RU2164419C2 - Mts gene, mutations of this gene and methods of diagnosis of malignant tumors using mts gene sequence - Google Patents

Abstract

FIELD: medicine, oncology, medicinal genetics. SUBSTANCE: invention relates to somatic mutations in gene of multifunctional tumor suppressor (MTS) in the case human neoplasia and using these mutations for diagnosis and prognosis of oncological diseases. Invention relates to mutations of MTS gene in embryonic line and using these mutations for diagnosis of diathesis to the following cancer forms: melanoma, ocular melanoma, leukosis, astrocytoma, glyoblastoma, lymphoma, glyoma, Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiosarcoma, squamous cell carcinoma, chronic lymphoid lympholeukosis and pancreas, ovary, uterine, testis, kidney, stomach, colon and straight intestine tumors also. Invention relates to also therapy of human neoplasia where mutation in MTS gene occurred including genetic therapy, substitution protein therapy and mimetics using. EFFECT: broadened arsenal of agents used for diagnosis and therapy of tumors. 53 cl, 9 tbl, 25 dwg

Description

 The invention relates to somatic mutations in the gene of a multifunctional tumor suppressor (MTS) in the case of human neoplasia and the use of these mutations for the diagnosis and prognosis of these diseases. The invention also relates to mutations of the MTS gene in the germline and the use of these mutations to diagnose a predisposition to such forms of cancer as melanoma, ocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiosarcoma squamous cell carcinoma, chronic lymphoid lymphocytic leukemia (CLL), as well as tumors of the pancreas, breast, brain, prostate, bladder, thyroid gland, ovaries, uterus, testes, kidneys, stomach, rim Noah and the rectum. The invention also relates to the treatment of those human neoplasias in which a mutation in the MTS gene occurred, including gene therapy, replacing protein therapy and the use of protein mimetics. Finally, the invention relates to screening for the treatment of tumors.

 Publications and other materials used to describe the state of the art, and in particular cases from practice, are incorporated by reference in this application, referred to in the subsequent text, and accordingly, a list of publications is attached to the description of the invention.

 Cancer genetics is complex and includes numerous dominant, positive regulators of the transformed state (oncogenes), as well as numerous recessive, negative regulators (tumor suppressor genes). More than a hundred oncogenes have been characterized. Less than a dozen suppressor genes have been identified, but their number is expected to exceed fifty (Knudson, 1993).

 The participation of so many genes underscores the complexity of the growth control mechanisms that function in the cell to maintain the integrity of normal tissue. This complexity manifests itself in another way. Thus, it has been shown that not one of the oncogenes is involved in the development of all, or at least most, human neoplasias. The most common oncogenic mutations are H-ras mutations found in 10-15% of all solid tumors (Anderson et al., 1992). The most common mutant tumor suppressor gene is the p53 gene, whose mutations are found in approximately 50% of all tumors.

 Without the “target” common to all transformed cells, the dream of a “magic pool” that can destroy or reverse cancer cells without damaging normal tissue remains unfounded. Hope for a new generation of specifically targeted antitumor drugs can be based on the ability to identify tumor suppressor and oncogen genes that play key roles in the control of cell division.

 To date, suppressor genes have been cloned and characterized, which determine sensitivity to the following types of neoplasia: 1) retinoblastoma (RB1); 2) Wilms tumors (WT1); 3) Li-Fraumeni tumors (TP53); 4) familial adenomatous polyposis (ARS); 5) neurofibromatosis type 1 (NF1); 6) neurofibromatosis type 2 (NF2); 7) von Hippel-Lindau syndrome (VHL); and 8) type 2A multiple endocrine neoplasia (MEN2A).

 Loci of suppressor genes that have been genetically mapped but not yet isolated include genes that determine the development of the following diseases: type 1 multiple endocrine neoplasia (MEN 1); Lynch Type 2 Cancer Families Syndrome (LCFS2); familial breast cancer (BRCA1); neuroblastoma (NB); basal cell nevus syndrome (BCNS); Beckwith-Weidemann syndrome (BWS); renal cell carcinoma (RCC); tuberous sclerosis 1 (TSC1) and tuberous sclerosis 2 (TSC2).

The suppressor genes described today encode products that resemble various types of proteins, including DNA-binding proteins (WT1), auxiliary transcriptional regulators (RB1), GTPase activating proteins (GAP _s ) (NF1), and cytoskeletal components (NF2), membrane-bound receptor kinases (MEN2A) and other products that do not have clear resemblance to known proteins (APC and VHL).

 In many cases, suppressor genes were originally identified through genetic studies that revealed their loss or mutation in some sporadic tumors.

 This gave reason to believe that regions of chromosomal aberrations may indicate the location of important suppressor genes involved both in cancer susceptibility and in sporadic neoplasias.

 One of the important features of the suppressor genes characterized today is that they are deleted with high frequency in certain types of tumors. Deletion often leads to the loss of one allele (the so-called heterozygous loss of LOH), but there is also a homozygous deletion of both alleles. In the case of LOH, the remaining allele is non-functional due to a preexisting inherited mutation or as a result of a secondary random mutation.

 Melanoma is a widespread tumor that affects one in a hundred Americans (American Cancer Society, 1992). Environmental factors, such as exposure to ultraviolet light, contribute significantly to the incidence of this neoplasia, but hereditary factors also play an important role. A gene for family melanoma (MLM) was mapped on chromosome 9p21 (Cannon-Albright et al., 1992; Nancarrow et al., 1993; Gruis et al., 1993; Goldstein et al., 1994). The presence of a single predisposing allele at the MLM locus increases the likelihood of melanoma in this individual by approximately 50 times. MLM belongs to an increasing family of genes suspected of being tumor suppressor genes. Melanoma predisposition is inherited as a dominant Mendelian trait, although predisposing mutations at the MLM locus appear to act as somatic recessive alleles as was first suggested by Knudson (1971). In an individual predisposed to melanoma and carrying one mutant MLM allele and one wild-type MLM allele, secondary mutations occur in dividing cells, leading to the loss or inactivation of a copy of wild-type MLM; this implements the inherited mutant allele MLM.

 Conversely, a single copy of the wild-type gene prevents the development of neoplasia.

 Chromosomal aberrations in the vicinity of MLM at the 9p21 locus have been widely characterized for tumors of several different types, including glioma cell lines, non-small cell lung cancer lines, and acute lymphoblastic leukemia lines (Olopade et al., 1992; Olopade et al., 1993; Lukeis et al., 1990; Diaz et al., 1988; Middleton et al., 1991; Fountain et al., 1992; Cheng et al., 1993; James et al., 1993).

 Thus, based on the frequency of chromosomal abnormalities at the 9p21 locus in non-melanoma tumor cells, it can be assumed that the MLM region contains a gene (or genes) that is involved at least in the progression of several different types of tumors. These events involve LOH, as well as a high frequency of homozygous deletions.

 Tissue cells have only three possibilities of their existence - they can grow and divide, not divide, but remain alive, or die through apoptosis. Tumors arise either as a result of unacceptable cell growth and division, or when the cells “refuse” to die, when they should do it.

 One of the mechanisms for controlling tumor growth should include direct regulation of the cell cycle. For example, genes that control the initiation of DNA replication are potential candidates for oncogenes or tumor suppressor genes, depending on the stimulating or inhibitory role in this process. Cycle progression of eukaryotic cells (phases G1, S, G2 and M) is regulated by the sequential formation, activation, and subsequent inactivation of a number of cyclin / cyclin-dependent kinase (Cdk) complexes. The participation in these processes of cyclin D's / Cdk2,4,5, cyclin E / Cdk2, cyclin A / Cdk2 and cyclin B / A / Cdk2 is shown. Cyclines D's and Cdk2, Cdk4 and Cdk5 participate in the transition from G1 to S, that is, at the stage when a decision is made in the cell about the start of DNA replication. Recently, additional elements of cell cycle control have been discovered. These are Cdk inhibitors (Cdk inhibitors, Cdl), which include Farl, p21, p40, p20 and p16 (Marx, 1994; Nasmyth & Hunt, 1993).

 Recently, a direct involvement in the regulation of the cell cycle of several oncogenes and suppressor genes has been shown. For example, one of the cyclins (proteins that accelerate DNA replication) was identified as an oncogene (Motokura et al., 1991; Lammie et al., 1991; Withers et al., 1991; Rosenberg et al., 1991), and a tumor suppressor Rb interacts with primary cyclin-binding partners, Cdks (Ewen et al., 1993). Identification of the locus of sensitivity to melanoma opens the way for genetic screening of patients with, for example, how much the risk of cancer increases when exposed to direct sunlight. MTS can also predispose to a large number of tumors of other localizations, which include (but not limited to) leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiosarcoma, squamous cell carcinoma, chronic lymphoid leukemia ( as well as tumors of the pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, uterus, testes, kidney, stomach, colon and rectum. In addition, since MTS affects the progression of tumors of several different types, MTS analysis may be useful in determining prognosis for cancer patients. So, MTS can serve as the basis for the development of very important diagnostic tests that determine the predisposition to such forms of neoplasia as melanoma, ocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin’s lymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiocarcinoma, chesha carcinoma, chronic lymphoid leukemia (CLL), as well as tumors of the pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, uterus, testes, kidney, stomach, colon, etc. pit of the intestine, and also predict the course of cancer. In addition, since MTS is involved in the progression of many types of tumors, direct or indirect use of the ability of MTS to suppress tumor growth for general antitumor therapy can be suggested. For example, restoring normal MTS function in a tumor cell can turn it into a non-tumor cell.

 The present invention relates to somatic mutations in the gene of a multifunctional tumor suppressor (MTS) in human malignancies and their use for the diagnosis and prognosis of cancer. The invention also relates to mutations in the MTS gene in the germline and their use for detecting a predisposition to many types of malignant tumors, such as melanoma, ocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiocarcinoma, squamous cell carcinoma, chronic lymphoblastic lymphocytic leukemia (CLL) and tumors of the pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, uterus, testes, kidney, gall ka, colon and rectum. In addition, the invention relates to the treatment of human malignant tumors that are characterized by a mutation in the MTS gene, including gene therapy, protein replacement therapy and the use of protein mimetics. And finally, the invention relates to the selection of drugs for the treatment of cancer.

 In FIG. 1A shows genealogy 3137. In all cases of melanoma, a sensitive haplotype was detected. In addition, cases of other malignant neoplasms in individuals with a sensitive haplotype are shown. The following notation is used: filled circles and squares indicate melanoma; partially filled circles and squares indicate other malignant tumors; / - means the death of the patient, * means that the sensitivity of the haplotype is unknown in the individual; ** mean that, apparently, the individual has a sensitive haplotype; the age of the studied patients is indicated by numbers. In addition, an established diagnosis is indicated.

 In FIG. 1B shows genealogy 3161. In all cases of melanoma, a sensitive haplotype was detected. For other malignant neoplasms, the sensitivity of the haplotype was not determined. The following notation is used: filled circles and squares indicate melanoma; partially filled circles and squares indicate other malignant tumors; / means death of the patient; = means the appearance of the disease in the pedigree; the number "3" in the pentagon means several marriages; the numbers indicate the age of the patient. In addition, an established diagnosis is indicated.

 In FIG. 1C, pedigree 3355 is shown. In all cases of melanoma, a sensitive haplotype was detected. For other malignant neoplasms, the sensitivity of the haplotype was not determined. The following notation is used: filled circles and squares indicate melanoma; partially filled circles and squares indicate other malignant tumors; / means death of the patient. In addition, an established diagnosis is indicated.

 In FIG. 1D shows pedigree 1771 showing cases of melanoma and other malignant tumors. When studying this pedigree, mutations in MTS were identified. The following notation is used: * means that the carrier of the mutation has been reliably identified; filled circles and squares indicate melanoma; partially filled circles and squares indicate other malignant tumors (colon cancer in this family tree); / means death of the patient; the numbers indicate the age of the patient. In addition, an established diagnosis is indicated.

 In FIG. Figure 2 shows YAC (Yeast Artificial Chromosome) and P1 clones in the region associated with IFNA-s and D9S171. The centromere is on the right. For P1 clones, arrows indicate the direction to the sequence of the T7 promoter in the vector. YACs grouped together correspond to clones that are similarly located relative to the STSs of the region. Presumably, these YACs are not identical. YACs A5, B11, C6 and F9 contain IFN-1 and IFN-s. YACs D1, F5 and E3 contain D9S126 and D9S171. Neither the proximal ends of YACs containing D9S171, nor the distal ends of YACs containing IFNA-s are shown. Distances are not to scale. Markers internal to IFNA-s and D9S171 are shown in FIG. 2. Markers that begin with "c" come from the terminal sequences of cosmids. Cosmids are not shown. The distances between c1b and c5.3 and between 760-L and D9S171 are unknown.

 Figure 3 shows a diagram of deletions observed in melanoma cell lines. Deletions are grouped into 12 classes based on a set of markers that are deleted. Eleven cell lines lost all of the markers shown in FIG. 3. This class is not listed. The number of deletions of each of the 12 other classes is indicated in the column marked with "# -lines". The location of the break points resulting from deletions in grades 1-10 is shown as falling on a marker adjacent to the deleted DNA; those. the last positive marker in the series indicates a deletion. For grades 11 and 12, deletion sites are indicated by filled triangles.

 In FIG. 4A shows a map of cosmid c5. The relevant STSs used for deletion analysis are the same for cosmids and P1 clones. The C1.b marker is located proximal to P1-1062 and is not shown. The transcriptional orientations of MTS1 and MTS2 are indicated by arrows.

 In FIG. 4B shows a restriction map and an STS cosmid map c5. The positions of the coding exons for MTSI and MTS2 are indicated by black rectangles. E1 and E2 are coding exon 1 and coding exon 2. “B” is BamHI, “S” is SalI, “R” is EcoRI and “R5” is EcoRV.

 5A and 5B compare the genomic sequence of MTS1 containing the 5'-untranslated region, exon 1 and part of intron 1, and the published sequence pl6 (Serrano et al., 1993). The start codon (underlined by a black line) is at position 891, and the splicing portion (marked by an arrow) is at position 1016. The MTS1 sequence shown in FIG. 5A-B is the sequence of SEQ ID NO: 3. The sequence p16 shown in FIG. 5B is the sequence of SEQ ID NO: 24.

 6A and 6B compare the genomic sequence of MTS1 containing exon 2 and part of intron 2 and the published sequence p16 (Serrano et al., 1993). The splicing sections (marked by arrows) are located up to position 192 and after position 498. The MTS1 sequence shown in FIG. 6A-B, is the sequence of SEQ ID NO: 4. The p16 sequence shown in FIG. 6A is identical to the nucleotide sequence 192-498 of SEQ ID NO: 4.

 7A and 7B compare the genomic sequence of MTS2 containing a portion of intron 1, exon 2 and intermediate regions, and the published sequence p16. The sequence of exon 2 is similar to exon 2 of MTS1 from nucleotide 273 to 580. The splicing site in MTS2 and p16 is indicated by arrows. The point at which the divergence begins is indicated by "o". The termination codon MTS2 is located in exon 2 at position 532 and is indicated by "*". The MTS2 sequence shown in FIG. 7B is the sequence of SEQ ID NO: 5. The p16 sequence shown in FIG. 7A is identical to the nucleotide sequence 192-498 of SEQ ID NO: 4.

 In FIG. Figure 8 compares the MTSI and MTS2 DNA sequences, including exon 2 and part of each surrounding intron. The positions of the 3'-spliced compound of intron 1 and the 5'-spliced compound of intron 2 in MTS1 are indicated by triangles. The discrepancy point near the 3'-end of coding exon 2 is indicated by an arrow. The shown sequence of MTS1 corresponds to the nucleotide sequence of 92-548 SEQ ID NO: 4. The following MTS2 sequence corresponds to nucleotide sequence 174-630 of SEQ ID NO: 5.

 Figure 9 shows deletions of various STSs in tumor cell lines. Positive and negative controls were included in each PCR. Cell lines in which only one or two STSs were deleted (e.g., class 21) were retested at least twice.

 In FIG. 10A-C shows the expression of MTS2 mRNA. Fig. 10A shows the relative level of transcripts of MTS2 mRNA (Clonetech) obtained from various human tissues. Line 1 - the brain; line 2 - mammary gland; line 3 - kidneys; line 4 - lungs; line 5 - lymphocytes; line 6 - ovaries; line 7 - pancreas; line 8 - prostate gland; line 9 - spleen; line 10 - stomach; line 11 - thymus. The origin of products with a different than expected molecular weight (see line 1) is unknown.

 10B shows the relative level of MTS2 transcripts in human lymphocytes as a function of time after mitogenic induction. Line 1 - 0 hours; line 2 - 1 hour; line 3 - 2 hours; line 4 - 4 hours; line 5 - 8 hours; line 6 - 16 hours; line 7 - 24 hours; line 8 - 32 hours; line 9 - 40 hours; line 10 - 48 hours; line 11 - 56 hours; line 12 - 64 hours. Most of the cells are in the S phase (40-50 hours after induction).

 In FIG. 10C reflects the level of MTS2 transcripts as a function of Rb status. Rb-positive cell lines are as follows: lane 1 — KIT (Hori et al., 1987); lane 2 - diploid human fibroblasts MRC5, passage 28; lane 3 - VWSCC2; lane 4, Brisotol 8; lane 7 - T24. RB-negative cell lines are as follows: lane 8 — MDA MB 468; lane 9 - 5637; lane 10 - C33A; lane 11 — SiHa; lane 12 — CaSki; lane 13 - WERI.

 In FIG. 11 shows the cDNA sequence (and corresponding polypeptide) for the 5'-untranslated region of MTS2. The beginning of exon 2 is at position 491 and is marked by an arrow. The sequence shown corresponds to the sequence of SEQ ID NO: 15, and the amino acid sequence corresponds to the sequence of SEQ ID NO: 16.

 In FIG. 12A and 12B show the cDNA sequence (and corresponding polypeptide) of MTS1E1-beta. Splicing sites are marked with arrows. The start of exon 2 is at position 335, and the exon starts at position 642. The sequence is shown as SEQ ID NO: 13, and the amino acid sequence is as shown in SEQ ID NO: 14.

 In FIG. Figure 13 shows a physical map of p16. The positions of exon 1-alpha (E1-alpha), exon 1-beta (E1-beta), exon 2 (E2) and exon 3 (E3) are indicated by filled rectangles. The localization of restriction sites EcoRI (R1), EcoRV (RV) and Sa1 I (S) was noted. Above the restriction map, the genomic cosmid clones c5 and P1 1063 are shown. Deletions in the cell lines A375 and SK-mel 93 are shown below the restriction map. The dashed line corresponds to the deleted DNA.

 In FIG. Figure 14 shows the correspondence between the human and mouse p16 beta transcript sequences. Capital letters designate identical nucleotides. Stop codons in p16 reading frame are underlined. The spliced compound between E1-beta and E2 is marked with V. The mouse beta sequence shown corresponds to SEQ ID NO: 25. The human beta sequence shown corresponds to nucleotide sequence 193-461 of SEQ ID NO: 13.

 In FIG. 15 shows the expression of an alpha transcript in cell lines that contain deletions of E1-beta. cDNA obtained using total RNA isolated from these samples. In amplification reactions of P16 transcripts, a radiolabeled primer was used. Equivalent volumes for amplification of alpha and beta were mixed and the products were separated in a denaturing 5% polyacrylamide gel: lane 1 — T cells at rest; lane 2 - cell line SK-mel 93; lane 3 - cell line A375.

 In FIG. 16 A-D shows the expression of P16 transcripts. In amplification reactions of P16 transcripts, a radiolabeled primer was used and the products were separated on a denaturing 5% polyacrylamide gel.

 FIG. 16A and 16D illustrate an experiment in which, before gel electrophoresis, reaction mixtures of alpha and beta from the same sample were mixed together. On figa shows the relative level of P16 transcripts in the RNA fraction obtained from various tissues and human cells. Lane 1 - the brain; lane 2 - mammary gland; lane 3 - kidneys; lane 4 - light; lane 5 - lymphocytes; lane 6 - ovaries; lane 7 - pancreas; lane 8 - prostate gland; lane 9 - spleen; lane 10 - stomach; lane 11 - thymus. In FIG. 16B shows the relative amount of beta transcript in human lymphocytes as a function of time after mitogenic induction: lane 1 to 0 hours; lane 2 - 1 hour; lane 3 - 2 hours; lane 4 - 4 hours; lane 5 - 8 hours; lane 6 - 16 hours; lane 7 - 24 hours; lane 8 - 32 hours; lane 9 - 40 hours; lane 10 - 48 hours; lane 11 - 56 hours; lane 12 - 64 hours.

 FIG. 16C shows the relative amount of alpha transcript in human lymphocytes as a function of time after mitogenic induction: bands mean the same as in FIG. 16B, however, the time point corresponding to 1 hour is omitted. The expression of other molecules was also analyzed, for which their influence on the course of the cell cycle is assumed and the formation of which is regulated at the transcriptional level during the cell cycle. According to preliminary results, levels of CDK4 GoS2 (molecules of unknown function, but whose transcription is induced when the resting T cells enter the cell cycle) increased with the induction of T cells (Russell and Forsdyke, 1991; Matsushime et al., 1992; Geng and Weinberg , 1993). In contrast, p27 RNA levels did not appear to change during the experiment (Tiyshima and Hunter, 1994; Kota et al., 1994).

 In FIG. 16D shows the level of P16 transcripts as a function of Rb status. Rb-negative cell lines: lane 1 - WERI; lane 2 - CaSki; lane 3 — SiHa; lane 4 - C334; lane 5 - 5637; lane 6 — MDA MB 468. Rb — positive cell lines: lane 12 — human diploid fibroblasts MRC5, passage 28; lane 13 — KIT (Hori et al., 1987).

 In FIG. 17 shows the MTS1 cDNA sequence, including non-coding regions of cDNA. Triangles indicate splicing connections. A breakdown in the sequence of the second splicing compound only makes it stand out and does not mean missing bases. The sequence shown corresponds to the sequence of SEQ ID NO: 36.

 The present invention relates to somatic mutations of the gene for multifunctional tumor suppressor (MTS) in human cancers and the use of these mutations for the diagnosis and prognosis of cancer. The invention also relates to germline mutations of the MTS gene and their use for the diagnosis of susceptibility to various forms of cancer, such as melanoma, ocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma, multiple myeloma, sarcoma, myosarcoma, celiac cholangiocarcinoma, lung carcinoma, chronic lymphoid lymphocytic leukemia (CLL), as well as tumors of the pancreas, breast, brain, prostate, bladder, thyroid, ovary, uterus, testes, kidneys, stomach, rim Noah and the rectum. The invention relates to the treatment of human malignant tumors that are characterized by a mutation in the MTS gene, including gene therapy, replacement protein therapy and the use of mimetic proteins. And finally, the invention relates to the selection of drugs for the treatment of cancer.

 The present invention claimed isolated polynucleotides representing the whole MTS locus or its part or a mutated MTS locus with a length of at least eight bases and not more than 100 thousand bases. Such polynucleotides may be antisense polynucleotides. The present invention also provides a recombinant construct containing a similar polynucleotide, for example, a recombinant construct useful for expression in transformed cells.

 In addition, the present invention provides methods for detecting a polynucleotide representing a portion of the MTS locus, or a product of its expression. A similar method may include the step of amplifying a portion of the MTS locus, as well as the step of creating a set of polynucleotides that are primers for amplifying the specified portion of the MTS locus. The method is applicable both for the diagnosis of cancer susceptibility and for the diagnosis and prognosis of cancer.

 The present invention also provides antibodies, preferably monoclonal antibodies, that specifically bind to an isolated polypeptide representing at least five amino acid residues encoded by the MTS locus.

 The present invention provides kits for detecting polynucleotides in samples representing a portion of the MTS locus. These kits are polynucleotides, complementary parts of the MTS locus, packaged in a suitable container and provided with instructions for use.

 The present invention also provides methods for producing polynucleotides, which are polymerizable polynucleotides, which form a sequence consisting of at least eight consecutive nucleotides of the MTS locus; methods for producing a polypeptide comprising polymerizable amino acids that form a sequence of at least five amino acids encoded by the MTS locus.

 In addition, the present invention provides drug screening methods for the treatment of tumors in order to identify drugs to restore the function of the MTS gene product.

 Finally, the present invention provides a strategy for creating gene therapeutic approaches for cancer cells. Such therapeutic agents may take the form of polynucleotides representing a part or a complete MTS locus, placed in the corresponding vectors or delivered to the target cell by other, more direct methods, in order to restore the function of the MTS protein. The therapeutic agents may also be in the form of polypeptides based on the partial or complete MTS protein sequence. They could functionally replace in vivo activity of MTS.

 An aspect of the present invention is the fact that the MTS locus (previously designated as the melanoma locus, MLM), which predisposes the individual to melanoma and other cancers, is the MTS1 gene, which is an inhibitor of Cdk, in particular Cdk4. This gene is designated MTS1. An aspect of the present invention is also the fact that the MTS locus contains a second coding sequence, MTS2, which is very similar to MTS1 in terms of sequences. It was also established that the MTS1 gene has two separate promoters - alpha and beta. When using the alpha promoter, RNA consists of exon 1-alpha, exon 2 and exon 3. It is designated as MTS1. When using the beta promoter, the resulting RNA consists of exon 1-beta, exon 2 and exon 3. It is designated as MTS1 El-beta. The discovery of the present invention is the fact that mutations of the MTS locus in the germline are indicative of a predisposition to melanoma and other cancers. Finally, it has been shown that somatic mutations at the MTS locus are associated with most, if not all, types of tumors and, thus, are a common indicator of cancer and can serve to predict cancer. Mutation events at the MTS locus can include deletions, insertions, and point mutations in both coding and non-coding sequences.

 The MLM locus was first genetically mapped to reveal a dramatic relationship between genetic markers and a predisposition to melanoma in several pedigrees in Utah and one pedigree in Texas (Cannon-Albright, 1992). The region identified by recombinants in the pedigrees is flanked by markers D9S736 and D9S171. Subsequently, these and other genetic markers were used to localize the gene by analyzing homozygous deletions in melanoma and non-melanoma tumor cell lines containing deletions. The minimum overlapping region of the deletion was flanked by IFNA-s and D9S171 markers. The YAC libraries (Yeast atrificial chromosomes - yeast artificial chromosome) were screened to identify the genomic clones surrounding these markers. Clones P1 isolated as a result of "walk on the chromosome" were continuous from IFNA-s to D9S171, with the exception of two spaces.

 Specific STS (site-tagged sequences) were obtained to construct a more detailed molecular map. Using these markers, as well as deletion analysis, the region of overlapping deletion was determined near markers c5.1 and c5.3 found in cosmid c5. The most frequently deleted marker was c5.3, which is closest to MTS.

 Analysis of the c5 marker for the presence of "CpG" islands showed that it contains at least one candidate gene in the MTS. The DNA sequences of EcoRI c5 fragments were determined, which were compared with sequences from the gene bank. Two different c5 regions were found to be similar to a previously identified gene encoding a human Cdk4 inhibitor, or p16 (Serrano et al., 1993). These two candidate genes were designated as MTS1 and MTS2. Screening cDNA libraries from lymphocytes, the embryonic brain, and normal mammary gland using a sample from exon 2 of MTS1 led to the discovery of an additional candidate named MTS1E1-beta.

 A detailed comparison of the c5 genomic sequences with the p16 mRNA sequence showed that MTS1 contains a fragment of 307 pairs of nucleotides identical to the part of the c16 coding sequence. This MTS1 fragment was flanked by recognizable splicing compound sequences. Further studies of MTS1 showed that it includes the complete coding sequence of p16 and two more introns. Intron 1 was localized at 126 base pairs "below" the site of the start of translation; intron 2 was localized at 11 base pairs "above" the site of the end of the broadcast. Two introns divide the p16 coding sequences into three regions, a 5 ′ region of 126 base pairs (encoding Exon 1), a middle region of 307 base pairs (encoding Exon 2), and a 3 ′ region of 11 base pairs (encoding Exon 3).

 MTS2 contained a region of DNA sequences that was almost identical to p16 in that part that extends from the 5'-end of coding exon 2 to approximately 200 nucleotides towards intron 2. However, the sequence similarity decreased to point 51 of the nucleotide pair “above” Intron 2 of MTS1, in which two sequences diverged completely. This position corresponds to the localization of the last codon MTS2. Comparison of the sequences of MTS1 and MTS2 showed that the similarity of the sequences of these two genes also extends 50 pairs of nucleotides “above” the 3'-spliced compound of intron 1. Thus, fragments of non-coding DNA turned out to be more conservative than some regions of the coding DNA. To exclude the possibility that sequence divergence is an artifact of cloning, PCR primers were designed to specifically amplify the sequence at the divergence point of MTS2. These primers amplified a fragment of the predicted size from cosmid P1 and genomic DNA. Thus, the diverged sequence located “below” the 3 ′ end of exon 2 of MTS2 is a bona fide genomic sequence.

 MTS1E1-beta contains exon 1, called exon 1-beta or E1-beta, which has a different sequence than that found in exon 1 of MTS1 and MTS2. MTS1E1 beta also contains exon 2 (E2) and exon 3 (E3), which are identical to exons 2 and 3 of MTS1. Exon 1-beta is located "above" exon 1 of MTS1 and does not contain any coding sequence. As a result, MTS1E1 beta encodes p10, for which the first ATG codon of exon 2 serves as the start of translation.

 MTS1 and MTS2 were examined for compliance with the genetic sensitivity of the MTS locus by analyzing the genomic DNA of individuals presumably carrying predisposing MLM alleles. DNA polymorphisms were found in exon 2 of MTS1 in one of eight individuals. The mutation was a replacement of one nucleotide, which led to the replacement of amino acids. This polymorphism segregated together in the predisposing MLM allele.

 The predominance of lesions in MTS1 (deletion and substitution of nucleotides) indicates that MTS1 or another closely located locus are involved in the formation of a tumor phenotype.

 Cells having similar lesions receive a selective advantage over those cells that do not.

 An alternative explanation that these lesions are accidental and not related to cell growth seems unconvincing for several reasons. First, the high correlation between the tumor phenotype and MTS1 mutations indicates a causal relationship between MTS1 mutations and tumor formation. Secondly, MTS1 determines susceptibility to melanoma and therefore acts independently as a tumor suppressor gene. Third, the biochemical role of p16 as a potent Cdk inhibitor is in good agreement with the model according to which MTS1 acts in vivo as a general inhibitor of the onset of DNA replication.

 In accordance with the diagnostic and prognostic methods of the present invention, changes in the wild-type MTS locus were detected. In addition, a method can be used to detect the wild-type MTS locus and confirm the lack of predisposition to neoplasia and neoplasia itself. The term "wild-type gene alterations" means all forms of mutations, including deletions, insertions, and point mutations in the coding and non-coding regions. Deletions can affect the whole or part of a gene. Point mutations can lead to stop codons, reading frame shifts, and amino acid changes. Somatic mutations include those that are found in any of the body tissues, for example, in tumor tissue, and are not inherited in the germ line. Germ line mutations can be found in any body tissue and inherited. If only one allele is somatically mutated, this indicates an early neoplastic state. A mutation of both alleles serves as an indication of a late neoplastic state. Thus, the detection of MTS mutations provides both diagnostic and prognostic information. The undeleted MTS allele (that is, found on the chromosome sister to the chromosome carrying the MTS deletion) can be investigated for other mutations, such as insertions, small deletions, and point mutations. It is believed that many mutations found in tumor tissue lead to a decrease in the expression of the MTS gene product. However, mutations leading to the formation of a non-functional product can also lead to a neoplastic state. Point mutation events can occur in regulatory regions, such as a gene promoter, which can lead to a loss or decrease in mRNA expression. Point mutations can also interfere with normal RNA processing, which will affect the expression of the MTS gene product, and may decrease translation efficiency and mRNA stability. Useful diagnostic methods include (but are not limited to) in situ fluorescence hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern blot hybridization, single chain conformational analysis, RNase protection test, allele-specific oligonucleotides (ASO), dot Blot hybridization and PCR-SSCP, which are described in more detail below.

 The predisposition to melanoma and other forms of cancer described in this application can be confirmed by examination of any human tissue regarding mutations of the MTS gene. For example, a person who has inherited a mutation in the germline MTS gene may be susceptible to cancer. This fact can be established by analyzing any body tissue. The easiest way is to take blood, followed by the isolation of DNA from blood cells. In addition, prenatal diagnosis can be performed by analyzing mutations in the MTS gene of fetal cells, placental cells, or amniotic fluid cells. Changes in the wild-type MTS allele, for example, by point mutation or deletion, can be detected by any of the methods described here.

 To detect changes in the wild-type MTS gene from a tissue, it is necessary to isolate a sample free of surrounding normal tissue. Methods for enriching tumor cells isolated from tissue preparations are well known. For example, a tissue sample may be taken from paraffin or cryostat sections. Cancer cells can be separated from normal by flow cytometry. These methods and other approaches to the separation of tumor and normal cells are well known. If the tumor tissue is largely contaminated with normal cells, the detection of mutations is difficult.

 A quick preliminary test for polymorphisms of DNA sequences can be carried out by analyzing a set of Southern DNA blots that are restricted with one or more endonucleases, preferably many endonucleases. Samples from normal individuals and samples taken from cancer patients should be presented on each blot. Blots containing hybridizing fragments that differ in length from the control ones (when hybridized with a sample representing the MTS locus) indicate a possible mutation. When using coarse restriction enzymes giving very large restriction fragments, pulse electrophoresis ("PFGE") is used.

 The detection of point mutations can be complemented by molecular cloning of the MTS allele (s) using well-known methods. Alternatively, gene sequences may be amplified “directly” from genomic DNA isolated from tumor tissue. The nucleotide sequence of the amplified DNA can then be determined.

 There are six well-known methods for more complex but indirect confirmation of the presence of a sensitive allele: 1) single-chain conformational analysis ("SSCA") (Orita et al., 1989); 2) denaturing gradient gel electrophoresis ("DGGE") (Wartell et al., 1990; Sheffield et al., 1989); 3) RNase protection test (Finkelstein et al., 1990; Kinszler et al., 1991); 4) allele-specific oligonucleotides ("ASOs") (Conner et al., 1983); 5) the use of proteins that recognize nucleotide mismatches, such as mutS-protein E. coli (Modrich, 1991); and 6) allele-specific polymerase chain reaction (PCR) (Rano & Kidd, 1989). For allele-specific PCR, primers are used that hybridize at their 3'-end with a single MTS mutation point. If this separate mutation is not present, then there is no amplification product. In addition, an Amplification Refractory Mutation System (ARMS) can also be used, as described in European Patent Application Publication No. 0332435 and Newton et al. , 1989. Insertions and deletions of genes can also be detected by cloning, sequencing and amplification. In addition, restriction fragment length polyformism samples (RFLPs) specific for a given gene or surrounding marker genes can be used to detect damage to an allele or insertion into a polymorphic fragment. This method is especially useful when screening a patient’s relatives for the presence of MTS mutations found in this patient. Other known methods for detecting insertions and deletions may be used.

 In the case of the application of the first three methods (i.e., SSCA, DGGE and the RNase protection test), a new electrophoretic band appears. SSCA detects a band that migrates differently, since changes in the sequence cause changes in the intramolecular compounds of single-stranded DNA bases. During the analysis by the RNase protection method, the mutant polynucleotide is cut into two or more two smaller fragments. DGGE shows differences in the rate of migration of mutant sequences compared to wild-type sequences in a denaturing gradient gel. When using an allele-specific nucleotide, an oligonucleotide capable of recognizing a specific sequence is constructed, and the conclusion about the results of the analysis is made by the presence or absence of a hybridization signal.

 In the case of the mutS test, the protein binds only to a sequence that has nucleotide mismatches in the heteroduplex between mutant sequences and wild-type sequences.

 As used herein, “mismatches” refers to hybridized nucleic duplexes in which both strands are not 100% complementary. The lack of complete homology may be due to deletions, insertions, inversions or substitutions. Mismatch detection can be used to detect point mutations in a gene or in its mRNA. Since these methods are less sensitive than sequencing, they are easier to carry out with a large number of samples of tumor material. An example of the method of "cutting mismatches" is the method of protection against RNase. In the context of the present invention, this method involves the use of a labeled ribo-probe that is complementary to the coding sequence of the wild-type human MTS gene. Riboprobes and mRNA or DNA isolated from tumor tissue are “annealed” (hybridized) together and then digested with the RNase A enzyme, which can detect some mismatches in the duplex structure of RNA. If RNase A detects a mismatch, it cuts at that location. Thus, when electrophoresis separates a hybridized RNA preparation in the event of mismatch and RNase A cutting, a product will be detected with a length shorter than the full RNA duplex length for a riboprobe and mRNA or DNA. Riboprobes do not have to have the full length of the MTS mRNA or represent the entire gene. If the riboprobe represents only the mRNA or gene segment, it is advisable to use a set of such samples for screening for mismatches of the entire mRNA sequence.

 Similarly, DNA probes can be used to detect mismatches using enzymatic or chemical cutting. For example, see Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986. Alternatively, mismatches can be detected by a shift in the electrophoretic mobility of "mismatched" duplexes compared to "coincident" duplexes. For example, see Cariello, 1988. Both in the case of the use of riboprobes and in the case of DNA probes, cellular mRNAs or DNA that are suspected of having mutations can be amplified by PCR (see below) before hybridization. Changes in the MTS gene DNA can also be detected by Southern hybridization, especially if these changes are large rearrangements, such as deletions and insertions.

 MTS gene DNA sequences amplified by PCR can be screened using allele-specific samples. These samples are nucleic oligomers, each of which contains a region of the MTS gene carrying a known mutation. For example, one of the oligomers may have 30 nucleotides in length and correspond to part of the sequence of the MTS gene. Using a set of such allele-specific samples, PCR products can be screened for previously identified mutations in the MTS gene. Allele-specific samples can be hybridized with amplified MTS sequences, for example, on nylon filters. Hybridization with a specific sample under severe conditions indicates the presence in the tumor tissue of the same mutation as in the allele-specific sample.

 The most specific test for the presence of mutations in a suspicious locus is a direct comparison of the genomic sequences of MTS from cancer patients with similar samples from the control population. Conversely, mRNA sequencing after amplification, for example by PCR, may eliminate the need to determine the exon structure of the candidate gene.

 When examining non-coding regions, such as introns, regulatory sequences located near or inside the MTS gene, mutations outside the MTS coding region can be detected in cancer patients. Indications that mutations in non-coding regions may be important follow from experiments using Northern blotting, in which RNA molecules of anomalous size or redundancy not found in control individuals were found in cancer patients.

 Impaired expression of MTS mRNA can be detected by any known method. These methods include Northern blotting, PCR amplification, and an RNase protection test. Reduced mRNA expression indicates abnormalities in the wild-type MTS gene. Changes in wild-type MTS genes can be detected by testing for abnormalities in the wild-type MTS protein. For example, monoclonal antibodies immunoreactive with the MTS protein can be used. Lack of antigen recognition indicates a MTS mutation. Antibodies specific for products of mutant alleles can be used to detect products of a mutant MTS gene. These immunological tests can be carried out by any suitable method.

 These methods include Western blot, immunohistochemical methods, and ELISA. Any approaches for detecting an altered MTS protein are appropriate for searching for changes in wild-type MTS genes. Functional approaches such as determination of protein binding can be used. For example, it is known that the MTS protein binds to Cdk, in particular Cdk4. Thus, the ability to bind to the wild-type MTS protein or to Cdk4 can be used. In addition, tests based on the biochemical properties of MTS can be used for its ability to inhibit Cdk, such as Cdk4, and the effect of MTS on the cell cycle of detection of a mutant product of the MTS gene indicates changes in the wild-type MTS gene.

 MUT mutant genes or products of this gene can be found in other biological samples taken from a patient, namely serum, feces, urine and sputum. The same methods described above for detecting mutant MTS genes or their products in tissues can be applied to other biological samples. Cancer cells desquamate from the tumor and are found in these biological samples. In addition, the MTS gene product can be secreted into the intercellular space and detected in these biological samples even in the absence of cancer cells. When screening these biological samples, a simple and quick diagnosis is possible for many forms of cancer. In addition, the results of the analysis of similar biological samples for mutant MTS genes or their products can be used to judge the effectiveness of chemo- and radiotherapy.

 The diagnostic methods described in this application are applicable to any cases of neoplasia for which the involvement of MTS in tumorigenesis is indicated. 9p chromosomal shoulder deletions or somatic mutations in the MTS region were observed in almost all of the tumors studied. The diagnostic method presented in the present invention is useful for clinicians and allows them to choose the appropriate course of treatment.

 The primer pairs described in the present invention are useful for determining the nucleotide sequence of a particular MTS allele by PCR. Pairs of single-stranded DNA primers can be hybridized with the sequences of the MTS gene or the sequences surrounding it on chromosome 9, for amplifying synthesis of the actual MTS gene. A complete set of these primers allows us to synthesize all nucleotides of the coding sequences of the MTS gene, i.e. exons. This set of primers allows you to synthesize both intron and exon sequences. Allele-specific primers may also be used. Such primers hybridize only with specific MTS mutant alleles, and thus amplification only occurs if a mutant allele is present as a template.

 To facilitate subsequent cloning of amplified sequences, primers can carry restriction sites at their 5'-ends. Thus, all primer nucleotides are derived from MTS sequences or sequences adjacent to this gene, with the exception of several nucleotides necessary for the formation of restriction sites. Restriction enzymes and their recognition sites on DNA are widely known. The primers themselves can be synthesized using standard methods. In general, primers can be synthesized using oligonucleotide synthesizers, commercially available devices. The MTS hidden reading frames are given in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO 15 and SEQ ID NO: 36, design specific primers are quite obvious.

 The nucleotide probes described herein may have a number of uses. They can be used in Southern hybridization with genomic DNA and in the RNase protection method to detect point mutations, as discussed above. These samples can be used to detect PCR amplification products. They are also useful for detecting mismatches with the MTS gene or with mRNA by other methods.

 In describing the present invention, the following definitions are used. "Amplification of polynucleotides" means the use of methods such as polymerase chain reaction (PCR, PCR), ligase amplification (or ligase chain reaction, LCR, LCR), as well as amplification methods based on the use of Q-beta replicase. These methods are well known and widely used in practice. For example, see US Pat. Nos. 4,683,195 and 4,683,202, as well as Innis et al. 1990 (PCR) and Wu et al., 1989a (LCR). Reagents and equipment for PCR are commercially available. The primers used to amplify the sequences of the MTS region are preferably complementary and, accordingly, specifically hybridize with the sequences of the MTS region or with sequences that flank the MTS region. Amplification MTS sequences can be directly sequenced. It is less preferred that the amplified sequences be cloned before sequencing. A method for direct cloning and sequencing of enzymatically amplified genomic fragments is described by Scharf, 1986.

 "Analyzed polynucleotide" and "Analyzed chain" mean a single or double stranded polynucleotide in which the desired sequence is expected and which can be represented in samples of various types, including biological samples.

 "Antibodies". The present invention describes polyclonal and / or monoclonal antibodies, or fragments thereof, or their immunological binding equivalents, which are capable of specifically interacting with MTS polypeptides, fragments of such polypeptides, or with polynucleotide sequences of the MTS region, especially from the MTS locus, or with fragments of such nucleotide sequences. The term “antibody” is used to mean both a homogeneous substance and a mixture similar to serum and containing many different homogeneous substances. Polypeptides can be synthetically prepared using a peptide synthesizer, conjugated to carrier molecules (for example, fissurella mollusk hemocyanin) and administered to rabbits for several months. Rabbit sera are tested for immunoreactivity with respect to the MTS polypeptide or fragments thereof. Monoclonal antibodies can be obtained by immunization of mice with natural polypeptides, fusion proteins or fragments of one or another. Monoclonal antibodies can be selected by ELISA and tested for specific immunoreactivity with the MTS polypeptide or fragments thereof (CM. Harlow & Lane, 1988). These antibodies can be used both for analytical studies and as therapeutic agents.

 Upon receipt of sufficient quantities of the desired polypeptide, it can be used for various purposes. A typical use example is the production of specific binding antibodies. These antibodies, monoclonal or polyclonal, can be obtained in vivo or in vitro by widely known methods.

 To obtain polyclonal antibodies, the appropriate target immune system, usually a mouse or rabbit, is chosen. Essentially, the purified antigen is introduced into the immune system by a method depending on the type of animal and a number of other parameters well known to immunologists. The usual routes of administration are intramuscularly, intraperitoneally, intradermally and into the paw pads. Of course, in addition to mice and rabbits, animals of other species can be used. Polyclonal antibodies are purified using known methods to achieve the desired specificity.

The strength of the immunological response is usually checked by immunological analysis. Typically, such an analysis involves some degree of purification of the source of antigen, for example, a substance produced by the same cells and in the same way as the original antigen. Numerous immunological assays are widely known. For example, see Harlow & Lane, 1988 or Coding, 1986. Using standard procedures, such as those described by Harlow & Lane, 1988 and Coding, 1986, monoclonal antibodies are obtained with an affinity of ^10-8 per mole, or preferably 10 ^-9 per mole, ^10-10 per mole or stronger. Acceptable animals must be selected and appropriate immunization procedures must be carried out. After the appropriate time has elapsed, the spleens are excised from the immunized animals and individual splenocytes are fused with immortalized myeloma cells under appropriate selective conditions. Hybrid cells are then cloned and the supernatants of individual clones are tested for the presence of antibodies specific for specific antigen epitopes.

 Other suitable methods include in vitro exposure of lymphocytes with antigenic polypeptides or selection of antibody libraries in phages or other similar vectors. See Huse et al., 1989. The polypeptides and antibodies provided in the present invention can be used with or without modification. Often, polypeptides and antibodies are labeled by covalent or non-covalent addition of a substance that gives a recognizable signal. There is a wide variety of labeling and conjugation methods described in both the scientific and patent literature. Acceptable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent agents, chemiluminescent agents, magnetic particles, etc.

 Among the patents devoted to the use of such marks, the following patents can be noted: US 3817837; 3,850,752; 3,939,350; 3996345; 4,277,437; 4,275,149 and 4,366,241. Recombinant immunoglobulins may also be prepared (see US Pat. No. 4,816,567).

 “Binding partner" means a molecule capable of binding a ligand with high specificity. Examples of such agents include an antigen and an antigen-specific antibody or enzyme and its inhibitor. In general, specific binding partners can bind with sufficient affinity to an immobilized copy of the analyte (complementary duplex chain in the case of polynucleotide hybridization) under isolation conditions. Such specific binding agents are known to those skilled in the art; these include, for example, biotin and avidin or streptavidin, IgG and protein A, as well as numerous well-known receptor-ligand pairs and complementary polynucleotide chains. In the case of complementary polynucleotide binding, the partners are usually at least 15 kilobases long, and may be about 40 kilobases long.

 “Biological sample” means a sample of tissue or fluid of an individual in which the desired polynucleotide or polypeptide is expected to be detected. Such samples include, but are not limited to, for example, plasma, serum, cerebrospinal fluid, lymph, secrets and secretions of the skin, respiratory tract, intestines, genitourinary tract, as well as tears, saliva, blood cells, tumor samples, various organs, tissues and constituent cell cultures obtained in vitro.

 As used herein, the term “diagnosis” or “prognosis” as applied to neoplasia means the following: 1) classification of damage as neoplasia; 2) establishing the severity of neoplasia; or 3) monitoring the progression of the disease before, during and after treatment.

 The term "coding" means the following. A polynucleotide, both natural and constructed using methods well known to those skilled in the art, is considered to encode a polypeptide if it is transcribed with the formation of messenger RNA (mRNA), which is then translated with the formation of the polypeptide or its fragment. The antisense chain is complementary to the indicated polynucleotide chain and, therefore, the sequence of the coding chain can be derived from it.

 The term "isolated or substantially purified." This concept is applied to a biological object, in particular a nucleic acid (RNA, DNA, mixed polymer), if it is essentially separated from other cellular components that naturally contaminate the native human nucleic acid sequence or protein, in particular from ribosomes, polymerases, many other genomic sequences and proteins. This concept refers to a nucleic acid or protein that is isolated from a natural microenvironment, in particular to isolates of recombinant or cloned DNA and its chemically synthesized analogues or analogues synthesized biologically in heterologous systems.

 The term “MTS allele” refers to the normal alleles of the MTS locus, as well as to alleles that have any changes that make the individual susceptible to the development of a malignant tumor of different localization, for example, melanoma, ocular melanoma, leukemia, astrocytoma, glioblastoma lymphoma, glioma, Hodgkin’s lymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiosarcoma, squamous cell carcinoma, chronic lymphoblastic lymphocytic leukemia (CLL), as well as tumors of the pancreas, breast, brain, prost tion prostate, bladder, thyroid, ovary, uterus, testis, kidney, stomach, colon and rectum. Such predisposing alleles are also called "MTS-sensitive alleles."

 The concepts of "MTS locus"; "MTS gene", "MTS nucleic acid" and "MTS polynucleotide" refer to polynucleotides, all of which are located in the MTS region and are highly likely to be expressed in normal tissues, with some alleles of these polynucleotides causing an individual's predisposition to develop melanoma and other malignant tumors, for example, ocular melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin’s lymphoma, multiple myeloma, sarcoma, myosarcoma, cholangiosarcoma, squamous cell carcinoma, chronic lymphoblastic lymphocytic leukemia (CLL), as well as tumors of the pancreas, breast, brain, prostate, bladder, thyroid gland, ovaries, uterus, testes, kidneys, stomach, colon and rectum. The term “MTS locus” is used interchangeably with the term “MLM locus” as previously proposed, and the use of the term “MTS locus” means the use of the term “MLM locus” in relation to a locus, gene, region, etc. Mutations at the MTS locus may be involved in the development and / or progression of other types of tumors. Partially, the locus was identified due to mutations that determine the individual's predisposition to the development of malignant tumors.

 These mutations affect the MTS region described below.

 The MTS locus includes coding sequences, intermediate sequences, and regulatory elements that control transcription and / or translation. The MTS locus also includes allelic variants of the DNA sequence.

 These terms, when referring to a nucleic acid, mean a nucleic acid that encodes an MTS polypeptide (including p16), a fragment, homologue or variant thereof, including, for example, fusion proteins or deleted proteins.

The nucleic acids described in the present invention are sequences that are derived from a natural MTS gene, or a substantially similar gene, or a gene having significant significant homology with the natural one, or based on a fragment of a natural gene. The coding sequence for the MTS polypeptide (MTS1) is SEQ ID NO: 1, the amino acid sequence of the MTS polypeptide MTS1) is
SEQ ID NO: 2. The coding sequence for the second MTS polypeptide (MTS1E1 beta) is SEQ ID NO: 13, and the corresponding amino acid sequence is SEQ ID NO: 14. The coding sequence of the third MTS polypeptide (MTS2) is SEQ ID NO: 15, and the corresponding amino acid sequence is SEQ ID NO: 16.

 The term “p16” is used interchangeably with the concepts of MTS1 and MTS1E1 beta and is used to denote the MTS1 locus that encodes p16 and the MTS1E1 beta locus that encodes p10. MTS1 and MTS1E1 beta are two forms of the same gene; the formation of different transcripts depends on the promoter used. MTS2 is a separate section of the MTS region and encodes p15.

 Polynucleotide compounds described in the present invention include RNA, cDNA, genomic DNA, synthetic molecules, mixed polymers, sense and antisense chains, which can be chemically or biochemically modified or may contain non-natural nucleotides, as well as nucleotide derivatives. The listed compounds can be easily obtained by specialists in this field of research. These modifications include, for example, labeling, methylation, substitution of one or more natural nucleotides with an analogue, internucleotide modifications, such as uncharged bonds (e.g. methylphosphonate, phosphotriether, phosphoamide, carbamate, etc.), charged bonds (e.g. , phosphothioate, phosphodithioate, etc.), additional groups (e.g., polypeptide), intercalating compounds (e.g., acridine, psoralen, etc.), chelators, alkylators, as well as modified bonds (e.g., alpha-anomeric ukleinovye acid, etc.). In addition, polynucleotide compounds include synthetic molecules that mimic the ability of polynucleotides to bind to a specific sequence through hydrogen bonds or other chemical interactions. These molecules are known in the art and include, for example, those compounds in which the peptide bonds are replaced by phosphate bonds in the backbone of the molecule.

 The present invention relates to recombinant nucleic acids consisting of a whole region of NTS or part thereof.

 The recombinant construct can autonomously replicate in the host cell. Alternatively, the recombinant construct may integrate into the chromosomal DNA of the host cell. Such a recombinant polynucleotide is a polynucleotide of genomic, cDNA, semisynthetic or synthetic origin and, by virtue of its origin or production method, 1) is not associated with all or part of the polynucleotide with which it is naturally associated; 2) associated with a polynucleotide other than that with which it is associated in nature; 3) does not occur in nature.

 Thus, the recombinant nucleic acids of the present invention are represented by sequences not found in nature. Although a wild-type sequence can be used, it will most often be changed, for example, by deletion, substitution or insertion.

 cDNA or genomic libraries of various origins can be screened as natural sources of nucleic acids of the present invention, or such nucleic acids can be obtained by amplification (for example, by PCR) of sequences contained in genomic DNA or other natural sources. The choice of cDNA libraries is usually correlated with a source of tissue rich in mRNA of a given protein. The use of phage libraries is preferred, but other libraries may be used. Clones of the appropriate library are plated on plates, transferred to a substrate for screening, denatured, and tested for the presence of desired sequences.

 The DNA sequences used according to the present invention typically contain about five codons (15 nucleotides), more likely about 7-15 codons, and most preferably about 35 codons. In addition, they may contain one or more introns. The indicated number of nucleotides usually provides the minimum length necessary to serve as a sample that will specifically hybridize to the MTS coding sequence.

 Techniques for working with nucleic acids are described, for example, by Sambrook et al., 1989 or Ausubel et al., 1992. The reagents used in the implementation of these techniques, for example, restriction enzymes, etc., are widely known and commercially available. For example, they are supplied by companies such as New England Biolabs, Boehringer Mannheim, Amersham, Promega Biotec, U.S. Biochemicals, New England Nuclear and others. Recombinant nucleic acid sequences used to produce the fusion proteins of the present invention may be derived from natural or synthetic sequences.

 Many natural gene sequences are obtained from various cDNA or genomic libraries using appropriate samples. For more information, see the Genebank of the US National Institute of Health.

 The term "MTS region" refers to the region of the ninth human chromosome contained in P1 clones P1-1062 and P1-1063. These P1 clones of E. coli NS 3529 were deposited with the American Cell Culture Collection, Rockville, Maryland, USA on March 16, 1994 and designated ATCC Nos. 69589 and 69590, respectively. The indicated region contains the MTS locus, including the MTS1, MTS2, and MTS1E1 beta genes.

 As used herein, the terms “MTS locus”, “MTS allele” and “MTS region” refer to double-stranded DNA containing a locus, allele or region, as well as single-stranded DNA containing a locus, allele or region.

 As used herein, the term “portion” of an MTS locus or region or allele refers to a region of minimum size, about eight nucleotides long, preferably about 15 nucleotides long, more preferably about 25 nucleotides long, and it can also be a region whose minimum size is at least at least about 40 nucleotides.

 The term “MTS protein” or “MTS polypeptide” refers to a protein or polypeptide that is encoded by the MTS locus (including the MTS1 polypeptide, MTS2 polypeptide and MTS1E1 beta polypeptide), as well as variants or fragments of the corresponding protein or polypeptide. The term "polypeptide" refers to polymers consisting of amino acids and their equivalents, and is not related to the length of these compounds. Thus, peptides, oligopeptides and proteins are encompassed by the term "polypeptide". This concept does not apply to modified polypeptides, for example, glycosylated, acetylated, phosphorylated, etc., however, it refers to polypeptides containing one or more amino acid analogues (including unnatural amino acids, etc.), polypeptides with substituted bonds, as well as with other well-known modifications of natural and non-natural origin. Typically, such polypeptides have at least 50% homology with the native MTS sequence, preferably 90% homology, and most preferably at least 95% homology. In addition, these polypeptides include proteins encoded by DNA that hybridizes to the MTS coding sequence under mild or severe hybridization conditions, as well as closely related polypeptides or proteins recognized by serum to the MTS protein (s).

 The length of the polypeptide sequences compared by homology, in General, is at least about 16 amino acids, usually about 20 amino acid residues, more often about 24 residues, most likely about 28 residues and preferably more than 35 residues.

 The term "functionally connected" refers to a specific arrangement of components that are thus connected in such a way that they can function as specified by the purpose of the experiment. For example, a promoter is operably linked to a coding sequence if it provides for its transcription or expression.

 "Samples." This term refers to polynucleotide polymorphisms associated with MTS alleles that are predisposed to various malignant tumors, or associated with most malignant tumors, and these polynucleotides are determined by hybridization with a polynucleotide probe, which forms a stable hybrid with a target sequence in severe and moderate conditions hybridization and washing. If it is assumed that the samples will be strictly complementary to the target sequence, stringent hybridization conditions should be used. The conditions may be less stringent if a slight degree of overlap is expected, for example, in the case of options where the sample is not completely complementary to the target. Hybridization conditions are chosen in such a way as to exclude surface or nonspecific binding and to minimize the effect of the background. Since neutral DNA polymorphisms, as well as mutations, are identified as a result of such determinations, additional studies are needed to confirm the identification of the MTS-sensitive allele. Samples for MTS alleles can be obtained based on the sequences of the MTS region or corresponding cDNA. Samples can be of any suitable length to span an entire or part of the MTS region and provide specific hybridization with the MTS region. If the target sequence contains a sequence identical to the sample, then the samples may be shorter, for example, on average about 8-10 nucleotide pairs, so that the hybrid will be relatively stable even under stringent hybridization conditions. If a certain degree of discrepancy is expected when using the sample, for example, if hybridization with a variant of the region occurs, then a longer sample with a given specificity can be used for hybridization with the target sequence.

 By breakdown is meant an isolated polynucleotide linked to a label or reporter molecule, which can be used to isolate other polynucleotide sequences having similarities detected by standard methods. Methods for preparing and labeling samples are described, for example, by Sambrook et al. , 1989 or Ausubel et al., 1992. Other similar polynucleotides can be selected using homologous polynucleotides. Alternatively, polynucleotides encoding data or similar polypeptides can be synthesized or selected based on the redundancy of the genetic code. Various codon substitutions can be made, for example, so-called “silent” substitutions (with the creation of various restriction sites). One of the goals of such substitutions may be to optimize expression in a particular system. Mutations can be introduced to modify the properties of polypeptides, in particular to alter ligand-binding affinities, interchain affinities, or circuit rates and degradation of polypeptides.

 Samples representing synthetic oligonucleotides or other polynucleotides of the invention may be obtained from natural or recombinant single or double stranded polynucleotides, or chemically synthesized. Samples can be labeled with nick translation, using Klenov fragments or other known methods.

 As samples, plots of nucleotide sequences representing at least eight, preferably 15 nucleotides, less often up to 6 kilobases, usually up to 1 kilobase of the nucleotide sequence encoding MTS, are applicable. Samples can also be used to detect MTS encoded RNA in cells and tissues.

 The term “modifications and fragments” as used in the present invention refers to polypeptides or fragments of MTS that are essentially homologous to the primary structural sequence, but which have (for example, in vivo or in vitro) chemical or biochemical changes or contain atypical amino acids. Such changes, for example, include acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, for example, with radionuclides, as well as various modifications obtained using enzymes, which are well known to specialists in this field of research. Various methods for labeling polypeptides, as well as substituents and labels used for these purposes, are known in the art. These include radioactive isotopes such as 32P, ligands that bind to labeled antiligands (e.g. antibodies), fluorochromes, chemiluminescent agents, enzymes, and antiligands that can serve as a specific binding partner for a labeled ligand. The choice of label depends on the required sensitivity, ease of conjugation with a primer, the necessary stability and availability of equipment. Labeling methods for polypeptides are known in the art. See, for example, Sambrook et al., 1989 or Ausubel et al., 1992.

 In addition to full-length polypeptides, the present invention also relates to biologically active fragments of these polypeptides. Significant biological activities include ligand binding ability, immunological activity, and other types of biological activity of MTS polypeptides. The immunological activity of a compound means, firstly, that this compound has immunogenic properties in relation to the immune system and, secondly, the presence of binding immunological epitopes in it, which ensures its functioning as a competitive or replacement antigen for the MTS protein epitope. As used herein, the term “epitope” means an antigenic determinant of a polypeptide. An epitope may contain three amino acids of a particular spatial conformation that are unique to a given epitope. In general, the epitope consists of at least five amino acids, more likely of 8-10 amino acids. Methods for determining the spatial conformation of such amino acids are well known in the art.

 For immunological purposes, tandem-repeating polypeptide segments with high antigenicity can be used as immunogens. Alternatively, such polypeptides can serve as very effective substituents in specific binding reactions. The preparation of antibodies specifically interacting with MTS polypeptides or fragments thereof is described below.

 The present invention also relates to fusion polypeptides containing MTS polypeptides and fragments thereof. Homologous polypeptides can be located between two or more MTS polypeptide sequences or between MTS sequences and a related protein. Similarly, heterologous fusions can be obtained with the expected combination of properties or activities of those proteins that were used for the fusion. For example, a binding ligand or other domains can be inserted between different, newly obtained fusion polypeptides or fragments thereof. Such homologous or heterologous fusion polypeptides may have, for example, altered binding strength or specificity. Fusion partners include immunoglobulins, bacterial beta-galactosidase, trp E, protein A, beta-lactamase, alpha-amylase, alcohol dehydrogenase and yeast mating factor. See, for example, Godowski et al., 1988.

 Fusion proteins are usually obtained by recombinant nucleic acid methods, as described below, or by chemical synthesis. Polypeptide synthesis techniques are described, for example, Merrifield, 1963.

 The term "protein purification" refers to various methods for the isolation of MTS polypeptides from biological material, for example, from cells transformed with recombinant nucleic acids encoding MTS. These methods are well known in the art. For example, polypeptides can be purified by immunoaffinity chromatography, for example, using antibodies obtained according to the present invention. Various protein purification methods are known in the art and are described, for example, by Deutscher, 1990 and Scopes, 1982.

 The terms "isolated", "essentially purified" and "essentially homogeneous" are used interchangeably with respect to a protein or polypeptide that is separated from the components associated with it in a natural material. A monomeric protein is considered to be substantially purified when at least about 60-70% of the sample is a specific polypeptide sequence. Essentially, the purified protein is usually 60 to 90% by weight in the protein sample, preferably 95%, and even more preferably about 99%.

 The purity and homogeneity of a protein is determined by many well-known methods, such as polyacrylamide gel electrophoresis, followed by visual detection of a specific polypeptide band after gel staining. For some purposes, higher purity can be achieved by high-performance liquid chromatography or other purification methods known to those skilled in the art.

 MTS protein is essentially free of the components associated with it in the natural sample when it is separated from the native impurities contained in the natural sample. Thus, a polypeptide chemically synthesized or synthesized in a cellular system other than that with which it is naturally associated can be substantially free of the natural components associated with the protein. A protein can also be substantially purified from its associated natural components using protein purification methods known in the art.

 A polypeptide obtained as an expression product of an isolated genetic or engineered sequence is denoted by the term “isolated polypeptide” as used herein, even if it is expressed in a homologous cell system. Polypeptides obtained chemically or expressed in a heterologous cell system are inherently isolated molecules.

 The term "recombinant nucleic acid" means a nucleic acid of a non-natural origin or a molecule obtained by artificial combination of two different fragments. Such an artificial combination is often carried out by chemical synthesis methods or by manipulating isolated nucleic acid fragments, for example, genetic engineering methods. So, the codon is often replaced by a degenerate codon encoding the same amino acid or conservative amino acid, creating or removing a recognition site. In contrast, by combining nucleic acid fragments with specific functions, the desired combination of functions can be obtained.

 "Regulatory sequences" means those sequences that are usually located within 10 kilobases from the coding region of the locus and which affect gene expression (including gene transcription, as well as translation, splicing, stability, and other characteristics of messenger RNA).

 "Homology or similarity in essence." A nucleic acid or a fragment thereof is “substantially homologous” (or “substantially similar”) to another nucleic acid if, when optimally positioned (with the appropriate insertions or deletions of nucleotides), it identifies the nucleotide sequence for at least 60% of the nucleotides, usually at least 70%, more often not less than 80%, preferably not less than 90% and even more preferably about 95-98% nucleotides.

 Alternatively, homology (or similarity) essentially takes place when a nucleic acid or fragment thereof hybridizes with another nucleic acid (or complementary strand) under certain hybridization conditions. In hybridization, selectivity occurs. Typically, hybridization selectivity is observed with a 55% homology of fragments of at least 14 nucleotides in length, preferably about 65%, preferably at least 75%, and even more preferably at least 90%. See Kanehisha, 1984. However, the length of the homologous comparison can be significant, in some cases it can exceed nine nucleotides, usually at least 20 nucleotides, usually at least 24 nucleotides, better than 28 nucleotides, even better 32 nucleotides and most preferably 36 or more nucleotides.

Hybridization of nucleic acids is affected by conditions such as salt concentration, temperature, the presence of organic solvents, and in addition, the composition of the bases, the length of complementary fragments, and the number of mismatches of nucleotide bases between hybridizing nucleic acids. All of these factors are well known in the art. Rigid temperature conditions mean a temperature above 30 ^° C, usually higher than 37 ^° C, and preferably above 45 ^° C. Rigid salt conditions mean a salt concentration of less than 1000 mM, usually less than 500 mM, and preferably less than 200 mM. However, a combination of parameters is more important than the value of one of them. See, for example, Wetmur & Davidson, 1968.

 Under certain conditions, samples can also hybridize with duplex DNAs to form higher-order triplexes or DNA complexes. The preparation of such samples and suitable hybridization conditions are known in the art.

 The terms “essentially homology” or “substantive identity” as applied to polypeptides indicate that the test polypeptide or protein exhibits at least 30% identity with the whole natural protein or part thereof, usually at least 70% identity and preferably at least 95% identity.

 A “substantially similar function” refers to the function of a modified nucleic acid or a modified polypeptide compared to a wild-type MTS nucleic acid or a wild-type MTS polypeptide. The modified polypeptide will be essentially homologous to the wild-type MTS polypeptide and, accordingly, will have essentially the same function, i.e. will inhibit Cdk, especially Cdk4. The modified polypeptide may have a modified amino acid sequence and / or may contain modified amino acids. In addition to the function of inhibiting Cdk, the modified polypeptide may have other beneficial properties, such as a longer half-life. The cdk inhibitory activity of the modified polypeptide may be essentially the same as that of the wild-type MTS polypeptide. In contrast, the Cdk inhibitory activity of the modified polypeptide may be higher than the activity of the wild-type MTS polypeptide. The modified polypeptide is synthesized using conventional methods, or it can be encoded with a modified nucleic acid and obtained by conventional methods. The modified nucleic acid is obtained by conventional methods. A nucleic acid with a function substantially similar to that of the wild-type MTS gene can serve to produce the modified protein described above.

 For polypeptides, homology is usually determined using sequence analysis software. See, for example, Sequence Analysis Software Package from Genetics Computer Group, University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 53705. Using computer programs for protein analysis, similar sequences are compared, measuring the degree of homology taking into account various substitutions, deletions and other modifications. Conservative substitutions usually include substitutions of the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; series, threonine; lysine, arginine; phenylalanine, tyrosine.

 A polypeptide "fragment", "site" or "segment" is a series of amino acid residues consisting of at least five to seven consecutive amino acids, often seven to nine consecutive amino acids, usually nine to 13 consecutive amino acids and most preferably 20 -30 or more consecutive amino acids.

 The polypeptides of the present invention, if soluble, can be bound to a solid substrate, such as nitrocellulose, nylon, column filling materials (e.g., Sepharose particles), magnetic beads, glass wool, plastic, metal, polymer gels, cells or other substrates. Such substrates may be, for example, in the form of beads, holes, rods or membranes.

 “Target region” means a region of a nucleic acid that must be amplified and / or detected. The term “target sequence” refers to the sequence with which a sample or primer forms a stable hybrid under certain conditions.

 In the embodiment of the present invention, unless otherwise specified, the usual methods of chemistry, molecular biology, microbiology, genetic engineering, genetics and immunology are used. See, for example, Maniatis et al., 1982; Sambrook et al. 1989; Ausubel et al., 1992; Glover, 1985; Anand, 1992; Guthrie & Fink, 1991. A general discussion of materials and methods for mapping human chromosomes, including mapping of chromosome 9p, is provided, for example, by White & Lalouel, 1988.

Preparation of recombinant or chemically synthesized acids: vectors, transformation, host cells
Large quantities of the polynucleotides of the present invention can be obtained by replication in an appropriate host cell. Natural or synthetic polynucleotides encoding the desired fragment must be inserted into recombinant polynucleotide constructs, typically DNA constructs capable of being introduced and replicated in prokaryotic or eukaryotic cells. Typically, polynucleotide constructs are designed for replication in a unicellular host, such as yeast or bacteria, but constructs designed to be introduced (with or without integration with the cell genome) into cultured mammalian, plant or eukaryotic cells of other origin can be obtained. Purification of the nucleic acids of the invention is carried out according to the methods described, for example, by Sambrook et al., 1989 or Ausubel et al., 1992.

 The polynucleotides of the present invention can be obtained by chemical synthesis, for example by the phosphoramidite method, as described by Beaucage & Carruthers, 1981, or by the triester method, as described by Matteucci et al., 1981. The synthesis can be carried out on commercial automatic oligonucleotide synthesizers. A double-stranded fragment can be obtained on the basis of a single-stranded chemical synthesis product or by synthesis of a complementary chain followed by annealing both chains together under appropriate conditions, or by completing the second complementary chain with DNA polymerase using the corresponding primer sequences.

 Polynucleotide constructs intended for introduction into prokaryotic or eukaryotic host cells must contain a replicable system recognizable by the host, including the target polynucleotide fragment encoding the desired polypeptide, as well as regulatory sequences for transcription and translation initiation, functionally linked to coding sequences. Expression vectors may include, for example, replication origin sites or autonomously replicating sequences (ARS); expression control sequences, promoter, enhancer; sites necessary for processing, such as a ribosome binding site, RNA splicing sites, polyadenylation sites, transcription terminators, and mRNA stabilization sequences.

 If necessary, secretion signals taken from the native MTS protein, other receptors, or other secreted polypeptides of the same or related species may be included. These signals allow the protein to pass through cell membranes and / or be fixed in them, while maintaining a functional topology, or secreted from the cell. These vectors can be obtained by standard recombination methods described, for example, by Sambrook et al., 1989, or Ausubel et al., 1992.

 The choice of the appropriate promoter and other important sequences of the vector is determined by the requirements of the functionality in this host, and under certain conditions, sequences associated with the MTS gene in nature can be selected. Examples of useful combinations of cell lines and expression vectors are given by Sambrook et al., 1989 or Ausubel et al., 1992; see also, for example, Metzger et al., 1988. Many convenient vectors can be obtained from suppliers such as Stratagene, New England Biolabs, Promega Biotech and others. In prokaryotic host cells, promoters such as trp, lac, phage promoters, tRNA promoters, and glycolytic enzyme promoters can be used. Convenient yeast promoters include promoters of metallothionein, 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase or glyceraldehyde-3-phosphate dehydrogenase, promoters of the enzymes responsible for the utilization of maltose and galactose. Convenient vectors and promoters for use in yeast are described by Hitzeman et al., EP 73675A. Promoters suitable for expression in mammalian cells include the early and late promoters of the SV40 virus (Fiers et al., 1978), the promoters of Moloni mouse leukemia virus, mouse breast cancer virus, avian sarcoma virus, adenovirus II, bovine papilloma virus or polyoma virus. In addition, an amplifiable gene (for example, a dehydrofolate reductase gene, DHFR) can be included in the construct, which allows multiple copies of the introduced gene to be obtained. For descriptions of acceptable enhancers and other sequences that affect expression, see also Enhancers and Eukaryotic Gene Expression, Cold Spring Harbor Press, Cold Spring Harbor, New York (1983).

 While such expression vectors can replicate autonomously, their replication in an integrated state in the genome of the host cell can be achieved.

 The expression and cloning vectors must contain a selective marker gene encoding the protein necessary for the survival and growth of the host cell transformed with the vector. The presence of this gene makes it possible to survive only those cells that express the insert. Typically, selective genes encode proteins that a) confer resistance to antibiotics or other toxic substances, such as ampicillin, neomycin, methotrexate, etc. b) complement auxotrophic deficiencies, or c) supply important nutrients not contained in the medium, for example, a gene encoding D-alanine racemase bacilli. The choice of an appropriate selective marker depends on the type of host cell used; acceptable markers for various hosts are well known.

 Vectors that contain a nucleic acid of interest can be transcribed in vitro, and the resulting RNA is introduced into the cell by one of the known methods, for example microinjection (see Kubo et al., 1988), or the vectors can be directly introduced into the cells using cell-type-dependent methods, for example, electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances. Microparticle bombardment of cells, lipofection, infection (when the vector is an infectious agent, such as a retrovirus), and other methods can be used. see Sambrook et al., 1989 and Ausubel et al. , 1992. The introduction of polynucleotides into the host cell by any of the known methods, including inter alia, is considered as "transformation". Cells into which the nucleic acids described above are introduced also mean the offspring of these cells.

 Large amounts of the nucleic acids and polypeptides of the present invention can be obtained by expressing MTS nucleic acids or fragments thereof using vectors in corresponding prokaryotic or eukaryotic cells. The most commonly used prokaryotic host cells are cells of different strains of Escherichia coli, although other prokaryotes, such as Bacillus subtilis or Pseudomonas, can be used.

 To obtain the proteins claimed in the present invention, mammalian cells and cells of other eukaryotes, for example, yeast, filamentous fungi, plants, insects, amphibians or birds, can be used. The growth of mammalian cells in culture per se is well known. See Jakoby and Pastan (eds.), 1979. Examples of widely used mammalian cell lines are VERO, HeLa, CHO (Chinese hamster ovary cells), W138, BHK, and COS cells, although in some cases cells of other lines may be more acceptable. for example, due to a higher level of expression, the desired nature of glycosylation or due to other features.

 Clones are selected using markers introduced into the structure. The marker gene may be in the same DNA molecule as the expressed gene, or maybe on another. The first option is preferable. In the case of a prokaryotic host, transformants can be selected, for example, by resistance to ampicillin, tetracycline or other antibiotics.

 A convenient marker can be the formation of a specific product only at a certain temperature (temperature-sensitive expression).

 Prokaryotic and eukaryotic cells transformed with the polynucleotides of the present invention can serve not only to produce nucleic acids and polypeptides, but also, for example, to study the properties of MTS polypeptides.

 Antisense polynucleotide sequences are useful for preventing or reducing the expression of the MTS locus, which is obvious to an experienced researcher. For example, polynucleotide vectors containing the whole MTS locus or part thereof, or other sequences from the MTS region (in particular, the flanking MTS locus) can be placed under the control of the promoter in the antisense orientation and introduced into the cell. Expression of such an antisense construct in the cell will interfere with transcription and / or translation and / or replication of MTS.

 Cyclin and Cdks are ubiquitous elements of the control of the cell cycle in eukaryotes. These proteins were originally found in yeast, then found in marine invertebrates, amphibians and mammals, including mice, rabbits and humans. Homologous genes responsible for cell cycle control were also found in animals of other species using samples and / or primers based on the gene sequence established for one biological species. Thus, samples and primers based on the MTS gene sequences described in the present invention are used to identify homologous sequences of the MTS gene and proteins in other species.

 The indicated proteins and sequences of the MTS gene were used for the diagnosis / prediction, therapy and screening of drugs in relation to the biological species from which they were isolated.

Uses: nucleic acid diagnostics and diagnostic kits
In order to establish the presence of the MTS allele, which determines the individual's susceptibility to cancer, a biological sample, such as a blood sample, is analyzed for the presence or absence of sensitive MTS alleles. To establish the presence of a tumor, to determine the progression in malignancy, or to obtain a prognostic indicator, analyze a biological sample from the site of damage for the presence or absence of neoplastic MTS alleles. The results of these analyzes and their interpretation are sent to the doctor for subsequent communication to the patient. This diagnosis can be carried out in diagnostic laboratories, or diagnostic kits can be sold to doctors or even patients for self-diagnosis.

 Initially, screening methods involved the amplification of significant MTS sequences, i.e., the use of PCR, followed by DNA sequencing. Another, more preferred embodiment of the present invention involves the use of a screening strategy that excludes the use of PCR. These screening methods imply the well-known two-step amplification labeling approach. Both strategies, using PCR and excluding it, make it possible to detect the desired sequences with high sensitivity.

 The most popular method today is target amplification. In this case, the target nucleic acid sequence is amplified by polymerases. The most preferred method of polymerase amplification is polymerase chain reaction (PCR). The PCR-based strategy and its application in the context of the present invention are shown in Example 1. PCR and other polymerase amplification methods can give more than a million-fold enrichment of the number of copies during amplification cycles. After amplification, the resulting nucleic acid can be sequenced or used as a substrate for DNA samples.

 When using DNA samples to detect target sequences (for example, when screening for tumor sensitivity), a biological sample intended for analysis, such as a blood sample, must be properly processed to isolate nucleic acids. To facilitate detection of the target sequence, nucleic acids isolated from the sample can be processed in various ways, for example, denatured, restriction, fractionated by electrophoresis or subjected to dot blotting. The target region in the analyzed nucleic acid must be at least partially single-stranded to form hybrids with the sequence of the sample. If this sequence is initially single-stranded, then denaturation is not required. However, if the sequence is double-stranded, it must be denatured. Denaturation is carried out by one of the known methods.

 The analyzed nucleic acid and the sample are incubated under conditions conducive to the formation of a stable hybrid between the target sequence of the sample and the desired sequence in the sample. The region of the sample that is used to bind to the analyzed nucleic acid can be made completely complementary to the target region of the 9p human chromosome. To prevent pseudo-positive results, hybridization should be carried out under stringent conditions. However, stringent hybridization conditions are necessary only if the samples are complementary to those regions of the chromosome that are unique in the genome. The stringency of hybridization conditions is determined by a number of factors affecting the stages of hybridization and subsequent washing. These include temperature, ionic strength, base composition, sample length, and formamide concentration. These factors are described, for example, by Maniatis et al., 1982 and Sambrook et al. , 1989. Under certain conditions for the detection of a target sequence, the formation of higher order hybrids, such as triplexes, quadruplexes, etc., may be desirable.

 The detection of hybrids formed is usually simplified by the use of labeled samples. Conversely, a sample may be unlabeled, but detected by direct or indirect binding to a labeled ligand. Acceptable labels, as well as methods for labeling samples and ligands, are well known and include, for example, radioactive labels incorporated by known methods (e.g. nick translation, random priming or curing), biotin, fluorescent groups, chemiluminescent groups (e.g. dioxetanes, partially triggered dioxetanes), enzymes, antibodies, etc. Variations of these basic schemes are diverse and are mainly aimed at facilitating the separation of the desired hybrids from the surrounding material and / or amplification of the signal of the labeled component. A number of such variations are described, for example, Mattews & Kricka, 1988; Landegren et al., 1988; Mittin, 1989 :; US Patent 4,868,105 and EPO Publication No. 225807.

 As indicated above, a screening strategy that excludes the use of PCR is included in the present invention. An exemplary procedure without PCR is shown in Example 15. According to this approach, a nucleic acid sample (or its analogue, for example, a methylphosphonate scaffold that replaces the normal phosphodiester) is hybridized with the target DNA. An enzyme may be covalently attached to the sample so that this covalent bond does not interfere with the specificity of hybridization. The enzyme-probe-conjugate-target nucleic acid complex is then separated from the free probe-enzyme conjugate and a substrate is added to detect the enzyme. Enzymatic activity is judged by the development of color or by registration of luminescence, which increases the sensitivity by a thousand to a million times. As an example of the preparation of the polygonucleotide alkaline phosphatase conjugate and its use as a hybridization test, the work of Jablonski et al., 1986 can be mentioned.

 The methodology of two-stage amplification with a label is also known. This method is based on the fact that a small ligand (for example, digoxygenin, biotin or the like) is conjugated to a nucleotide probe capable of specifically binding to MTS. An example of a probe on MTS1 is a nucleotide probe corresponding to nucleotides 448 to 498 of the sequence SEQ ID NO: 4. Allele-specific probes are also included in this example and overlap predisposing mutations summarized in Table 3 and somatic mutations in tumors summarized in Table 5.

 In one case, a small ligand conjugated to a nucleotide probe is specifically recognized by an antibody-enzyme conjugate. For example, a nucleotide probe is conjugated to digoxygenin. Hybridization is detected by an antibody-alkaline phosphatase conjugate that activates a chemiluminescent substrate. Nucleic acid labeling methods relevant to the context of this invention are described by Martin et al., 1990.

 In another case, a small ligand is recognized by a second ligand-enzyme conjugate that is capable of forming a specific complex with the first ligand. Well-known embodiments of this approach are biotin-avidin interactions. Methods for labeling nucleic samples and their use in tests based on the use of biotin-avidin are described by Rigby et al., 1977 and Nguyen et al., 1992.

 The use of a mixture of nucleotide probes capable of detecting MTS genes is included in the context of the present invention. Thus, in one example, more than one probe complementary to MTS1, in particular 2, 3 or 5 different nucleotide probes, was used to detect MTS1 in a cell sample. In another example, more than one complementary MTS1 probe was used to detect mutations in the MTS1 gene sequences in a patient, in particular a mixture of probes capable of binding to allele-specific mutations identified in a population of patients with MTS1 disorders. With this approach, any number of probes can be used, which should preferably consist of probes corresponding to major gene mutations predisposing an individual to breast cancer. Some possible samples claimed in the present invention have allele-specific mutations shown in Table. 3 and Table 5.

Methods of use: peptide diagnostics and diagnostic kits
Neoplastic conditions can be diagnosed based on changes in the wild-type MTS polypeptide. Such changes can be detected by sequencing using standard methods. It is preferable to use antibodies (polyclonal or monoclonal) to detect differences (or lack thereof) in MTS peptides. In a preferred embodiment of the invention, the antibodies immunoprecipitate MTS proteins from solution, or these antibodies react with the MTS protein on Western or immunoblots of polyacryamide gels. In another embodiment, the antibodies recognize the MTS protein in paraffin or frozen tissue sections by immunocytochemical methods. Methods for the preparation and purification of antibodies are widely known and any of these methods can be selected to achieve the objectives of the present invention. An enzyme-linked immunosorbent method (ELISA), a radioimmunological method (RIA), an immunoradiometric method (IRMA), and an immunoenzymatic method (lEMA), including a sandwich method using monoclonal and / or polyclonal antibodies, can be used to detect MTS peptides and mutations of these peptides. Examples of the sandwich method are described by David et al., In US Patent NN 43761100 and 4486530, incorporated by reference and are given in Example 18.

Uses: screening compounds
The present invention is applicable for screening compounds using Cdk polypeptides or binding fragments thereof in any of a variety of drug screening procedures.

 It is preferable to use Cdk4. The Cdk peptide or fragment thereof used in this test may be in the form of a solution, fixed on a solid substrate, or located on the cell surface. In one of the methods for screening substances based on competitive binding, eukaryotic or prokaryotic host cells are used that are stably transformed with recombinant polynucleotides and expressing the polypeptide or its fragments. These cells in a living or fixed state can be used in standard binding experiments. You can determine, for example, the formation of complexes between the Cdk polypeptide or its fragment with the analyte, or to determine the extent to which the analyte affects the formation of the complex between the Cdk polypeptide (or its fragment) and the MTS polypeptide (or its fragment).

 Thus, the present invention provides drug screening methods based on binding a test drug to a Cdk polypeptide and to determine: 1) the presence of a complex between a test agent and a Cdk polypeptide or fragment thereof, or 2) the presence of a complex between a Cdk polypeptide or its fragment and ligand .

 Cdk activity is also measured to establish the ability of the test agent to inhibit Cdk and, accordingly, regulate the cell cycle. In such competitive assays, the Cdk polypeptide or fragment thereof is usually labeled. The free Cdk polypeptide or fragment thereof is separated from the one bound in the protein / protein complex, and the amount of free (i.e., unbound) label is an indication of the binding of the test agent to Cdk or an indication of the interference of the former with the binding of Cdk: MTS. Small portions of the MTS polypeptide (mimetic peptides) are analyzed by this method to identify those peptides that have Cdk inhibitory activity.

 Another high-throughput method for screening compounds having affinity for Cdk polypeptides is described by Heisen (Geysen, European Patent Application N 84/03664, published September 13, 1984). Briefly, a large number of different peptides that need to be tested are synthesized on a solid substrate, such as plastic sticks or some other surface. These peptides are allowed to react with the Cdk polypeptide and washed. The bound Cdk polypeptide is then detected by known methods.

 Purified Cdk can be applied to tablets for use in the aforementioned drug screening methods. However, non-neutralizing antibodies to the polypeptide can be used to immobilize Cdk in the solid phase.

 The present invention also claims the use of competitive screening tests in which neutralizing antibodies capable of specifically binding a Cdk polypeptide compete with a test compound for binding to a Cdk polypeptide or fragment thereof. Thus, antibodies are used to detect any peptide having one or more common antigenic determinants with a Cdk polypeptide.

 Other drug screening methods involve the use of eukaryotic cell lines or cells (similar to those described above) that have a non-functional MTS gene. Similar cell lines or cells are defective with respect to cell cycle control at the Cdk level. Such cells are grown in the presence of a test substance. The cell growth rate is determined and, based on these data, a conclusion is drawn on the ability of the test substance to regulate the cell cycle. One way to determine growth rate is to determine the biological activity of individual Cdk, preferably Cdk4.

Uses: rational drug design
The goal of rational drug design is to create structural analogues of biologically active polypeptides or small molecules with which they interact (for example, agonists, antagonists, inhibitors) to obtain drugs that, for example, are more active or stable forms of polypeptides that enhance or inhibit in vivo function of the polypeptide. See, for example, Hodgson, 1991.

 In one approach, the three-dimensional structure of the polypeptide of interest (e.g., p16 or Cdk4) or the Cdk4-pl6 complex is first determined using x-ray crystallography, computer simulation, or more often, a combination of these methods. Less commonly, useful information regarding the structure of a polypeptide can be obtained by modeling the structure of homologous proteins. An example of rational drug design is the development of human immunodeficiency virus (HIV) protease inhibitors (Erickson et al., 1990). In addition, peptides can be analyzed in an alanine test (Wells, 1991). According to this method, the amino acid residue is replaced with Ala and the effect of such a replacement on the activity of the peptide is determined. Thus, each amino acid of the peptide is analyzed to determine its important regions.

 It is also possible to obtain target-specific antibodies selected in a functional test, followed by determination of their crystal structure. In principle, this approach can provide basic information for rational drug design.

 Crystallography of proteins can be avoided by obtaining anti-idiotypic antibodies to functional, pharmacologically active antibodies. As a double mirror image, the binding site of anti-idiotypic antibodies should be an analogue of the original receptor. Anti-idiotypic antibodies can then be used to identify and isolate peptides from banks of chemically or biologically derived peptides. Selected peptides serve as the basis for drug design.

 Thus, preparations can be designed that have, for example, increased MTS activity or stability, or that act as inhibitors, agonists, antagonists, and other MTS activities. Due to the availability of cloned MTS sequences, the MTS polypeptide can be obtained in quantities sufficient for analytical procedures such as x-ray crystallography. In addition, the MTS protein sequence disclosed in the present invention allows computer simulation to be used with or instead of X-ray crystallography.

Methods of application: gene therapy
The present invention provides a method for restoring the function of wild-type MTS in a cell carrying mutant MTS alleles. The restoration of such a function should suppress the neoplastic growth of recipient cells. The wild-type MTS gene (or part of this gene) can be introduced into the cell via a vector providing extrachromosomal persistence of the gene in the cell. In this case, the gene will be expressed in the cell without integration into the chromosome. If a gene fragment is introduced and expressed in a cell carrying mutant MTS alleles, then this fragment should encode that part of the MTS protein that is necessary for neoplastic cell growth.

 A more preferable situation is when the wild-type MTS gene or its fragment is introduced into the cell in such a way that provides recombination with the endogenous mutant MTS gene present in the cell. Such recombination requires two recombination events to correct the mutant MTS gene. Vectors are known both for introducing genes for the purpose of recombination and for maintaining them in an extrachromosomal state, and the choice of such vectors is quite wide. Such methods of introducing DNA into cells as electroporation, calcium phosphate coprecipitation and viral transduction are well known and the choice of a specific method of introduction is entirely within the competence of the researcher. Cells transformed with the wild-type MTS gene can serve as a model system for studying tumor remission and therapeutic approaches that promote such remission.

 As previously discussed, the MTS gene or its fragment can be used in gene therapy to increase the number of expression products of these genes in tumor cells. A similar approach is especially applicable in the case of cancerous and precancerous cells, in which the level of the MTS polypeptide is reduced compared to normal cells. This approach can also be used to increase the level of MTS expression in those tumor cells in which the mutant gene is expressed at the “normal” level, but the expression product is not fully functional.

 Gene therapy should be carried out according to generally accepted methods, for example, as described by Friedman in Therapy of Genetic Diseases, T. Friedman, ed. Oxford University Press (1991), pp. 105-121. Cells from a patient's tumor should be initially analyzed using the methods described above to determine the level of production of the MTS polypeptide in these cells. A viral or plasmid vector is prepared in which a copy of the MTS gene is linked to expression control elements and is capable of replication in tumor cells. Suitable vectors are described, for example, in US Pat. No. 5,252,479 and published PCT application WO 93/07282. Then the vector is injected into the patient locally into the tumor or systemically so that it can penetrate into the tumor cells of possible metastases. If the transfected gene does not integrate permanently into the genome of each tumor target cell, treatment may be repeated periodically. Since MTS polypeptides are involved in cell cycle control, it is preferable that the MTS gene be introduced along with its own regulatory elements to avoid constitutive expression of MTS polypeptides in all cells that receive this gene as a result of the described procedure.

 Known gene transfer systems can be used to implement the claimed invention for gene therapy purposes. These systems include both viral and non-viral transfer methods. Many viruses have been used as vectors for gene delivery: papovaviruses (e.g., SV40, Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988, Gorziglia and Kapikian, 1992; Quantin et al., 1992; Rosenfeild et al., 1992; Wilkinson et al., 1992; Stratford-Perricaudet et al., 1990), vaccinia virus (Moss, 1992), adeno-associated virus (Muzyczka, 1992; Ohi et al., 1990), herpes viruses, including HSV and EBV (Margolskee, 1992; Johnson et al., 1992; Fink et al., 1992; Breakfeild and Geller, 1987; Treese et al., 1990) and avian retroviruses (Brandyopadhyay and Temin, 1984; Petropoulos et al., 1992), mouse (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann and Baltimore, 1985; Miller et al., 1988) and of human origin (Shimada et al., 1991 ; Helseth e t al., 1990; Page et al., 1990; Buchschacher and Panganiban, 1992). Most methods of human gene therapy are based on the use of inactivated mouse retroviruses.

 Currently known non-viral gene transfer methods include chemical approaches such as calcium phosphate coprecipitation (Graham and van der Eb, 1973; Pellicer et al., 1980); mechanical methods, for example microinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster et al., 1981; Constantini and Lacy, 1981); membrane fusion transfer via liposomes (Felgner et al., 1987; Wang and Huang, 1989; Kaneda et al., 1989; Stewart et al., 1992; Nabel et al., 1990; Lim et al., 1992); direct DNA capture and DNA transfer using receptors (Wollf et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al., 1989b; Wollf et al., 1991; Wagner et al., 1990; Wagner et al., 1991; Gotten et al., 1990; Curiel et al., 1991a; Curiel et al., 1991b). Virus-mediated gene transfer can be combined with direct in vivo gene transfer using liposomes, which allows the delivery of viral vectors to tumor cells, rather than to non-dividing surrounding cells.

 In addition, cells producing a retroviral vector can be injected into tumors (Culver et al., 1992), which creates a constant source of viral particles. The specified method was tested on patients with inoperable brain tumors.

 In an approach combining biological and physical methods of gene transfer, plasmid DNA of any size binds to polylysine-conjugated antibodies specific for an adenovirus hexon protein, and the resulting complex is coupled to an adenoviral vector. Then cells are infected with this trimolecular complex. The adenoviral vector provides effective binding, internalization and degradation of the endosome before degradation of the bound DNA occurs.

 It has been shown that DNA / liposome complexes can be used for direct gene transfer in vivo. While in the case of the standard liposome preparation method, the in vivo gene transfer process is nonspecific, in vivo DNA transfer and expression in tumors have been proven, for example, by direct in situ administration (Nabel, 1992).

Methods of use: peptide therapy
Peptides having MTS activity can be delivered to cells with lost MTS alleles or carrying mutant MTS alleles. The sequences of the MTS proteins are described (SEQ ID NO: 2, SEQ ID NO: 14 and SEQ ID NO: 16). A protein can be obtained by expressing cDNA in bacteria, for example, using known expression vectors. Alternatively, the MTS polypeptide can be extracted from MTS-producing mammalian cells. In addition, synthetic chemistry approaches can be used to synthesize MTS protein. Any of these methods allows you to get declared in the present invention, the drug consisting of MTS protein. This drug is largely free from impurities of other human proteins. This goal is most easily achieved with in vitro synthesis and synthesis in microorganisms.

 Active MTS molecules can be introduced into cells by microinjection or via liposomes. Some of these active molecules can be captured by cells actively or by diffusion. Extracellular use of the MTS gene product may be sufficient to have an effect on tumor growth. Delivery of molecules with MTS activity should lead to a partial reversal of the neoplastic state. Other molecules with MTS activity (e.g., peptides, drugs, or organic compounds) can also be used to achieve this reversion. Modified peptides having a very similar function are also applicable to peptide therapy.

Methods of application: transformed hosts
Cells and animals carrying the mutant MTS allele can be used as model systems for the study and testing of substances from which a potential therapeutic effect can be expected. For cultivation, epithelial cells are usually used. Somatic or germ line cells can be obtained from patients with MTS mutations.

 In contrast, a cell line with a mutation of the MTS allele can be constructed as described above. After the test substance is added to the cells, a neoplastic transformed cell phenotype is determined. Any of the parameters of neoplastic cell transformation, including substrate-independent growth, tumorigenicity in nude mice, invasiveness or dependence on growth factors, can be characterized. The methods for this are well known.

 Animals for testing therapeutic agents can be selected after mutagenesis carried out on whole animals, germ line cells or zygotes. Such mutagenesis involves the introduction of mutant alleles of MTS, usually an animal of a different species, or the introduction of damaged homologous genes. In contrast, the endogenous MTS gene (s) of animals can be damaged by insertion or deletion or the introduction of other genetic damage using known methods (Capecchi, 1989; Valancius and Smithies, 1991; Hasty et al., 1991; Shinkai et al., 1992; Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992; Donehower et al., 1992). After administration of the test substances to the animals, tumor growth should be monitored. If the tested substances prevent or inhibit the growth of tumors, then these substances are possible candidates for the role of therapeutic agents in relation to the forms of cancer indicated in this application.

 The present invention is illustrated by the following examples, which in no way limit its use. Used standard methods or methods specifically described below.

Example 1. Materials and Methods
A. Genealogy of MTS
In FIG. 1A-1D, Pedigrees 3137, 3161, 3355, and 1771 are shown, respectively. The frequency of cancer in these pedigrees is indicated on the drawings. In all melanomas in genealogy 3137, a sensitive haplotype was found, which was found in the analysis of this genealogy in tumors of a different origin.

 All melanomas in genealogies 3161 and 3355 had a sensitive haplotype. MTS mutations were identified in tumors in the 1771 family tree.

B. Tumor cell lines
Seventy-six cell lines of melanoma were obtained from the Ludwig Cancer Institute (Memorial Sloan Kettering Cancer Center), 8 melanoma and 5 non-melanoma cell lines were obtained from the American Cell Collection (ATCC).

C. Isolation and analysis of DNA from tumor cell lines
DNA was isolated from tumor cells by adding 3 ml of lysis buffer (0.1 M NaCl; 0.1 M Tris HCl pH 8.0; 5 mM EDTA; 0.5% SDS) to approximately 10 million cells. The mixture was stirred on a Vortex shaker and incubated at 65 ^° C. for 30 minutes. Then added 0.5 ml of 8M KOAc, the mixture was stirred and incubated in
ice 30 minutes After centrifugation (five minutes at 10,000 · g), the supernatant was precipitated with an equal volume of 95% ethanol and centrifuged again (15 minutes at 10,000 · g). DNA was resuspended in 50-200 ml of H ₂ O.

D. Polymerase chain reaction (PCR)
50 ng of the template was added to 30 pM of each oligonucleotide primer in 20 ml of the reaction mixture, which contained 0.1 mM dNTPs, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2 mM MgCl ₂ , 0.01% gelatin and 1 Amplitaq polymerase unit ( Perkin-Elmer). Samples were subjected to 35 cycles on a Perkin-Elmer 9600 thermosikler at 94 ^° C. for 10 seconds, at 55 ^° C. for 10 seconds and at 72 ^° C. for 10 seconds. After electrophoresis in 1.5% agarose (SeaKem) or 3% NuSieve agarose (3: 1) (FMC BioProducts), the polymerase chain reaction product was visually detected by staining with ethidium bromide.

E. Artificial yeast chromosomes (YACs)
Artificial yeast chromosomes (YACs) containing MTS region markers were obtained by screening YACCN libraries using IFNA, D9A171 and D9S126 probes using the PCR conditions described above. Yeast strains containing YACs were grown at 30 ^° C for three days under vigorous stirring in ANS medium (10 g / L casein acid hydrolyzate; 1.7 g / L basic yeast nitrogen; 5 g / L ammonium sulfate; 20 mg / L adenine hemisulfate ; 2% glucose; pH 5.8). Yeast DNA was isolated as described by Ausubel et al., 1992.

F. Creating a phage library
Yeast genomic DNA containing YAC DNA was subjected to complete hydrolysis with BamHI restriction enzyme, then using T4 DNA ligase (Boehringer-Mannheim) ligated with BamHI-restricted phage arms EMBL3 (Promega) and packaged in vitro using Gigapack II (Stratene) extracts. The phage were grown in E. coli cells of strain C600. Recombinant phages containing human DNA were identified by hybridization with human Cot-1 DNA labeled with 32-P (GIBCO-BRL). Phages containing human sequences included in the YAC vector (terminal clones) were identified by screening with PCR fragments containing the left or right shoulder of the artificial yeast chromosome. Hybridization and washing were performed under standard conditions (Middleton et al., 1991). Selected positive plaques, which were purified by triple reseeding.

 Phage DNA was isolated on Qiaex columns (Qiagen).

G. Creation of a cosmid library
The yeast genomic DNA containing YAC DNA was partially hydrolyzed by Sau3A restriction enzyme and fractionated by linear sucrose gradient (10-40%) as described by Maniatis et al., 1982. SuperCos 1 cosmid vector (Stratagene) was prepared according to the manufacturer's instructions, mixed with the DNA insert at a ratio of 4: 1 (insert: vector), treated with ligase and packaged in vitro as described above.

 Cosmids were introduced into the host cells of DH57 alpha and scattered at a density of 02,000 colonies per 15 cm dish. Colony hybridization was performed as described above and as described by Maniatis et al., 1982.

N. P1 clones
P1 clones spanning the MTS region were obtained from Genome Systems, Inc. , St. Louis, Missouri, by screening using STS obtained as described in this application. DNA from these clones was isolated by alkaline hydrolysis (Birnboim and Doly, 1979), and then purified by centrifugation in a density gradient of cesium chloride (Maniatis et al., 1982).

I. Getting STS (site-tagged sequences)
STS was obtained by sequencing 1.0 mg of P1, cosmid or template DNA with oligonucleotides complementary to sequences that flank the cloning site in P1 vector (pSacBII), SuperCos 1 vector or EMBL3 vector. Sequencing was performed using the ABI 373A DNA sequencing system with the PRISM Ready Reaction DyeDeoxy Terminator Cycle (ABI) sequencing kit. STS were designed so that, if possible, have a length of about 20 kilobases and Tm close to 60 ^o C.

J. Germinal MTS1 gene mutations in pedigrees susceptible to melanoma
The genomic DNA of carrier individuals was isolated from blood by standard methods. The primers were designed to amplify the coding exons 1 or 2 of the MTS1 gene or the coding exon 2 of the MTS2 gene using 20 ng of DNA in each sample.

 A standard buffer was used for PCR with the exception that DMSO was added to a final concentration of 5%. Cyclic sequencing reactions were performed on amplified products using alpha-P32-dATP and using primers located at different points in the sequence. Sequencing products were analyzed by electrophoresis in 6% denaturing polyacryamide gels, sequentially applying all reaction mixtures (A) to the gel, then all reaction mixtures (B), etc. All polymorphisms were confirmed by sequence analysis of the opposite chain.

Example 2. Localization of MTS using genetically linked markers
A set of markers associated with MTS was used to analyze tumor cell lines for homozygous deletions in the 9p21 region. These markers were initially used to show a predisposition to melanoma in 10 pedigrees in Utah and in one pedigree in Texas (Cannon-Albright et al., 1992). The indicated set of markers included a sequence from the alpha interferon gene cluster (IFNA) (Kwiatkowski & Diaz, 1992), which served as the most distal marker; a proximal marker (D9S104) and four additional intermediate markers (D9S171, D9S126, D9S161 and D9S169) (Cannon-Albright et al., 1992). Genetic studies allow you to arrange these markers in the following sequence: D9S171, D9S126, D9S161, D9S169. The IFNA marker consists of an oligonucleotide primer pair that amplifies two wild-type genomic DNA fragments: a 138-150 base pair polymorphic fragment containing a poly (CA) region, and an invariant 120 base pair fragment (IFNA-s). The localization of IFNA-s in relation to IFNA-1 remains unknown.

 Using genetic markers, five non-melanoma tumor cell lines having deletions were analyzed. In each of the cell lines, homozygous deletions of at least one of the studied markers were detected (Table 1). When using markers D9S161, D9S169 and D9S104 homozygous deletions were not detected. The minimum overlapping region of the identified deletions was flanked by IFNA-1 and D9S171; this suggests that in the region between these two markers is located the gene (s) involved (s) in tumor suppression, possibly MTS. The genomic region between D9S171 and IFNA-1, especially adjacent to IFNA-s, was subsequently investigated in more detail.

Example 3. Genomic clones of the MTS region
To obtain genomic clones adjacent to the IFNA-s region, YACN CERN libraries were screened (Cohen et al., 1993). Eleven YAC clones were identified as containing the D9S171 marker and five as containing IFNA-s (Fig. 2). Three YAC clones (C9, C6, F9) were subcloned into the phage and one YAC clone (C6) was subcloned into the cosmid vector. Not obtained YAC clones containing both D9S171 and IFNA-s simultaneously (Figure 2). These clones made it possible to obtain STSs internal to the known genetic markers, as well as to implement the chromosome walk procedure described below.

 To create an independent source of genomic DNA in order to construct a continuous genomic map of a given region and to facilitate the creation of STS, a “chromosome walk” was carried out in P1 clones from IFNA-s towards D9S171, then back from D9S171 to IFNA-s, and from the ends of the YAC C6 in both directions. As a result, a total of 27 P1 clones were isolated (Fig. 2). The ordered P1 clones formed a series of contigs spanning the distance from INFA-s to D9S171 with two passes. Clones P1, along with some phage and cosmid clones, were used to obtain a detailed map of the MTS region.

Example 4. A detailed analysis of the structure of the MTS region
Additional markers were required to obtain a more detailed molecular map of the MTS region. DNA sequences obtained from genomic clones were used to construct PCR primers for STS. These STSs, in turn, served to order P1 clones and YAC clones. A total of 54 STS from the region between IFNA-s and D9S171 served as the basis for creating a detailed physical map of the MTS region (Figure 2). The indicated primary STS sequences were deposited in the Genome Database.

 A set of new markers spanning sections from IFNA-s to D9S171 was used to analyze 84 melanoma cell lines for homozygous deletions in the MTS region. Homozygous deletions were found in 52 lines (Fig. 3). Some of the deletions were extensive; for example, in 13 lines areas including both 816.7 and 760-L were lost.

 For the purposes of MTS localization, the most informative cell lines were divided into two groups (Fig. 3): I) containing only c5.1 deletion (class 11); and II) containing only c5.3 deletion (class 12). Only 5 melanoma lines fell into these categories. In all detected cases of deletions, these deletions were simple, that is, there were no indications of multiple deletion acts in the region between IFNA-s and D9S171. If we take into account the presence of lines in which deletions overlap near markers c5.1 and c5.3, then there is no need to create a complete physical map of the region from IFNA-s to D9S171.

Example 5. Identification of cosmids c5 and P1, colonies P1062 and P1063 as containing MTS
A. Location of genetic markers
Analysis of YAC clones and deletions in tumor lines yielded results that coincided with the genetic position of the markers: IFNA, D9S171, D9S126. Three YAC clones contained D9S171 and D9S126, and four YAC clones contained IFNA-1 and IFNA-s (Fig. 2). None of them contained D9S171 and IFNA together. Therefore: 1) IFNA-1 and IFNA-s are closely related and 2) are connected D9S126 and D9S171. These results are confirmed by deletions in cell lines. In most cell lines that lost D9S171, D9S126 was also absent. Conversely, the U-87 line, positive for the D9S171 and D9S126 markers, lost IFNA-1 and IFNA-s (Table 1). In one of the melanoma cell lines, SK-mel-5, IFNA-s, D9S171 and D9S126 were absent, but IFNA-1 was detected. Thus, IFNA-1 must be removed from IFNA-s. In another melanoma cell line, SK-Mel-Zan, IFNA-1, IFNA-s and D9S171 were deleted, but not D9S126, which gives reason to put D9S171 between IFNA-s and D9S126. Together, these data confirm the location of the markers given in Table. 1.

 The gene family of human alpha-interferon consists of more than 23 genes and pseudogenes located on the 9p chromosome. This gene cluster has been cloned and divided into 10 binding groups (Henco et al., 1985). Binding groups were partially ordered by analysis of deletions of various alpha-interferon sequences in glioma cell lines (Olopade et al., 1992). In glioma line H4, IFNA-1 and IFNA-s are lost. It also lacks the binding group IV sequences (i.e., alpha 13, alpha 6, and alpha 20). In the glioma line A172, there are both IFNA-1 and binding group IV, but the sequences from the binding groups I (i.e. alpha 1, alpha 19), III (i.e. alpha 8) and IX (i.e. alpha 2) and IFNA-s. The distal border of deletions in A172 was determined within the same P1 length from IFNA-s. Thus, the binding groups I, III, and IX should be located proximal to the point 85 kb from IFNA-s.

B. Physical distance between genetic markers
The available results do not accurately determine the distance between IFNA-s and D9S171, because it was not possible to isolate the YAC clone containing both markers. Furthermore, based on STS mapping, it was shown that none of the five YAC clones spanning sequences distal to IFNA-s matched any of the 11 YAC clones proximal to IFNA-s. Considering that YAC SERN inserts average 500 kilobases, it can be considered that the distance between IFNA-s and D9S171 is not less than this value.

 The area between IFNA-s and c5.1 overlaps with nine steps in the P1 library. If we take the average value of each step equal to half of the P1 insert, then the distance between c5.1 and IFNA-s is approximately 400 kilobases. Thus, the tumor suppressor gene is closely related to c5.1, which is often lost in melanoma lines, and is located in 400 kilobases proximal to IFNA-s.

C. Deletion in tumor lines
Homozygous deletions of the 9p21 region were found in 57% of the examined melanomas. Fourteen tumor lines had deletions that extended to the proximal side from 760-L, and 16 lines had deletions extending beyond 816.7 to the distal side. If we accept the causal significance of deletions, that is, deletions of a gene (s) in this area are involved in the formation of a tumor phenotype, then the suppressor gene (or suppressor genes) should lie between 760-L and 816.7. The smallest deletions capture markers c5.1 and c5.3. Of all the markers studied, c5.3 was deleted in the largest number of lines, namely 51. Therefore, the most likely location of the suppressor gene is near c5.3, since this is the most frequently deleted marker. Four lines had deletions only c5.3 (class 12) and one - only c5.1 (class 11). Both of these markers are represented in cosmid c5. Thus, it is likely that the suppressor gene includes the sequences represented in cosmid c5. P1 clones P1062 and P1063 contained sequences found in and adjacent to cosmid c5. Thus, as will be shown below, P1062 and P1063 contained the entire MTS region.

 The results given in the Examples are consistent with preliminary genetic studies of MTS, which showed that the region between IFNA-s and D9S126 is the most likely localization of MTS (Cannon-Albright et al., 1992). Recent genetic studies have clarified the localization of MTS using a (CA) polymorphic repeat lying between IFNA-s and c5.3 at P1-452 (Fig. 2). Analysis of the recombinant chromosome using this marker allowed MTS to be placed proximal to P1-453. Thus, MTS is mapped in the region of homozygous deletions in melanoma cell lines.

 These results support the suggestion that the tumor suppressor locus, MTS, is located near c5.3.

 All lines with deletions lost the same DNA sequences despite the fact that the set of deletions was limited to c5.1 or c5.3 (classes 11 and 12). There are no indications of overlapping deletions in the used set of cell lines other than deletions presented in cosmid c5. Thus, there is no reason to build a more complex scheme, for example, with the participation of the second locus-suppressor in 9p21, remote from c5.1 and c5.3.

 The fact that homozygous deletions of the 9p21 region are observed in tumors of many different types indicates that a localized gene or suppressor genes can be expressed in a variety of tissues. The suppressor gene can be similar to the p53 gene in that it can be involved in the development of different types of tumors (see below). Other types of tumors are also found in melanoma-prone families (Nancarrow et al., 1993; Bergman et al., 1990). A thorough deletion analysis of various types of tumors using c5.1 and c5.3 (shown below) confirmed the importance of the tumor suppressor gene in the development of neoplasias other than melanoma.

 Some of the observed homozygous deletions remove several genetic markers. Fountain et al. described homozygous deletions of 2-3 megabases in length on chromosome 9p21 in two different melanoma lines (Bergman et al., 1990). In this study, at least one line, SK-MEL-5, had deletions extending from the most distal of the studied markers, IFNA-1, and capturing D9S126. This area is too large to be represented in a single YAC clone. The predominance of extended deletions indicates that the region surrounding MTS lacks the genes necessary for cell viability.

Example 6. Isolation of candidate MTS genes
The previous Examples described the results of a "walk on the chromosome" in the MTS region using YAC and P1 clones. Detailed STS mapping based on cosmid c5 sequences showed small non-overlapping deletions of c5 sequences in five different melanoma lines. These results suggest that the tumor suppressor gene, presumably MTS, is at least partially located in c5 sequences.

 Another indication that c5 contains at least one gene was obtained by analyzing the frequency (CpG) of dinucleotides in c5 cosmid and adjacent cosmids. In mammals, almost all housekeeping genes and almost half of tissue-specific genes are associated with regions enriched in (CpG) dinucleotides (Bird, 1989; Larsen et al., 1992). Thus, the presence of “CpG” islands is an indicator of the presence of genes. Cosmids c5, c12, c57, and c59 were restricted with endonucleases EagI, BssHI, and SacII, i.e. enzymes whose recognition sites contain two (CpG) pairs. Only cosmids c5 and c12 contained restriction sites for these enzymes. Cosmid c5 had one EagI site, at least 10 BssHI sites, and at least 12 Sacl sites. The presence of CpG islands in cosmid c12 and overlapping cosmid c12 suggests that cosmid c5 has at least one MTS candidate gene.

 To search for MTS, the nucleotide sequences of EcoRI restriction fragments of cosmid c5 were determined. When these sequences were compared with sequences obtained from the Genebank, two different c5 regions were identified that showed significant similarity to the sequence of the previously identified human cyclin-dependent kinase 4 inhibitor gene or p16 (Serrano et al., 1993). These two candidate MTS genes were designated MTS1 and MTS2. MTS1 was localized near the end of cosmid c5 closest to the telomere of chromosome 9p, and MTS2 was localized near the centromere end of c5 (See Fig. 4B). Cosmid maps showing the positions of MTS1 and MTS2, as well as maps of the P1 clones 1062, 1063 and 1069 are shown in FIG. 4A.

 A detailed comparison of the genomic sequence of MTS1 from c5 cosmid with the p16 mRNA sequence showed that MTS1 contains a 307 base pair fragment identical to that of the p16 coding sequence. This fragment of the nucleotide sequence of MTS1 is flanked by recognizable sequences of splicing compounds. It was further shown that MTS1 contains the complete coding sequence p16 and two more introns (Figs. 5A and 5B and Figs. 6A and 6B). Intron 1 was localized at 126 base pairs "below" the site of the start of translation; intron 2 was localized at 11 base pairs "above" the site of the end of the broadcast. Two introns divide the coding sequence of MTS1 into three regions: the 5 ′ region of 126 base pairs (encoding exon 1), the middle region of 307 base pairs (encoding exon 2), and the 3 ′ region of 11 base pairs (encoding exon 3). SEQ ID NO: 3 represents the nucleotide sequence of the 5'-region, exon 1 and part of intron 1 of MTS1. SEQ ID NO: 4 represents the nucleotide sequence of part of intron 1, exon 2, and part of intron 2 of MTS1.

 MTS2 contains a DNA sequence that is almost identical to p16, which extends from the 5 ′ end of coding exon 2 to approximately 211 nucleotides towards nitron 2 (FIG. 7A). However, sequence similarity decreases to the 51st pair of nucleotides “above” MTS1 intron 2, which corresponds to the localization of the last MTS2 codon (Fig. 8). Comparison of the sequences of MTS1 and MTS2 shows that the similarity of the sequences of these genes is found approximately 40 nucleotides "higher" than the 3'-spliced compound of intron 1. Thus, fragments of non-coding DNA turned out to be more conservative than some regions of the supposedly coding DNA. To exclude the possibility that sequence divergence was an artifact of cloning, PCR primers were designed to specifically amplify the sequences at the divergence point of MTS2. Using these primers, a fragment of the predicted size from cosmids, P1 clones, and genomic DNA was amplified. Thus, the diverged sequence located near the 3 ′ end of exon 2 in MTS2 is a bona fide genomic sequence. SEQ ID NO: 5 is the nucleotide sequence of a portion of intron 1, exon 2, and intron 2 of MTS2. SEQ ID NO: 15 represents the cDNA sequence of MTS2.

 The discovery of two closely related genes in cosmid c5 suggests that there are other related genes in this region. To test this possibility, Southern blotting of restriction fragments of cosmids c5, c12, c59, Pls 1063 and human genomic DNA was performed. As a sample in these experiments, we used a fragment containing most of exon 2 of MTS1, including the region common with MTS2. Two positive EcoRI fragments were detected in genomic DNA and in cloned DNA. This result confirms the presence in the genome of two p16-like genes, MTS1 and MTS2. It also confirms the already known existence of MTS1E1-beta, an alternative form of MTS1 containing exons 2 and 3, but not exon 1 of MTS1.

Example 7. Isolation and structure of MTS1E1 beta
Isolation of MTS1E1 Beta
Clones containing MTS1E1 beta were isolated by selecting hybrids using the complete MTS1 cDNA as a sample and using the standard cDNA library screening procedure. The cDNA library was screened using a sample obtained on the basis of exon 2 of MTS1. One million clones were screened from each library obtained from the embryonic brain, normal mammary gland and lymphocytes, respectively. A hybridizing cDNA clone was isolated from a lymphocyte library. The clone was sequenced and revealed the presence of E1-beta. In addition, it contained exon 2 (E2) and exon 3 (E3) MTS1. Hybridization-positive cDNA clones were isolated by incubation of cDNA obtained from ovarian tissue with cosmid c5. The cosmid was labeled with biotin and "made" single-stranded. Next, conditions were created for the formation of hybrids between c5 and cDNA, and then the biotinylated plasmid was "captured" using magnetic particles coated with streptavidin. The cDNAs thus selected were separated from the cosmid, amplified by PCR, cloned and sequenced. Clones of cDNA were similar to those isolated from cDNA libraries in that they contained E1-beta, E2, and E3. None of the clones contained the previously described exon 1 (see SEQ ID NO: 3). The sequence of MTS1E1-beta is described in the number SEQ ID NO: 13.

MTS1E1 Beta Structure
MTS1 and MTS1E1-beta are two forms of the same gene: exons 2 and 3 are found in both forms, but they have different first exons. MTS1 contains an alpha form (E1 alpha) that encodes the first 43 amino acids of the p16 protein encoded by MTS1.

 MTS1E1-beta contains the beta form (E1-beta) of exon 1. The exon structure of the p16 gene was determined by comparing the sequences of composite cDNA clones with the genomic regions from which they were obtained (Fig. 13). A combination of genomic blots, sequence analysis of the genomic region containing P16, and extended PCR were used to determine the positions of P16 exons (Fig. 13). The p16 gene occupies approximately 30 kilobases of genomic DNA. E1-beta is closest to the 5'-end, and in general the order of exons is as follows: E1-beta, E1-alpha, E2 and E3.

 Translation of the E1-beta in the p16 reading frame (extrapolated from the reading frame used for exons 2 and 3) opens a stop codon, which is only 10 codons “above” the splicing connection between the E1-beta and E2. The position of the stop codon was confirmed by sequence analysis of both genomic and cDNA. The first possible initiating codon “below” this stop codon is located immediately behind the splicing joint E1 / E2 in the 3 ′ direction. This potential start codon is flanked by sequences that do not resemble the Kozak consensus sequence (Kozak, 1989). When translated in the p16 reading frame, the transcript of the E1 beta p16 gene would encode a 105 amino acid protein.

 Subsequent analysis of cDNA beta showed that it contained an extended reading frame, different from that used for p16 encoding. The indicated reading frame (ORF2) overlaps E1-beta and extends to 67 amino acids in E2. A full reading frame can encode a 180 amino acid protein. However, the reading frame remains open from the 5'-end of E1-beta and therefore may be incomplete. Statistical analysis shows that an ORF of this size is unlikely to occur by chance in DNA consisting of random sequences (P = 0.003). However, for a given composition of the beta transcript, the probability is much higher (P = 0.16). The hypothetical polypeptide is not similar to any of the described proteins.

 The determination of the evolutionarily conserved parts of E1-beta may explain which sequences are important for fulfilling the functions of this protein. Using a modified RACE method called “capture of -RACE hybrids” (HCR) (see Example 12), murine p16 cDNAs were isolated that were compared to human p16 cDNAs. One type of murine p16 cDNA (beta) had exons equivalent to human E1-beta and E2. Another type (alpha type) contained the equivalent of E1 alpha coupled to E2. Exons E1-alpha and E2 mice were 70% identical to their human counterparts. The nucleotide sequences of human and mouse E1-beta exons were 51% identical (Fig. 14), and mouse E1-beta exon also had stop codons in the p16 reading frame. The human and mouse polypeptides predicted based on the nucleotide sequence located in the 5'-direction from the stop codon were completely different. Therefore, it is unlikely that stop codons in the p16 reading frame are sequencing artifacts.

 Given the uncertainty of the role of El-beta, the authors analyzed the similarities between the beta transcripts of mouse and human for all three reading frames. The mouse and human beta transcripts contained a large open reading frame (ORF2), different from that used for p16 encoding (ORF1). The putative polypeptides encoded by ORF2 were 40% identical. However, this coincidence was reduced to 28% with a stricter comparison of ORF2 peptides in the part encoded by E1-beta. In contrast, mouse and human p16 sequences coincide 67%. In addition, the polypeptides of the putative ORF2 E2 matched to the same extent (42%) as the putative polypeptides of the third reading frame E2 (ORF3). These results indicate that ORF2 was not selectively supported and does not appear to encode a protein. The secondary structure of mouse and human beta RNAs was also compared. No similarities were found. Together, these results show that beta transcript is necessary for the functioning of p16 in mice and humans; if it is translated, then the encoded protein, apparently, begins with the first methionine exon 2.

Example 8. Mutation MTS1 in germline
To answer the question of whether MTS1 or MTS2 correspond to the genetically sensitive MTS locus, the genomic DNA of eight individuals, presumably carrying the melanoma-prone MTS allele, was analyzed (Cannon-Albright et al., 1992). The exon DNA sequences were amplified in each case using oligonucleotide primers (Table 2) derived from intron sequences specific for MTSI or MTS2.

 Exon 1 of the MTS1 gene was amplified using primers 1F and 1108R and then sequenced using primer 1108R. Exon 2 of the MTS1 gene was amplified using 42F and 50R primers, reamplified using 89F and 50R primers, and then sequenced using 89F and 50R primers.

 DNA sequencing of these genomic fragments revealed polymorphisms in two of the eight individuals. Similar polymorphisms were not found in any of the other samples, indicating their low prevalence in the population. In order to show the relationship of polymorphisms with the MTS chromosome, and not with a different homologue, the genomic DNA of other individuals from each pedigree studied carrying an allele predisposing to melanoma was analyzed. In each case, polymorphisms segregated with the predisposing MTS allele. A codon 93 mutation (gly ---> trp) was detected in an individual (12821) from genealogy 3012. It was also found in a sibling carrier melanoma patient (13183) and a healthy cousin carrier (14917), but not in a healthy carrier siblings (13184). A codon 118 mutation (val ---> asp) was found in an individual (15635) in the 1771 pedigree. It was also found in a melanoma patient with a cousin-nephew (10205), a sick cousin carrier (11414) and an affected cousin of an individual 10205 (10146), but not in the unexposed non-carrier uncle of the individual 10205 (10120).

 Polymorphisms were single nucleotide substitutions causing amino acid substitutions (Table 3), which led to the replacement of a large hydrophobic residue with a small hydrophilic or a charged amino acid with a neutral one.

 Exon 2 of the MTS2 gene did not show polymorphism in eight tested samples. Therefore, at least in this group of pedigrees, the MTS2 gene does not cause a predisposition to melanoma. The similarity of MTS2 with MTS1 suggests that the former is involved in the development of other types of cancer. It is also possible that MTS2 is a non-functional gene.

 The detection of MTS1 mutations in germline but not MTS2 in melanoma-prone individuals is consistent with an analysis of homozygous deletions in melanomas.

Example 9. Detection of MTS in tumor cell lines
Since deletions in the 9p21 region are high in various types of tumors, cell lines obtained from 12 different types of tumors were analyzed for the presence or absence of MTS 1. To analyze genomic DNA from tumor cells lines for the presence or absence of the desired fragment (Fig. 4A, 4B and 9), a set of STSs (sequence-tagged sites) covering the gene was used. The results of this study suggest that MTS1 was deleted in a large number of tumor lines (Table 4). Homozygous deletions were found in all tested types of tumors with the exception of tumors of the rectum and neuroblastoma. The percentage of deletions ranged from 25% in the case of lung cancer and leukemia to 94% in astrocytomas. Altogether, homozygous deletions were detected in 135 of the 290 cell lines tested. This result corresponds to a minimum estimate of the frequency of homozygous deletions in tumor lines, since the STS used for the analysis do not cover the entire gene. Thus, individual small deletions could remain undetected. In addition, the approach used does not detect damage such as insertions, deletions of several nucleotides or nucleotide substitutions.

 For a more accurate estimate of the total number of cell lines having MTS1 mutations, 34 lines in which no obvious homozygous deletions of the MTS1 sequence were found were subjected to a more thorough analysis. Sequences comprising ~ 97% of the coding sequence of MTS1 were amplified and screened for polymorphism. In 14 out of 34 melanomas analyzed (Table 5), eighteen somatic mutations were found in exon 2 or exon 1 of the MTS1 gene. Three of these mutations were reading frame shifts, 7 were nonsense mutations, 4 were missense mutations, and 4 were silent mutations. Three of the four lines containing silent mutations had additional mutations and 16 of the 18 mutations were localized in coding exon 2. Only one line had exclusively hemi- or homozygous polymorphisms, and the remaining homologous chromosomes had unreduced deletions. The only heterozygous line had two different silent mutations. This observation confirms the assumption that each homologue undergoes an independent mutational event.

 Based on the DNA sequence and deletion analysis of the MTS1 gene, it can be concluded that at least 75% of the melanoma lines contain the mutant MTS1 gene or both gene homologues have lost.

 The high frequency of damage to MTS1 (deletions and nucleotide substitutions) indicates that MTS1 or a closely related locus are involved in the development of a tumor phenotype. Cells with this kind of damage receive a selective advantage over cells that do not have such damage. An alternative explanation, such damage being accidental and not related to cell growth, seems unlikely for a number of reasons. First, the high correlation between the tumor phenotype and MTS1 mutations indicates a causal relationship between MTS1 mutations and tumor formation. Secondly, MTS1 determines susceptibility to melanoma and thus independently functions as a tumor suppressor gene. Third, the biochemical function of p16 as a potent Cdk inhibitor is in good agreement with the model according to which p16 acts in vivo as a general inhibitor of the onset of DNA replication.

 It is possible that mutations or loss of the MTS1 gene is the result of cell growth in culture. However, a high percentage of primary leukemia cells also has homozygous deletions in the alpha interferon gene cluster, a gene family located less than 500 kb from MTS1 (Diaz et al., 1990). Early studies of deletion indicated that alpha interferon gene deletions constantly capture markers located behind MTS1 toward the centromere (Weaver-Feldhaus et al., 1994). Since homozygous deletions of the MTS1 region are observed in both primary tumor cells and cultured cell lines, it is unlikely that deletions observed in tumor cell lines are an exclusively artifact of cell growth in culture. Nevertheless, the question of whether MTS1 mutations occur during tumor progression can be further resolved by analyzing samples of primary tumors.

The role of MTS1 in vivo
For normal division, eukaryotic cells need a transition through two critical points in the life cycle: a transition from phase G1 to phase S when DNA synthesis begins, and a transition from phase G2 to phase M when mitosis begins. In mammals, the mechanism of control of cell division consists of many elements, mostly interconnected (see Sherr, 1993). Cdk can serve as the basis of the control apparatus, since they regulate the phosphorylation of a number of key substrates, which in turn include the transition from G1 to S and from G2 to M. The transition from G1 to S is, apparently, the most critical “decision point”, since it occurs in the cell cycle first. Four types of Cdk (Cdk2-5) that can be involved in controlling the transition from GI to S, as well as a number of positive regulators of these Cdk (cyclins C, D1-3, E), are identified. Recently, several negative regulators have been identified: p16, p15, p18, p20, p21 and p27 (Xiong et al., 1993; Serrano et al., 1993; Gu et al., 1993; E1-Diery et al., 1993; Harper et al., 1993; Hannon and Beach, 1994; Polyak et al., 1994b; Toyshima and Hunter, 1994; Guan et al., 1995). These negative regulators appear to act by inhibiting the kinase activity of Cdk.

 Some of the cell cycle regulators are involved in the development of tumors in humans (see Hunter and Pines, 1994). p20 inhibits Cdk2 and possibly other Cdk, while p16 (also called MTS1, CDKN2, or INK4a) inhibits Cdk4 but does not inhibit Cdk2 in vitro (Serrano et al., 1993).

 In vitro studies and interactions with p53 have made p21 a common inhibitor of all Cdk (Xiong et al., 1993). Thus, in vitro p16 is obviously more specific than p21.

 Each of these inhibitors seems to prevent entry into the S phase. In addition, cyclin D1 or CDK4 is expressed above normal in some breast carcinomas, and the p16 gene is mutated or deleted in a large number of cell lines and primary tumors (Buckley et al., 1993; Caldas et al., 1994; Kamb et al., 1994b; Mori et al., 1994; Tam et al, 1994a). From the above results, it follows that individual cyclins and CDK are proto-oncogenes, and p16 (MTS1) is the tumor suppressor gene. The biochemical properties of p15, p18, p21 and p27 indicate that they can also be tumor suppressors, but a detailed mutational analysis of their genes in tumors or cell lines has not yet been performed. The results presented here indicate the functioning of MTS1 in vivo as an inhibitor of cell division.

 The p16 gene (MTS1), located in the 9p21 segment of the ninth human chromosome, is particularly interesting because it is mutated or homozygously deleted in many types of tumors and tumor cell lines (Caldas et al., 1994; Kamb et al., 1994b; Mori et al. 1994; Nobori et al., 1994). In addition, MTS1 mutations segregate with a predisposition to melanoma in several pedigrees for which 9p21-linked sensitivity to melanoma is shown (Hussussian et al., 1994; Kamb et al., 1994a). However, the role of MTS1 in the occurrence of hereditary and sporadic cancer remains unclear. In several pedigrees with a high melanin frequency and high LOD values for 9p21 markers, no mutations were found in the MTS1 coding sequences. In addition, the predominance of homozygous deletions of MTS1 in tumors and cell lines is atypical for inactivation of tumor suppressor genes and may indicate the location of another gene (or genes) also involved in tumor formation in the vicinity of MTS1.

 Recent studies indicate the existence of mitogenic and anti-mitogenic signals that affect the cell cycle, at least by partially regulating the activity of CDK inhibitors (Firpo et al., 1994; Hannon and Beach, 1994; Kato et al., 1994; Polyak et al., 1994a; Slingerland et al., 1994).

 For example, beta-TGF-induced cell cycle arrest may be due to activation of p15 and p27. Conversely, p27 can be inhibited during IL-2-induced mitogenic activation of resting T-lymphocytes. Relatively little is known about the regulation of MTS1. Many recent publications indicate that MTS1 levels can be determined by the Rb protein (Serrano et al., 1993; Li et al., 1994a; Tam et al., 1994b; Parry et al., 1995). These and other observations (Serrano et al., 1995) allow us to construct a model of the action of MTS1, according to which MTS1 inhibits CDK4 / 6 and thereby prevents phosphorylation of Rb. The Rb protein, in turn, is involved in feedback and limits the levels of MTS1.

 These results provide genetic evidence of the important role of MTS1 in cell cycle regulation. Moreover, it follows from these data that the target of MTS1 in vivo is one of the main factors of tumorigenesis. If MTS1 inhibits Cdk4 in vivo and does not inhibit Cdk2, Cdk4 can be a very likely candidate for oncogenes. The predominance of MTS1 gene mutations indicates that Cdk4 may function as a general activator of cell division in most (if not all) cells. Further biochemical studies of the effects of MTS1 on various Cdk will help to determine the hierarchy of Cdk activity in both normal and transformed cells. By analogy with p16, p21 acts as a common inhibitor of various Cdk and its gene can be mutated or lost in a large percentage of tumors.

 If MTS1 is a common tumor suppressor active in most normal cells, germline mutations of this gene should predispose to the emergence of tumors other than melanoma. For example, a germline mutation of the p53 gene, similar to that found in Li-Fraumeni syndrome, increases the likelihood of many types of tumors, including childhood sarcoma, breast cancer (Malkin et al., 1990). Early work described an unusually high incidence of pancreatic cancer in some families susceptible to melanomas (Bergman et al., 1990; Nancarrow et al., 1993). These observations are consistent with evidence that homozygous MTS1 deletions are found in pancreatic cancer cell lines.

 It is possible that the genetics of MTS1-related predisposition to tumors may differ from the genetics of MTS1 somatic cells. For example, extended deletions that can remove many kilobases of DNA in the surrounding MTS1 regions are unlikely to be fixed in the gene pool due to their selective adverse effects. However, such mutations may be beneficial for transformed somatic cells, possibly because they remove many genes. This assumption is consistent with the existence of a second gene, very similar to MTS1 and called MTS2. MTS2 is located approximately 12 kilobases "above" the first exon of MTS1, the first exon MTS2 is located approximately 2.5 kilobases "above" the second exon of MTS2. MTS2 can function like MTS1. Deletions that remove MTS1 and MTS2 can provide a cell growth advantage over cells in which one of these genes is inactivated. On the contrary, these genes can act in the "non-overlapping" or "partially overlapping" types. These capabilities require further careful study of cells.

Example 10. Mutation analysis of MTS1E1-beta
The widespread distribution of homozygous deletions that inactivate p16 in tumor cell lines, as well as the absence of p16 mutations in pedigrees with 9p21-associated melanomas, suggest that there is another gene (or genes) in the vicinity of p16 that is also involved in tumor formation (Cairns et al. 1994; Spruck et al., 1994). If E1-beta encodes a protein that regulates cell growth, then these genes in the case of sporadic and / or familial cancers may contain mutations that might not have been detected by earlier studies.

The genetic characteristics of pedigrees with a high incidence of melanomas have been published previously (Cannon-Albright et al., 1992). The isolation of genomic DNA from melanomas (Kamb et al., 1994a) and cell lines (Liu et al., 1995) has also been described. Amplification of El-beta by polymerase chain reaction was carried out with a direct primer (5'-AGTCTGCAGTTAAGG-3'SEQ ID NO: 33) and a reverse primer (5'-GGCTAGAGGCGAATTATCTGT-3'SEQ ID NO: 34) for 30 cycles under the following conditions : 3 s at 97 ^o C, 10 s at 65 ^o C, 20 s at 75 ^o C. Amplification mixtures were diluted 100 times and amplified repeatedly under the same conditions with the same forward primer and another reverse primer (5'-CACCAAACAAAACAAGTGCCG- 3'SEQ ID NO: 35). PCR products were fractionated by agarose gel electrophoresis and extracted with Qiagen beads (Qiagen, lnc.), And then sequenced using the Cyclist Sequencing kit (Stratagene) and the above direct primer (SEQ ID NO: 33).

 Neither in 24 cell lines obtained from 4 types of tumors (Table 6), nor in six "melanoma" genealogies, whose members showed a significant coincidence of haplotypes (Cannon-Albright et al., 1992) and in which P16 mutations were not previously detected (Kamb et al., 1994a) no E1-beta sequence variants were found. These experiments show that E1-beta mutations do not necessarily accompany tumor progression and are not responsible for the 9p21-associated sensitivity to melanomas in these pedigrees.

Example 11. Screening for MTS2 mutations
MTS2 Mutation Screening in Cell Lines
The predominance of homozygous deletions that remove MTS1 in tumor-derived cell lines suggests that another gene or genes are located near MTS1 that may also be involved in tumor development. If the MTS2 gene is involved in the development of sporadic cancer, then mutations must be present in tumor cell lines. Therefore, to identify mutations in the coding sequences of MTS2, a set of tumor cell lines was investigated.

 Exons 1 and 2 of MTS2 amplified PCR as described by Kamb et al. (1994a). Two primers, namely 2E1.F1 (5'-AGGGAGAGTGTCG-TTAAG-3 'SEQ ID NO: 19) and 2E1. R2 (5'-AGACTCCTGTACAAATCTAC-3 'SEQ ID NO: 20) was used to produce exon 1. Two primers, namely 89F (SEQ ID NO: 12) and 50R (SEQ ID NO: 11) were used to produce exon 2. After amplification, the DNA products were separated on a 1% agarose gel and then extracted using Qiagen beads (Qiagen, Inc.). Products were sequenced using a Cyclist Sequencing kit (Stratagene) with primer 2E1.F1 for exon 1 and 89F for exon 2.

 MTS2 coding sequences were screened for mutations in a set of cell lines derived from tumors of the bladder, lung, kidney, glioma, astrocytoma, and melanoma. All these types of tumor cell lines had a high frequency of homozygous deletions of MTS1 and MTS2 (Kamb et al., 1994b). Cell lines derived from melanoma, lung, kidney, and bladder tumors have been shown to have MTS1 point mutations or MTS1 reading frame shift mutations (Liu et al., 1995). Glioma and astrocytoma cell lines did not have similar MTS2 mutations. The individual cell lines used in these screening experiments were selected from the group of lines for which the absence of homozygous deletions in the MTS1 and MTS2 sequences was shown (Kamb et al., 1994b).

 No mutations were found in any of the 58 studied cell lines in the MTS2 coding sequences (see Table 7). Based on previous studies of MTS1 in these cell lines, it can be assumed that the set of lines used should contain about 8 MTS1 mutations per line, obtained from tumors of the bladder, lung, kidney, and also melanoma (Liu et al., 1995). Thus, somatic mutations of MTS2 were not detected during screening of this set of tumor cell lines.

MTS2 Mutation Screening in Pedigrees
There remained the possibility that the MTS2 gene is responsible for sensitivity to melanoma in those melanoma-prone pedigrees in which there are no mutations in the MTS1 coding sequences. The genetic characteristics of melanoma-prone pedigrees have been published previously (Cannon-Albright et al., 1992). The genomic DNA of members of these families was isolated from lymphocytes, which were obtained from blood samples according to the standard method (Kamb et al., 1994a). Screening was performed as described above for mutations in the MTS2 coding sequences in six pedigrees with high LOD values for the 9p21-associated susceptibility to melanoma, but in which no MTS1 mutations were found (see Table 8) (Kamb et al., 1994a). No MTS2 mutations were detected. Thus, these experiments did not confirm the involvement of MTS2 damage in the hereditary development of melanoma, although only on the basis of these experiments, such a possibility cannot be completely excluded.

Example 12. Expression of MTS1 and MTS1E1 Beta mRNA
Two p16 promoters
Two different forms of p16 mRNA can be obtained in two different ways. Firstly, mRNA transcription can start from two different promoters and, secondly, mRNAs formed during transcription from one promoter can then be alternatively spliced to form two different transcripts.

The existence of two different promoters for the alpha and beta transcripts is evidenced by the fact that the alpha transcript is formed in cell lines even when the E1-beta sequence located “above” is deleted. The cell lines A375 and SK-mel 93 contain a deletion with one break point between E1-alpha and E1-beta (Fig. 13). A clear location of the proximal rupture point in both cell lines has not been established, but it was at least 85 kilobases “above” the 5'-end of E1-beta. Using reverse transcriptase PCR using alpha-specific primers, it was shown that alpha transcript is expressed in both of these cell lines (Fig. 15). The reverse transcriptase PCR procedure was as follows: cDNA was synthesized based on total RNA (Sambrook et al., 1989) isolated from T cells, cell lines or human tissues (Clonetech). To initiate cDNA synthesis, randomly selected nine nucleotide seeds and reverse transcriptase Superscript II (Bethesda Research Laboratories) were used. The cDNA yield was determined by the incorporation of alpha-32P-dATP (Amersham) during the synthesis reaction (0.1 Ci / mmol), followed by detection of the amount of radioactive nucleotide contained in the final product. The level of p16 alpha and beta transcripts was analyzed by PCR using alpha and beta specific forward primers and reverse primers from E2 in two productive amplification cycles. In the initial amplification cycle, 2 ng of cDNA was amplified using an alpha-specific AS primer. 1 (5'-CAACGCACCGAATAGTTACG-3 'SEQ ID NO: 26) and beta-specific primer BS. 1 (5'-TACTGAGGAGCCAGCGTCTA-3 'SEQ ID NO: 27) and X2R140'(5'-TACTGAGGAGCCAGCGTCTA-3'SEQ ID NO: 22). The reactions were carried out in a Perkin-Elmer 9600 thermo-cycler for 20 cycles under the following conditions: 97 ^° C. 0.43 s; 65 ^o C 0.410 s and 75 ^o C 0.420 s. The reaction mixtures were diluted 100-fold and re-amplified using AS.1 or BS.1 and OX42B (5'-CGTGTCCAGGAAGCCC-3 'SEQ ID NO: 23). The oligonucleotide OX42B was radioactively labeled at the 5'-end (Sambrook et al., 1989) using gamma-32P-dATP (DuPont). PCR conditions were similar to those described above, except that 15 cycles were performed. To eliminate contamination of genomic DNA, PCR products blocked the splicing compound E1-alpha or E2 / E2. Then they were separated by electrophoresis in denaturing 5% polyacrylamide gel. Dried gels were exposed with X-Omat (Kodak) film overnight.

 The results of the studies suggest that the alpha transcript is read from a promoter that is not related to the 5'-sequences of E1-beta. An alternative explanation is that ectopic promoter sequences are fused to E1 alpha by fission. However, this is unlikely, since A375 and SK-mel 93 are independently derived cell lines. The exact location of the alpha promoter is not known, but the RNase protection test shows that transcription is initiated “above” p16 of the initiating codon, and this distance is at least 440 kilobases. Thus, the human p16 gene has a complex structure: two independent promoters P-alpha and P-beta initiate the synthesis of two partially overlapping transcripts with different coding capacities.

The expression pattern of P16
The functional activity of genes can be determined by the nature of their expression in various tissues. To identify the nature of p16 expression, a set of cDNA samples obtained from 11 types of tissues were screened by PCR using alpha and beta primers (Fig. 16A-D). Both forms of the p16 transcript were found in all the tissues studied, but there were some differences. For example, in the spleen, the ratio between alpha and beta forms was shifted towards beta. In contrast, the indicated ratio in the tissues of the mammary gland shifted towards alpha. The results are consistent with studies that detect deletions and point mutations of p16 in cell lines originating from many different types of tissues (Kamb et al., 1994b; Liu et al., 1995), indicating a specific role for p16 in these tissues.

 Given the biochemical significance of p16 as an inhibitor of Cdk4 and Cdk6, shown in vitro (Serrano et al., 1993; Li et al., 1994a; Perry et al., 1995), p16 expression was analyzed in cells that underwent a cell cycle. Human peripheral blood lymphocytes (PBLs) were stimulated with phytohemagglutinin (PHA) and interleukin-2 (IL-2) and cells were taken at different times after stimulation. Cells were then examined by flow cytometry to determine the stage of the cell cycle, by reverse transcriptase PCR to determine the relative level of p16 gene expression, and by Western blotting to determine the amount of p16 protein. PBLs were isolated from blood obtained from healthy adult donors and partially purified by ficoll-hypak gradient flotation (Bogum, 1968). Then, the lymphocytes were purified by reverse flow elution, as previously described by Elstad et al., (1988). These authors determined that the cell population thus obtained contains 98% purified T and B cells. Cells were then grown in RPMI medium (Gibco) supplemented with 10% calf fetal serum.

 Non-dividing cells were stimulated with 10 μg / ml PHA (Sigma) and 10 U / ml IL-2 (Sigma). Cell cycle progression was monitored by flow cytometry. RNA was isolated from primary T cells using the RNAzol B kit (CINNA / BIOTECX Laboratories, Inc.) as recommended by the manufacturer. Reverse transcriptase PCR was quantified using serial dilutions of T cell cDNA obtained after stimulation. The amount of cDNA in an undiluted sample was determined by the dilution at which the target was no longer amplified. Although the results of various experiments using PCR were consistent with each other, dilution experiments showed that changes in RNA levels can only be detected if they differ by more than four times. Similarly studied samples of Rb + and Rb- cell lines. Human actin was easily detected and detected in similar amounts in each cDNA sample (cell lines, tissues, or T cells).

 The relationship between the two forms of the p16 transcript changes significantly as the cell cycle progresses (Fig. 16B-C). Initially, the beta form is detected in small quantities, but after 30-40 hours after stimulation, its level begins to increase. At this time, the expression level of the alpha form remains relatively constant, possibly increasing slightly. Flow cytometry showed that a change in the ratio of alpha and beta forms correlates with the release of cells from the Go phase and entry into the S phase. Quantification of reverse transcriptase PCR was performed in matrix dilution experiments. It was found that reverse transcriptase PCR is sensitive to more than four-fold increase in the level of transcripts. It is estimated that beta form induction was at least ten times that of alpha form.

 Thus, as T cells enter the cell cycle, the relative content of the two forms of the p16 transcript changes in them, so this ratio changes towards beta.

 In addition, the authors investigated the level of p16 expression in T cells undergoing a cell cycle. Protein was isolated from cells at various times after mitogenic induction and analyzed by Western blotting. The level of p16 protein was determined using antibodies to p16 interacting with 20 terminal amino acids of the whole polypeptide. As the cells left the Go phase, the content of p16 in them remained relatively constant. Thus, during the cell cycle, the level of the p16 transcript (alpha transcript) and the p16 protein did not change relatively. Other authors have noted a moderate increase in p16 during the S phase (Tam et al., 1994b). The inventors did not note the accumulation of p16, which may reflect differences in the regulation of p16 in various types of cell lines or may be due to the existence of certain problems in detecting a two- or three-fold increase in the protein content or the corresponding cDNA.

The expression of p16 in tumor cell lines
Previous studies have suggested that Rb affects p16 expression (Serrano et al., 1993; Li et al., 1994a; Parry et al., 1995). The inventors investigated the effect of the Rb status of cells on the expression of beta form mRNA (Fig. 16 D). cDNA was obtained using a set of cell lines, five of which expressed wild-type Rb protein, and six showed non-functional Rb protein (Parry et al., 1995). As expected, the alpha transcript was detected only in Rb-negative lines. However, the beta transcript was present in both Rb-positive and Rb-negative lines. Thus, in contrast to the alpha form, expression of the beta form mRNA is independent of the mutation process in the Rb gene in tumor cell lines.

 There is evidence that the p16 gene belongs to the multigenic family (Guan et al., 1995). By analogy with other multigenic families, the components of this family should be responsible for excessive functional activity, as well as for various functions or for functions performed at different times in different tissues.

 Thus, taking into account the low level of p16 protein and the apparent lack of regulation of p16 by Rb, it can be assumed that p16 does not affect the regulation of the cell cycle in T lymphocytes. However, since beta-transcript formation is substantially induced during T-cell stimulation, and since p16 is deleted in a large percentage of T-cell tumors (Hebert et al., 1994), it seems likely that p16 plays a significant role in human T cells. A significant effect of Rb on p16 is observed only in transformed and tumor-derived cell lines. Perhaps p16 is regulated in wild-type tissues in a slightly different way.

E1-beta is a conservative and regulated region of p16
Although the role of the beta transcript is unclear, research results
suggest its importance for the functioning of the p16 locus for some reason. First, E1-beta is a conserved sequence in the mouse genome; secondly, the relative content of beta transcript is regulated both at the tissue-specific level and depending on the stage of the cell cycle, and thirdly, two cell lines have homozygous deletions that remove E1-beta, but not E1-alpha. These data indicate that E1-beta is necessary for the functioning of wild-type p16.

 mouse beta cDNAs were isolated and compared to human MTS1e1 beta form. Mouse cDNA clones were obtained using a modified hybrid selection technique called RACE Hybrid Capture (HCR). Mouse enriched poly A + RNA was isolated from the tissues of the thymus and mammary gland. The first cDNA strand in the synthesis reaction (Sambrook et al., 1989) was obtained using random oligomers of 12 nucleotides and Superscript II reverse transcriptase (Bethesda Research Laboratories). After the second strand synthesis, cDNAs were anchored by ligation of the 5'-ends of the cDNA with the specific double-stranded oligonucleotide dsRP.2 (5'-TGAGTAGAATTCTAACGGCCGTCATTGTTC-3 'SEQ ID N0: 28). The 5'-end of the second cDNA strand was the only phosphorylated DNA end in the ligation reaction. After ligation, the “anchored” cDNA was purified by fractionation on a Sepharose CL-4B column. The "anchored" cDNA was amplified using a p16-specific reverse primer (5'-AGCGTGTCCAGGAAGCCTTC-3 'SEQ ID NO: 29) and a set of RP.2 (RP.B) (5'-TGAGTAGAATTCTAACGGCCGTCATTG-3' SEQ ID NO: 30) followed by capture of a biotinylated gene-specific oligonucleotide (5'ACTGCGAGGACCCCACTACCTTCTCC-3 'SEQ ID NO: 31) "above" the reverse primer used in the first amplification. The captured cDNA was then again amplified by RP. B and the gene-specific reverse primer (5'-GAACGTTGCCCATCATCATC-3 'SEQ ID NO: 32) "above" the oligonucleotide used in the "capture". The product obtained as a result of the described manipulations was purified by gel electrophoresis, cloned and sequenced. P16 mouse oligonucleotides were determined by cloning and sequencing a mouse genomic clone that contained sequences that weakly hybridized to a human E2-based probe.

 Comparison of human and mouse beta transcripts suggests that E1 beta does not encode a protein. Only the p16 reading frame sequence in E2 was highly conserved. Thus, if the beta transcript is translated, it seems likely that protein synthesis begins in E2 and occurs in the same reading frame as p16. The hypothetical polypeptide has a calculated molecular weight of the order of 10 kDa and retains 2 3/4 of the four hairpin repeats present in p16. However, p16 contains only 2 1/2 hairpin repeats (Hannon and Beach, 1994) and other proteins fit and function with only one or two repeats. Whether the p10 molecule exists in vivo and whether it serves as a Cdk4 / 6 inhibitor remains to be investigated.

Beta RNA Function
If the role of the beta transcript is to inhibit cell growth, then one should expect the presence of disruptive E1 beta mutations in tumor cell lines. This is confirmed by the existence of two melanoma lines with deletions that remove E1-beta, but at the same time the formation of an alpha transcript continues. In these cell lines, the p16 coding sequence is a wild type sequence. However, in the 25 studied tumor cell lines at the E1-beta locus, no minor genetic damage was found (e.g., nucleotide substitutions). Thus, it is difficult to conclude that E1-beta is the target of homozygous deletions. If the E1-beta exon does not encode a protein, minor genetic damage may not be enough to impair its function. Alternatively, the target of the above deletions may be any other gene. For example, it is possible that the p15 gene (MTS2) serves as a relevant target for deletions in the melanoma cell lines mentioned above. From this point of view, E1-beta can be easily deleted, since it is closer to p15 than E1-alpha. However, since the inventors were not able to detect point mutations in the p15 gene of various cell lines, and because they did not have cell lines containing deletions that specifically delete p15 (Kamb et al., 1994b), this explanation seems implausible.

 Genetic studies suggest that p16 and Rb are components of a growth regulation system that is often inactivated during tumor progression. If the role of the beta transcript is to upregulate cell growth, it may be part of another system that mutates independently of p16 and Rb. This may explain the fact that deletions that specifically break E1-beta are found only in Rb-negative cell lines. Based on the nature of the expression of E1-beta, with a high degree of probability we can conclude that E1-beta plays a role in cells that are actively going through the life cycle. A definite judgment on the value of E1-beta can be made after studies of its expression in vivo.

Example 13. Expression of MTS2 mRNA
RNA was isolated from cell lines or primary T cells using the RNAzol B kit (CINNA / BIOTECX Laboratories, Inc.), As recommended by the manufacturer. cDNA was synthesized using total RNA (Sambrook et al., 1989) using a randomly selected nine-nucleotide primer. The cDNA yield was determined by the incorporation of alpha-32P-dATP (Amersham) during the synthesis reaction (0.1 Ci / mmol), followed by detection of the amount of radioactive nucleotide contained in the final product.

The expression of MTS2 was determined by PCR with reverse primers in two consecutive amplification rounds. In the first round, 2 ng of cDNA was amplified using E1F (5'TGAGGGTCTGGCCAGC-3 'SEQ ID NO: 21) and X2. R140 '(5'-AGCACCACCAGC-GTGTC-3 / SEQ ID NO: 22). Amplification reactions were carried out in a Perkin-Elmer 9600 thermo-cycler for 20 cycles under the following conditions: 97 ^° C, 3 s; 65 ^o C, 10 s; 75 ^o C, 20 s. The reaction mixtures were diluted 100 times and reamplified using E1F and X2B (5'-CGTGTCCAGGAAGCCC-3 'SEQ ID NO: 23). X2B oligonucleotide was radioactively labeled at the 3'-end (Sambrook et al., 1989) of gamma-32P-dATP (DuPont). The PCR conditions were similar to those described above, with the exception that 15 cycles were performed. The resulting products were separated by electrophoresis in denaturing 5% polyacrylamide gel. Dried gels were exposed with X-Omat (Kodak) film overnight.

MTS2 expression in various tissues
MTS2 has been found to be expressed in many types of tissues, including those that give rise to tumors with homozygous deletions of MTS2 (see Fig. 10A). However, there are some differences between these tissues. For example, while the MTS2 transcript is easily detected in lung tissue, it is not found in the tissues of the prostate and brain. In contrast, the expression of the closely spaced MTS1 gene is detected in all tissues examined. It is not known whether tissue-specific differences in MTS2 RNA levels reflect the needs of different tissues in the MTS2 protein.

MTS2 expression as the cell cycle progresses
If MTS2 regulates important transitional stages in the cell cycle, its expression level changes as the cell cycle progresses. For example, in normally dividing cells, p21 mRNA redundancy varies as a function of a specific phase of the cell cycle (Li et al., 1994b). In order to determine whether or not transcription of MTS2 is regulated as the cell cycle progresses, resting T cells stimulated PHA and IL-2 and were examined at various stages after stimulation (see Fig. 10B). There was no obvious relationship between the level of MTS2 expression as the cells left the Go phase and passed through the phases of the cell cycle. In contrast, as expected, the expression of the control gene, Cdk4, changed (Matsushime et al., 1992). Thus, there was no evidence in favor of the fact that the level of expression of MTS2 mRNA in normally dividing cells as they pass through the cell cycle is not the same.

 It is believed that the MTS1 protein is involved in one of the growth regulation pathways in which the Rb retinoblastoma protein is involved (Serrano et al., 1993; Guan et al., 1995; Serrano et al., 1995). Comprehensive evidence has been obtained in recent studies that the expression of MTS1 is controlled, at least in part, by the Rb protein (Li et al., 1994a; Parry et al., 1995). The biochemical similarities between MTS1 and MTS2 suggest that MTS2 could also be regulated by Rb. This possibility was investigated by comparing the levels of MTS2 mRNA in Rb-positive and Rb-negative cell lines. No correlation was found between the Rb status and the MTS2 RNA level (see Fig. 10C). This suggests that the Rb status of the cell line does not significantly affect the redundancy of the MTS2 transcript. Thus, in contrast to MTS1, the expression of MTS2 may not be dependent on Rb.

Example 14. Ectopic expression of MTS1 and MTS2
By polymerase chain reaction with primer MTS1.F (5'AAA GGA TCC ATT GCC ACC ATG GAG CCG GCG GCG GGG AGC AGC ATG GAG CCT TCG GCT 3 ') (SEQ ID NO: 17) and primer E3.R (5' TTT GAA TTC AAT CGG GGA TGT CTG 3 ') (SEQ ID NO: 18), a 483 bp MTS1 fragment was obtained. MTS1.F primer was designed to contain restriction sites near the 5'-end for subsequent cloning, as well as Kozak's consensus sequence (Kozak, 1987).

The cDNA from the mammary gland served as template DNA for this reaction. The resulting fragment was inserted into the pcDNA3 expression vector (In Vitrogen), which was then digested with Eco RI and Bam HI. The pcDNA3 vector contains the cytomegalovirus promoter (CMV) and carries ampicillin and neomycin resistance genes. The resulting recombinant pcDNApl6 vector was introduced by electroporation into HS294T cells. HS294T cells are derived from melanoma and have a homozygous deletion of both MTS1 and MTS2. HS294T cells were grown on DMEM (Gibco) supplemented with 10% fetal calf serum, unnecessary amino acids, sodium pyruvate and L-glutamate. Cells were grown at 37 ^° C in an atmosphere of 5% CO ₂ . HS294T cells were co-transformed at a 1: 4 ratio with a pSS vector (Stratagene) reporting hygromycin resistance to the cells and pcDNA3 expression vector (Invitrogen) containing the MTS1 coding sequence under the control of the CMV promoter, or pcDNA3 without insertion.

 The coding region of MTS2 was likewise cloned into the pcDNA3 vector to form a Kozak sequence, and the resulting recombinant pcDNApl5 vector was introduced into HS294T cells. MTS2 cDNA obtained as described in Example 13 was used in these experiments. Plasmid pSS (Stratagene) containing a selective marker of hygromycin resistance was used for cotransfection with pcDNApl6 or pcDNAl5. This plasmid in an amount of 20 μg per cuvette was added by electroporation. Electroporation conditions were as follows: 800 μl cell suspension at a concentration of 1.5 million cells per milliliter; capacitor capacity - 500 microfarads; voltage - 400 volts. In control experiments, a mixture of plasmids pcDNa3 and pSS was used. Electroporated cells (about 400 μl) were seeded on Petri dishes in medium with hygromycin (300 μg / ml). Colonies (tricks) were counted on the 14th day. The results are presented in Table. 9.

 When a construct containing the MTS2 coding sequence under the control of the CMV promoter was introduced into HS294T cells, colony formation decreased by a factor of seven, which is comparable in effect to the ectopic expression of MTS1. This result indicates that ectopic expression of MTS2 is sufficient to inhibit cell growth. It is not clear whether the transformed cells are stopped in the G1 phase, which would result from the ectopic expression of MTS1, or whether their growth was stopped in another way. From the data tab. 9, it can be concluded that expression of p15 or p16 in a cell line in which p15 or p16 were not expressed before transformation (due to homozygous deletion) inhibits cell growth. The exact mechanism of this phenomenon is unclear, but it is possible that expression of p15 or p16 stops cell division or kills the cell. These results indicate the action of p15 and p16 in vivo as suppressor proteins of bona fide tumors and indicate their possible therapeutic use.

 For many reasons, MTS2 is an attractive candidate as a tumor suppressor gene. It has significant sequence similarity with MTS1, binds to CDK and inhibits their action in vitro, and ectopic expression of MTS2 inhibits cell growth in vivo. These results indicate the possibility that, in spite of the biochemical similarity between MTS2 and MTS1, the functions of the two proteins in vivo can vary significantly. Two features of MTS2 indicate this: MTS2 rather than MTS1 is induced by TGF-beta (Hannon and Beach, 1994) and 2) unlike MTS1, the transcription of MTS2 is not dependent on Rb. It is possible that MTS2 is not involved in tumorigenesis at all. In contrast, MTS2 may participate in tumor suppression in a different way than MTS1. The components of this process are unknown, but it can be assumed that some of them can mutate at a significantly higher frequency than MTS2 in somatic tissues.

 Thus, the absence of somatic mutations of MTS2 does not exclude its important role in tumor suppression. As mentioned above, ectopic expression of MTS2 inhibits cell growth, which is compatible with the role of MTS2 as a tumor suppressor. A constant level of expression of MTS2 during the cell cycle, its induction of TGF-beta indicates the value of MTS2 at G1 stop and not necessarily its participation in determining the time of other events of the cell cycle. Conversely, regulation of MTS1 expression by Rb indicates a possible role for MTS1 as an oscillator of the cell cycle. It is important to study in vivo the function of MTS2 as a growth regulator and to establish methods of its action.

Example 15. Two-stage analysis of the presence of MTS in the sample
A patient's DNA sample was processed according to the method described by Antonarakis et al. (1985). DNA was fractionated by electrophoresis on a 1% agarose gel and transferred onto a nylon filter (Southern blot). DNA was “sewn” to the filter by ultraviolet irradiation at 150 mJ (GS Gene Linker, Bio-Rad).

 A MTS sample corresponding to nucleotides 448-498 of the sequence SEQ ID NO: 4 was subcloned into the pTZ18U vector. Phagemides transformed E. coli cells of strain MV1190 infected with helper phage M13KO7 (Bio-Rad, Richmond, CA). Single-stranded DNA was isolated by a standard method (see Sambrook, et al., 1989).

Blots were prehybridized for 15-30 minutes at 65 ^° C. in 7% SDS in 0.5 M NaPO ₄ . The methods used below are described by Nguyen et al., 1992. Blots were hybridized overnight at 65 ^° C. in 7% SDS, 0.5 M NaPO ₄ with 25-50 ng / ml single-stranded DNA sample. After hybridization, the filters were washed twice for 30 minutes in 5% SDS, 40 mm NaPO ₄ at 65 ^o C and then twice for 30 minutes in 1% SDS, 40 mm NaPO ₄ at 65 ^o C.

The blots were then rinsed in phosphate buffered saline (PBS, pH 6.8) for 5 min at room temperature and incubated in a 0.2% solution of casein in PBS at room temperature, and then rinsed in PBS for 5 min. This was followed by preincubation for 5-10 min in a shaking water bath at 45 ^o C with hybridization buffer of the following composition: 6M urea, 0.3 M NaCl and 5x Denhardt's solution (Sambrook et al., 1989). The buffer was removed and replaced with fresh hybridization buffer (calculated at 50-75 μl per square centimeter of filter) containing 2.5 nM covalently linked oligonucleotide-alkaline phosphatase conjugate with a nucleotide sequence complementary to the universal primer site (UP-AP, BioRad). Hybridization lasted 20-30 minutes at 45 ^° C, and after hybridization, blots were washed twice for 10 minutes in 6M urea, 1x standard citrate solution (SSC), 0.1% SDS at 45 ^° C and once for 10 minutes in 1x SSC , 0.1% Triton X-100. The blots were then rinsed for 10 min at room temperature in 1x SSC.

Blots were incubated for 10 minutes at room temperature while shaking in a substrate buffer consisting of 0.1 M diethanolamine, 1 mM MgCl ₂ , 0.02% sodium azide, pH 10.0. Separate filters were placed in sealed bags with substrate buffer and 0.2 M AM PPD (3- (2'-spiroadamantane) -4-methoxy-4- (3'-phosphoryloxy) phenyl-1,2-dioxethane , disodium salt, Bio-Rad). After 20 minutes of incubation at room temperature with shaking, the excess AMPPD solution was removed. Blots were exposed with x-ray film overnight. Positive bands indicated the presence of MTS.

Example 16. Obtaining polyclonal antibodies against MTS
MTS coding sequence segments were expressed in E. coli as a fusion protein. The superexpressed protein was purified by gel elution and used to immunize rabbits and mice according to a protocol similar to that described by Harlow and Lane, 1988. It was shown that antibodies also against various other proteins are formed with this method of immunization (see, for example, Kraemer et al., 1993).

 Briefly, a fragment of the MTS coding sequence was cloned as a fusion protein in the plasmid PET5A (Novagen Inc., Madison, WI). The cloned MTS sequence included amino acids 448-498 of the sequence SEQ ID NO: 4. Following IPTG induction, overexpression of the expected molecular weight fusion protein was confirmed by SDS / PAGE. Protein was isolated from the gel by electroelution. Identification of the protein as an MTS fusion product was confirmed by protein sequencing from the N-terminus. The purified protein was then used to immunize rabbits. Animals were immunized with 100 μg of protein in complete Freund's adjuvant and boosted twice at 3-week intervals, the first time with 100 μg of immunogen in incomplete Freund's adjuvant, and the second 100 μg of immunogen in PBS. Serum containing antibodies was collected after two weeks.

 Antibodies against mutant forms of MTS were obtained according to the same protocol. These antibodies, together with antibodies to wild-type MTS, were used to detect and determine the relative level of mutant forms in various tissues and biological fluids.

Example 17. Obtaining monoclonal antibodies specific for MTS
Monoclonal antibodies were prepared according to the following protocol. Mice were immunized with an immunogen consisting of intact MTS or MTS peptides (mutant or wild type) conjugated with glutaraldehyde or EDC with fissurella mollusk hemocyanin. The immunogen was mixed with adjuvant. Each mouse was injected four times from 10 to 100 μg of the immunogen, and after the fourth injection, blood samples were taken to determine the presence of antibodies to the immunogen in serum. Serum titer was determined using ELISA or RIA. Mice in the serum of which antibodies to the immunogen were detected were selected to obtain hybridomas.

 Spleens were taken from immune mice and unicellular suspensions were prepared from them (see Harlow and Lane, 1988). Cell fusion was performed as described by Kohler and Milstein, 1975. P3.63.3 myeloma cells (American Cell Culture Collection, Rockville, MD) were fused to immune splenocytes using polyethylene glycol as described by Harlow and Lane, 1988. 200,000 cells were seeded per well in 96-well culture plates. Supernatants from cells in which hybrid cell growth was observed were examined for the presence of MTS-specific antibodies using ELISA or RIA with mutant wild-type MTS or MTS. Cells from positive cells were grown and subcloned to confirm monoclonality.

 Clones of the desired specificity were propagated and cultured as ascites in the body of mice or on systems with hollow fibers to obtain sufficient quantities of antibodies for their characterization and development of the test.

Example 18. Savdwich method for detecting MTS
Monoclonal antibodies are adsorbed on a solid phase carrier (tablets, tubes, beads or microcarriers). It is preferable to adsorb antibodies on the surface of the wells of 96-well ELISA plates. A 100 μl sample of material (serum, urine, tissue cytosol) containing MTS peptide / protein (wild-type or mutant) is added to the antibodies bound on a solid phase support. Samples are incubated for 2 hours at room temperature. Then the samples are decanted, and the solid phase carrier is washed with buffer to remove unbound material. Then, second antibodies (to another determinant of the peptide / MTS protein) are added to the solid phase carrier in a volume of 100 μl. The second antibodies, usually labeled 125-1, with an enzyme, fluorophore or chromophore, are applied to the solid phase and incubated for 2 hours at room temperature. Then the second antibodies are removed and the solid phase is washed with buffer to remove unbound material.

 The amount of bound label is determined which is proportional to the amount of MTS peptide / protein present in the sample. Separate determinations are made using monoclonal antibodies specific for wild-type MTS, as well as monoclonal antibodies specific for MTS of each mutant type.

 It should be understood that the described methods and compositions can be used in various embodiments of the claimed invention, only part of which is disclosed in this description. The specialist is clear that there may be other embodiments of the invention that do not change its essence. Thus, the above examples are illustrative and cannot be construed as limiting the claimed invention.

Claims (53)

 1. Isolated DNA encoding a multifunctional tumor suppressor (MTS) polypeptide having the amino acid sequence of SOQ ID NO: 2, 14 or 16 described herein, or a modified form of said polypeptide that is functionally equivalent to or associated with a predisposition to cancer, including melanoma, leukemia, astrocytoma, glioblastoma, lymphoma, glioma, Hodgkin's lymphoma, chronic lymphoid lymphocytic leukemia (CLL), tumors, pancreas, breast, thyroid gland, ovaries, uterus, testes, kidney , Stomach and rectum.  2. The isolated DNA according to claim 1, having the nucleotide sequence of SEQ ID NO: 1, 3, 4, 5, 13, 15 or 36 specified in the description, or the corresponding RNA.  3. Isolation of DNA according to claim 1, which is DNA with an allelic variant of the nucleotide sequence of SEQ ID NO: 1, 3, 4, 5, 13, 15 or 36 specified in the description, or the corresponding RNA.  4. The isolated nucleic acid according to claim 1, which is a DNA with a mutated formula of the nucleotide sequence of SEQ ID NO: 1, 3, 4, 5, 13, 15 or 36 or the corresponding RNA.  5. The isolated nucleic acid according to claim 4, characterized in that the mutation is a deletion, a torn allele, a nonsense mutation, a frame shift mutation or a missense mutation.  6. The isolated DNA according to claim 4, selected from the group consisting of (a) SEQ ID NO: 1, having nucleotide A at position 241; (b) SEQ ID NO: 1 having a T nucleotide at position 148; (c) SEQ ID NO: 1 having nucleotide A at position 418; (d) SEQ ID NO: 1, in which nucleotides 226-270 are deleted; (e) SEQ ID NO: 1 having a T nucleotide at position 214; (f) SEQ ID NO: 1 having nucleotide A at position 306; (q) SEQ ID NO: 1, in which nucleotides 148-155 are deleted; (h) SEQ ID NO: 1 having a T nucleotide at position 317; (i) SEQ ID NO: 1 having nucleotide A at position 305; (j) SEQ ID NO: 1 having nucleotide A at position 124; and (k) SEQ ID NO: 1 in which nucleotides 104-105 are deleted, or the corresponding RNA.  7. An isolated nucleic acid according to any one of claims 1 to 6, characterized in that it is a DNA containing regulatory sequences of the MTS gene.  8. The isolated DNA according to claim 2 or 3, characterized in that the nucleotide sequence represented by SEQ ID NO: 1, 3, 4, 5, 13, 15 or 36, or its allelic variant are functionally associated with regulatory sequences having a mutation which in vivo inhibits or prevents the expression of the MTS polypeptide.  9. An isolated nucleic acid containing at least 15 adjacent DNA nucleotides according to any one of claims 4 to 6, having mutations in the nucleotide sequences represented by SEQ ID NO: 1, 3, 4, 5, 13, 15 or 36.  10. The isolated DNA according to any one of the preceding paragraphs, embedded in a replicative cloning vector having a replicon operating in a host cell.  11. The isolated DNA according to any one of claims 1 to 9, embedded in an expression vector, characterized in that said isolated DNA is functionally linked to the corresponding control sequences.  12. Hybridization test for determining in a sample DNA with a nucleotide sequence selected from the nucleotide sequences of SEQ ID NO: 1, 3, 4, 5, 13, 15 or 36, their allelic variants and their mutated forms or RNA corresponding to the specified DNA, representing an isolated DNA containing a portion of the nucleotide sequence of the DNA according to any one of claims 1 to 6.  13. The sample according to p. 12, characterized in that the DNA sequence of the specified sample is part of the nucleic acid sequence according to any one of claims 4 to 6, having a mutation in the nucleotide sequence represented by SEQ ID NO: 1, 3, 4, 5, 13, 15 or 36.  14. The culture strain of host cells transformed with the vector of claim 10 or 11.  15. The method of obtaining the polypeptide, which is an MTS polypeptide with the amino acid sequence depicted by SEQ ID NO: 2, 14 or 16, or a modified form of the specified polypeptide according to claim 1, which consists in culturing a host cell containing an expression vector encoding the specified a polypeptide under suitable conditions; and regenerating said polypeptide.  16. A method of producing a human MTS polypeptide that is substantially free of other human proteins, wherein said polypeptide has the amino acid sequence depicted by SEQ ID NO: 2, 14 or 16, or an amino acid sequence substantially homologous to the wild-type MTS polypeptide whose amino acid sequence is depicted SEQ ID NO: 2, 14 or 16, and said polypeptide has a function substantially similar to that of the wild-type MTS polypeptide.  17. An antibody capable of specifically binding to one or more polypeptides mentioned in the method of claim 16.  18. The antibody according to 17, characterized in that it is a monoclonal antibody.  19. A pair of single-stranded oligonucleotide primers for determining the nucleotide sequence of the MTS gene using a nucleic acid amplification reaction, the sequence of these primers being made from 9p21 human chromosome and the use of these primers in the nucleic acid amplification reaction leads to the synthesis of DNA and / or RNA, the corresponding part or the whole MTS gene sequences.  20. The pair of primers according to claim 19 for detecting the full or part of the sequence of the MTS gene with the nucleotide sequence represented by SEQ ID NO: 1, 3, 4, 5, 13, 15 or 36, and / or their mutated form.  21. A method for diagnosing or predicting a lesion of a mammalian tissue, comprising detecting a change in the gene of a multifunctional tumor suppressor (MTS) or in the regulatory sequence of the MTS gene in a sample of affected tissue of a specified subject, the change indicating neoplasia of the specified tissue.  22. The method according to item 21, wherein the sequence of the MTS gene of the specified sample is compared with the sequence of one or more sequences of the wild-type MTS gene selected from the sequences of SEQ ID NO: 1, 3, 4, 5, 13, 15 and their allelic wild type options.  23. The method according to item 21, wherein the level and / or sequence of the product of expression of the MTS gene in the specified sample is examined.  24. The method according to item 23, wherein the specified expression product is an mRNA.  25. The method according to paragraph 24, wherein the mRNA of the specified sample is in contact with a sample based on the MTS gene under conditions suitable for hybridization of the specified sample with RNA corresponding to the specified MTS gene, and then the hybridization of the specified sample is determined.  26. The method according to item 21 or 22, characterized in that the sample based on the MTS gene is in contact with genomic DNA isolated from the specified sample under conditions suitable for hybridization of the specified sample with the specified gene, and then the hybridization of the specified sample is determined.  27. The method according to claim 25 or 26, wherein said sample is a mutated allele-specific sample.  28. The method according to item 23, wherein the specified expression product is a polypeptide encoded by the MTS gene in the specified sample.  29. The method according to p, characterized in that the polypeptide is detected immunoblotting or immunocytochemistry.  30. The method according to p. 28, characterized in that the change in the protein of the wild-type MTS gene is detected by determining the binding interactions between the MTS gene protein isolated from the specified tissue and the cyclin-dependent Cdk kinase.  31. The method according to p, characterized in that the change in the protein of the MTS gene is determined by determining the inhibition of the biochemical activity of Cdk.  32. The method according to item 30 or 31, characterized in that Cdk is a Cdk4.  33. The method according to item 21 or 23, characterized in that it establishes changes in the regulatory regions of the MTS gene in the specified sample.  34. The method according to item 21 or 22, characterized in that detect changes in the sequence of the MTS gene in the specified sample by the shift of the electrophoretic activity of single-stranded DNA of the specified sample in non-denaturing polyacrylamide gels.  35. The method according to item 21 or 22, characterized in that the whole or part of the MTS gene of the specified sample is amplified and the specified amplified sequence is sequenced.  36. The method according to item 21 or 23, characterized in that oligonucleotide primers are used to identify the specific mutant MTS allele in said sample by nucleic acid amplification.  37. The method according to p. 21 or 22, characterized in that clone the whole or part of the MTS gene of the specified sample in order to obtain a cloned sequence, and the specified cloned sequence is sequenced.  38. The method according to any one of paragraphs.21-24, characterized in that mismatches between the molecules of (1) the MTS gene of the genomic DNA or MTS mRNA isolated from the specified sample, and (2) a nucleotide probe complementary to the wild-type human MTS region are detected when molecules (1) and (2) hybridize with each other to form a duplex.  39. The method according to any one of paragraphs. 21 to 24, characterized in that the amplification of the sequences of the MTS gene of the specified sample and determine the hybridization of the amplified sequences with one or more nucleotide samples that contain the sequence of the wild-type MTS gene or the sequence of the mutated MTS gene having a mutation.  40. The method according to item 21 or 22, which consists in detecting in situ hybridization of the MTS gene of the specified sample from one or more nucleotide samples that contain the sequence of the wild-type MTS gene or the sequence of the mutated MTS gene having a mutation.  41. The method according to any one of paragraphs.21 to 40, characterized in that the change is detected by screening for a deletion mutation.  42. The method according to any one of paragraphs.21 to 40, characterized in that the change is detected by screening for a point mutation.  43. The method according to any one of paragraphs.21 to 40, characterized in that the change is detected by screening for insertion.  44. A nucleic acid selected from the group consisting of a nucleic acid encoding a wild-type MTS polypeptide and a nucleic acid substantially homologous and functionally equivalent to it or its parts, which can be used in gene therapy to restore the function of the wild-type MTS gene or MTS functions similar to those of the wild type in a cell that has lost the indicated gene function or has an altered gene function due to a mutation in the MTS gene.  45. The nucleic acid according to item 44, which is a regulatory sequence of the MTS gene.  46. The nucleic acid according to claim 44 or 45, characterized in that it is integrated into the genome of the specified cell.  47. A molecule of a wild-type MTS polypeptide, a polypeptide that is practically homologous and has a similar function to a wild-type MTS polypeptide, functional parts and molecules that mimic the function of a wild-type MTS polypeptide that can be used in peptide therapy to restore the function of MTS- wild-type gene in a cell that has lost its gene function or has an altered gene function due to a mutation in the MTS gene.  48. A method for screening potential anticancer drugs, which comprises (a) mixing a suitable cyclin-dependent kinase (Cdk) solution with a definition of what is suspected to be an anti-cancer drug, Cdk, cyclin, Cdk substrate and labeled ATP; (b) determining the amount of phosphorylated substrate; and (c) comparing the amount of phosphorylated substrate in step (b) with a control sample obtained in accordance with step (a) wherein said control sample contains an MTS polypeptide instead of said anticancer drug.  49. A method for screening potential anti-cancer drugs, which comprises mixing a cyclin-dependent kinase (Cdk), an MTS polypeptide and a compound presumably an anti-cancer drug, and determining the amount of bound MTS polypeptide to Cdk.  50. A method for screening potential anti-cancer drugs, which comprises mixing a cyclin-dependent Cdk kinase and a compound presumably an anti-cancer drug, and determining the biological activity of Cdk.  51. The method according to any one of claims 48 to 50, wherein Cdk is Cdk4.  52. A method for screening potential anti-cancer drugs, which comprises growing a transformed eukaryotic host cell containing an altered MTS gene in the presence of a compound presumably an anti-cancer drug, and determining the growth rate of said host cell.  53. The method according to paragraph 52, wherein the growth rate of the specified host cell is determined by the biological activity of the cyclin-dependent kinase (Cdk).

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