are incoφorated herein by reference.

It has been found that pRb2/pl30, as well as pl07 and pRb, act as negative regulators of cell cycle progression, blocking the cells in the GI phase (Goodrich et al , Cell 67: 293-302 (1991); Zhu et al , Genes Dev. 7: 1111-1125 (1993); Claudio et al , Cancer Res. 54:5556-5560 (1994); and Zhu et al , EMBO J. 74:1904-1913 (1995)). However, the three proteins exhibit different growth suppressive properties in selected cell lines, suggesting that although the different members of the retinoblastoma protein family may complement each other, they are not fully functionally redundant (Claudio et al , supra).

The mechanisms by which these three proteins exert their control on cell cycle progression are not fully understood but likely include complex formation and modulation of the activity of several transcription factors (Sang et al. , Mol. Cell Differ. 3: 1-29 (1995)). The most studied of these complexes is the one with the E2F family of transcription factors. E2F's are heterodimeric transcription factors composed of E2F-like and DP-like subunits that regulate the expression of genes required for progression through G ₀ /G, S phase of the cell cycle (Lan Thangue, N.B. , Trends Biochem. Sci. 19: 108-114 (1994)). The three proteins bind and modulate the activity of distinct

E2F/DP1 complexes in different phases of the cell cycle (Sang et al. . supra; Chellapan <?t tf/. , Cell 65: 1053-1061 (1991); Shirodkar et al. , Cell 66: 157-166 (1992); Cobrinik et al. , Genes Dev. 7:2392-2404 (1993); Hijmans et al. , Mol. Cell. Biol. 75:3082-3089 (1995); and Vairo et al . Genes Dev. 9:869-881

( 1995)). This suggests distinct roles for these related proteins in the regulation of the cell cycle.

It has been demonstrated that the growth suppressive properties of pRb2/pl30 are specific for the GI phase. D-type cyclins, as well as transcription factor E2F-1 and ElA viral oncoproteins, were able to rescue pRb2/pl30-mediated GI -growth arrest in tumor cells. This suggests that, like other Rb family proteins, the phosphorylation of pRb2/pl30 is controlled by the cell cycle machinery, and that pRb2/pl30 may indeed be another key Gl-S phase regulator. Claudio et al . Cancer Res. 56, 2003-2008 (1996). The association of pRb with transcription factors, such as E2F, has been shown to occur by interactions at a region known as the "pocket region" (Raychaudhuri et al. , Genes Develop. , 5 1200-1207 (1991)). Recently, pl07 has also been shown to exert such a binding profile (Cao et al , Nature, 355 176-179 (1992)). Domains A and B, along with a spacer, are believed to correspond with the "pocket region" in the pRb2/pl30 gene described herein. Moreover, mutations have been found in the pocket region for several human cancers where a lack of function for the pRb protein is thought to be involved in the acquisition of the transformed phenotype (Hu et al , EMBO J., 9 1147- 1153 (1990); Huang et al , Mol. Cell Biol , 10: 3761-3769 (1990)). The Rb, pl07, and pRb2/pl30 proteins may play a key role in cell cycle regulation in that all three proteins interact with several cyclin/cdk complexes. pRb can be regulated by cyclin/cdk complexes, such as cyclin A/cdk2, cyclin E/cdk2 and cyclin D/cdk4, even if stable interaction between pRb and cyclin A/cdk2 or cyclin A/cdk2 has not been found in vivo (MacLachlan et al , Eukaryotic Gene Exp. 5: 127-156 (1995)). On the other hand, both pl07 and pRb2/pl30 stably interact in vivo with cyclin E/cdk2 and cyclin A/cdk2 complexes (Li et al , supra; Ewen et al, Science 255:85-87 (1992); and Faha et al . Science 255:87-90 (1992)). These complexes may be responsible for the existence of different phosphorylated forms of pRb, pl07 and pRb2/pl30 in the various phases of the cell cycle (Chen et al . Cell

55: 1193-1198 (1989); De Caprio et al , Proc , Natl. Acad. Sci. USA 89: 1795- 1798 (1992): and Beijersbergen et al. , Genes Dev. 9: 1340-1353 (1993)). In that pRb"s functional activities are enhanced by these phosphorylations. it is likely that pRb2/pl30 is also affected in the same manner by this post-translational modification. Since pRb2/pl30 demonstrates similar, even if not redundant, functional properties to pRb, it is proposed that pRb2/pl30 acts, like pRb, as a tumor suppressor gene. It has also been found that pRb2/pl30 maps on the long arm of chromosome 16. This finding reinforces the notion of pRb2/pl30 as a tumor suppressor gene. Chromosome 16 is a region frequently reported to show loss of heterozygosity (LOH) in several human neoplasias, such as breast, ovarian, hepatocellular and prostatic carcinomas (Yeung et al, Oncogene 5:3465-3468 (1993)). Chromosome 16, and specifically pRb2/pl30, has also been implicated in a rare human skin disease known as hereditary cylindromatosis (HR). HR has been reported as mapping to loci on chromosome 16ql2-ql3. In that the pRb2/pl30 gene maps to chromosome 16ql2-ql3, it has been put forth as a likely candidate for the tumor suppressor gene involved with the onset of this disease. Biggs et al , Nature Genetics 11 :441-443 (December 1995).

There is a need for improved methods for identification of individuals at risk for cancer, and for the detection and evaluation of cancers.

Because the pRb2/pl30 gene is a tumor suppressor gene and because it maps to a chromosomal region known to be associated with various carcinomas, there is a need for a method to screen individuals for mutations in this gene. There is also a need to identify sequence polymorphisms in this gene. It is believed that mutations, both within the exon coding sequences and the exon-intron junctions, can occur that will affect pRb2/pl30's function. Direct DNA sequence analysis of individual exons taken from genomic DNA extracted from rumors has been used successfully to identify mutations of the p53 gene in ovarian carcinomas and the Rb gene in retinoblastoma tumors. Milner et al . Cancer Research 53: 2128-2132 (1993); Yandell et al . N.E.J.

Medicine 321 : 1689-1695 (1989). However, direct sequencing of exons is an undesirable approach because it is a time intensive process. An understanding of the genomic structure of the pRb2/pl30 gene will enable those skilled in the an to screen a patient's DNA for polymorphisms and sequence mutations in the pRb2/pl30 gene. Identification of sequence mutations will also enable the diagnosis of carriers of germline mutations of the pRb2/pl30 gene and enable prenatal screening in these cases.

B. Gynecologic Cancers

Gynecologic cancers include cancers of the uterus, ovary, cervix, vagina, vulva, and fallopian tube as well as gestational trophoblastic disease. Cancers of the uterus include endometrial carcinomas and uterine sarcomas.

Endometrial cancer is the most common malignancy of the female genital tract. Although this neoplasm is frequently diagnosed at an early stage (75 percent in stage I), approximately 20 percent of the patients will die of the disease, half of which were diagnosed at stage I (Pettersson, Annual Report On The Results Of Treatment In Gynecological Cancer, Radiumhemmet, Stockholm, vol. 22: 65-82; Braly, Gynecol Oncol 58: 145-7 (1995)). The ability to identify patients with a more aggressive disease is crucial to planning an adequate treatment for each case. With this purpose in mind, several pathologic tumor features have been considered so far, including histologic type, grade of differentiation, depth of myometrial invasion, lymph nodal metastases and extra- uterine spread (MacMahon, Gynecol Oncol 2: 122 (1974); Chambers et al , Gynecol Oncol 27: 180-8 (1987)). Unfortunately, none of these factors allows a sufficiently accurate stratification of the patients. Such parameters have also questionable reproducibility.

There is great need for a simple laboratory test which is a consistent predictor of clinical outcome in endometrial cancer. What is needed is a prognostic method which can, at an early disease stage, identify the aggressiveness of an individual patient's disease, before initiation of therapy.

Ovarian cancer is the leading cause of gynecologic cancer death in the United States. Most ovarian malignancies are epithelial carcinomas, with a minority of mmors arising from the germ or stromal cells. In ovarian cancers, the degree of cellular differentiation (histologic grade) is an important independent predictor of both response to treatment and overall survival. Ovarian cancers frequently exhibit chromosomal alterations. The pRb2/pl30 gene maps to human chromosome 16ql2.2, which is one region that is frequently altered in human ovarian cancers. There is a need for improved methods of grading ovarian mmors. The improved methods would be useful in the diagnosis of disease, in selection of treatment, and as prognostic indicators.

C. Lung Cancer

Lung cancer is the greatest single cause of cancer-related deaths in Western countries. Selecting an appropriate course of therapy for lung cancer requires an accurate determination of the cancer's malignant potential. This determination is typically made by "grading" the tumor. The grading of mmors is typically carried out by examination of the character and appearance of tumor sections by skilled pathologists. A significant problem in the use of histologic criteria when determining the prognosis and types of treatment for lung cancer is the degree of interobserver and intraobserver variability in reading the same specimens. Determinations are necessarily subjective. In addition, there is heterogeneity within the tumor itself in both primary and metastatic sites. It may become necessary to obtain the opinion of several pathologists to reach a consensus on individual tumor grade.

There is a need for a simple laboratory test which is more consistently predicative of the malignant potential of an individual patient's lung tumor than the present subjective pathological analysis of tumor samples.

Detection of latent cancers before the appearance of lung lesions would allow therapeutic intervention at the earliest stages of the disease, thereby maximizing the prospects for a positive therapeutic outcome. It would also be

desirable, through a simple genetic test, to identify disease free individuals who are at risk of lung cancer. Such a screening test would be most advantageous for those individuals who, through environmental exposure to carcinogens or through family history of cancer, may be at risk for developing lung cancer. There is a need for a simple laboratory test which can be used to augment other forms of lung cancer diagnosis and to identify individuals with latent lung cancers. There is also need for a test to screen individuals for a predisposition to lung cancer.

Summary of the Invention The present invention relates to the human pRb2/pl30 gene and pRb2/pl30 protein, and their use as molecular markers in methods for the diagnosis and prognosis of cancer and for prediction of a predisposition to cancer. According to a preferred embodiment of the invention, the cancer is a gynecologic cancer or a non-small cell lung cancer. According to a most preferred embodiment of the invention, the cancer is endometrial carcinoma, ovarian cancer, a squamous cell carcinoma of the lung, or adenocarcinoma of the lung.

It is an object of the invention to provide a method for determining a prognosis in a patient afflicted with cancer comprising determining the expression level of the pRb2/pl30 gene in a sample from the patient. A decreased level of pRb2/pl30 gene expression in, the sample is indicative of an unfavorable prognosis.

Another object of the invention is to provide a method for detecting or identifying a cancerous disease state in a tissue comprising determining the expression level of the pRb2/pl30 gene in a sample of the tissue. Evaluation is advantageously conducted by determining the level of pRb2/pl30 expression in the sample, and comparing the expression level in the sampled tissue with the pRb2/pl30 expression level in normal, non-cancerous tissue. A decreased pRb2/pl30 expression level is indicative of the presence

of cancer. This method may be used to detect cancer in an individual not otherwise displaying a visible lesion.

A further object of the invention is to provide a method for identifying disease free individuals at risk for cancer, or individuals at risk for the recurrence of cancer following treatment, comprising determining the level of expression of the pRb2/pl30 gene in tissue sampled from an individual and comparing the pRb2/pl30 expression level in the sampled tissue with a normal pRb2/pl30 expression level. A decreased level of pRb2/pl30 expression is indicative of the likelihood of disease or disease recurrence. In the case of endometrial cancer, a method is provided for identifying the risk of recurrence following hysterectomy, and for evaluating the need for further treatment such as radiation therapy or chemotherapy.

Another object of the invention is to provide a method for grading a cancer comprising determining the level of expression of the pRb2/pl30 gene in a sample of tissue from a patient suffering from cancer. The expression level in the sampled tissue is compared with the expression level in normal tissue.

The degree of the decrement in expression level in the cancer sampled tissue as compared to the normal tissue is indicative of the pathological grade of the cancer. A larger decrement indicates a more aggressive disease state. It is an object of the invention to provide a DNA segment consisting essentially of an intron of the pRb2/pl30 gene, or an at least 15 nucleotide segment thereof.

Another object of the invention is to provide an amplification primer of at least 15 nucleotides consisting essentially of a DNA segment having a nucleotide sequence substantially complementary to a segment of a pRb2/pl30 intron exclusive of the splice signal dinucleotides of said intron.

A further object of the invention is to provide methods for identifying polymorphisms and mutations in an exon of a human pRb2/pl30 εene.

One embodiment of the invention includes a method for amplifying and identifying polymoφhisms and mutations in an exon of a human pRb2/pl30 gene, which method comprises:

(a) treating, under amplification conditions, a sample of genomic DNA containing the exon with a primer pair comprising a first primer which hybridizes to the promoter region or to an intron upstream of said exon and a second primer which hybridizes to an intron or to the 3'-noncoding region, said treatment producing an amplification product containing said exon;

(b) determining the nucleotide sequence of said amplification product to provide the nucleotide sequence of said exon; and

(c) comparing the sequence of said exon obtained in step b to a sequence for the sequence of a corresponding wild type exon. Each primer of the PCR primer pair consists of an amplification primer of at least 15 nucleotides consisting essentially of a DNA segment from the promoter region, from a pRb2/pl30 intron exclusive of the splice signal dinucleotides, or from the 3'-noncoding region.

The amplification primer described above has a nucleotide sequence substantially complementary to the 3'-noncoding region, the promoter region given as SEQ ID NO: 113, or an intron having a nucleotide sequence selected from the group consisting of SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61 , SEQ ID NO:62. SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65. SEQ ID NO:66, SEQ ID NO:67. and SEQ ID NO:68.

In a preferred embodiment, the amplification primer as described above has a nucleotide sequence selected from the group consisting of SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71 , SEQ ID NO:72. SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77. SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81. SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86. SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95. SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105. SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: l l l , and SEQ ID NO: 112.

Another embodiment of the invention includes a method for identifying polymoφhisms and mutations in an exon of a human pRb2/pl30 gene, which method comprises:

(a) forming a polymerase chain reaction admixture by combining in a polymerase chain reaction buffer, a sample of genomic DNA containing said exon, a primer pair comprising a first primer which hybridizes to the promoter region or to an intron upstream of said exon and a second primer which hybridizes to the 3'-noncoding region or to an intron downstream of said exon. a mixmre of one or more deoxynucleotide triphosphates, and a compound capable of radioactively labeling said primer pair, and a DNA polymerase;

(b) subjecting said admixture to a plurality of polymerase chain reaction thermocycles to produce a pRb2/pl30 amplification product;

(c) denaturing said pRb2/pl30 amplification product:

(d) electrophoretically separating said denatured pRb2/pl30 amplification product:

(e) exposing the electrophoretically separated product of step d to a film to produce a photographic image; and (e) comparing the mobility of the bands in said photographic image of said pRb2/pl30 amplification product to a electrophoretically separated amplification product for a corresponding wild type exon.

In another embodiment, the invention includes a method for identifying mutations in a human chromosomal sample containing an exon of a human pRb2/pl30 gene, which method comprises:

(a) forming an admixture by combining in a buffer, a chromosomal sample containing said exon, a primer pair comprising a first primer which hybridizes to the promoter region or to an intron upstream of said exon and a second primer which hybridizes to the 3'-noncoding region or to an intron downstream of said exon, a mixture of one or more deoxynucleotide triphosphates including at least one deoxynucleotide triphosphate that is labeled, and a DNA polymerase;

(b) subjecting said admixture to a temperamre and time sufficient to produce a pRb2/pl30 amplification product; and

(c) visualizing said pRb2/pl30 amplification product with a fluorochrome conjugate specific to said label; and

(d) comparing the visualized pRb2/p 130 amplification product obtained in step a to a visualized amplification product for a corresponding wild type exon.

These and other objects will be apparent to those skilled in the an from the following discussion.

Description of the Figures

Figure 1 is a plot of the probability of survival of 100 patients with endometrial carcinoma (all stages) who were characterized as having either pRb2/pl30-positive or pRb2/pl30-negative mmors.

Figure 2 is a plot of the probability of survival in the same 100 patients with endometrial carcinoma, as stratified by stage and pRb2/pl30 expression.

Figure 3A is a schematic representation of the human pRb2/pl30 gene. Exons are represented by open rectangles, while the introns are represented by hatched vertical bars. Exons 10-13, 14-16, and 17-20 represent domain A, a spacer, and domain B, respectively.

Figure 3B is a schematic representation of the human pRb2/pl30 genomic clones derived from the PI and λ phage libraries. Figure 4 is the nucleotide sequence (SEQ ID NO: 4) of the 5 ' end and 5' upstream region of the human pRb2/pl30 gene showing the transcription stan site (→) and the sequence complementary to a primer utilized for a primer extension analysis (underlined). Position + 1 is assigned to the A of the ATG translation start codon (bold and underlined). The sequences corresponding to the Spl factor recognition motif are boxed. Also boxed are the sequence motifs corresponding to the MyoD and Kerl transcription factors. The nucleotides beginning at position 1 through position 240 correspond to exon 1 of pRb2/pl30. The lowercase letters beginning at position 241 represent the first ten nucleotides of intron 1. Figure 5 shows the products of a primer extension experiment done to identify the transcription start site for the human pRb2/pl30 gene. Cytoplasmatic RNA was hybridized overnight to an oligonucleotide complementary to the twenty four nucleotides beginning at position -22 of Figure 4 (SEQ ID NO:4). Lane M contains molecular-weight markers (φ l74

DNA/Hae III. Promega). Lanes 1 and 2 contain the primer-extended product of pRb2/pl30 from HeLa cells and tRNA as template, respectively.

Figure 6 illustrates two alleles containing exon 20 of the pRb2/pl30 gene in the nucleus of a peripheral blood lymphocyte visualized through the use of the PRINS technique.

Detailed Description of the Invention

A. Abbreviations and Definitions

1 . Abbreviations bp base pairs

BSA Bovine Serum Albumin dATP deoxyadenine triphosphate dCTP deoxycytosine triphosphate dGTP deoxyquenosine triphosphate

DIG DNA Digoxigenin-labeled DNA

DIG-dUTP Digoxigenin-deoxyuridine triphosphate

DNA deoxyribonucleic acid dTTP deoxythymine triphosphate

EDTA ethylene dinitrolotetraacetic acid

FITC fluorescein isothiocyanate

PCR polymerase chain reaction

PHA phytohemagglutinin

PRINS oligonucleotide-PRimed IN Situ synthesis

RNA ribonucleic acid

SDS sodium dodecyl sulfate

SSC standard saline citrate

SSCP single-strand conformation polymoφhism

TBE buffer mixmre of 0.09 M tris. 0.09 M boric acid, and 2.5 mM EDTA

2. Definitions

"Allele" refers to one or more alternative forms of a gene occupying a given locus on a chromosome.

"Affected tissue" means tissue which, through visual or other examination, is believed to contain a puφorted cancerous or precancerous lesion.

"Amplification product" refers to a nucleic acid segment produced by amplification procedures such as PCR, SSCP, and PRINS, which product is complementary to the template segment amplified. "Downstream" identifies sequences which are located in the direction of expression, i.e., on the 3 '-side of a given site in a nucleic acid.

"Endometrial cancer" or "endometrial carcinoma" means a polypoid growth arising in the endometrium.

"Expression", with respect to the pRb2/pl30 gene, means the realization of genetic information encoded in the gene to produce a functional RNA or protein. The term is thus used in its broadest sense, unless indicated to the contrary, to include either transcription or translation.

"Expression level", with respect to the pRb2/pl30 gene, means not only an absolute expression level, but also a relative expression level as determined by comparison with a standard level of ρRb2/pl30 expression.

"Genomic DNA" refers to all of the DNA sequences composing the genome of a cell or organism. In the invention described herein it includes the exons, introns, and regulatory elements for the pRb2/pl30 gene.

"Grading", with respect to a tumor sample, means a classification of the perceived degree of malignancy. In grading tumor samples, a pathologist or other observer evaluates the degree of differentiation (e. g. grade 1 , well differentiated, grade 2, moderately differentiated, grade 3, poorly differentiated) of the tissue.

"Gynecologic cancer" means a tumor arising in the uterus, ovary, cervix, vagina, vulva, or fallopian mbe, as well as gestational trophoblastic disease.

"Hybridization" means the Watson-Crick base-pairing of essentially complementary nucleotide sequences (polymers of nucleic acids) to form a double-stranded molecule.

"3'-noncoding region" means those nucleic acid sequences downstream of the termination codon.

"Non-small cell lung cancer" (NSCLC) means all forms of lung cancer except small cell lung cancer (SCLC). In particular, by non-small cell lung cancer is meant the group of lung cancers including squamous cell carcinomas, adenocarcinomas, bronchiolo-alveolar carcinomas and large cell carcinomas.

"Polymorphic" refers to the simultaneous occurrence in the population of genomes showing allelic variations. As used herein the term encompasses alleles producing different phenotypes, as well as proteins for which amino acid variants exist in a population, but for which the variants do not destroy the protein's function.

"Primer" refers to an oligonucleotide which contains a free 3' hydroxyl group that forms base pairs with a complementary template strand and is capable of acting as the starting point for nucleic acid synthesis by a polymerase. The primer can be single-stranded or double- stranded, however, if in double-stranded form, the primer is first treated in such a way so as to separate it from its complementary strand. ^M pRb2/pl30 gene" means the gene which encodes the pRb2/pl30 protein, the cDNA of which is set forth as SEQ ID NO: l , and all allelic variations and mutants thereof.

^M pRb2/pl30 intron" as used herein means a wild type intron segment of the pRb2/pl30 gene, as well as any allelic variations thereof.

"pRb2/pl30 protein" means the translation product of the pRb2/pl30 gene, including all allelic variations and mutants thereof. The pRb2/pl30 amino acid sequence is set forth as SEQ ID NO: 2.

"Prognosis" is used according to its ordinary medical meaning, that is, the prospect of recovery from a disease.

"Splice junction" or "exon-intron junction" refers to the nucleotide sequence immediately surrounding an exon-intron boundary of a nuclear gene. As used herein the term includes the sites of breakage and reunion in the mechanism of RNA splicing. "Splice signal dinucleotide" refers to the first two nucleotides

(5 '-terminal) or the last two nucleotides (3 '-terminal) of an intron. In highly conserved genes the 5 '-terminal dinucleotide is GT and the 3 '-terminal dinucleotide is AG. Alternatively, the '-terminal dinucleotide and the 3'- terminal dinucleotide are referred to as the "donor" and "acceptor" sites, respectively.

"Substantially complementary nucleotide sequence" means, as between two nucleotide sequences, a relationship such that the sequences demonstrate sufficient Watson-Crick base-pair matching to enable formation of a hybrid duplex under hybridization conditions. It is not required, however. that the base-pair matchings be exact.

Downstream" identifies sequences which are located in the direction of expression, i.e. , on the 3 '-side of

"Upstream" identifies sequences which are located in the direction opposite from expression, i.e. on the 5'-side of a given site in a nucleic acid.

The present invention provides methods for the identification of individuals at risk for cancer, and for the detection and evaluation of cancers.

These methods are of two basic types: methods based on determination of pRb2/pl30 expression levels, and methods based on determination of the genomic structure of pRB2/pl30.

B. Methods Based on Determination of pRb2/p!30 Expression Levels

The present invention provides improved methods, based on pRb2/pl30 expression levels, for the diagnosis and prognosis of cancers including but not limited to gynecologic cancers and non-small cell lung cancers. Among the gynecologic cancers to which these methods may be applied are ovarian cancer and endometrial cancer. 1. Gynecologic Cancers

Early ovarian cancer is frequently asymptomatic, or produces only mild symptoms which might be ignored by the patient. The majority of ovarian mmors have spread beyond the ovary, and frequently beyond the pelvis, at the time of diagnosis. Improved methods for the diagnosis and prognosis of ovarian cancer will be useful in treatment selection, and should have a favorable effect on patient outcomes. The present invention rests on the discovery that in ovarian cancer tissue, there is a correlation between the expression of pRb2/pl30 and tumor grade.

Endometrial cancer often follows a favorable course, however a considerable proportion of these cases behave poorly and ultimately die of the disease. Cunently used surgical-pathologic parameters do not always allow the identification of this subset of patients. According to the F.I.G.O. criteria for staging in endometrial cancer, surgical procedure should always include peritoneal washing, abdominal hysterectomy, bilateral salpingo-oophorectomy and systematic pelvic and paraaortic lymphadenectomy. Indeed, this operation is often unnecessarily "radical" and potentially dangerous to patients with mmors limited to the uterine coφus. This observation becomes more relevant if it is considered that patients with endometrial cancer very often present also cardiovascular disease, diabetes mellitus, hypertension and severe obesity (Wingo et al. , Am J Obstet Gynecol 152:803-8 (1985), which are known risk factors for morbidity from abdominal surgery (DiSaia et al , "Adenocarcinoma Of The Uterus" In: Clinical

Gynecologic Oncology, St. Louis: Mosby-Year Book, p. 156-93 (1993). On the other hand, in the obese or patients at high surgical risk total hysterectomy can be easily and safely performed by the vaginal technique (Massi et al . Am J Obstet Gynecol 174: 1320-6 (1996); Pitkin, Obstet Gynecol 49:567-9 (1977); Peters et al.. Am J Obstet Gynecol 146:285-90 (1983)). With this in view, the relative pRb2/pl30 expression, assayed according to the present invention may be used in the selection of candidates for a less aggressive surgical treatment, without decreasing their chance of cure, as well as being helpful for the identification of high risk patients, to whom every surgical effort should be attempted and post-surgical treatment given.

Normal cells of the endometrium express a relatively high level of pRb2/pl30 protein. The present invention rests on the discovery of a highly statistical inverse correlation between the expression of pRb2/pl30 in tissues from endometrial cancer patients and the eventual clinical outcome following treatment. Decreased levels of pRb2/pl30 are significantly associated with a poor survival. The study results reported herein indicate that the risk of dying of endometrial carcinoma is increased almost fivefold in patients whose mmors were pRb2/pl30 negative, regardless of the mmor stage or grade of differentiation. Tissue with the greatest malignant potential expresses little or no pRb2/pl30. Accordingly, a sample is contacted with an antibody specific for pRb2/pl30 protein. In the case of endometrial cancer, the sample may typically comprise endometrial tissue, and may specifically comprise an endometrial mmor. The amount of antibody bound by the sample may be determined relative to the amount of antibody bound by a sample of normal endometrial tissue. The difference in the amount of antibody bound by the normal and test samples is indicative of the patient's prognosis. The endometrial carcinoma study described in Example 1 concurrently tested a known molecular prognostic indicator, i.e. , DNA index, various classic clinical-pathologic parameters and pRb2/pl30 expression. Decreased levels of pRb2/pl30 were significantly

associated with a poorer survival. The expression of pRb2/pl30 thus represents an independent predictor of clinical outcome in endometrial carcinoma. Well known risk factors, such as F.I.G.O. stage and mmor ploidy were also confirmed as independent prognosticators, although of minor strength. The pRb2/pl30 expression was significantly correlated with mmor ploidy and patient age. in that pRb2/pl30 negativity was associated with aneuploidy (P=0.001) and with age > 65 years (P =0.008), in accordance with the known negative impact of such feamres on survival in endometrial cancer (DiSaia et al. ,Am J Obstet Gynecol 151: 1009-15 (1985); Susini et al . Am J Obstet Gynecol 170:527-34 (1994); Massi et al . Am J Obstet Gynecol 174: 1320-6 (1996)). However, it is noteworthy that mmor ploidy resisted as an independent prognostic variable by multivariate analysis. Stratification by pRb2/pl30 status and ploidy allowed identification of patient subgroups with significant differences in survival (data not shown). A trend toward correlation was also found between pRb2/pl30 status and another major prognostic indicator such as grade of differentiation, where pRb2/pl30 negativity was more frequent among moderately and poorly differentiated mmors (P=0.06). Furthermore, concerning grade of differentiation, stratification by pRb2/pl30 status revealed significant differences in survival within each grade group (data not shown). Expression of pRb2/pl30 was not correlated with mmor stage; pRb2/pl30 negative mmors were equally distributed among different mmor stages, thus indicating that this feamre is typical of certain mmors, from their onset in early stages.

Thus, the pRb2/pl30 expression level may serve as a convenient molecular marker to replace or augment conventional prognostic techniques. An important advantage of the use of pRb2/pl30 expression over classical surgical pathologic parameters as a prognostic factor is that the former can be determined at the time of the initial diagnosis, before any therapy is initiated. For patients not previously treated by radiotherapy or chemotherapy, low levels

of pRb2/pl30 can be used to identify tumors with a tendency to behave aggressively.

An early accurate determination of the aggressiveness of disease in an individual patient is a necessary pan of designing a course of treatment. In cases where the test method of the invention identifies a poor prognosis, adjuvant therapy, such as radiation therapy or chemotherapy, may be initiated. This more aggressive treatment should increase the patient's chance of survival. The pRb2/pl30 expression level, even in early stages of the disease, is reflective of the malignant potential of the patient's carcinoma and the aggressiveness of the ensuing disease course. This form of "molecular based" prognosis can be evaluated more consistently than conventional prognostic factors which are based upon subjective evaluations of histological type, grade of differentiation, depth of myometrial invasion, degree of lymph nodal metastases, extra-uterine spread, and the other factors upon which endometrial carcinoma prognoses are presently based. 2. Lung Cancer

In the case of lung cancer, a sample of lung tissue is removed from an individual by conventional biopsy techniques which are well-known to those skilled in the art. The sample is generally collected by needle biopsy. See, for example, Cancer: Principles & Practice of Oncology, V. T. DeVita, Jr. et al , eds. 3rd edition (1989), J. B. Lippincott Co. , Philadelphia, PA, p. 616-619, incoφorated herein by reference (transcarinal needle biopsy and transthoracic percutaneous fine-needle aspiration biopsy). For identification of lung lesions as comprising NSCLC, the sample is taken from the disease lesion. The disease lesion is first located by x-ray or other conventional lung lesion imaging techniques known to those skilled in the an. For testing for latent NSCLC or NSCLC predisposition, the tissue sample may be taken from any site in the lung. Tissue with the greatest malignant potential expresses little or no pRb2/pl30. Normal lung tissue cells express a high level of pRb2/pl30 protein. The pRb2/pl30 expression level in the cells of the patient lung mmor

tissue can be compared with the level in normal lung tissue of the same patient, or with the level in the lung tissue of a normal control group.

Non-small cell lung cancer (NSCLC) includes squamous cell carcinomas, adenocarcinomas, bronchiolo-alveolar carcinomas and large cell carcinomas. A highly significant statistical inverse correlation has been established between the expression of pRb2/pl30 in tissues from non-small cell lung cancers and the tissues' pathological grading by skilled pathologists.

Thus, the pRb2/pl30 expression level may serve as a convenient molecular marker to replace or augment conventional mmor grading. Accurate mmor grading is a necessary part of designing a course of treatment for the individual patient. Grading is reflective of the malignant potential of the mmor in question and thus the aggressiveness of the ensuing disease course. The generation of vital mmor grade information is made easier, by relying on pRb2/pl30 as a molecular surrogate for more subjective observations concerning mmor histology. This form of "molecular-based" grading can be performed more consistently than conventional pathological grading which is based upon subjective evaluations by expert pathologists. pRb2/pl30 expression levels may also serve as a convenient molecular marker for the presence of active or latent NSCLC, or predisposition to NSCLC. Lung lesions may be identified as non-small cell lung carcinomas

(NSCLCs) by showing a decrement in the expression of pRb2/pl30 in the lesion compared to the level of pRb2/pl30 in normal, non-cancerous control lung tissue. Similarly, the level of pRb2/pl30 expression in lung tissue of individuals with no apparent lung lesion but other symptoms of lung cancer, or in disease-free individuals, indicates latent NSCLC or risk of NSCLC, respectively. Early diagnosis of NSCLC, even before the appearance of visible lung lesions, will permit earlier initiation of treatment and increased survival. According to the practice of the invention, an at least about one- third decrement in pRb2/pl30 expression level in an affected lung tissue sample, in comparison with normal controls, indicates that the lesion is an NSCLC.

Similarly, a pRb2/pl30 expression decrement of about one-third or greater in lung tissue of patients who are free of lung lesions but manifest other potential lung cancer symptoms such as sputum cytology irregularities, coughing or bronchitis, is indicative of pre-lesion NSCLC. An about one-third or greater pRb2/pl30 expression decrement in lung tissue of otherwise healthy individuals manifesting no symptoms of lung cancer is believed indicative of a risk of future NSCLC. Decrements in pRb2/pl30 expression of about one-half or greater are even more indicative of NSCLC disease or NSCLC predisposition.

According to one aspect of the invention, individuals who are disease free are evaluated for risk in contracting NSCLC. The test method may be used to identify individuals at risk of developing NSCLC from among populations exposed to environmental carcinogens, e.g. asbestos workers, miners, textile workers, tobacco smokers and the like, and from among families having a history of NSCLC or other forms of cancer. 3. Methods for Deteπnining Expression Levels

According to the practice of the present invention, a sample of affected tissue is removed from a cancer patient by conventional biopsy techniques which are well-known to those skilled in the art. The sample is preferably obtained from the patient prior to initiation of radiotherapy or chemotherapy. The sample is then prepared for a determination of pRb2/pl30 expression level.

Determining the relative level of expression of the pRb2/pl30 gene in the tissue sample comprises determining the relative number of pRb2/pl30 RNA transcripts, particularly mRNA transcripts in the sample tissue, or determining the relative level of the corresponding pRb2/ρl30 protein in the sample tissue. Preferably, the relative level of pRb2/pl30 protein in the sample tissue is determined by an immunoassay whereby an antibody which binds pRb2/pl30 protein is contacted with the sample tissue. The relative pRb2/pl30 expression level in cells of the sampled mmor is conveniently determined with respect to one or more standards. The standards may comprise, for example.

a zero expression level on the one hand and the expression level of the gene in normal tissue of the same patient, or the expression level in the tissue of a normal control group on the other hand. The standard may also comprise the pRb2/pl30 expression level in a standard cell line. The size of the decrement in pRb2/pl30 expression in comparison to normal expression levels is indicative of the future clinical outcome following treatment.

Methods of determining the level of mRNA transcripts of a particular gene in cells of a tissue of interest are well-known to those skilled in the art. According to one such method, total cellular RNA is purified from the effected cells by homogenization in the presence of nucleic acid extraction buffer, followed by centrifugation. Nucleic acids are precipitated, and DNA is removed by treatment with DNase and precipitation. The RNA molecules are then separated by gel electrophoresis on agarose gels according to standard techniques, and transferred to nitrocellulose filters by, e.g., the so-called "Northern" blotting technique. The RNA is immobilized on the filters by heating. Detection and quantification of specific RNA is accomplished using appropriately labelled DNA or RNA probes complementary to the RNA in question. See Molecular Cloning: A Laboratory Manual, J. Sambrook et al. , eds. , 2nd edition, Cold Spring Harbor Laboratory Press, 1989, Chapter 7, the disclosure of which is incoφorated by reference.

In addition to blotting techniques, the mRNA assay test may be carried out according to the technique of in situ hybridization. The latter technique requires fewer mmor cells than the Northern blotting technique. Also known as "cytological hybridization" , the in situ technique involves depositing whole cells onto a microscope cover slip and probing the nucleic acid content of the cell with a solution containing radioactive or otherwise labelled cDNA or cRNA probes. The practice of the in situ hybridization technique is described in more detail in U.S. Patent 5,427,916, the entire disclosure of which is incoφorated herein by reference.

The nucleic acid probes for the above RNA hybridization methods can be designed based upon the published pRb2/pl30 cDNA sequence of Li et al . Genes Dev. 7: 2366-2377 (1993), the entire disclosure of which is incoφorated herein by reference. The nucleotide sequence is reproduced herein as SEQ ID NO: 1. The translation initiation codon comprises nucleotides 70-72 of SEQ ID NO: l . The translation termination codon comprises nucleotides 3487-3489.

Either method of RNA hybridization, blot hybridization or in situ hybridization, can provide a quantitative result for the presence of the target RNA transcript in the RNA donor cells. Methods for preparation of labeled DNA and RNA probes, and the conditions for hybridization thereof to target nucleotide sequences, are described in Molecular Cloning, supra, Chapters 10 and 11 , incoφorated herein by reference.

The nucleic acid probe may be labeled with, e.g. , a radionuclide such as ³² P, ¹ C, or ³⁵ S; a heavy metal; or a ligand capable of functioning as a specific binding pair member for a labelled ligand, such as a labelled antibody, a fluorescent molecule, a chemolescent molecule, an enzyme or the like.

Probes may be labelled to high specific activity by either the nick translation method or Rigby et al , J. Mol. Biol 113: 237-251 (1977) or by the random priming method, Fienberg et al . Anal. Biochem. 132: 6-13 (1983). The latter is the method of choice for synthesizing ³² P-Iabelled probes of high specific activity from single-stranded DNA or from RNA templates. Both methods are well-known to those skilled in the art and will not be repeated herein. By replacing preexisting nucleotides with highly radioactive nucleotides, it is possible to prepare ³² P-labelled DNA probes with a specific activity well in excess of IO ⁸ cpm/microgram according to the nick translation method. Autoradiographic detection of hybridization may then be performed by exposing filters on photographic film. Densitometric scanning of the filters provides an accurate measurement of mRNA transcripts.

Where radionuclide labelling is not practical, the random-primer method may be used to incoφorate the dTTP analogue 5-(N-(N-biotinyl-epsilon- aminocaproyl)-3-aminoallyl)deoxyuridine triphosphate into the probe molecule. The thus biotinylated probe oligonucleotide can be detected by reaction with biotin binding proteins such as avidin, streptavidin, or anti-biotin antibodies coupled with fluorescent dyes or enzymes producing color reactions.

The relative number of pRb2/pl30 transcripts may also be determined by reverse transcription of mRNA followed by amplification in a polymerase chain reaction (RT-PCR), and comparison with a standard. The methods for RT-PCR and variations thereon are well known to those of ordinary skill in the art.

According to another embodiment of the invention, the level of pRb2/pl30 expression in cells of the patient tissue is determined by assaying the amount of the corresponding pRb2/pl30 protein. A variety of methods for measuring expression of the pRb2/pl30 protein exist, including Western blotting and immunohistochemical staining. Western blots are run by spreading a protein sample on a gel, using an SDS gel, blotting the gel with a cellulose nitrate filter, and probing the filters with labeled antibodies. With immunohistochemical staining techniques, a cell sample is prepared, typically by dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visually detectable, such as enzymatic labels, florescent labels, luminescent labels, and the like.

According to one embodiment of the invention, tissue samples are obtained from patients and the samples are embedded then cut to e.g. 3-5 μm, fixed, mounted and dried according to conventional tissue mounting techniques. The fixing agent may advantageously comprise formalin. The embedding agent for mounting the specimen may comprise, e.g. , paraffin. The samples may be stored in this condition. Following deparaffinization and rehydration. the samples are contacted with an immunoreagent comprising an antibody specific

for pRb2/pl30. The antibody may comprise a polyclonal or monoclonal antibody. The antibody may comprise an intact antibody, or fragments thereof capable of specifically binding pRb2/pl30 protein. Such fragments include, but are not limited to, Fab and F(ab') ₂ fragments. As used herein, the term "antibody" includes both polyclonal and monoclonal antibodies. The term "antibody" means not only intact antibody molecules, but also includes fragments thereof which retain antigen binding ability.

Appropriate polyclonal antisera may be prepared by immunizing appropriate host animals with pRb2/pl30 protein and collecting and purifying the antisera according to conventional techniques known to those skilled in the art. Monoclonal antibody may be prepared by following the classical technique of Kohler and Milstein, Nature 254:493-491 (1975), as further elaborated in later works such as Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analysis, R. H. Kennet et al , eds., Plenum Press, New York and London (1980).

Substantially pure pRb2/pl30 for use as an immunogen for raising polyclonal or monoclonal antibodies may be conveniently prepared by recombinant DNA methods. According to one such method, pRb2/pl30 is prepared in the form of a bacterially expressed glutathione S-transferase (GST) fusion protein. Such fusion proteins may be prepared using commercially available expression systems, following standard expression protocols, e.g. , "Expression and Purification of Glutathione-S-Transferase Fusion Proteins" , Supplement 10, unit 16.7, in Current Protocols in Molecular Biology (1990). Also see Smith and Johnson, Gene 67: 34-40 (1988); Frangioni and Neel, Anal. Biochem. 210: 179-187 (1993). Briefly, DNA encoding for pRb2/pl30 is subcloned into a pGEX2T vector in the correct reading frame and introduced into E. coli cells. Transformants are selected on LB/ampicillin plates; the plates are incubated 12 to 15 hours at 37°C. Transformants are grown in isopropyl-0- D-thiogalactoside to induce expression of pRb2/pl30-GST fusion protein. The cells are harvested from the liquid cultures by centrifugation. The bacterial

pellet is resuspended and the cell pellet sonicated to lyse the cells. The lysate is then contacted with glutathione-agarose beads. The beads are collected by centrifugation and the fusion protein eluted. The GST carrier is then removed by treatment of the fusion protein with thrombin cleavage buffer. The released pRb2/pl30 protein is recovered.

As an alternative to immunization with the complete pRb2/pl30 molecule, antibody against pRb2/pl30 can be raised by immunizing appropriate hosts with immunogenic fragments of the whole protein, particularly peptides corresponding to the carboxy terminus of the molecule. The antibody either directly or indirectly bears a detectable label.

The detectable label may be attached to the primary anti-pRb2/pl30 antibody directly. More conveniently, the detectable label is attached to a secondary antibody, e.g. , goat anti-rabbit IgG, which binds the primary antibody. The label may advantageously comprise, for example, a radionuclide in the case of a radioimmunoassay; a fluorescent moiety in the case of an immunofluorescent assay; a chemiluminescent moiety in the case of a chemiluminescent assay; or an enzyme which cleaves a chromogenic substrate, in the case of an enzyme- linked immunosorbent assay.

Most preferably, the detectable label comprises an avidin-biotin- peroxidase complex (ABC) which has suφlus biotin-binding capacity. The secondary antibody is biotinylated. To locate pRb2/pl30 antigen in the tissue section under analysis, the section is treated with primary antiserum against pRb2/pl30, washed, and then treated with the secondary antiserum. The subsequent addition of ABC localizes peroxidase at the site of the specific antigen, since the ABC adheres non-specifically to biotin. Peroxidase (and hence antigen) is detected by incubating the section with e.g. H ₂ O ₂ and diaminobenzidine (which results in the antigenic site being stained brown) or H ₂ O ₂ and 4-chloro- 1 -naphthol (resulting in a blue stain).

The ABC method can be used for paraffin-embedded sections, frozen sections, and smears. Endogenous (tissue or cell) peroxidase may be quenched e.g. with H ₂ 0 ₂ in methanol.

The level of pRb2/pl30 expression in mmor samples may be compared on a relative basis to the expression in normal tissue samples by comparing the stain intensities, or comparing the number of stained cells. The lower the stain intensity with respect to the normal controls, or the lower the stained cell count in a tissue section having approximately the same number of cells as the control section, the lower the expression of the pRb2/pl30 gene, and hence the higher the expected malignant potential of the sample.

In the examples which follow, a polyclonal antibody raised against pRb2/pl30, designated ADL1 was utilized. The specificity of the antibody has been confirmed by Western blot analysis, (Pertile et al , Cell Growth & Diff 6: 1659-64 (1995); Claudio et al , Cancer Res 56:2003-8 (1996)), as well as by immunoprecipitation of the antibody with the in vitro translated forms of the cDNAs coding for pRb2/pl30 and for the other retinoblastoma related proteins, pRb/pl05 and pl07. The ADL1 antibody was able to immunoprecipitate only the in vitro translated form of the pRb2/pl30 protein (Baldi e al , Clin Cancer Res 2: 1239-45 (1996).

C. Methods Based on Determination of the Genomic Strucmre of pRB2/p!30

The genomic strucmre of the human pRb2/pl30 gene is described herein. The pRb2/pl30 genomic DNA has been cloned and sequenced. The pRb2/pl30 gene has been mapped to the long arm of chromosome 16, an area previously reported to show loss of heterozygosity (LOH) for human neoplasias. The putative promoter for pRb2/pl30 has been identified, cloned and sequenced. The complete intron-exon organization of the gene has been elucidated. The pRb2/pl30 gene contains 22 exons and 21 introns, spanning over 50 kb of genomic DNA. The length of the individual exons ranges from

65 bp to 1517 bp, while the length of individual introns ranges from 82 bp to

9837 bp. The organization of these exons and introns are shown in Figure 3A.

The location and size of each exon and intron of pRb2/pl30, as well as the nucleotide sequences at the exon-intron junctions are shown below in Table 7. (SEQ ID NOS: 6-47). The exon sequences are shown in upper case letters, while the intron sequences are in lower case letters. The superscript numbers correspond to the nucleotide positions of the exon-intron boundaries on SEQ ID NO: l.

All the exons were completely sequenced and no discrepancies were found in comparing the genomic sequence of the exons and the cDNA sequence previously reported. Li, Y. et al . Genes 7:2366-2377 (1993). The exon-intron boundaries were determined by comparing the sequence of the genomic DNA described herein to the published cDNA sequence of Li et al. , supra. The exon-intron boundaries were identified as the positions where the genomic DNA sequence diverged from that of the cDNA.

With the exception of exon 22, the largest of all the exons (1517 bp in length), the exons found were relatively small, with the shortest, exons 4 and 7, comprising only 65 nucleotides each. Exons 10 through 20 code for the region of the pRb2/pl30 protein which form the "pocket region" . Exons 10 through 13 and 17 through 20 translate to Domain A and Domain B, respectively. Exons 14, 15, and 16 code for the region of the pRb2/pl30 protein, known as the "spacer. " The spacer lies between Domains A and B.

The introns have been completely sequenced. The shortest intron, intron 16, lying between exons 16 and 17, is only 82 bp in length, whereas the largest intron, intron 21 , spans 9837 bp. Intron 21 is located between exons 21 and 22. The complete sequences for the introns are given as SEQ ID NOS: 48-68. All of the intron sequences of pRb2/pl30 conform to the GT-AG rule found to be characteristic of other human genes. Breathnach, R. et al , Annu. Rev. Biochem. 50:349-383 (1981). This rule identifies the generic sequence of an intron as GT AG. Introns having this generic form are

characterized as conforming to the GT - AG rule. The two dinucleotides. GT and AG, known as the "splice signal dinucleotides," act as signals for splicing out the introns during the processing of the pRb2/pl30 mRNA. Point mutations in splice signal dinucleotides have been associated with aberrant splicing in other genes in vivo and in vitro. See generally, Genes V, B. Lewin. Oxford University Press, pp. 913-916, New York (1994) and Yandell et al , supra at p. 1694. Thus, it is important to identify any mutations to the splice signal dinucleotides or other sequences that are excluded from the RNA transcript during splicing. The pRb2/p 130 genomic strucmre and intron sequences described herein may be used to delineate mutations and rearrangements associated with mmor formation. The genomic strucmre and intron sequences herein may also be used to screen for naturally occurring polymoφhisms at the nucleotide level. Knowledge of a specific single polymoφhism can be used to eliminate a mutation in pRb2/pl30 as a causative factor in a mmor if the puφorted mutation displays the same pattern as the polymoφhism. Knowledge of polymoφhisms in pRb2/pl30 can be used to determine the genetic linkage of an identical mutation, and in turn, the tracing of parental origin and family histories without the need for time for time intensive sequencing if mutation is of germline origin. These polymoφhisms can then be utilized for the development of diagnostic approaches for human neoplasias. However, it should be noted that not all polymoφhisms are of equal utility in these applications. It is preferable to seek out mutations in the exons, as these mutations are most likely to lead to mmor development. Further, because the coding regions of the gene are generally more stable and less likely to mutate over time, it follows that polymoφhisms in the exon region are typically less common. The detection of a polymoφhism in the exon region of pRb2/pl30 would enable screening of both genomic DNA and cDNA.

In the examples that follow, several screening methods are exemplified to identify pRb2/pl30 mutations and polymoφhisms.

1. Transcriptional Control of pRb2/p!30

There is evidence that mmor suppressor gene products directly interact with transcription factors, such as MyoD, which regulate not only cell growth, but also cell differentiation. Sang et al , supra at p. 8. Mutations in the sequence region motifs for these transcription factors would be expected to effect the function of the mmor suppressor genes. Accordingly, in addition to identifying the genomic strucmre of the pRb2/pl30 gene, additional experiments were conducted to define the 5 '-flanking promoter sequence of pRb2/pl30. Part of the putative promotor sequence for pRb2/pl30, along with the entire sequence of the first exon and the beginning of the first intron is shown in Figure 4 (SEQ ID NO:4). The full sequence for the putative promoter region is given in SEQ ID NO: 113.

To characterize the pRb2/pl30 promoter, a primer extension analysis was performed to locate the transcription initiation site. The protocol for the prime-extension analysis is given in the examples that follow. A twenty four nucleotide segment (SEQ ID NO: 114) containing the antisense-strand sequence 26 to 50 nucleotides upstream from the putative ATG codon (See Fig. 4) was end-labeled and used as a primer for an extension reaction on cyctoplasmatic RNA from HeLa cells. As shown in Fig. 5, a major extended fragment of 78 bp was detected (lane 1) from the primer extension done with HeLa cells as the template. The additional bands detected by the primer extension analysis could represent additional initiation sites. This finding (lane 1) is consistent with a transcription initiation site 99 nucleotides upstream of the start codon. On the contrary, there was no primer extension product observed when tRNA was used as a template (lane 2). The probable position of the identified transcription initiation site within the promoter sequence is indicated by the arrow in Fig. 4. The primer extension analysis was repeated three times, and similar results were produced in each instance.

The putative transcription factor-binding sites were identified by their similarity to consensus sequences for known transcription factor-binding

sites by use of the SIGNAL SCAN program. A description of this program is included in the examples that follow. The most recognizable sequence motifs are for the transcription factors Spl (two sites), Kerl and MyoD. Fig. 4 shows the location of these motifs. Kerl is involved in keratinocyte-specific transcription, while MyoD is involved in myogenesis. Leask et al , Genes Dev. 4: 1985-1998 (1990); Weintraub, H. , Cell 75: 1241-1244 (1993). The presence in the promoter region for pRb2/pl30 of these sequence motifs supports a hypothesis of an involvement of this gene in the complex pathways regulating differentiation of specific cell systems. 2. Detection of Mutations in pRb2/p!30

The present invention provides a method for amplifying the genomic DNA of pRb2/pl30 and for screening polymoφhisms and mutations therein. The assay methods described herein can be used to diagnose and characterize certain cancers or to identify a heterozygous carrier state. While examples of methods for amplifying and detecting mutations in pRb2/pl30 are given, the invention is not limited to the specific methods exemplified. Other means of amplification and identification that rely on the use of the genomic DNA sequence for pRb2/pl30 and/or the use of the primers described herein are also contemplated by this invention. Generally, the methods described herein involve preparing a nucleic acid sample for screening and then assaying the sample for mutations in one or more alleles. The nuclei acid sample is obtained from cells. Cellular sources of genomic DNA include culmred cell lines, or isolated cells or cell types obtained from tissue (or whole organs or entire organisms). Preferably, the cell source is peripheral blood lymphocytes. Methods of DNA extraction from blood and tissue samples are known to those skilled in the art. See, for example, Blin et al, Nuc. Acids Res. 3:2303-2308 (1976); and Sambrook et al , Molecular Cloning :A Laboratory Manual, Second Edition, pp. 9.16-9.23. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), the entire disclosure of which is incoφorated herein by reference. If the patient sample

to be screened is in the form of double- stranded genomic DNA. it is first denamred using methods known to those skilled in the an Denaturation can be carried out either by melting or subjecting the strands to agents that destabilize the hydrogen bonds, such as alkaline solutions and concentrated solutions of formamide or urea

In one embodiment of the invention, prior to screening the genomic DNA sample, the pRb2/pl30 genomic DNA sample is amplified by use of the polymerase chain reaction (PCR), using a primer pair, a buffer mixmre, and an enzyme capable of promoting chain elongation Methods of conducting PCR are well known to those skilled in the art. See, for example, Beutler et al , U.S. Patent No. 5,234,811 , or Templeton, N.S. , Dιag. Mol Path 1(1).58- 72 (1992), which are incoφorated herein by reference as if set forth at length The amplification product produced from PCR can then be used to screen for mutations using the techniques known as Single Strand Conformational Polymoφhism (SSCP) or Primed In-Situ DNA synthesis (PRINS). Of course, mutations can also be identified through the more laborious task of sequencing the gene isolates of a patient and comparing the sequence to that for the corresponding wild type pRb2/pl30 segment

PCR is carried out by thermocycling, i e. , repeated cycles of heating and cooling the PCR reaction mixmre, within a temperamre range whose lower end is 37°C to 55°C and upper end is around 90°C to 100°C. The specific temperamre range chosen is dependent upon the enzyme chosen and the specificity or stringency required. Lower end temperatures are typically used for annealing in amplifications in which high specificity is not required and conversely, higher end temperatures are used where greater stringency is necessary. An example of the latter is when the goal is to amplify one specific target DNA from genomic DNA. A higher annealing temperamre will produce fewer DNA segments that are not of the desired sequence Preferably, for the invention described herein, the annealing temperamre is between 50°C and 65 °C Most preferably, the annealing temperamre is 55 °C

The PCR is generally performed in a buffered aqueous solution, i. e. . a PCR buffer, preferably at a pH of 7-9, most preferably about 8. Typically, a molar excess of the primar is mixed with the buffer containing the template strand. For genomic DNA, this ratio is typically 10 ⁶ : 1 (primer: template). The PCR buffer also contains the deoxynucleotide triphosphates (dATP, dCTP, dGTP, and dTTP) and a polymerase. Polymerases suitable for use in PCR include, but are not limited to, E. coli DNA polymerase I, the Klenow fragment of E. coli DNA polymerase I, T4 DNA polymerase, T7 DNA polymerase, Taq DNA polymerase (Thermus aquaticus DNA polymerase I), and other heat-stable enzymes which will facilitate the formation of amplification products.

The primers used herein can be naturally occurring oligonucleotides purified from a nucleic acid restriction digest or produced synthetically using any suitable method, which methods are known to those skilled in the art. The primers used herein can be synthesized using automated methods.

Because a mutation can occur in both the exon itself and the splice junction, it is necessary to design primers that will ensure that the entire exon region to be analyzed is amplified. To amplify the entire exon, the oligonucleotide primer for any given exon must be designed such that it includes a portion of the complementary sequence for the promoter region, for the 3 '- noncoding region, or for the introns flanking the exon to be amplified, provided however that the primer sequence should not include the sequence for the splice signal dinucleotides. It is important to exclude the complementary sequence for the splice signal dinucleotides from the primer in order to ensure that the entire region, including the splice signal dinucleotide, is amplified. Including the complementary sequences to the splice signal dinucleotides could result in an amplification product that "plasters over" the splice junction and masks any potential mutation that could occur therein. It should be noted, however, that the introns flanking the exon are not limited to the introns immediately adjacent

to the exon to be amplified. The oligonucleotide primer can be designed such that it includes a portion of the complementary sequence for the introns upstream or downstream from the exon to exon to be amplified. In the latter instance, the amplification product produced would include more than one exon. Preferably at least 20 to 25 nucleotides of the sequence for each flanking intron are included in the primer sequence.

The primers used herein are selected to be substantially complementary to each strand of the pRb2/pl30 segment to be amplified. There must be sufficient base-pair matching to enable formation of a hybrid duplex under hybridization conditions. It is not required, however, that the base-pair matchings be exact. Therefore, the primer sequence may or may not reflect the exact sequence of the pRb2/pl30 segment to be amplified. Non-complementary bases or longer sequences can be interspersed into the primer, provided the primer sequence retains sufficient complementarity with the segment to be amplified and thereby form an amplification product.

The primers must be sufficiently long to prime the synthesis of amplification products in the presence of a polymerizing agent. The exact length of the primer to be used is dependent on many factors including, but not limited to, temperamre and the source of the primer. Preferably the primer is comprised of 15 to 30 nucleotides, more preferably 18 to 27 nucleotides, and most preferably 24 to 25 nucleotides. Shorter primers generally require cooler annealing temperatures with which to form a stable hybrid complex with the template.

Primer pairs are usually the same length, however, the length of some primers was altered to obtain primer pairs with identical annealing temperatures. Primers of less than 15 bp are generally considered to generate non-specific amplification products.

According to one embodiment of this invention, SSCP is used to analyze polymoφhisms and mutations in the exons of pRb2/pl30. SSCP has the advantages over direct sequencing in that it is simple, fast, and efficient.

The analysis is performed according to the method of Orita et al.. Genomics 5:874-879 (1989), the entire disclosure of which is incoφorated herein by reference. The target sequence is amplified and labeled simultaneously by the use of PCR with radioactively labeled primers or deoxy nucleotides. Neither in situ hybridization nor the use of restriction enzymes is necessary for SSCP. SSCP detects sequence changes, including single-base substitutions (point mutations), as shifts in the electrophoretic mobility of a molecule within a gel matrix. A single nucleotide difference between two similar sequences is sufficient to alter the folded strucmre of one relative to the other. This conformational change is detected by the appearance of a band shift in the mmor DNA, when compared with the banding pattern for a corresponding wild type DNA segment. Single base pair mutations can be detected following SSCP analysis of PCR products up to about 400 bp. PCR products larger than this size must first be digested with a restriction enzyme to produce smaller fragments.

In another embodiment of the invention, sequence mutations in pRb2/ρl30 can be detected utilizing the PRINS technique. The PRINS method represents a versatile technique, which combines the accuracy of molecular and cytogenetic techniques, to provide a physical localization of the genes in nuclei and chromosomes. See Cinti et al , Nuc. Acids Res. Vol 21 , No. 24: 5799- 5800 (1993), the entire disclosure of which is incoφorated herein by reference. The PRINS technique is based on the sequence specific annealing of unlabeled oligodeoxynucleotides in situ. The oligodeoxynucleotides operate as a primer for in situ chain elongation catalyzed by Taq I polymerase. Labeled nucleotides, labeled with a substance such as biotin or Digoxigenin, act as substrate for chain elongation. The labeled DNA chain is visualized by exposure to a fluorochrome-conjugated antibody specific for the label substance. Preferably, the label is Digoxigenin and the fluorochrome conjugated antibody is anti-Digoxigenin-FITC. This results in the incoφoration of a number of labeled nucleotides far greater than the number of nucleotides in the primer

itself. Additionally, the specificity of the hybridization is not vulnerable to the problems that arise when labeled nucleotides are placed in the primer. The bound label will only be found in those places where the primer is annealed and elongated. Neither the SSCP nor the PRINS technique will characterize the specific nature of the polymoφhism or mutation detected. If a band shift is detected through use of SSCP analysis, one must still sequence the sample segment and compare the sequence to that of the corresponding wild type pRb2/pl30 segment. Similarly, if the absence of one or both of the alleles for a given exon segment is detected by the PRINS technique, the sequence of the segment must be determined and compared to the nucleotide sequence for the corresponding wild type in order to determine the exact location and nature of the mutation, i.e. , point mutation, deletion or insertion. The PRINS technique is not capable of detecting polymoφhisms. Protocols for the use of the SSCP analysis and the PRINS technique are included in the examples that follow.

The PRINS method of detecting mutations in the pRb2/pl30 gene may be practiced in kit form. In such an embodiment, a carrier is compartmentalized to receive one or more containers, such as vials or test mbes, in close confinement. A first container may contain one or more subcontainers, segments or divisions to hold a DNA sample for drying, dehydrating or denaturing. A second container may contain the PRINS reaction mixmre, which mixmre is comprised of a PCR buffer, a DIG DNA labeling mixmre, a polymerase such as Taq I DNA polymerase, and the primers designed in accordance with this invention (see Example 7, Table 8). The DIG DNA labeling mixmre is comprised of a mixmre of labeled and unlabeled deoxy nucleotides. Preferably, the labeled nucleotides are labeled with either biotin or Digoxigenin. More preferably, the label is Digoxigenin. A third container may contain a fluorochrome conjugated antibody specific to the label. The fluorochrome conjugated antibody specific for Digoxigenin is anti-

Digoxigenin-FITC. Suitable conjugated fluorochromes for biotin include avidin- FITC or avidin Texas Red. The fourth container may contain a staining compound, preferably Propidium Iodide (PI). The kit may further contain appropriate washing and dilution solutions.

Examples

The following examples illustrate the invention. These examples are illustrative only, and do not limit the scope of the invention.

Example 1 Expression of pRb2/pl30 in Endometrial Carcinoma A. Patients and Tumors

Between September 1988 and December 1994, 196 patients with previously untreated endometrial carcinoma were seen at the Department of Obstetrics and Gynecology, University of Florence, Italy. To avoid concern for the possibility radiation affecting molecular analyses, the patients who received preoperative irradiation were excluded. In 175 cases surgery was the first treatment. Paraffin-embedded tissue blocks containing the most representative portion of the mmor were available in 104 of these cases; four patients were lost to follow up, leaving a total of 100 patients. Patients' ages ranged from 46 to 84 years with a median age of 64 years. Histologic slides were reviewed to assess histologic type, grade of differentiation and depth of myometrial invasion. The stage was evaluated by microscopic analysis of the surgical specimen according to the 1988 International Federation of Gynecology and Obstetrics (FIGO) classification (Gynecol Oncol 35: 125 (1988). Table 1 summarizes the clinical and pathological characteristics of the study group.

B. Surgical Treatment

Surgical treatment included total hysterectomy in 95 cases and extended hysterectomy in five cases. Bilateral salpingo-oophorectomy was

always associated. Pelvic and paraaortic lymphadenectomy were performed at the surgeon ^' s discretion, but not systematically. Overall. 43 patients underwent lymphadenectomy. The omenmm was removed when appropriate (four cases).

Table 1. Clinical And Pathological Feamres Of 100 Patients In Which pRb2/pl30 Expression Was Tested.

Feature Number of Patients

Age

< 65 yr 52

65 yr 48

FIGO stage

I 68

II 15

III 14

IV 3

Histologic type

Adenocarcinoma 74

Adenosquamous 17

Adenoacanthoma 4

Papillary serous 4

Clear cell 1

Grade of differentiation

Well differentiated 44

Moderately differentiated 26

Poorly differentiated 25

Not evaluable 5

Depth of myometrial invasion

< 50% 41

> 50% 59

Adjuvant treatment

None 57

Radiotherapy 37

Chemotherapy 6

C. Tumor Specimen Collection

For all 100 patients, a tumor specimen was taken fresh from a site regarded to be representative of the lesion immediately after hysterectomy. Each mmor sample was later divided into two parts: one for flow cytometry and the other for histological analysis.

P. Adjuvant Therapy

Forty-three of the 100 patients received adjuvant treatment. Of the 43 patients receiving adjuvant treatment, 37 received radiotherapy and 6 received chemotherapy. Poor grade of differentiation, deep myometrial invasion ( > 50 percent) and mmor outside the uterine coφus (stage > I) were the major criteria for receiving adjuvant treatment. The irradiated patients (37 patients) received 56Gy on the whole pelvis. Chemotherapy (six patients) was given, when possible, in cases with more advanced disease (stage III-IV). The chemotherapy regimen included cisplatin (60 mg per square meter of body surface area) in combination with cyclophosphamide (600 mg per square meter

of body surface area) and epirubicin (60 mg per square meter of body surface area), every 21 days, for six cycles.

E. Follow-up And Evaluation Of Results

After completing the treatment, patients were seen every three months for the first two years, every four months during the third and fourth years, and every six months thereafter. Recurrence was considered as any documented relapse of the mmor either in the pelvis or systemic. Disease-free interval was calculated from the date of the operation. Patients with residual disease after surgery or who recurred within three months from the date of the operation were not considered free of disease and therefore excluded from the disease-free analysis, but not from the actuarial survival calculation. Patients with deaths from causes other than endometrial cancer were considered as lost to follow-up and therefore -their survival times were censored at the date of death. Follow-up data were available for all 100 patients, with a median of 48 months (range 20 to 86 months). Disease-free interval and actuarial survival were the end-points of the study .

F. Flow Cytometric Analysis Of DNA Index

For flow cytometry, a suspension of mmor cells was obtained by mincing the sample with a lancet and scissors in phosphate-buffered saline. The cell suspension was filtered by a 50 micrometer mesh of polyacrylamide, fixed in 70 percent ethanol, and stored at -4°C until assayed. Prior to DNA analysis the ethanol was removed by centriguation (1500 revolutions/min for ten minutes); the pellet was then resuspended and washed twice in phosphate- buffered saline. The RNA was removed by digestion with ribonuclease (Serva, 0.1 mg/ml in phosphate-buffered saline) for 30 minutes at 37°C. the nuclei were washed in phosphate-buffered saline, and DNA was stained with 40 mg propidium iodide (Becton Dickinson) and 1 gm sodium citrate per liter in distilled water. Human female lymphocytes were added to the samples before

enzymatic treatment and staining, and they were used as the DNA diploid standard. The DNA analyses were performed with an Elite flow cytometer (Coulter Coφoration, Hialeah, Fla.) provided with a 15 mW Argon laser, at a wavelength of 488 mm. Data were expressed as DNA histograms. The DNA ploidy was given by the DNA index, defined as a proportion of the modal DNA values of the mmor G ₀ and G[ cells (peak channel) to the DNA content of the diploid standard. The histograms were based on measurement of more than 10,000 cells and resulted, in general, in a good resolution with a coefficient of variation of three to six percent. Calculation of DNA index was done by processing each histogram in the computer-assisted program Multicycle Autofit, version 2.00 (Phoenix Flow Systems, San Diego, CA).

All cases with DNA index value of 1 (±0.04) were classified as diploid and others as aneuploid.

G. Antibody Rabbit polyclonal immune serum, designated ADL1 , was prepared against pRb2/pl30 according to the procedure of Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Laboratory Press (1988), Chapter 5, the disclosure of which is incoφorated herein by reference. Rabbits were immunized with a conjugate comprising the peptide Glu-Asn-His-Ser-Ala- Leu-Leu-Arg-Arg-Leu-Gln-Asp-Val-Ala-Asn-Asp-Arg-Gly-Ser-His- Cys (SEQ ID NO: 3) coupled to keyhole limpet hemocyanin (KLH). The peptide corresponds to the carboxy terminus of the pRb2/pl30 protein. Briefly, rabbits were immunized with the SEQ ID NO:3-KLH conjugate by subcutaneous injection once every two weeks until a total of three injections were given. The initial injection (primary immunization) comprised 1 mg SEQ ID NO:3-KLH conjugate in 500 μl PBS, plus 500 μl of complete Freund's adjuvant. The second and third injections (boosts) comprised 500 μg of the conjugate in 500 μl PBS. plus 500 μl of complete Freund's adjuvant. The rabbits were bled after

the third injection. Subsequent boosts, with the same composition as the second and third injections, were given once a month.

H. Immunohistochemical Analysis

Sections of each mmor specimen were cut to 5-micrometer, mounted on glass and dried overnight at 37 °C. All sections were then deparaffinized in xylene, rehydrated through graded alcohol series and washed in phosphate-buffered saline. This buffer was used for all subsequent washes and for the dilution of the antibodies. Sections were quenched in 0.5 percent hydrogen peroxide and blocked with diluted ten percent normal goat anti-rabbit serum. Slides were then incubated for one hour at room temperamre with the ADL1 immune serum at a dilution of 1 : 1000, then incubated with diluted goat anti-rabbit biotinylated antibody (Vector, Burlingame, Calif.) for 30 minutes at room temperamre. After washing in phosphate-buffered saline, the slides were processed by the ABC method (Vector) for 30 minutes at room temperamre. Diaminobenzidine (Sigma, St. Louis) was used as the final chromogen, and hematoxylin as the nuclear counterstain. Negative controls for each tissue section consisted of substitution of the primary antibody with the conesponding pre-immune serum. Moreover, preincubation of the antibody with an excess of the corresponding immunizing antigen, blocked the immunocytochemical reaction, thus confirming the specificity of the ADL1 antibody for pRb2/pl30 (data not shown).

All the samples were processed under the same conditions. In each experiment, normal uterine tissue was also included as a control. The results of pRb2/pl30 immunostaining were independently inteφreted by three observers who had no previous knowledge of the clinical outcome of each patient. The level of concordance, expressed as the percentage of agreement between the observers was 90 percent (90 of 100 specimens). In the remaining specimens the score was obtained from the opinions of the two investigators in agreement. The results were expressed as percentage of positive cells. In each

mmor sample, at least 20 high power fields were randomly chosen and 2.000 cells were counted. The pRb2/pl30 immunostaining was mostly nuclear, but a few specimens also exhibited cytoplasmatic staining. This pattern of immunoreactivity could be referred to microstructural alterations caused by the fixing and embedding procedures, or might reflect differences in the levels of expression and in the localization of this antigen during the various phases of the cell cycle, as has already been shown at the molecular level (Claudio et al. , Cancer Res 56: 2003-8 (1996).

I. Cellular Reactivity Cutoff Point To evaluate the prognostic value of pRb2/pl30 expression, the patients' disease-free and actuarial survival durations were compared after dividing them into two groups using different cutoff points of percent pRb2/pl30 positivity. The P values were significant for poor disease-free and actuarial survival when a cutoff point of 40 percent or fewer reactive cells was used (P = 0.003 and P< 0.001, respectively). The level of significance decreased to P = 0.02 and P = 0.01 , respectively, with a cutoff point of 50 percent positivity and became insignificant with a cutoff point of 60 percent or higher positivity. Consequently, subsequent survival analyses were carried out using a 40 percent reactivity cutoff point. A similar approach to identify optimal cutoff points has been used in immunohistochemical smdies utilizing p53 expression and bcl-2 expression (Shim et al. , J Natl Cancer Inst 88: 519-29 (1996); Silvestrini et al , J Clin Oncol 14: 1604-10 (1996)).

J. Statistical Analysis

Fisher's exact test was used to evaluate the association between pRb2/pl30 expression and the other prognostic variables (Fienberg, The

Analysis Of Cross-Classified Categorical Data, MIT Press, Cambridge, Mass. :

Zelterman et al , "Contingency Tables In Medical Smdies" . NEJM Books 293-

310 (1992)). Disease-free interval and actuarial survival were calculated

according to the Kaplan-Meier method (Kaplan et al. , Am Stat Assoc 53: 457- 81 (1958)) and evaluated by the log-rank test (Miller, Survival Analysis . pp. 44- 102. John Wiley, New York (1981)). Univariate Cox analysis was used to assess the effect of each prognostic variable on disease-free interval and survival. A multivariate analysis (Cox proportional-hazards regression, with forward selection of variables) (Cox, J R Stat Soc 34: 187-220 (1972)) was performed to estimate which of the possible risk factors yielded independent prognostic information. Data analysis was performed with the SPSS statistical package, release 5.0.1 (SPSS Inc. , Chicago, IL).

K. Results

A brown stain indicated the presence of pRb2/pl30 in mmor cells. The specimens were characterized as having no detectable staining, staining in only a few positive cells (about ten percent), staining in more than 40 percent of the cells, or intense staining in the majority of cells. Tumors with immunostaining in more than 40 percent of cells were considered to be positive for pRb2/pl30.

In normal uterine samples, strong immunoreactivity was detected for pRb2/pl30 in all endometrial and endocervical epithelial cells. Of the 100 endometrial adenocarcinomas examined, five showed immunoreactivity for pRb2/pl30 in 20 percent or fewer cells, 15 had reactivity in 30 percent of the cells and nine had staining in 40 percent of the cells. These, 29 mmors (29 percent) were considered pRb2/pl30 negative. The remaining 71 mmors were scored as 50 percent positivity in 11 cases, as 60 percent positivity in 49 cases and with staining in over 70 percent of the cells in four cases. These 71 mmors (71 percent) were considered pRb2/pl30 positive.

The DNA index values showed a diploid type in 73 cases and an aneuploid type in 27 cases. The DNA index of the aneuploid mmors was hypodiploid in one case, hypertetraploid in four cases; the remaining 22 cases

had a modal DNA content in the diploid to tetraploid range (1 < DNA index < 2).

L. Association Of pRb2/p!30 Expression With Clinical And Pathological Feamres. The expression of pRb2/pl30 was inversely correlated with patients' age: in patients younger than 65 years pRb2/pl30 negative mmors were nine of 52 (17.3 percent) in contrast with 20 of 48 in patients aged 65 years or older (41.6 percent) (P = 0.008). Immunostaining for pRb2/pl30 was more frequently negative among patients with aneuploid mmors (13 of 27; 48.1 percent) than among those with a diploid pattern (16 of 73; 21.9 percent) (P = 0.001). Tumors negative for pRb2/pl30 were more frequent among patients with poorly or moderately differentiated carcinomas, but this association was not statistically significant (P = 0.06). The level of expression of pRb2/pl30 did not differ significantly between patients with mmors limited to the uterine coφus (stage I) and those in whom the mmor had spreads outside the coφus uteri (stage > I), (P = 0.4). No significant difference in the incidence of pRb2/pl30 negativity was found among the histologic types, nor among patients with different degrees of myometrial infiltration.

Expression of pRb2/pl30, mmor ploidy, FIGO stage and grade of differentiation were significantly correlated with disease-free interval and actuarial survival, by Univariate Cox analysis, as shown in Table 2. Other clinico-pathological feamres, including age, histologic type and depth of myometrial invasion were not associated with the outcome (data not shown). As shown in Figure 1, patients with pRb2/pl30 negative mmors had a significantly reduced disease-free interval and survival (P=0.001 and P< 0.0001 , respectively); the five-year survival probability was 52.0 percent in patients with such mmors, in contrast with 92.5 percent in patients with pRb2/pl30 positive mmors.

Table 2. Significant Predictors Of Clinical Outcome In 100 Patients With Endometrial Carcinoma, According

To Cox Univariate Analysis For Disease-free Interval And Actuarial Survival.

Variable Recurrence 95% P Value Death 95% P Value Rate Ratio Confidence Rate CI +

Interval Ratio pRb2/ _P 130 positive 1 1 negative 4.83 1.70 - 13.64 0.003 6.68 2.32 - 19.27 <0.0001

FIGO stage

I 1 1

> I 5.42 1.86 - 15.77 0.002 5.08 1.78 - 14.51 0.002

Ploidy status 00 diploid 1 1 aneuploid 3.43 1.24 - 9.51 0.01 5.94 2.14 - 16.42 <0.001

Grade of differen tiation (1 = well differentiated, 2= moderately differentiated, 3 = poorly differentiated)

1 1 1

2 7.73 1.54 - 38.67 0.01 13.88 1.65 - 0.01 1 16.27

3 7.45 1 .43 - 38.78 0.01 18.36 2.23 - 0.007 151. 10

Table 3 shows the results of Cox proportional-hazards regression analysis in which the response to pRb2/pl30 immunostaining, tumor ploidy, FIGO stage and grade of differentiation were tested simultaneously to estimate the rate ratios for the occurrence of death from disease in patients with endometrial cancer. Negative immunostaining for pRb2/pl30 resulted as the strongest independent predictor of poor outcome. Patents with pRb2/pl30 negative tumors had a significantly higher rate ratio for dying due to disease (4.91) than patients with pRb2/pl30 positive tumors. Multivariate analysis revealed that tumor spread outside the corpus uteri (stage > I) and aneuploidy were also associated with a higher probability of death from disease, whereas grade of differentiation yielded no independent prognostic information. By the combined use of pRb2/pl30 expression and FIGO stage, a more accurate definition of risk of death was possible.

Figure 2 presents Kaplan Meier survival estimates according to these stratified risk groups. The following is the comparison between the groups by the log-rank test:

Stage I, pRb2/p 130- Positive versus Stage > I, pRb2/pl30-Positive: difference not significant; Stage I, pRb2/pl30-Positive versus Stage I, pRb2/pl30-Negative: P = 0.01; Stage I, pRb2/pl30-Negative versus Stage > I, pRb2/pl30-Negative: P = 0.005;

Stage > I, pRb2/pl30-Positive versus Stage > I, pRb2/pl30-Negative: P = 0.003; Stage > I, pRb2/pl30-Positive versus Stage I, pRb2/pl30-Negative: difference not significant.

Table 3. Results Of Cox Proportional-Hazards Regression Analysis For Survival Data.

Variable Rate Ratio 95% Confidence P Value* Interval

pRb2/pl30

positive 1 negative 4.91 1.66 - 14.54 0.004

FIGO stage

I 1

> I 4.18 1.43 - 12.23 0.009

Ploidy status

Diploid 1

Aneuploid 3.36 1.17 - 9.62 0.02

* Chi-square of the model, P < 0.001

Example 2 Expression of pRb2/pl30 in Ovarian Cancer

A. Tumors

Sixty archived (formalin fixed and paraffin-embedded) epithelial carcinoma specimens were obtained from the Department of Pathology at Pennsylvania Hospital. The specimens included Grade 1 , Grade 2, and Grade 3 tumors.

B. Immunohistochemistrv

Immunohistochemical staining was performed using an automated immunostainer (Ventana ES, Ventana Medical Systems, Tucson. AZ) and a

Peroxidase-DAB immunodetection kit (Ventana Medical Systems). Five micron sections were cut from each tumor specimen. The sections were mounted on slides and air-dried. The sections were deparaffinized in xylene and hydrated through a graded alcohol series into water. A polyclonal anti-RB2 primary antibody was applied at a dilution of 1 :500 for 30 minutes at 37°C. The slides were then incubated with a biotinylated goat anti-rabbit antibody for 30 minutes. The slides were then incubated with a horseradish peroxidase conjugated-avidin. Hydrogen peroxide was used as the oxidizing substrate, and diaminobenzidine (DAB) was used as the chromagen. The slides were counterstained with hematoxylin, dehydrated, and mounted. The intensity of pRb2/pl30 immunostaining was evaluated.

C. Results

The preliminary results are shown in Table 4. These results suggest that as the grade of tumor increases, less expression of the pRb2/pl30 protein is detected. The pRb2/pl30 expression level may therefore be useful in grading and as a prognostic indicator in human epithelial ovarian cancer.

Table 4. Immunohistochemical Detection Of pRb2/pl30 In Human Epithelial Ovarian Carcinoma Specimens

Grade of Tumor Intensity of Immunostaining

Negative + + + + + +

Grade 1 20% 40% 40% 0%

Grade 2 50% 33 % 17% 0%

Grade 3 37% 26% 23% 14%

Example 3 Expression of pRb2/pl30 in Lung Cancer, Series I

A. Antibody Against pRb2/p!30

The rabbit polyclonal immune serum designated ADL1. as described in Example IG, was used in these studies.

B. Antibody Against p!07

Rabbit polyclonal immune serum was prepared against pl07 (ADL2) by immunizing rabbits with a bacterially expressed GST-pl07 fusion protein. Expression of the fusion protein was performed according to the procedure reported by Smith and Johnson, Gene 67:31-40 (1988) and Frangioni and Neel, Anal. Biochem. 270. 179-187 (1993). Rabbits were immunized with the fusion protein by subcutaneous injection once every two weeks until a total of three injections were given. The initial injection (primary immunization) comprised 500 μg protein in 500 μl PBS, plus 500 μl of incomplete Freund's adjuvant. The second and third injections (boosts) comprised 100 μg of the protein in 500 μl PBS, plus 500 μl of incomplete Freund's adjuvant. The rabbits were bled after the third injection. Subsequent boosts, with the same composition as the second and third injections, were given once a month.

C. Antibody Against pRb/p!05 An anti-pRb/pl05 monoclonal antibody (XZ 77), prepared as described by Hu et al , Mol. Cell. Biol 11:5192-5199 (1991), was used in these studies.

P. Tissue Samples

Lung tissue specimens from 51 patients with surgically resected lung cancer were obtained from patients who had not received chemo- or radiotherapy prior to surgical resection. The samples consisted of 39 squamous cell carcinomas and 12 adenocarcinomas. Histological diagnosis and grading

were performed by a skilled lung pathologist. Samples were graded on the scale of 1-2-3 with "3" representing the most malignant disease and " 1 " representing the least malignant disease. Normal lung tissue samples containing the stratified columnar epithelia of trachea, bronchi and adjacent glands were obtained either from biopsy or autopsy performed within 10 hours of the patient's death.

E. Immunohistochemistrv

Sections from each lung tissue specimen were cut at 3-5 μm, mounted on glass and dried overnight at 37 °C. All sections were then deparaffinized in xylene, rehydrated through a graded alcohol series and washed in phosphate-buffered saline (PBS). The same buffer was used for all subsequent washes and for dilution of antibodies.

Tissue sections for pRb2/pl30 and pl07 detection were sequentially quenched in 0.5 % hydrogen peroxide and blocked with diluted 10% normal goat anti-rabbit serum (Vector Laboratories). The slides were incubated for 1 hour at room temperature with the rabbit polyclonal immune serum (ADL1) raised against pRb2/pl30 at a dilution of 1 :2000, or the ADL2 antibody against pi 07 at a dilution of 1 :500. The slides were then incubated with diluted goat anti-rabbit biotinylated antibody (Vector Laboratories) for 30 minutes at room temperature.

Sections for pRb/pl05 detection were heated twice in a microwave oven for 5 min each at 700 W in citrate buffer (pH6), were quenched sequentially in 0.5 % hydrogen peroxide, and were blocked with diluted 10% normal horse anti-mouse serum (Vector Laboratories, Inc. ) The monoclonal mouse anti-human pRb/pl05 antibody XZ77 (at a dilution of 1 :500) was added and incubated for 120 min. at room temperature. After being washed in PBS, the slides were incubated with diluted horse anti-mouse biotinylated antibody (Vector Laboratories. Inc.) for 30 min. at room temperature.

Slides were processed by the so-called "ABC" method according to the instructions of the biotinylated antibody manufacturer (Vector Laboratories) for 30 minutes at room temperature. Diaminobenzidine was used as the final chromagen, and hematoxylin as a nuclear counterstain. Negative controls for each tissue section consisted of substitution of the primary antibody with pre-immune serum for ADL1 and ADL2, or leaving out the primary antibody for XZ77.

Three pathologists scored the expression of pRb2/pl30 protein as the percentage of positively stained nuclei on a scale of 0-1-2-3: 0 = undetectable level of expression; 1 = low expression level (1-30% cells stained positive); 2 = medium expression level (30-60% cells stained positive); 3 = high expression level (60-100% cells stained positive). The normal lung tissue samples comprising the stratified epithelia of the trachea, bronchi and adjacent glands were strongly stained, indicating a high expression level.

F. Results

The results are shown in Table 5. TABLE 5

Sample Type Grading pRb2/pl30 pl07 pRb/pl05

No. Level Level Level

1 squamous 3 0 2 3

2 squamous

3 squamous 1 3 3

4 squamous 1 3 3

5 squamous 2 2 2

6 squamous 2 3 ~>

7 squamous 3 1

8 squamous 2 3

9 squamous 2 1

10 squamous 2 3 1

1 1 squamous 2 3 2

12 squamous 1 3 3

13 squamous 3 1 1

14 squamous 1 3 3

15 squamous 3 0 3

16 squamous 2 2 2

17 squamous 2 3 2

18 squamous 2 1 2

19 squamous 1 3 3

20 squamous 3 1 1

21 squamous 2 3 2

22 squamous 3 2 3

23 squamous 2 3 3

24 squamous 2 3 1

25 squamous 2 3

26 squamous 1 3

27 squamous 3 1

28 squamous 2 3

29 squamous 1 3

0 squamous 1 3

1 squamous 1 2

2 squamous 2 3 1 2

33 squamous 3 3 1 3

34 squamous 2 3 1

35 squamous 2 0 1 2

36 squamous 2 3 1 1

37 squamous 2 3 1 1

38 squamous 1 3 1 3 3 399 ssqquuaammoouuss 3 1 1 0

40 adenocarc ma 3 0 2 2

41 adenocarcinoma 1 2 1 2

42 adenocarcinoma 2 1 2 1

43 adenocarcinoma 2 1 1 2

44 adenocarcinoma 2 0 2 1

45 adenocarcinoma 2 1 1 2

46 adenocarcinoma 1 2 1 2

47 adenocarcinoma 3 0 2 2

48 adenocarcinoma 1 2 1 2 49 adenocarcinoma 3 0 ?

50 adenocarcinoma 2 1

51 adenocarcinoma 2 0 1 2

Statistical Analysis

The data from Table 5 were analyzed using the Jonkheere- Terpstra test and STATXACT statistical software (Cytel Software Corp. , Cambridge, MA) determine whether there is a relationship between tissue grade and protein expression level.

A statistically significant inverse relationship was found between the pathological grading and the expression of pRb2/pl30 in squamous cell carcinomas (p < .0001) and adenocarcinomas (p < .004). Although a statistically significant inverse relationship was found between pathological grading and the expression of pRb/pl05 in squamous cell carcinomas (p =0.004), no such relationship was found between pRb/pl05 expression and grading of adenocarcinomas.

Example 4 Expression of pRb2/pl30 in Lung Cancer, Series II

A. Lung Cancer Specimens

One hundred and fifty eight lung cancer specimens were obtained from patients that underwent a surgical resection (lobectomy or pneumonectomy) in the Departments of Thoracic Surgery of the V. Monaldi Hospital and of the II University of Naples (Italy) between January 1995 and April 1996. Specimens were obtained only from patients who had not received chemo- or radiotherapy prior to surgical resection.

The histological diagnoses and classifications of the tumors were based on the WHO criteria, and the postsurgical pathologic TNM stage was determined using the guidelines of the American Joint Committee on Cancer.

The routine histopathological evaluation of the 158 tumor specimens analyzed was performed independently of the pRb2/pl30 immunostaining. Thirty two tumors were adenocarcinomas. 118 were squamous

carcinomas, 4 were carcinoids and 4 were small cell lung cancers. Eighty seven tumors (55.1 %) were classified as stage I, 43 tumors (27.1 %) were classified as stage II and 28 tumors (17.7%) were classified as stage Ilia. The adenocarcinomas and squamous carcinomas were classified by grade, as shown in Table 6.

B. Immunohistochemistrv

Sections of each specimen were cut at 3-5 μm, mounted on glass and dried overnight at 37 °C. All the sections were then deparaffinized in xylene, rehydrated through a graded alcohol series and washed in PBS. This buffer was used for all subsequent washes and for the dilution of the antibodies. Sections were heated twice in a microwave oven for five minutes each at 700 W in citrate buffer (pH 6), sequentially quenched in 0.5% hydrogen peroxide and blocked with diluted 10% normal goat anti-rabbit serum. Slides were then incubated for one hour at room temperature with rabbit polyclonal immune serum raised against pRb2/pl30 at a dilution ranging from 1 :500 to 1 : 1500, then incubated with diluted goat anti-rabbit biotinylated antibody (Vector Laboratories) for 30 minutes at room temperature. After washings in PBS, the slides were processed by the ABC method (Vector Laboratories) for 30 minutes at room temperature. Diaminobenzidine was used as the final chromogen, and hematoxylin as the nuclear counterstain. Negative controls for each tissue section were obtained by substituting the primary antibody with pre-immune serum.

All samples were processed under the same conditions. Three pathologists (A. Baldi, G.G. Giordano and F. Baldi) evaluated the staining pattern of the protein separately and scored it for the percentage of positive nuclei: score 1 , less than 10% of positive cells (low to undetectable level of expression); score 2, from 10% to 50% of positive cells (medium level of expression); score 3, more than 50% of positive cells (high level of expression). The level of concordance, expressed as the percentage of agreement between the

observers was 90% (142 of 158 specimens). In the remaining specimens the score was obtained from the opinions of the two investigators in agreement. At least 20 high power fields were chosen randomly and 2000 cells were counted. This coded score was preferred to facilitate the statistical analyses.

C. Statistical Analysis

Statistical analyses, using the chi square test, were performed to evaluate the significance of associations between the different variables of the considered tumors (histological type and grading, evidence of metastasis, pRb2/pl30 expression levels). A p value < .05 was considered statistically significant.

D. Results pRb2/pl30 immunostaining was mostly nuclear, but some specimens clearly exhibited cytoplasmatic staining with a low to absent background. Immunohistochemical staining patterns of the tumors can be summarized as follows: 50 specimens (31.6%) showed low to undetectable levels of pRb2/pl30 (score 1), 73 specimens (46.2%) exhibited medium pRb2/pl30 expression levels, while high levels of expression were detected in 35 specimens (22.2%). The small number of small cell lung cancers and carcinoids included in this study did not allow statistical analysis in these histological groups. All the SCLCs specimens exhibited low to undetectable pRb2/pl30 expression levels, while a high level of expression of this protein was recognized in all carcinoids.

Statistical analyses revealed that pRb2/pl30 expression did not correlate with tumor stage or with TNM status (p = n.s.). However, a negative significant relationship was found between pRb2/pl30 expression level and the histological grading (p < .0001). The correlation between histological grade and pRb2/pl30 expression is shown in Table 6.

TABLE 6

pRb2/pl30 Level

Type Grade No. 1 2 3

Squamous 1 13 2 0 11

Squamous 2 42 8 28 6

Squamous 3 63 30 27 6

Adenocarcinoma 1 8 0 2 6

Adenocarcinoma 2 27 4 16 2

Adenocarcinoma 3 2 2 0 0

The mean follow-up period was too short to allow a detailed analysis of the disease free and the overall survival time of the patients.

However, in looking at the development of metastasis in the patients, we found a significant inverse relationship between metastasis and the expression of pRb2/pl30 (p < .0001).

Example 5 Isolation and Characterization of Genomic Clones

A. Isolation of Genomic Clones

To isolate the entire human pRb2/pl30 gene, a human PI genomic library (Genome System Inc. , St. Louis, MO) was screened by using two primers made from the published cDNA sequence, Li et al , Genes Dev. 7:2366-2377 (1993). The sequences for the primers used to isolate the genomic clones are GTATACCATTTAGCAGCTGTCCGCC (SEQ ID NO: 116) and the complement to the sequence GTGTGCCATTTATGTGATGGCAAAG (SEQ ID NO: 115).

One of the clones identified upon screening the PI genomic library (clone no. 1437, Fig. 3B) was confirmed by Southern blot hybridization to contain a pan of the pRb2/pl30 gene. To obtain the additional 5' flanking sequence of the pRb2/pl30 gene containing the putative promoter region, a human placenta genomic DNA phage library (EMBL3 SP6/T7) from Clontech, Palo Alto, CA was screened with a cDNA probe according to the method of Sambrook et al. , Molecular Cloning :A Laboratory Manual, Second Edition, pp. 12.30-12.38, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), the entire disclosure of which is incorporated herein by reference. The cDNA probe, labeled with [γ- ³² P], corresponded to the first 430 bp after the start codon of the published cDNA sequence, Li et al. , supra. Of the two positive clones obtained, one, identified as < SCR3 (Fig. 3B), was determined to contain the 5' flanking region of the pRb2/pl30 gene.

B. Identification of Exon/intron Boundaries To precisely characterize the position of the exons and the exon/intron boundaries in the genomic DNA, a set of oligonucleotide primers were used to sequence the genomic DNA clones. The primers were synthesized based upon the cDNA nucleotide sequence of pRb2/pl30 such that they annealed to the genomic DNA at roughly 150 bp intervals. The exon intron boundaries were identified from those positions in which the genomic DNA sequence differed from that of the published cDNA sequence.

C. Sequencing of Clones

Sequencing of the recombinant clones was carried out in part by automated DNA sequencing using the dideoxy terminator reaction chemistry for sequence analysis on the Applied Biosystem Model 373 A DNA sequencer and, in part, by using a dsDNA Cycle Sequencing System kit purchased from GIBCO BRL, Gaithersburg, MD, according to the instructions of the manufacturer.

D. Synthesis of Oligonucleotide Primers

All oligonucleotide primers used herein were synthesized using Applied Biosystems DNA-RNA synthesizer Model 394, using beta-cyanoethyl phosphoramidite chemistry.

E. Results of the Genomic Clones Characterization

The human pRb2/pl30 gene consists of 22 exons and 21 introns and spans more than 50 kb of genomic DNA. The organization of these exons and introns are shown approximately to scale in Figure 3 A. The location and size of each exon and intron of pRb2/pl30, as well as the nucleotide sequences at the exon-intron boundaries are shown in Table 7 (SEQ ID NOS: 6-47). The exons range in size from 65 to 1517 bp in length. The introns, which range in size from 82-9837 bp in length, have been completely sequenced. The nucleotide sequences are given as SEQ ID NOS.48-68.

Example 6 Characterization of Transcriptional Control Elements

A. Cell Culture and RNA Extraction

The human HeLa (cervix epithelioid carcinoma) cell line was obtained from the American Type Culture Collection and maintained in culture in Dulbecco's modified Eagle medium (DHEM) with 10% fetal calf serum (FCS) at 37°C in a 10% CO ₂ -containing atmosphere. Cytoplasmatic RNA was extracted utilizing the RNAzol B method (CINNA/BIOTECX, Friendswood, TX).

TABLE 7

Exon-Intron Boundaries of the Human pRb2/pl30 Gene

Exon No. (bp) 5' Donor sequence 3' Acceptor sequence Intron No. (bp)

1(240) ACGCTGGAG ³⁰⁹ gtgcgctcgc tcttttacag ^3l0 GGAAATGAT 1(4220) (SEQ ID NO:6) (SEQ ID NO: 7 ) (SEQ ID NO:66)

2(131) AGAGCAGAG ⁴⁴⁰ gtaactatgt ttaataccag^CTTAATCGA 2(3507) (SEQ ID NO:8) (SEQ ID NO:9) (SEQ ID NO:67)

3(201) GAAACAGCG^gtaggttttc tcccccaaag^GCGACAGCC 3(3865) (SEQ ID NO: 10) (SEQ ID NO: 11) (SEQ ID NO:48)

UJ

4(65) ATGCAAAAG ⁷⁰⁶ gtaagaaaat aatcctgcag ⁷⁰⁷ GTAATTTCC 4(4576) (SEQ ID NO: 12) (SEQ ID NO: 13) (SEQ ID NO:49)

5(129) ATTTTAAAG ⁸³⁵ gtaggtttgt acaccatag ⁸³⁶ GCTTATCTG 5(1618) (SEQ ID NO: 14) (SEQ ID NO: 15) (SEQ ID NO:50)

6(161) GAAAAAAAG^gtttgtaagt ttcatcatag" ⁷ CTCCTTAAG 6(92) (SEQ ID NO: 16) (SEQ ID NO: 17) (SEQ ID NO:51)

7(65) AGAGAGTTT ^106, gtgagtactt ttcctatag ^I062 TAAAGCCAT 7(889) (SEQ ID NO: 18) (SEQ ID NO: 19) (SEQ ID NO:52)

8(187) TTTGACAAG ^,248 gtgagtttag ttttctttag ¹²⁴⁹ TCCAAAGCA 8(4586) (SEQ ID NO: 20) (SEQ ID NO:21) (SEQ ID NO:53)

19(107) AAGATAGAA ²⁹⁵⁰ gtgggatctt ctggctgcag ^295l CCAGTAGAG 19(572) (SEQ ID NO: 42) (SEQ ID NO:43) (SEQ ID NO: 64)

20(202) C AGGC A A AT ³ ' "gtaagtatga tttttaaacag ³¹⁵⁴ ATGGGATGC 20(901) (SEQ ID NO:44) (SEQ ID NO:45) (SEQ ID NO: 65)

21(165) CCTTCAAAG ^33,8 gtgagcctaa cccaccatag ³³¹⁹ AGACTGAGA 21(9837) (SEQ ID NO:46) (SEQ ID NO:47) (SEQ ID NO.68)

22(1517) to the polyadenylation signal

Os CJ»

B. Primer Extension Analysis

To characterize the pRb2/pl30 promoter, a primer extension analysis was performed to locate the transcription initiation site. The primer for this analysis was an oligonucleotide, 5 'ACCTC AGGTGAGGTGAGGGCCCGG 3' (SEQ ID NO: 114), complementary to the pRb2/pl30 genomic DNA sequence starting at position -22 (See Fig. 4, SEQ ID NO:4). The primer was end labeled with [7 ³² P]ATP and hybridized overnight with 20 μg of HeLa cytoplasmatic RNA at 42 °C. The primer-annealed RNA was convened into cDNA by avian myeloblastosis virus reverse transcriptase in the presence of 2 mM deoxy nucleotides at 42 °C for 45 minutes. The cDNA product was then analyzed on 7% sequencing gel containing 8 M urea. The position of the transcription start site was mapped from the length of the resulting extension product.

C. SIGNAL SCAN Program Several of the transcription factor-binding motifs were identified through the use of SIGNAL SCAN VERSION 4.0. SIGNAL SCAN is a computer program that was developed by Advanced Biosciences Computing Center at the University of Minnesota, St. Paul, MN. This program aids molecular biologists in finding potential transcription factor binding sites and other elements in a DNA sequence. A complete description of the program can be found in Prestridge, D.S. , CABIOS 7: 203-206 (1991), the entire disclosure of which is incorporated herein as if set forth at length.

SIGNAL SCAN finds sequence homologies between published signal sequences and an unknown sequence. A signal, as defined herein, is any short DNA sequence that may have known significance. Most of the known signals represent transcriptional elements. The program does not interpret the significance of the identified homologies; interpretation of the significance of sequences identified is left up to the user. The significance of the signal

elements varies with the signal length, with matches to shon segments having a higher probability of random occunence.

D. Results of the Primer Extension Analysis And SIGNAL SCAN

Figure 5 shows the results of the primer extension analysis done to locate the transcription initiation site for pRb2/pl30. A major extended fragment of 78 bp was detected (lane 1) from the primer extension done with HeLa Cells as the template. The probable position of the identified transcription start site is indicated by the arrow in Fig. 4. Putative transcription factor-binding sites were identified by their similarity to consensus sequences for known transcription factor-binding sites. The sequence motifs corresponding to Spl , Kerl, and MyoD are also indicated in Fig. 4.

Example 7 Detection of Heterozygous Mutations By PCR

A. Preparation of Genomic DNA The genomic DNA used herein was obtained from human peripheral blood lymphocytes. The samples were prepared by the methods of Sambrook et al, Molecular Cloning :A Laboratory Manual Second Edition, pp. 9.16-9.23, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

B. Synthesis Of PCR Primers

The PCR primers used herein were synthesized as described in Example 5D. The specific primer sequences used and their annealing temperatures are given in Table 8. as SEQ ID NOS: 69 to 112.

Table 8

Size Of

Annealing PCR

Exon Temperature Product Amplified Sequence Of Primer (5 '-3') (°C) (bp)

Exon 1 TTCGCCGTTTGAATTGCTGC 55 359 (SEQ ID NO:93) Exon l(rev) ACCGGTTCACACCAACTAGG

(SEQ ID NO: 94)

Exon 2 GAGATAGGGTCATCATTGAAAC 55 206

(SEQ ID NO: 95)

Exon 2(rev) CATTAGCCATACTCTACTTGT (SEQ ID NO:96)

Exon 3 GCTAATTTAACTCTGTAACTGC 55 327

(SEQ ID NO:97)

Exon 3(rev) CACTGCAGCACAGACTAATGTGT (SEQ ID NO:98) Exon 4 TCTCTCCCTTTAACTGTGGGTTT 55 245

(SEQ ID NO:99)

Exon 4(rev) GGAGTTGACGAGATTAATACCTG (SEQ ID NO: 100)

Exon 5 CTCTGTAACTGCTTATAATCCTG 55 235

(SEQ ID NO:69)

Exon 5(rev) CTAGGAAACCTGTACAACTCC (SEQ ID NO: 70)

Exon 6 GGCTTATTGTGTGCTGATATC 55 289

(SEQ ID NO:71) Exon 6(rev) AGAGATCCTTAAGTCGTCATG

(SEQ ID NO: 72)

Exon 7 CATGACGACTTAAGGATCTCTT 55 196

(SEQ ID NO: 101)

Exon 7(rev) CTCAGTTTCCAGAGTACAAAC (SEQ ID NO: 102)

Exon 8 CAGTTTCTGTGAGAGAGTACA 55 283

(SEQ ID NO:73)

Exon 8(rev) GGCTTACCTGCTCCTGTATTT (SEQ ID NO:74)

Exon 9 GTGAATTAAAGTCTTTCTGGCC 55 277

(SEQ ID NO: 103)

Exon 9(rev) ATCTTAGAAAGCAGACAGGGC (SEQ ID NO: 104)

Exon 10 GAGACATTTTATCCCCTTGTG 55 289

(SEQ ID NO: 105) Exon lθ(rev) TCCATGCCTCCAGTCTAAAGT

(SEQ ID NO: 106)

Exon 11 GAGGAGGAATGGGCCTTTATT 55 244

(SEQ ID NO:75)

Exon 11 (rev) AACCCACAGAATAGGGCAGGA (SEQ ID NO: 76)

Exon 12 CACTTAAGTTGCACTGGGTA 55 273

(SEQ ID NO: 107)

Exon 12(rev) CAACAGGAAGTTGGTCTCATC (SEQ ID NO: 108) Exon 13 TAAAAGGAAGAGCGGCTGTTT 55 378

(SEQ ID NO: 109)

Exon 13(rev) TTAAACCTAACTGCCACCCTC (SEQ ID NO: 110)

Exon 14 GGATACTGGCATTCTGTGTAAC 55 197

(SEQ ID NO.77)

Exon 14(rev) ATTTCCAGATAGTAAGCCCCA (SEQ ID NO: 78)

Exon 15 AGCTTGGACGGAAGTCAGATC 55 413

(SEQ ID NO:79) Exon 15(rev) TCTAGCCAAACCTCGGGTAAC

(SEQ ID NO: 80)

Exon 16 AATTGTAAACCTCTGCCC 55 394

(SEQ ID NO: 81)

Exon 16(rev) ATTTCCCAAGCTCATGCT (SEQ ID NO: 82)

Exon 17 AGCATGAGCTTGGGAAAT 55 277

(SEQ ID NO: 83)

Exon 17(rev) TGAAGACCTATCTTTGCC (SEQ ID NO: 84)

Exon 18 GTTCACAGAGCTCCTCACACT 55 230

(SEQ ID NO:85)

Exon 18(rev) AGGCCACAGAGTCAACTATGG (SEQ ID NO: 86)

Exon 19 AGGTCCTATCACCAAGGGTGT 55 250

(SEQ ID NO: 87) Exon 19(rev) GCTTAGTTACTTCTTCAAGGC

(SEQ ID NO:88)

Exon 20 GTAGCTGTTCCCTTTCTCCTA 55 364

(SEQ ID NO: 89)

Exon 20(rev) CCTCAACACTCATGAGAGTGA (SEQ ID NO: 90)

Exon 21 TGGTTTAGCACACCTCTTCAC 55 325

(SEQ ID NO:91)

Exon 21 (rev) GCTTAGCACAAACCCTGTTTC (SEQ ID NO: 92) Exon 22 CTGAGCTATGTGCATTTGCA 55 232

(SEQ ID NO: 111)

Exon 22(rev) AAGGCTGCTGCTAAACAGAT (SEQ ID NO: 112)

C. PCR Amplification

The sample DNA was amplified in a Perkin-Elmer Cetus thermocycler. The PCR was performed in a 100 μl reaction volume using 2.5 units of recombinant Taq DNA-polymerase and 40 ng of genomic DNA. The reaction mixture was prepared according to the recommendations given in the Gene Amp DNA Amplification kit (Perkin-Elmer Cetus). The reaction mixture consisted of 50 mM/1 KCI, lOmM/1 Tris-HCI (pH 8.3), 1.5 mM MgCl. 200 μM each deoxynucleotide triphosphate and 1 μM of each primer. Thirty five (35) PCR cycles were carried out, with each cycle consisting of an initial denaturation step at 95 °C for one minute, one minute at the annealing temperature (55 °C). an extension step at 72°C for one minute, and followed by

a final incubation period at 72 °C for seven minutes. Suitable annealing temperatures are shown in Table 8 for each of the primers designed in accordance with this invention. Minor adjustments in the annealing temperatures may be made to accommodate other primers designed in accordance with this invention.

D. Amplification Products of PCR

The size of the amplification products produced by PCR are shown in Table 8 above. The lengths of the PCR products ranged from 196 bp to 413 bp.

E. Sequencing of PCR Products

Sequencing of the amplification products of pRb2/pl30 can be conducted according to the method set forth in Example 5C above. Sequencing can also be performed by the chain termination technique described by Sanger et al , Proc. Nat'l Acad. Sc , U.S.A. 74:5463-5467 (1977) or Sambrook et al , Molecular Cloning: A Laboratory Manual, Second Edition, pp. 13.42- 13.77, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) with appropriate primers based on the pRb2/pl30 genomic sequence described herein.

Example 8 Detecting Mutations By SSCP Analysis

A. General Methods

The SSCP analysis was performed according to the methods of Orita et al , Genomics 5: 874-879 (1989) and Hogg et al , Oncogene 7: 1445- 1451 (1992), each of which is incorporated herein by reference. For the SSCP analysis, amplification of the individual exons was, in some experiments, performed as described in Example 7 with the exception that 1 μCi of [ ^: P]dCTP (3000 Ci mmol ') was added to the mixture in order to obtain a

labeled product. A 10% aliquot of the PCR-amplified product was diluted with a mixture of 10-20 μl of 0.1 % SDS and 10 mM EDTA. Following a 1 : 1 dilution with 95% formamide. 2mM EDTA, 0.05% bromophenol blue, and 0.05 % xylene cyanol loading solution (United States Biochemicals. OH), the diluted sample was run on a 6% non-denaturing gel. The DNA was electrophoresed in TBE (0.09 M Tris base, 0.09 M boric acid and 2.5 mM EDTA) running buffer at constant wattage at room temperature. The gel was dried on filter paper and exposed to X-ray film for 12 to 72 hours without an intensifying screen. Polymoφhisms and mutations were detected by observing a shift in the electrophoretic mobility pattern of the denatured PCR-amplified product relative to a conesponding wild type sample or normal tissue sample from the same patient. Once a band shift was identified, the segment was sequenced to confirm the exact nature of the polymoφhism or mutation.

B. Detection Of pRb2/p!30 Gene Mutations In the CCRF-CEM Cell Line

DNA was extracted from the CCRF-CEM line (human lymphoblastoid cells), and amplified. For the amplification, 50 μl of the PCR reaction mix containing 4 ng of genomic DNA, 0.2 mM of each deoxynucleotide triphosphates, 2 U of Taq polymerase and 0.4 μM of each primer were used. Fifty-Five cycles of denaturation (95°C, 1 minute), annealing (55°C, 1 minute) and extension (72°C, 1 minute) were carried out in a thermal cycler. The SSCP analysis was performed using an MDE mutation detection kit (AT Biochem). The PCR products were heated to 95 °C for two minutes and placed directly on ice for several minutes. The samples were run through the MDE gel at 8 Watts constant power for eight hours at room temperature, in 0.6X TBE running buffer. The gel was stained for 15 minutes at room temperature in a 1 μg/ml ethidium bromide solution, made in 0.6X TBE buffer, and placed on a UV-transilluminator to visualize the bands. Exon

20 showed a different migration relative to the control, suggesting the presence of mutations.

The sequences of the PCR products were determined by automated DNA sequencing, using dideoxy-terminator reaction chemistry. Two point mutations were identified: ACC to GCC at position 2950 of SEQ ID

NO: l , resulting in a threonine to alanine substitution; and CCT to CGT at position 3029 of SEQ ID NO: l , resulting in a proline to arginine substitution.

C. Detection of pRb2/p!30 Gene Mutations in Other Cell Lines

Using the SSCP and DNA sequencing methods described above, mutations in the pRb2/pl30 gene were identified in the following human tumor cell lines:

Jurkat cell line (human leukemia, T-cell lymphoblast): point mutations in exon 22;

K562 cell line (human chronic myelogenous leukemia, erythroblastoid cells): point mutations in exon 22, deletion in exon 21 ;

Molt-4 cell line (human T-cell leukemia, peripheral blood lymphoblast): point mutations in exon 21 , mutation(s) in exon 22;

Daudi cell line (human thyroid lymphoma, lymphoblast B cell): point mutations and insertion in exon 19, point mutations and insertions in exon 21, mutations(s) in exon 22;

Cem cell line (lymphoblastoid cell line, T-lymphocytes): mutation(s) in exon 20, point mutations and insertions in exon 22;

Saos-2 cell line (human primary osteogenic sarcoma): point mutations and insertions in exon 21 , point mutations and insertion in exon 22; U2-Os cell line (human primary osteogenic sarcoma): point mutations in exons 19 and 21 , point mutation and insertion in exon 22;

MG63 cell line (human osteosarcoma): point mutations in exon 19;

Hos cell line (human osteogenic sarcoma. TE85): point mutations in exon 19; insertions in exon 22;

U1752 cell line (human lung tumor): point mutations in exon 19, point mutations and insertion in exon 21 , point mutation and insertion in exon 22;

H69 cell line (human lung tumor): point mutations in exon 21 , point mutations and insertions in exon 22;

H82 cell line (human lung tumor): point mutations in exon 21 ; and Hone cell line (human nasopharyngeal carcinoma): mutations and insertion in exon 21 , mutation(s) in exon 22.

P. Detection of pRb2/p!30 Gene Mutations in Primary Tumors

Using the SSCP and DNA sequencing methods described above, mutations in the pRb2/pl30 gene were identified in the following primary human tumors:

13 NPC primary tumor (human nasopharyngeal carcinoma): point mutations in exon 21 , point mutation and insertions in exon 22; and

5 NPC primary tumor (human nasopharyngeal carcinoma) : point mutations and insertion in exon 22.

Example 9

Detecting Mutations By The PRINS Technique

The PRINS technique was performed according to the method of Cinti et al , Nuc. Acids Res. Vol. 21 , No. 24: 5799-5800 (1993) using human peripheral lymphocytes as the source of genomic DNA. The oligonucleotide primers were designed such that they included portions of the introns flanking exon 20. The sequences of the primers utilized to amplify exon 20 are listed in Table 8 above (SEQ ID NOS: 89 and 90).

Human fixed metaphase chromosomes or inteφhase nuclei from PHA stimulated peripheral blood lymphocytes were spread onto glass slides and allowed to air dry for ten days. The DNA was dehydrated in an ethanol series (70%, 90% , and 100%) and then denatured by heating to 94°C for 5 minutes. Using a reaction mixture containing 200 pmol of each oligonucleotide primer, 5 μl of 10 X PCR Buffer II (AmpliTaq, Perkin-Elmer), 2 μl DIG DNA labeling mixture (1 mM dATP, ImM dCTP, ImM dGTP, 0.65 mM dTTP, 0.35 mM DIG-dUTP, Boehringer-Mannheim) and 2 Units of Taq I DNA polymerase (AmpliTaq, Perkin-Elmer), the samples were incubated for 10 minutes at 55 °C and for 30 minutes at 72 °C. Suitable annealing temperatures for other primers designed in accordance with this invention are shown in Table 8. The samples were then washed two times in 2 X SSC (pH 7.0) and in 4 X SSC (pH 7.0) for 5 minutes at room temperature. The DNA samples were then placed in a solution of 4 X SSC and 0.5 % Bovine Serum Albumin (BSA) (pH 7.0), incubated at room temperature for 45 minutes with anti-Digoxigenin-FITC (Boehringer-Mannheim), and diluted 1: 100 in 4 X SSC and 0.5% BSA (pH 7.0). After washing the samples in 4 X SSC and 0.05% Triton X-100, the samples were counterstained with 1 μg/ml Propidium Iodide (PI).

The slides were examined under a Confocal Laser Scanning Microscope (CLSM Sarastro, Molecular Dynamics). The FITC and PI signals were detected simultaneously, independently elaborated and the final projections were superimposed with a Silicon Graphic Computer Personal IRIS-4D/20 workstation.

Figure 6 shows the results of a PRINS reaction on normal human inteφhase nuclei. The bright spots correspond to a DNA segment containing exon 20 of pRb2/pl30. This individual is homozygous for the presence of exon 20 of pRb2/pl30. Had there been a mutation in exon 20 of this individual, either one or both of these areas would have been diminished in intensity or not visible in its entirety. To determine the exact nature of this mutation, the

patient ^' s pRb2/pl30 DNA segment would be sequenced by methods known to those skilled in the art and compared to a wild type sample of pRb2/pl30 DNA All the reterences discussed herein are incoφorated by reference Some or all of the reagents, compositions, and supplies needed to carry out the methods, procedures, and techniques disclosed herein may be provided in the form of a kit Such kits are another embodiment of the present invention

One skilled in the art will readily appreciate that the present invention is well adapted to carry out the ends and advantages mentioned, as well as those inherent therein The nucleic acids, compositions, methods. procedures, and techniques described herein are presented as representative ot the preferred embodiments, and are intended to be exemplary and not limitations on the scope of the invention The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as defining the scope of the invention

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(l) APPLICANT: Thomas Jefferson University

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(vi) CURRENT APPLICATION DATA:

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(viii) ATTORNEY/AGENT INFORMATION:

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(A) TELEPHONE: (215) 568-8383

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(2) INFORMATION FOR SEQ ID NO:l:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4853 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: cDNA

(IX) FEATURE :

(A) NAME/KEY: CDS

(B) LOCATION: 70..3489

(xi) SEQUENCE DESCRIPTION- SEQ ID NO: 1 : TTCGCCGTTT GAATTGCTGC GGGCCCGGGC CCTCACCTCA CCTGAGGTCC GGCCGCCCAG 60

GGGTGCGCT ATG CCG TCG GGA GGT GAC CAG TCG CCA CCG CCC CCG CCT 108

Met Pro Ser Gly Gly Asp Gin Ser Pro Pro Pro Pro Pro 1 5 10

CCC CCT CCG GCG GCG GCA GCC TCG GAT GAG GAG GAG GAG GAC GAC GGC 156 Pro Pro Pro Ala Ala Ala Ala Ser Asp Glu Glu Glu Glu Asp Asp Gly 15 20 25

GAG GCG GAA GAC GCC GCG CCG TCT GCC GAG TCG CCC ACC CCT CAG ATC 204 Glu Ala Glu Asp Ala Ala Pro Ser Ala Glu Ser Pro Thr Pro Gin lie 30 35 40 45

CAG CAG CGG TTC GAC GAG CTG TGC AGC CGC CTC AAC ATG GAC GAG GCG 252 Gin Gin Arg Phe Asp Glu Leu Cys Ser Arg Leu Asn Met Asp Glu Ala 50 55 60

GCG CGG CCC GAG GCC TGG GAC AGC TAC CGC AGC ATG AGC GAA AGC TAC 300 Ala Arg Pro Glu Ala Trp Asp Ser Tyr Arg Ser Met Ser Glu Ser Tyr 65 70 75

ACG CTG GAG GGA AAT GAT CTT CAT TGG TTA GCA TGT GCC TTA TAT GTG 348 Thr Leu Glu Gly Asn Asp Leu His Trp Leu Ala Cys Ala Leu Tyr Val 80 85 90

GCT TGC AGA AAA TCT GTT CCA ACT GTA AGC AAA GGG ACA GTG GAA GGA 396 Ala Cys Arg Lys Ser Val Pro Thr Val Ser Lys Gly Thr Val Glu Gly 95 100 105

AAC TAT GTA TCT TTA ACT AGA ATC CTG AAA TGT TCA GAG CAG AGC TTA 444 Asn Tyr Val Ser Leu Thr Arg He Leu Lys Cys Ser Glu Gin Ser Leu 110 115 120 125

ATC GAA TTT TTT AAT AAG ATG AAG AAG TGG GAA GAC ATG GCA AAT CTA 492 He Glu Phe Phe Asn Lys Met Lys Lys Trp Glu Asp Met Ala Asn Leu 130 135 140

CCC CCA CAT TTC AGA GAA CGT ACT GAG AGA TTA GAA AGA AAC TTC ACT 540 Pro Pro His Phe Arg Glu Arg Thr Glu Arg Leu Glu Arg Asn Phe Thr 145 150 155

GTT TCT GCT GTA ATT TTT AAG AAA TAT GAA CCC ATT TTT CAG GAC ATC 588 Val Ser Ala Val He Phe Lys Lys Tyr Glu Pro He Phe Gin Asp He 160 165 170

TTT AAA TAC CCT CAA GAG GAG CAA CCT CGT CAG CAG CGA GGA AGG AAA 636 Phe Lys Tyr Pro Gin Glu Glu Gin Pro Arg Gin Gin Arg Gly Arg Lys 175 180 185

CAG CGG CGA CAG CCC TGT ACT GTG TCT GAA ATT TTC CAT TTT TGT TGG 684 Gin Arg Arg Gin Pro Cys Thr Val Ser Glu He Phe His Phe Cys Trp 190 195 200 205

GTG CTT TTT ATA TAT GCA AAA GGT AAT TTC CCC ATG ATT AGT GAT GAT 732 Val Leu Phe He Tyr Ala Lys Gly Asn Phe Pro Met He Ser Asp Asp 210 215 220

TTG GTC AAT TCT TAT CAC CTG CTG CTG TGT GCT TTG GAC TTA GTT TAT 780 Leu Val Asn Ser Tyr His Leu Leu Leu Cys Ala Leu Asp Leu Val Tyr 225 230 235

GGA AAT GCA CTT CAG TGT TCT AAT CGT AAA GAA CTT GTG AAC CCT AAT 826 Gly Asn Ala Leu Gin Cys Ser Asn Arg Lys Glu Leu Val Asn Pro Asn 240 245 250

TTT AAA GGC TTA TCT GAA GAT TTT CAT GCT AAA GAT TCT AAA CCT TCC 876 Phe Lys Gly Leu Ser Glu Asp Phe His Ala Lys Asp Ser Lys Pro Ser 255 260 265

TCT GAC CCC CCT TGT ATC ATT GAG AAA CTG TGT TCC TTA CAT GAT GGC 924 Ser Asp Pro Pro Cys He He Glu Lys Leu Cys Ser Leu His Asp Gly 270 275 280 285

CTA GTT TTG GAA GCA AAG GGG ATA AAG GAA CAT TTC TGG AAA CCC TAT 972 Leu Val Leu Glu Ala Lys Gly He Lys Glu His Phe Trp Lys Pro Tyr 290 295 300

ATT AGG AAA CTT TAT GAA AAA AAG CTC CTT AAG GGA AAA GAA GAA AAT 1020 He Arg Lys Leu Tyr Glu Lys Lys Leu Leu Lys Gly Lys Glu Glu Asn 305 310 315

CTC ACT GGG TTT CTA GAA CCT GGG AAC TTT GGA GAG AGT TTT AAA GCC 1068 Leu Thr Gly Phe Leu Glu Pro Gly Asn Phe Gly Glu Ser Phe Lys Ala 320 325 330

ATC AAT AAG GCC TAT GAG GAG TAT GTT TTA TCT GTT GGG AAT TTA GAT 1116 He Asn Lys Ala Tyr Glu Glu Tyr Val Leu Ser Val Gly Asn Leu Asp 335 340 345

GAG CGG ATA TTT CTT GGA GAG GAT GCT GAG GAG GAA ATT GGG ACT CTC 1164 Glu Arg He Phe Leu Gly Glu Asp Ala Glu Glu Glu He Gly Thr Leu 350 355 360 365

TCA AGG TGT CTG AAC GCT GGT TCA GGA ACA GAG ACT GCT GAA AGG GTG 1212 Ser Arg Cys Leu Asn Ala Gly Ser Gly Thr Glu Thr Ala Glu Arg Val 370 375 380

CAG ATG AAA AAC ATC TTA CAG CAG CAT TTT GAC AAG TCC AAA GCA CTT 1260 Gin Met Lys Asn He Leu Gin Gin His Phe Asp Lys Ser Lys Ala Leu 385 390 395

AGA ATC TCC ACA CCA CTA ACT GGT GTT AGG TAC ATT AAG GAG AAT AGC 1308 Arg He Ser Thr Pro Leu Thr Gly Val Arg Tyr He Lys Glu Asn Ser 400 405 410

CCT TGT GTG ACT CCA GTT TCT ACA GCT ACG CAT AGC TTG AGT CGT CTT 1356 Pro Cys Val Thr Pro Val Ser Thr Ala Thr His Ser Leu Ser Arg Leu

415 420 425

CAC ACC ATG CTG ACA GGC CTC AGG AAT GCA CCA AGT GAG AAA CTG GAA 1404 His Thr Met Leu Thr Gly Leu Arg Asn Ala Pro Ser Glu Lys Leu Glu 430 435 440 445

CAG ATT CTC AGG ACA TGT TCC AGA GAT CCA ACC CAG GCT ATT GCT AAC 1452 Gin He Leu Arg Thr Cys Ser Arg Asp Pro Thr Gin Ala He Ala Asn 450 455 460

AGA CTG AAA GAA ATG TTT GAA ATA TAT TCT CAG CAT TTC CAG CCA GAC 1500 Arg Leu Lys Glu Met Phe Glu He Tyr Ser Gin His Phe Gin Pro Asp 465 470 475

GAG GAT TTC AGT AAT TGT GCT AAA GAA ATT GCC AGC AAA CAT TTT CGT 1548 Glu Asp Phe Ser Asn Cys Ala Lys Glu He Ala Ser Lys His Phe Arg 480 485 490

TTT GCG GAG ATG CTT TAC TAT AAA GTA TTA GAA TCT GTT ATT GAG CAG 1596 Phe Ala Glu Met Leu Tyr Tyr Lys Val Leu Glu Ser Val He Glu Gin 495 500 505

GAA CAA AAA AGA CTA GGA GAC ATG GAT TTA TCT GGT ATT CTG GAA CAA 1644 Glu Gin Lys Arg Leu Gly Asp Met Asp Leu Ser Gly He Leu Glu Gin 510 ^* 515 520 525

GAT GCA TTC CAC AGA TCT CTC TTG GCC TGC TGC CTT GAG GTC GTC ACT 1692 Asp Ala Phe His Arg Ser Leu Leu Ala Cys Cys Leu Glu Val Val Thr 530 535 540

TTT TCT TAT AAG CCT CCT GGG AAT TTT CCA TTT ATT ACT GAA ATA TTT 1740 Phe Ser Tyr Lys Pro Pro Gly Asn Phe Pro Phe He Thr Glu He Phe 545 550 555

GAT GTG CCT CTT TAT CAT TTT TAT AAG GTG ATA GAA GTA TTC ATT AGA 1788 Asp Val Pro Leu Tyr His Phe Tyr Lys Val He Glu Val Phe He Arg 560 565 570

GCA GAA GAT GGC CTT TGT AGA GAG GTG GTA AAA CAC CTT AAT CAG ATT 1836 Ala Glu Asp Gly Leu Cys Arg Glu Val Val Lys His Leu Asn Gin He 575 580 585

GAA GAA CAG ATC TTA GAT CAT TTG GCA TGG AAA CCA GAG TCT CCA CTC 1884 Glu Glu Gin He Leu Asp His Leu Ala Trp Lys Pro Glu Ser Pro Leu 590 595 600 605

TGG GAA AAA ATT AGA GAC AAT GAA AAC AGA GTT CCT ACA TGT GAA GAG 1932 Trp Glu Lys He Arg Asp Asn Glu Asn Arg Val Pro Thr Cys Glu Glu 610 615 620

GTC ATG CCA CCT CAG AAC CTG GAA AGG GCA GAT GAA ATT TGC ATT GCT 1980 Val Met Pro Pro Gin Asn Leu Glu Arg Ala Asp Glu He Cys He Ala 625 630 635

GGC TCC CCT TTG ACT CCC AGA AGG GTG ACT GAA GTT CGT GCT GAT ACT 2028 Gly Ser Pro Leu Thr Pro Arg Arg Val Thr Glu Val Arg Ala Asp Thr 640 645 650

GGA GGA CTT GGA AGG AGC ATA ACA TCT CCA ACC ACA TTA TAC GAT AGG 2076 Gly Gly Leu Gly Arg Ser He Thr Ser Pro Thr Thr Leu Tyr Asp Arg 655 660 665

TAC AGC TCC CCA CCA GCC AGC ACT ACC AGA AGG CGG CTA TTT GTT GAG 2124 Tyr Ser Ser Pro Pro Ala Ser Thr Thr Arg Arg Arg Leu Phe Val Glu 670 675 680 685

AAT GAT AGC CCC TCT GAT GGA GGG ACG CCT GGG CGC ATG CCC CCA CAG 2172 Asn Asp Ser Pro Ser Asp Gly Gly Thr Pro Gly Arg Met Pro Pro Gin 690 695 700

CCC CTA GTC AAT GCT GTC CCT GTG CAG AAT GTA TCT GGG GAG ACT GTT 2220 Pro Leu Val Asn Ala Val Pro Val Gin Asn Val Ser Gly Glu Thr Val 705 710 715

TCT GTC ACA CCA GTT CCT GGA CAG ACT TTG GTC ACC ATG GCA ACC GCC 2268 Ser Val Thr Pro Val Pro Gly Gin Thr Leu Val Thr Met Ala Thr Ala 720 725 730

ACT GTC ACA GCC AAC AAT GGG CAA ACG GTA ACC ATT CCT GTG CAA GGT 2316 Thr Val Thr Ala Asn Asn Gly Gin Thr Val Thr He Pro Val Gin Gly 735 740 745

ATT GCC AAT GAA AAT GGA GGG ATA ACA TTC TTC CCT GTC CAA GTC AAT 2364 He Ala Asn Glu Asn Gly Gly He Thr Phe Phe Pro Val Gin Val Asn 750 755 760 765

GTT GGG GGG CAG GCA CAA GCT GTG ACA GGC TCC ATC CAG CCC CTC AGT 2412 Val Gly Gly Gin Ala Gin Ala Val Thr Gly Ser He Gin Pro Leu Ser 770 775 780

GCT CAG GCC CTG GCT GGA AGT CTG AGC TCT CAA CAG GTG ACA GGA ACA 2460 Ala Gin Ala Leu Ala Gly Ser Leu Ser Ser Gin Gin Val Thr Gly Thr 785 790 795

ACT TTG CAA GTC CCT GGT CAA GTG GCC ATT CAA CAG ATT TCC CCA GGT 2508 Thr Leu Gin Val Pro Gly Gin Val Ala He Gin Gin He Ser Pro Gly 800 805 810

GGC CAA CAG CAG AAG CAA GGC CAG TCT GTA ACC AGC AGT AGT AAT AGA 2556 Gly Gin Gin Gin Lys Gin Gly Gin Ser Val Thr Ser Ser Ser Asn Arg 815 820 825

CCC AGG AAG ACC AGC TCT TTA TCG CTT TTC TTT AGA AAG GTA TAC CAT 2604 Pro Arg Lys Thr Ser Ser Leu Ser Leu Phe Phe Arg Lys Val Tyr His 830 835 840 845

TTA GCA GCT GTC CGC CTT CGG GAT CTC TGT GCC AAA CTA GAT ATT TCA 2652 Leu Ala Ala Val Arg Leu Arg Asp Leu Cys Ala Lys Leu Asp He Ser 850 855 860

GAT GAA TTG AGG AAA AAA ATC TGG ACC TGC TTT GAA TTC TCC ATA ATT 2700 Asp Glu Leu Arg Lys Lys He Trp Thr Cys Phe Glu Phe Ser He He 865 870 875

CAG TGT CCT GAA CTT ATG ATG GAC AGA CAT CTG GAC CAG TTA TTA ATG 2748 Gin Cys Pro Glu Leu Met Met Asp Arg His Leu Asp Gin Leu Leu Met 880 885 890

TGT GCC ATT TAT GTG ATG GCA AAG GTC ACA AAA GAA GAT AAG TCC TTC 2796 Cys Ala He Tyr Val Met Ala Lys Val Thr Lys Glu Asp Lys Ser Phe 895 900 905

CAG AAC ATT ATG CGT TGT TAT AGG ACT CAG CCG CAG GCC CGG AGC CAG 2844 Gin Asn He Met Arg Cys Tyr Arg Thr Gin Pro Gin Ala Arg Ser Gin 910 915 920 925

GTG TAT AGA AGT GTT TTG ATA AAA GGG AAA AGA AAA AGA AGA AAT TCT 2892 Val Tyr Arg Ser Val Leu He Lys Gly Lys Arg Lys Arg Arg Asn Ser 930 935 940

GGC AGC AGT GAT AGC AGA AGC CAT CAG AAT TCT CCA ACA GAA CTA AAC 2940 Gly Ser Ser Asp Ser Arg Ser His Gin Asn Ser Pro Thr Glu Leu Asn 945 950 955

AAA GAT AGA ACC AGT AGA GAC TCC AGT CCA GTT ATG AGG TCA AGC AGC 2988 Lys Asp Arg Thr Ser Arg Asp Ser Ser Pro Val Met Arg Ser Ser Ser 960 965 970

ACC TTG CCA GTT CCA CAG CCC AGC AGT GCT CCT CCC ACA CCT ACT CGC 3036 Thr Leu Pro Val Pro Gin Pro Ser Ser Ala Pro Pro Thr Pro Thr Arg 975 980 985

CTC ACA GGT GCC AAC AGT GAC ATG GAA GAA GAG GAG AGG GGA GAC CTC 3084 Leu Thr Gly Ala Asn Ser Asp Met Glu Glu Glu Glu Arg Gly Asp Leu 990 995 1000 1005

ATT CAG TTC TAC AAC AAC ATC TAC ATC AAA CAG ATT AAG ACA TTT GCC 3132 He Gin Phe Tyr Asn Asn He Tyr He Lys Gin He Lys Thr Phe Ala 1010 1015 1020

ATG AAG TAC TCA CAG GCA AAT ATG GAT GCT CCT CCA CTC TCT CCC TAT 3180 Met Lys Tyr Ser Gin Ala Asn Met Asp Ala Pro Pro Leu Ser Pro Tyr 1025 1030 1035

CCA TTT GTA AGA ACA GGC TCC CCT CGC CGA ATA CAG TTG TCT CAA AAT 3228 Pro Phe Val Arg Thr Gly Ser Pro Arg Arg He Gin Leu Ser Gin Asn 1040 1045 1050

CAT CCT GTC TAC ATT TCC CCA CAT AAA AAT GAA ACA ATG CTT TCT CCT 3276 His Pro Val Tyr He Ser Pro His Lys Asn Glu Thr Met Leu Ser Pro 1055 1060 1065

CGA GAA AAG ATT TTC TAT TAC TTC AGC AAC AGT CCT TCA AAG AGA CTG 3324 Arg Glu Lys He Phe Tyr Tyr Phe Ser Asn Ser Pro Ser Lys Arg Leu 1070 1075 1080 1085

AGA GAA ATT AAT AGT ATG ATA CGC ACA GGA GAA ACT CCT ACT AAA AAG 3372 Arg Glu He Asn Ser Met He Arg Thr Gly Glu Thr Pro Thr Lys Lys 1090 1095 1100

AGA GGA ATT CTT TTG GAA GAT GGA AGT GAA TCA CCT GCA AAA AGA ATT 3420 Arg Gly He Leu Leu Glu Asp Gly Ser Glu Ser Pro Ala Lys Arg He 1105 1110 1115

TGC CCA GAA AAT CAT TCT GCC TTA TTA CGC CGT CTC CAA GAT GTA GCT 3468 Cys Pro Glu Asn His Ser Ala Leu Leu Arg Arg Leu Gin Asp Val Ala 1120 1125 1130

AAT GAC CGT GGT TCC CAC TGA GGTTAGTCTC TTGTATTAAA CTCTTCACAA 3519 Asn Asp Arg Gly Ser His *

1135 1140

AATCTGTTTA GCAGCAGCCT TTAATGCATC TAGATTATGG AGCTTTTTTC CTTAATCCAG 3579

CTGATGAGTT ACAGCCTGTT AGTAACATGA GGGGACATTT TGGTGAGAAA TGGGACTTAA 3639

CTCCTTCCAG TGTCCTTAGA ACATTTTAAT TCATCCCAAC TGTCTTTTTT TCCCTACCAC 3699

TCAGTGATTA CTGTCAAGGC TGCTTACAAT CCAAACTTGG GTTTTTGGCT CTGGCAAAGC 3759

TTTTAGAAAT ACTGCAAGAA ATGATGTGTA CCCAACGTGA GCATAGGAGG CTTCTGTTGA 3819

CGTCTCCAAC AGAAGAACTG TGTTTCAAGT TCAATCCTAC CTGTTTTGTG GTCAGCTGTA 3879

GTCCTCATAA AAAGCAAAAC AAAAATTAGG TATTTTGTCC TAAAACACCT GGTAGGAGTG 3939

TGTGATTTTT TGCATTCCTG ACAAAGGAGA GCACACCCAG GTTTGGAGGT CCTAGGTCAT 3999

TAGCCCTCGT CTCCCGTTCC CTTTGTGCAC ATCTTCCCTC TCCCCATTCG GTGTGGTGCA 4059

GTGTGAAAAG TCCTTGATTG TTCGGGTGTG CAATGTCTGA GTGAACCTGT ATAAGTGGAG 4119

GCACTTTAGG GCTGTAAAAT GCATGATTTT GTAACCCAGA TTTTGCTGTA TATTTGTGAT 4179

AGCACTTTCT ACAATGTGAA CTTTATTAAA TACAAAACTT CCAGGCTAAA CATCCAATAT 4239

TTTCTTTAAT GCTTTTATAT TTTTTTAAAA TGTTAAAACC CCTATAGCCA CCTTTTGGGA 4299

ATGTTTTAAA TTCTCCAGTT TTTTGTTATA TAGGGATCAA CCAGCTAAGA AAAGATTTTA 4359

AGTCAAGTTG AATTGAGGGG ATTAATATGA AAACTTATGA CCTCTTCCTT TAGGAGGGAG 4419

TTATCTAAAA GAAATGTCTA TTAAGGTGAT ATATTTAAAA ATATTTTTGG GTGTTCCTGG 4479

CAGTTTAAAA AAATTGGTTG GAGAATTTAG GTTTTTATTA GTACCATAGT ACCATTTATA 4539

CAAATTAGAA AATGTTATTT AACAGCTGAA TTATCTATAC ATATCTTTAT TAATCACTAT 4599

TGTTCCAGCA GTTTTCAAGT CAAATTAATA ATCTTATTAG GGAGAAAATT CAATTGTAAA 4659

TTGAATCAGT ATAAACAAAG TTACTAGGTA ACTTCATATT GCTGAGAGAA ATATGGAACT 4719

TACATTGTTC AATTAGAATA GTGTTCTCCC CAAATATTTA TAAAACTTCT CAAGATACTG 4779

CTACGTGTAA TTTTATATGA AGATAAGTGT ATTTTTCAAT AAAGCATTTA TAAATTAAAA 4839

AAAAAAAAAA AAAA 4853

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1140 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Pro Ser Gly Gly Asp Gin Ser Pro Pro Pro Pro Pro Pro Pro Pro

1 5 10 15

Ala Ala Ala Ala Ser Asp Glu Glu Glu Glu Asp Asp Gly Glu Ala Glu 20 25 30

Asp Ala Ala Pro Ser Ala Glu Ser Pro Thr Pro Gin He Gin Gin Arg 35 40 45

Phe Asp Glu Leu Cys Ser Arg Leu Asn Met Asp Glu Ala Ala Arg Pro 50 55 60

Glu Ala Trp Asp Ser Tyr Arg Ser Met Ser Glu Ser Tyr Thr Leu Glu 65 70 75 80

Gly Asn Asp Leu His Trp Leu Ala Cys Ala Leu Tyr Val Ala Cys Arg 85 90 95

Lys Ser Val Pro Thr Val Ser Lys Gly Thr Val Glu Gly Asn Tyr Val 100 105 110

Ser Leu Thr Arg He Leu Lys Cys Ser Glu Gin Ser Leu He Glu Phe 115 120 125

Phe Asn Lys Met Lys Lys Trp Glu Asp Met Ala Asn Leu Pro Pro His 130 135 140

Phe Arg Glu Arg Thr Glu Arg Leu Glu Arg Asn Phe Thr Val Ser Ala 145 150 155 160

Val He Phe Lys Lys Tyr Glu Pro He Phe Gin Asp He Phe Lys Tyr 165 170 175

Pro Gin Glu Glu Gin Pro Arg Gin Gin Arg Gly Arg Lys Gin Arg Arg 180 185 190

Gin Pro Cys Thr Val Ser Glu He Phe His Phe Cys Trp Val Leu Phe

195 200 205

He Tyr Ala Lys Gly Asn Phe Pro Met He Ser Asp Asp Leu Val Asn 210 215 220

Ser Tyr His Leu Leu Leu Cys Ala Leu Asp Leu Val Tyr Gly Asn Ala 225 230 235 240

Leu Gin Cys Ser Asn Arg Lys Glu Leu Val Asn Pro Asn Phe Lys Gly 245 250 255

Leu Ser Glu Asp Phe His Ala Lys Asp Ser Lys Pro Ser Ser Asp Pro 260 265 270

Pro Cys He He Glu Lys Leu Cys Ser Leu His Asp Gly Leu Val Leu 275 280 285

Glu Ala Lys Gly He Lys Glu His Phe Trp Lys Pro Tyr He Arg Lys 290 295 300

Leu Tyr Glu Lys Lys Leu Leu Lys Gly Lys Glu Glu Asn Leu Thr Gly 305 310 315 320

Phe Leu Glu Pro Gly Asn Phe Gly Glu Ser Phe Lys Ala He Asn Lys 325 330 335

Ala Tyr Glu Glu Tyr Val Leu Ser Val Gly Asn Leu Asp Glu Arg He 340 345 350

Phe Leu Gly Glu Asp Ala Glu Glu Glu He Gly Thr Leu Ser Arg Cys 355 360 365

Leu Asn Ala Gly Ser Gly Thr Glu Thr Ala Glu Arg Val Gin Met Lys 370 375 380

Asn He Leu Gin Gin His Phe Asp Lys Ser Lys Ala Leu Arg He Ser 385 390 395 400

Thr Pro Leu Thr Gly Val Arg Tyr He Lys Glu Asn Ser Pro Cys Val 405 410 415

Thr Pro Val Ser Thr Ala Thr His Ser Leu Ser Arg Leu His Thr Met 420 425 430

Leu Thr Gly Leu Arg Asn Ala Pro Ser Glu Lys Leu Glu Gin He Leu 435 440 445

Arg Thr Cys Ser Arg Asp Pro Thr Gin Ala He Ala Asn Arg Leu Lys 450 455 460

Glu Met Phe Glu He Tyr Ser Gin His Phe Gin Pro Asp Glu Asp Phe 465 470 475 480

Ser Asn Cys Ala Lys Glu He Ala Ser Lys His Phe Arg Phe Ala Glu 485 490 495

Met Leu Tyr Tyr Lys Val Leu Glu Ser Val He Glu Gin Glu Gin Lys 500 505 510

Arg Leu Gly Asp Met Asp Leu Ser Gly He Leu Glu Gin Asp Ala Phe 515 520 525

His Arg Ser Leu Leu Ala Cys Cys Leu Glu Val Val Thr Phe Ser Tyr

530 535 54 0

Lys Pro Pro Gly Asn Phe Pro Phe He Thr Glu He Phe Asp Val Pro 545 550 555 560

Leu Tyr His Phe Tvr Lys Val He Glu Val Phe He Arg Ala Glu Asp 565 570 575

Gly Leu Cys Arg Glu Val Val Lys His Leu Asn Gin He Glu Glu Gin 580 585 590

He Leu Asp His Leu Ala Trp Lys Pro Glu Ser Pro Leu Trp Glu Lys 595 600 605

He Arg Asp Asn Glu Asn Arg Val Pro Thr Cys Glu Glu Val Met Pro 610 615 620

Pro Gin Asn Leu Glu Arg Ala Asp Glu He Cys He Ala Gly Ser Pro 625 630 635 640

Leu Thr Pro Arg Arg Val Thr Glu Val Arg Ala Asp Thr Gly Gly Leu 645 650 655

Gly Arg Ser He Thr Ser Pro Thr Thr Leu Tyr Asp Arg Tyr Ser Ser 660 665 670

Pro Pro Ala Ser Thr Thr Arg Arg Arg Leu Phe Val Glu Asn Asp Ser 675 680 685

Pro Ser Asp Gly Gly Thr Pro Gly Arg Met Pro Pro Gin Pro Leu Val 690 695 700

Asn Ala Val Pro Val Gin Asn Val Ser Gly Glu Thr Val Ser Val Thr 705 710 715 720

Pro Val Pro Gly Gin Thr Leu Val Thr Met Ala Thr Ala Thr Val Thr 725 730 735

Ala Asn Asn Gly Gin Thr Val Thr He Pro Val Gin Gly He Ala Asn 740 745 750

Glu Asn Gly Gly He Thr Phe Phe Pro Val Gin Val Asn Val Gly Gly 755 760 765

Gin Ala Gin Ala Val Thr Gly Ser He Gin Pro Leu Ser Ala Gin Ala 770 775 780

Leu Ala Gly Ser Leu Ser Ser Gin Gin Val Thr Gly Thr Thr Leu Gin 785 790 795 800

Val Pro Gly Gin Val Ala He Gin Gin He Ser Pro Gly Gly Gin Gin 805 810 815

Gin Lys Gin Gly Gin Ser Val Thr Ser Ser Ser Asn Arg Pro Arg Lys 820 825 830

Thr Ser Ser Leu Ser Leu Phe Phe Arg Lys Val Tyr His Leu Ala Ala 835 840 845

Val Arg Leu Arg Asp Leu Cys Ala Lys Leu Asp He Ser Asp Glu Leu 850 855 860

Arg Lys Lys He Trp Thr Cys Phe Glu Phe Ser He He Gin Cys Pro

865 870 875 880

Glu Leu Met Met Asp Arg His Leu Asp Gin Leu Leu Met Cys Ala He 885 890 895

Tyr Val Met Ala Lys Val Thr Lys Glu Asp Lys Ser Phe Gin Asn He 900 905 910

Met Arg Cys Tyr Arg Thr Gin Pro Gin Ala Arg Ser Gin Val Tyr Arg 915 920 925

Ser Val Leu He Lys Gly Lys Arg Lys Arg Arg Asn Ser Gly Ser Ser 930 935 940

Asp Ser Arg Ser His Gin Asn Ser Pro Thr Glu Leu Asn Lys Asp Arg 945 950 955 960

Thr Ser Arg Asp Ser Ser Pro Val Met Arg Ser Ser Ser Thr Leu Pro 965 970 975

Val Pro Gin Pro Ser Ser Ala Pro Pro Thr Pro Thr Arg Leu Thr Gly 980 985 990

Ala Asn Ser Asp Met Glu Glu Glu Glu Arg Gly Asp Leu He Gin Phe 995 1000 1005

Tyr Asn Asn He Tyr He Lys Gin He Lys Thr Phe Ala Met Lys Tyr 1010 1015 1020

Ser Gin Ala Asn Met Asp Ala Pro Pro Leu Ser Pro Tyr Pro Phe Val 1025 1030 1035 1040

Arg Thr Gly Ser Pro Arg Arg He Gin Leu Ser Gin Asn His Pro Val 1045 1050 1055

Tyr He Ser Pro His Lys Asn Glu Thr Met Leu Ser Pro Arg Glu Lys 1060 1065 1070

He Phe Tyr Tyr Phe Ser Asn Ser Pro Ser Lys Arg Leu Arg Glu He 1075 1080 1085

Asn Ser Met He Arg Thr Gly Glu Thr Pro Thr Lys Lys Arg Gly He 1090 1095 1100

Leu Leu Glu Asp Gly Ser Glu Ser Pro Ala Lys Arg He Cys Pro Glu 1105 1110 1115 1120

Asn His Ser Ala Leu Leu Arg Arg Leu Gin Asp Val Ala Asn Asp Arg 1125 1130 1135

Gly Ser His *

1140

(2) INFORMATION FOR SEQ ID NO: 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 amino acids

(C) STRANDEDNESS:

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(v) FRAGMENT TYPE. C- erminal

(xi) SEQUENCE DESCRIPTION SEQ ID NO.3 :

Glu Asn H s Ser Ala Leu Leu Arg Arg Leu Gin Asp Val Ala Asn Asp 1 5 10 15

Arg Gly Ser His Cys 20

(2) INFORMATION FOR SEQ ID NO: :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 561 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 312..551

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

CAGCCCTGTT GAATGTTCTC ACGGTGGGGA GGTACGTGTT TAAAATACGG GGAAGGTGCT 60

TTTATTTCAC CCCTGGTGAA ACTAGGGGAG CTAATTTTTT TAAACATGAT TTTTGTCCCC 120

CTTGAACCGC CGGCCTGGAC TACGTTTCCC AGCAGCCCGT GCTCAAGACT ACGGGTGCCT 180

GCAGGCGGTC AGCGTCGTTT GCGACGGCGC AGACGCGGTG CGGGCGGCGG ACGGGCGGGC 240

GCTTCGCCGT TTGAATTGCT GCGGGCCCGG GCCCTCACCT CACCTGAGGT CCGGCCGCCC 300

AGGGGTGCGC T ATG CCG TCG GGA GGT GAC CAG TCG CCA CCG CCC CCG CCT 350 Met Pro Ser Gly Gly Asp Gin Ser Pro Pro Pro Pro Pro 1 5 10

CCC CCT CCG GCG GCG GCA GCC TCG GAT GAG GAG GAG GAG GAC GAC GGC 398 Pro Pro Pro Ala Ala Ala Ala Ser Asp Glu Glu Glu Glu Asp Asp Gly 15 20 25

GAG GCG GAA GAC GCC GCG CCG TCT GCC GAG TCG CCC ACC CCT CAG ATC 446 Glu Ala Glu Asp Ala Ala Pro Ser Ala Glu Ser Pro Thr Pro Gin He 30 35 40 45

CAG CAG CGG TTC GAC GAG CTG TGC AGC CGC CTC AAC ATG GAC GAG GCG 494 Gin Gin Arg Phe Asp Glu Leu Cys Ser Arg Leu Asn Met Asp Glu Ala 50 55 60

GCG CGG CCC GAG GCC TGG GAC AGC TAC CGC AGC ATG AGC GAA AGC TAC 542 Ala Arg Pro Glu Ala Trp Asp Ser Tyr Arg Ser Met Ser Glu Ser Tyr 65 70 75

ACG CTG GAG GTGCGCTCGC 561

Thr Leu Glu

(2) INFORMATION FOR SEQ ID NO: 5 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 80 amino acids

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

(ii) MOLECULE TYPE: protein

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

Met Pro Ser Gly Gly Asp Gin Ser Pro Pro Pro Pro Pro Pro Pro Pro

1 5 10 15

Ala Ala Ala Ala Ser Asp Glu Glu Glu Glu Asp Asp Gly Glu Ala Glu 20 25 30

Asp Ala Ala Pro Ser Ala Glu Ser Pro Thr Pro Gin He Gin Gin Arg 35 40 45

Phe Asp Glu Leu Cys Ser Arg Leu Asn Met Asp Glu Ala Ala Arg Pro 50 55 60

Glu Ala Trp Asp Ser Tyr Arg Ser Met Ser Glu Ser Tyr Thr Leu Glu 65 70 75 80

(2) INFORMATION FOR SEQ ID NO:6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACGCTGGAGG TGCGCTCGC 19

(2) INFORMATION FOR SEQ ID NO: 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 7 ^• TCTTTTACAG GGAAATGAT 19

(2) INFORMATION FOR SEQ ID NO: 8 :

U) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8 : AGAGCAGAGG TAACTATGT 19

(2) INFORMATION FOR SEQ ID NO: 9 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9 : TTAATACCAG CTTAATCGA 19

(2) INFORMATION FOR SEQ ID NO: 10:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GAAACAGCGG TAGGTTTTC 19

(2) INFORMATION FOR SEQ ID NO: 11:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 11: TCCCCCAAAG GCGACAGCC ₁ 9

(2) INFORMATION FOR SEQ ID NO: 12: li) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12 : ATGCAAAAGG TAAGAAAAT 19

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO: 13: AATCCTGCAG GTAATTTCC 19

(2) INFORMATION FOR SEQ ID NO: 14:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 14: ATTTTAAAGG TAGGTTTGT 19

(2) INFORMATION FOR SEQ ID NO: 15:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

'11) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: ACACCATAGG CTTATCTG 18

(2) INFORMATION FOR SEQ ID NO: 16:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GAAAAAAAGG TTTGTAAGT 19

(2) INFORMATION FOR SEQ ID NO: 17:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: TTCATCATAG CTCCTTAAG 19

(2) INFORMATION FOR SEQ ID NO: 18:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:18: AGAGAGTTTG TGAGTACTT 19

(2) INFORMATION FOR SEQ ID NO: 19: ) SEQUENCE CHARACTERISTICS.

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear ii) MOLECULE TYPE. DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: TTCCTATAGT AAAGCCAT (2) INFORMATION FOR SEQ ID NO: 20:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: TTTGACAAGG TGAGTTTAG 19

(2) INFORMATION FOR SEQ ID NO: 21:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: TTTTCTTTAG TCCAAAGCA 19

(2) INFORMATION FOR SEQ ID NO:22:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:

GATTCTCAGG TTAGTTTGA 19

(2) INFORMATION FOR SEQ ID NO:23

(1/ SEQUENCE CHARACTERISTICS.

(A) LENGTH- 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE. DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: CCTTTTTTAG GACATGTTC 19

(2) INFORMATION FOR SEQ ID NO:24:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: DNA (genomic)

(x ) SEQUENCE DESCRIPTION: SEQ ID NO:24: GTGCTAAAGG TAATTGTGC 19

(2) INFORMATION FOR SEQ ID NO: 25:

U) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: ATTTCTACAG AAATTGCCA 19

(2) INFORMATION FOR SEQ ID NO:26:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: GATTTATCTG TGAGTAAAA 19

(2) INFORMATION FOR SEQ ID NO:27:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27: ATTTTATAGG GTATTCTG 18

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.-28: TTTTATAAGG TATTTCCCA 19

(2) INFORMATION FOR SEQ ID NO: 9.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 29: TTTATTTCAG GTGATAGAA 19

(2) INFORMATION FOR SEQ ID NO: 30:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

«D) TOPOLOGY linear (11/ MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION SEQ ID NO 30: TGTGAAGAGG TGAAAATCA 19

(2) INFORMATION FOR SEQ ID NO:31:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS- double

(D) TOPOLOGY- linear

(n) MOLECULE TYPE. DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TCTTCATAGG TCATGCCA 18

(2) INFORMATION FOR SEQ ID NO:32:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS double

(D) TOPOLOGY linear

( i) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: TTGGAAGGAG TAAGTTTAA 19

(2) INFORMATION FOR SEQ ID NO: 3:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE- DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO.33. TTGACCCCTA GGCATAACAT 20

(2) INFORMATION FOR SEQ ID NO-34

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

[ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34: CTGTGCAAGG TAAGGAAGG 19

(2) INFORMATION FOR SEQ ID NO: 35 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: CTGTCACTAG GTATTGCCA 19

(2) INFORMATION FOR SEQ ID NO: 36:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: TTTAGAAAGG TAATTTTTC 19

(2) INFORMATION FOR SEQ ID NO: 37:

(l) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

TATCTCCTAG GTATACCAT 19

(2) INFORMATION FOR SEQ ID NO:38:

(u SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:38: ATGGCAAAGG TGAGTACCA 19

(2) INFORMATION FOR SEQ ID NO: 39:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 39: GTTTGCCAGG TCACAAAA 18

(2) INFORMATION FOR SEQ ID NO:40:

(1) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:40: CGGAGCCAGG TAACTACAT 19

(2) INFORMATION FOR SEQ ID NO:41:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:41 : TTCTCTAAAG GTGTATAGA ₁ 9

(2) INFORMATION FOR SEQ ID NO:42:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 42. AAGATAGAAG TGGGATCTT 19

(2) INFORMATION FOR SEQ ID NO:43:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: CTGGCTGCAG CCAGTAGAG 19

(2) INFORMATION FOR SEQ ID NO:44:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 4: CAGGCAAATG TAAGTATGA 19

(2) INFORMATION FOR SEQ ID NO:45:

(l) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- double

(D) TOPOLOGY linear (11! MOLECULE TYPE DNA (genomic)

(xi) SEQUENCE DESCRIPTION- SEQ ID N0-45: TTTTTAAACA GATGGGATGC 20

(2) INFORMATION FOR SEQ ID NO:46:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE. DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46: CCTTCAAAGG TGAGCCTAA 19

(2) INFORMATION FOR SEQ ID NO:47:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 19 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: CCCACCATAG AGACTGAGA 19

(2) INFORMATION FOR SEQ ID NO:48:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3865 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:48: GTAGGTTTTC TTGTTGGTTC ATCAGGAATA CACATTAGTC TGTGCTGCAG TGTTGATATT 60 CTGCTAGGTT TTTTTTTTCT GGTTTTAAAA AAGAAATAAG ATTTAAAAAA TCTTTTTCCT 120

CAGTCGTTTT CTTTTAATGA TGCTTCCGGG GCTTCACATT GTGGGTTAGC CATGAAGAGT 180

GGCTTTCACA TATTGCTAAA TGTATACAGG TCTGTGTTTC TATAAACTAC ATGTGTCTTA 240

TTTCATTTTA TTATTATTTA CCTCCTCAGT GATCCTTGTT CTGAAACCTT CCTTTTTCAT 300

TTAAGCAACA AAAAATGCAG ACTGTACAAG TCAGACTTAG GGATTTTCAC CCTTTCGCCG 360

CCTTGGAGAG TTCTGTATCT GTATCTGGAT ATATATATTT TTTATTGCGC AGGGGCCATG 420

CTAATCAATG TATTGTTCCA ATTTTAGTAT ATGTGCTGCC GAAGGGAGCA CTGCCCTAGA 480

TATAGATCAC TATATTAACC ACTATATTTT CTACTAGTGA TTATATAGAC TATTTTATGT 540

CAAACTGAGT AATAAATAAT CCCCTTGAAA TGACTTCTCT ATGTATTTTG ATGTTTATAA 600

TGAATTCAGA ATAGAGAGAC TGGATTGGGA AAAGACAGGA GAACTGAAAC TATTATGAAT 660

TTGTGCTTTC TGATCACTTC TGCAAAGTCT ATAAGCATGC TCTGACTCAG TGTTTTCTAC 720

CTTTCCTGAT AGATAAAGGC AGTTATGGAA TACACATTTT CCTTCTTTAT CATTGAAAGT 780

TTTTTCATAA AGTAGAAATG AAAATTCTAA CAATTAAAAA AATGTTGACA AGAAAAGTAA 840

AGGGAAAGGA GTTAAAATTA TTTGGCTAGA ATAAATAATG TTTGCTTCTC TTTAAATATA 900

AAAGTTTTCC CAGACTGTGA AGGATGTTTA CATTAAGTGT AACCTTTTAA AAATAAAATG 960

GAATGACAAA CCAGGAGGAA AAAAAATTTA AAAAAACTAG AACTATTTAC ATTTTAATAT 1020

AGATGGCACC ACTGATACAG AAGCATCTGG TCTAGCTCAC TTACAGTTTT GGGGAATTGA 1080

CTATTTAAAA TGAAGCATTC TGAGCCAGGC GGGTTGGCTC ACGCCTGTAA TCCCAGCACT 1140

TTTATGAGGC TGAGGCAGGC GAATCACCTG AGTTCAGGAG TTCAATACCA GCCTGGCCAA 1200

CGTGGCAAAA CCCCGTCTCT ACTAAAAATA CAAAAATTAG CTGTGCATGG TGGTGCATGC 1260

CTATAATCCC AGCTACTCGG GAGGCTGAGT CAGTTGAATC CCTTGAACCG AGAAGCAGAG 1320

GTTGTGAGCC AAGATCGTAC CATTGCATTC GAGCCTGGGC GACAGAATGA AACTCCATCT 1380

CATAAATAAA TAAATAAACT AATAAAATGA CATATTCTCC TAGCACTTTG GGAGGCCGAG 1440

GCAGGTGGAT TGCTGGAGGT CAGGAGTTCA AGACTAGCTT GGCCAATGTG CCAAAACCCC 1500

ATTTCCATTA AAAATACAAA AATTAGGCAG GTATGGTGGT GTGTGCCTGT TGTCCCAGTT 1560

ACTTGAGGGC TGAGGCAGGT GAATCACTTG AACCCAGGAG TCGGAGGTTT CAGTGAGCTG 1620

CGATCGCGCC AATGCACTCC AGCTTAGGTG ACAGAGTGAG ACTTCGTCTC CAAATAAATA 1680

AATAAAAAAT GAAGTATTCT AAAGGTTTGA ATAGAAGCTT TGTACTGAGT CTGAGTGAGG 1740

CCAATGTGAT CATTTATGGG AAGATATCTT CTTTGTTTGG AGTATCTGGA AAATAATTTC 1800

AGATTGCACT TGTTTTGCTA TTTCTTAGGA TATATATACT ACCTAATTCT AATTAAGAGA 1860

ATTTTAAAAG GCCATGTGCA GTGGCTCACA CCTGATCCCC AGCACTTTGG GAGGCTGAAG 1920

TGGACAGATC ACTTGAGCCC AGGAGTTTGA GACCAGCCTG GACAGTATGG CGAAACTTCA 1980

TCTCCACAAA AAATACAAAA ATTAGCTTGG AGTGGTGGCG CACACCTGTG GTCCCAGCTA 2040

CTGGGGAGGC TGGAGGTGGG GGGATCACTT GAGCCTGGGA GGTTGAGGCT GCAGTGAGCT 2100

GTGCTCATAC CACTGTACTC CAGTTTGGGT GACAGAGCAA GACCTTGTTT CAAAAAAAAA 2160

AAAAAAAAGT AAATCACTTT ATTAGAGATT TTACATTTTA ATCACTTTGT ATACTTTCTG 2220

TTAGCTCTTT CTGTTAACTA TAGTCATAAT GTATAGCACT TACTGAGCAT TTACTTTGGG 2280

GCAGGGACTC TTAAGACTTC AATATGTATT ACTTCAGTTA ATCCCTCTGA CAACCTTGTG 2340

ATACTCATAC TATTGTTAGA TAGAGAAAAT TAACCGCAGA GAGGTTAAGT AATTTGGCCA 2400

GGGTCGCACA ACCAAGCGTG GAGTTCTTAT TGAAACTGAC TGCGGGAACC CATGTGCTTT 2460

ACTGTGACTA TATACTGCAT CTCTCACACA CTATCTGAAA ATGTGTCACT ATTTGTTTAG 2520

CACTTATCCA CAGGAAATAC TGTCAGGTAT TATGTAGGAC ACAAGCATTT TTTAAAACAC 2580

CAAACCCCAC AGTTTTTGTT TTCTGAGAGC TTACAGTACA GTCAGCGAGA TGAGGCAGGT 2640

ATGAAGATTC CAGTGCATGC AATGCAGTGT GTTATAAAAG TCCCATGACT ACCAGAGGGA 2700

ATACAGATGT AAAACTTAGG AGGAAAAGAA ATCACTCTGG ATGAGCCAGT CAGGTAAGTT 2760

TACATGGAAT AAGTAGAAAT GGGTCTTGAA AGATGGGTAC GAGTTTGATA GGTGAATTTG 2820

AAGATACAGA TAGCACCTTC TGTGTAGAGG AAACAAGAAA AGACAAAAGC AGTAAAGCAA 2880

GAAGAAATGT GGGAGGTTAG TCAAGTTTTT TTTTCTAGAA TTCTCAAGTT GTAGAGCCAG 2940

AATTAAGAGT AGCTTAAGTG TTAAGCTAAA AAAAATTGAA TTTTATTTTG GTAGGCAACT 3000

AAAACTAGAA ATAGTTTATC ATGCGCCTAT GGTAGAGAGG ATACTTTTAA AAGCAGAACA 3060

CTGACATTTA ATCCTTGCCA TGGAGTGGTG AACTAAGTAC AGTATTGTAC CCAAGTAGAG 3120

TAATCTTTTG ACAGATGAAA TGACTAAGGC CCAGGTGAGC AAGTGTACCC TAGCTAATGG 3180

CAGTGCTGGA ACTAAATCTA ATCTAATCTT CTCCACGGAA TTTCGTTCTT CTGGGCACCT 3240

TGTTAGAATA AGGCTGTTGG GAGGTGGAGA CCACAGATTT CTTGTCTAAA AGTTGTCAGA 3300

GGTTTTGGTA GAAAAGCCAA GCTTAAAGCA GGTCTGAAAC TTGGCAGACT ACTTGGCAAT 3360

ATACAACAGG TACTCTTAAT GGATGGAAGT ATAAGGAATT ATAGGAAGCT CATAATTTAC 3420

ATTAAAAAGG CCTTTTGTGA TTTGATATAG TCTGGAATAT CTTTAAGGAG GGAGGGAGGG 3480

ATACAGGTCA TTAGCTATGA TAAAGGAGAA AAAAATAAGG ACATATCTGA CTGCATATAG 3540

TGGTCCTGAA TCAGCATAGC ATTGCTGTGT CATCGAAAGA ACTATTTTTA TTCATTTTAT 3600

TTTCCACCTC ACCTATCTTG CCTTCACAAA ACTTTAAAAG ATTCTTTAAG AATTTTCTTT 3660

TCTTTGAGAT GGGCTCTTTC CCTGGTACCC AGCTATTTCC TACCAATATT TTGTTAAGGC 3720

AGAACGTCCA CGTTTTCCAT GTGAAGCTGA ATCTGTTGTC TCTCCCTTTA ACTGTGGGTT 3780

TTATTTTACA CCTGATTTAT AATCATTTGG GATTTTTTTT TCTGATCTTC TGGTGTCTCG 3840

TGACTGGGGT TTTCTTCCCC CAAAG 3865 (2) INFORMATION FOR SEQ ID NO:49 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4576 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:

GTAAGAAAAT AGTAATATTT ATTTAGATTT AATATGTCTA TTTACATTAC CAGGTATTAA 60

TCTCGTCAAC TCCTAATATG TATCAGGAAA AGATTTCCAC TGAAAATTTT CTCAAGGGTT 120

TTAATCCTAG ATTCTTTTTT AAGTATTGCC TTTCCATCAA AGGATCTATT GGATTTCTTT 180

ACAATATCCA AATCTCTCTT ATTAAATGGA AAGTCCATTA ACTTCGTTGT ATACAACATC 240

TTTCCTACCC AAAGCTACTC TCCTCAAATT ATGAGCTGAA AACACATAAT CCTGTATATG 300

CTTGTATTGC GAACTCTATC TTCCATGAGA TGTATCTTAT TTAGTCTGAG CGCAATTACT 360

GATCAACCTC AGAGCTGTTC AGATTTTTTT GTGTGTCTTG TTCACATAAG TATACTTAGT 420

CAAATGCTTT TATATACTAT TTATTTTCTT TCCCTTTTTT CTTGTCTCAT TTAACCTACC 480

CAAGGTCTGC ATTCAGTGAA ATACATGTCT CTATTATTTT TTGTCCTTTT TGTATTTATT 540

TATTTATTTA TTTATTTGAG ATGGAATCTC ATTCTGTCTC CCAGGGCTAG ATTGTAGTGG 600

CACAATCTCG GCTCACTGCA GGCTACACCT CCCAGGTTCA AGTAATTCTC CTGCCTCAGC 660

CTCCCGAATA GCCGTGATTA CAGGCGCCCA CCACCATGCC CAGCTAATTT TTGTGTTTTC 720

AGTAGAGATG GGGTTTCACC ATGTTGGCCA GGCTGGTCTC AAACTCCTGA CCTCAGGTGA 780

TCTGCCTGCC CTGGCCTCCC ACAGTGCTGG GATTATAGGC ACGAGCCACT GCGTCCAGCA 840

CCTTAGTATC TTTCTATGTA GAACGAATGC TCCCAGGTAG ATGGGAAAGT GCAGATATAT 900

TATTATGTAG TCAGCTCCTG TATACCATGT GGCTTGGCCT TCGTCACTAA GATGGCTCAC 960

TCTGAATGCA AAGTTATCAC AGAGTCTTAG GTGCTGGAAG GAGTTGCACA GGTATCACTG 1020

AGACTCTCAT TATTAGATTA ACTAGCTTAA CTTACTTTAT TTTTTTTTGA GATGGAGTCT 1080

CACTCTGTTG CCCAGGCTGG AGTGCAGTGG TGCGATCTCG GCCCACTGCA ACCTCTGCTG 1140

CCCGGGTTCA AGCGATCTCC TGCCTCAGCC TCCCGAGTAG CTGGGATTAC AGGTGCCTGC 1200

CACTGTGCCC GGCTAATTTT TTGTCGTTTT AGTAGACACG GAGTTTCACC ATCTTGGCCA 1260

GGCTGGCCTT GAACTCCTGA CCTCGTGATC CACCTGCGTC AGCCTCCCAA AGTGCTGGGC 1320

TTACAGGCGT GAGCCATCGC ACCCAGCCTA GCTTAACTCA GTTACTTTAT TTTCTATTTT 1380

TATTTTTATT TTTGACACAG GATCTTGCTC TGTTGCCCAG GCTGGAGTGC AGTGGTATGA 1440

TCTCTGCTCA CTGCAACCTC CGCCTCTTGT GTTCAAGTTG ATTCTTGTGG CTCAGCCTCT 1500

TGAGTAGCTG GGATTGCAGG CATGCACCAT TATACCTGGC TAATTTTTGT ATTTTTAGTA 1560

GTGTTGGGGT TTTGCCATGT TGGCCAGGGT GGTCTCGAAC TCCTGACCTC AAGTGATCTG 1620

CCACCTCGGC CTCCCAAAGT GTTGGGATTA CAGGTGTTGA GCCACCATGC TCAATCAGCT 1680

TAGTTACTTT AAAGATTAGG CAGCTGAGCC CAGAAACTAG CTGCTGGGAA CAAAGCTAAG 1740

ATTGAACTCA GATCTCCTGG TTCCTGGTTC TTAGTTTCAT ACTGGCTGTG AAGGCCTCTG 1800

GGAAGAATGT GTTACATTGT TGGTCTCCAG GTTTGATTTG TCCTGGTCCC TCTCTGGCTA 1860

ATTAGGGTGA GAGCCGCCAT CCTTCCTTCC CTGAGCTGCA TGCTTGATTC AAGAGAAAAA 1920

TCTTTCTTTT GTCATACATG ACACTGGCAT GTTTCTTTAA TGATGATAAA GGCGACATGA 1980

TCAGTGGCAT GAAATAAAGG TTTTGGAGTA TATAAACCAT TTTTACAGCG GCTACAAATT 2040

TTAGAATGTG TGACTGCTAT TATGTATGAT GGTAATCTTT TCATATGATT GTATTGGGCA 2100

AGTATGTCTC ATTTCTAGGG TTTTTATCTG TTTTGTTTGT CTTTTATGGC ATATGTGTAC 2160

TTAGAAGTAA ATATAGTTGG TACTATATAT AATATGTACA ATACAATAAA AAATAATTTC 2220

ATTGTCCTTA TTTTGTTCTC ACTGGACCTG TTGGGGTGGT TTTTTCTCTG TAATTAACTC 2280

AGTGTTTGAC TTTTATCTCA TTAATTCAGT TTATAATAAT TCCACCTTAA GAACCTTTGT 2340

GGATTGGGCA TGTTGGCGTA TGCCTGGAAC CTAGCTACTT GGGAAGTTGA AGTGGGAAGC 2400

GGAGGCTGCA GTGAGCTGAG ATTGCACCTC CAGTTTGGGC GAATTTGAGA CCGTGTTTCG 2460

AAAAAAAAAA AAAAAAAAAA AGAAACTTGG TCCTTTCACA GTCCACCACT GTGATCTTTT 2520

ATAATACACG ATGATCTTTT TCTAATAGTC ATTTAATTGC TTTAATTCAG TTCTCATTTA 2580

TTTGGGGGAA AGGTGTACTC TTTTATAGCC ACCTTTCTAA TGACAAATAA GCCAACTCTG 2640

GAGATGAAAC ATTTCTATTT ACTTGTTATC TTTGTTGATT AAAAGATAAA ATACCTCACA 2700

AAGTCAGATT TATTTGTAAG GTCAGGATTT GAAATAGAAA ATACGTCATG TTGAGAGAGT 2760

CCTAGAATTT AATTTAAATT AGATTCTGAT CTTTAGGGGC ATTTCAGCTT TTTATTAGAT 2820

GTTACGAGTA CTGTTTTTTT TTTTTTTTTT TTTGCCTTCT ATGGCAAGTG CACACCAGTA 2880

ACAAGTTTAG GCTTGTTGGT GTGATGGGCT TTGTAGCTTG AAATCAGTAG GTGCTACTTA 2940

CTTACTTTTT TACACATGAG GAACCAAGTA TATTTTAATA TTAAACCTCT TTATAGGAGA 3000

GCCAAGCAAG TTGGTTTGGC TGTATCAATG CGCAGTTTGA TGTGGTGATT ATCGTTTGCC 3060

TGCTTTGGCA GAGGAGGATT TTTTTTTCTC TTTAGTTCAT TTAAGTTGAT TTGTTGAATG 3120

TTTCCATCTA AACAAAAAAG AATTGCTTTG TATACGCTGA GGTAAGTGGT AACTTTCTTT 3180

GGAGGAACAG AGAGAAAGGG AAACCTGAAA CAAAACTGCA GGTGTGTGTG TGTGTGTACA 3240

TGTACACTTG GGTAGGCGTT AAGTGTGAAA TGCTGAGGTT TGGAAATAAT TCTTCATATG 3300

TATGTTAGCT TATTTAAATT GAATTTATCT GATGATACAA GAATGTAAAA TCACCATGAA 3360

GCATACATGT GCAGTGTTTA ACTAAAAAAG GATGGGCTTG AAGTTATAAA ATAACTAGAA 3420

ATAATTCTTA ATTTCTAGAA AATTAAGATA ATAATAAAAT GGTTTAACTA CACGTAAAAA 3480

TGTGTTCAGT GTTAGAGTTC AACCAGCACT GCAGAAAATT ACATGTTTCT GTCAGTTTAG 3540

GTTTTTGATT TCTTATTTCC CTGTTACCAA GCATCAGCAA TTATTCTTGG GATTATTAGC 3600

CCTGGAATTG AAAGATATTT AATGGTACTC CTGTTGCATT AATTTGTCTG AGTTTATGTA 3660

GAAAAGTATT AAAAATGTTA CTGTTGGAGT CTGATAAAAA GTTCTGGTCT TTTAAAAATA 3720

TGTGTATGAG AAATAGCATG AACTCAGGAG GCAGAGCTTG CAGTGAGCTG AGATCGTGCC 3780

ACTGCACTCC AGCCTGGGCG ACAGTGAGAC TCCATCTCAA AAAAAAAAAA TGTATATGAG 3840

AATAATTAAG TGAATTATTT TTTCGGCTGT CTCCTAAGTA TTTCTAATAA TTTTCATGAC 3900

AGAAAAATGT TTTCATGCAA AACAATTTCC TTACAGTTTG AGATAATTTA TAAATGTTTT 3960

GTGTTCAGAA TTTTCAAAGA AAAGACCAAT GATAAAGTTT TATTCAGCTA CTAGGTATTT 4020

AATAAACACT TAATGATGAA TGGCATTTTT AGTAAAGTTA TAGTTTTCAC TAAGCTGTTA 4080

GACATTTATT AATTTATTAA AGGCCAGGCA TGGTGGTTTA CACCTGTAAT CCTAGCACTT 4140

TGGGAGGCCA AGGCAGAAGG ATCACTTGAG TCCAGGAGTT CAAGACCAGC CTGGGCAACA 4200

TAGCAAGACT CCATCTCTAA AAAAAGTTTT TAAATTAGCC ATGTGTGGTG GCGTGTACCT 4260

GTAATTTGCA GCTGCCCAGG AGGCTGAGAC AGGAAGCCCT TGAGCCCAAG AGGTTGAGGG 4320

TGCAGTGAGC CATGATCATA CCACTGTACT CCAGCCTGGG TGACCCACCA AGACTCTGTC 4380

TCTTGAAATA AATAAATAAA GAAATTTATT AAGATATTAG AGTAATATGT CGGATGTAAA 4440

TTTGCCAAAA CACTTATTGT AATGAGTCAA TTTTGTACAA TTGTTTTGTA ATGTCATAAT 4500

AAGAAAGGAA GAAATTTTTT AAAAATGTTA CAAAGTCAAT GCTAATTTAA CTCTGTAACT 4560

GCTTATAATC CTGCAG 4576 (2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1618 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

GTAGGTTTGT AAATCAAAGA TTTTTGGGCA ATCTGCGTTT CTGTGTTATG TTTACCCTTG 60

GAGTTGTACA GGTTTCCTAG CATCAGTATT TTGAAGAGCT CCTGTCATTA CGGCTATCCA 120

GGGTACTTAT AACTAAGAGT CAAGCTGCCT GTAAAAATAT TTTTGGATAA ACAGTTGCAG 180

ATACCACAAA GTTTAAAGTC TTAAATGACA ACTTCAAGAA GTTTCTGAAA TATATACTCA 240

ACAAGGAGAA GGCATTTAGA AACTCAGAGT TGCGAAGATG ACATTAAAGC CGATAATGTT 300

TCCTACATTG GCAAACTTTG TGCCTGACAC ATTGTAGGAG ATCAAAAAGA ATTTGTTGAA 360

AGAATCTTAC TTCAAATTTT GGTACAGAAG AATAGTTATG GTTCTAAAAT AAAGAAAATG 420

AACTTTCATC TTTTAAACTA ACAGATATAT GGAAATGATG ATTTTGGCAT TGCATTTAAT 480

AGAACTTAGG TATATAATTT CTATGAATGA TAAACAGTTA CAAGCCCAAA TTATGATTTA 540

CAAAGCAAAT ATTAAAAAGT ATGTATAGAG TTAAAATAAA TATTGCTGCT GCTATTTGAG 600

TAATATTGTA ATAGGATTCT GGGTGATTCT CAGTTTGGAG GTAATTTCAG TTAAAATTTC 660

AGCTTGTCTA TCAAGGTAGA TTTTTAAAAT TAGTGGAGTT CAGTTGCTCC TGGTATGGTA 720

AATTTAATGT TCCTCATCTT CTTTTCTGTT CTTTCTCTCA TTTCTATCAT AACTCCCTTG 780

TATATTCCCA AAAAGCTGCT TCCTTTCACT TTTATCTTTT TTTGGTTTTA AATTAAAAAG 840

AATTTTTTTT TTGGAGACAG GGTCTCACTC TGTCACCCAG GTTGGGATGC AGTGGTGAAA 900

TCACAATTCA CTGCAGCCTC AATCTCCTGG GCTCAGATGA TCCTCTCATC TCAGCCTCCC 960

AGGTAGCTGG GACTACAGAC ATACACCACC ACACCCAGTT AATTTTTTTG TATTTTTCAG 1020

TATAGATGAG GTTTCACCAT GTTTCCTGGG TTGTCTCAAA CTCCTGGACT CAAGCGATGT 1080

ACCCACCTTG GCCTCCCAAA GTGGATTATA GGAATGGAGC CACTATGCCC AACCTTTACC 1140

TCTTTTATTT TTAGTTGATT TTTTTTCTTT TGTGCTGAGT CTAGGGCAAG AATAAATTGT 1200

AAACTAGTAT GAAATACATC TAATACATTC AAATTAAAGA TATAAATATC TGAACAGTGT 1260

AATTTTTTAA AGTGGTGTTT TTTGTTTAAA AGTAGACTTA CTTGCAAAGT TGTATTTTGT 1320

GGTTTTTAGA TCTTAGTATC CTAAAATTTG ATTACCTAAA ATTTAAGTTT TAAGTTTCCC 1380

TTAACCATCT CTACATAAAT AATTGAATAA CTGAAATCTT TCGAGTAATG ATACACTTTA 1440

CTTCTATTTG CCATTTTTTG ACAAATTCTT AGTGTTGAAA TAGGCCCATA TATACTGTTT 1500

CCTATACATT TGTATGCTAA GTGGTATACT GATTATACTC TATGTTTTAC ATTTTAGTTT 1560

ATTACAAATT GGCTTATTGT GTGCTGATAT CTCTGTTTTG TGATTCTATA CACCATAG 1618 (2) INFORMATION FOR SEQ ID NO:51:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 92 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

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

GTTTGTAAGT AGCAAAGAAA TAACGTGAAA ATGTTTTCTG GAGAAAAACT TGATTTAACA 60

TGACGACTTA AGGATCTCTT CTTTCATCAT AG 92

(2) INFORMATION FOR SEQ ID NO: 52

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 889 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 52:

GTGAGTACTT CTGTATAAAA TGTTTTAATA TTTTAAATTG TATACTTAGG AAACTTCAGA 60

AGTTAGTGTT TTTATTGTTT GTACTCTGGA AACTGAGAAT ATGTTTTGTG AGAGAATACA 120

GGGAAGCAAA AATTCTGTCA CCTAAATATA AGCACACTTT TTAAATGTGT TCAAAATTGT 180

ATGGCTGTCT CCGAAGTTTC TTTAAGCTTC TGGATTATAA ATTCTGAAAT AAATTCTCTG 240

GGAACTATAT GGGTGAAAAT TGATGATGTG TAAGTGTGGA AAGTCTTCAG GGGTGCCTAG 300

AGCAGCTAGA CAGATAGTTA AGCTTCTCAC CGGAAGTTGC ACCTACCAGC AGCTGAAACA 360

CTGTCAGCAA AAATACTTGT CCTGTGTGAT GGATGAGCTT GGGGATAGCA GGATTACATG 420

TGATACTATC CAGTTTTTGT TTTGTTTTGT TTTTTGAGAT GGAGTCTCGC TGTGTCGCCC 480

AGGCTGGAAT GCAGTGGCAT GATCTCGGCT CACTGCAACC TCTGCCTCCC AGGTTCAAGC 540

GATTCTTCTG CCTCAGCCTC CTGAGTAGCT GTGAATACAG GCACGTGCCA CCATGCCCAG 600

CTAATTTTTG TATTTTTAGT AGAGACAGGG TTTCACCATA TTGGCCAGGC TGGTCTCAAA 660

CTCCTGACTT CGTGACCACC TGCCTCAGCC TCCCAAAGTG CTGGGATTAC AGACGGGAGC 720

TACTGCACCC AGCTATACTA TCCAGTTCTT ATAACTACAA GTTACCCTAC CAAAGTTTAA 780

CTTTCCAAAA AACTATTAGA ACTTTTAGTA AATAAAAAAA TGAAATAATT AATTGAAATG 840

GCAGTTTCTG TGAGAGAGTA CATTTTGTCT GTATTTGTTT TTCCTATAG 889

(2) INFORMATION FOR SEQ ID NO: 53:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4586 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53: GTGAGTTTAG CCATGCCAGA AGAGTAGAAA TACCAGGAGC AGGTAAGCCA GGGGTTCTTT 60

TTTATTTGGG TAATTTCATO TTTGTGTTTT ACTTGCCTAC AGTATGAAGG AGAAAATTCT 120

CATCATACTT CTCTTAATTG AAAAAGGTAT CTCTATGATA TTTGCTTTGT TAATATCAAC 180

TTTCATTCAT TTTAGTGAGG TCTGAGAAAA GAAATTAATA TAAATTTAAA ACAAATGTGT 240

CATGCTGATA ATTGTTGGTT TTAAAAAGAT GGGCCAGTAA TATATGGTCT TATATGTAGT 300

GAACATAGTG TAGGCATTTA GAAAGTGATA ATTGACCTGA CTGGGGCCTT CATTTAAGAG 360

ACTGGAGTAA AATGAGGATC TACAGTCTTT AAGAAAATTC TTTCAAACTG AATTTCAGGA 420

CCACGTGGTA TTATTTCTAA CAGACACTTA GAGTGATGCA GGCCAAGAGT TTCCCTCCTG 480

CTATGTGGTG GAACAGAAAA CACCAAACTT CTGGAAAGTG CCACCAGGGG AAACACTGGG 540

TAATCCAAGG GCCAGTTCAC CTGGATAGTG AGCTGCTTCA GACTTGAGAC TGGTCTGCTT 600

ATTCATTCAA CAGATATTCC TAAAGCATTT TATATGTCAG GTTGTGTCCT GGACACTGGA 660

GATAAAGCAG TGAACAAAAT AACCACGAGA ACCCTGTTCT AAAGAAGCTT ATATTCCAGT 720

GTGGGGAGAT GGACAGGAGA TAAACAAGTA AATATATAGT ATGTTGGGTG ATGATAGATG 780

AAGAAAATAG AGTAGTAATA CAAAATATTG AGGGGAGGGG AGAATGGGAT GGCTGGGCTG 840

TGGTAGGTAA GGTGGTTGGG AACGGTGTCA CACACCAGAA GTAAGTGAGG AAGCAAGCCA 900

TATGAATAGC TGGGTAAATG TATTTGAAGC TGAGAGCATA ACAAATGCAA AGCCATGAGG 960

TTGGAACAGG ATTAGCTTTT TGGAGGAACA GTGAGAATGC TAGTGTGGTA GGAATAGAGT 1020

GAGGGAAAAA GTGGTAAGAA GTGACGGGAG GCCAGGTGTG ATGGCTCATA CTTGTAATCC 1080

TAGCACATTG GGAGACTGAG GCAGAAGACT GCCTGAGCCC AGGAGTTCAA GACTAGTCTG 1140

GGCAACAAAG TGAGACCCCG TCTCTACATA AAATATTAAT ACAAAAAATA AGCTGGCCAT 1200

GGTTGTGTCC ACCTGTGGGC CCAGCTACTT GCGAGGCTGA GTTAGGAGGA TTCGTTGAGC 1260

CCAGGAGTTC CAGGCTGCAG TGAGCCGTGA TCGCGTCACT GCCCTCCAGC CTGGGTGACA 1320

GAGCAAGAGC CTGTCTTTAA AAAAAAAGAA AAAAAGAAGA AGAAAAAGAA ATGCAGGGAA 1380

GAGGGAACAA GAGAGCCAGA CAGACCGTGT AGGCTTTGGA AGCCATCGTA AGGACTTTTG 1440

CTTCTGCTCT GATTGAGGTG AAAGCCATTA AGAGGGTTAT TAAGAGGAGT GACTGATTTA 1500

CATTTTTAAA GGTCTTCTGG GAAAGTGGGA TTAGAGGCAA GGGTGGAAGT AGGGAGTTAA 1560

GAAGCTATTG GAATGATTCT GGCAATAGTT TATGGTGGCT TGCTTCAGAA AATGGTTTGT 1620

AGCTGGGCCA TATTTTGGAG ATGGCACCCA CAGGATTTAC CGAGGGTTTG TATCTAGGGT 1680

ATGAGAAAAA GAGAACAGTG ATGTCTCCAG TTGGGTGAAT GATATAAAAG CTAAAATCCT 1740

GACAAGTGCC TGTAATGTTG TAAGTTATCT GGCCCTGGCT CTCTCTGAAT TCATCTACTT 1800

TCCTCCCTCC TCACCCACTT ATGCCACATT AACCTCCTTT TTTGTTCTTC AGATATGCCA 1860

GGCATGCCTG CAACACAAAG CCTTTGCCTT TGCAATTCCC TCTGCCTAAA CTGTATTGCT 1920

TCAAGAGATT CATGTGGCTT CCTTCTCACT TCATTCTGGT CTCTGATAAC CCAACTGCTA 1980

TGTCAATAAT AACCACAACA TCCTCCCCAA CCCTCAGGAC TTCTTTTCCC CCTGACTCTG 2040

CTTGCTAGTG TTTCTCTTCG TATTTATCAC TGTCTGACAG TAAGTACGGA CGTACGTACA 2100

AAAGAATTGT TTATTACCTG TCTCCTTGCA TTAGAATATA AGCTTCACCA AGGCTGTGAC 2160

CAGTGTTGTA TGCAGCGCTT GGCACATAGT AAACATTCGG GGAACATTTA CTACTGAAAT 2220

TTATTAACCA GGGAACAAGT CTGGGGGAAC GGGAATCAAC AAGTTACGGT TATTACCATG 2280

TTAAATTACA GATGTCTTTT AAGCATCCTA CTAGAGAAGT TGAATACACA CTTGAGGTAT 2340

ACAAGACAGG AGTTCACAGT TCACACTACA GGTTAGGGGT TGTGTATATA TGTCCTGGGG 2400

TCATCAGGGT GGGTACAGAT AGCCTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT 2460

TTTTTTTTTG AGATGGATCT CGCTCTTCAC CCAGGGTGGA GTGCAGTGGT GCAATCTTGG 2520

CTGCAGCTGT GACCTGTGCC ACGGTGGGTT GCAAGGGATT CTCCTGCCTC AGCGTCGTGA 2580

GTAGCTGGGA TTACAGGTGC CTGCCACCAT GCCCAGCTAA TTTTTTGTGA TTTTTGAGTA 2640

GAAACGGCAT TTCACCATCT TGGCTAGTCT GATCTTGACT CCTGCCCTCA TGATCTTCCC 2700

ACCTCGACTT CCTGAAGTGC TGGGATTATA GGCGTGAGCC ACCATACCCA GCCGTAGATG 2760

GCTGTTAAAG CTATAAAATG AGGAGGGATT ACTTAGAGGT ATGAATTGAG AGAGAATACA 2820

AGAGGTCTAA GGACAAAGCT CAGGGTCACT CCAAATTTTG TAAGTCTTCA TTTGGAGATG 2880

GAACATCCTA ATATTTTTAA GATACCGACT TAATATTTGC ACCCAAGTTA AAGATCCTCT 2940

TGATCAGAAT GAACAGGAAG CTTTAAGCTA AGCACAGTGC TACCAAGAAG CACCATGTTG 3000

ACCTTGAGGA CTCTGGCAGG AAGCTGTTTG TGGTTGTCAC ACCTAGTTTC CTCTGTGAAA 3060

CTACTGCTGC CTGTGGGTGA TGTGGTTATA TGCTGCTGGC TGCTGTTGAT TCTCCTGTTT 3120

GTGTACAAGG TGTTTTTCCC TCCCAGTACC TCCCAATGTA GGCATCGGTT CATGCACAGT 3180

GAAGTAGTTG CCTGCGAGAA ACCTTGTAAG GCAGGGAGCA GCCTTTTGAA TGCAATAATC 3240

TACCCGAATC ATTTTAATGA CTTAATTATA GAATGAATTT CTTTGAGACA AAGTGAAAGT 3300

CTTAGTTGTA TTACACTTTT AGACATAGAG GAGACATGTA GGTTTGTTTC TGTATACAGT 3360

AAATTTCTGT GCTTTTCTAT ATCTTATGAA ACTTGAATAG TTGGCTCTGT TGCCAGGTGA 3420

AAGTTTTGCT AGGTTTTTTA GGAAATTAGG ATGAGTACAT TTAAGACACA GGGAAATTTT 3480

ATCTTGAATA GTAAAAGACA TTGTTAAGCT ATCGATTCCT TTCAGAGTTT ATTTGGAAAA 3540

TCAGAGAGAT GTTTTACTGG CTCCTTTGAC ACCAAGTCAC ATCTTCTCCT AATTTATTGT 3600

GAAGAATGTT GACATTAACT TATTTCTCTG AAGACCTGTC TACCTTAGGG GGCTGTTCTG 3660

CATCAAGTTG CCTTTTTAGG GGATGTACAA CTTATTATCT OTCTCTGAAG CAAATATGAA 3720

TATTTGGATG GTGGGTGTAT TAATTCATTT TAACACTGCT GATAAAGACA TGCCCCAAAC 3780

TGGGGAACAA AAAGAGGTTT AATTGGACTT TACAGTTCCA CATGACTGGG GAGTCCTCAG 3840

AATCATGGTG TGAGACGAAA GGCACTTCTT AGGTGGCGGT GGCAAGAGAA AAATGAGGCA 3900

GAAGCAAAAG TGGAAACCCC TGATAAGACC GTCAGATCTC GCGAGACGTA TTCACTATCA 3960

CAAGAATAGG ACGGGAAAGA CTGGCCTCCA TAATTCAATT ACCTCCCACT GGGTGCCTCA 4020

CACAGCACAT GGGAATTCTG GGAAAAACAA TTCAATGGGA GGCTTCGATG CAGACATAGC 4080

CAAACCATAT CAGTAGGCTT TTGTTAAATC ATGGATTTTT TTTGGAACCA AATTTAATCA 4140

CAATTTTCTT TTATCTTTGA GTGTCTCCCA AAATAGCAGT AGATGGGAAT TGTGAAATTC 4200

TGTTTCTCAG AGCTGAGAAT AATCTTAATT TTTCAGGTGA GCAGAATGCT TATCTTTGCC 4260

TCCGAGCATA AGTTTTACAA GAGGGTATGT AGGGAGCTGT ACCTTATTTT AGAGTTTTAA 4320

CTTTTAAGAG ACAAACTTTT AGTTAGCTAA AATACAAATT ATTCTTTCAC ACCTTCGTCT 4380

TCACATGGAT ATTGGCGGCT CTTAATGCTG TTATGTTTAA ATTCCAAAGA ATGGTGACAT 4440

TTGAGTCACT AAAATTTATT GATATTGTAA AGATAAAGTC TATCTGGCTT GAAGTCCCAT 4500

TTGTGAAGTG AATTAAAGTC TTTCTGGCCT AAAATAATGT TCTTTAAAAA ATGTTTATTA 4560

ATTCTGTGTA ATTTTTTTTT CTTTAG 4586 (2) INFORMATION FOR SEQ ID NO: 54:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2127 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION ^' : SEQ ID NO: 54:

GTTAGTTTGA GCCCTGTCTG CTTTCTAAGA TTTGGTTATT GACCATTTTC CAATTTCCTA 60

TTCTTTCATT ATTAATGCCT TAATTCACCC ATGAATAATT TTTTATCAAT TGTATACTCA 120

GTCCTGTTGT GAGTCTATAG AGGACCTAGC AATAAGATGT ATAAGTGGAA GATCTTCTTT 180

CCTTAGATTT CTTTAATATA ATACAAGACA CAGTAACTAA TAACACCAGA CAGTGTAGAG 240

TAAAACACAA AAGTGTCTTA TTGCCAACTG TTCTTTCAAG ATTTCAGGGA GTGGTGACGT 300

GGCGGCGGGG GGAAGCTCAG TGATGATGGG AATAATTGTC AAAGGACTTT ATGAAGAGGG 360

TTGACCTGAG GTAAGTTCTG AAGGGTGACT CAGATTTGCC AAGATTAATA GAGTTCCACA 420

TGTTCATAAA GCAGGACAAA AACCACTGTA ACTTTTGTAA GCTCTATAAA ACATCCTTAT 480

CCTGGAAAGG AAGTTGACTG CATTTAGCTC CTTTGATCTC CCTGAGACTG GTAGGAATAT 540

CATTGAGTTT TAATTAAAAG CCCAGTAGGC TGAATCTCAT CATCTTATGC ATAACCTTTG 600

GCAAGTTGAT TTGAAAAGCT ACCTCCAAGG TCCCTCTCAG TCCTAAAACC TTATGATATG 660

ATAACGTTGA CCCAAAAGGA CCCCATTTCT TTTCTGATGA TGGTATATCA AGAAGACCCT 720

ATATGTACAC ATAAGTAATT TCCCACTCAT AGCCAGGCTT CTTAAATGCC AACTACTTTT 780

CCTTTAACAT TTCAGTGAAG TCTGCTTTAT TCATAAACTT GATTGTGATT TATACTCAAC 840

AAGTTATATC TCTGTGGCCT CTTCCTGAGT CATGTTTTTC AGATGCACCT TGTTTGGCTT 900

GAATTTAGAA GCATTTCGTA AATACATTTC AGAAGCCATC TTAATCTCTG TGTCTTCCAG 960

ATCGCTTTAC AGTTTCTAAC TAGGCATAAC AGCATTTTAA ATCTTAGGGA CCATTAGTGG 1020

GGTTAAATAA TTATTACCAG TAAATACTAG GTAAAATAAA GGGTGCTATT TTTGCTGAAA 1080

GGTATGTGTG CGTGTGTTCC CAGAAAAATT CTGCTTGTAT ATGTATTCAG TAGTTATCTC 1140

TAGCAGGACT GTAATTGATT TCTATTCTCT TTATAATTTT TTAAACTTGC TTCATTTTCA 1200

CAAAGAATAT GTATATAATT ATATATATAT TTGTGATCAA GATAAAAACA GTTGTTACAA 1260

AAAGCTTACA TGGTGATAAT TTGTATAATG CTTCTGGATT GAACATATAT TGCTCCCTAA 1320

TAATAGAAAG ACTGAAGTAA ACCTCGTTGG CGGGAAAAAA ATGTAGAATG CCAGGAACAG 1380

TTTATGTGAG TCTGTAGTAT GGGTTTTACA CCCCTTCATT CTATTTTCTT CCAGGTGTTC 1440

TTAATGGGAG TTTTACTGTC CTCTAGGGAA ATAGTTAAGG GCAAGTTTGG GATAATCAGT 1500

GACTGGGGAT GTGTAGGACA GGTGGGGGAC AGTCATAGAT ATCGAATGGG CCCAGGCCAA 1560

GGTTGCTAAA CTTCCTGCAC TGAAAGGTGT ATCCCCGGCC GGGCGAAGTG GTTCATTCCT 1620

GTAATCCTAA CACTTTGGGA GCCTGAGGCA AGTGGATCAC TTGAGGCCAG GAGTTCGAGA 1680

CCAGCCTGGC CAACATGGTG AAACCCCATC TCTACTGAAA ATACAAAAAT TAGCTGGGCG 1740

TGGTGGCAGG TGCCTGCAGT TCCAGCTACT TTGGAGGCTG AGGCAGGAGA ATCACTTGAA 1800

CCTGGGAGGT GGAGGTTGCA GTGAGCCAAG ACTGCATCAC TGCATTCCAT CCTGGGTGAA 1860

AGAGCGAGAC TCTGTCTCAA AAAAAATATA TATATATAAA AATAAAAGGT GTAGCTCCCA 1920

CAAGAAAAGT TTTTTTTTTT TCATTCAAAC TGGTAATACC ACCACCTTTG AAAAGGAAGT 1980

ATGGGATCTC TTGGATTAAT TTGGGAAGTG TATAGTTTCT GTTCAGAGTG TTTTATATTT 2040

ACATGTTAGT GAAATTATAG AGACATTTTA TCCCCTTGTG ACTTGACAAG ACCTTTAAAT 2100

TATGTTATTT CTCATTACCT TTTTTAG 2127 (2) INFORMATION FOR SEQ ID NO: 55:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 716 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

!ιi) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

11

GTAATTGTGC TAAAGGTAAG GTTTAACATT GTTATTCTGC TTCCATGTTT GAAGTTTAAC 60

TAAATGGAGT CATTTCTTAC TAACTAAGAA AGATGAGGAA AAGATTTATG ACTTTAGACT 120

GGAGGCATGG ATATGGCTGT CCAATTTTTC TGGTCAACCA ACTGATTTCT GAGCCCTTCT 180

CAGTAAGATA GAAATTTTAG AATGGTATCT TTATTATATT GGACTACTGA TGCTTCCCTA 240

TCTGCAAATC TTTAGGTTTC CCTTGTAAAC TGGAAATTAA ATAGAAGTGT AGTGATTCTT 300

CAACATATTG AGAATAAGGA CAGGAGATAT CACTGTTATG GGCGGAAACC TGGGCTAGGA 360

ATTGTTTGCT GTCAGGAATT GGAACTAAGT AGGTGTGGAC TAGTAAGCCA ATTACATACC 420

TCTTAGCATT GGTCTGTTTT GTTCCAACAT AGAGGAAAAA AAAGGGTGTT AGTCTTAAAT 480

GATATTACAG TTCCTTATGT GCCAATTTCA TTTAATAATT TTAGAAAAAT GTGACTGTTA 540

CCATGAAGAA AATTAAGGTA TCTTAGGGAT AATTAAAACA CCAATCATAA GAAGTGTGCA 600

TATCTAAAGT ATTGGGTTGG TTTTGAATTT TATTTTGTGA GTAAAGGAGG AGGAATGGGC 660

CTTTATTTTC TTTGTGTTCC AATTTTGTGG GGGTTTTTTT TTTATTATTT CTACAG 716 (2) INFORMATION FOR SEQ ID NO: 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 837 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

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

GTGAGTAAAA TAACCAATGT ATTGATCAGC ACAATGAAAC ATAATTTCCT TCCTGCCCTA 60

TTCTGTGGGT TGTTTCCTTA CTTTATATAT AGTCTCCTTT CATACACAAA AGTTTTTAAT 120

TTTGATGAAA TCCAATATAT TTTTTCACTA GTTGCCTGTG CTTTCGTTTC ATGTATGTAT 180

GTATGTATGT ATTTACCTAT TCGAGATGGA GTCTCGCGCT GTCGCCAAGG CTGGAGTGTA 240

GTGGCACGAT CTCGGCTCAA TGCAACCTCC GCCTCCTGGG TTCAAGCAAT TCTCCTGCCT 300

CAGCCTCCCA AATAGCTGGG ATTATAGGCA TGTGCCACCA TGCCCAGCTC ATTTCTGTCT 360

TTTTCGTAGA GATGGCGTTT AGTCATGTTG GGCAGGATGT TCTCGAACTC CAGACCTCAT 420

GTGGACCACA TTCCTTGTGC TCCCAGAGTG CTAGTATTAC AGCTGTGAGC CACCCATGCC 480

TTGCCTGTTG CCTGTGCCTT TGGCTCTTCA ATAACTTTTA TTTATAACAT CTTTGCCCTG 540

TCATTGTTCT TCTAAGCATC AGTGTGTGTG TATTTTGGTT AGAGATGTAA TCTCTTTTAA 600

GATACATTTT ATATAGGTAA GGTTTTAAAA TTCTCATACA TTCCTTTTAT ATATTTCCTC 660

TACTAAAAAA TGGGCTTTAT TTATATAATT AAGAAAGGTT TTGTAAGAAA ATAAGGACAC 720

ACTTTGCACT CACTCAGAAA ATGAGACTTT CTTTGGTATT TTCACTTAAG TTGCACTGGG 780

TATGAAATGA CTTTTTAGAC TAAGTAGATG TTTCTAATGC TGTACTTTAT TTTATAG 837 (2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1081 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:

GTATTTCCCA AAAAATATGA TACTAATGGG GATATTGTAG ATGAGACCAA CTTCCTGTTG 60

TTAGTCATTT AGTTCAAGTT AACATCTAAG AACATTTATT CTGTTTCTAT TTACATAGTT 120

AATCTCTACT TGTGGAGTAG AAAAGAAATA GAATCTTAAG ACCTATGTAA ATTCTTTTAA 180

TATTGTATGA AAGATCTATT TTGGGTAAAA GCTTCGATTC CTCTCTATCT AATAAAAGTT 240

TTTAGAATAC TGTGATTTTT ATGAGCTGAG AAGGCTTAAA AAAAGTAGCA CACATGTCAC 300

TAGCTAATCT TGTATAGCAG CCTTTCCTTA TCTTATGAAA ATTAAATACC ATTGAAAATG 360

TCAGAAAAAA AATAAAAAGT TGTCTTTCAT GTGTTACAGA GAGGCATAGA GTTAAAAGCA 420

TTGATTTGGT AGCTAGTTCT TCCCCCTCCG GAGATGGAGT CTTGCTCTGT CGCCCAGCGT 480

GGAGTGCAGT GGCGCCATCT CAGCTCACAG AAAGCTCCAC CTCCTGGGTT CACGCCATTC 540

TCCTGCCTCA GCCTGCCGAG TAGCTGGGAC TACAGGCGGC CGCCACCACA CCCGGCTAAT 600

TTTTTGTATT TTTAGCAGAG ACGGGGTCTA CACCGTGTTA GCCAGGATGG TACTCGATCT 660

CCTGACCTCG TGATCCTGCC CGCCACGGCC CCCCAGAGTG CTGGGATTAC AGGCTGGTAG 720

CTATTTCCTT GATACTGACT TAGCATATGA GTTTATGCTT AACTCTCATA AGATAGACGA 780

AACTAATTTT TATAGTGGCA TAGATTAAAT GTTTAGAGAT TTTTATATGA AATTTTAAGA 840

GTAATGTTTT TCAACCTCAA TGTACAAAAC ATGTATTTTA TTAAAAAATT TTGAAATACA 900

TCACAATGTA AACCATTTTA TATAATTCAT AGTTTGAACT ATAATTATTT ACAAAGACAG 960

TAAAAGGAAG AGCGGCTGTT TCAAAATAAT ACTTCAACTT GTAATTTTGA CTAATTTCTT 1020

GTCTAAATAT TTAAAAAATA TTTAATAATT ATTCAGTGAA CCAAGACATT TTTTATTTCA 1080

1081

(2) INFORMATION FOR SEQ ID NO:58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1455 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (11) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

GTGAAAATCA ACATCTTTTT ATGAGAAAAA TACATCAATA TCTAATCTAT TAATAATCCT 60

TTTGGGGATG GGAGGGTGGC AGTTAGGTTT AATATGTTAT AATTACACCT TGTTATGAGA 120

AAAATCTTGG ACTGTAACGT CCCTCTCTAC CCACAAATTG GGAAGGTGCC AAGAGACCAA 180

AGAATGACTC AGACAAGTCC AGCTCGGCAA GTACATAACG TCTATTAAGA CTTACATATG 240

GAGGAGGCAG AGGTGGTGGG GAAAAATAAA AGACTTATAT ACAGGGTACT CCTAGGTAGC 300

AGCAGGACAG CTCTAGAGAT CCTCGCTACC TCCCATCGCT AAGCTGCTTT TAAGCTAATT 360

TTCTGGCTCT TTGCCTACTA TGTGTGTGCA CGATGGGACT GTTTTCCTTG GTAGTTTCTC 420

AGATCTTCTC TGGGATGTTG GGGTTCTCAG GGACACCTGT TCCTTGGCTG GGCACCATGG 480

CCTTGGCTCA CTGCCTAGCC TTCAGGGTTT AGGCAGCAGA CATACACCCT TAAGTAAGGT 540

AGGTGACCTG TCACATTTCA CCCCATGTCA AAGAGGAAAC GAGTCAGATA ATTTGTGGTT 600

GCCCTAAGAT TTTGGTGACA GAGTAAAAAT TCAGTGTTCT TTCTTGATTT CCTTACCAAG 660

TTTCTTTCCC ATAGAGCAGT GGTCCATCCT TTTTGGCACC AAGGACCAGT TTCATGGAAG 720

ACAATTTTTC CATGGACAGG GTTGGGGGTT GGAGAGATTT TGGGATGATT CATCTGCCTT 780

ACATTTATTG CACACTTTAT TTCTATTATT ATTACGTGGT AATATATAAT GAAATAATTA 840

TACAACTCAC CAAAATGTAG AGTCAGTGGG AGCCCTGAGC TTGTTTTCCT GCAACTAGAT 900

GGTCCCATCT GGGGGCGGTG GGAGACAGTG ACAGATCAGC AGGCATTAGA TTCTCATAAG 960

GAGCATGCAA CCTAGATCCC TTATGTGTGC AGTTCACAAT AGGGTTCACA CTCCTGTGAG 1020

AATCTAATGC CACCACTAAT CTGACAGGAG GCCAGCACAG GCGGCAATGT GAGCGATGGG 1080

GAGCAGCTTT ACATACAGAT GAAGCTTTGC TCGGATGCTC ACTGCCTGCT GCTCACCTCC 1140

TGCTATGTTG CCCAGTTCCT AACAGGGTCC ATGGCCCAGG GGTTGGGGAC TCCTGCTTTA 1200

GAGTGGTTGA TATTCAAACT CCTCTCCAAA CCAGTCAATG AAGTTTGACT CATATTTAGT 1260

ATCCAATTAC AAGGTTTTGA ATTTTTTGAC TGCCAAAAGT TTTTTTTTTA ACTTTATTAT 1320

TAAAATGGGA AAGACAGCTG ATTTTATTTA GATGGAATAA TTGTTAAGAT ACTTCTTCTG 1380

CCTTAGATTA CTATTGTATT TGTAATTAAA GTGCTCGTTT GGATACTGGC ATTCTGTGTA 1440

ACCAATTCTT CATAG 1455

(2) INFORMATION FOR SEQ ID NO: 59:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2741 base pairs

*B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:

GTAAGTTTAA CAATACTAGG AGAATATCTT GGGGCTTACT ATCTGGAAAT TTAAATTTCA 60

TCTAACCCTA CAAGTGAAGT TAATAGGGTA TACATAGAAG AAAATATTCT ATGCATTTTG 120

GTACCCATGG ATCACTTAAA AGAAGGGCCT TTAAAGACTA AGAACACAGG AAAATGCATG 180

ATATAACAGG TATCTTTTAA AAAGGATAGA CTGCTTTATT TATTTATTTA TTTATTGAGA 240

CAGAGTCTTG CTCTGTCACT CAAGCTGGAG TGCAGTGGCC CAATCTCAGC TCACTGCAAC 300

CTCTGCCTGC CGGGTTCAAG CGATTCTCAT GCCTCAGCCT CCTGAGTAGC TGGGACTACA 360

GGCATGCGCC ACCACGCCTA GCTAATTTTT GTATTTTTAG TAGAGAAGGG GTTTTGCCAT 420

ATTGGCCAGG CTGGCCTTGA ACTCCTGACC TCAAGTGATC CGTCTACCTC GTTCTCCCAA 480

AGTGTTGGAA TTACAGGCAT GGGCACCGTG CCCGGCTGAC TGCTGTATAT TTAATATGAT 540

CCCTATTTTT AAAGTGTATG TTTATTTATG AGCATACAAA ATAGTGGAAA TGGAAAAACC 600

AAACTGTTAA GATCATTGTT GGGTGAATGA ATTCCTGGTG ATTTCTGTAA AATTTTTAAG 660

GCAAATACAT ATTACTTTTA AAATCAGAAA TAGAAAAGCC TTCTTAAAGA TAGAGCTGCA 720

TGATCCAGTT AGGTATAGAC AAGCCAGTGA GTTAAGACAA CTGAGTATGT TCCACTTTGT 780

TGAGCTGTGC TACCCTAGTT AATGTGACAT TAGTGCTGGC CCAAGAAATA CAGAAAAGGG 840

CAGTTTTGCT ATCTATCTGG TTTATATTTT TTAGGCAGCT GCTTAGAAGA TCTGCAAGGT 900

GAAAGGTTTT AGTTTACATA TGTGAGATAG AACTACTTTT TTAAAGAGCA ATTCAGTAAA 960

TCCAGAGAGT TCTAAATCCT TGGATCCAAT TAAAAGAATA TTGTTATTTG TAGATCAGTT 1020

TTATAATGTA ATTGATAAGA ACTGGCTATA GAAGGAATAC CAGTTTTAAA GTCAGGATTC 1080

ACTCTAGGCT GGGCATGGTG GCTCATGCCT GTAATCCCAG CACTGTGGGA GACCTAGTGG 1140

GGAGGATCAC TTGAGCCCCG GAGTTCAAGA CCATCCTGGG CAACATAGCA AGATACCATC 1200

TCTACCCCCA ACCCCCCCAA AAAAATCACT CTAAGTGTAT ACTTAATACA CATGGATGAT 1260

CCTTATGAAA AGTCCTCATT TTTGAAAGAT CTGAGAGCTG GTCTTTCTTA GTCTATTTTT 1320

GTAGAATTTT CCGTTCCCTA ATCTACAGAT TAGGAAGACT TGACGTTAAC TTCATTTTCA 1380

ATGTCTTACC ACTTGCTCAG TTTTCCTGAG ATCTCTTGAT ATTTTATGGA GGAGAAATGA 1440

TCATAATCTA TTCTTTGCTG ATTCTGCAGC TTTGTACCAA ATACAAACTC AGTAAGTTTA 1500

TTTACTTTTG TATCATCTGG AAATAGAAAT GTTAAGCCAC AGTTTGTTAG GATTTACTCC 1560

TATCAGTACT TCTTACAAAC TTTGCTATGT ATATTTTAAA TTTTAAAAAC ACTCTGATGC 1620

ACAGCTCTTA GAAGTGGACA CAGAAGAAGG AAGAAATGCT TCTCAAAAAT TCAGACATTG 1680

GTGTGAATAC TTAAAAATAG ACTAAGCCAT AATGGGTTGT GTACCACTGA ATCATACACT 1740

TAAAAATGGT TGAATGGTAA ATTTTATGTT ATATATATAA CCACAATTTT AAAAAACTAG 1800

CCTGTAATAC CAGCATTTTG GGAGGCCAAG GCGGGTGGAT CACCTGAGGT CAGGAGTTCG 1860

AGACCAGCCT GGCCAACATG GTGACCTCAT CTCTACTAGG GAGGCGGAAA GTAGCCATGC 1920

CGTGTGGCAT ATGCCTCTAA TCCCAGTTAC TTGGGAGGCT GAGGCGCAAG AATCACTTAA 1980

ACCCAGGAGG CAGAGGTTGT AGTGAACCGA GATCAGGCTA CTGCACTCCA GCCTGGGTGA 2040

TAGAGTAAGA CTCTGTCAAA AAATAAATAG TAACAATTTG CCCCAAACCA TTGAATTGTA 2100

TAATTTAAGT AGATGAAATT TATGGTATAT AAACTGTTTT AAAAAAATAA ATTATGCTTA 2160

ACTGAATCCA AATCATGCAT GTCCACCTTG CTTAAGAACA TTATTGAGTT TTAATAATTT 2220

TTTATATGTG GAAAAAGACA GAGATCCAAA TTGATAAAAC CGGTGGCGGC GGAATGCTCC 2280

TAGATGACAT ACTACCAATC AGGTCCCCTT ATCAAGTAGT GGCTCTGTAG TAAAATCACA 2340

TCTTACATGA GTGGTAGGTA GAAAGTGGAT ATGATAGAAA ATATTATAGA AAAATATAAT 2400

ATAGAAAAAT AGGGTAATTC CTTAAATTGC CCCTAAATCA TGAAGGTTCT TTAGTAGTGG 2460

AAGACAGAGT CAGGTCTGAT TTGGGAAAGG GGGCGTGGAG AAAGGAACAC TGCAAGACAC 2520

AAAATTCCGT TTTAAAATTT TGCTCTCAGT AGTGTTCACT GAACACGAAT GAAAGTTCAC 2580

TAATGAATAT AGGTAAGATT AGACTTCTGT AATTCTTGTT TGCTTTTTGA ATTATGAAGT 2640

ATTTCAAACA CTGTAGTTAT TTTTTAACAT AAGAGCTTGG ACGGAAGTCA GATCTGAGTC 2700

TCCTTGAGTT AAATGCTTTG TTTGATTTGT TTTGACCCTA G 2741 (2) INFORMATION FOR SEQ ID NO:60

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 197 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:60:

GTAAGGAAGG CAGAGTTGGA TATTGAGTTC CTTCTCTGTG GCATGTATTG AAAAGTTACC 60

CGAGGTTTGG CTAGAGTGAC ATAGGGGACA GAGGAGTGAT GGGGAGAGAG GGTTTGGGAG 120

AGCAGAAATT GTAAACCTCT GCCCGGAGAA CCTCTTATTA TCAACATTTT CTTCATGCTT 180

TTTTTCTCTG TCACTAG 197

:) INFORMATION FOR SEQ ID NO: 61

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 82 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS. double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: GTAATTTTTC ACATACCTTA TCAGAGCATG AGCTTGGGAA ATACAAGTGT TAAACAAAGT 60 TTGAAATGTT TTTATCTCCT AG 82

(2) INFORMATION FOR SEQ ID NO:62:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1079 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:

GTGAGTACCA TTTGGAATTG TAAAGGCAAA GATAGGTCTT CATTACTGAG TAACATTTTT 60

TAACCACTGT CTTGAGATAC AGTTTACATG CTCTATAATT CACCTATTTA AAATGCACAA 120

CTAAATGGGT CTTAGTATAT TCACAGATAT GTGCAATACT CACCACAATT TTAGAACATA 180

ATATCCCATT GTATAGTTAT ATGAGAGTAT TTTTATCCAT TCATTAGCTA ATGTATATTT 240

CAGTTGTTTC TACTTGGGGC ATATATGCAT AATACCACTA TTAGCATTTG TGTTTGGGTT 300

TTGGTATAGA CATGTATTTT CATTTCTCTA GGGTATATAC CTAGGAATGG GCTGCTGGGT 360

CATACATTAA CTGTGTTTTA CCTATTTAGG GAATTGCTAG ATTGGTTCTC CAAAGTACTG 420

TACCATCTTA CACTTACACA GCAGTATAAT AAAGATTTTA GTTTCTCCAC TATCTCATTA 480

ACACTTACTA TCTTACTTTG TTTAAATAAC TTATTGAGGA GAAATTCACA TAACATAAAA 540

TTAATTGGGT _T TTTCTTTTC TTTTGGGAGA TGTTGTTTCA TTCTTGTCAC CCAGGCTGGA 600

GTGCAGTGGT GCATCTCAGC TCACTGCAAC CTCTGCCTCC CAGGTTCAAG CGATTCTCCT 660

GTCGTAGCCT CCCGAGTAGC TGGGATTACA GCCATGTGCC ACCACGCCTG GCTAATTTGG 720

GGATTTTTAG TAGAGATGGG GTTGACCATG TTGGCCAGGC AGGTCTCAAA CTCCTGACCT 780

CAGGTGATCT GCCCACCTCG GTCTCCCAAA GTGCTGGGAT TACAGGTGTG AACCACCGCA 840

CCTGGCCTCT AAGTCTTGAT TCACATACTA TAGACTCCTA TTGTTTTTAT TGAATTTTAA 900

TAGATATTCT TGAATCGATG TATCTTCATT TGCTATATGC CGTTAATACC ATTTCCAGAG 960

ACTTTAAATA GCTTTTATAT AATTTTCACC CCTTTTACTG GGCAGCAGGT TCACAGAGCT 1020

CCTCACACTA TTATGGTGGT AGTTGCTATG TCTCTCAGAG CACTCTTGCT GTTTGCCAG 1079 (2) INFORMATION FOR SEQ ID NO:63:

(l) SEQUENCE CHARACTERISTICS-

(A) LENGTH: 659 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:63:

GTAACTACAT TTTCTCTATG GGCTGCAAAA TAAAGCTTAT AGTCTGTGAT GAATACAAAA 60

AATTACCCAT AGTTGACTCT GTGGCCTTTT TTCCAAGATA AACACCTGGG ACTCTACTTA 120

AGGAAGTTTC TACTTTAATC TTTATTCTTG ATGTCACATG TTGATTAAGG TCTCTTTTCC 180

TCAAAAGGCA ACAATGTTAA ATATTTCATT GCCTTCTTAA TTCAGAAAAA TCACAAGATA 240

GGAATTAAGA AGTTACTTGG TTTCTATGTC ACCTTTCATT CTGGTTTAGT AAACATACTG 300

TAGGTTTAAC CAAGAGAATG TCACATGGAA ATTTAAAACC CACTTCGACT TTATTACCAT 360

TCATCTCTGA GAGGCAAATC GGCCAGATCT GTGTATCTTA CTTAGAATGA CTTGACATTA 420

TGGTTGGGTG CTGTCACTGC AGTGTAGTAC TGCAGGTAGT ACTTGGCATG TGATGCTAGA 480

TGGGCTCTGA TTGAATCCTG GATCTGTTAT AATTTGAGTT ATGTTTCTCA ACCTGTTCTG 540

AGGACAACTA TTGCTATACA GGTTATTGTG AAAACCAAGT AACATATGTG AAGGTCCTAT 600

CACCAAGGGT GTGCTCAACA AATACTAGTT TATGTCCCCT CCTCATTGTT TCTCTAAAG 659 (2) INFORMATION FOR SEQ ID NO.-64:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 572 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

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

GTGGGATCTT TGTGAACTAC AAGACAAAAT TAGGAGCTTT TCTTACTTTT TAGGCCTTGA 60

AGAAGTAACT AAGCATTACT AAATGAAATA ACTATAGAAA CTATGAAAGT GTTTTATAGA 120

TCAGTAAACC ATATTCTAGC TGGCAAAACT GTCCATTACA TAGCTTTGGG GCACAATATT 180

ATGTAACATA TTTCTCCAGG AGAATTAGAG CTTTCAGGGA GGAATCTGCT TGCCTGAGTT 240

CCAGAAAGGT CTGATATGTC AATTGGAACC ATGCTATGGA AATACCATCC CCTGCCTGTC 300

TGCTTTGTAC CACTTAGTAC AGGGCTTAGG TCCTAGAAAA TTTGGTGTAA CTTATTAATG ^' 360

GACACTACTC AGAAAGCCCT TGCTATGGTT ATGGCATAGG GAGAAAGTTA ATATCCTAGC 420

TGAGCTTTGC TTTTTGGTGT GAAGAACAGA GTGCCTATTC ACTGTTATTA GCAAGTAGTG 480

CAGGTAGCTG TTCCCTTTCT CCTACTTTTA AAAAATTAAA ACAGTCACTA TTAGCAGCCT 540

TTGTTCGACA GCCTTGGTTC TCCTGGCTGC AG 572 (2) INFORMATION FOR SEQ ID NO:65:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 901 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(XI) SEQUENCE DESCRIPTION: SEQ ID NO:65: GTAAGTATGA CAGGGATTAT TTCATACTTT TCTCACTCAT GAGTGTTGAG GAATCATTTA 60

TGATTTATAT ATGGACCATT CACCTGGTCC GTATATAAAC TAGTTTTGGC CAGGTGTGGT 120

GGCTCACACC TGTAATCCTA GCACTTTGGG AGGCCGAGGA GGGTAGATCA CTTGAGGTCA 180

GGAGTTCAAG ACCAGCCTGG CCAACGTGGC AAAACCCAGT CTCTACTAAA CATACAAAAA 240

TGAGCTGGGC GTGGTGGCAC ACACTTGTAA TCCCAGCTAC TCTGGGGGCT GAGGCAGGAG 300

AATTGTCTGT ACATGGAAGG CGGCGGCTGT AGTGACCTGA CATTGTGCCA CTGCACTCCA 360

GCTTGGGTGA CAGAACAAGA CTCTGTCTCA TCACTAAGCT AGCTCTACAA ACACTTCTCT 420

TATGTACAAT GAGGAAGTCT GTAATCTACC TAACCAATAT AAATTCTACT GTTGTCAAGC 480

ATCAACCGAG TAAGATTGTA TTTGGAGTCC CCGCAAAGTA TAGTAGTACA AGAGGCAGGC 540

TACATGGGTT CAAATTTCCC AGTACTTAAC AGTGGTGGTA ACCCTGCAAA TCATTAAATT 600

TTCTCTGTAC CTCATTTCCT CATATATAAA ATGGGAATAT AACTAGTTCC TAGCATATGG 660

GGTTGTTGTA AGGATGACAT GACATAATGT ATAAAAATTG CTTACAATAA TAACTGGCAC 720

AAACTAAGCA CTTAAGGTTT GCTATTAGAA TATTTTTCTT TAGGTTAAGT TATTGCTAAA 780

ACATCACTCT GTCATTCATA AAACTACTGG TTTAGCACAC CTCTTCACTC AATAATCATT 840

TTCAGTAAAA ATAATTATAA ATTTTTTTTC TTAGAATTAC TGATTTTTTT TTTTTAAACA 900

G 901 (2) INFORMATION FOR SEQ ID NO: 66-

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4220 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:

GTGCGCTCGC GGGCGGAGGG GCGCTTCCGG CCTAGTTGGT GTGAACCGGT GCCTTCCGAG 60

CCGTGTCGCG CGCCTCGAGA GACTCTCGGG CGGGTTGCGG GCTCCCAGCC CCGAGAGGGG 120

TGGGGACTCC CTCTGCGCTA TTCCGAGGCT CTTAGCCGCT CCGAGGGTTA ACCCGCTCTC 180

GCCGGGCTTT CCTGCGGCTT CCGAATGGGG AACGTGTCTT GCCCTAAAGT AGCACAGCAA 240

GGTTGAGATC GCGTTGGGGC CCCGTTGAGG AAAATGGGTG TGTGTGGTCC ATCTGACCCC 300

CCGCCCGTCT TGTTAGTAGA ATGAACTAGT GTCGTTGTCA AGACCACACG GACAAGGGGA 360

GGGGACTTGC CCTTATTTGC ACCGCGATTA ACCGGGTTGT GGCACCTGGG TCTCCACGCG 420

TCTCCGTCTG TTCGCTTCCC CCTGTTAACC AAATTGCCTT TGCCCTGGCG TTGCGGGCGT 480

TTGAGTCAAC GTGCTGATGC GTTTTGGGCT GTGTTTACGT CTGTGTAAAC AAATTAATAC 540

TCATTTCCCC CCAGGCCATA TGAAATGAGC CCACCGCCGA CCCGGATGTT TACACATGCC 600

CCCATTTGTC ACTACGATCA GGACTGTGGC TACCTCCAGG GCTTTTTGGT CACCCCGCGC 660

ATTGCACAGG ACTCCTGTTG TCGTCGCCAT CCGGGTGTGT TAGGTCGCAG CCTTCGGCAC 720

AGGGCTTGCA CCATGACAAA AATGGCCATT CTAGCCAGTG AGTGTCAGCT TTGTATGCAC 780

CTCCCCTTCA TGGGCCAATG GGAAGTGACA CGGAAGTACG GATTGTTTAT CACCTGTTTG 840

ACTGTGTGTG TGGCATTTAA ACCTGAGGCC ATTTGATTTC TCAAGTCGTT TTATAATTAA 900

TTTGTACAAA GAGTCGGGCA AATACGTCCA GGATGCAAAG CCTAACGAAG GTATTATTTA 960

AATATGATGT TTTTGGCTAT GTGTACTGAT GACTGAGGTT ATTTTTAATT TGTATTTGCA 1020

TTAATACAAT TTTAATTCAA TTACTAGTTC CCTCTTTGAA TTGTTAGGTC TGCACAACAT 1080

ACTGTATGGT GGCTTTACAA CCCGACAGAC CTGAAACCGC TGAAAAAGTT CAGTATGGTG 1140

ATCTCTAAAC TGGAGATATT TGTGTTTACC TCACAGAGCT GTTCTGAAGA TTAAATAAGG 1200

CAATAATGTA GTTTCTGGCA CATAAAGCAC CCATATGGAC AGTGTTTTCA AGTTTACTAA 1260

GCTCTTTGTA TATTTACATG ATCTGGCTGA GTAAGCTATG TTCCTATTCA TCTCTCAGTG 1320

CCTTTCTGTA GTCTGGCAAA GAGAAGGACT GGTTGGCTTT TTATGTTGTT TTTTGTTTTT 1380

TGGGTTTTTT TTTGGTAAAT GGCCTTAAAG GCTTCCAAAC AAGCTCTTAT TTTACCCTCA 1440

AGATAATCCT GTAAATCAGA TAGAACAAGC ATTATCGCCA TTTATTTGAG GTATTTCAAC 1500

TCATAGCAGT TAAGTTGTAT GAAGTCTAGT GATACATGAG CAAGTATCAC GTAATAGCTG 1560

GTTAGTAAAT TATTTTTGAA ATCATGTTTG ATTACTCAAT TCTTTTGATT ACTGAGACTT 1620

TAGTTTCAGC TTCTTAGCCC AGTTTATCAG TAAATGATTT ACTCAGTAAA ATATTCATCA 1680

AATATTTCTT GAGCACCTAT TACTTGCTAC ACATTGTTCT AGGTGCTGGA TATAGAGCAC 1740

AAACTGCTCT TGTGGGGCTT ACAGTGAGGT ACGCTGTGAC AATATGGGAT GTCATTCTCA 1800

TGGGAGTGCA AGGGTAAAAT AAAGCTCTTA TGATGTTTAA TACAGAATAC TGGTTATGGA 1860

ATTTTAACTT GATTTCTTGT ATTTTCTGTG CATTTTTAAC CTGTAACTCA TTCTCACAGT 1920

CCTCAGCCAA GAAAATGCAG CCTCTGAGAC TGTTAAGTAA TTTCCCCACT GTGTTATAGC 1980

TACTGTATGG CAGAGCCGGA ATTTGAAACC AGATCTATTT GACCCTAGAA GATGTGACCA 2040

TGAGATGTTA ATTTTGAGGA TAACTTTTTT AGTATTATGG AATTTTCAAC ATATATTTTT 2100

TAGGACCAAA GATAAACTAG GCACAGAGTC TACTCTTTGC ATAAATTATT TAAAAGAGCT 2160

TCGCGCTCCA TTTTGTCATC TAAGCACTGT AAAATTCTCA CAAGACTAAT _T CTTCTTTTT 2220

AGGAACGATA TAGTTGTAAA CTTTCTATTT TTTTTCTTTT TTTTTTCTCC CTCCACCATC 2280

CAAGTAGTTG TGAATTTTCT AGAGCCAAAA TAGAACACTA TAGATTATCT TTTAAACCCT 2340

TTATTGAAGC AGAGGATAAT GCTGTGACCG ACTTAACTTT ATGCTTTCTA AGAGATATTG 2400

ATATAGTAGA GAAATGCAGT AGTTATGCAT CTCCGTTTGC TTTTACATCA TAAATCAAGA 2460

ATATTATGAA ACCATCTCCC AGAGATATAT GTGATACACA GATCTTGGCT GTTTTTTTTT 2520

TTTACAAAAG TAACATCTAT GCTATTGATA CATATAAGTG GGTTTGTAAG ACAGTCTATG 2580

TGTAAATGTG AAAAAAGGAA GAATTTCCAG TTCTTCTCAT TTTCATTTAG ACCAGTAATG 2640

AATACAGTGA AGCTAAAGGA CATCTTCCAT CCTTCCTCGC TTTTATAGGG AGAGGAAAGT 2700

TGTATCACTT CTTGAGTAAA AAGAATTGTG ACGATCTTTT ACAAACAATG CCTTAAAAAT 2760

TATTATTTTT GAATGATATG TGGTAGTGGG ATCCACAATA GTCTCATTTG GTTATACAAA 2820

TAAATTTTAT GTATTCATGT ATGTGTTTTG ATTAGGTATA AAATTAGTGG CTGAATATCC 2880

ATTCAAGCTT AATTTTGTAT TTCTATCACT TTTGTAGATT TTGAGCAAGA TTAAAAATAT 2940

AAACAATAGG CCAGGCGCAG GGGCTCACGC CTGTAATCCC AGCACTTTGG GAGGTCTAGG 3000

TGGGCGAGTC ACGAGGTCAG GAGATCAAGA CCATCCTGGC TAACACATTG AAACCCAGTC 3060

TGCTACTAAA AATACAAAAA ATTAGCTGAG CGTGGTGGTG GGCACCTGTA GTCCCAGCTA 3120

CTCAGGAGGC TGAGGCAGGA GAATGGTGTG AACCTGGGAG GCAGAGCTTG GAGTGAGCCA 3180

AGATGGAGCC ACTGTACTCC AGCCTGGGTG ACACAGTGAG ACTCCATCTC AAAAAAAATA 3240

AAAAATAAAT AAAAATAAAC AATAATATTG TTTGCATTAC TATGGCTATA TAGCAAATTG 3300

CCTTAAAACT TAGGGGCAGA AAGCAATTTG TTTTGGTCAC AGGTTCTGTG AGTAAGGAAT 3360

TCAGGCTGGG GACAGTGTGG ATGTCATGTT TCTGCGTCAA AATGACTGGT ACCTCACCTG 3420

GAAGACTTGA GCAACTAGGT ACTGGCACAG CTGGAGCTCG TTGGGCATCT CTGTATGTTT 3480

GTTCCATGTG GTCTCACCAG CATGGTGATC CAGGGTAGGT AAATTGTTAC ATGCTGGTTC 3540

AGGACTCCGA AGGCACATGT CCTAAGAGAG AGAACCAAGT GGAATCTATA GTGCGTTGTA 3600

TAATCTTTTA GAATTACATA GTTTCAGTTG TACCTGTGCA ATTATTGATA GAGACAGTTA 3660

ATCAGTGTGA GGGAACACAG ACCCTTGCCC AGGTCCAAGG TGAGGGAACC CTCTGTACCT 3720

GTCAGTGGAA TAATGTTAAT GTCACATTAT AAGAAGAGCC TGACGGGGCT GGGTAGAGTG 3780

GCTCACACCT GTAATCCCAG CACTTTGGAA GACCAAGGCG GATGGATCAC TTGAGGCCAG 3840

GAGTTCAAGA CCAGCCTGGG CGACATGACA AAACCCTGTC TCGACCAAGA AAACATAGAA 3900

TTAGCCAGGT ATGGTGGCGC ACTTCTGTAG TCCCAGCTAC TTGGGAGACT GAGGTAGGAG 3960

GAGTGCTTGA ACCTGGGAGG TGGAGGTTTC AGTGAGCCAA GATTGCGCCA CTGCACTCCA 4020

GCCTGGGTGA CAGAGCAAGA TTCCATCTCC GAGAGAAAAA AAAAAAAAAA AAAAAAAGAG 4080

CGTATGAGAT AGGGTCATCA TTGAAACTAA GTTTCCCACA AAAATATAAA CAACACTTTC 4140

AATTTAAACA TACTTTTAAA AATATTGAAA TATTTATATG TAGCTTTTTA ACTGAAAATC 4200

AATTTTCTTT TCTTTTACAG 4220

(2) INFORMATION FOR SEQ ID NO:67:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3507 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:

GTAACTATGT TAGAGTTTGA CAAGTAGAGT ATGGCTAATG TAAGCTCATA AATCATAGTG 60

ATAGTAAGAA TTATCTCTGC TCATCATTTC CTGAGCATTT GTACCTGTGG ACTGGCGAAA 120

TTAGATGCTA AAACTAGCAT CTAATGATTT TCCTCCTCTA TATCACAGTT AATATCCATT 180

ATATTTTACT TCTTTGGTGA AAATATTTAA ATTTTAATGT TTTAGGCACT TGTATGGCAG 240

AATTTATTTT TAAAGTTTAG GACATTGTGT AATATTGGGA GAAATGAAGG ATATTGAGAA 300

ACTTTAGGAG ATACTCCAAG TTGAAAAGGT AAATAAAATA TTATTTGCTA TTATACTTAG 360

CAAATATGTG CACAGGACTT GTGGTCTTAA TATAAATGGA ACATGTAAGT ATTTCTCAGT 420

TTCCTGTTTG GAGGATAAAT GACATGATTA TAATCCATTT TAGAAAGGGT CAAATATGTT 480

TAAAAGAAGA GGCAGAAATT GCTTTATCTG TTGTGTAATT AAATTGATTA CATTTATTTT 540

TTGTGCCTTT TAGGTGAATT TTCTTACATG GCTTATTAAA GATAAGTGGA AAAATGATGT 600

TTAGCATTTT GGGGGAAATT ACCACTGTCA AAATTTATGG AGTTAATGGT TAAAAAATCA 660

CTTACTAAAT AAAAAAATTA ACTGGGTGTG GTTGTGCATA CCTGCAGGCC TAGCTACTTG 720

GGAGGCTGAG ATGGGAGGAT CACTTGAGCC CTGAATGATG GAGCAGCACT GCACTCCAGC 780

CTGGGCCACA GAGCAAGACC TTGTCTCCAA AAAAAAAAAA AAAAAAGAAG GTTACTATTA 840

AAATAATTAG CAGGCTGGGG GCGGTGGCTC ACACTTGTAA TCCCAGTAAT CCCAGCACTT 900

TGGAGGCCAA GGTGTGTGGA TCACTTGAGG TCAAGAATTG GAGATCAGCC TGGCCAATAT 960

GGTGAAACCC CGTCTCAACT AAAAATACAA AAATTAGCCG AGTGTGGTGA CATGCGCCTG 1020

TAATCTTAGC TACTCAGGAA GCTGAGTCAG GAAAATCACT TGAGCCCAGG AGGCACAGGT 1080

TGCAGTGAGC ACTATTGCAC TCCAGCCTGG GTGACAAGAG CGAGACTCCA TCTCAAAACA 1140

AATAAATAAA ATAAAATAAT TCACAATGTC ATGTTTTAGC TGACATTGTG AATTTTAGTA 1200

ATCTTTTTTT AACCTTTAAC TCCATCCTGA GTTACATTGA CCAAAGAAAT CAGTATCTAG 1260

AATTATATCA GGGAACTACT AACAGGGTTA ATAAAATGAA TAAAGAACAT GACTTCACAA 1320

AGGTTATAAT TCACATAGCT AATAGATACA GGAAGAGATA TTCACTGTCA CTAATAAAGA 1380

CTTTCAAAGT AGAAAGATAA CATTTCATTC TGTTTTTTTT GAGATGGAGT CTTGCTGTTT 1440

CACCCAGGCC AGGGTGCAGG GGCGTGATCT CAGCTCATTG CAGCGTGTGC GTCCCAGGTT 1500

CAAATGATTC TCCCGCTGTG GCCTCCCAAG TAGCTGGGAT TACAGATGCG CACCACCACA 1560

CCTGGCTAAT TTTTTGTATT TTTAGTAGAG ACGGGTTTCA CCATGTTGGC CAGGCTGGTT 1620

TCCAACTCCT GACCTCAGGT GATCCACCCG CCTTGGACTC CCAAAGTGCT GGCATTACAG 1680

GTGTGAGCCA CCATGCCTGG CCAACATTTT ATTCTTATCA TTGGGAAAAT TTGAAGTCTG 1740

GTATACCAAG TTTGGTCACT GTACAGGGAA ACAGGAACTC TATTTTTTTT ATTTTTCAGT 1800

TCTTTTTTTT TTTTTTTTTT TTTTTTTGAG ATGGAGTCTC ACTCTGCTGC CCAGGCTGGA 1860

GTGCAGTAGC TCAATCTCTA CTCACTGCAA CCTCCACTTC CCAGGTTCAG GTGATTCTCA 1920

TGCTTCAGCC TCCCGGAGTA GCTGGGATAA AGGCACATAC CACTATACCT GACTAATTTT 1980

TGTATTTTTT GTGGAGACCA GGTTTCACCG TGTTGACCAG GCTAGTCTCG AACTCCTGAC 2040

CTCAAGTGAT CTACCTGCCT CGGTCTCCCA AAGTGCTGGG ATTACAGGCA TGAGCCACTG 2100

CGCTCAGGCA GGAACTCTAT ATTGCTGGTG TACATTGGTG AGAGTCAAAA TTGACACAAC 2160

TACTTTACTA GCAAATTTGG TGGTATTTAG TAATATTGAA GGTGCACATT CTCTTACTGT 2220

ACTTCTTGGA GTAGTCCCCA AAGAAACTCC TGCACACATG TATAAGGATG TTTTCATTAC 2280

AACATGTTTT GTTATCATGG AATATTAGAA ACAACCTAAA TTTCCATTGG TTGGGGAGTG 2340

AATGCAAAAA GTCATTGTAT GTTCATATGA AAGAATGTTT TTAGCAATTA AAATGAATAT 2400

ATCTTACATA TCAACATTAA TGTCAGAAAC ATTATTGAGT GTGAAAAAGC AAGTTGCAGA 2460

ATACCACTGA AGTATGATAG CATTTATATA AAATGTAAAA ACACGTAATA AGATATTGCT 2520

TATTGTTTAC ACATACATGT GTATGTGTAG TAAGTGTGAA AACATAGGAA GGATTAAGAC 2580

CAACTTTGGA ATGGTTTTTA TCTTTGGGGT AGAAGGGTAA GGATGGGATT AGGGAGGAGT 2640

ATAAAATGGT AATTTTGACT GTTTCTTTTT CTTTTTCTTT TTCTTTTTTG AGACAGAGTC 2700

TCGCATTGTC GCCAGGCTGG AGTGCAGTGG COTGATCTCG GCTCACTGCA ACCTCCGCCT 2760

CCCAGGTTTA AGTGATTTTC CTGCCTCAGC CTCCTGAGTA GCTGGGATTA CAGGTGCCCG 2820

CCACCACGCC CAGCTAATTT TTTGTATTTT TAGTAGAGAT CGGGTTTTAC CATGTTGGCC 2880

ATGCTGGTTT CAAACTCCTG ACCTTGTGAA TCTCCCACCT CGGCCTCCCA AAGTGCTGGG 2940

ATTACAGGTG TGAGCTACTG CGCCTAGCCT TGACTGCTTT TATAGTGTTG CTAGTTTAAA 3000

AAAAAATCTG AAGTGGCAGG AGGAGGTGGC TCACACCTGT AATCACAGTG TTCTAGGAAG 3060

CCAAAGTAGG AGGATCACTC AAGCCCAGGA GTCTGCGGTG AGCTGTGATC TTGCCACTGA 3120

ACTCCAACAT GGGTGATAGA ACGAAACCCT ATCTCTTACA AAAACAAAAA CGACAAAATT 3180

TATTTAATAT ATTAACATTT AAAAAATCTG GCAGTGAACC AACGTGAATG TTGGTTAGGT 3240

TACTCTTGTT AATTTTGGTT TGTATTTTCA AATATTTCAT AGTTAACAAA TACTTTAGGT 3300

AACCTAAACA AAATGGATTA GGAGGATCAG AGGAATATAC CAATCTGTAA GAAATTAAGC 3360

TAGTCAGAGA CATGAGTTGT GATTTTATTT CACTGTCTAA AAGTAATATA ATTTAATGCG 3420

ATAATATTGA TTTACTTTTG AATACTTACT TTTGTATACT TTAGCCTTAT GTTAATTATG 3480

AAATATCTTG TTTGTCTTTA ATACCAG 3507 (2) INFORMATION FOR SEQ ID NO:68:

(l! SEQUENCE CHARACTERISTICS:

(A) LENGTH: 9837 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(il) MOLECULE TYPE: DNA (genomic)

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

GTGAGCCTAA CATCAATCTT GGCCTTTACT AACCTCAAAA TGCTTCAGAT GCTAGAAACA 60

GGGTTTGTGC TAAGCTTAGG CACTCATTAG AGTGATGAGA GCTGCCAGGG AGCAGTGATC 120

AGTCAGTCCT CATGAAGCAA AACCCAGGGT TGTTTTGTTT TTTGCCTTTT TTGAGGGGGA 180

GGGGGTGGAA TTTAAGGGTG GGAAACAGGG CAAGGGATTT TGATTCTTTT TATTCCCTCT 240

CCTATTTGTA CATTTTGGTG TAAACCTGAA ATTGATTTCT TACCAAAGGC CTGTTTCTGG 300

GACAGGCAGT GTCCTCAGGA GTCTGGCTAA TGGGAGAAGT TGACATTTTT GACATTGCAG 360

TTCAATAGTC ATATTAGCAC AGATGTATGT GGCAACAGCC ACCTCATTCT AAGAAGGGGA 420

AGGAAGCTTG AGTCAGGCCT TAATGTTGAA AAGTCAGGGA GCTGTTGAGG TATGGAAGGG 480

CACTCAGCAG GAAGCAGGTT AAGGGGAAGA AAACAGTGTC CTTGAGGCAG ACAGTGATTC 540

AAAGCTTAAT TACGGGCATC ATGCTATGTT AGCGAGTGGA ACTGGATTGT GACGGCCCTT 600

ACATAATGAG ATTTTTATTG ATAAAGGTTG CTTAGAGGCT GGGCGTTGTG GCTCACACCT 660

GTACTCCCAA CACTTTGGGA GGCCACAGTG GGCAGATCAC CTGAGGTCAG GAGTTCATGA 720

CCAGGCTAGT CAACACGGTG TAAACCTCAT CTCTATTAAA AATACAAAAA TTAGCTGGGT 780

GTGGTGGAAT GCACCTGTAA TCCCAGCTAC TCGGGAGGCT AAGGCAGGAA AATAGCTTGA 840

ACCCAGGAGG TGGAGGTTGC AGTGAGCAGA GCATTGCGCC ATTGCACTCC AGCCTGGGTG 900

ACAAAAGCGA AACTCACTGT CTCAAAAAAA AAAAAAAACC GGTTGCTTAG AAATACACAT 960

TTTTTTTTGG CCTGAACTCT TCAAAAAAAG GTCAGTATGG TAAGAGGACG GGGAAGGTTT 1020

CGTAGAGGAG ACTAGGGAGA CACGACATCC AAATGCAATG CATGATTCTT GACCCTGCAT 1080

AGGAAATCGT CGTTATAAAG GACATTTTGA GGAAAATTTG AATGTGGGCT TTAGTGTATT 1140

TTTTTTTTTA AAGTTTCTTT GGTGTTGATG ATGTCTAGCA GATTATGTAG GAGACTGTGC 1200

TGAAAAGTAT TCAGAGGTAA AGTGTCCCAG TGTCTGCAGC TTACTTTCAA ACGGGTTGGT 1260

TGCAATATAT TTAGGTAGGG AGAGAGTGAA AGTAACTCTT AGACATTAAT GATTGATAAG 1320

TGGCTGTTCA GTGTACTATT TTTTTCAACT CTTTGTAGGC TTGCAATCTT TTAAAAAGTT 1380

GAGGAAAACA GTCCGGGTGC AGTGCCTCAC GCCTGTAATC CCAACATTTT GGCAGGCTGG 1440

GATGGGAAAA TTGCTTGAGG CCAGAATTTG GAAAACGGCT CAGGCAACAT AAAACCCCAT 1500

CCCTACAACA AATAAAAATT AGCTGAGCAT GGTGCCATGC ACCTGTAGTT GTATCTACTC 1560

AGGAGGCTGA GCCCAAAATT TCAAGGCTGC GGTGAGCTAT GGTCGTGCCA CCACACTCCA 1620

GCCTGGGCAA TAAATTGAGA AACCCTGTCT GTTTGGAAAA AAAAGTTGAG GAAAACAATT 1680

AAACAATAAC AGCAAAAATC TGTTATAAAA TGTAATAATG GGCCAGGTGT GGTGGCTCAT 1740

GCCTGTAATC CCACCACTTT GGGAGGCCGA AATGGGTGGA TCACCTGAGG TCAGGAGTTC 1800

AAAATCAGCT TGGCCAACAT GGTGAAACCC CATCTCTGCT AAAATTACAA AAAAATTAGC 1860

TGGGTGCGGT GGCGCACACC TGTAATCCCA GATACTCAGG AGGCTGAGGC AGGAGAATCG 1920

CTTGAACCCA GGAGGCGGAG GTTGCAGTGA GCCGAGATCG TGCCACTACA CTCCAGCCTG 1980

GGCAACAGAG CCAGACTCTG TCTCAAAAAA AAAAAAAAGT TTAATTCACG CAGAGCCAGC 2040

TGAACGGCAG ACAGGAGTTT GGTTATTCAA ATCAGCCTAC CAGAAAATTC GGAGACTGGG 2100

GTTTTTAAAG AATGACTTGG CGGGTAGGGG GCCAGGGATT GGCGAATGCT AATTTGTCAG 2160

GTGGGAGGTG AAATCACAGG GGGTTGAAGT GGGCTCTTGC TGTCTTCTGT TACTGAGTGG 2220

AATTGCAGAA CTTGTTGAGC CAGATTATGG TCTGAGTGGC GCCAGCTAGT GCATTGGAAT 2280

GCGCGGTCTG AAAAGTATCT CCAGCACCAA TCTTAGGTTT TACAATAGTG ATGTTATCCC 2340

TGAGAGCAAT TGGGGAGGTC AGGAATCTTA TAGCCTCTGG CTGCAAGCCT CCTAAATCAT 2400

AATTTCTAAT CTTGTGGCTA ATTTGTTAGT TCTACAAAGG CAGACTGATC CCCAGGCAAG 2460

AATGGGGTTT GTTTTTGGAA AGGACTGTTA CAATCTTTGT TTCAAAGTGA AATTAGAAAT 2520

TAAATTCCTC CTGTAGTTAG TTAGGTCTTC GCCCAGGAAT GAACAAGGGC AGCTCGGAAG 2580

TGAGAAGCGT GGAGTCATTT AGGTCAGATC CCTTGCACTG TCATAACTTT CTCACTGTTA 2640

GGATTTTTGC AAAGGCAGTT TCGTGAACGT ACAGAGACAG GCCCTTGCTA TTATCCCTAT 2700

TTTTTAGATA AGGATATCCA GGCGATGAGG AAGTTTTACT TCTGGGAACA GCCTGGATAC 2760

GAAACCTTCA CACGTCAGTG TCTTTTGGGA CATTTTCTCG TCAGTACAGC CCTGTTGAAT 2820

GTTCTCACGG TGGGGAGGTA CGTGTTTAAA ATGCGGGGAA GGTGCTTTTA TTTCACCCCT 2880

GGTGAAACTA GGGGAGCTAA TTTTTTTAAA CATGATTTTT GGCCCCCTTG AACCGCCGGC 2940

CTGGACTACG TTTCCCAGCA GCCCGTGCTC AAGACTACGG GTGCCTGCAG GCGGTCAGAG 3000

TCGTTTGCGG CGGCGCAGGC GCGGTGCGGG CGGCGGACGG GCGGGCGCTT CGCCGTTTGA 3060

ATGGCTGCGG GCCCGGGCCC TCACCTCACC TGAGGTCGGC CGCCCAGGGG TGCGCTATGC 3120

CGTCGGGAGG TGACCAGTCG CCACCGCCCC CGCCTCCCCC TCCGGCGGCG GCAGCCTCGG 3180

ATGAGGAGGA GGAGGACGAC GGCGAGGCGG AAGACGCCGC GCCGCCTGCC GAGTCGCCCA 3240

CCCCTCAAAG CCGAATTCTG CAGATATCCA TCACACTGGC GGCCGCTCGA GCATGCATCT 3300

AGAGGGCCCA ATTCGCCCTA TAGTGAGTCG TATTACAATT CACTGGCCGT CGTTTTACAA 3360

CGTCGTGACT GGGAAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG CACATCCCCC 3420

TTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT CGCCCTTCCC AACAGTTGCG 3480

CAGCCTGAAT GGCGAATGGA CGCGCCCTGT AGCGGCGCAT TAAGCGCGGC GGGTGTGGTG 3540

TTACGCGAGC GTGACCGCTA CACTTGCCAG CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT 3600

CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TCCCCGTCAA GCTCTAAATC GGGGGCTCCC 3660

TTTAGGGTTC CGATTTAGTG CTTTACGGCA CCTCGACCCC AAAAAACTTG ATTAGGGTGA 3720

TGGTTCACGT ATTGGGCCAT CGCCCTGATA GACGGTTTTT CGCCCTTTGA CGTTGGGAGT 3780

CCACGTTCTT TAATAGTGGA CTCTTGTTCC AAACTGGAAC AACACTCAAC CCTATCTCGG 3840

TCTATTCTTT TGATTTATAA GGGATTTTGC CGATTTCGGC CTATTGGTTA AAAAATGAGC 3900

TGATTTAACA AAAATTTAAC GCGAATTTTA ACAAAATTCA GGGCGCAAGG GCTGCTAAAG 3960

GAAGCGGAAC ACGTAGAAAG CCAGTCCGCA GAAACGGTGC TGACCCCGGA TGAATGTCAG 4020

CTACTGGGCT ATCTGGACAA GGGAAAACGC AAGCGCAAAG AGAAAGCAGG TAGCTTGCAG 4080

TGGGCTTACA TGGCGATAGC TAGACTGGGC GGTTTTATGG ACAGCAAGCG AACCGGAATT 4140

GCCAGCTGGG GCGCCCTCTG GTAAGGTTGG GAAGCCCTGC AAAGTAAACT GGATGGCTTT 4200

CTTGCCGCCA AGGATCTGAT GGCGCAGGGG ATCAAGATCT GATCAAGAGA CAGGATGAGG 4260

ATCGTTTCGC ATGATTGAAC AAGATGGATT GCACGCAGGT TCTCCGGCCG CTTGGGTGGA 4320

GAGGCTATTC GGCTATGACT GGGCACAACA GACAATCGGC TGCTCTGATG CCGCCGTGTT 4380

CCGGCTGTCA GCGCAGGGGC GCCCGGTTCT TTTTGTCAAG ACCGACCTGT CCGGTGCCCT 4440

GAATGAACTG CAGGACGAGG CAGCGCGGCT ATCGTGGCTG GCCACGACGG GCGTTCCTTG 4500

CGCAGCTGTG CTCGACGTTG TCACTGAAGC GGGAAGGGAC TGGCTGCTAT TGGGCGAAGT 4560

GCCGGGGCAG GATCTCCTGT CATCCCACCT TGCTCCTGCC GAGAAAGTAT CCATCATGGC 4620

TGATGCAATG CGGCGGCTGC ATACGCTTGA TCCGGCTACC TGCCCATTCG ACCACCAAGC 4680

GAAACATCGC ATCGAGCGAG CACGTACTCG GATGGAAGCC GGTCTTGTCG ATCAGGATGA 4740

TCTGGACGAA GAGCATCAGG GGCTCGCGCC AGCCGAAACT GTTCGCCAGG CTCAAGGCGC 4800

GCATGCCCGA CGGCGAAGGA TCTCGTCGTG ACCCATGGCG AATGCCTGCT TGCCGAATAT 4860

CATGGGTGGA AAAATGGCCG CTTTTCTGGG ATTCATCGAA CTGGTGGCCG GGCTGGGTGT 4920

GGCGGACGCT ATCAGGACAT AGCGTTGGCT ACCCGTGATA TTGCTGAAGA GCTTGGCGGC 4980

GAATGGGCTG ACCGCTTCCT CGTGCTTTAC GGTATCGCCG CTCCCGATTC GCAGCGCATC 5040

GCCTTCTATC GCCTTCTTGA CGAGTTCTTC TGAATTGAAA AAGGAAGAGT ATGAGTATTC 5100

AACATTTCCG TGTCGCCCTT ATTCCCTTTT TTGCGGCATT TTGCCTTCCT GTTTTTGCTC 5160

ACCCAGAAAC GCTGGTGAAA GTAAAAGATG CTGAAGATCA GTTGGGTGCA CGAGTGGGTT 5220

ACATCGAACT GGATCTCAAC AGCGGTAAGA TCCTTGAGAG TTTTCGCCCC GAAGAACGTT 5280

TTCCAATGAT GAGCACTTTT AAAGTTCTGC TATGTGGCGC GGTATTATCC CGTATTGACG 5340

CCGGGCAAGA GCAACTCGGT CGCCGCATAC ACTATTCTCA GAATGACTTG GTTGAGTACT 5400

CACCAGTCAC AGAAAAAGCA TCTTACGGAT GGCATGACAG TAAGAAGAAT TATGCAGTGC 5460

TGCCATAACC ATGAGTGATA ACACTGCGGC CAACTTACTT CTGACAACGA TCGGAGGACC 5520

GAAGGAGCTA ACCGCTTTTT TGCACAACAT GGGGGATCAT GTAACTCGCC TTGATCGTTG 5580

GGAACCGGAG CTGAATGAAG CCATACCAAA CGACGAGCGT GACACCACGA TGCCTGTAGC 5640

AATGGCAACA ACGTTGCGCA AACTATTAAC TGGCGAACTA CTTACTCTAG CTTCCCGGCA 5700

ACAATTAATA GACTGGATGG AGGCGGATAA AGTTGCAGGA CCACTTCTGC GCTCGGCCCT 5760

TCCGGCTGGC TGGTTTATTG CTGATAAATC TGGAGCCGGT GAGCGTGGGT CTCGCGGTAT 5820

CATTGCAGCA CTGGGGCCAG ATGGTAAGCC CTCCCGTATC GTAGTTATCT ACACCGACGG 5880

GGAGTCAGGC AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA 5940

TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT GATTTAAAAC 6000

TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT TGATAATCTC ATGACCAAAA 6060

TCCCTTAACG TGAGTATTCG TTCCACTGCA GCGTCAGACC CCGTAGAAAA GATCAAAGGA 6120

TCTTCTTGAG ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG 6180

CTACCAGCGG TGGTTTGTTT GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT 6240

GGCTTCAGCA GAGCGCAGAT ACCAAATACT GTTCTTCTAG TGTAGCCGTA CGTAGGCCAC 6300

CACTTCAAGA ACCTCTGTAC CACCGCCTAC ATACCTCGCT CTGCTAATCC TGTTACCAGT 6360

GGCTGCCGCC AGTGGCGATA AGTCGTGTCT TACCGGGTTG GACTCAAGAC GATAGTTACC 6420

GGATAAGGCG CAGCGGTCGG GCTGAACGGG GGGTTCGTGC ACACAGCCCA GCTTGGAGCG 6480

AACGACCTAC ACCGAACTGA GATACCTACA GCGTGAGCTA TGAGAAAGCG CCACGCTTCC 6540

CGAAGGGAGA AAGGCGGACA GGTATCCGGT AAGCGGCAGG GTCGGAACAG GAGAGCGCAC 6600

GAGGGAGCTT CCAGGGGGAA ACGCCTGGTA TCTTTATAGT CCTGTCGGGT TTCGCCACCT 6660

CTGACTTGAG CGTCGATTTT TGTGATGCTC GTCAGGGGGG CGGAGCCTAT GGAAAAACGC 6720

CAGCAACGCG GCCTTTTTAC GGTTCCTGGC CTTTTGCTGG CCTTTTGCTC ACATGTTCTT 6780

TCCTGCGTTA TCCCCTGATT CTGTGGATAA CCGTATTACC GCCTTTGAGT GAGCTGATAC 6840

CGCTCGCCGC AGCCGAACGA CCGAGCGCAG CGAGTCAGTG AGCGAGGAAG CGGAAGAGCG 6900

CCCAATACGC AAACCGCCTC TCCCCGCGCG TTGGCCGATT CATTAATGCA GCTGGCACGA 6960

CAGGTTTCCC GACTGGAAAG CGGGCAGTGA GCGCAACGCA ATTAATGTGA GTTAGCTCAC 7020

TCATTAGGCA CCCAGGCTTT ACACTTTATG CTTCCGGCTC GTATGTTGTG TGGAATTGTG 7080

AGCGGATAAC AATTTCACAC AGGAAACAGC TATGACCATG ATTACGCCAA GCTATTTAGG 7140

TGACACTATA GAATACTCAA GCTATGCATC AAGCTTGGTA CCGAGCTCGG ATCCACTAGT 7200

AACGGCCGCC AGTGTGCTGG AATTCGGCTT AAAGGTAGGC GGATCTGGGT CGACTCTAGG 7260

CCTAAATGGC CATTTAGGTG ACACTATAGA AGAGCTCGAG GACAACAGAA AATCTTAGTG 7320

AACATGTTTT ATGGGAAAAT TTTATATACA ACATCAAAAG CACAATCCGT AAAATACTGT 7380

TAAAATGGAT TTTATCAAAA TGAATAATTT CTGCTATTTG AGACACTGTT AAGAGAATTA 7440

AAAAACCAGC CATAGACTAT TAGAAAATCT GTACACGTTC CATATCTGAT GAAGCATTTG 7500

TATATCTACA GTATCTAAAG AATTCTCAAA ATTCAGTAGG AAAACCACCA AATGTAAAAG 7560

TGGGCAAAAG ATTTGAACAC ACTTCACCCA TTACATGCCT GTTAGAATGG CTAAAATCCA 7620

AAAAGTGACA AATCGTAAGT TCTGACAACA ATGTGGAACA ATTTTACATA TTGCTGGTGT 7680

GAACGCAAAA TGGCATCGCC ACTGTGGAAA GTTGTTTCTT AAACATACCA TTATACAACC 7740

AGCAATCTCA TTCCTAGGTA TTTACACAAA TGAAATGGAA ACTTATGTTT AGACAAAATC 7800

ACGTACATGA CTGTTTATAG TGACTTTCTT CCTAATTGCC AAAAAGTGGG AAACAACCCA 7860

AACGTCCTTC AGCTGGTGAA TGCATATAAA TAAGCTGTGG TGCATCCAGA CAATCGACTG 7920

CTACTTTGCA ATAAAAAGGA ACTGATATAT TCAATGTAGA TAAATCTCAA ATGCATCAAT 7980

GCTTAAGTGA AAGACACTGG ATTCAGTAGG CTACTTATGA TTCCATTTCT GTGACATTGT 8040

GGAAAAGGCA AAACTATTGG ACAAGAACAT CAGTGGTGGT TTGGGATAGG CTGACAAGGG 8100

AGTATGAGGG ATTTTTTCAG AGGAACAGTT TTATCCGACT GTAGGTATTT CTAGCACAGA 8160

ATTGGGAGTC TGTCCAGTAA AATGATAGCG ATTATTAGAC TCTTGGTTGG AGAAAGATTT 8220

GTCATCTTGA CGTAATAGGT GATAGCTGAA ACTTACGGGG AGAATATTAC AAAGCAAGGA 8280

GGGGGAGAAT ATTCCCAAGC AAGAAGTAGC TTATGTCTAG AACCAATCTA TAACGTACTA 8340

ACATTTAGAC TACTATGAGG GGATAATTAT CAAATACTAT ACAAGATCAG TTAAGATGAA 8400

GACTGATCAT TAGTGATACT TGACAGAGCA GTGTCAGTGC ACTGGTATGA CTTGTTGAGA 8460

AATAAATTAT GGTAGCATTG CTTATACACA ATTAACGATG TATACAGTAA GACAGTGTGA 8520

GAAATATTCA AGCAAATGGG AGACCGCAGA GATACCAAAT GCAGACCAGA CTCTTAGGAG 8580

GCAAGAAGGG GGCTAGAAAA AGAATTGAAG GAAAGCTTTC TTCAGATGCT TAAGATTTTG 8640

TGGCCAGGTG CAGTGGCTCA TGCCTGTTCC CAGCACATTA GGAGGCCCAA AGCAGGAGGA 8700

TTGCTTGAGC CCAGGAATTC AAGACCAGCT TGGACAACAT AGTGCAACCC CATTTCTATT 8760

GGTAATTAAA AAAAAAAAAA AATGAAAAAC ACTTGTGAAG GTACATCTGT TGATAATAAA 8820

GAACACTGAT TTTCATTAAA ACCCCCAAAA CATTTATTAC TTTAAAGAAT AAAAATAACA 8880

AGTGTCATGA TAAAATATGT CTGGGATTTG TTTTAAAATA ATCTGGGGAA TGGAAGTGAA 8940

TCAGAGTATA AATCAAGCAA GGCTGGCCAA ACATGCTGAA GTAGAGGAAT AGGTATGTGA 9000

GGATGCATTA TGCTTCTCTA CTTTTGTATG TTTACAATTT CCCTATAATA GATATCTGTG 9060

AATTTGCTTA GTATGCTTTC TGTAAGCAAA CATGGATGAA GCAGCACATG AAAAAGAATT 9120

TTAACCAACA AACTAGCAGA AATAATGTGA CAGACGACTT TTAGAGGCTT TGGAGAAACT 9180

GAATGCTAAA GGTGCTGTAC AGCCAGCCCC AGTCTTTCTG ACATTCTGGC AGTGTCTTTC 9240

TCAATTGCAG CTCCTCATCT GAGCCACTGT CCAGAAAATA ATTTGAGTAA CTTTAATCCT 9300

CAATTCTCCC AAGGATAGTA CCATTCTAGA TCTTACTAAT TTATTAGCTA CAATGGATAC 9360

CTTAGGGGGG GATTAAGGCC TACTTTTCTA GTGAAATCCC AGTTGAGAAT GGCTGCTAAA 9420

AACTGAGTAA CATTAGACTG AAAGAAAGGG AATATTGTAT AAAGTTGTAC TTTGAAAAAG 9480

AGAAAAAGAT GTGTCTAAGT GACTATCAGA TAGCAATGTA ATGCTCCCTA ATTGTAAAAA 9540

AAATCACAAA TTTGTGAACT CACGAATTAT AGACATGTAT AATTGACCTA CAGGTCAAGA 9600

AGTGCCTGTG GAAGAGCTTG TTAAAAATAG AACTACTCAG CCCCTTCTCA AATAGCCATC 9660

GGCCTCAGCC ATCTGGAAAG TAAAGTTGGC AGGTTATGTA ACTTAGTGTT TCTTTTACTC 9720

TGTAGATGTG TTCAAACTCT TCCAGGTAAA CTGCTTAACT CATTTGAGAT TCTTTGACTA 9780

ATACTGAGCT ATGTGCATTT GCATTTTGAA AAATTATGTA TCTTTTTCCC ACCATAG 9837 (2) INFORMATION FOR SEQ ID NO:69:

(l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (3) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69 CTCTGTAACT GCTTATAATC CTG 23

(2) INFORMATION FOR SEQ ID NO: 70

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE- other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70: CTAGGAAACC TGTACAACTC C 21

(2) INFORMATION FOR SEQ ID NO:71:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71: GGCTTATTGT GTGCTGATAT C 21

(2) INFORMATION FOR SEQ ID NO:72:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION SEQ ID NO:72 :\GAGATCCTT AAGTCGTCAT G 2 ₁

(2) INFORMATION FOR SEQ ID NO.73:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE- nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY: linear

(in MOLECULE TYPE- other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73: CAGTTTCTGT GAGAGAGTAC A 21

(2) INFORMATION FOR SEQ ID NO: 74:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74: GGCTTACCTG CTCCTGTATT T 21

(2) INFORMATION FOR SEQ ID NO: 75:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75: GAGGAGGAAT GGGCCTTTAT T 21

(2) INFORMATION FOR SEQ ID NO: 76:

(li SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY: linear

(in MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION- SEQ ID NO-76 AACCCACAGA ATAGGGCAGG A 21

(2) INFORMATION FOR SEQ ID NO.77

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH- 22 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION- SEQ ID NO: 77: GGATACTGGC ATTCTGTGTA AC (2) INFORMATION FOR SEQ ID NO .78

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION SEQ ID NO: 78: ATTTCCAGAT AGTAAGCCCC A 21

(2) INFORMATION FOR SEQ ID NO: 79-

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79- AGCTTGGACG GAAGTCAGAT C 21

(2) INFORMATION FOR SEQ ID NO: 80-

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY: linear

(11) MOLECULE TYPE: other nucleic acid

(x ) SEQUENCE DESCRIPTION- SEQ ID NO: 80: TCTAGCCAAA CCTCGGGTAA C 21

(2) INFORMATION FOR SEQ ID NO: 81:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:81: AATTGTAAAC CTCTGCCC IB

(2) INFORMATION FOR SEQ ID NO: 82:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ll) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82: ATTTCCCAAG CTCATGCT 18

(2) INFORMATION FOR SEQ ID NO: 83:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83: AGCATGAGCT TGGGAAAT 18

(2) INFORMATION FOR SEQ ID NO: 84: (l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

[ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84 TGAAGACCTA TCTTTGCC (2) INFORMATION FOR SEQ ID NO: 85:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85: GTTCACAGAG CTCCTCACAC T 21

(2) INFORMATION FOR SEQ ID NO:86:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86: AGGCCACAGA GTCAACTATG G 21

(2) INFORMATION FOR SEQ ID NO: 87:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87

AGGTCCTATC ACCAAGGGTG T ₂₁

(2) INFORMATION FOR SEQ ID NO: 88:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88: GCTTAGTTAC TTCTTCAAGG C 21

(2) INFORMATION FOR SEQ ID NO: 89:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89: GTAGCTGTTC CCTTTCTCCT A 21

(2) INFORMATION FOR SEQ ID NO: 90:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90: CCTCAACACT CATGAGAGTG A 21

(2) INFORMATION FOR SEQ ID NO: 1:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION- SEQ ID NO-91 TGGTTTAGCA CACCTCTTCA C (2) INFORMATION FOR SEQ ID NO: 92:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(i ) MOLECULE TYPE: other nucleic acid

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 92: GCTTAGCACA AACCCTGTTT C 21

(2) INFORMATION FOR SEQ ID NO: 93:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93: TTCGCCGTTT GAATTGCTGC 20

(2) INFORMATION FOR SEQ ID NO: 94 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94 : ACCGGTTCAC ACCAACTAGG 20

(2) INFORMATION FOR SEQ ID NO: 95-

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY: linear (11) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION. SEQ ID NO: 95. GAGATAGGGT CATCATTGAA AC 22

(2) INFORMATION FOR SEQ ID NO:96:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96 : CATTAGCCAT ACTCTACTTG T 21

(2) INFORMATION FOR SEQ ID NO: 97:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97: GCTAATTTAA CTCTGTAACT GC 22

(2) INFORMATION FOR SEQ ID NO: 98 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98- CACTGCAGCA CAGACTAATG TGT 23

(2) INFORMATION FOR SEQ ID NO- 99-

SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY, linear

( i) MOLECULE TYPE, other nucleic acid

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 99: TCTCTCCCTT TAACTGTGGG TTT 23

(2) INFORMATION FOR SEQ ID NO: 100:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO: 100: GGAGTTGACG AGATTAATAC CTG 23

(2) INFORMATION FOR SEQ ID NO: 101:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 22 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO- 101- CATGACGACT TAAGGATCTC TT 22

(2) INFORMATION FOR SEQ ID NO: 102:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE-, nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

⁽ Xl ⁾ SEQUENCE DESCRIPTION: SEQ ID NO.102:

CTCAGTTTCC AGAGTACAAA C (2) INFORMATION FOR SEQ ID NO 103

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH- 22 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY linear

(n) MOLECULE TYPE other nucleic acid

(xi) SEQUENCE DESCRIPTION- SEQ ID NO: 103 GTGAATTAAA GTCTTTCTGG CC 22

(2) INFORMATION FOR SEQ ID NO 104

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE, other nucleic acid

(xi) SEQUENCE DESCRIPTION- SEQ ID NO: 104- ATCTTAGAAA GCAGACAGGG C 21

(2) INFORMATION FOR SEQ ID NO- 105:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH 21 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS- single

(D) TOPOLOGY, linear

(ii) MOLECULE TYPE- other nucleic acid

(xi) SEQUENCE DESCRIPTION SEQ ID NO: 105- GAGACATTTT ATCCCCTTGT G 21

(2) INFORMATION FOR SEQ ID NO: 106

(l) SEQUENCE CHARACTERISTICS

(A) LENGTH 21 base pairs

(B) TYPE nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY linear

(n) MOLECULE TYPE other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106: TCCATGCCTC CAGTCTAAAG T 21

(2) INFORMATION FOR SEQ ID NO: 107:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(Xl) SEQUENCE DESCRIPTION: SEQ ID NO:107: CACTTAAGTT GCACTGGGTA 20

(2) INFORMATION FOR SEQ ID NO: 108:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108: CAACAGGAAG TTGGTCTCAT C 21

(2) INFORMATION FOR SEQ ID NO:109:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109: TAAAAGGAAG AGCGGCTGTT T 21

(2) INFORMATION FOR SEQ ID NO: 110:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 21 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear (11) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO-110- TTAAACCTAA CTGCCACCCT C 21

(2) INFORMATION FOR SEQ ID NO:111:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111: CTGAGCTATG TGCATTTGCA 20

(2) INFORMATION FOR SEQ ID NO: 112:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 20 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112: AAGGCTGCTG CTAAACAGAT 20

(2) INFORMATION FOR SEQ ID NO: 113:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2461 base pairs

(B) TYPE: nucleic ac d

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113: TGCCCGCCTT GGCCTCCCAA CGTGTAGGGA TTACAGGCGT GAGTCACCGC GCCTTGCCAA 60 ATTATTTATT ATTATTTTTT GGAGACAGGG TCTCTGTTGC CCAAGCTGTA GTGGTATGGC 120

C _A CAGTTCAC TGCAGACTCC CCAGGATTAG GCGTTCCTCC CACCTCAGTC TCCCAAGTAG 180

CTAGGATTAC AGGCGTCTAC CACCACTCTG GGTTAATTTT TCTATTTTTT GGAGAGACAG 240

GGTTTCACTA TGTCGCCCAG GCTGGACCTC GAACTCCTOT CTCAAGCAGC CCCCCCACCT 300

CGCCTCCCAA AGTGCTGGAT TTACAGGTGT GATCCACAAC GTCCAGCCTA TATACTTAAG 360

ATACTTCTAA ACCATTTGTG TTCAACTTCT GTTCTTGCCC CATAGTCACC TTGAGACTCA 420

TCACTTAGCC AACTCCAAAA GCATTGCTGA TTACTGTGAA TTTTACTAAG GTTTTCTTAA 480

GAGGGTTCCA TTGTCTCAAA ATTGTTCCTG AAATATCCTG TTACCTGTCT ACCTGATTTT 540

CTCCTATCTT CAGAGTTCCA TTTCCTGTCC TCCCGCCTGT CATTATACCT TCCATAAGCC 600

CCTACTTTTG TCCCAGCACT TTTCCCTCTG TCAGTTTACA TATCCCACCA AGCAAAACAA 660

AAATAGCAAA ACAGTAATGC CTTCTGAATC CTCAAATTGC TCAATCCTCA GATTGCTCCT 720

CAATCTGGAA AATGTTTTAT ATCAAGCCCA TTTATAAATC AAGGATTGGC AATTTAAAAA 780

ATTAAAATAA AGAAAGGAGA ATTGGAAATA AAATGAATTG GCTGGGCACG GTGGCTCACG 840

CCTGTAATCC CAGAACTTTG GGAGGCCGAG GTGGGTGGAT CACTTGAGGT CAGGAGTGCG 900

AGACCAGCCT GGCCAACATG GTGAAACCCT GCCTGTTCTG AAAATCCAAA AATCAGCTGG 960

GTGCGGCGGC GCACACCTGT AATCCCAGAT ACTCAGGAGG CTGAGGCAGG AGAATCGCTT 1020

GATCCCAGGA GGCGGAGGTT GCAGCGAGCC GAGATCGTGC CACTACACTC CAGTCTGGCC 1080

AACAGAGCCA GACTCTGTCT CACAAAAAAA AAAAAGTTTA ATTCACGGAG AGCCAGCTGA 1140

ACGGCAGACA GGAGTTTGGT TATCCAAATC AGCCTACCAG AAATTGGAGA CTGGGGTTTT 1200

TAAAAGAATG ACTTGGCGGG TAGGGGCCCA GGGATTGGCG AATGCTAATT TGTCAGGTGG 1260

GAGGTGAAAT CACAGGGGGT TGAAGTGGGC TCTTGCTGTC TTCTGTTACT GAGTGGAATT 1320

GCAGAACTTG TTGAGCCAGA TTATGGTCTG AGTGGCGCCA GCTAGTGCAT CGGAATGCGC 1380

GGTCTGAAAA GTATCTCCAG CACCAATCTT AGGTTTTACA ATAGTGATGT TATCCCTGAG 1440

AGCAATTGGG GAGGTCAGGA ATCTTATAGC CTCTGGCTGC AAGCCTCCTA AATCATAATT 1500

TCTAATCTTG TGGCTAATTT GTTAGTTCTA CAAAGGCAGA CTGATCCCCA GGCAAGAATG 1560

GGGTTTGTTT TTGGAAAGGA CTGTTACAAT CTTTGTTTCA AAGTGAAATT AGAAATTAAA 1620

TTCCTCCTGT AGTTAGTTAG GTCTTCGCCC AGGAATGAAC AAGGGCAGCT CGGAAGTGAG 1680

AAGCGTGGAG TCATTTAGGT CAGATTCCTT GCACTGTCAT AACTTTCTCA CTGTTAGGAT 1740

TTTTGCAAAG GCAGTTTCGT GAACGTACAG AGACAGGCCC TTGCTATTAT CCCTATTTTT 1800

TAGATAAGGA TATCCAGCCG ATGAGGAAGT TTTACTTCTG GAACAGCCTG GATACGAAAC 1860

CTTCACACGT CAGTGTCTTT TGGACATTTT CTCGTCAGTA CAGCCCTGTT GAATGTTCTC 1920

ACGGTGGGGA GGTACGTGTT TAAAATACGG GGAAGGTGCT TTTATTTCAC CCCTGGTGAA 1980

ACTAGGGGAG CTAATTTTTT TAAACATGAT TTTTGTCCCC CTTGAACCGC CGGCCTGGAC 2040

TACGTTTCCC AGCAGCCCGT GCTCAAGACT ACGGGTGCCT GCAGGCGGTC AGCGTCGTTT 2100

GCGACGGCGC AGACGCGGTG CGGGCGGCGG ACGGGCGGGC GCTTCGCCGT TTGAATTGCT 2160

GCGGGCCCGG GCCCTCACCT CACCTGAGGT CCGGCCGCCC AGGGGTGCGC TATGCCGTCG 2220

GGAGGTGACC AGTCGCCACC GCCCCCGCCT CCCCCTCCGG CGGCGGCAGC CTCGGATGAG 2280

GAGGAGGAGG ACGACGGCGA GGCGGAAGAC GCCGCGCCGT CTGCCGAGTC GCCCACCCCT 2340

CAGATCCAGC AGCGGTTCGA CGAGCTGTGC AGCCGCCTCA ACATGGACGA GGCGGCGCGG 2400

CCCGAGGCCT GGGACAGCTA CCGCAGCATG AGCGAAAGCT ACACGCTGGA GGTGCGCTCG 2460

C 2461 (2) INFORMATION FOR SEQ ID NO.114.

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114: ACCTCAGGTG AGGTGAGGGC CCGG 24

(2) INFORMATION FOR SEQ ID NO: 115:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: other nucleic acid

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115. GTGTGCCATT TATGTGATGG CAAAG 25

(2) INFORMATION FOR SEQ ID NO:116:

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 base pairs

(B) TYPE, nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: other nucleic acid

( l) SEQUENCE DESCRIPTION: SEQ ID NO-116: GTATACCATT TAGCAGCTGT CCGCC 25