CN1128935A - Wild-type multiple tumour suppressor (MTS) gene and mutant sequences-useful in diagnosis, prognosis and therapy of human cancer e.g. melanoma or leukaemia - Google Patents

Abstract

The present invention relates to somatic mutations in the Multiple Tumor Suppressor (MTS for short) gene in human cancers and their use in the diagnosis and prognosis of human cancer. The invention further relates to germ line mutations in the MTS gene and their use in the diagnosis of predisposition. The invention also relates to the therapy of human cancers which have a mutation in the MTS gene, including gene therapy, protein replacement therapy and protein mimetics. Finally, the invention relates to drugs for cancer therapy.

Description

Many TIFs gene, its sudden change and with the method for this gene order diagnosis cancerous swelling and the relation of related application

The present invention is patent application No.08/251, the part renewal application of 938 (applications on June 1st, 1994), 08/215,088 (application on March 18th, 1994) and 08/214,581 (application on March 18th, 1994), and above-mentioned patent application is incorporated herein by reference.Patent application No.08/251,938 is again patent application No.08/227, the part renewal application of 369 (applications on April 14th, 1994), and patent application No.08/227,369 is again patent application No.08/214, the part renewal application of 582 (applications on March 18th, 1994), these patent applications also are incorporated herein by reference.Background of invention

The present invention relates to the somatic mutation of many TIFs in people's cancerous swelling (Multiple TumorSuppressor, be called for short MTS) gene and in the diagnosis of people's cancerous swelling and the application aspect the prognosis.The invention still further relates to kind system (germline) sudden change and the application aspect the tendency of diagnosis cancerous swelling generation thereof of mts gene, these cancerous swellings comprise the cancerous swelling of melanoma, ophthalmomelanoma, leukemia, astrocytoma, spongioblastoma, lymphoma, glioma, Hodgkin lymphomas, Huppert's disease, sarcoma, muscle tumor, cholangiocarcinoma, dermoid cancer, chronic lymphocytic leukemia (CLL) and pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, uterus, testis, kidney, stomach, colon and rectum.The invention still further relates to the treatment of people's cancerous swelling of mts gene sudden change, comprising gene therapy, protein replacement treatment and protein analogue (mimetics).At last, the invention still further relates to the medicine that screening is used for the cancerous swelling treatment.

Be used for illustrating background of the present invention herein, especially be used for implementing to provide extra details publication and other materials, in this application in conjunction with quoting as a reference, and point out in the article below for convenience's sake, and be categorized in respectively in the appended document inventory.

Cancerous swelling genetics is very complicated, relate to positive regulator a plurality of dominance, conversion conditions (oncogene) and a plurality of recessiveness, down regulator (tumor suppressor gene).Determined to surpass 100 kinds oncogene.Certified tumor suppressor gene is less than 12 kinds, but this number can increase to above 50 kinds (Knudson, 1993) according to estimates.

Involve multiple gene so and emphasized the complexity of the adjusting and controlling growth mechanism that in cell, plays a role for the integrality of keeping normal structure.This complexity also embodies by another kind of mode.Up to now, also there is not individual gene to participate in the process of all or most people's cancerous swellings.Modal oncogene mutation is in the H-ras gene, all solid tumors 10-15% in have this kind sudden change to find (Anderson et al., 1992).The tumor suppressor gene that mutation frequency is the highest is the p53 gene, undergos mutation in all tumours of about 50%.The target at all transformants that neither one is common, just can not search out can destruction or inverse cancer cell and don't damage Normocellular " magic bullet ".The hope of a new generation's specificity guiding type antineoplastic is just placed on and can be identified tumor suppressor gene or the oncogene that plays generally effect in cell divising regulatory.

The tumor suppressor gene of having cloned and having determined influences the neurological susceptibility of following cancerous swelling: 1) retinoblastoma (RB1); 2) WilmShi knurl (WT1); 3) Li-Fraumeni (TP53); 4) familial adenomatous polyposis disease (APC); 5) I type neurofibromatosis (NF1); 6) II type neurofibroma (NF2); 7) von Hippel-Lindau syndrome (VHL) and 8) 2A type MEA (MEN2A).

Determined genetic map but also do not have the locus of separated TIF to comprise following gene: I type MEA (MEN1); II type Lynch cancer family syndrome (LCFS2); Familial mastocarcinoma (BRCA1); Neuroblastoma (NB); Basal cell naevus syndrome (BCNS); Beckwith-Wiedemann syndrome (BWS); Clear-cell carcinoma (RCC); I type tuberous sclerosis (TSC1) and II type tuberous sclerosis (TSC2).Tumor suppressor gene coded product and multiple proteins type have similitude qualitatively at present, comprising DNA in conjunction with albumen (WT1), complementary transcript regutation protein (RB1), GTP enzyme activation albumen (being called GAP again) (NF1), cytoskeleton component (NF2), membrane-bound receptor kinases (MEN2A), other coded product and known protein matter do not have tangible similitude (APC and VHL).

In most of the cases, the tumor suppressor gene of differentiating at first by genetic research shown in some accidental tumour be disappearance or sudden change.This result hint, the zone of chromosome abnormality can show the position of related important tumor suppressor gene in the genetic predisposition of cancerous swelling and accidental cancerous swelling.

One of feature of several tumor suppressor genes of Que Dinging is that they are in some tumor type high frequency ground disappearance up to now.Disappearance usually relates to and loses an allelomorph, promptly so-called loss of heterozygosity (loss of heterozygosity is called for short LOH), but also relate to two allelic homozygous deletions (homozygous deletion).For LOH, remaining allelomorph is considered to inoperative, its reason or because existing genetic mutation, or because secondary accidental sudden change.

Melanoma is a kind of common cancerous swelling, has 1 people to get this disease (American Cancer Society, 1992) among per 100 Americans.Environmental influence plays an important role for melanomatous as being exposed to ultraviolet ray, but heredity also is decisive factor.A gene of familial melanoma is MLM, has been located in chromosome 9p21 (Cannon-Albright etal., 1992; Nancarrow et al., 1993; Gruis et al., 1993; Goldsteinet al., 1994).If the MLM locus has a tendentious allelomorph, then suffering from melanomatous possibility increases about 50 times.MLM is a member in the growing tumor suppressor gene family of number.Melanomatous tendentiousness is carried out heredity by the dominance mendelian character, but tendentious MLM sudden change is considered to be rendered as somatic Recessive alleles by the mode that is proposed by Knudson (1971) at first.In carrying a wild type and allelic tendentiousness individuality of saltant MLM, the cell of division has passed through secondary catastrophic event, this incident relates to losing or inactivation of MLM wild type copy, thereby exposes heritable saltant MLM allelomorph.On the contrary, single wild type copy gene can prevent malignant tumour.

In several different tumor types, be determined widely near the chromosome abnormality the MLM of 9p21 place, comprise glioma cell line, non-small cell lung cell-line and acute lymphoblast leukaemia system (Olopade et al., 1992; Olopade et al., 1993; Lukeis et al., 1990; Diaz et al., 1991; Middleton et al., 1991; Fountainet al., 1992; Cheng et al., 1993; James et al., 1993).Therefore, according to the frequency of 9p21 chromosome abnormality in non-melanoma tumor cell, the gene that (or a plurality of) participate in several at least different tumor type progress may be contained in the MLM zone.These incidents relate to LOH and high-frequency homozygous deletion.

Cell in the tissue is having only three kinds of important selections in life: growth and division, not growing but keeping lives, perhaps apoptosis.Unsuitable growth and division or cell are when this is dead and not deadly all can cause tumour.A kind of mechanism of controlling tumor growth is the direct regulation and control cell cycle.For example, genes that those control decisions cause dna replication dnas are strong candidates of oncogene or tumor suppressor gene, and it depends on that they rise in this process stimulate or inhibitory action.At cell cycle (G ₁, S, G ₂With the M phase) in, eukaryotic process is controlled by formation in turn, activation and the subsequent inactivation of the kinases of a series of cyclins (cyclin)/cyclin dependent (cyclin-dependent kinase is called for short Cdk) compound.Show cyclin D ＇ s/Cdk2,4,5, cyclin E/Cdk2, cyclin A/Cdk2 and cell periodic protein B/A/Cdk2 relate to this process.Cyclin D ＇ s and Cdk2, Cdk4 and Cdk5 relate to from G ₁To the transformation of S, promptly when cell is grown and determine whether to begin dna replication dna.Also found other the cell cycle regulating factor recently.These factors are Cdk inhibitor (Cdkinhibitors are called for short CkI), comprising Farl, p21, p40, p20 and p16 (Marx, 1994; Nasmyth ﹠amp; Hunt, 1993).

Recent findings, several oncogenes and tumor suppressor gene are participated in the cell cycle directly.For example, a kind of in the cyclin protein of dna replication dna (promote) participates in (Motokura et al., 1991 as oncogene; Lammie et al., 1991; Witherset al., 1991; Rosenberg et al, 1991), and TIF Rb and main cyclin gametophyte are Cdk have an effect (Ewen et al., 1993).Differentiate that melanomatous susceptible gene seat will open the path of individual inheritance screening, thereby can assess for example owing to being exposed to the high-risk rate that causes cancerous swelling under the sunlight.MTS also makes the people tend to take place a large amount of other cancerous swellings, and these cancerous swellings include, but are not limited to: the cancerous swelling of leukemia, astrocytoma, spongioblastoma, lymphoma, glioma, Hodgkin lymphomas, Huppert's disease, sarcoma, muscle tumor, cholangiocarcinoma, dermoid cancer, chronic lymphocytic leukemia and pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, uterus, testis, kidney, stomach, colon and rectum.In addition, because MTS influences the progress of several different tumor types, so it can be used for determining cancerous swelling patient's prognosis.Therefore, MTS can be used as the basis of the very important diagnostic test of development, people can predict cancerous swelling takes place (as melanoma, ophthalmomelanoma, leukemia, astrocytoma, spongioblastoma, lymphoma, glioma, the Hodgkin lymphomas, Huppert's disease, sarcoma, muscle tumor, cholangiocarcinoma, dermoid cancer, chronic lymphocytic leukemia and pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, the uterus, testis, kidney, stomach, the cancerous swelling of colon and rectum) tendency, and people can also predict the prognosis of cancerous swelling.In addition, because MTS relates to the process of many tumor types, so MTS can suppress the means that the tumor growth ability provides general anticancer swollen treatment directly or indirectly because of it.For example tumour cell is reverted to and have normal MTS function, it can be changed over non-pernicious.Summary of the invention

The present invention relates to the somatic mutation of many TIFs (Multiple TumorSuppressor, the be called for short MTS) gene in people's cancerous swelling and in the diagnosis of people's cancerous swelling and the application aspect the prognosis.The invention still further relates to the germ line mutation and the application aspect the tendency of many kinds of cancerous swellings generations of diagnosis thereof of mts gene, these cancerous swellings comprise the cancerous swelling of melanoma, ophthalmomelanoma, leukemia, astrocytoma, spongioblastoma, lymphoma, glioma, Hodgkin lymphomas, Huppert's disease, sarcoma, muscle tumor, cholangiocarcinoma, dermoid cancer, chronic lymphocytic leukemia and pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, uterus, testis, kidney, stomach, colon and rectum.The invention still further relates to the treatment of people's cancerous swelling of mts gene sudden change, comprising gene therapy, protein replacement treatment and protein analogue.At last, the present invention relates to screen the medicine that is used for the cancerous swelling treatment.The summary of accompanying drawing

Figure 1A shows kindred 3137.All melanoma patients all carry susceptible haplotype (haplotype).Other cancerous swellings in the individuality that carries the susceptible haplotype have also been indicated.Illustrate that symbol is as follows: solid circles or square expression melanoma; Part solid circles or other cancerous swellings of square expression; "/" expression is dead; " * " expression does not know whether this individuality carries the susceptible haplotype; As if " * * " represents that this individuality carries susceptible haplotype and " 35 " expression if check under the ill situation or the age during diagnosis.

Figure 1B shows kindred 3161.All melanoma patients all carry the susceptible haplotype.Other cancerous swellings are not determined haplotype.Illustrate that symbol is as follows: solid circles or square expression melanoma; Part solid circles or other cancerous swellings of square expression; "/" expression is dead; "=" is illustrated in other local appearance the among the kindred; Age when " 3 " expression in the pentagon has repeatedly marriage and " 35 " expression as if inspection under the ill situation or diagnosis.

Fig. 1 C has shown kindred 3355.All melanoma patients all carry the susceptible haplotype.Other cancerous swellings are not determined haplotype.Illustrate that symbol is as follows: solid circles or square expression melanoma; Part solid circles or other cancerous swellings of square expression; Dead and " 35 " expression of "/" expression is if check under the ill situation or the age during diagnosis.

Fig. 1 D has shown kindred 1771 and melanoma, and a situation arises with other cancerous swellings.In this kindred, on MTS, differentiate a sudden change.Illustrate that symbol is as follows: the carriers of mutation that " * " expression is confirmed; Solid circles or square expression melanoma; Part solid circles or square expression other cancerous swellings (being colon cancer in this kindred); Dead and " 35 " expression of "/" expression is if check under the ill situation or the age during diagnosis.

Fig. 2 is that yeast artificial chromosome (YAC) and P1 clone the zone of both sides in abutting connection with IFNA-s and D9S171.The kinetochore is on the right side.For the P1 clone, arrow has been pointed out the direction of T7 promoter sequence in this carrier.The YAC that combines represents, according in the zone to the location of sequence-tagged site (STS), these clones are similar.These YAC are assumed to be it is incomplete same.YACA5, B11, C6 and F9 contain IFN-1 and IFN-s.YACD1, F5 and E3 contain D9S126 and D9S171.Do not demonstrate the near-end and the far-end that contains the YAC of IFNA-s of the YAC that contains D9S171.Distance is not strictly drawn in proportion.In Fig. 2, marked the mark that is positioned at IFNA-s and D9S171 inside.Mark with " c " beginning is derived and is got from cosmid (cosmid) end sequence.Do not mark cosmid.Between c1.b and the c5.3 and the distance between 760-L and the D9S171 not quite clear.

Fig. 3 is observed disappearance figure in melanoma cell series.According to the mark of a series of disappearances, disappearance is divided into 12 classes.11 cell-lines have lacked whole marks of being painted among the figure, and this type do not mark.The representative number of each class is listed in " # system " column in other 12 classes.The disappearance breakpoint location of 1-10 class is depicted in the adjacent mark of DNA that falls within and lack, and is the last positive mark in a series of marks that extend to the disappearance place.For the 11st class and the 12nd class, the position of disappearance marks with solid triangle.

Fig. 4 A is cosmid c5 figure.Being used for the relevant STS of deletion analysis and cosmid and P1 thereof is marked together.The c1.b mark is positioned at the near-end of P1-1062 and does not mark.The transcriptional orientation of MTS1 and MTS2 marks with arrow.

Fig. 4 B is restriction map and the STS figure of cosmid c5.Mark the position of the coding exon of MTS1 and MTS2 with thick line." E1 " and " E2 " represents " coding exons 1 " and " coding exon 2 " respectively." B " is BamHI, and " S " is SalI, and " R1 " is that EcoRI and " R5 " are EcoRV.

Fig. 5 A and 5B contain the MTS1 genome sequence of 5 ＇ untranslated region territories, exons 1 and part introne 1 and have announced comparison between the p16 sequence (Serrano et al., 1993).Initiation codon (band underscore) is positioned at 891, and stitching position (arrow) is positioned at 1016.In the MTS1 sequence shown in Fig. 5 A-B is SEQ ID NO:3.In the p16 sequence shown in Fig. 5 B is SEQ ID NO:24.

Fig. 6 A and 6B are the comparisons that contains between the MTS1 genome sequence of part introne 1, exon 2 and part intron 2 and the p16 sequence of having announced (Serrano et al., 1993).Stitching position (arrow) is before 192 and after 498.In the MTS1 sequence shown in Fig. 6 A-B is SEQ ID NO:4.Identical in the p16 sequence shown in Fig. 6 A with the nucleotide 192-498 position of SEQ ID NO:4.

Fig. 7 A and 7B contain part introne 1, " exon 2 " and the MTS2 genome sequence of sequence and the comparison between the p16 sequence announced subsequently." exon 2 " sequence is very similar between nucleotide 273-580 position to the exon 2 of MTS1.Stitching position among MTS2 and the p16 marks with arrow.The site of beginning divergent (divergence) is used ^{" 0 "}Expression.The terminator of MTS2 is positioned at 532 of exon 2 and uses " * " expression.In the MTS2 sequence shown in Fig. 7 A-B is SEQ ID NO:5.Identical in the p16 sequence shown in Fig. 7 A with the nucleotide 192-498 position of SEQ ID NO:4.

Fig. 8 is the comparison of dna sequence dna between MTS1 and the MTS2, comprises exon 2 and part intron on every side.For MTS1,3 ＇ stitching positions of introne 1 and 5 ＇ stitching positions of intron 2 mark with triangle.The divergence point of the 3 ＇ end of close coding exon 2 marks with arrow.Shown MTS1 sequence is corresponding to the nucleotide 92-548 position of SEQ ID NO:4.Shown MTS2 sequence is corresponding to the nucleotide 174-630 position of SEQ ID NO:5.

Fig. 9 has shown the deletion condition in the tumor cell line of different STS.Each PCR experiment comprises positive control and negative control, and the cell-line (for example the 21st class) that one or two STS disappearance only arranged twice of retest at least.

Figure 10 A-C is the expression of MTS2 mRNA.Figure 10 A has shown and is deriving from the level relatively that different people organizes the MTS2 transcript among the RNA (Clonetech): road 1-brain; Road 2-breast; Road 3-kidney; Road 4-lung; Road 5-lymphocyte; Road 6-ovary; Road 7-pancreas; Road 8-prostate; Road 9-spleen; Road 10-stomach; Road 11-thymus gland.Have to be different from and expect start from some unknown cause (the seeing 1) of product of molecular weight.Figure 10 B has shown aspect the function of time after induced fission the transcript level of relative MTS2 in the human lymphocyte: road 1-0 hour; Road 2-1 hour; Road 3-2 hour; Road 4-4 hour; Road 5-8 hour; Road 6-16 hour; Road 7-24 hour; Road 8-32 hour; Road 9-40 hour; Road 10-48 hour; Road 11-56 hour; Road 12-64 hour.Between 40-50 hour, most cells are in the S phase after inducing.The transcript level of MTS2 when Figure 10 C has shown with the Rb state as function.Rb ⁺Cell-line is: road 1-KIT (Hori et al., 1987); Road 2-dliploid human fibroblasts MRC5, passage 28; Road 3-UMSCC2; Road 4-Bristol 8; Road 5-ZR75; Road 6-HaCaT; Road 7-T24.Rb ^-Cell-line is: road 8-MDA MB 468; Road 9-5637; Road 10-C33A; Road 11-SiHa; Road 12-CaSki; Road 13-WERI.

Figure 11 is the cDNA sequence (and encoded polypeptides) that comprises the MTS2 in 5 ＇ untranslated region territories.The initial of exon 2 is positioned at 491, and represents with arrow.The cDNA sequence is SEQ ID NO:15, and amino acid sequence is SEQ ID NO:16.

Figure 12 A and 1 2B have shown the cDNA sequence (and encoded polypeptides) of MTS1E1 β.Splicing site is represented with arrow.The initial of exon 2 is positioned at 335, and exon 3 is since 642.The cDNA sequence is SEQ ID NO:13, and amino acid sequence is shown in SEQID NO:14.

Figure 13 is the physical map in p16 zone.Exons 1 α (E1 α), exons 1 β (E1 β), exon 3 (E2) and exon 3 (E3) are represented with solid box.Also marked the restriction enzyme site of Eco RI (RI), Eco RV (RV) and Sal I (S).Above restriction map is genomic clone cosmid c5 and P1 1063.Below be disappearance among cell-line A375 and the SK-mel93.Dotted line is represented the DNA that lacks.

Figure 14 is the arranging situation of mouse and people's p16 β transcript sequence.Capitalization is represented identical nucleotide.The terminator that p16 reads in the frame marks with underscore.Splicing between E1 β and the E2 (is v) represented with caret.Mouse β sequence is SEQ ID NO:25.People β sequence is identical with the nucleotide 193-461 position among the SEQ ID NO:13.

Figure 15 is the expression of alpha transcriptional thing in the cell-line that contains E1 β disappearance.With from shown in separate the sample total RNA and derive and obtain cDNA.In reaction, add radiolabeled primer with amplification p16 transcript.The α of equivalent volumes and β amplified production, product separates in 5% denaturing polyacrylamide gel: the T cell that road 1-is static; Road 2-cell-line SK-mel93; Road 3-cell-line A375.

Figure 16 A-D is the expression of p16 transcript.In reaction, add radiolabeled primer with amplification p16 transcript, and product separates in 5% denaturing polyacrylamide gel.In Figure 16 A and 16D, before electrophoresis, will mix from the α and the beta response thing of common sample.Figure 16 A has shown the level relatively of organizing the p16 transcript among the RNA from different people: road 1: brain; Road 2: breast; Road 3: kidney; Road 4: lung; Road 5: lymphocyte; Road 6: ovary; Road 7: pancreas; Road 8: prostate; Road 9: spleen; Road 10: stomach; Road 11: thymus gland.Figure 16 B has shown aspect the function of time after induced fission the relative quantity of β transcript in the human lymphocyte: road 1:0 hour; Road 2:1 hour; Road 3:2 hour; Road 4:4 hour; Road 5:8 hour; Road 6:16 hour; Road 7:24 hour; Road 8:32 hour; Road 9:40 hour; Road 10:48 hour; Road 11:56 hour; Road 12:64 hour.Figure 16 C has shown aspect the function of time after induced fission the relative quantity of alpha transcriptional thing in the human lymphocyte, and each road is identical with Figure 16 B, but has shortened 1 hour.Also analyzed the expression of other molecules, these molecules or may influence the process of cell cycle, perhaps the transcriptional level in the cell cycle is conditioned.Consistent with former result, in case inducing T cell, Cdk4 and GoS2 (molecule that a kind of function is not clear, but when static T cell entered the cell cycle, it was transcribed and is induced.) level just increase (Russell and Forsdyke, 1991; Matsushime et al, 1992; Gengand Weinberg, 1993).On the contrary, as if p27 rna level does not change (Toyoshima and Hunter, 1994 in experimentation; Kato et al., 1994).The transcript of p16 when Figure 16 D has shown with the Rb state as function.Rb-cell-line is: road 1:WERI; Road 2:CaSki; Road 3:SiHa; Road 4:C33A; Road 5:5637; Road 6:MDAMB 468.Rb ⁺Cell-line is: road 7:T24; Road 8:HaCaT; Road 9:Zr75; Road 10:Bristol 8; Road 11:UMSCC2; Road 12: dliploid human fibroblasts MRC5; Road 13, KIT (Hori et al., 1987).

Figure 17 is the cDNA sequence of MTS1 that comprises the non-coded portion of cDNA.Triangle is represented splicing place.Dotted line only is to emphasize this splicing place in the sequence of second splicing place, does not represent to lack base.This sequence is SEQ ID NO:36.Detailed Description Of The Invention

The present invention relates to the somatic mutation of many TIFs (MTS) gene in people's cancerous swelling and in the diagnosis of people's cancerous swelling and the application aspect the prognosis.The invention still further relates to the germ line mutation and the application aspect the tendency of the various cancerous swelling generation of diagnosis thereof of mts gene, these cancerous swellings comprise the cancerous swelling of melanoma, ophthalmomelanoma, leukemia, astrocytoma, spongioblastoma, lymphoma, glioma, Hodgkin lymphomas, Huppert's disease, sarcoma, muscle tumor, cholangiocarcinoma, squamous cell knurl, chronic lymphocytic leukemia and pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, uterus, testis, kidney, stomach, colon and rectum.The invention still further relates to the treatment of people's cancerous swelling of mts gene sudden change, comprising gene therapy, protein replacement treatment and protein analogue.At last, the present invention relates to screen the medicine that is used for the cancerous swelling treatment.

The invention provides a kind of polynucleotide of separation, it contains the mts gene seat of all or part of mts gene seat or sudden change, and its length is preferably at least 8 bases and to be no more than about 100 kilobase right.This polynucleotide can be the antisense polynucleotide.The present invention also provides a kind of recombination to construct thing that contains the polynucleotide of this separation, for example is applicable to the recombination to construct thing of expressing in transformed host cells.

The present invention also provides to detect in analyte and has contained the polynucleotide of part mts gene seat or the method for its expression product.This method also can comprise the step of amplification part mts gene seat, and also can comprise provides the primer of a cover as this part mts gene seat of amplification again.This method can be used to diagnose the tendency of suffering from cancerous swelling or diagnosis or the prognosis that is used for cancerous swelling.

The present invention also provides isolated antibody, preferably is monoclone antibody, they can be specifically with separate, contain at least 5 polypeptide combinations by mts gene seat amino acids coding residue.

The present invention also provides the kit that is used for detecting at analyte the polynucleotide contain part mts gene seat, and this kit comprises and is packaged in the appropriate containers and polynucleotide and the operation instruction complementation of part mts gene seat.

The polynucleotide method that the present invention also provides preparation to contain polymerization nucleotide, thus the nucleotide sequence that contains at least 8 continuous mts gene seats produced; The method for making of the polypeptide that contains polymeric amino acid also is provided, has contained at least 5 by mts gene seat amino acids coding thereby produce.

In addition, the invention provides the method that screening is used for the medicine of cancerous swelling treatment, so that identify medicine suitable, that can recover mts gene product function.

At last, the invention provides at the required means of the gene therapy of cancerous swelling cell.These treatment reagent can utilize the polynucleotide that contains all or part of mts gene seat, and be placed on suitable carriers or it sent and pass into this target cell with more direct method, thus the function of recovery MTS protein.Treatment reagent also can utilize the polypeptide based on part or all of MTS protein sequence.Thereby make it on function, substitute the activity of MTS in vivo.

The present invention finds that the mts gene seat (being called melanoma (MLM) locus in prior art) that makes individual tendency suffer from melanoma and other cancerous swellings is the gene of coding MTS1, has found that it is the especially inhibitor of Cdk4 of Cdk.This gene is called as MTS1 herein.The present invention finds that also the mts gene seat contains second coded sequence that is called as MTS2, and it and MTS1 are closely similar on partial sequence.The present invention finds that also the MTS1 gene has two promotor-α and β.When using the α promotor, the mRNA that obtains is made of exons 1 α, exon 2 and exon 3.This is called as MTS1.When using the β promotor, the mRNA that obtains is made of exons 1 β, exon 2 and exon 3.This is called as MTS1E1 β.The present invention finds that the sudden change of mts gene seat represents to exist the tendency of suffering from melanoma and other cancerous swellings in kind of system.At last, the present invention finds that the somatic mutation of mts gene seat is relevant with great majority (if not whole words) tumor type, is the general mark of cancerous swelling or cancerous swelling prognosis therefore.The catastrophic event of mts gene seat relates to disappearance, insertion and the point mutation in coded sequence and the non-coding sequence.

The MLM locus is to use genetic method the earliest, by the kindred of several Utah States and remarkable chain (Cannon Albright, 1992) of locating of kindred between genetic marker and melanoma tendency of a Texas.Both sides by the zone that recon defined in the kindred are D9S736 and D9S171.Subsequently, these and other genetic marker is used to gene location, promptly contains homozygous deletion situation in the melanoma of disappearance and the non-melanoma cell series by analysis.The both sides of minimum disappearance overlapping region are IFNA-s and D9S171.Screening YAC library is to differentiate the genomic clone around these marks.The P1 clone is separated by the part of chromosome walking (chromosomal walk), and except two intervals are arranged, in abutting connection with IFNA-s to D9S171.(specific sequence-tagged site is STS) to make up more detailed molecular linkage map in preparation distinguished sequence sign site.Use these marks and deletion analysis, in cosmid 5 (c5), find to be the middle absence of heart overlapping region of both sides with mark c5.1 and c5.3.The mark that deletion frequency is the highest is c5.3, and it is very near MTS.

Whether exist " CpG " island to the analysis showed that it contains the candidate gene of at least one MTS to c5.Determined c5 the EcoRI fragment dna sequence dna and compare with the sequence of GenBank.Identify the c5 zone of two uniquenesses, they are the regional similar of p16 (Serrano et al., 1993) with the gene of the coding people Cdk4 inhibitor of differentiating in the past.These two candidate genes are called as MTS1 and MTS2.Use cDNA library, identify another candidate gene MTS1E1 β from probe screening lymphocyte, fetal brain and the normal breast of MTS1 exon 2.

In detail relatively disclosing of c5 genome sequence and p16mRNA sequence, MTS1 contains a 307bp fragment, and this fragment is identical with part p16 coded sequence.The both sides of this nucleotide fragments among the MTS1 are discernible splicing sequence.To further studies show that of MTS1, it comprises whole coded sequences and two introns of p16.Introne 1 is positioned at 126bp place, downstream, translation initiation position; Intron 2 is positioned at 11bp place, downstream, translation termination position.These two introns are divided into the 5 ＇ zones (coding exons 1) of 3 zone: 126bp, the zone line (coding exon 2) of 307bp and the 3 ＇ zones (coding exon 3) of 11bp with the coded sequence of p16.

MTS2 contain one with p16 dna sequence dna much at one, it extends about 200bp from the 5 ＇ end of coding exon 2 to intron 2.Yet the similitude of sequence weakens at the 51bp place, intron 2 upstream of MTS1, and two kinds of sequences are divergent fully herein.This is corresponding to the position of the final codon of MTS2.The sequence of MTS1 and MTS2 comparison shows that, the sequence similarity between two kinds of genes also extends nearly 50 nucleotide from the upstream of 3 ＇ splicings of introne 1.Therefore, noncoding DNA is more conservative than some zone of the coding DNA of supposition.In order to get rid of sequence divergence in the coding DNA is because the possibility of clone's illusion, and the PCR primer of the sequence divergence point of MTS2 is crossed in design amplification specifically.These primers amplify the fragment of pre-sizing from cosmid P1 and genomic DNA.Therefore, be positioned at genome sequence really near the divergent sequence of 3 ＇ of MTS2 exon 2 end.

MTS1E1 β contains exons 1, and this exon is called exons 1 β or E1 β, and it has the sequence of the exons 1 that is different from MTS1 and MTS2.MTS1E1 β also contains exon 2 (E2) and the exon 3 (E3) identical with exon 3 with the exon 2 of MTS1.Exons 1 β be positioned at MTS1 exons 1 the upstream and do not contain any coded sequence.As a result, MTS1E1 β coding translation initiation position is positioned at the p10 at first ATG place of exon 2.

Utilize exon 2, carry the correlation that the allelic genes of individuals group of MLM tendentiousness DNA tests MTS1 and MTS2 and genetic predisposition locus MTS by analyzing supposition.Have in 8 individualities in the MTS1 exon 2 of body one by one and identify dna polymorphism.This sudden change is single nucleotide subsitution, causes an amino acid to change.This polymorphism is separated together with MLM tendentiousness allelomorph.

Damage among the MTS1 (disappearance and nucleotide subsitution) advantage shows that MTS1 or certain closely linked locus cause tumor phenotypes.The cell that is subjected to these damages has selective advantage than the cell that does not sustain damage.The another kind of explanation thinks that these damages are and the irrelevant random event of cell growth, and this explanation is impossible.Reason is, at first, has causality in the high correlation hint between tumor phenotypes and the MTS1 sudden change between MTS1 sudden change and tumour formation.Secondly, MTS1 influences melanomatous neurological susceptibility, so it has independently the meaning as tumor suppressor gene.Moreover, as the biochemical function of the potent inhibitor p16 of Cdk and MTS1 in vivo as the initial general inhibitor of dna replication dna and the pattern that plays a role conform to well.

According to diagnosis of the present invention and method of prognosis, can detect the variation of wild type mts gene seat.In addition, this method can also wild type mts gene seat and confirmation lack tendentiousness or neoplasia is carried out by detecting." change of wild type gene " comprises the sudden change of form of ownership, is included in disappearance, insertion and the point mutation of coding region and non-coding region.Disappearance can be whole gene or be the disappearance of portion gene.Point mutation can cause terminator, frameshift mutation or amino acid replacement.Somatic mutation is to occur over just some tissue as the sudden change in the tumor tissues, can heredity in kind of system.Germ line mutation finds in can a bodily tissue in office and is hereditary.If only have a single allelomorph to be somatic mutation, explanation is to be in early stage neoplasia state so.Yet if two allelomorph all suddenly change, explanation is to be in neoplasia in late period state so.Therefore, the discovery of MTS sudden change can provide diagnosis and prognosis information.Can screen the MTS allelomorph that do not have disappearance (promptly as the MTS allelomorph that carries on sister's chromosome of MTS deletion), with the sudden change that determines whether other as insert, little disappearance and point mutation.It is believed that many sudden changes of finding cause the mts gene product to be expressed and descend in tumor tissues.But, cause the sudden change of non-functional gene outcome also can produce cancerous swelling.The point mutation incident can occur in the regulation and control zone as in the promotor of gene, thus the disappearance or the decline that cause mRNA to express.Point mutation also can destroy suitable RNA processing, thereby causes disappearance that the mts gene product expresses or the decline that causes mRNA stability or translation efficiency.

Useful diagnostic techniques comprises; but be not limited to: fluorescence in situ hybridization (FISH), direct dna sequencing, pulse alternative field gel elec-trophoresis (PFGE) analysis, Southern engram analysis, single stranded conformational analysis (SSCA), ribalgilase (RNase) protection mensuration, allele specific oligonucleotide oligonucleotides (ASO), dot blot analysis and polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP), hereinafter will describe in more detail.

The tendency of suffering from cancerous swelling such as melanoma and other cancerous swellings of pointing out herein can be passed through the sudden change of the mts gene of anyone tissue of test and determine.For example, a heredity has kind of a people who is the MTS sudden change will easily suffer from cancerous swelling.This point can be determined from the DNA in any bodily tissue of this individuality by test.The most simply, can extract blood and from haemocyte extracting go out DNA.In addition, whether there is the sudden change of mts gene can carry out pre-natal diagnosis by detecting fetal cell, placenta cells or amniotic fluid.For example by point mutation or disappearance and the allelic change of wild type MTS that causes can detect with arbitrary means described herein.

In order in tissue, to detect the change of wild type mts gene, this separate tissue must be gone out and do not contain normal surrounding tissue.The method of organizing goods that enrichment contains tumour cell is as known in the art.For example, can be from the segment of paraffin or cryostat chorista.Can also cancerous swelling cell and normal cell be separated with flow cytometry.These technology and other are as known in the art with the technology that tumour cell and normal cell separate.If tumor tissues is by the normal cell severe contamination, Tu Bian detection will become more difficult so.

A kind of initial analysis method fast that detects dna sequence polymorphism be observe the one or more restriction enzymes of a series of usefulness, more preferably be Southern trace with the DNA of a large amount of digestion with restriction enzyme.Every trace sheet contains a series of normal individual and a series of cancerous swelling case.Demonstrate hybridized fragment the Southern trace (when with near or the sequence that contains the mts gene seat during as probe hybridization, on length, can difference be arranged with contrast DNA.) show and may have a sudden change.If use in the time of to produce the restriction enzyme of very large restriction fragment, can use pulse alternative field gel elec-trophoresis (PFGE) so.

The detection of point mutation can be carried out the allelic molecular cloning of MTS and this equipotential gene sequencing is realized with techniques well known in the art.Perhaps, can be with prior art to from the direct amplification gene sequence of the genomic DNA goods of tumor tissues.And then the dna sequence dna of definite extension increasing sequence.

Have 6 kinds known, more complete but still be not that direct method of testing can be determined the allelic existence of neurological susceptibility: 1) single stranded conformational analysis (SSCA) (Orita et al., 1989); 2) denaturing gradient gel electrophoresis (DGGE) (Wartell et al., 1990); 3) (Finkelstein et al., 1990 are measured in the RNase protection; Kinszler et al., 1991); 4) allele specific oligonucleotide oligonucleotides (ASO) (Conner el al., 1983); 5) use protein such as the Escherichia coli mutS protein (Modrich, 1991) and 6 of discerning the nucleotide mispairing) allele specific oligonucleotide PCR (Rano ﹠amp; Kidd, 1989).For allele specific oligonucleotide PCR, use the primer that can suddenly change and hybridize with specific MTS at its 3 ＇ end.If specific MTS sudden change does not exist, then do not observe amplified production.Can also use as disclosed amplification in people's (1989) such as european patent application No.0332435 and Newton article should not suddenly change system (Amplification Refractory Mutation System, ARMS).The insertion of gene and disappearance can also detect by clone, order-checking and amplification.In addition, can also use at this gene or the restriction fragment length polymorphism of marker gene (RFLP) probe on every side, so that assess allelic change or insertion with the polymorphic bands form.Whether this method has identical MTS for the relatives that screen diseased individuals is suddenlyd change particularly useful.The additive method that also can use detection as known in the art to insert and lack.

In first three methods (be SSCA, DGGE and RNase protection are measured), a new electrophoretic band appears.SSCA detects the different band of migration, because sequence variation causes the difference of base pairing in the single chain molecule.The RNase protection relates to is cut into two or more littler fragments with the saltant polynucleotide.DGGE uses denaturing gradient gel, detects the mobility of the saltant sequence different with wild-type sequence.In allele specific oligonucleotide oligonucleotides is measured, design can the detection specificity sequence oligonucleotides, whether analyze by the existence that detects hybridization signal then.In mutS measured, protein only combined with the heteroduplex sequence that contains the nucleotide mispairing that is formed by saltant and wild-type sequence.

According to the present invention, tramp is the nucleic acid of heterozygosis, is not 100% complementation between the two strands.Disappearance, insertion, inversion or displacement also cause the disappearance of whole autoploidy.Mispairing detects the point mutation that can be used for detecting gene or its mRNA product.Though these technology are lower than the sensitivity of order-checking, for a large amount of tumor samples, its operation is more easy.The example that technology is cut off in mispairing is the RNase Protection Code.In practice of the present invention, this method relates to the mark riboprobe that uses with the complementation of people's wild type mts gene coded sequence.This riboprobe and from tumor tissues isolated mRNA or DNA anneal together (hybridization), subsequently with enzyme ribonuclease A (RNase A) digestion that can detect some mispairing in the double-stranded RNA structure.If RNase A detects mispairing, it just cuts off it in the mispairing position.Therefore, the RNA product of annealing separates in running gel matrix, if mispairing is detected and cuts off by RNase A, can observe a RNA product so, and it is littler than double-stranded RNA total length, that formed by riboprobe and mRNA or DNA.Riboprobe needs not to be the MTS mRNA or the gene of total length, and it can be their fragment.If riboprobe only contains the fragment of MTS mRNA or gene, need to screen whole mRNA sequence so and whether have mispairing with these a large amount of probes.

By similar mode,, can use dna probe to detect mispairing by the cut-out of enzyme process or chemical method.As referring to Cotton et al., 1988; Shenk et al., 1975; Novack et al., 1986.Perhaps, the change by the mispairing two strands electrophoretic mobility double-stranded with respect to correct pairing detects mispairing.As referring to Cariello, 1988.With riboprobe or dna probe, before hybridization, contain the cell mRNA or the DNA of sudden change with PCR (seeing below) amplification, detect the variation of mts gene DNA with the Southern hybrid method, be big rearrangement as lacking and during insertion especially when changing.

Also can use the mts gene dna sequence dna of pcr amplification with allele specific oligonucleotide probe screening.These probes are nucleic acid oligomers, and every kind contains a zone of carrying the mts gene sequence of known mutations.For example, an oligomer can be about 30 nucleotide, and corresponding to a part of mts gene sequence.By using one group of this allele specific oligonucleotide probe, just can screen, thereby determine in mts gene, whether there is fixed mutation type with pcr amplification product.For example can on nylon leaching film, hybridize with the MTS sequence and the allele specific oligonucleotide probe of amplification.With specific probe hybridization taking place is illustrated in the sudden change that exists in this tumor tissues with allele-specific probe same type under tight hybridization conditions.

For the clearest and the most definite method of testing of suddenling change in the candidate gene seat is directly to compare cancerous swelling patient and contrast crowd's genome MTS sequence.Perhaps, people can check order to mRNA with PCR method amplification back, thereby needn't determine the exons structure of candidate gene.

Cancerous swelling patient can be by detecting near the mts gene or inner non-coding region such as intron and regulating and controlling sequence detect in the sudden change outside the MTS coding region.The sudden change of non-coding region is the vital Blot experiment from Northern that indicates in early days, and this experiment discloses with contrasting individuality and compares, and the unusual or abundant mRNA molecule of molecular size is arranged in the canker people.

The change that MTS mRNA expresses can detect with techniques well known in the art.Comprising Northern engram analysis, pcr amplification and RNase Protection Code.The minimizing that mRNA expresses shows that change has taken place the wild type mts gene.Can also detect the change of wild type mts gene by the change of screening wild type MTS protein.For example, can use the immunoreactive monoclone antibody that has at MTS to screen tissue.Lack corresponding antigen and just represent to have the MTS sudden change.Also can use the special antibody of mutant allele product is detected saltant mts gene product.These immunologic assays can carry out in mode easily as known in the art.Comprising: Western trace, immunohistochemistry are measured and enzyme linked immunosorbent assay (ELISA) (ELISA).The method that any detection MTS protein changes may be used to detect the change of wild type mts gene.Can function of use measure, determine method as combined with protein.For example, known MTS protein especially combines with Cdk4 with Cdk.Therefore can measure binding ability with wild type MTS protein or Cdk4.In addition, can measure the biochemical function of MTS, to Cdk as to the inhibit feature of Cdk4 and the regulating and controlling effect of cell cycle.Search out saltant mts gene product and just represent to exist the change of wild type mts gene.

Can also other the human sample such as serum, ight soil, urine and saliva in detect saltant mts gene or gene outcome.The technology of saltant mts gene or gene outcome in the above-mentioned identical detection tissue can be applied to other human sample.Thereby the cancerous swelling cell can split away off from tumour and appear at these human samples.In addition, itself can be secreted into extracellular space the mts gene product, thereby found in originally not having these human samples of cancerous swelling cell.By these human samples are screened, can carry out early diagnosis to many kinds of cancerous swellings.In addition, can more early stagely by whether existing saltant mts gene or gene outcome to monitor the progress of chemotherapy or radiotherapy in the testing human sample.

Diagnostic method of the present invention also is applicable to any tumour that MTS plays a role in tumour takes place.In nearly all detected tumour, all observe in the somatic mutation in MTS zone or the disappearance of chromosome arm 9p.Diagnostic method of the present invention can make them determine suitable therapeutic scheme for the healthcare givers of great use.

Primer of the present invention is to being used for determining the allelic nucleotide sequence of a certain specific MTS by PCR.The single stranded DNA primer is to annealing with the sequence inner or on every side of the mts gene on the chromosome 9p, so that cause the synthetic amplification of the DNA of mts gene itself.These a whole set of primers can synthesize the nucleotide of all mts gene coded sequences (being exon).More preferably, a cover primer can synthesize intron and exon sequence.Also can use allele specific oligonucleotide primer.This primer is only annealed with specific MTS mutant allele, thereby can only amplify with the product of mutant allele as template.

In order to promote the clone of extension increasing sequence subsequently, primer can contain the restriction enzyme site sequence at its 5 ＇ end.Therefore except that minority formed the required nucleotide of restriction enzyme site, all primer nucleotide was from the sequence of MTS sequence or close MTS.These enzymes and restriction enzyme site are as known in the art.Primer itself can be synthetic with techniques well known in the art.Generally can use commercially available oligonucleotide synthesizer to prepare primer.According to the specific primer of the design of the MTS open reading frame shown in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:13, SEQ ID NO:15 and the SEQ ID NO:36 is that those of skill in the art institute is competent.

Nucleic acid probe provided by the invention can be used for many purposes.As mentioned above, they can be used for the Southern of genomic DNA hybridization and be used for the RNase Protection Code suddenling change with test point.These probes can also be used to detect pcr amplification product.Use other technologies, they can also be used to detect the mispairing of mts gene or mRNA.Definition

The present invention uses following definition:

" amplification of polynucleotide " adopted such as polymerase chain reaction (PCR), ligation amplification and (or is called ligase chain reaction, LCR) with based on the amplification method that uses the Q-beta replicase.These methods are known and are widely used in the art.For example referring to United States Patent (USP) 4,683,195 and 4,683,202 and Innis et al., 1990 (PCR) and Wuet al., 1989a (LCR).Be used to carry out reagent and the hardware commercialization of PCR.The primer of mts gene sequence of being used to increase preferably is complementary to and specifically with the MTS regional sequence or define the regional sequence hybridization of target region.The MTS sequence that produces with amplification method can directly check order.Perhaps, the sequence of amplification can be cloned before sequence analysis, but this method is inadvisable slightly.To describing by Scharf (1986) with the direct clone of the genomic fragment of enzyme process amplification and the method for sequencing analysis.

" analyzed polynucleotide " and " analyzed chain " refers to strand or double-stranded polynucleotide, it by suspicious comprise one section target sequence and be present in various dissimilar samples comprise in the biological sample.

" antibody ".The present invention also provide can be specifically with the MTS polypeptide or its fragment combines or with the polymerized nucleoside acid sequence in MTS zone particularly the polyclone that combines of mts gene seat or its part and/or monoclone antibody and fragment thereof with and immunity in conjunction with equivalent.Term " antibody " refers to the molecule entity of homogeneous or mixture such as the serum product of being made up of multiple different molecule entity.Polypeptide can synthesize and be coupled to carrier molecule (as key hole a fabulous creature, said to be like a turtle and blow poisonous sand in man's face hemocyanin) on peptide synthesizer, inject the rabbit several months then.Tested in rabbit serum is to the immunoreactivity of MTS polypeptide or fragment.Monoclone antibody can prepare by protein and peptide, fused protein or its fragment are injected mouse.Screen monoclone antibody with ELISA, test the specific immune reactivity of itself and MTS polypeptide or its fragment then.Referring to Harlow ﹠amp; Lane, 1988.These antibody can be used for analyzing and as medicine.

In case obtain the required polypeptide of q.s, just can use it for various uses.Typical purposes is the antibody that is used for the combination of production specificity.These antibody can be polyclone or monoclone antibody, and can produce in external or body with techniques well known in the art.

For producing polyclonal antibody, can select suitable target immune system, typically mouse or rabbit.By the mode of knowing by method that is applicable to animal and immunologist that other parameters limited, the antigen of basic purifying is supplied with immune system.Typical injection position is palmula, intramuscular injection, intraperitoneal injection or hypodermic injection.Certainly, also can substitute mouse or rabbit with other animals.Use techniques well known in the art purifying polyclonal antibody then, regulate required specificity again.

Immune response is analyzed with immunoassays usually.These immunoassays usually relate to carries out to a certain degree purifying to a kind of antigen source, this antigen source produce by identical cell and this antigen source in antigen identical.Various method of immunity are as known in the art.As referring to Harlow ﹠amp; Lane, 1988 or Goding, 1986.

Typically, with standardization program such as Harlow ﹠amp; Lane (1988) or the described program of Goding (1986), can make affinity is 10 ^-8M ^-1Or more preferably be 1O ^-9To 10 ^-100M ^-1Or higher monoclone antibody.In brief, can select suitable animal for use, adopt required immunization protocol then.Through reasonable time, take out the spleen of animal, under suitable alternative condition, the myeloma cell of individual splenocyte and immortalization is merged then.Subsequently, by the clone and separate cell, and each clone's of test supernatant is to determine whether to produce the suitable specific antibody at required antigen zone.

Other suitable technique relate to external lymphocyte are exposed to antigenic polypeptide, perhaps select the antibody library in phage or the similar substrates.Referring to Huse et al., 1989.Polypeptide of the present invention and antibody can be modified or without polishing the use.Polypeptide and antibody usually can be by covalently or non-covalently being connected a kind of material that detectable signal is provided and mark.A large amount of different label and interconnection techniques are known, and are widely reported in science and patent documentation.Suitable label comprises radionuclide, enzyme, substrate, co-factor, inhibitor, fluorescer, chemiluminescence agent, magnetic-particle etc.The patent of relevant these labels of use comprises United States Patent (USP) 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241.Equally, can also produce recombination immunoglobulin (referring to United States Patent (USP) 4,816,567).

" binding partners " refers to a kind of molecule that can combine with ligand molecular with high specificity, as antigen and antigen-specific antibodies or enzyme and its inhibitor.Normally, thus specific binding partner must carry out combination in fixedly analyte copy/complementation double-stranded (when carrying out the polymerized nucleoside acid hybridization) under the separation condition with enough affinity.Specific binding partners is to know in this area, for example comprises vitamin h and avidin or strepavidin (streptavidin), IgG and a-protein, countless known receptor-ligand conjugates and complementary polynucleotide chain.In the polynucleotide binding partners of complementation, gametophyte length is normally at least about 15 bases, and length can be 40 bases at least.Polynucleotide can be made of DNA, RNA or synthetic nucleic acid analog.

" biological sample " refers to contain from certain individuality, dubiously the tissue or the humoral sample of analyzed polynucleotide or polypeptide, includes, but are not limited to: for example blood plasma, serum, spinal fluid, lymph liquid, skin appearance, respiratory tract, enteron aisle and reproduction-the urinary tract, tears, saliva, haemocyte, tumour, organ, tissue and cell in vitro are cultivated the sample of composition.

As used herein, term " diagnosis " or " prognosis " when being used for the context that relevant tumour forms, are used to indicate 1) the neoplasia damage is classified 2) determine neoplastic seriousness or 3) and before the treatment, among or monitor the progress of disease afterwards.

" coding ".If when certain polynucleotide in its native state or the method that those of skill in the art know in this area when operation, produced mRNA or polypeptide or its fragment thereby can transcribe and/or be translated, so then claim certain polynucleotide " coding " polypeptide.Antisense strand is the complement of this nucleic acid, can derive coded sequence from it.

" separation " or " pure substantially "." separation " or " pure substantially " nucleic acid (as RNA, DNA or mixed polymer) is the nucleotide that separates with other cell components of natural human sequence who follows it under nature or protein (as ribosome, polymerase, a lot of other people genome sequence and protein) basically.This term comprises nucleotide sequence or its protein that takes out from its environment that exists naturally, and comprises the analog of DNA separator reorganization or the clone and chemosynthesis or pass through the allos system and biosynthetic analog.

" MTS allelomorph " refers to the allelomorph of normal mts gene seat and the allelomorph that carries variation, and these variations make individual tendency suffer from cancerous swelling at many positions.These cancerous swellings comprise the cancerous swelling of melanoma, ophthalmomelanoma, leukemia, astrocytoma, spongioblastoma, lymphoma, glioma, Hodgkin lymphomas, Huppert's disease, sarcoma, muscle tumor, cholangiocarcinoma, dermoid cancer, chronic lymphocytic leukemia and pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, uterus, testis, kidney, stomach, colon and rectum.This tendentiousness allelomorph also is called as " MTS susceptible allelomorph ".

" mts gene seat ", " mts gene ", " MTS nucleic acid " or " MTS polynucleotide " all refers to be positioned at the polynucleotide in MTS zone, they can be expressed in normal tissue, wherein, some allelomorph can make individual tendency suffer from melanoma and other cancerous swellings, for example ophthalmomelanoma, leukemia, astrocytoma, spongioblastoma, lymphoma, glioma, the Hodgkin lymphomas, Huppert's disease, sarcoma, muscle tumor, cholangiocarcinoma, dermoid cancer, chronic lymphocytic leukemia and pancreas, breast, brain, prostate, bladder, thyroid gland, ovary, the uterus, testis, kidney, stomach, the cancerous swelling of colon and rectum.In this article, the mts gene seat can exchange with title MLM locus in this area and use, and uses " MTS " to use " MLM " as locus, gene, zone etc. in order to comprise.The sudden change of mts gene seat relates to the initiation and/or the progress of other tumours.This locus is partly represented by the sudden change that causes individual tendency to suffer from cancerous swelling.These sudden changes are arranged in the following MTS zone of this paper.The mts gene seat comprises the regulating and controlling sequence that coded sequence, intermediate sequence and control are transcribed and/or translated.The mts gene seat comprises all allelic mutant dna sequences.

When these terms are used for nucleic acid, be meant coding MTS polypeptide (comprising p16), fragment, homologue or the variant nucleic acid of (for example comprising fusion or disappearance albumen).Nucleic acid of the present invention have or derived from or be similar to the sequence of natural MTS encoding gene, perhaps have basically and natural MTS encoding gene or its homeologous sequence.The coded sequence of MTS polypeptide (MTS1) is shown in SEQ ID NO:1, and the amino acid sequence of MTS polypeptide (MTS1) is shown in SEQ ID NO:2.The coded sequence of second kind of MTS polypeptide (MTS1E1 β) is shown in SEQ ID NO:13, and amino acid sequence corresponding is shown in SEQ ID NO:14.The coded sequence of the third MTS polypeptide (MTS2) is shown in SEQ ID NO:15, and amino acid sequence corresponding is shown in SEQ ID NO:16.Term p16 can exchange use with MTS1 and MTS1E1 β, is used to mean the MTS1 of coding p16 and the MTS1E1 β of coding p10.MTS1 and MTS1E1 β are two kinds of forms of a gene, and these two kinds of forms depend on any transcribing of using in two kinds of promotors.MTS2 is a unitary part in the MTS zone, its p15 that encodes.

Polynucleotide of the present invention comprises RNA, cDNA, genomic DNA, synthesized form and mixed polymer, can be that justice or antisense strand are arranged, and can be with chemistry or biochemical method modified or contain nucleotide base non-natural or that derive, these are conspicuous for the those of skill in the art in this area.For example these modifications comprise, mark, methylate, replace modification between one or more natural nucleotides, the nucleotide such as uncharged key with analog and connect the key that (as methyl-phosphonate, phosphotriester, phosphoamide thing, carbamate etc.), charged key connect (as thiophosphate, phosphorodithioate etc.), pendant moiety (as polypeptide), intercalator (intercalator) (as acridine, psoralen etc.), chelating agent, alkylating agent and modification and be connected (as α anomerization (anomeric) nucleic acid etc.).Also comprise synthetic molecule, the polynucleotide that this molecular energy combines with specified sequence by hydrogen bond and the simulation of other chemical interactions.This molecule is to know in this area, for example comprises those molecules with peptide bond replacement phosphate bond in molecular skeleton.

The invention provides the recombinant nucleic acid that comprises all or part of MTS zone.The recombination to construct thing can self-replacation in host cell.Perhaps, the recombination to construct thing is integrated in the chromosomal DNA of host cell.That the polynucleotide of this reorganization comprises is genomic, the polynucleotide in the semisynthetic or synthetic source of cDNA, and this polynucleotide presents because of its source or operation: 1) do not link to each other with all or part of polynucleotide coupled under native state; 2) be connected in polynucleotide not coupled under native state or 3) do not exist natively.

Therefore, the invention provides and contain the recombinant nucleic acid that there is sequence in non-natural.Although can use wild-type sequence, wild-type sequence usually is carried out change, as passing through disappearance, displacement or inserting.

Can use dissimilar cDNA or genomic library to screen, also can obtain these nucleic acid by using in genomic DNA or other natural origins, the increase sequence of existence of technology such as PCR as natural nucleus acid source of the present invention.The cDNA storehouse of selecting is usually corresponding to the tissue source of the mRNA that is rich in desired protein.General phage library is preferable, but also can use other libraries.The clone in library is applied on flat board, is transferred to the enterprising row filter of basement membrane, sex change and utilize probe in detecting whether to have required sequence.

Be used for dna sequence dna of the present invention and contain at least 5 codons (15 nucleotide), more generally at least 7-15 codons, the most at least 35 codons usually.Can there be one or more introns.The number of nucleotide normally can with the required minimum length of the successful probe that the MTS coded sequence is hybridized specifically about.

The technology of related nucleic acid operation is for example at Sambrook et al., has widely in 1989 or Ausubelet al., 1992 and describes.Use the reagent of these technology, as restriction enzyme etc. is to know in this area, and can from supplier such as New EnglandBioLabs, Boehringer Mannheim, Amersham, Promega Biotec, U.S.Biochemicals, New England Nuclear and in a large number other suppliers buy.The recombinant nucleic acid sequence that is used for producing fusion of the present invention can be derived from sequence natural or synthetic and be got.Many natural gene orders can obtain from genomic library or from different cDNA with suitable probe.Referring to GenBank, National Institutes of Health.

" MTS zone " refers to clone at P1 the part of the human chromosome 9 that finds among P1-1062 and the P1-1063.These P1 clones that are arranged in Escherichia coli NS3529 are deposited in American type culture collection on March 16th, 1994, and (Rockville, MarylandUSA), its preserving number is respectively ATCC No.69589 and 69590.The mts gene seat is contained in this zone, comprises MTS1, MTS2 and MTS1E1 β gene.

As used herein, term " mts gene seat ", " MTS allelomorph " and " MTS zone " all refer to contain the double-stranded DNA in this locus, allelomorph or zone and contain this locus, allelomorph or regional single stranded DNA.

As used herein, " part " mts gene seat or zone or allelomorph are defined as having at least about 8 nucleotide, or preferably about 15 nucleotide or, and can be the minimal amount that has at least about 40 nucleotide more preferably at least about the minimal amount of 25 nucleotide.

" MTS protein " or " MTS polypeptide " refers to by mts gene seat encoded protein matter or polypeptide (comprising MTS1 polypeptide, MTS2 polypeptide and MTS1E1 beta polypeptides), its variant protein or its fragment.Term " polypeptide " refers to polymer of amino acid or its equivalent, does not refer to have the product of length-specific; Therefore, peptide, oligopeptides and protein all are included in this definition of polypeptide.The modification of polypeptide such as glycosylation, acetyl groupization, phosphorylization etc. do not got rid of in this term.This definition comprises and contains one or more amino acid analogues polypeptide of (for example comprising alpha-non-natural amino acid etc.), has and replace key and other natural or non-natural modified polypeptides as known in the art.Usually, this peptide species have at least about 50%, preferably greater than about 90%, more preferably at least about 95% with the autoploidy of natural MTS sequence.Also comprise by under the tight or low stringent condition of height with the coded protein of DNA of MTS code nucleic acid hybridization, and use closely-related polypeptide or the protein that obtains at the antiserum of MTS protein.

The peptide sequence length that is used for the comparison autoploidy is usually at least about 16 amino acid, usually at least about 20 residues, and more generally at least about 24 residues, typically at least about 28 amino acid, and more preferably greater than about 35 residues.

" can operate continuous " and be meant that a kind of like this coordination, the residing relation of wherein said component make them to bring into play function by the mode of expection.For example, transcribe or express if promotor can cause one section coded sequence, then this promotor can be operated and is connected in coded sequence.

" probe ".Can detect by the hybridization reaction with certain polymerized nucleoside acid probe with the polynucleotide polymorphism of the MTS allelic association that causes easily suffering from some cancerous swelling or most of cancerous swellings, this probe can form stable heterozygote tight with this target sequence to medium tight hybridization and wash conditions.If the complete and target complement sequence of expection probe can use stringent condition so.If there is some mispairing in expection, when for example Yu Qi variant and probe are not complementary fully, can reduce the stringency of hybridization so.Non-specific/accidental combination that the condition of selecting should be eliminated promptly should reduce background.Because this demonstration can be determined neutral dna polymorphism and sudden change, so, also need further to analyze to show the allelic disappearance of MTS susceptible.

Being used for the allelic probe of MTS can obtain from MTS zone or its cDNA.Probe can have any appropriate length, and what it can be across the MTS zone is all or part of, and can be specifically and the MTS area hybridization.If target sequence contains the sequence identical with probe, probe can be lacked so, as is about 8-30 base-pairs, because heterozygote also is metastable under stringent condition.If the mispairing of some degree is arranged between expection and the probe, if promptly suspect probe can with the variation area hybridization, can adopt probe long, that can produce necessary specific hybrid with target sequence so.

Probe comprises the separation polynucleotide that is connected in label or reporter molecule, and can use standard method to be used to the polymerized nucleoside acid sequence that separates other, have sequence similarity.For the preparation and the mark of probe, see also Sambrook et al., 1989 or Ausubelet al., 1992.Other similar polynucleotides can be selected by the polynucleotide that uses homology.Perhaps, the encode polynucleotide of these polypeptide or similar polypeptide can be synthesized by the genetic codon that utilizes Feng Yu or be selected.Can introduce different codon displacements, for example reticent expression that changes (thereby producing different restriction enzyme sites) or optimize certain specific system.The performance of sudden change be can introduce, the affinity of part combination, affinity, polypeptide degraded or the conversion ratio (turnover rate) of interchain perhaps can be changed with modified polypeptide.

Probe of the present invention contains synthetic oligonucleotide or other polynucleotides, and they can perhaps synthesize by chemical mode derived from strand or double-stranded polynucleotide naturally occurring or reorganization.Probe can be inserted method or other methods as known in the art are carried out mark by nick translation, Klenow.

The polymerized nucleoside acid sequence of preferably selecting coding MTS for use is as probe, and this probe has at least about 8 nucleotide, normally at least about 15 nucleotide and right less than about 6 kilobase, normally less than the right polymerized nucleoside acid sequence part of about 1.0 kilobase.Probe can also be used for determining whether to exist the mRNA of MTS of encoding at cell or tissue.

For MTS polypeptide or its fragment, the present invention also provides " protein modification form or fragment ", the basic homology of they primary structure sequences, but for example comprise, body is interior or external by chemistry and biochemical modified forms, and mixes non-common amino acid whose form.These modifications comprise acetyl groupization, carboxylated, phosphorylization, glycosylation, ubiquitination (ubiquitination), as carrying out the modification of mark and various enzymes with radionuclide, these all are that those of skill in the art in this area can understand easily.Method and a large amount of different substituting group that is used for this purposes or labels of a large amount of different labeling polypeptides are that this area is known, comprising radioisotope as ³²P, part, fluorogen, chemiluminescence agent, the enzyme that can combine and the anti-part of ligand specificity that serve as a mark in conjunction with the pairing member with the anti-part (as antibody) of mark.The selection of label depend on required sensitivity, with simplicity, desired stability and the getable checkout equipment of primer coupling.The method of labeling polypeptide is to know in this area.For example referring to Sambrook et al., 1989 or Ausubel et al., 1992.

Except the polypeptide of total length basically, the present invention also provides the polypeptide fragment with biologic activity.Important biologic activity comprises the biologic activity of ligand-binding activity, immunologic competence and other MTS polypeptide.Immunologic competence comprises the immunogene function in the target immune system, and has for the immune epitope of combination with as the competitor of MTS protein epitope or replace antigen.As used herein, " epi-position (claiming antigenic determinant again) " refers to the antigenic determinant of polypeptide.Epi-position can contain three epi-positions exclusive be in amino acid in the space conformation.Usually, an epi-position is by at least 5 such amino acid, more commonly by at least 8-10 such amino acid formations.The method of determining these amino acid whose space conformations is to know in this area.

For immunologic purposes, can use polypeptide fragment that series connection repeats as antigen, thereby producing high antigenic protein.Perhaps, this polypeptide antigen is as the extremely effectively competitor of specificity combination.Production process at the antibody of MTS polypeptide or its fragment is hereinafter described specifically.

The present invention also provides the fused polypeptide that contains MTS polypeptide and fragment thereof.The polypeptide of homology is merged.Can make up the fusion of allos equally, it has the composite performance or the activity of derived protein.For example, can be between different new fused polypeptide or fragment " exchange " ligand binding domain or other domain.The fused polypeptide of this homology or allos can show for example changed in conjunction with vigor and specificity.Fusion partner comprises immunoglobulin, bacteria beta-galactosidase, trpE, a-protein, beta-lactamase, α-Dian Fenmei, alcohol dehydrogenase and yeast α conjugative element.For example referring to Godowski et al., 1988.

Typically, fusion can make with recombinant nucleic acid method (shown in hereinafter) or with chemical synthesis.The technology that is used for synthetic polypeptide is at Merrifield, description arranged in 1963.

" protein purification " refers to that these methods are to know in this area with MTS polypeptide isolated various distinct methods from the other biological material as from the nucleic acid cell transformed of using the coding MTS that recombinates.For example, can use immunoaffinity chromatography to come purified polypeptide with antibody as providing among the present invention.Various protein purification method is to know in this area, comprising at Deutscher, and 1990 and Scopes, the method described in 1982.

Term " separation ", " pure substantially " and " basic homogeneity " can exchange use, all are used to describe the protein or the polypeptide that have separated with the component of following it under native state.When the sample when about 60-75% had single peptide sequence, this monomeric protein was pure substantially.Substantially pure protein typically contains the 60-90%W/W that has an appointment, more generally about 95% and more preferably surpass about 99% protein example.The purity of protein or homogeneity can be with the method representations of knowing in multiple this area, as polyacrylamide gel electrophoresis or after gel is dyeed single polypeptide band in the observing protein sample.For some situation, can provide higher resolution to be used for purifying with the means of knowing in high performance liquid chromatography (HPLC) or other this areas.

When MTS protein separated with the natural pollutant of following it under native state, then this MTS protein just had been substantially free of with its natural relevant component.Therefore, with the synthetic polypeptide of chemical method or in the cell system different with the cell of natural this polypeptide of generation synthetic polypeptide be substantially free of natural relevant component with it.Can also use the purified technology of protein of knowing in this area, by separation protein be gone up substantially and do not contained and its natural relevant component.

As used herein, be " polypeptide that separates " as the polypeptide that produces with exercisable gene order expression product that separates, even in the cell type of homology, express.Synthetic form or be exactly the molecule that separates with the molecule of allos cellular expression itself.

" recombinant nucleic acid " is natural non-existent nucleic acid, or with the nucleic acid of two sequence fragments that separate by artificial combination formation itself.This artificial combination perhaps realizes by the nucleic acid fragment that separates is carried out manual operation (for example passing through genetic engineering technology) normally by the chemosynthesis means.Typically, when needs are introduced or removed a recognition sequence site, normally replace a certain codon with coding amino acid whose Feng Yu codon identical or conservative.Perhaps, the nucleic acid fragment with required function can be merged the required function combination of generation.

" regulating and controlling sequence " refer to those be usually located within certain locus 10Kb coding region, influence the gene expression sequence of (comprising translation, splicing, stability of gene transcription and mRNA etc.).

" basic homology or similar ".If when arranging (having suitable nucleotide inserts or lack) best with other nucleic acid (or its complementary strand), the same degree of nucleotide sequence has the nucleotide base at least about 60%, normally at least about 70% nucleotide base, more generally at least about 80%, preferably at least about 90%, during more preferably at least about 95-98% nucleotide base, we claim this nucleic acid or its fragment and another nucleic acid " basic homology " (or " similar substantially) so.

Perhaps, under the hybridization conditions that nucleic acid or its fragment are being selected can with another nucleic acid (or its complementary strand) or with a certain chain or the hybridization of its complementary strand, so just have basic homology or similarity.Have more when optionally hybridizing when taking place to lack fully, exist the selectivity of hybridization than specificity.Typically, when in one section interval, have at least about 14 nucleotide autoploidy at least about 55%, preferably at least about 65%, more preferably at least about 75%, during best at least about 90% autoploidy, can take place optionally to hybridize.Referring to Kanehisa, 1984.As described herein, the comparison length of autoploidy can be carried out at longer section.Often be at least about 9 length of nucleotides in certain embodiments, normally at least about 20 nucleotide, more generally at least about 24 nucleotide, typically at least about 28 nucleotide, more typically at least about 32 nucleotide, and more preferably at least about 36 nucleotide or more.

Except the factor affecting such as number that are subjected to nucleotide base mispairing between base composition, complementary strand length and the hybrid nucleic acid, nucleic acid hybridization also is subjected to the influence such as factors such as salinity, temperature or organic solvents, and this point is that the those of skill in the art in this area know.Tight temperature condition generally includes and surpasses 30 ℃ temperature, typically surpasses 37 ℃, more preferably above 45 ℃.Tight salinity is generally less than 1000mM, typically less than 500mM, more preferably less than 200mM.Yet the combination of these parameters is more even more important than any single parameter.For example referring to Wetmur ﹠amp; Davidson, 1968.

Probe sequence can also be hybridized with double-stranded DNA under given conditions specifically, thereby forms three strands or other more senior DNA compounds.The preparation of these probes and suitable hybridization conditions are well known in the art.

When being used for polypeptide, term " basic homology " or " basic identical " interested polypeptide of expression or protein are compared with complete naturally occurring protein or its part, identical, normally identical, more preferably identical at least about 30% at least about 95% at least about 70%.

" similar substantially function " refers to the modification of nucleic acids for wild type MTS nucleic acid or wild type MTS polypeptide or the function of modifying protein.Modified polypeptide promptly suppresses Cdk and especially suppresses Cdk4 basically with wild type MTS homologous peptide and have identical functions basically.Modified polypeptide can have different amino acid sequences and/or contain modified amino acid.Except suppressing the Cdk function, modified polypeptide can also have other performance, such as the longer half life period.The activity of the inhibition Cdk of modified polypeptide is active identical with wild type basically.Perhaps, the activity of the specific activity wild type of the inhibition Cdk of modified polypeptide is higher.Modified polypeptide is synthetic with routine techniques, perhaps produces with the modification of nucleic acids coding and with routine techniques.Modification of nucleic acids prepares with routine techniques.The nucleic acid of substantially similar wild type mts gene function can produce above-mentioned modifying protein.

Typically, the autoploidy of polypeptide is determined with sequence analysis software.For example referring to GeneticsComputer Group (University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wisconsin 53705) sequence analysis software bag (Sequence Analysis Software Package).Protein analysis software mates similar sequence by measuring various displacements, disappearance and the autoploidy of other modification aspect appointments.Typically the displacement of conservative comprises the displacement of listing in down in the group: glycine, alanine; Valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; Lysine, arginine and phenyl alanine, tyrosine.

Polypeptide " fragment ", " part " be meant at least about 5-7 continuous amino acids, normally at least about 7-9 continuous amino acids, typically at least about 9-13 continuous amino acids, best at least about one section amino acid residue of 20-30 or more continuous amino acids.

Polypeptide of the present invention, if it is soluble, can be coupled to solid phase carrier, for example NC Nitroncellulose, nylon, post choke material (as Ago-Gel (Sepharose) pearl), magnetic beads, glass fiber, plastics, metal, polymer gel, cell or other substrate.These carriers can be forms such as pearl, groove (well), gage or film.

" target region " refers to be amplified and/or detected nucleic acid region.The sequence of term " target sequence " indication will form stable heterozygote with probe or primer at desired conditions.

Unless indicate in addition, chemistry, molecular biology, microbiology, recombinant DNA, genetics and immunologic routine techniques are adopted in enforcement of the present invention.For example referring to Maniatis et al., 1982; Sambrook et al., 1989; Ausubel et al., 1992; Glover, 1985; Anand, 1992; Guthrie ﹠amp; Fink, 1991.Be used for the people's gene collection of illustrative plates and comprise the technology of human chromosome 9p collection of illustrative plates and the generality discussion of material, can be referring to White ﹠amp; The article of Lalouel (1988).Preparation nucleic acid reorganization or chemosynthesis; Carrier, conversion, host cell

A large amount of polynucleotide of the present invention can produce by duplicating in suitable host cell.Natural or the synthetic polymerized nucleoside acid fragment of the required fragment of encoding can be integrated in the reorganization polynucleotide construction, is generally the DNA construction.This construction can be introduced in protokaryon or the eukaryotic and duplicate therein.Usually, the polynucleotide construction is adapted at duplicating in unicellular host such as yeast or the bacterium, (integrates or is not integrated in the genome) but also can be introduced in the mammal or plant or other eukaryotic cell lines of cultivation.To the purification of nucleic acids that produces with the inventive method,, description is arranged in 1989 or Ausubel et al., 1992 at Sambrook et al..

Polynucleotide of the present invention can also produce with the method for chemosynthesis, for example Beaucage ﹠amp; Caruthers, 1981 described phosphoramidite methods or Matteucci etal., 1981 described triester methods, and can on oligonucleotide synthesizer that be purchased, automatic, carry out.Obtain double-stranded fragment from the single stranded product basis of chemosynthesis, promptly under appropriate condition, make two chain annealing then, perhaps use archaeal dna polymerase and suitable primer sequence to add complementary strand by synthetic complementary strand.

In order to introduce in protokaryon or the eucaryon host and the polynucleotide construction of preparation can contain the dubbing system that an energy is discerned by the host, this system comprises the polymerized nucleoside acid fragment of the required polypeptide of encoding, and preferably contains and operationally be connected in transcribing and the translation initiation regulating and controlling sequence of peptide coding fragment.Expression vector contain replication origin or self-replacation sequence (autonomously replicating sequence, ARS) and expression control sequenc, promotor, enhancer and necessary machining information site such as ribosome bind site, RNA splicing site, polyadenylation site, transcription terminator and mRNA critical sequences.Suitable words, can contain from natural MTS protein or from other acceptors or from the secretion signal of the secrete polypeptide of identical or relevant species, thereby make protein can by and/or stay cell membrane, and therefore obtain its functional topological structure or from cell, secrete away.These carriers can be with the preparation of the standard recombinant technique known in this area, for example can be referring to Sambrook et al., and 1989 or Ausubel et al., 1992.

Should select suitable promotor and other necessary carrier sequences that it can be played a role in the host.Suitable, can select for use and the natural relevant sequence of mts gene.The exercisable Sambrook et al. that is combined in of cell-line and expression vector has description in 1989 or Ausubelet al., 1992, can also be referring to Metzger et al., 1988.Many useful carrier are to know in this area, and can obtain from supplier such as Stratagene, NewEngland Biolabs, Promega Biotech etc.Promotor such as trp, lac and phage promoter, tRNA promotor and glycolytic ferment promotor can be used for prokaryotic hosts.Useful Yeast promoter comprises metallothionein (metallothionein), glycerol 3-phosphate acid kinase or other glycolytic ferments such as enolase or glyceraldehyde-3-phosphate dehydrogenase, undertake the promoter region of enzyme of utilizing maltose and galactose etc.Be suitable for the carrier of Yeast expression and promotor also at Hitzeman et al., EP 73, further describe among the 675A.Suitable non-natural mammalian promoter comprises from SV40 early stage and late promoter (Fierset al., 1978) or from the promotor of Moloney murine leukemia virus, mouse tumour virus, Avian sarcoma virus, adenovirus II, bovine papilloma virus or polyoma.In addition, thereby construction can be connected in the gene (as dihyrofolate reductase (DHFR)) that can increase can produce multi-copy gene.For suitable enhancer and other expression control sequenc, also can be referring to Enhancers and Eukaryotic Gene Expression, Cold Spring HarborPress, Cold Spring Harbor, New York (1983).

Although these expression vectors can self-replacation, they also can duplicate by using the method for knowing in this area be inserted in the genome of host cell again.

Expression and cloning vector may contain selective marker gene, and the protein of this gene code is the survival of carrier transformed host cells or grows necessary.This gene guarantees to have only the host cell of expressing this insertion to grow.The typical following protein of gene code of selecting: a) provide protein to resistances such as antibiotic or other toxicants such as ampicillin, neomycin, methotrexates; B) complementary auxotrophic protein or c) protein of the important nutrient component that does not have in the complex medium, for example gene of bacillus D-alanine racemase be provided.Select suitable selective marker gene to depend on used host cell, and various different hosts' appropriate flags gene is well known in the art.

The carrier that contains nucleic acid interested can be in in-vitro transcription, for example pass through injection (referring to T.Kubo et al. with the method for knowing then, 1988) RNA that obtains is introduced host cell, perhaps also can carrier directly be introduced host cell with the method for knowing in this area, this depends on the type of host cell.These methods comprise: electroporation; Adopt the transfection of calcium chloride, rubidium chloride, calcium phosphate, diethyl amino ethyl group (DEAE)-glucan or other materials; Microparticle bombardment (microprojectile bombardment); Lipofection (lipofection); Infect (when carrier is infectious agent, for example retroviruse genome) and additive method.Usually referring to Sambrook et al., 1989 or Ausubel et al., 1992.Can be with any method as known in the art, particularly above-mentioned method.The process of polynucleotide being introduced host cell is referred to herein as " conversion ".The cell of having introduced above-mentioned nucleic acid also comprises the offspring of this cell.

A large amount of nucleic acid of the present invention and polypeptide can be prepared by carrier or the expression of other expression vectors in compatible protokaryon or eukaryotic host cell.The most frequently used prokaryotic hosts is a coli strain, although other prokaryotes are arranged, as hay bacillus or pseudomonas (genus).

Mammal or other eukaryotic host cell as the host cell of yeast, filamentous fungi, plant, insect or amphibian or birds, also can be used to produce protein of the present invention.The cultivation propagation of mammalian cell is that everyone knows in this area.Referring to Jakoby and Pastan (editor), 1979.The example of mammalian host cell line commonly used is VERO and Hela cell Chinese hamster ovary (CHO) cell and W138, BHK and COS cell-line.But those skilled in the art know that other cell-line also is suitable, be used to provide more efficiently express, required glycosylation form or other characteristics.

According to the mode of vector construction, select the clone by marker gene.Marker gene is positioned on the identical or different dna molecular, preferably on identical dna molecular.In prokaryotic hosts, can be by to selecting transformant such as ampicillin, tetracycline or other antibiotic resistances.The specific product that produces according to temperature sensitivity also can be used as suitable label.

The protokaryon or the eukaryotic that transform with polynucleotide of the present invention not only can be used to produce nucleic acid of the present invention and polypeptide, also can be used to study the character of MTS polypeptide.

Antisense polymerized nucleoside acid sequence can be used for preventing or weakening the expression of mts gene seat, and this point is that the those of skill in the art in this area are to understand.For example, can will contain all or part of mts gene seat sequence or other polymerized nucleoside acid vectors from MTS regional sequence (especially being positioned at the sequence of mts gene seat both sides) place under the control of antisense orientation promotor, and introduce cell.In cell, the expression of this antisense construct thing can disturb MTS to transcribe and/or duplicate.

Cyclin (Cycline) and Cdk are the cell cycle regulating factors that extensively exists in the eucaryote.These protein find in yeast at first, and have comprised among mouse, rabbit and the people oceanic invertebrate, amphibian and mammal and finding.By using probe and/or primer, can in other species, identify the cell cycle control gene of homology based on a kind of species gene sequence.Therefore, the probe of mts gene sequence disclosed herein and primer can be used for mts gene sequence and protein in other species discriminating homologies.At the species of isolating these materials, available these mts gene sequences and protein carry out diagnosis/prognosis as herein described, treatment and drug screening.Using method: diagnostic nucleic acid and diagnostic kit

To make individual MTS allelomorph of easily suffering from cancerous swelling in order detecting whether to exist, can to prepare biological sample such as blood, analyze whether there is neurological susceptibility MTS allelomorph then.In order to detect the malignant development that whether has neoplasia or previous damage or the sign of diagnosis, can prepare the biological sample of damage, analyze whether to exist then and cause neoplastic MTS allelomorph.The result of these tests and explanation can be supplied with healthcare facility, thereby tell the individuality of tested person.These diagnosis can be undertaken by the diagnostic test chamber, perhaps can make diagnostic kit and sell to healthcare facility or individual for OBD.

Originally, screening technique relates to by the relevant MTS sequence of pcr amplification, carries out dna sequence analysis then.In another preferred example of the present invention, screening technique is a kind of non-PCR scheme.This screening technique comprises the two step mark amplifying techniques of knowing in this area.The screening scheme of PCR and non-PCR can both detect target sequence with very high sensitivity.

The most frequently used method is the amplification of target at present.Use the polymeric enzymatic amplification target nucleic acid sequence.A kind of amplification reaction method particularly preferred, that polymerase drives is polymerase chain reaction (PCR).A kind of preferred PCR scheme that is within the scope of the invention provides in embodiment 1.Because polymerase drives amplification cycles, so the amplification analytical method of polymerase chain reaction and the driving of other polymerases increases more than 1,000,000 times the copy number.In case be amplified, the nucleic acid that obtains can be used to check order or as the substrate of dna probe.

(for example when the neurological susceptibility of screening cancerous swelling) can be handled biological sample to be analyzed (for example blood and serum) and go out nucleic acid with extracting when using probe to detect existing of target sequence.Sample nucleic acid can be prepared with diverse ways, thereby promotes the detection of target sequence.For example sex change, restrictive diges-tion, electrophoresis or dot blot.The target region of analyzed nucleic acid must be the strand state to small part usually, so that form heterozygote with the target sequence of probe.As infructescence itself is strand, does not need sex change so.But, be double-stranded as infructescence, sequence may need sex change so.Can carry out sex change with the various technology of knowing in this area.

Impel to form between the target sequence of supposing in the target sequence of probe and the analyte in meeting and stablize under the condition of heterozygote, analyzed nucleic acid and probe are incubated.The probe area that combines with analyte can be made into the target region of human chromosome 9p complementary fully.Therefore, in order to prevent false positive, need high stringent condition.Have only the complementary time side of chromosomal region single in probe and genome to use high stringent condition.The stringency of hybridization is determined by the numerous factor in hybridization and the washing process, comprising temperature, ion strength, base composition, probe length and formamide concentration.These factors are at Maniatis et al., summary are arranged in 1982 and Sambrook etal., 1989.In some cases, the formation of more senior heterozygote such as tripolymer, the tetramer etc. also can be used as the method that detects target sequence.

If there is heterozygote, the detection of the heterozygote of Xing Chenging realizes by the usage flag probe usually so.Perhaps, probe is unlabelled, but can detect by combining specifically with the part of direct or indirect mark.Suitable label and be used for label probe and the method for part is to know in this area, comprising the radioactively labelled substance that mixes with known method (as nick translation, random priming and transphorylation method (kinasing)), vitamin h, fluorophor, chemiluminescent groups (, especially triggering the attitude dioxetane), enzyme, antibody etc. as dioxetane (dioxetane).Change under this basic fundamental framework is to know in this area, promotes heterozygote to be detected separated and/or amplify change from the signal of mark part comprising those from exogenous material.Numerous change forms are summarized in Matthews ﹠amp; Kricka, 1988; Landegren et al., 1988; Mittlin, 1989; United States Patent (USP) 4,868 is in 105 and EPO publication No.225,807.

As mentioned above, the present invention provides non-PCR screening assays suitably.Representational non-PCR program is provided in embodiment 15.In this program, with the DNA target hybridization of nucleic acid probe (or analog, for example common di-phosphate ester of usefulness methyl-phosphonate skeleton replacement) with low concentration.This probe has the enzyme that links to each other with its covalency, and the covalently bound specificity that can not disturb hybridization reaction.Then this enzyme probe-conjugate-target nucleic acid compound can separate with free probe-enzyme conjugates, adds substrate then and carries out the enzyme detection.Can increase 10 by colour developing variation or sensitivity ³-10 ⁶Fluorescence output quantity doubly and observe enzymic activity.For the preparation of oligodeoxynucleotide-alkaline phosphatase enzyme conjugates and as the purposes of hybridization probe, can be referring to Jablonski et al., 1986.

Two step mark amplifying techniques are to know in this area.These analytical methods are based on such principle: with little part (as digoxigenin (digoxigenin), vitamin h etc.) be connected in can be specifically with nucleic acid probe that MTS combines on.At the representative probe of MTS1 position corresponding to nucleotide 448-498 among the SEQ ID NO:4.Allele specific oligonucleotide probe is also among the design scope of this example, and representational allele specific oligonucleotide probe comprises and contains tendentiousness sudden change that is summarized in table 3 and the probe that is summarized in the tumour somatic mutation of table 5.

In an example, be connected in little part on the nucleic acid probe by antibody-enzyme conjugates specific recognition.In this example, digoxigenin is connected on the nucleic acid probe.Detect hybridization with the antibody that chemical luminous substrate is changed-alkaline phosphatase enzyme conjugates.The method that is used for this example amplifying nucleic acid probe of mark can be referring to Martin et al., 1990.In another example, little aglucon can be discerned by another compound with it specifically aglucon-enzyme conjugates.An interaction that well-known example is vitamin h-antibiotin in this case.The purposes that the method for labeling nucleic acid probe and they are analyzed based on vitamin h-antibiotin, referring to Rigby, et al., 1977 and Nguyen, et al. (1992).

Same is the mixture that nucleic acid probe analysis of the present invention can be adopted the nucleic acid probe that can detect mts gene among design scope of the present invention.Therefore, detect from cell sample in the example that MTS1 exists at one, adopts multiple and the probe MTS1 complementation, especially this organizes different probes and can be 2,3 or 5 kind of different nucleic acid probe sequence.In another example, in order in patient, to detect the sudden change that whether has the mts gene sequence, can use the probe of more than one and MTS1 complementation, this mixture contains the probe that can suddenly change and combine with allele-specific, and these sudden changes are to identify in the patient crowd who has the MTS1 sudden change.In this example, can use the probe of any number, and preferably include corresponding to, make individuality tend to suffer from the probe of the main gene mutation of mastocarcinoma.Candidate probe within some scope of the invention comprises the probe of the allele-specific sudden change of listing in table 3 and 5.Using method: peptide diagnosis and diagnostic kit

The change that the neoplasia situation of damage can take place according to wild type MTS polypeptide and being detected.This change can be determined by sequence analysis with conventional technology.More preferably, use antibody (polyclone or monoclone antibody) to detect the difference of MTS polypeptide or the shortage of MTS polypeptide.In a preferred embodiment of the invention, antibody goes out MTS protein immunoprecipitation from solution, and in the Western of polyacrylamide gel trace or Western blotting with the MTS proteins react.In another preferred embodiment, by using immunocytochemical technique, antibody can detect MTS protein from paraffin or frozen tissue section.The technology that is used for obtaining with antibody purification is well-known in this area, thereby can select for use arbitrary such technology to realize preparation of the present invention.

The preferred example that detects MTS or its mutation method comprises enzyme linked immunosorbent assay (ELISA) (enzyme linked immunosorbent assays, ELISA), radiommunoassay (RIA), immunoradiometric assay (IRMA) and IEA (IEMA), comprise the sandwich assay that is used for monoclonal and/or polyclonal antibody.The sandwich assay example is in people's such as David U.S. Patent No. 4,376,110 and 4,486, description (these documents are incorporated herein by reference) arranged in 530, and is example with embodiment 18.Using method: drug screening

The present invention is particularly useful for screening compounds, promptly passes through to use Cdk polypeptide or its binding fragment and screening compounds in various drug screening technologies.Preferably use Cdk4.The Cdk polypeptide that uses in test or its fragment can be in the free state in the solution, perhaps are fixed in certain solid phase carrier, perhaps are positioned at cell surface.A kind of drug screening method preferably in competition in conjunction with in measuring, the protokaryon or the eukaryotic host cell that adopt reorganization polynucleotide with express polypeptide or its fragment stably to transform.This cell perhaps is in free state or is in fixed form, may be used in the standard binding analysis.For example people can measure between Cdk polypeptide or its fragment and tested reagent whether form compound, perhaps detect the degree that the compound that forms between Cdk polypeptide or its fragment and MTS polypeptide or its fragment is disturbed by reagent that tested.

Therefore, the invention provides the method for screening of medicaments, it comprises certain reagent is contacted with Cdk polypeptide or its fragment, measures with the method for knowing in this area then: 1) whether have the compound or 2 that is formed by this reagent and Cdk polypeptide or its fragment) whether there is a compound that forms by Cdk polypeptide or its fragment and aglucon.Can also measure the activity of Cdk, to determine whether this reagent can suppress Cdk, promptly whether can regulate the cell cycle.In this competitive binding assay, Cdk polypeptide or its fragment are labeled.With free Cdk polypeptide or its fragment from protein: separate the protein complex, the amount of free (promptly not compound) label just is respectively the measured value that tested reagent is incorporated into Cdk, or it disturbs the measured value of Cdk:MTS polypeptide combination.Can so that determining, which have Cdk and suppress active by the little peptide (peptide mimics) of this methods analyst MTS polypeptide.

The method of another kind of drug screening can have the compound of binding affinity that comprehensive screening is provided with the Cdk polypeptide for suitable, and this method (publication on September 13rd, 1984) in the european patent application No.84/03664 of Geysen has detailed description.In brief, synthetic a large amount of different little peptide test compounds on solid-phase matrix such as plastics pin or other surfaces are then with peptide test compounds and reaction of Cdk polypeptide and washing.Then detect the Cdk polypeptide of combination with the method for knowing in this area.

The Cdk of purifying can directly be coated in dull and stereotyped going up for use in above-mentioned drug screening technology.But, also can use and catch the antibody that is fixed in Cdk polypeptide on the solid phase at the nonneutralizing antibody of polypeptide.

The present invention also provides the purposes of competitive drug screening assay determination method.Wherein can compete in conjunction with neutralizing antibody and the test compounds of Cdk specifically, contention combines with Cdk polypeptide or its fragment.In this method, can use antibody to detect to have any peptide of one or more Cdk polypeptide antigen determinants.

The technology of another kind of drug screening relates to use and has the eukaryotic host cell or the cell-line (for example above-mentioned) of no function mts gene.There is defective on these host cell systems or the Cdk level of cell in cell cycle regulating.Under the medical compounds existence condition, these host cell systems or cell growth.The growth rate of measuring host cell is to determine whether compound can cell cycle regulation.A kind of method of measuring growth rate is to measure Cdk, the biologic activity of Cdk4 preferably.Using method: reasonably drug design

Reasonably the purpose of drug design is to produce interested analogue with biologically active polypeptides, or with the small molecule structure analog (for example agonist, antagonist, inhibitor) of its reaction, so that the medicine of designing is active higher or more stable polypeptide, perhaps this medicine can strengthen or disturb peptide function in vivo.For example referring to Hodgson, 1991.In one approach, people to pass through the X-ray diffraction Laue method earlier, by computer model design or the most typically determine the three-dimensional structure of proteins of interest matter (for example, p16 or Cdk4) or Cdk16-p16 compound by multimedia combination.Can obtain the uncommon useful information of relevant certain peptide structure by modelling based on the homologous protein structure.Reasonably an example of drug design is the exploitation (Erickson et al., 1990) of human immunodeficiency virus (HIV) protease inhibitors.In addition, can use alanine scanning method (Wells, 1991) to analyze peptide (as p16 or Cdk4).In this technology,, determine the influence of this displacement then to the peptide activity with Ala replacement amino acid residue.Each amino acid residue of peptide is all analyzed in this way, to determine the important area of peptide.

By the selection of functional examination, can also separate the target specific antibody, untie its crystal structure then.Theoretically, this method can obtain a drug core (pharmacore), and drug design subsequently can be based on this.By producing, just might walk around the protein crystal analysis at the anti-id AB (anti-id) that antibody function, that pharmacologic activity is arranged is arranged.As the mirror image of a mirror image, the binding site that can estimate anti-id AB is the analog of initial acceptor.Then, anti-id AB can be used for differentiating and isolate required peptide from the peptide library of chemistry or biological method generation.At this moment, the peptide of selecting can be used as drug core.

Therefore, people can design the higher or more stable medicine of MTS activity, perhaps can design the medicine as MTS activity inhibitor, agonist, antagonist etc.Because existing clone's MTS sequence, thus the MTS polypeptide of q.s can be produced, so that carry out analysis such as X-ray diffraction crystal.In addition, the knowledge of MTS protein sequence provided herein adopts computer models design to replace the people of X-ray diffraction crystal to those and those use the people of these two kinds of methods to play directive function.Using method: gene therapy

According to the present invention, provide to carrying the allelic cell of saltant MTS wild type MTS is provided the method for function.Provide a kind of like this function can suppress the tumor growth of recipient cell.The wild type mts gene or the portion gene that are arranged in carrier can be introduced cell, the gene of introducing is in outside the chromosome.In this case, express outside the chromosome of gene in cell.If portion gene is introduced into and express in carrying the allelic cell of saltant MTS, then portion gene should be able to be encoded and be made cell carry out the required part MTS protein of non-neoplasia growth.Such a case more preferably: wild type mts gene or its part be introduced into mutant cell and and cell in the endogenous saltant mts gene that exists recombinate.Dual group of incident need take place in this reorganization, thereby causes the correction of mts gene sudden change.Thereby be used for the gene introducing being recombinated or maintaining extrachromosomal carrier, be to know in this area, and can use any suitable carriers.DNA is introduced the method for cell, and for example electroporation, coprecipitation of calcium phosphate and virus transduction all are to know in this area, and the selection of method is that those skilled in the art can both accomplish.Can be with wild type mts gene cell transformed as studying the model system that cancerous swelling disappears and studies this pharmacotherapy of disappearing of promotion.

As mentioned above, can in gene therapy method, adopt mts gene or its fragment (where applicable), so that increase the quantity of this gene expression product in the cancerous swelling cell.This gene therapy is specially adapted to cancerous swelling cell and preceding cancerous swelling (pre-cancerous) cell, because compare with normal cell, the level of MTS polypeptide descends or shortage in this cell.This gene therapy can also be used for increasing at other tumour cells the expression of mts gene, and mutated genes is expressed by " normally " level in these cells, but gene outcome does not but have function fully.

Gene therapy can be carried out with generally accepted method, for example the method for Friedman described in Therapy For Genetic Disease (T.Friedman, ed., OxfordUniversity Press (1991), pp.105-121).Can analyze with above-mentioned diagnostic method earlier from the patient's tumor cell, to determine the producing MTS polypeptide in the tumour cell.Preparation contains the virus or the plasmid vector of the mts gene copy that is connected in expression regulation element and can duplicates in tumour cell then.The carrier that is suitable for is to know in this area, for example at United States Patent (USP) 5,252,479 and the application WO 93/07282 that publishes of PCT in disclosed.Then vector injection can be gone into patient, or carry out at knub position partly, or general is carried out (in order to enter the tumour cell that may be transferred to other positions).If the gene of transfection permanently is not integrated into the genome of each target tumour cell, need so to treat termly.Because MTS polypeptide and the regulation and control of cell cycle are closely related, therefore preferably mts gene and the controlling element of himself are introduced together, thereby avoided taking in the constitutive expression of MTS polypeptide in the cell of this gene at all.

Gene transfer system as known in the art may be used to as implementing gene therapy method of the present invention.Comprise virus and non-viral transfer method.A large amount of virus is as gene transfer vector, comprising papovavirus (as SV40, Madzak et al., 1992), adenovirus (Berkner, 1992; Berkner et al., 1988; Gorziglia andKapikian, 1992; Quantin et al., 1992; Rosenfeld 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 virus (Margolskee, 1992 that, comprise herpes simplex virus (HSV) and Epstein-Barr virus (EBV); Johnson et al., 1992; Fink et al., 1992; Breakfield and Geller, 1987; Freese et al., 1990) and from birds (Brandyopadhyay and Temin, 1984; Petropoulos et al., 1992), mouse (Miller, 1992; Miller et al., 1985; Sorge et al., 1984; Mann andBaltimore, 1985; Miller et al., 1988) and people (Shimada et al., 1991; Helseth et al., 1990; Page et al 1990; Buchschacher andPanganiban, 1992) retroviruse.Most human gene therapy's scheme is based on the mouse retroviruse of attenuation.

Non-viral gene transfer method as known in the art comprises chemical method, for example coprecipitation of calcium phosphate (Graham and van der Eb, 1973; Pellicer et al., 1980); Mechanical means, for example microinjection (Anderson et al., 1980; Gordon et al., 1980; Brinster et al., 1981; Constantini and Lacy, 1981); The film that is undertaken by liposome merges mediation transfer (Felgner et al., 1987; Wang andHuang, 1989; Kaneda et al., 1989; Stewart et al., 1992; Nabel etal., 1990; Lim et al., 1992) and direct DNA takes in and receptor-mediated DNA shifts (Wolff et al., 1990; Wu et al., 1991; Zenke et al., 1990; Wu et al., 1989b; Wolff et al., 1991; Wagner et al., 1990; Wagner et al., 1991; Cotten et al., 1990; Curiel et al., 1991a; Curiel et al., 1991b).Virus-mediated gene transfer can be sent to pass and directly carry out vivo gene transfer and use with liposome, makes this transfer instruct viral vectors to enter tumour cell rather than not somatoblast on every side.Perhaps, the cell-line that produces retrovirus vector can be injected into tumour (Culver et al., 1992), the injection of this cell can provide the carrier granular source continuously.This technology has been approved for suffers from the brain tumor patient that can not undergo surgery.

In conjunction with biology and physics gene transfer method, with the plasmid DNA of any size with the special polylysine coupling antibody of adenovirus hexon is mixed, the compound of formation is connected in adenovirus vector.Use this three molecular complex infection cells then.Before double-stranded DNA destroys, adenovirus vector combination effectively, internalization (internalization) and degraded endosome (endosome).

Show that liposome/DNA compound can mediate direct vivo gene transfer.Although for the liposome of standard, the gene transfer process is nonspecific, and report has been arranged, and in direct original position application process (Nabel 1992) afterwards, takes in there is local body at the tumor focus position and expresses.Using method: peptide treatment

Peptide with MTS activity can be offered and carry saltant MTS allelomorph or lack the allelic cell of MTS.Herein disclosed is the sequence (SEQ ID NO:2, SEQ ID NO:14 and SEQ ID NO:16) of MTS protein.Use known expression vector, the expression by the cDNA sequence in the bacterium produces protein.Perhaps, the MTS polypeptide can go out from the mammalian cell that produces MTS in extracting.In addition, can use chemical synthesising technology to synthesize the wherein any technology of MTS protein can both provide and contain MTS protein, preparation of the present invention.Said preparation is substantially free of the human protein of other kinds.By in microorganism or external synthetic, can realize this purpose easily by the utmost point.

Can or use liposome that active MTS molecule is introduced cell by microinjection.Perhaps, some bioactive molecule can be taken on one's own initiative or by diffusion by cell.Use the mts gene product in the extracellular and be enough to influence growth of tumor.Provide and have the MTS bioactive molecule and can part reverse the neoplasia state.Also can use other molecules (for example peptide, medicine or organic compound) to realize this reverse with MTS activity.Can also use modified polypeptide to carry out the peptide treatment with basic similar functions.Using method: host transformed

Similarly, carry allelic cell of saltant MTS and animal and can be used as model system, may become the material of therapeutic agent with research and test.Typically, these cells are epithelial cells of cultivating.These cells can separate from have the individuality that somatic cell or kind are the MTS sudden change and get.Perhaps as mentioned above, can pair cell system carry out engineeredly, make it to carry the allelic sudden change of MTS.Testing drug is applied to after the cell, and the neoplasia of measuring cell transforms phenotype.Any proterties to the neoplasia transformant can be assessed, comprising anchorage independent growth (anchorage-independent growth), in nude mice oncogenicity, pair cell invasion and to the dependence of growth factor.Wherein the mensuration of any proterties is to know in this area.

After all animals being carried out mutagenesis or germ line cell or zygote handled, can select to be used to test the animal of therapeutic agent.These processing comprise inserts saltant MTS allelomorph (usually from another kind of animal) and inserts the homologous gene that destroys.Perhaps, can use routine techniques (Capecchi, 1989; Valancius and Smithies, 1991; Hasty et al.m 1991; Shinkai et al., 1992; Mombaerts et al., 1992; Philpott et al., 1992; Snouwaert et al., 1992; Donehower et al., 1992) destroy the endogenous mts gene of animal by insertion or deletion mutation or other hereditary change.After test substances is applied to animal, must the assessment growth of tumor.If test substances prevents or suppresses growth of tumor that this test substances is the candidate therapeutic agent that is used for the treatment of cancerous swelling described herein so.

The present invention sets forth in conjunction with the following example.These embodiment only are used to set forth the present invention, and do not limit the present invention in any way.What adopt is standard technique of knowing in this area or the technology that specifies hereinafter.Embodiment 1 material and method

The A.MTS pedigree

Figure 1A-1D has shown kindred 3137,3161,3355 and 1771 respectively.A situation arises to have shown cancerous swelling among these kindreds in the drawings.Melanoma all among the kindred 3137 are all carried the neurological susceptibility haplotype, and other carry the cancerous swelling of neurological susceptibility haplotype but also marked this kindred.All melanoma in kindred 3161 and 3355 are all carried the neurological susceptibility haplotype.Identify the sudden change of MTS in the cancerous swelling in kindred 1771.

B. tumor cell line

Obtain 76 melanoma cell series from Ludwig Institute for Cancer Research, Memorial Sloan-Kettering Cancer Center, obtain 8 melanoma cell series and 5 non-melanoma cell series from American type culture collection (ATCC).

C. the preparation of tumor cell line DNA and analysis

DNA isolation process from cell-line: to 3 milliliters of lysis buffer (0.1 MNaCl; 0.1M TrisHCl pH 8.0; 5mM EDTA; Add about 1 * 10 0.5%SDS) ⁷Cell, vortex vibrates and is incubated 30 minutes at 65 ℃ then.Add 0.5ml8M KOAc, mixed reactant is incubated 30 minutes then on ice.After centrifugal (10,000 * g, 5 minutes), with isopyknic 95% precipitation with alcohol supernatant, and centrifugal once more (10,000 * g, 15 minutes).DNA is suspended in the 50-200 ml water again.

The D.PCR reaction

50ng template and every kind of Oligonucleolide primers of 30pmol are added in the 20ml reactant mixture, and this mixture contains 0.1 mM dNTP, 10mM Tris-HCl (pH8.3), 50mM KCl, 2mM MgCl ₂, 0.01% gelatin and 1 Amplitaq of unit polymerase (Perkin-Elmer).Sample carries out 35 circulations by 94 ℃ 10 seconds, 55 ℃ 10 seconds and 72 ℃ of orders of 10 seconds on Perkin-Elmer 9600 thermal cyclers.Observe product subsequently by 1.5% agarose (SeaKem) or 3: 1 agaroses (FMC BioProducts) of 3%NuSieve electrophoresis, and with ethidium bromide staining.

E.YAC

Use above-mentioned PCR condition, with IFNA, D9S171 and D9S126 screening CEPHYAC library, obtain to contain the MTS zone marker the yeast artificial chromosome (Yeastartificial chromosomes, YAC).The yeast strain that contains YAC is 30 ℃, (10g/l caseic hydrolysate-acid of AHV medium; 1.7g/l yeast nitrogenous base; 5g/l ammonium sulfate; 20mg/l hemisulfic acid adenine; 2% glucose; PH=5.8) grew 3 days by acutely shaking in.Press Ausubel et al., 1992 described methods prepare cerevisiae dna.

F. the structure of phage library

Contain the pastoris genomic dna BamHI catapepsis of YAC DNA, use T4 dna ligase (Boehringer-Mannheim) to be inserted into the EMBL3 bacteriophage arm (Promega) of BamHI digestion then, carry out external the packing with Gigapack II extract (Stratagene).Phage is grown on the coli strain C600.By with ³²The people C of P mark ₀The hybridization of t-1DNA (GIBCO-BRL) identifies the recombinant phage that contains people DNA.Screen with the PCR fragment that contains YAC left arm or right arm sequence, identify the phage (terminal clone) that contains the human sequence who is connected in yac vector.Hybridization and washing are carried out under standard conditions (Middleton et al., 1991).The picking positive plaque is by dull and stereotyped purifying 3 times.With Qiaex post (Qiagen) preparation phage DNA.

G. cosmid library construction

Partly digest the pastoris genomic dna that contains YAC DNA with Sau3A, separate each component then by size on linear (10-40%) saccharose gradient, as Maniatis et al., 1982 is described.Explanation by manufacturer prepares SuperCos 1 viscous plasmid vector (Stratagene), and (insertion was sub: carrier) mix, handle with ligase, and pack external as mentioned above in 4: 1 by mass ratio with inserting sub-DNA then.Cosmid is introduced DH5 α host cell, carry out flat board by 2000 clones' of each 15 centimetres of culture dishes cultivation density then and cultivate.As mentioned above with as Maniatis et al., 1982 describedly carry out colony hybridization.

The H.P1 clone

Screen by the STS with the methods described herein preparation, from GenomeSystems, (St.Louis Missouri) obtains to clone across the P1 in MTS zone Inc..Use alkaline lysis, use caesium chloride density gradient centrifugation technology (Maniatis et al., 1982) from these clones, to isolate DNA (Birnboim and Doly, 1979) then,

The generation of I.STS

With with the oligonucleotides of the cloning site both sides sequence complementation of P1 carrier (pSacBII), SuperCos 1 carrier or EMBL3 carrier, to 1.0mg P1, cosmid or template DNA order-checking, obtain STS.Order-checking is carried out with PRISM fast reaction dyestuff deoxidation terminator cycle sequencing kit (PRISM Ready ReactionDyeDeoxy Terminator Cycle Sequencing Kit (ABI)) on ABI 373A dna sequencing instrument.STS is designed to length as far as possible near 20 base-pairs, and Tm is as far as possible near 60 ℃.

J. the germ line mutation of MTS1 among the melanoma tendentiousness kindred

The genomic DNA that from blood, prepares carrier's individuality with standard method.Primer be designed in the introne position in case from MTS1 amplification coding exons 1 or 2, perhaps from MTS2, amplify coding exon 2 (using 20ng DNA) for each sample.Is 5% except adding dimethyl sulfoxide (DMSO) (DMSO) to final concentration, and standard buffer solution is used in the PCR reaction.α-p is used in the cycle sequencing reaction ³²-dATP carries out the product that gets with the primer amplification that is arranged in the sequence diverse location.The order-checking product is analyzed on 6% denaturing polyacrylamide gel, and sample is gone up in all (A) reactions together, is (C) reaction or the like then.All polymorphisms can be determined by the sequence analysis of relative chain.Embodiment 2 usefulness genetic linkage marks are determined the position of MTS

In order to analyze the homozygous deletion that tumour cell ties up to the 9p21 zone, adopt the known and chain mark of MTS of a cover.These marks are used for showing significantly chain (LOD value=12.7) be inclined to melanoma at first in the kindred of 10 Utah States and 1 Texas kindred (Cannon-Albright et al., 1992).These marks comprise from alpha-interferon genes bunch (IFNA) (Kwiatkowski ﹠amp; Diaz, 1992) sequence, proximal marker (D9S104) and other 4 marks (D9S171, D9S126, D9S161 and D9S169) (Cannon-Albright et al., 1992) that are positioned at wherein of (it is the distal-most end mark of having tested).According to genetic research, the linear precedence of these intervenient marks is: D9S171, D9S126, D9S161, D9S169.The IFNA mark is by amplifying the Oligonucleolide primers of two fragments to constituting from wild type gene group DNA, these two fragments are: one is the polymorphic bands (IFNA-1) that contains poly CA section, about 138-150 base-pair, and another is the constant fragment (IFNA-s) of about 120 base-pairs.IFNA-s is not clear with respect to the position of IFNA-1.

5 non-melanoma tumor cell that contain disappearance of previous report are to analyze with genetic marker.The result discloses, and has at least a kind of mark of testing to have homozygous deletion (table 1) in each cell-line.Do not identify homozygous deletion when using D9S161, D9S169 or D9S104.Overlapping Minimum Area is defined by IFNA-1 and D9S171 in these disappearances.This hint, the gene that relates to tumor suppression is contained in the zone between these two marker gene, may be MTS.Therefore then study near the zone of genome area, especially IFNA-s between IFNA-1 and the D9S171 in great detail.

Table 1

With detecting homozygous deletion in the tumor cell line with the chain genetic marker of MTS

Mark tumour IFNA-1 IFNA-2 D9S171 D9S126 D9S161 D9S169 D9S104 clone U-138----+++U-118----+++U-87--+++++A-172+-+++++H4----+++to annotate: all clones derive from ATCC, are glioma or neuroblastoma. The genomic clone in embodiment 3MTS zone

In order to obtain the genomic clone of IFNA-s peripheral region, (Cohen et al., 1993) are screened in CEPH YAC library.Identify 11 YAC and 5 YAC that contain IFNA-s that contain the D9S171 mark.Do not isolate the YAC (Fig. 2) that contains D9S171 and IFNA-s simultaneously.3 yac clones (C9, C6, F9) subclone is gone into phage, another YAC (C6) subclone is gone into viscous plasmid vector.These steps and other cosmids clone provide a kind of approach easily to produce the STS that is positioned at known genetic marker inside, and have accelerated following chromosome walking.

In order to be provided for making up the independent source of the required genomic DNA of the continuous gene picture group in this zone, and, can in the P1 clone, begin to extend, extend and extend and the beginning chromosome walking to both direction to IFNA-s in reverse direction from YAC C6 end from D9S171 to D9S171 from IFNA-s for assist in generating STS.As the part of this chromosome walking, isolate 27 P1 clones (Fig. 2) altogether.That these P1 that arrange have in order formed is continuous, extend to D9S171 and have the assembly (assembly) of two breach from IFNA-s.P1 clone and several phage and cosmid clone are used to produce the fine structure map in MTS zone.The fine-structure distribution in embodiment 4MTS zone

In order to make up more detailed MTS zone Molecular Graphs, also need extra mark.Use the PCR primer that is designed for STS from the dna sequence dna of genomic clone.These STS are used for assisting P1 and yac clone are carried out the order arrangement.54 STS of total from zone between IFNA-s and the D9S171 are main bases (Fig. 2) of setting up detailed MTS zone physical map.These STS primer sequences are transfused to Genome Database.

Be used to test 84 melanoma cell series from IFNA-s to the new mark of a cover that D9S171 extends, to determine whether homozygous deletion is arranged in the MTS zone.Amount to 52 cell-lines homozygous deletion zone (Fig. 3) is arranged.Several disappearances are on a large scale, and for example the zone of 13 cell-lines disappearance comprises 816.7 and 760-L.

For MTS is positioned, can provide the tumor cell line of information to be divided into two groups (Fig. 3): i) to contain the cell-line (11 class) of independent c5.1 disappearance and ii) contain the cell-line (12 class) that independent c5.3 lacks.Add up to 5 melanoma cell series and belong to these two groups.Detect in the situation of disappearance at all, the seemingly simple disappearance of disappearance there is no indication that promptly multiple disappearance incident takes place the zone between IFNA-s and D9S171.It is the disappearance overlapping region at center with mark c5.1 and c5.3 that these cell-lines of carrying disappearance have been described one altogether, thereby needn't be to making complete physical map from IFNA-s to the zone of D9S171.Embodiment 5 differentiates that cosmid c5 and P1 contain bacterium colony P1062 and the P1063 of MTS

A. the layout of genetic marker

To the analysis of yac clone and to the analysis that lacks in the tumor cell line and the result consistent with the layout of genetic marker: IFNA, D9S171, D9S126.3 YAC contain D9S171 and D9S126, and 4 YAC contain IFNA-1 and IFNA-s (Fig. 2).Neither one YAC contains D9S171 and IFNA simultaneously.This hint: i) IFNA-1 and IFNA-s close linkage and ii) D9S126 and D9S171 are chain.These results can confirm with the disappearance of cell-line.The cell-line of great majority disappearance D9S171 also lacks D9S126.On the contrary, cell-line U-87 although D9S171 and D9S126 are positive, lacks IFNA-s and IFNA-1 (table 1).A melanoma cell series is SK-MEL-5 disappearance IFNA-s, D9S171 and D9S126, but does not lack IFNA-1.Therefore, IFNA-1 must be positioned at the far-end of IFNA-s.Another melanoma cell series SK-Mel-Zan disappearance IFNA-1, IFNA-s and D9S171, but do not lack D9S126, so D9S171 is between IFNA-s and D9S126.In a word, these discoveries are supported in the order arrangement of the mark that provides in the table 1.

Human's gene family is by being positioned at constituting above 23 genes and pseudogene on the chromosome 9p.This gene cluster has been cloned and has been divided into 10 chain group (Henco etal., 1985).By disappearance loss assays, partial ordered (Olopade et al., 1992) have been carried out to these chain group to the different sequence of alpha-interferon in the glioma cell line.Glioma cell line H14 disappearance IFNA-1 and IFNA-s.It also lacks the sequence of chain group of IV (for example α 13, α 6 and α 20).Glioma cell line A172 contains IFNA-1 and chain group of IV, but lacks chain group of I (for example α 1 and α 19), III (for example α 8) and IX (for example α 2) and IFNA-s.This analysis result places IFNA-1 the far-end of chain group of I, III and IX and IFNA-s.The far-end border of A172 disappearance is set within the P1 length of IFNA-s far-end.Therefore, chain group of I, III and IX are inevitable near being positioned at from IFNA-s far-end certain place a bit less than 85kb.

B. the physical distance between the genetic marker

The above results can't be made accurately the distance between IFNA-s and the D9S171 and being estimated, because can not isolate the YAC that contains these two marker gene.In addition, according to the collection of illustrative plates of making of STS, from IFNA-s to 5 YAC that far-end extends neither one YAC with overlap to 11 YAC of proximal extension from D9S171.The average length of inserting son because of CEPHYAC is within 500kb, so that the distance between IFNA-s and the D9S171 should have at least is so big.

Zone between IFNA-s and the c5.1 is covered by 9 step sections of moving one's steps among the P1 clone.Suppose that each step section average out to P1 inserts half length of son, the distance between c5.1 and the IFNA-s is about 400kb so.Therefore, in melanoma cell series often disappearance, must be positioned at the about 400kb of IFNA-s near-end with the closely linked tumor suppressor gene of c5.1.

C. the disappearance of tumor cell line

In the melanoma tumour of 57% test, found the homozygous deletion in 9p21 zone.14 tumor cell lines contain the disappearance that extends through 760-L in near-end one side, and 16 cell-lines contain and extend beyond the disappearance that is positioned at far-end one side 816.7.Suppose that disappearance is a cause, promptly cause the phenotype of tumour in the disappearance of this regional gene, tumor suppressor gene is inevitable between 760-L and 816.7 so.Minimum disappearance relates to mark c5.1 and c5.3.In the mark of all tests, the disappearance of c5.3 is arranged in a lot of cell-lines (51).Therefore, the most probable position of tumor suppressor gene is very near C5.3, because C5.3 lacks the most frequent mark.4 cell-lines contain independent C5.3 disappearance (12 class), and a cell-line lacks C5.1 (11 class) separately.These two marks all are present on the same cosmid c5.Therefore, might tumor suppressor gene comprise sequence from cosmid c5.P1 clone P1062 and P1063 contain the sequence of finding and center on cosmid in c5.Therefore, as further described below, P1062 and P1063 contain complete MTS zone.

Result in the foregoing description was with in the past consistent to the genetic research of MTS, and former studies show that, the zone between IFNA-1 and D9S126 is the position (Cannon-Albright et al., 1992) that the MTS most probable exists.Nearest genetic research has utilized the polymorphism (CA) between IFNA-2 on the P1-452 and C5.3 to repeat to limit the position (Fig. 2) of MTS.Use this mark to carry out the recombinant chromosome analysis, the result places the P1-453 near-end with MTS.Therefore, MTS is located in the zone of homozygous deletion in melanoma cell series bunch.

These results support such viewpoint: have TIF locus MTS in the somewhere near C5.3.All cell-lines that contain disappearance all have common disappearance DNA zone, are defined to the cell-line (11 and 12 class) of C5.1 or C5.3 except those disappearances.Except those are arranged in the non-overlapped disappearance of cosmid c5, do not have evidence to show in this group cell-line and have non-overlapped disappearance.Therefore, have no reason to propose more complicated mechanism, for example relate to away from another of C5.1 and C5.3 and be positioned at the mechanism of the TIF locus of 9p21.

Have this observed result hint of homozygous deletion of 9p21 in many tumor types, the tumor suppressor gene that is arranged in the there may be expressed at multiple different tissue.Therefore, on the development process this point that participates in the polytype tumour, tumor suppressor gene may be similar (referring to following further data) with the p53 gene.Cancerous swelling (Nancarrow et al., 1993 of other types in family, had been reported with melanoma tendency; Bergman et al., 1990).With C5.1 and C5.3 various cancerous swelling type is carried out deletion analysis (seeing below) completely, illustrated the importance of this tumor suppressor gene in the tumour except that melanoma.

Some observation on to homozygous deletion removed many genetic markers.People such as Fountain report, the homozygous deletion of chromosome 9p21 has extended 2-3Mb (Bergman et al., 1990) in two different melanoma cell series.In this research, at least one cell-line SK-MEL-5 contains from the distal-most end mark IFNA-1 beginning of test and the disappearance by D9S126, and this zone is obviously too greatly so that can not be present on the single YAC.The big disappearance hint of preponderating is at the MTS peripheral region shortage pair cell necessary gene of surviving.The separation of embodiment 6MTS candidate gene

In the above embodiments, near the result that YAC and P1 chromosome walking analyze MTS has been described.It is bright to carry out fine structure collection of illustrative plates test card with the STS that derives from the c5 sequence, has the disappearance of little, non-overlapped c5 sequence in 5 different melanoma cell series.This result shows that possible tumor suppressor gene (may be MTS) to small part is arranged in c5.

Show that further c5 contains the evidence of at least one gene from the analysis to (CpG) dinucleotide frequency in c5 and near the cosmid.In mammal, nearly all daily gene and all tissue-specific genes of nearly half and unusual abundant zone relevant (Bird, 1989 of (CpG) dinucleotides; Larsen et al., 1992).Therefore, exist this " CpG island " just to show and have gene.Restriction endonuclease EagI, the BssHI and SacII digestion cosmid c5, c12, c57 and the c59 that contain two (CpG) matched sequences with identification.Have only cosmid c5 and c12 to contain the recognition site of these enzymes.Cosmid c5 contains an EagI site, at least 10 BssHI sites and at least 12 SacII sites.Have CpG island hint in c5 and overlapping cosmid c12, in fact c5 contains at least one MTS candidate gene.

In order to seek MTS, determine dna sequence dna from the EcoRI fragment of cosmid c5.When the sequence with these sequences and GenBank compares, identify two distinguished c5 zones, they and kinases 4 (cyclin-dependent kinase 4 that identified in the past, coding people cyclin dependent, Cdk4) regional similar (Serrano et al., 1993) of inhibitor or p16 gene.These two candidate genes that gene is MTS are called MTS1 and MTS2.MTS1 is positioned near the end of the most close chromosome 9p of cosmid c5 telomere, and MTS2 is positioned near the end of c5 kinetochore.See Fig. 4 B.The cosmid figure that provides among Fig. 4 A has shown the position of MTS1 and MTS2 and P1 clone P1062, P1063 and P1069.

From carefully relatively disclosing of the MTS1 genome sequence of c5 and p16mRNA sequence, MTS1 contain one section 307bp, with the identical sequence of part p16 coded sequence.The both sides of this MTS1 nucleotide section are discernible splicing sequence.To further studies show that of MTS1, it comprises complete p16 coded sequence and two introns (Fig. 5 A and 5B and Fig. 6 A and 6B).Introne 1 is positioned at 126bp place, downstream, translation initiation position; Intron 2 is positioned at translation termination position upstream 11bp place.These two introns are divided into the 5 ＇ zones (coding exons 1) of 3 zone: 126bp, the zone line (coding exon 2) of 307bp and the 3 ＇ zones (coding exon 3) of 11bp with the MTS1 coded sequence.SEQ IDNO:3 is the nucleotide sequence of 5 ＇ zones, exons 1 and the part introne 1 of MTS1.SEQ ID NO:4 is the nucleotide sequence of part introne 1, exon 2 and the part intron 2 of MTS1.

MTS2 contains almost the dna sequence dna zone identical with p16, and it extends about 211bp (Fig. 7 A) from the 5 ＇ end of coding exon 2 to intron 2.Yet sequence similarity descends at 51bp place, MTS1 intron upstream, and this place is corresponding to the position (Fig. 8) at the last codon place of MTS2.The sequence of MTS1 and MTS2 relatively (Fig. 8) shows that intergenic sequence similarity upstream extends about 40 nucleotide from the 3 ＇ splice point that contain son 1.Therefore, noncoding DNA is partly more conservative than the coding DNA zone of some supposition.In order to get rid of sequence divergence in the coding DNA is because clone's this possibility that illusion caused, and design can amplify the PCR primer of crossing over MTS2 sequence divergence point specifically.These primers amplify the fragment of pre-sizing from cosmid, P1 and genomic DNA.Therefore, the divergent sequence of 3 ＇ of close MTS2 exon 2 end is genome sequence really.SEQ ID NO:5 has listed the part introne 1 of MTS2, the nucleotide sequence of " exon 2 " and " intron 2 ".SEQ ID NO:15 has listed the cDNA sequence of MTS2.

In cosmid c5, there are two gene hints that are closely related, may have other related genes in this zone.In order to confirm this possibility, with cosmid c5, c12, c59 and P1 1063 and 1060 and human gene group DNA's digestion with restriction enzyme product carry out the Southern trace.Survey these traces with the fragment that contains most of MTS1 exon 2 (comprising the zone total) with .MTS2.In cloned DNA and genomic DNA with probe in detecting to two an EcoRI fragment.This result with have two p16 genoids at genome, promptly MTS1 and MTS2 are consistent.Also with at present the known MTS1E1 β that exists is consistent, and MTS1E1 β is the another kind of form of MTS1, and the exon 2 and 3 that it contains MTS1 does not still contain exons 1.The separation of embodiment 7MTS1E1 β and structure MTS1E1 β separate

As probe and the conventional cDNA library screening technology of use, select to isolate the clone who contains MTS1E1 β with complete MTS1 cDNA by hybridization.Conventional cDNA library screening is to carry out with the probe that derives from the MTS1 exon 2.To from fetal brain, normal breast and lymphocytic each library, screen 1,000,000 clones.From the lymphocyte library, isolate the cDNA clone of hybridization.The clone shows through order-checking and contains E1 β.It also contains exon 2 (E2) and the exon 3 (E3) of MTS1.Cultivate together by the cDNA and the cosmid c5 that will derive from ovary tissue, isolate the cDNA clone who gets with the hybridization back-and-forth method.This cosmid is with biotin labeling and make single stranded form.Allow form heterozygote between c5 and the cDNA, catch biotin labeled cosmid with the magnetic-particle that is coated with strepavidin then.The cDNA that selects is eluted from cosmid, uses pcr amplification, cloning and being checked order.The clone that this cDNA clone gets with separating with the library screening method is similar, because they contain E1 β, E2 and E3.Described exons 1 (seeing SEQ ID NO:3) before wherein the neither one clone contains.The sequence of MTS1E1 β cDNA is listed in SEQ ID NO:13.

The structure of MTS1E1 β

MTS1 and MTS1E1 β are two kinds of forms of a gene: these two kinds of forms are all used exon 2 and 3, but have the first different exons.MTS1 contains alpha form (E1 α), and its coding is by preceding 43 amino acid of the p16 protein of MTS1 coding.MTS1E1 β contains the beta form (E1 β) of exons 1.By compound cDNA cloned sequence is likened to the clone from genome area, determine the exons structure (Figure 13) of p16 gene.The genome area that contains p16 is carried out genome Southern trace, sequence analysis and long PCR, to determine the position (Figure 13) of p16 exon.The p16 gene is striden the genomic DNA of 30kb approximately.E1 β is the part that exon leans on 5 ＇ end most, and order is E1 β, E1 α, E2 and E3.

E1 β reads translation in the frame (knowing by inference from the coding exon 2 of p16 and 3 used reading frames) at p16 and discloses, and 10 codon places only, the stitching position upstream of terminator between E1 β and E2 of reading in the frame are arranged.The position of this terminator is confirmed by genome and cDNA sequence analysis.First the potential initiation codon that is positioned at this terminator downstream is the reading frame at p16, near the 3 ＇ end of E1/E2 splicing site.The both sides sequence of this potential initiation codon and total Kozak sequence (Kozak, 1987) are not quite similar.If in p16 reads frame, translate, the E1 β transcript of p16 gene 105 amino acid whose protein of will encoding so.

Other analyses to β cDNA disclose, and it has the great opening that is in the framework different with coding p16 framework and reads frame (ORF).This ORF (being called ORF2) extends through E1 β and stretches into 67 amino acid places of E2.180 amino acid whose protein of the ORF codified of total length.Yet this reading frame is open at E1 β 5 ＇ end, may be incomplete therefore.The statistical analysis hint, the ORF of this size can not be by accidental produce (P=0.003) of the DNA that random sequence constitutes.Yet if provide the base composition of β transcript, possibility is big (P=0.16).The polypeptide of prediction and any protein of describing in the past are all dissimilar.

Identify the evolution conservative part of E1 β, can provide relevant which sequence the vital clue of its function.With terminal rapid amplifying method (RACE) technology of the cDNA of improvement, be called heterozygote and catch RACE (Hybrid Capture RACE HCR) (sees embodiment 12), isolates mouse p16cDNA, and compares with people p16cDNA.One type mouse p16cDNA (β type) has exon and the E2 equivalent with people E1 β equivalence.Second type (α type) contains the E1 α equivalent that is connected in E2.E1 α and E2 mouse exon and people's homologue has 70% homogeny.Mouse and people's E1 β exon nucleotide sequence has 51% homogeny (Figure 14), and mouse E1 β exon also contains the terminator that is in the coding p16 reading frame.People who derives from the nucleotide sequence of terminator 5 ＇ and the polypeptide of mouse are divergent fully.Therefore, the terminator of reading in the frame at p16 can not be the order-checking illusion.

Consider the uncertainty of E1 β effect, we have analyzed the similitude of reading mouse and people β transcript in the frames at all three kinds.In the reading frame different with the reading frame (ORF1) of coding p16, the β transcript of people and mouse all contains big ORF (ORF2).Derive, have a homogeny of 40% by ORF 2 encoded polypeptides.But, if with the ORF2 peptide relatively be defined in the coded part of E1 β, they only have 28% homogeny.On the contrary, mouse and people's p16 sequence has 67% homogeny.In addition, according to polypeptide that ORF2 derived among the E2 and the polypeptide of being derived according to the reading frame of the 3rd among the E2 (ORF3) similar (42%).These results hint, ORF2 does not optionally keep, thereby coded protein not.Also compared the secondary structure of going into, do not found surprising similitude with mouse β RNA.In a word, these result's hints, the β transcript is that the p16 function is needed, because it all exists in mouse and people; If it is translated, first methionine place that encoded protein matter may be in exon 2 is initial so.Germ line mutation among the embodiment 8MTS1

Whether corresponding in order to measure MTS1 or MTS2 with genetic predisposition locus MTS, the genomic DNA (Cannon-Albright et al., 1992) that carries the allelic individuality of MTS tendentiousness from 8 supposition is analyzed.Use Oligonucleolide primers (table 2) that every kind of sample is increased, amplify the exon dna sequence dna derived from MTS1 or MTS2 specificity intron sequences.

Table 2

The primer of exon among the screening MTS1

Primer SEQ ID NO: exon genes

1F??????????????6???????????????1??????????MTS1

1108R???????????7???????????????1??????????MTS1

42F?????????????8???????????????2??????????MTS1

551R????????????9???????????????2??????????MTS1

21F?????????????10??????????????2??????????MTS2

50R?????????????11??????????????2??????????MTS2

89F?????????????12??????????????2??????????MTS2

With the exons 1 of primer 1F and 1108R amplification MTS, then with primer 1108R order-checking.With the exon 2 of primer 42F and 551R amplification MTS, then with primer 42F and 551R order-checking.Exon 2 with 21F and 50R amplification MTS2 increases with primer 89F and 50R again, checks order with primer 89F and 50R then.

The dna sequence dna of these genomic fragments discloses among in 8 individualities 2 and has polymorphism.Polymorphism is not present in other any sample, this hint, and they are not common in the crowd.In order to show that polymorphism is and MTS chromosome rather than chain with other homologous chromosome, to analyzing from carrying allelic other the individual genomic DNAs of tendentiousness among each kindred.In each case, polymorphism is all separated with MTS tendentiousness allelomorph.In kindred 3012 individuality (12821), found the sudden change (gly → trp) of codon 93.This also finds in ill carrier compatriot (13183) and not ill carrier cousin (14917), but does not find in not ill non-carrier compatriot (13184).The sudden change of discovery codon 118 in kindred 1771 individuality (15635) (val → asp).This also finds in ill carrier's one-level cousin (10146) of ill carrier's one-level cousin (relationship between cousins of the same paternal grandfather (table) brother and sister) (10205), ill carrier's one-level cousin (relationship between cousins of the same paternal grandfather (table) brother and sister) (11414) and 10205, but does not find in 10205 not ill non-carrier's uncle (or uncle) (10120).

Polymorphism is single nucleotide subsitution, and it causes amino acid whose variation (table 3).Displacement relates to residue big, hydrophobicity and replaces little, hydrophilic residue, perhaps uses charged aminoacid replacement neutral amino acid.

Table 3

The tendentiousness kind is the MTS sudden change

Sudden change coding effect location ^*

G→T???????????gly→trp?????????????277

T→A???????????val→asp?????????????353

*: the position of sudden change in SEQ ID NO:1 dna sequence dna.

In 8 samples analyzing, do not find the polymorphism of MTS2 exon 2.This hint, in these kindreds, MTS2 can not cause easily suffering from melanoma at least.Because MTS2 is similar to MTS1, so MTS2 may be relevant with the cancerous swelling of other types.Might MTS2 be non-functional gene also.

Easily suffering from the melanomatous individuality, in MTS1 and do not find in MTS2 that germ line mutation is consistent with the result of melanoma homozygous deletion analysis.The analysis that whether MTS exists in embodiment 9 tumor cell lines

Because so whether the 9p21 of many tumor types high frequency disappearance is to existing MTS1 to analyze from 12 kinds of dissimilar cell-lines.Use one is enclosed within the sequence-tagged site (STS) that distributes at regular intervals in the gene and tests the fragment (Fig. 4 A, 4B and 9) whether the genomic DNA of tumor cell line exists expection.This result of study hints, has the disappearance (table 4) of MTS1 in the tumor cell line of high percentage.Homozygous deletion is present in the tumor type of all tests, except colon and neuroblastoma cell system.25% to the height to astrocytoma in 94% of the percentage of disappearance from be low to moderate lung cancer and leukemia.In a word, detect homozygous deletion in 135 cell-lines in the cell-line of 290 tests.Can obtain carrying the minimum estimated value of the tumor cell line percentage of disappearance from this number, not cover whole gene because be used for the STS of this analysis.Therefore some little disappearance may not be detected.In addition, for example insertion of minority nucleotide or disappearance equivalent damage and nucleotide subsitution, this method also can omission.

Table 4

Homozygous deletion tumor type clone number disappearance number disappearance % melanoma 99 57 58 leukaemia 41 25 lung cancer 59 15 25 spongioblastoma 10 00 carcinoma of urinary bladder 15 5 33 kidney 95 56 astrocytoma 17 16 94 colon cancer 20 00 mastocarcinoma 10 6 60 oophoroma 72 29 glioma 35 25 71 osteosarcoma 53 60 in different tumor types amount to 290 135 47%

In order to improve estimation, whether 34 cell-lines that do not show tangible MTS1 sequence homozygous deletion are existed the more careful detection of MTS1 damage carrying out to the cell-line sum that contains the MTS1 sudden change.The sequence that contains nearly 97%MTS1 coded sequence is amplified and analyzes polymorphism.Observe 18 kinds of somatic mutations (table 5) of MTS1 exons 1 or exon 2 in 14 in 34 melanoma.3 kinds in these sudden changes is frameshift mutation, and 7 kinds is nonsense mutation, and 4 kinds is that missense mutation and 4 kinds are silent mutations.Contain 3 mutant forms that also contain other in 4 cell-lines of silent mutation, and in 18 kinds of sudden changes 16 kinds are positioned at coding exon 2 place.Except that a cell-line, the polymorphism that narrows or isozygoty is all contained specially in all cells system, hints that other homologous chromosome also has disappearance.Two different non-silent mutations are contained in single hybrid cell system, and this discovery shows that every chromosome has all experienced independently catastrophic event.According to MTS1 dna sequence dna and deletion analysis, at least 75% melanoma cell series contains saltant MTS1 or this gene of disappearance on two homologous chromosomes.

Table 5

Somatic cell MTS sudden change in the tumour

Cell-line sudden change coding effect location ^*

SK-M-ste G → A does not have 240

G→A???????????gly→ser???????241

SK-M-swi C → T arg → termination 148

SK-M-ris??????G→A???????????ala→thr???????418

SK-M-beh lacks 5 base frameshit 266-270

SK-M-178 C → T arg → termination 214

SK-M-sta G → A trp → termination 306

SK-M-uti C → T arg → termination 214

SK-M-EML131 lacks 8 base frameshit 148-155

C → A does not have 147

SK-M-koz??????C→T???????????pro→leu???????317

(het. ^*) C → T do not have 213

C → T arg → termination 214

SK-M-kra??????C→T???????????pro→leu???????317

SK-M-kuu C → T does not have 354

SK-M-mar G → A trp → termination 305

SK-M-whi C → T gln → termination 124

SK-M-adl lacks 2 base frameshit 104-105

(het.) the position * * Het. that suddenlys change in SEQ ID NO:1 dna sequence dna of * represents " heterozygote ", refers to exist in sample wild type and saltant sequence.Damage advantage among the MTS1 (disappearance and nucleotide subsitution) expression is that MTS1 or certain closely linked locus cause tumor phenotypes.The cell that sustains damage has selective advantage than the cell that does not sustain damage.The another kind of explanation thinks that these damages are and the irrelevant random event of cell growth, and this explanation is impossible.Reason is that at first, there is causality in the high correlation hint between tumor phenotypes and the MTS1 sudden change between MTS1 sudden change and tumour formation.Secondly, MTS1 influences melanomatous neurological susceptibility, so it has independently the meaning as tumor suppressor gene.Moreover the pattern that plays a role as the initial general inhibitor of dna replication dna in vivo as the biochemical function of the strong inhibition p16 agent of Cdk and p16 conforms to well.

Sudden change that might MTS1 or disappearance are the results of cell growth when cultivating.Yet a high proportion of primary leukemia cell also contains the homozygous deletion of alpha-interferon genes bunch, and this gene cluster and MTS1 are at a distance of not enough 500kb (Diaz et al., 1990).Disappearance in the past studies show that the disappearance of alpha-interferon genes always relates to those marks (Weaver-Feldhaus et al., 1994) that extends beyond MTS1 to the kinetochore.Because the homozygous deletion in MTS1 zone is present in primary tumor cell and the cultured cell system, therefore in tumor cell line observed disappearance can not be purely when cultivating the cell growth cause.Yet, in the development of tumour MTS1 sudden change when this problem appears and will be by the analysis of original tumor sample is provided best answer.MTS1 effect in vivo

In all eukaryotics, cell division need be through two vital commit points: from the transformation of G1 to S, it is synthetic to begin DNA this moment, and from the transformation of G2 to M, begins mitosis this moment.In mammal, control fissional mechanism and relate to multiple component, wherein many is (referring to the Sherr, 1993) that are correlated with.Cdk may be the core in the control composition, because they are regulated and control by making a large amount of crucial substrate phosphorylations, these substrates then cause from G1 to S and from the transformation of G2 to M.May be even more important commit point from G1 to the transformation of S, because it occurs in the cell cycle at first.Up to now, identified 4 kinds and participated in control from the Cdk (Cdk2-5) of G1 to S and the cover positive regulatory factor (cyclin C, D1-3, E) of these Cdk.Also identify several negative regulatory factors recently, comprise p16, p15, p18, p20, p21 and p27 (Xiong et al., 1993; Serrano et al., 1993; Gu et al., 1993; El-Dierv et al., 1993; Harper et al., 1993; Hannon and Beach, 1994; Polyak etal., 1994b; Toyoshima and Hunter, 1994; Guan et al., 1995).These negative regulatory factors seemingly work by the kinase activity that suppresses Cdk.Some cell cycle regulating factor relates to people's cancerous swelling (can with reference to Hunter and Pines, 1994).P20 suppresses Cdk2 and may other Cdk, and p16 (being also referred to as MTS1, CdkN2 or INK4a) suppresses Cdk4 but in vitro test, do not suppress Cdk2 (Serranoet al., 1993) significantly.Based on the interaction of in vitro study and itself and p53, p21 is considered to the general inhibitor (Xiong et al., 1993) of all Cdk.Therefore external, p16 has more specificity than p21.In these inhibitor each all stops cell to enter the S phase.Equally, cyclin D1 or Cdk4 overexpression in some mastocarcinoma, and the p16 gene is (Bucklev et al., 1993 of sudden change or disappearance in a large amount of cell-lines and former generation tumour; Caldas et al., 1994; Kamb et al., 1994b; Mori et al., 1994; Tam et al., 1994a).These results hint, some cyclin and Cdk are proto-oncogenes and p16 (MTS1) is the TIF gene.P15, p18, the biochemistry behavior of p21 and p27 shows that they also are TIFs, but does not also have report detailed, relevant their genes mutation analysis in tumour or cell-line.Result provided herein has provided MTS1 in vivo as the evidence of cell division inhibitor.

The p16 gene (MTS1) that is positioned at No. 9 chromosome 9p21 of people section is especially interesting, because it suddenlys change or homozygous deletion (Caldas et al., 1994 in the cancerous swelling of a high proportion of some type with in derived from the cell-line of tumour; Kamb et al., 1994b; Mori etal., 1994; Nobori et al., 1994).In addition, in several known kindreds that carry the chain melanoma neurological susceptibility of 9p21, the MTS1 sudden change separates (Hussussian et al., 1994 with suffering from melanomatous tendentiousness; Kamb et al., 1994a).Yet,, also have some an open questions for the effect of MTS1 in inheritance and sporadic cancerous swelling.Several to the 9p21 mark have high LOD value, easily suffer from melanomatous kindred and in the MTS1 coded sequence, find sudden change.And MTS1 homozygous deletion advantage also is atypical for the tumor suppressor gene inactivation in tumour or the cell-line, thereby hint exists other to participate in the gene that cancerous swelling forms near MTS1.

Nearest report hint, some mitogenesis and the process of antimitotic effect of signals cell cycle, this influence is (Firpo et al., 1994 of being undertaken by the activity of regulation and control Cdk inhibitor at least in part; Hannon and Beach, 1994; Kato etal., 1994; Polyak et al., 1994a; Slingerland et al., 1994).For example, the inhibition of beta induced cell cycle of TGF can mediate by activating p15 and p27.On the contrary, in the lymphocytic mitogenesis process of the static T that IL-2 induces, p27 is by negative regulation.It is less relatively that what know is the regulation and control of MTS1.The evidence that nearest many reports provide shows that the horizontal component ground of MTS1 is by Rb protein regulation (Serrano et al., 1993; Li et al., 1994a; Tam et al., 1994b; Parry et al., 1995).These and other discovery (Serrano et al., 1995) has caused setting up the model of a MTS1 effect, and wherein MTS1 suppresses Cdk4/6, thereby prevents the phosphorylation of Rb.Rb participates in feedback loop subsequently, the level of restriction MTS1.

These results provide hereditary evidence for MTS1 plays outstanding effect in the regulation and control of cell cycle.In addition, these result's hints, the target of MTS1 is the principal element that tumour forms in the body.If MTS1 suppresses Cdk4 rather than Cdk2 in vivo, Cdk4 is the strong candidate gene of oncogene so.Ubiquity sudden change hint in MTS1, Cdk4 is fissional universal activator in great majority (if not whole words) cell.The hierarchical relationship that the further biochemical research of different Cdk influences is helped to understand fully Cdk activity in normal cell and transformant about MTS1.Similar to p16, if p21 is the general inhibitor of Cdk, its gene also can lack in a high proportion of tumour or suddenly change so.

If MTS1 is the general TIF that is in activated state in most of normal cells, estimate that so the germ line mutation of MTS1 can cause the easily cancerous swelling of trouble except that melanoma.The germ line mutation of p53 gene for example as the sudden change of finding, can increase the possibility of suffering from multiple cancerous swelling in Li-Fraumeni syndrome, comprising the childhood sarcoma, mastocarcinoma (Malkin et al., 1990).Discovering in the past easily suffered from the melanomatous family incidence of disease of cancer of pancreas high unusually (Bergman et al., 1990 at some; Nancarrow et al., 1993).This observed result exists the homozygous deletion of MTS1 consistent with discovery in pancreatic tumor cell system.Might the heredity of MTS1 tendentiousness be different with the heredity of MTS1 somatic cell.For example, because the inferior position of selecting, the big disappearance that the MTS1 peripheral region lacks thousands of base pair dnas may not be present in the people's gene storehouse.Yet this disappearance may be favourable in the somatic cell that transforms, and this may be because they have removed a plurality of genes.This possibility gene extremely similar with having another and MTS1 is that MTS2 is consistent.MTS2 is positioned at about 12kb place, upstream of MTS1 exons 1, and first exon of MTS2 is positioned at about 2.5kb place, upstream of second exon of MTS2.MTS2 is by bringing into play its function with the similar mode of MTS1.With only make two genes in the sudden change of any inactivation compare, the disappearance that MTS1 and MTS2 are removed can provide bigger growth vigor to cell.Perhaps, these two genes are to play a role in different or cell category that part is identical.These possibilities require further study.The mutation analysis of embodiment 10MTS1E1 β

Because inactivation p16 homozygous deletion is preponderated in derived from the cell-line of tumour, and 9p21 chain, easily suffer from the sudden change that melanomatous kindred does not have p16, so cause other personages to propose near p16, to exist other the gene (Cairnset al., 1994 that relate to cancerous swelling formation equally; Spruck et al., 1994).If related protein in the growth of E1 β coding regulating cell, these sequences just may contain and be present in sudden change in sporadic and/or the familial cancerous swelling, that missed in early stage research so.Therefore, easily suffer from the sudden change of screening E1 β among melanomatous kindred the cell-line that gets deriving and at some from various tumour.

Have report (Cannon-Albrightet al., 1992) about the hereditary capacity of easy trouble melanoma family.(Kamb et al. 1994a) and from the process of the middle isolation of genomic DNA of cell-line (Liu et al., 1995) is existing describes from the melanomatous kindred of easy trouble.With forward primer (5 ＇-AGTCTGCAGTTAAGG-3 ＇ SEQ ID NO:33) and reverse primer (5 ＇-GGCTAGAGGCGAATTATCTGT-3 ＇ SEQ ID NO:34) E1 β is carried out pcr amplification, under following condition, carries out 30 circulations: 97 ℃ 3 seconds, 65 ℃ 10 seconds, 75 ℃ 20 seconds.With 100 times of amplified reaction product dilutions, under same reaction condition, use same forward primer to increase once more then with different reverse primer (5 ＇-CACCAAACAAAACAAGTGCCG-3 ＇ SEQ ID NO:35).The PCR product is electrophoresis on 1% Ago-Gel, and (Qiagen Inc) carries out extracting to use the Qiagen pearl then.The product that obtains checks order with Cyclist sequencing kit (Stratagene) and above-mentioned forward primer (SEQ ID NO:33).

One group from 4 kinds of tumours (table 6), amount in 24 cell-lines, and carry tangible haplotype between the ill family member jointly but before research (Kamb et al., 1994a) do not find 6 melanoma kindred (Cannon-Albright etal. that p16 suddenlys change, 1992) in, do not detect the series jump body of E1 β.These test hint, and in the process of tumour, E1 β sudden change is not common incident, and they with these kindreds in the chain melanoma neurological susceptibility of 9p21 have nothing to do.

Table 6

The cell-line of screening E1 β sudden change

Number of types

Lung (cancer) 3

Bladder (cancer) 7

Glioma 9

Melanoma 5

Amount to ¹24

Annotate: ¹Do not show the sudden change (Liu et al., 1995) that these cell-lines contain homozygous deletion in the p16 zone or carry the p16 coded sequence in the past.According to former result (Liuet al., 1995), in bladder and melanoma group, should there be the cell-line of similar number and type to show 4 point mutation at the p16 code sequence.11 couples of MTS2 of embodiment carry out screen mutation

MTS2 sudden change in the cell-line

In cell-line derived from tumour, remove the homozygous deletion advantage hint of MTS1, near MTS1, there is gene other, that relate to cancerous swelling formation.If the MTS2 gene relates to sporadic cancerous swelling, in tumor cell line, may contain sudden change so.Therefore, the sudden change of screening MTS2 coded sequence in one group of tumor cell line.

Press the described method of people such as Kamb (1994a), by the exons 1 and 2 of pcr amplification MTS2.Use primer that 2E1.F1 (5 ＇-AGGGAAGAGTGTCGTTAAG-3 ＇ SEQ ID NO:19) and 2E1.R2 (5 ＇-AGACTCCTGTACAAATCTAC-3 ＇ SEQ ID NO:20) are obtained exons 1.Use primer that 89F (SEQ IDNO:12) and 50R (SEQ ID NO:11) are obtained exon 2.After the amplification, the DNA product is electrophoresis on 1% Ago-Gel, and (Qiagen Inc) carries out extracting to use the Qiagen pearl then.The product that obtains checks order to exons 1 with Cyclist sequencing kit (Stratagene) and primer 2 E1.F1, with primer 89F and 50R exon 2 is checked order.

One group derived from carcinoma of urinary bladder, glioma, astrocytoma, kidney, lung cancer and melanomatous cell-line in, the sudden change of screening MTS2 coded sequence.All these cell-line types all contain to high-frequency MTS1 and MTS2 homozygous deletion (Kamb et al., 1994b).Cell-line derived from melanoma, kidney, lung cancer and carcinoma of urinary bladder contains MTS1 point mutation or frameshift mutation (Liu et al., 1995).Yet the cell-line of glioma and astrocytoma does not contain such MTS2 sudden change.The special cells system that is used for these screening tests be from one group showed in the past the cell-line of not carrying MTS2 and MTS1 sequence homozygous deletion select (Kamb et al., 1994b).

All there is not in the MTS2 coded sequence, to find MTS2 sudden change (table 7) in 58 cell-lines of screening any.According to former research to MTS1 in these cell-line types, the MTS1 sudden change (Liu et al., 1995) that should containing in this group cell-line has an appointment 8 is confined to carcinoma of urinary bladder, melanoma, lung cancer and kidney group.Therefore, from this group tumor cell line, do not obtain the evidence of relevant MTS2 somatic mutation.

Table 7

The screening of MTS2 sudden change in the cell-line

Polymorphism 1

The number of variations type coding effect that cell-line type # screening number # changes

Astrocytoma 20

Bladder (cancer) 41 G → A; C → A does not have

Glioma 60

Melanoma 17 2 G → A; C → A does not have

Kidney (cancer) 41 G → A; C → A does not have

Lung (cancer) 10 1 G → A; C → A does not have

Cellule lung (cancer) 72 G → A; C → A does not have

Non-small cell lung (cancer) 80

Amount to 58 7

(Kamb et al. 1994a) is positioned at introne 1 to 1 these common polymorphisms, the 3 ＇ acceptor sites that close nucleotide position-27 (C to A) and-103 (G to A) locate.Screening MTS2 sudden change in the kindred

In the chain easy trouble melanoma kindred of the 9p21 of no MTS1 sequence encoding mutant, the MTS2 gene may be the reason that causes the melanoma neurological susceptibility, and this possibility attracts people that it is understood.Have report (Cannon-Albright et al., 1992) about the hereditary capacity of easy trouble melanoma family.Use standardization program, utilize isolated lymphocyte from whole blood, separate genomic DNA from the member of family (Kamb et al., 1994a).As mentioned above, the chain easy trouble melanoma of the 9p21 with high LOD value and before research (Kamb et al. 1994a) does not find among the kindred of MTS1 sudden change (seeing Table 8) sudden change of screening MTS2 coded sequence.Do not detect the MTS2 sudden change.Therefore, these tests do not provide the MTS2 damage to cause the melanomatous evidence of inheritance, although only this possibility can't be got rid of according to these limited tests.

Table 8

Easily suffering from screening MTS2 germ line mutation among the melanomatous kindred

Kindred LOD value case sum carries the number of haplotype

3346????????5.97???????21?????????????21

3137????????1.9????????17?????????????21

1764????????1.04???????4??????????????4

3006????????0.19???????6??????????????3

3161???????-0.01???????10?????????????8

Two p16 promotors of expression of 3343-0.53 10 8 embodiment 12MTS1 and MTS1E1 β RNA

Two kinds of multi-form may formation of p16mRNA by two kinds of different approach.Transcribing can be initial from different promotors, and perhaps mRNA can derive from same promotor, carries out different splicings then and produces multi-form transcript.

Because even when the E1 of upstream β sequence deletion, alpha form also can be transcribed in cell-line, so explanation exists independently alpha transcriptional and β transcripting promoter.Cell-line A375 and SK-mel contain a kind of disappearance, and there is a breakpoint (Figure 13) in this disappearance between E1 α and E1 β.The breakpoint of this near-end is not also accurately located in these two cell-lines, but is positioned at 85kb place at least, E1 β 5 ＇ end upstream.Carry out reverse-transcription polymerase chain reaction (RT-PCR) with α-Auele Specific Primer, these two kinds of cell-lines are express alpha transcript (Figure 15) all.The program of RT-PCR is as follows: from the synthetic cDNA (Clontech) of total RNA (Sambrooket al., 1989) of T cell, cell-line or people's separate tissue.The cDNA reaction is adopted 9 polymers to cause the synthetic of DNA and is used Superscript II reverse transcriptase (Betheda ResearchLaboratories).By with α ³²P-dATP (Amersham) mixes synthetic reaction thing (0.1Ci/mmol), measures then and is incorporated into the quantity of the radioactive nucleus thuja acid in the end product, thereby calculate the cDNA productive rate.Use PCR method, use α or β, analyze p16 α and p16 β transcript level by continuous two-wheeled amplification specifically from the forward primer and half nested reverse primer of E2.In amplification for the first time, with the cDNA template of α-Auele Specific Primer AS.1 (5 ＇-CAACGCACCGAATAGTTACG-3 ＇ SEQ ID NO:26) or β-Auele Specific Primer BS.1 (5 ＇-TACTGAGGAGCCAGCGTCTA-3 ＇ SEQ ID NO:27) and X2.R140 ＇ (5 ＇-AGCACCACCAGCGTGTC-3 ＇ SEQ ID NO:22) amplification 2ng.Reaction is on Perkin-Elmer 9600 thermal cyclers, carries out 20 circulations by following condition: 97 ℃ 3 seconds; 65 ℃ 10 seconds; 75 ℃ 20 seconds.100 times of product dilutions use primer AS.1 or BS.1 and X2B (5 ＇-CGTGTCCAGGAAGCCC-3 ＇ SEQ ID NO:23) to increase once more then.The X2B oligomer is at its 5 ＇ end γ ³²P-dATP (DuPont) carries out mark (Sambrook etal., 1989).The PCR condition is as above still only carried out 15 circulations.Because genomic DNA pollutes the problem that causes, the PCR product is striden E1 α or E1 β/E2 splice point in order to eliminate.Product by electrophoresis on 5% denaturing polyacrylamide gel separately.The gel of draining spends the night with X-OMAT (Kodak) exposure.

Result's hint, the alpha transcriptional thing is from being independent of the promotor transcriptional start of E1 β 5 ＇ sequences.The another kind of explanation is that disappearance causes dystopy promoter sequence and E1 α to merge.As if but this is impossible, because A375 and SK-mel93 are the cell-line of independent separate separately.The accurate position of α promotor is not clear, but the RNase protection the analysis showed that it is positioned at p16 upstream from start codon 440bp place at least.Therefore, people p16 gene is complicated, and it has two kinds of partly overlapping transcripts.These two kinds of transcripts have different coding potentiality, and by different promotor P _αAnd P _βProduce.

The expression way of p16

The clue of related gene function is from the analysis to gene expression way in different tissues.In order to determine the expression way of p16, preparation one cover cDNA sample screens (Figure 16 A-D) with α and β Auele Specific Primer then from 11 tissues.In the tissue of all detections, all detect the p16 transcript of two kinds of forms, although there are some differences.For example, in spleen, the ratio between α and the beta form tends to β.On the contrary, this ratio tends to α in breast.These expression datas conform to some research, and these are discovered in the cell-line from many histological types, all have disappearance and point mutation (Kamb et al., the 1994b of p16; Liu et al., 1995), this hint p16 has effect in multiple tissue.

Consider the biochemical function of p16, promptly in external inhibitor (Serrano et al., 1993 that show as Cdk4 and Cdk6; Li et al., 1994a; Parry et al., 1995), can the expression of p16 in the analysis of cells when the cell cycle.Add interleukin 2 (IL-2) stimulation human peripheral blood lymphocyte (PBL) with phytolectin (PHA), harvesting in the different time after stimulation.These cells, are analyzed determining the level relatively of p16 gene expression with RT-PCR determining the residing phase of the cycles of cell with flow cytometry analysis, and analyze level with definite p16 protein with the Western blotting.Extract blood from the donor of growing up normally, therefrom isolate peripheral blood lymphocyte then and partial purification by suspension in Ficoll-Hypaque gradient (Boyum, 1968).Be further purified lymphocyte by former described adverse current analysis by elutriation (Elstad et al., 1988) again.The original author estimates that Zhi Bei cell mass 98% is pure B and T cell in this way.The cell of purifying is growth in the RPMI (Gibco) that adds 10% calf serum, induces akinete with 10 μ g/mlPHA (Sigma) and 10U/ml IL-2 (Sigma).Monitor the progress of cell cycle with flow cytometer.By the described method of manufacturer, (CINNA/BIOTECX Laboratories Inc.) isolates RNA from former generation T cell with RNazol B.Make a series of dilution with isolated T cell cDNA after inducing, thereby determine the amount of PT-PCR.Determine the extension rate when target no longer is amplified, thereby determine to be present in the quantity of the target cDNA in the undiluted sample.Although the result from different PCR test conforms to, dilution test shows, has only the variation that just can detect rna level when rna level difference during greater than 4 times.Derive from Rb ⁺And Rb ^-The cDNA sample of cell-line is all analyzed in this way.For various cDNA samples (no matter coming self-organizing, cell-line still is the T cell), all detect human actin easily, and their content is identical substantially.

(Fig. 6 B-C) takes place to change significantly in the ratio of two kinds of form p16 transcripts in the cell cycle.Originally, the B form is less, but after stimulation 30-40 hours, its level begins to rise.Simultaneously, the expression of alpha form is kept relatively stable, and perhaps having only slightly increases.By using flow cytometer to find, to enter the cell of S phase relevant with leaving the G0 phase in the variation of ratio.Detect the amount of RT-PCR with the template dilution test.These tests show that RT-PCR can detect 4 times of transcriptional levels or bigger variation.Estimate beta inducedly to be at least 10 times.Therefore, when cell entered the cell cycle, the relative quantity that they change two kinds of p16 transcripts made the variation of ratio tend to β.

We have also detected when the T cell passes through the cell cycle, the level of p16 protein expression.Different times isolated protein after mitogenesis is induced carries out the Western engram analysis to the protein that separates then.Use level at complete peptide C-20 amino acid whose p16 TPPA p16 protein of end.When cell leaves G0 during the phase, the level of p16 protein is kept relatively stable.Therefore, p16 coding RNA (alpha transcriptional thing) and p16 protein both keep relatively stable in the cell cycle.Other scholars report is arranged, S phase p16 level have gentle increasing (Tam et al., 1994b).We do not observe the increase of p16, and this may reflect the difference of p16 regulation and control in different cell types, or because have problems when detection protein (or cDNA) level increases by 2-3 times.

The expression of p16 in tumor cell line

Studies show that in the past, Rb influences expression (Serrano et al., 1993 of p16; Liet al., 1994a; Parry et al., 1995).We have tested the influence (Figure 16 D) that the Rb state is expressed β mRNA in the cell.From one group of cell-line, prepare cDNA, 5 Rb protein that contain wild type wherein, 6 contain non-functional Rb protein (Parry etal., 1995).Just as expected, only in Rb negative cells system, detect the alpha transcriptional thing.But, in the Rb positive and Rb negative cells system, all detect the β transcript.Therefore, opposite with α, the expression of β RNA is irrelevant with the mutation status derived from Rb in the cell-line of tumour.

Evidence suggests that p16 is the member (Guan et al., 1995) of multigene family.Similar to other multigene families, the member of this family may carry out the function that repeats, different function or carry out function in different temporary or tissue specificity modes.Therefore, consider the low-level of p16 protein and obviously do not have the regulation and control of Rb that possible p16 is cell cycle regulation in the T lymphocyte not to p16.Yet because when inducing T cell, the β transcript is induced significantly, and again because p16 is (Hebert et al., 1994) of disappearance in the cell-derived tumour of a high proportion of T, as if therefore, p16 plays an important role in the human T-cell.Only in viral cell transformed system or cell-line, observe the obvious influence of Rb to p16 derived from tumour.Perhaps, p16 is regulated and control by other means in wild type tissue.

E1 β is the conservative of p16 and regulation and control part

Although the effect of β transcript is not clear, the result shows that it is most important for the function of p16 locus, because: (i) E1 β guards in mouse; (ii) the relative amount of β transcript regulate and control by tissue specificity and the mode that depends on the cell cycle and (iii) the homozygous deletion that carries of two cell-line remove E1 β, rather than E1 α.These result's hints, E1 β is that wild type p16 performance function is necessary.

The β cDNA that separates mouse, and with people's MTS1E1 β relatively.Catch the improvement crossbred option program separation mouse cDNA clone of RACE (HCR) with being called crossbred.From the breast of mouse and thymus gland, isolate and be rich in poly-A ⁺RNA.Article one, the synthetic reaction of cDNA chain (Sambrook et al., 1989) adopts at random 12 polymers and SuperscriptII reverse transcriptase (Bethesda Research Laboratories).After the second chain is synthetic, the cDNA anchor is lived by specific double-stranded oligomer (double-stranded RP.2) (5 ＇-TGAGTAGAATTCTAACGGCCGTCATTGTTC-3 ＇ SEQ ID NO:28) being connected in its 5 ＇ end.The 5 ＇ end of second cDNA chain is unique by the DNA end of phosphorylation in coupled reaction.After coupled reaction, the cDNA that anchor is lived is by at SepharoseCL-4B post wash-out and purifying.The cDNA that anchor is lived increases with p16 specific reverse primers (5 ＇-AGCGTGTCCAGGAAGCCTTC-3 ＇ SEQ ID NO:29) and nested type RP.2 (RP.B) (5 ＇-TGAGTAGAATTCTAACGGCCGTCATTG-3 ＇ SEQ ID NO:30), subsequently be positioned at the used reverse primer upstream end that increases for the first time, biotin labeled gene specific oligonucleotides (5 ＇-ACTGCGAGGACCCCACTACCTTCTCC-3 ＇ SEQ ID NO:31) catches.The cDNA that catches is increased once more, wherein use RP.B and be positioned at the capture oligo upstream end, gene specific reverse primer (5 ＇-GAACGTTGCCCATCATCATC-3 ＇ SEQ ID NO:32).The product that obtains is by gel-purified, clone and order-checking.Check order and determine the sequence of mouse p16 oligonucleotides by the clone with to musculus cdna group clone (contain under low stringency with people E2 probe hybridization sequence).

Mouse β transcript and people are compared, result's hint, E1 β is coded protein not.The sequence that only contains p16 reading frame among the E2 is strict conservative.Therefore, as if if the β transcript is translated, so protein is can be from E2 initial and according to translating with the same reading frame of coding p16.The calculating molecular weight of the polypeptide of deriving is 10kDa, and keeps 23/4 in 4 ankyrins (ankyrin) repetitive sequence among the p16.Yet p15 only contains 31/2 ankyrin repeat (Hannon and Beach, 1994), and other Protein Folding and performance function have only 1 or 2 repetition.Whether the p10 molecule is present in the body and whether it suppresses Cdk4/6 and also need further test.

The function of β RNA

If the role of β transcript is a cell growth inhibiting, we can find to interrupt the mutant form of E1 β in derived from the cell-line of tumour so.What conform to this viewpoint is two melanoma cell series, and their disappearance is E1 β to be removed still still can continue the express alpha transcript.The p16 coded sequence is a wild type in these cell-lines.Yet, in one group of 25 tumor cell line, do not find the medium and small genetic damage of E1 β (for example base replacement).Therefore, the E1 β that also is difficult to draw a conclusion is the target of homozygous deletion.If E1 β exon is coded protein not, so little genetic damage is not enough to interrupt its function.Perhaps, the target of above-mentioned disappearance may be other genes.For example, might in these melanoma cell series, p15 gene (MTS2) be the related objective of disappearance.In this viewpoint, E1 β disappearance is because compare its more close P15 with E1 α purely.Yet,, be to contain the disappearance of removing p15 specifically (Kamb et al., 1994b), therefore this explanation is seemingly impossible because there is not cell-line again because we can not detect the point mutation of P15 in many cell-lines.

Heredity evidence hint, p16 and Rb are the members in the adjusting and controlling growth approach, this path in the tumour generating process often by inactivation.If the role of β transcript is the growth of negative regulation cell, it may be that another must be a part that is independent of the path of p16 and Rb sudden change so.The disappearance that so just can be interpreted as what specificity interruption E1 β only just can be observed in Rb-cell-line.As if according to the expression way of E1 β, E1 β works in active circulating cells.Clear and definite conclusion about E1 β role awaits analyzing its expression in vivo.The expression of embodiment 13MTS2mRNA

By the described method of manufacturer, (CINNA/BIOTECXLaboratories Inc.) isolates RNA from cell-line or former generation T cell with RNazol B.From the synthetic cDNA of total RNA (Sambrook et al., 1989), wherein adopting at random, 9 polymers cause the synthetic of DNA.By with α ³²P-dATP (Amersham) mixes synthetic reaction thing (0.1Ci/mmol), measures then and mixes the quantity of the radioactive nucleus thuja acid in the end product, thereby calculate the cDNA productive rate.Use PCR method, make half nested (heminested) reverse primer, analyze the expression of MTS2 by continuous two-wheeled amplification.In amplification for the first time, with the cDNA template of E1F (5 ＇-TGAGGGTCTGGCCAGC-3 ＇ SEQ ID NO:21) and X2.R140 ＇ (5 ＇-AGCACCACCAGCGTGTC-3 ＇ SEQ ID NO:22) amplification 2ng.Reaction is on Perkin-Elmer 9600 thermal cyclers, carries out 20 circulations by following condition: 97 ℃ 3 seconds; 65 ℃ 10 seconds; 75 ℃ 20 seconds.100 times of product dilutions use primer E1F and X2B (5 ＇-CGTGTCCAGGAAGCCC-3 ＇ SEQ ID NO:23) to increase once more then.The X2B oligomer is at its 5 ＇ end γ ³²P-dATP (DuPont) carries out mark (Sambrook etal., 1989).The PCR condition is as above still only carried out 15 circulations.Product separates by electrophoresis on 5% denaturing polyacrylamide gel.The gel of draining spends the night with X-OMAT (Kodak) exposure.

The expression of MTS2 in different tissues

Find that MTS2 expresses in many types of organizations, comprise that those MTS2 homozygous deletions cause tumor type (seeing Figure 10 A).But, in different tissues, still have some difference.For example, the MTS2 transcript can be detected easily in lung, but can not detect in prostate and brain tissue.On the contrary, in the cell of all detections, can detect the MTS1 expression of gene that is closely related.Whether this tissue specificity difference of present not clear MTS2RNA level has reflected the organizing specific sexual demand to MTS2 protein.

The expression of MTS2 in the cell cycle

If the great shift in the MTS2 cell cycle regulation, its expression meeting changed in the cell cycle so.For example, in the proper splitting cell, the abundance of p21mRNA can change with the difference in cell cycle in each stage (Li et al., 1994b).Whether in the cell cycle, regulated and control in order to test transcribing of MTS2, stimulate static human T-cell with PHA and IL2, and monitor (seeing Figure 10 B) in post-stimulatory different phase.When cell leaves the G0 phase when cell cycle in each stage, do not detect the significant change of MTS2 expression.On the contrary, the expression of controlling gene Cdk4 is by people's expection change (Matsushime et al., 1992).Therefore, the evidence of the differential expression aspect of MTS2mRNA is not also found during about the cycle by the proper splitting cell.

Propose, MTS1 protein participates in adjusting and controlling growth approach (Serrano et al., 1993 that relate to Retinoblastoma Protein Rb; Guan et al., 1995; Serranoet al., 1995).Nearest research provides strong and detailed evidence, supports MTS1 to be subjected to regulation and control (Li et al., the 1994a of Rb at least in part; Parry et al., 1995).The similitude hint of the biochemistry aspect between MTS1 and the MTS2, MTS2 also may be subjected to the regulation and control of Rb.This possibility can be confirmed by comparing the level (concentration) of MTS2mRNA in Rb positive cell line and Rb negative cells system.Do not detect the correlation (seeing Figure 10 C) between Rb state and the MTS2RNA level.This hint, the Rb state of cell-line also influence the abundance of MTS2 transcript indistinctively.Therefore, opposite with MTS1, the expression of MTS2 is independent of Rb.The ectopic expression of embodiment 14MTS1 and MTS2

By polymerase chain reaction, use primer MTS1.F (5 ＇-AAA GGA TCCATT GCC ACC ATG GAG CCG GCG GCG GGG AGC AGC ATGGAG CCT TCG GCT-3 ＇) (SEQID NO:17) and E3.R (5 ＇-TTTGAA TTC AAT CGG GGA TGT CTG-3 ＇) (SEQ ID NO:18) to amplify the 483bp fragment of MTS1.The primer MTS1.F of design contains restriction endonuclease sites near 5 ＇ end so that later clone, and contains Kozak consensus sequence (Kozak, 1987).Be used for the cDNA of the template DNA of this reaction from breast tissue.The fragment that produces is inserted the expression vector pcDNA3 (In Vitrogen) that has digested with EcoRI and BamHI.PcDNA3 contains the codon of cytomegalovirus (CMV) promotor and coding ampicillin and neomycin resistance.The recombinant vector pcDNAp16 that produces enters cell-line HS294T by electroporation again.HS294T is from melanoma and contain MTS1 and the homozygous deletion of MTS2.HS294T the improved Eagle medium of the Dulbecco that is added with 10% calf serum, dispensable amino acid, Sodium Pyruvate and L-glutamate (DMEM) (Gibco) in the growth.Cell is grown in 5% carbonic acid gas at 37 ℃.HS294T and pSS (Stratagene) are in 1: thus 4 ratio is carried out cotransformation and is given transformant with hygromycin resistance, meanwhile, HS294T with contain pcDNA3 (Invitrogen) expression vector of the MTS1 coded sequence that is inserted in CMV promotor downstream, or do not contain the pcDNA3 carrier cotransformation that inserts son.

The coded sequence of MTS2 similarly is cloned into pcDNA3, similarly also forms the Kozak consensus sequence, thereby produce pcDNAp15, then it is imported HS294T.Wherein, use the MTS2 cDNA of preparation in embodiment 13.

To contain the plasmid pSS (Stratagene) and pcDNAp16 or pcDNAp15 while cotransformation of hygromycin resistance selected marker, every sample cell contains 20 microgram pSS in electroporation.The condition of electroporation is every sample cell 800 microlitre cells (1.5 * 10 ⁶Cells/ml) and 500 microfarads (μ F) electric capacity, 400 volts.Carry out check experiment with pcDNA3 and pSS.Cell behind the electroporation (about 400 microlitres) is placed the culture dish that contains 300 mcg/ml hygromycin.After 14 days colony is counted.The results are shown in table 9.

Table 9

pcDNAp15????????pcDNAp16

Plasmid colony/dull and stereotyped colony/flat board

pcDNA3＋pSS????????26.6±4.8???????17.2±0.15

pcDNAp15＋pSS??????3.8±0.8???????????--

pcDNAp1?6＋pSS????????--???????????1.1±0.5

When the construction that contains the total length MTS2 coded sequence that is blended in the CMV promotor was transformed into HS294T, compared with the control, it suppressed colony formation with seven times ratio, therefore can be complementary with the MTS1 ectopic expression.This result shows that the ectopic expression of MTS2 is enough to suppress the growth of cell.Whether not clear cell transformed is to be parked in the G1 phase (causing as the MTS1 ectopic expression), and perhaps its growth otherwise is terminated.Conclusion lacks in the cell-line of p15 or p16 expression (herein being because homozygous deletion) at script as can be drawn from Table 9, and p15 or p16 overexpression meeting suppress the growth of cell.Accurate mechanism is not clear, but may be because p15 or p16 overexpression stop cell division or cell killing.These experimental datas hint, p15 and p16 in vivo really as tumor suppressor protein, so p15 and p16 may have therapeutical uses.

For a variety of reasons, MTS2 is the strong candidate gene of tumor suppressor gene.It has widely and the MTS1 sequence similarity, and it combines with Cdk and suppress its activity external, and the ectopic expression cell growth inhibiting of MTS2 in the body.The above results causes existing such possibility: although there is the similitude of biochemistry aspect between MTS2 and the MTS1, two kinds of protein have function very inequality in vivo.Two features hint of MTS2, situation may be such: i) be MTS2 rather than MTS1 by TGF beta induced (Hannon and Beach, 1994) with ii) different with MTS1, as if transcribing of MTS2 have nothing to do with Rb.Possible MTS2 does not relate to tumour and forms.Perhaps, MTS2 participates in a tumor suppression approach different with the approach that relates to MTS1.The various factors in this approach are not clear, but can imagine, wherein some factor can be undergone mutation with the frequency more much higher than MTS2 in somatic cell.In this viewpoint, the somatic mutation that lacks MTS2 can not be got rid of its key player in tumor suppression.As mentioned above, the ectopic expression cell growth inhibiting of MTS2, this role is that TIF conforms to MTS2.The MTS2 expression is stable in the cell cycle, and it is beta induced by TGF, these hints, and in the stopping of G1 phase, MTS2 plays a role, and opportunity of each incident in the cell cycle regulation necessarily.On the contrary, the expression of MTS1 is subjected to the regulation and control of Rb, and this expression MTS1 works in the cell cycle changes.Therefore be necessary to test the function of MTS2, and analyze the approach of MTS2 performance function as adjusting and controlling growth molecule in the body.The MTS that exists in the embodiment 15 two-step method test sample

Method (1985) according to people such as Antonarakis is handled patient samples, separates by 1% Ago-Gel, goes to then on the nylon membrane for the Southern engram analysis.Film is when 150mJ, and it is crosslinked (Bio-Rad) to carry out UV with GS gene junction (GS Gene Linker).MTS probe corresponding to nucleotide 448-498 position among the SEQ ID NO:4 is gone into pTZ18U by subclone.Phasmid (phagemid) is transformed into M13K07 helper phage (Bio-Rad, Richmond, CA) the Escherichia coli MV1190 of Gan Raning.Separate single stranded DNA with standardization program (seeing Sambrook, et al., 1989).

Trace is at 65 ℃, 7% dodecyl sodium sulfate (SDS), 0.5M NaPO ₄Middle prehybridization 15-30 minute.This method is identical with described methods of people (1992) such as Nguyen.Trace is at 65 ℃, 7%SDS, 0.5M NaPO ₄In spend the night with 25-50ng/ml. ssDNA probe hybridization.Wash after the hybridization, comprising at 65 ℃, 5%SDS, 40mM NaPO ₄Middle washing 30 minutes is total to secondary, then at 65 ℃, 1%SDS, 40mM NaPO ₄Middle washing 30 minutes, secondary altogether.

Then, trace is incubated 30-60 minutes with containing 0.2% caseic PBS with (pH6.8) rinsing at room temperature 5 minutes of phosphate buffer (PBS) under room temperature, rinsing 5 minutes in PBS then.Then, trace in 45 ℃, was cultivated 5-10 minutes with hybridization buffer (6M urea, 0.3M NaCl and 5X Denhardt ＇ s solution (seeing Sambrook, etal., 1989)) in shaking water-bath in advance.Remove buffer solution, and replace 50-75 microlitre/cm ²Fresh hybridization buffer adds oligonucleotides-alkaline phosphatase enzyme conjugates that the 2.5nM covalency links to each other, wherein nucleotide sequence and universal primer position (UP-AP, Bio-Rad) complementation.Trace was 45 ℃ of hybridization 20-30 minutes, and the washing after the hybridization is in 45 ℃ of washings 10 minutes, secondary in 6M urea, 1 * standard citric acid salt solution (SSC), 0.1%SDS; Then at 1 * SSC, 0.1%Triton ^X-100 washing 10 minutes, once.Trace was at room temperature used 1 * SSC rinsing 10 minutes.

Trace is in shaking water-bath, under room temperature, at substrate buffer solution (0.1M diethanol amine, 1mM MgCl ₂, 0.02% sodium azide, pH10.0) in cultivated 10 minutes.(disodium salt Bio-Rad) is placed the heat-seal bag together for 3-(2 ＇-spiral shell adamantane)-4-methoxyl group-4-(3 ＇-phosphoryl oxygen base) phenyl-1,2-dioxetane for each trace and substrate buffer solution and 0.2mM AMPPD.At room temperature, shake insulation after 20 minutes, remove unnecessary AMPPD solution.Trace spends the night with the exograph exposure.Positive band represents to exist MTS.The polyclonal antibody that embodiment 16 produces at MTS

The fragment of MTS coded sequence is expressed as fusion in Escherichia coli.The protein of high expressed carries out purifying with the gel elution method, and by being similar to described program immunity rabbit of Harlow and Lane (1988) and mouse.Shown that this program can produce the antibody (for example referring to Kraemer, et al., 1993) at various other protein.

In brief, with MTS coded sequence section as plasmid PET5A (Novagen, Inc., Madison, WI) fusion in is cloned.The MTS integration sequence comprises the amino acid corresponding to 448-498 among the SEQ ID NO:4.After inducing with IPTG, confirm to have the Expression of Fusion Protein of estimated molecular weight by SDS/PAGE.Fusion is purified into from gel with electroelution method.Identifying protein by the protein sequencing at the N end is the MTS fusion product.Then, the protein of purifying is used as immunogene and removes immunize rabbit.Rabbit carries out immunity with 100 micrograms of protein and complete Freund adjuvant, carries out twice again by 3 weekly intervals then, for the first time with 100 microgram immunogenes and incomplete Freund adjuvant, for the second time with 100 microgram immunogene and PBS.Two weeks were collected the serum that contains antibody after this.

Repeat this program, to obtain antibody at the mts gene mutated form.These antibody, and at the antibody of wild type MTS are used to detect the existence and the relative level of mutated form in different tissues and biological fluid.The monoclone antibody that embodiment 17 produces at MTS

Produce monoclone antibody with following scheme.Use contains the immunogen immune mouse of complete MTS or MTS peptide (wild type or saltant), and this MTS or MTS peptide are connected in key hole  hemocyanin with glutaraldehyde or EDC.These are to know in this area.

Immunogene and adjuvant are mixed.Each mouse is accepted 4 times 10-100 immunogenic injections of microgram, after the 4th injection, gathers blood sample to determine whether contain at immunogenic antibody in the serum from mouse.Determine serum titer with ELISA or RIA.Select and show the mouse of existence in the serum, be used to produce hybridoma at immunogenic antibody.

From immune mouse, take out spleen, preparation single-cell suspension liquid (seeing Harlow andLane, 1988).Basically use the described method fusion cell of Kohler and Milstein (1975).In brief, press the described method of Harlow and Lane (1988), (American type culture collection, Rockville MD) merge with immune spleen cell with P3.65.3 myeloma cell with polyethylene glycol.By 2 * 10 ⁵The density of cells/well places cell in the tissue culturing plate in 96 holes.Check whether each hole has the growth of cell, and the supernatant in the hole of growth is passed through ELISA or RIA, test to determine existing of MTS specific antibody with wild type or saltant MTS target protein.To the cell in the positive hole proceed to cultivate and subclone to obtain and to confirm monoclonal system.

Have required specific being cloned in the mouse and continue to breed and growth, thereby produce the usefulness of enough antibody for research and analysis as ascites or in the hollow fibre system.The sandwich assay of embodiment 18MTS

Monoclone antibody is connected in solid phase surface, for example plate, test tube, pearl or particle.Preferably, antibody is attached to the hole surface of 96 hole elisa plates.The 100 microlitre samples (for example serum, urine, organize cytosol) that will contain MTS peptide/protein (wild type or saltant) add to insolubilized antibody.Sample at room temperature is incubated 2 hours.Then remove sample liquids, wash solid phase to remove uncombined material with buffer solution.Second kind of monoclone antibody (at the different antigenic determinant of MTS peptide/protein) added to solid phase.This antibody is with detection molecules (I for example ¹²⁵, enzyme, fluorophor, chromophore) mark.Solid phase and second kind of antibody at room temperature are incubated 2 hours.Remove second kind of antibody, wash solid phase to remove uncombined material with buffer solution.

Measure the quantity of binding label quantitatively, the quantity of MTS peptide/protein is in direct ratio in it and the sample.Re-use to the special monoclone antibody of wild type MTS and to the special monoclone antibody of various MTS sudden changes and analyze.

Clearly, method of the present invention and content can be used for various embodiment, wherein have only sub-fraction to be disclosed in this.Those of skill in the art in this area know, and also have other avatar (or embodiment), and these all are to belong within the scope of the invention.Therefore, the above embodiments only are used for purposes of illustration, are not limited to purpose.

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List of patents and patent applications U.S. Patent No.3, 817,837 U.S. Patent No.3, 850,572 U.S. Patent No.3, 939,350 U.S. Patent No.3, 996,345 U.S. Patent No.4, 275,149 U.S. Patent No.4, 277,437 U.S. Patent No.4, 366,241 U.S. Patent No.4, 376,110 U.S. Patent No.4, 486,530 U.S. Patent No.4, 683,195 U.S. Patent No.4, 683,202 U.S. Patent No.4, 816,567 U.S. Patent No.4, 868,105 U.S. Patent No.5, 252,479 EPO Publication No.225, 807 European Patent Application Publication No.0332435 Geusen, European Patent Application No.84/03664, 1984. Sep.13th issue Hitzeman et al., EP 73,675 A PCT Application Publication No. WO 93/07282 ...

Sequence table (1) general information:

(i) applicant: Kamb, Alexander

(ii) denomination of invention: mts gene, its somatic mutation and with the method for this gene order diagnosis, prognosis and treatment cancerous swelling

(iii) sequence number: 36

(iv) address:

(A) address: Venable, Baetjer, Howard ﹠amp; Civiletti, LLP

(B) street: 1201 New York Avenue, Suite 1000

(C) city: Washington

(D) state: DC

(E) country: the U.S.

(F) postcode: 20005

(v) computer-reader form:

(A) recording medium type: floppy disk

(B) calculator: IBM PC compatible type

(C) operating system: PC-DOS/MS-DOS

(D) software: PatentIn Release#1.0, version #1.30

(vi) the application's data:

(A) application number: US

(B) applying date:

(C) classification:

(vii) application materials formerly:

(A) application number: US 08/251,938

(B) applying date: 01-06 month-1994

(vii) application materials formerly:

(A) application number: US 08/215,088

(B) applying date: 18-03 month-1994

(vii) application materials formerly:

(A) application number: US 08/214,581

(B) applying date: 18-03 month-1994

(vii) application materials formerly:

(A) application number: US 08/227,369

(B) applying date: 14-04 month-1994

(vii) application materials formerly:

(A) application number: US 08/214,582

(B) applying date: 18-03 month-1994

(viii) lawyer/agent's information:

(A) name: Saxe, Stephen A.

(B) registration number: 38,609

(C) reference/number of documents: 24884-109348-PCT-2

(ix) communication information:

(A) phone: 202-962-4848

(B) fax: the information of 202-962-8300 (2) SEQ ID NO:1:

(i) sequence signature:

(A) length: 447 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(ix) feature:

(A) title/retrieval symbol: CDS

(B) position: 1..447

(xi) sequence description: SEQ ID NO:1:ATG GAG CCT TCG GCT GAC TGG CTG GCC ACG GCC GCG GCC CGG GGT CGG 48Met Glu Pro Ser Ala Asp Trp Leu Ala Thr Ala Ala Ala Arg Gly Arg1 5 10 15GTA GAG GAG GTG CGG GCG CTG CTG GAG GCG GTG GCG CTG CCC AAC GCA 96Val Glu Glu Val Arg Ala Leu Leu Glu Ala Val Ala Leu Pro Asn Ala

20??????????????????25??????????????????30CCG?AAT?AGT?TAC?GGT?CGG?AGG?CCG?ATC?CAG?GTC?ATG?ATG?ATG?GGC?AGC????144Pro?Asn?Ser?Tyr?Gly?Arg?Arg?Pro?Ile?Gln?Val?Met?Met?Met?Gly?Ser

35??????????????????40??????????????????45GCC?CGA?GTG?GCG?GAG?CTG?CTG?CTG?CTC?CAC?GGC?GCG?GAG?CCC?AAC?TGC??????192Ala?Arg?Val?Ala?Glu?Leu?Leu?Leu?Leu?His?Gly?Ala?Glu?Pro?Asn?Cys

50??????????????????55??????????????????60GCC?GAC?CCC?GCC?ACT?CTC?ACC?CGA?CCC?GTG?CAC?GAC?GCT?GCC?CGG?GAG??????240Ala?Asp?Pro?Ala?Thr?Leu?Thr?Arg?Pro?Val?His?Asp?Ala?Ala?Arg?Glu65???????????????????70??????????????????75??????????????????80GGC?TTC?CTG?GAC?ACG?CTG?GTG?GTG?CTG?CAC?CGG?GCC?GGG?GCG?CGG?CTG??????288Gly?Phe?Leu?Asp?Thr?Leu?Val?Val?Leu?His?Arg?Ala?Gly?Ala?Arg?Leu

85??????????????????90??????????????????95GAC?GTG?CGC?GAT?GCC?TGG?GGC?CGT?CTG?CCC?GTG?GAC?CTG?GCT?GAG?GAG??????336Asp?Val?Arg?Asp?Ala?Trp?Gly?Arg?Leu?Pro?Val?Asp?Leu?Ala?Glu?Glu

100?????????????????105?????????????????110CTG?GGC?CAT?CGC?GAT?GTC?GCA?CGG?TAC?CTG?CGC?GCG?GCT?GCG?GGG?GGC??????384Leu?Gly?His?Arg?Asp?Val?Ala?Arg?Tyr?Leu?Arg?Ala?Ala?Ala?Gly?Gly

115?????????????????120?????????????????125ACC?AGA?GGC?AGT?AAC?CAT?GCC?CGC?ATA?GAT?GCC?GCG?GAA?GGT?CCC?TCA??????432Thr?Arg?Gly?Ser?Asn?His?Ala?Arg?Ile?Asp?Ala?Ala?Glu?Gly?Pro?Ser

The information of 130 135 140GAC ATC CCC GAT TGA 447Asp Ile Pro Asp *, 145 (2) SEQ ID NO:2:

(i) sequence signature:

(A) length: 149 amino acid

(B) type: amino acid

(D) topological structure: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:2:Met Glu Pro Ser Ala Asp Trp Leu Ala Thr Ala Ala Ala Arg Gly Arg1 5 10 15Val Glu Glu Val Arg Ala Leu Leu Glu Ala Val Ala Leu Pro Asn Ala

20??????????????????25??????????????????30?Pro?Asn?Ser?Tyr?Gly?Arg?Arg?Pro?Ile?Gln?Val?Met?Met?Met?Gly?Ser

35???????????????????40???????????????????45Ala?Arg?Val?Ala?Glu?Leu?Leu?Leu?Leu?His?Gly?Ala?Glu?Pro?Asn?Cys

50??????????????????55??????????????????60Ala?Asp?Pro?Ala?Thr?Leu?Thr?Arg?Pro?Val?His?Asp?Ala?Ala?Arg?Glu65???????????????????70??????????????????75??????????????????80Gly?Phe?Leu?Asp?Thr?Leu?Val?Val?Leu?His?Arg?Ala?Gly?Ala?Arg?Leu

85??????????????????90??????????????????95Asp?Val?Arg?Asp?Ala?Trp?Gly?Arg?Leu?Pro?Val?Asp?Leu?Ala?Glu?Glu

100?????????????????105?????????????????110Leu?Gly?His?Arg?Asp?Val?Ala?Arg?Tyr?Leu?Arg?Ala?Ala?Ala?Gly?Gly

115?????????????????120?????????????????125Thr?Arg?Gly?Ser?Asn?His?Ala?Arg?Ile?Asp?Ala?Ala?Glu?Gly?Pro?Ser

The information of 130 135 140Asp Ile Pro Asp *, 145 (2) SEQ ID NO:3:

(i) sequence signature:

(A) length: 1149 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(ix) feature:

(A) title/retrieval symbol: intron

(B) position: 1..890

(ix) feature:

(A) title/retrieval symbol: exon

(B) position: 891..1016

(ix) feature:

(A) title/retrieval symbol: intron

(B) position: 1017..1149

(xi) sequence description: SEQ, ID, NO:3:TCCCCCGCCC, GTWTTAAWTA, AACCTCATCT, TTCCAGAGTC, TGTTCTTATA, CCAGGAAATG, 60TACACGTCTG, AGAAACCCTT, GCCCCAGACA, GTCGTTTTAC, ACGCAGGAGG, GGAAGGGGAG, 120GGGAAGGAGA, GAGCAGTCCT, TTTCTCCAAA, AGGAATCCTT, NGAACTAGGG, TTTCTGACTT, 180AGTGAACCCC, GCGYTCCTGA, AAATCAWGGG, TTGAGGGGGT, AGGGGGACAC, TTYCCTAGTC, 240GYACAGSTKA, TTTCGMTYCT, CGGTGGGGCT, CTCACAMCTA, GGAAAGAATW, GTTTTGCTTT, 300TTCTTATGAT, TAAAAGAAGA, AGCCATACTT, TTCCCTATGA, CACCAAACAC, CCCGATTCAA, 360TTTGGCAGTT, AGGAAGGTTG, TATCGCGGAG, GAAGGAAACG, GGGCGGGGGC, GGATTTCTTT, 420TTTAACAGAG, TGAACGCACT, CAAACACGCC, TTTGCTGGCA, GGCGGGGGGA, GCGCGGCTGG, 480GAGCAGGGGA, GGCCGGAGGG, CGGTGTGGGG, GGCAGGTGGG, GAGGAGCCCA, GTCCTCCTTC, 540CTTGCCAACG, CTGGCTCTGG, CGAGGGCTGC, TTYCGGCTGG, TGCCCCCGGG, GGAGACCCAA, 600CCTGGGGCGA, CTTCAGGGGT, GCCACATTCG, CTAAGTGCTC, GGAGTTAATA, GCACCTCCTC, 660CGAGCACTCG, CTCACAGCGT, CCCCTTGCCT, GGAAAGATAC, CGCGGTCCCT, CCAGAGGATT, 720TGAGGGACAG, GGTCGGAGGG, GGCTCTTCCG, CCAGCACCGG, AGGAAGAAAG, AGGAGGGGCT, 780GGCTGGTCAC, CAGAGGGTGG, GGCGGACCGC, GTGCGCTCGG, CGGCTGCGGA, GAGGGGGAGA, 840GCAGGCAGCG, GGCGGCGGGG, AGCAGCATGG, AGCCGGCGGC, GGGGAGCAGC, ATGGAGCCTT, 900CGGCTGACTG, GCTGGCCACG, GCCGCGGCCC, GGGGTCGGGT, AGAGGAGGTG, CGGGCGCTGC, 960TGGAGGCGGT, GGCGCTGCCC, AACGCACCGA, ATAGTTACGG, TCGGAGGCCG, ATCCAGGTGG, 1020GTAGAGGGTC, TGCAGCGGGA, GCAGGGGATG, GCGGGCGACT, CTGGAGGACG, AAGTTTGCAG, 1080GGGAATTGGA, ATCAGGTAGC, GCTTCGATTC, TCCGGAAAAA, GGGGAGGCTT, CCTGGGGAGT, 1140TTTCAGAAC, 1149, (2) SEQ, ID, the information of NO:4:

(i) sequence signature:

(A) length: 1187 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(ix) feature:

(A) title/retrieval symbol: intron

(B) position: 1..191

(ix) feature:

(A) title/retrieval symbol: exon

(B) position: 192..498

(ix) feature:

(A) title/retrieval symbol: intron

(B) position: 499..1187

(xi) sequence description: SEQ, ID, NO:4:GAATTCATTG, TGTACTGAAG, AATGGATAGA, GAACTCAAGA, AGGAAATTGG, AAACTGGAAG, 60CAAATGTAGG, GGTAATTAGA, CACCTGGGGC, TTGTGTGGGG, GTCTGCTTGG, CGGTGAGGGG, 120GCTCTACACA, AGCTTCCTTT, CCGTCATGCC, GGCCCCCACC, CTGGCTCTGA, CCATTCTGTT, 180CTCTCTGGCA, GGTCATGATG, ATGGGCAGCG, CCCGAGTGGC, GGAGCTGCTG, CTGCTCCACG, 240GCGCGGAGCC, CAACTGCGCC, GACCCCGCCA, CTCTCACCCG, ACCCGTGCAC, GACGCTGCCC, 300GGGAGGGCTT, CCTGGACACG, CTGGTGGTGC, TGCACCGGGC, CGGGGCGCGG, CTGGACGTGC, 360GCGATGCCTG, GGGCCGTCTG, CCCGTGGACC, TGGCTGAGGA, GCTGGGCCAT, CGCGATGTCG, 420CACGGTACCT, GCGCGCGGCT, GCGGGGGGCA, CCAGAGGCAG, TAACCATGCC, CGCATAGATG, 480CCGCGGAAGG, TCCCTCAGGT, GAGGACTGAT, GATCTGAGAA, TTTGTACYCT, GAGAGCTTCC, 540AAAGCTCAGA, GCATTCATTT, TCCAGCACAG, AAAGTTCAGC, CCGGGAGACC, AGTCTCCGGT, 600CTTGCGCTCA, GCTCACGCGC, CAATGCGGTG, GGACGGCCTG, AGTCTCCCTA, TGCGCCCTGC, 660CSCGCACAGC, GCGGCAAATG, GGAAATAATC, CCGAAATGGA, CTTGCGCACG, TGAAAGCCCA, 720TTTTGTACGT, TATACTTCCC, AAAGCATACC, ACCACCCAAA, CACCTACCCT, CTGCTAGTTC, 780AAGGCCTAGA, CTGCGGAGCA, ATGAAGACTC, AAGAGGCTAG, AGGTCTAGTG, CCCCCTCTTC, 840CTCCAAACTA, GGGCCAGTTG, CATCSACTTA, CCAGGTCTGT, TTCCTCATTT, GCATACCAAG, 900CTGGCTGGAC, CAACCTCAGG, ATTTCCAAAC, CCAATTGTGC, GTGGCATCAT, CTGGAGATCT, 960CTCGATCTCG, GCTCTTCTGC, ACAACTCAAC, TAATCTGACC, CTCCTCAGCT, AATCTGACCC, 1020TCCGCTTTAT, GCGGTAGAGT, TTTCCAGAGC, TGCCCCAGGG, GGTTCTGGGG, ACATCAGGAC, 1080CAAGACTTCG, CTGACCCTGG, CAGTCTGTGC, ACCGGAGTTG, GCTCCTTTCC, CTCTTAAACT, 1140TGTGCAAGAG, ATCCCTATAG, TGAGTCGTAT, TATNCGGCCG, CGAATTC, 1187, (2) SEQ, ID, the information of NO:5:

(i) sequence signature:

(A) length: 1244 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(ix) feature:

(A) title/retrieval symbol: intron

(B) position: 1..273

(ix) feature:

(A) title/retrieval symbol: misc-RNA

(B) position: 274..529

(D) other information :/lime light=" show corresponding to the outer of SEQ ID NO:4

Son "

(xi) sequence description: SEQ, ID, NO:5:GATCATCACT, TTACCATCAA, CTTTCTTGTC, TCTGAACGTT, TAGAGAATAA, AATGGCATTT, 60AATTGGTVCT, GAGTWTAACC, TGAAGGTGGG, GTGGGAAAGT, GGWTTGCATC, AGCAADTGAA, 120GAAACACCAG, ACATCAGAGA, CCTGAACACC, TCTGCACTGG, GTGAAAACTT, GGCAATTAGG, 180TGTTTCTTTA, AATGGCTCCA, CCTGCCTTGC, CCCGGCCGGC, ATCTCCCATA, CCTGCCCCCA, 240CCCTGGCTCT, GACCACTCTG, CTCTCTCTGG, CAGGTCATGA, TGATGGGCAG, CGCCCGCGTG, 300GCGGAGCTGC, TGCTGCTCCA, CGGCGCGGAG, CCCAACTGCG, CAGACCCTGC, CACTCTCACC, 360CGACCGGTGC, ATGATGCTGC, CCGGGAGGGC, TTCCTGGACA, CGCTGGTGGT, GCTGCACCGG, 420GCCGGGGCGC, GGCTGGACGT, GCGCGATGCC, TGGGGTCGTC, TGCCCGTGGA, CTTGGCCGAG, 480GAGCGGGGCC, ACCGCGACGT, TGCAGGGTAC, CTGCGCACAG, CCACGGGGGA, CTGACGCCAG, 540GTTCCCCAGC, CGCCCACAAC, GACTTTATTT, TCTTACCCAA, TTTCCCACCC, CCACCCACCT, 600AATTCGATGA, AGGCTGCCAA, CGGGGAGCGG, CGGAAAGCCT, GTAAGCCTGC, AAGCCTGTCT, 660GAGACTCACA, GGAAGGAGGA, GCCGACCGGG, AATAACCTTC, CATACATTTT, TTTCTTTGTC, 720TTATCTGGCC, CTCGACACTC, ACCATGAAGC, GAAACACAGA, GAAGCGGATT, TCCAGGGATA, 780TTTAGGAGTG, TGTGACATTC, CAGGGGTCGT, TTGNTTTTCA, GGGTTTTCTG, AGGGAAAGTG, 840CATATGAAAT, CCTTGACTGG, ACCTGGTGGC, TACGAATCTT, CCCGATGGAT, GAATCTCCCA, 900CTCCAGCGCT, GAGTGGGAGA, AGGCAGTGAT, TAGCACTTGG, GTGACGGCAG, TCGATGCGTT, 960CACTCCAATG, TCTGCTGAGG, AGTTATGGTG, AACCCACAAC, TTAGGCCCTA, GCGGCAGAAA, 1020GGAAAACCTG, AAGACTGAGG, ACAAAGTGGA, GGAGGGCCGA, GGTGGGCTTC, AGTATGTCCC, 1080CNNCGGCGCT, TTAGTTTGAG, CGCATGGCAA, GTCACATGCG, TAAACGACAC, TCTCTGGAAG, 1140CCCTGGAGAC, CCTCGCCCAA, CTCCACCAGA, TAGCAGAGGG, GTAAGAGAGG, ATGTCCAAGC, 1200GACGACAGAT, GCTAAAATCC, CTGGATCACG, ACGCTGCAGA, GCAC, 1244, (2) SEQ, ID, the information of NO:6:

(i) sequence signature:

(A) length: 19 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:6:CAGCACCGGA GGAAGAAAG 19 (2) SEQ ID NO:7:

(i) sequence signature:

(A) length: 20 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: be

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:7:GCGCTACCTG ATTCCAATTC 20 (2) SEQ ID NO:8:

(i) sequence signature:

(A) length: 20 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:8:GGAAATTGGA AACTGGAAGC 20 (2) SEQ ID NO:9:

(i) sequence signature:

(A) length: 19 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: be

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:9:TCTGAGCTTT GGAAGCTCT 19 (2) SEQ ID NO:10:

(i) sequence signature:

(A) length: 21 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:10:GATCATCACT TTACCATCAA C 21 (2) SEQ ID NO:11:

(i) sequence signature:

(A) length: 19 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: be

(vi) originate at first:

(A) organism: people (xi) sequence description: the information of SEQ ID NO:11:GGGTGGGAAA TTGGGTAAG 19 (2) SEQ ID NO:12:

(i) sequence signature:

(A) length: 20 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:12:TGAGTTTAAC CTGAAGGTGG 20 (2) SEQ ID NO:13:

(i) sequence signature:

(A) length: 1131 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(ix) feature:

(A) title/retrieval symbol: CDS

(B) position: 338..655

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List of patents and patent applications U.S. Patent No.3, 817,837 U.S. Patent No.3, 850,572 U.S. Patent No.3, 939,350 U.S. Patent No.3, 996,345 U.S. Patent No.4, 275,149 U.S. Patent No.4, 277,437 U.S. Patent No.4, 366,241 U.S. Patent No.4, 376,110 U.S. Patent No.4, 486,530 U.S. Patent No.4, 683,195 U.S. Patent No.4, 683,202 U.S. Patent No.4, 816,567 U.S. Patent No.4, 868,105 U.S. Patent No.5, 252,479 EPO Publication No.225, 807 European Patent Application Publication No.0332435 Geusen, European Patent Application No.84/03664, 1984.Sep.13th issue Hitzeman et al., EP 73,675 A PCT Application Publication No. WO 93/07282 ...

Met?Met?Met?Gly?Ser?Ala

150?????????????????155CGA?GTG?GCG?GAG?CTG?CTG?CTG?CTC?CAC?GGC?GCG?GAG?CCC?AAC?TGC?GCC????????403Arg?Val?Ala?Glu?Leu?Leu?Leu?Leu?His?Gly?Ala?Glu?Pro?Asn?Cys?Ala

160?????????????????165?????????????????170GAC?CCC?GCC?ACT?CTC?ACC?CGA?CCC?GTG?CAC?GAC?GCT?GCC?CGG?GAG?GGC????????451Asp?Pro?Ala?Thr?Leu?Thr?Arg?Pro?Val?His?Asp?Ala?Ala?Arg?Glu?Gly

175?????????????????180?????????????????185TTC?CTG?GAC?ACG?CTG?GTG?GTG?CTG?CAC?CGG?GCC?GGG?GCG?CGG?CTG?GAC????????499Phe?Leu?Asp?Thr?Leu?Val?Val?Leu?His?Arg?Ala?Gly?Ala?Arg?Leu?Asp

190?????????????????195?????????????????200GTG?CGC?GAT?GCC?TGG?GGC?CGT?CTG?CCC?GTG?GAC?CTG?GCT?GAG?GAG?CTG????????547Val?Arg?Asp?Ala?Trp?Gly?Arg?Leu?Pro?Val?Asp?Leu?Ala?Glu?Glu?Leu

205?????????????????210?????????????????215GGC?CAT?CGC?GAT?GTC?GCA?CGG?TAC?CTG?CGC?GCG?GCT?GCG?GGG?GGC?ACC????????595Gly?His?Arg?Asp?Val?Ala?Arg?Tyr?Leu?Arg?Ala?Ala?Ala?Gly?Gly?Thr220?????????????????225?????????????????230?????????????????235AGA?GGC?AGT?AAC?CAT?GCC?CGC?ATA?GAT?GCC?GCG?GAA?GGT?CCC?TCA?GAC????????643Arg?Gly?Ser?Asn?His?Ala?Arg?Ile?Asp?Ala?Ala?Glu?Gly?Pro?Ser?Asp

240?????????????????245?????????????????250ATC?CCC?GAT?TGA?AAGAACCAGA?GAGGCTCTGA?GAAACCTCGG?GAAACTTAGA????????????695Ile?Pro?Asp??*

255TCATCAGTCA CCGAAGGTCC TACAGGGCCA CAACTGCCCC CGCCACAACC CACCCCGCTT 755TCGTAGTTTT CATTTAGAAA ATAGAGCTTT TAAAAATGTC CTGCCTTTTA ACGTAGATAT 815AAGCCTTCCC CCACTACCGT AAATGTCCAT TTATATCATT TTTTATATAT TCTTATAAAA 875ATGTAAAAAA GAAAAACACC GCTTCTGCCT TTTCACTGTG TTGGAGTTTT CTGGAGTGAG 935CACTCACGCC CTAAGCGCAC ATTCATGTGG GCATTTCTTG CGAGCCTCGC AGCCTCCGGA 995AGCTGTCGAC TTCATGACAA GCATTTTGTG AACTAGGGAA GCTCAGGGGG GTTACTGGCT 1055TCTCTTGAGT CACACTGCTA GCAAATGGCA GAACCAAAGC TCAAATAAAA ATAAAATTAT 1115TTTCATTCAT TCACTC 1131 (2) SEQ ID NO: 14 information about:

(i) sequence signature:

(A) length: 106 amino acid

(B) type: amino acid

(D) topological structure: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:14:Met Met Met Gly Ser Ala Arg Val Ala Glu Leu Leu Leu Leu His Gly 15 10 15Ala Glu Pro Asn Cys Ala Asp Pro Ala Thr Leu Thr Arg Pro Val His

20??????????????????25??????????????????30Asp?Ala?Ala?Arg?Glu?Gly?Phe?Leu?Asp?Thr?Leu?Val?Val?Leu?His?Arg

35??????????????????40??????????????????45Ala?Gly?Ala?Arg?Leu?Asp?Val?Arg?Asp?Ala?Trp?Gly?Arg?Leu?Pro?Val

50??????????????????55??????????????????60Asp?Leu?Ala?Glu?Glu?Leu?Gly?His?Arg?Asp?Val?Ala?Arg?Tyr?Leu?Arg65???????????????????70??????????????????75??????????????????80Ala?Ala?Ala?Gly?Gly?Thr?Arg?Gly?Ser?Asn?His?Ala?Arg?Ile?Asp?Ala

85??????????????????90??????????????????95Ala?Glu?Gly?Pro?Ser?Asp?Ile?Pro?Asp??*

The information of 100 105 (2) SEQ ID NO:15:

(i) sequence signature:

(A) length: 751 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(ix) feature:

(A) title/retrieval symbol: CDS

(B) position: 335..751

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: CGGGCAGTGA GGACTCCGCG ACGCGTCCGC ACCCTGCGGC CAGAGCGGCT TTGAGCTCGG 60CTGCGTCCGC GCTAGGCGCT TTTTCCCAGA AGCAATCCAG GCGCGCCCGC TGGTTCTTGA 120GCGCCAGGAA AAGCCCGGAG CTAACGACCG GCCGCTCGGC CACTGCACGG GGCCCCAAGC 180CGCAGAAGGA CGACGGGAGG GTAATGAAGC TGAGCCCAGG TCTCCTAGGA AGGAGAGAGT 240GCGCCGGAGC AGCGTGGGAA AGAAGGGAAG AGTGTCGTTA AGTTTACGGC CAACGGTGGA 300TTATCCGGGC CGCTGCGCGT CTGGGGGCTG CGGA ATG CGC GAG GAG AAC AAG 352

Met?Arg?Glu?Glu?Asn?Lys

110GGC?ATG?CCC?AGT?GGG?GGC?GGC?AGC?GAT?GAG?GGT?CTG?GCC?AGC?GCC?GCG???????400Gly?Met?Pro?Ser?Gly?Gly?Gly?Ser?Asp?Glu?Gly?Leu?Ala?Ser?Ala?Ala

115?????????????????120?????????????????125GCG?CGG?GGA?CTA?GTG?GAG?AAG?GTG?CGA?CAG?CTC?CTG?GAA?GCC?GGC?GCG???????448Ala?Arg?Gly?Leu?Val?Glu?Lys?Val?Arg?Gln?Leu?Leu?Glu?Ala?Gly?Ala

130?????????????????135?????????????????140GAT?CCC?AAC?GGA?GTC?AAC?CGT?TTC?GGG?AGG?CGC?GCG?ATC?CAG?GTC?ATG???????496Asp?Pro?Asn?Gly?Val?Asn?Arg?Phe?Gly?Arg?Arg?Ala?Ile?Gln?Val?Met145?????????????????150?????????????????155?????????????????160ATG?ATG?GGC?AGC?GCC?CGC?GTG?GCG?GAG?CTG?CTG?CTG?CTC?CAC?GGC?GCG???????544Met?Met?Gly?Ser?Ala?Arg?Val?Ala?Glu?Leu?Leu?Leu?Leu?His?Gly?Ala

165?????????????????170?????????????????175GAG?CCC?AAC?TGC?GCA?GAC?CCT?GCC?ACT?CTC?ACC?CGA?CCG?GTG?CAT?GAT???????592Glu?Pro?Asn?Cys?Ala?Asp?Pro?Ala?Thr?Leu?Thr?Arg?Pro?Val?His?Asp

180?????????????????185?????????????????190GCT?GCC?CGG?GAG?GGC?TTC?CTG?GAC?ACG?CTG?GTG?GTG?CTG?CAC?CGG?GCC???????640Ala?Ala?Arg?Glu?Gly?Phe?Leu?Asp?Thr?Leu?Val?Val?Leu?His?Arg?Ala

195?????????????????200?????????????????205GGG?GCG?CGG?CTG?GAC?GTG?CGC?GAT?GCC?TGG?GGT?CGT?CTG?CCC?GTG?GAC???????688Gly?Ala?Arg?Leu?Asp?Val?Arg?Asp?Ala?Trp?Gly?Arg?Leu?Pro?Val?Asp

210?????????????????215?????????????????220TTG?GCC?GAG?GAG?CGG?GGC?CAC?CGC?GAC?GTT?GCA?GGG?TAC?CTG?CGC?ACA???????736Leu?Ala?Glu?Glu?Arg?Gly?His?Arg?Asp?Val?Ala?Gly?Tyr?Leu?Arg?Thr225?????????????????230?????????????????235?????????????????240GCC?ACG?GGG?GAC?TGA???????????????????????????????????????????????????751Ala?Thr?Gly?Asp??*

The information of 245 (2) SEQ ID NO:16:

(i) sequence signature:

(A) length: 139 amino acid

(B) type: amino acid

(D) topological structure: linearity

(ii) molecule type: protein

(xi) sequence description: SEQ ID NO:16:Met Arg Glu Glu Asn Lys Gly Met Pro Ser Gly Gly Gly Ser Asp Glu 15 10 15Gly Leu Ala Ser Ala Ala Ala Arg Gly Leu Val Glu Lys Val Arg Gln

20??????????????????25??????????????????30Leu?Leu?Glu?Ala?Gly?Ala?Asp?Pro?Asn?Gly?Val?Asn?Arg?Phe?Gly?Arg

35??????????????????40??????????????????45Arg?Ala?Ile?Gln?Val?Met?Met?Met?Gly?Ser?Ala?Arg?Val?Ala?Glu?Leu

50??????????????????55??????????????????60Leu?Leu?Leu?His?Gly?Ala?Glu?Pro?Asn?Cys?Ala?Asp?Pro?Ala?Thr?Leu?65??????????????????70??????????????????75??????????????????80Thr?Arg?Pro?Val?His?Asp?Ala?Ala?Arg?Glu?Gly?Phe?Leu?Asp?Thr?Leu

85??????????????????90??????????????????95Val?Val?Leu?His?Arg?Ala?Gly?Ala?Arg?Leu?Asp?Val?Arg?Asp?Ala?Trp

100?????????????????105?????????????????110Gly?Arg?Leu?Pro?Val?Asp?Leu?Ala?Glu?Glu?Arg?Gly?His?Arg?Asp?Val

115?????????????????120?????????????????125Ala?Gly?Tyr?Leu?Arg?Thr?Ala?Thr?Gly?Asp??*

The information of 130 135 (2) SEQ ID NO:17:

(i) sequence signature:

(A) length: 57 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:17:AAAGGATCCA TTGCCACCAT GGAGCCGGCG GCGGGGAGCA GCATGGAGCC TTCGGCT 57 (2) SEQ ID NO:18:

(i) sequence signature:

(A) length: 24 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: be

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:18:TTTGAATTCA ATCGGGGATG TCTG 24 (2) SEQ ID NO:19:

(i) sequence signature:

(A) length: 19 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:19:AGGGAAGAGT GTCGTTAAG 19 (2) SEQ ID NO:20:

(i) sequence signature:

(A) length: 20 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: be

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:20:AGACTCCTGT ACAAATCTAC 20 (2) SEQ ID NO:21:

(i) sequence signature:

(A) length: 16 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:21:TGAGGGTCTG GCCAGC 16 (2) SEQ ID NO:22:

(i) sequence signature:

(A) length: 17 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: be

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:22:AGCACCACCA GCGTGTC 17 (2) SEQ ID NO:23:

(i) sequence signature:

(A) length: 16 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: DNA (genome)

(iii) suppose: not

(iv) antisense: be

(vi) originate at first:

(A) organism: people

(xi) sequence description: the information of SEQ ID NO:23:CGTGTCCAGG AAGCCC 16 (2) SEQ ID NO:24:

(i) sequence signature:

(A) length: 144 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(iv) antisense: not

(xi) sequence description: the information of SEQ ID NO:24:AATTCGGCAC GAGGCAGCAT GGAGCCTTCG GCTGACTGGC TGGCCACGGC CGCGGCCCGG 60GGTCGGGTAG AGGAGGTGCG GGCGCTGCTG GAGGCGGTGG CGCTGCCCAA CGCACCGAAT 120AGTTACGGTC GGAGGCCGAT CCAG 144 (2) SEQ ID NO:25:

(i) sequence signature:

(A) length: 395 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(iv) antisense: not

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25: AAGAGAGGGT TTTCTTGGTA AAGTTCGTGC GATCCCGGAG ACCCAGGACA GCGTAGCTGC 60GCTCTGGCTT TCGTGAACAT GTTGTTGAGG CTAGAGAGGA TCTTGAGAAG AGGGCCGCAC 120CGGAATCCTG GACCAGGTGA TGATGATGGG CAACGTTCAC GTAGCAGCTC TTCTGCTCAA 180CTACGGTGCA GATTCGAACT GCGAGGACCC CACTACCTTC TCCCGCCCGG TGCACGACGC 240AGCGCGCGAA GGCTTCCTGG ACACGCTGGT GGTGCTGCAC GGGTCAGGGG CTCGGCTGGA 300TGTCCGCGAT GCCTGGGGTC GCCTCCCGCT CGACTTCGCC CAAGAGCGGG GACATCAAGA 360CATCGTGCGA TATTTGCGTT CCGCTGGGTG CTCTT 395 (2) SEQ ID NO: 26 information about:

(i) sequence signature:

(A) length: 20 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(xi) sequence description: the information of SEQ ID NO:26:CAACGCACCG AATAGTTACG 20 (2) SEQ ID NO:27:

(i) sequence signature:

(A) length: 20 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(xi) sequence description: the information of SEQ ID NO:27:TACTGAGGAG CCAGCGTCTA 20 (2) SEQ ID NO:28:

(i) sequence signature:

(A) length: 30 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: other nucleic acid

(xi) sequence description: the information of SEQ ID NO:28:TGAGTAGAAT TCTAACGGCC GTCATTGTTC 30 (2) SEQ ID NO:29:

(i) sequence signature:

(A) length: 20 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(xi) sequence description: the information of SEQ ID NO:29:AGCGTGTCCA GGAAGCCTTC 20 (2) SEQ ID NO:30:

(i) sequence signature:

(A) length: 27 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: other nucleic acid

(xi) sequence description: the information of SEQ ID NO:30:TGAGTAGAAT TCTAACGGCC GTCATTG 27 (2) SEQ ID NO:31:

(i) sequence signature:

(A) length: 26 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(xi) sequence description: the information of SEQ ID NO:31:ACTGCGAGGA CCCCACTACC TTCTCC 26 (2) SEQ ID NO:32:

(i) sequence signature:

(A) length: 20 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(xi) sequence description: the information of SEQ ID NO:32:GAACGTTGCC CATCATCATC 20 (2) SEQ ID NO:33:

(i) sequence signature:

(A) length: 15 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(iv) antisense: not

(xi) sequence description: the information of SEQ ID NO:33:AGTCTGCAGT TAAGG 15 (2) SEQ ID NO:34:

(i) sequence signature:

(A) length: 21 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(iv) antisense: be

(xi) sequence description: the information of SEQ ID NO:34:GGCTAGAGGC GAATTATCTG T 21 (2) SEQ ID NO:35:

(i) sequence signature:

(A) length: 21 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: strand

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(iv) antisense: be

(xi) sequence description: the information of SEQ ID NO:35:CACCAAACAA AACAAGTGCC G 21 (2) SEQ ID NO:36:

(i) sequence signature:

(A) length: 947 base-pairs

(B) type: nucleic acid

(C) characteristic of stock: two strands

(D) topological structure: linearity

(ii) molecule type: cDNA

(iii) suppose: not

(iv) antisense: not

(vi) originate at first:

(A) organism: people

(ix) feature:

(A) title/retrieval symbol: composite character

(B) position: 151

(D) other information :/lime light=" splicing site acceptor. "

(ix) feature:

(A) title/retrieval symbol: composite character

(B) position: 458

(D) other information :/lime light=" splicing site acceptor. "

(xi) sequence description: SEQ, ID, NO:36:ATGGAGCCGG, CGGCGGGGAG, CAGCATGGAG, CCTTCGGCTG, ACTGGCTGGC, CACGGCCGCG, 60GCCCGGGGTC, GGGTAGAGGA, GGTGCGGGCG, CTGCTGGAGG, CGGTGGCGCT, GCCCAACGCA, 120CCGAATAGTT, ACGGTCGGAG, GCCGATCCAG, GTCATGATGA, TGGGCAGCGC, CCGAGTGGCG, 180GAGCTGCTGC, TGCTCCACGG, CGCGGAGCCC, AACTGCGCCG, ACCCCGCCAC, TCTCACCCGA, 240CCCGTGCACG, ACGCTGCCCG, GGAGGGCTTC, CTGGACACGC, TGGTGGTGCT, GCACCGGGCC, 300GGGGCGCGGC, TGGACGTGCG, CGATGCCTGG, GGCCGTCTGC, CCGTGGACCT, GGCTGAGGAG, 360CTGGGCCATC, GCGATGTCGC, ACGGTACCTG, CGCGCGGCTG, CGGGGGGCAC, CAGAGGCAGT, 420AACCATGCCC, GCATAGATGC, CGCGGAAGGT, CCCTCAGACA, TCCCCGATTG, AAAGAACCAG, 480AGAGGCTCTG, AGAAACCTCG, GGAAACTTAG, ATCATCAGTC, ACCGAAGGTC, CTACAGGGCC, 540ACAACTGCCC, CCGCCACAAC, CCACCCCGCT, TTCGTAGTTT, TCATTTAGAA, AATAGAGCTT, 600TTAAAAATGT, CCTGCCTTTT, AACGTAGATA, TAAGCCTTCC, CCCACTACCG, TAAATGTCCA, 660TTTATATCAT, TTTTTATATA, TTCTTATAAA, AATGTAAAAA, AGAAAAACAC, CGCTTCTGCC, 720TTTTCACTGT, GTTGGAGTTT, TCTGGAGTGA, GCACTCACGC, CCTAAGCGCA, CATTCATGTG, 780GGCATTTCTT, GCGAGCCTCG, CAGCCTCCGG, AAGCTGTCGA, CTTCATGACA, AGCATTTTGT, 840GAACTAGGGA, AGCTCAGGGG, GGTTACTGGC, TTCTCTTGAG, TCACACTGCT, AGCAAATGGC, 900AGAACCAAAG, CTCAAATAAA, AATAAAATTA, TTTTCATTCA, TTCACTC, 947

Claims (105)

1. a separated DNA is characterized in that, the cDNA by coding MTS polypeptide constitutes basically.

2. separated DNA as claimed in claim 1 is characterized in that, its coding MTS1 polypeptide.

3. separated DNA as claimed in claim 1 is characterized in that, its coding MTS2 polypeptide.

4. separated DNA as claimed in claim 1 is characterized in that, its coding MTS1E1 beta polypeptides.

5. separated DNA as claimed in claim 1 is characterized in that, this MTS polypeptide contains at the amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:14 or the SEQ ID NO:16.

6. a separated DNA is characterized in that, the DNA by 15 nucleotide among the cDNA that has 15 claims 1 at least constitutes basically.

7. separated DNA, it is characterized in that, constitute by the DNA that is selected from down group basically: have DNA in the sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:13, SEQ ID NO:1 5 and the SEQ ID NO:36.

8. separated DNA as claimed in claim 7 is characterized in that, has the sequence shown in the SEQID NO:1.

9. separated DNA as claimed in claim 7 is characterized in that, has the sequence shown in the SEQID NO:3.

10. separated DNA as claimed in claim 7 is characterized in that, has the sequence shown in the SEQID NO:4.

11. separated DNA as claimed in claim 7 is characterized in that, has the sequence shown in the SEQID NO:5.

12. separated DNA as claimed in claim 7 is characterized in that, has the sequence shown in the SEQID NO:13.

13. separated DNA as claimed in claim 7 is characterized in that, has the sequence shown in the SEQID NO:15.

14. separated DNA as claimed in claim 7 is characterized in that, has the sequence shown in the SEQID NO:36.

15. isolated DNA molecule, it is characterized in that this dna molecular is the mutant form of the MTS nucleotide sequence shown in Fig. 5 (A and B), Fig. 6 (A and B), Fig. 7 (A and B), Fig. 8, Figure 11, Figure 12 (A and B), SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQID NO:13, SEQ ID NO:15 or the SEQ ID NO:36.

16. a pair of single stranded DNA primer, it is used for determining by polymerase chain reaction the nucleotide sequence of mts gene, it is characterized in that, the sequence derived from human chromosome 9p21 of this primer, also cause synthetic DNA when wherein, in polymerase chain reaction, using this primer with total length or part mts gene sequence.

17. primer as claimed in claim 16 is right, it is characterized in that, this mts gene sequence is listed in SEQ ID NO:1.

18. primer as claimed in claim 16 is right, it is characterized in that, this mts gene sequence is listed in SEQ ID NO:3.

19. primer as claimed in claim 16 is right, it is characterized in that, this mts gene sequence is listed in SEQ ID NO:4.

20. primer as claimed in claim 16 is right, it is characterized in that, this mts gene sequence is listed in SEQ ID NO:5.

21. primer as claimed in claim 16 is right, it is characterized in that, this mts gene sequence is listed in SEQ ID NO:13.

22. primer as claimed in claim 16 is right, it is characterized in that, this mts gene sequence is listed in SEQ ID NO:15.

23. primer as claimed in claim 16 is right, it is characterized in that, this mts gene sequence is listed in SEQ ID NO:36.

24. a separated DNA is characterized in that, it is selected from down group:

(a) nucleotide position 241 is the SEQ ID NO:1 of A;

(b) nucleotide position 148 is the SEQ ID NO:1 of T;

(c) nucleotide position 418 is the SEQ ID NO:1 of A;

(d) the SEQ ID NO:1 of nucleotide 266-270 disappearances;

(e) nucleotide position 214 is the SEQ ID NO:1 of T;

(f) nucleotide position 306 is the SEQ ID NO:1 of A;

(g) the SEQ ID NO:1 of nucleotide 148-155 disappearances;

(h) nucleotide position 317 is the SEQ ID NO:1 of T;

(i) nucleotide position 305 is the SEQ ID NO:1 of A;

(j) nucleotide position 124 is the SEQ ID NO:1 of A; With

(k) the SEQ ID NO:1 of nucleotide 104-105 disappearances.

25. nucleic acid probe with the complementation of people's wild type mts gene sequence.

26. nucleic acid probe as claimed in claim 25 is characterized in that, it and the exon hybridization that is selected from down group: the nucleotide 891-1016 of SEQ ID NO:3 and the nucleotide 192-498 of SEQ IDNO:4.

27. the nucleic acid probe of a mts gene sequence complementation that changes with the people is characterized in that it is hybridized with the part mts gene, this part mts gene is selected from down group:

(a) nucleotide position 241 is the SEQ ID NO:1 of A;

(b) nucleotide position 148 is the SEQ ID NO:1 of T;

(c) nucleotide position 418 is the SEQ ID NO:1 of A;

(d) the SEQ ID NO:1 of nucleotide 266-270 disappearances;

(e) nucleotide position 214 is the SEQ ID NO:1 of T;

(f) nucleotide position 306 is the SEQ ID NO:1 of A;

(g) the SEQ ID NO:1 of nucleotide 148-155 disappearances;

(h) nucleotide position 317 is the SEQ ID NO:1 of T;

(i) nucleotide position 305 is the SEQ ID NO:1 of A;

(j) nucleotide position 124 is the SEQ ID NO:1 of A; With

(k) the SEQ ID NO:1 of nucleotide 104-105 disappearances.

28. a reproducible cloning vector is characterized in that, it contains just like the described DNA isolation of arbitrary claim in the claim 1-14 and the replicon that can operate in host cell.

29. an expression system is characterized in that, it contain operationally be connected in suitable regulating and controlling sequence as the described DNA isolation of arbitrary claim in the claim 1-14.

30. the recombinant host cell that transforms with the expression system of claim 29.

31. a method that produces reorganization MTS polypeptide is characterized in that, is included in can produce effectively under the condition of this MTS polypeptide, cultivates the cell of claim 30.

32. a people MTS polypeptide formulations, it is substantially free of other people protein, it is characterized in that, the amino acid sequence of this protein is corresponding to the sequence shown in SEQ ID NO:2, SEQ IDNO:14 or the SEQ ID NO:16.

33. people MTS polypeptide formulations, it is substantially free of other people protein, it is characterized in that, this amino acid sequence of polypeptide basically with the amino acid sequence homologous shown in SEQ ID NO:2, SEQ IDNO:14 or the SEQ ID NO:16, and this polypeptide has and the similar substantially function of wild type MTS polypeptide.

34. an antibody is characterized in that, it can with mammal MTS polypeptide generation immune response, and basically not with mammiferous other polypeptide generation immune responses.

35. antibody as claimed in claim 34 is characterized in that, it is a monoclone antibody.

36. in mammal, tissue damage diagnosed or the method for prognosis for one kind, it is characterized in that, comprise: change at the somatic cell that detects wild type mts gene or its expression product from the isolated tissue of this mammal, this change represents that promptly there is neoplasia in this tissue.

37. method as claimed in claim 36 is characterized in that, this mts gene is MTS1.

38. method as claimed in claim 36 is characterized in that, this mts gene is MTS2.

39. method as claimed in claim 36 is characterized in that, this expression product is the mRNA of MTS1.

40. method as claimed in claim 36 is characterized in that, this expression product is the mRNA of MTS2.

41., it is characterized in that the change of wild type mts gene mRNA is by detecting from the mRNA and the mts gene probe hybridization of this tissue sample as claim 39 or 40 described methods.

42. method as claimed in claim 36 is characterized in that, is to detect in the regulation and control zone of mts gene to change.

43. method as claimed in claim 36 is characterized in that, the change of wild type mts gene is to detect by observing single stranded DNA change of electrophoretic mobility in non-denaturing polyacrylamide gel.

44. method as claimed in claim 36 is characterized in that, the change of wild type mts gene is by detecting from genomic DNA and the mts gene probe hybridization that this separate tissue goes out.

45. method as claimed in claim 44 is characterized in that, mts gene probe and the exon hybridization that is selected from down group: the nucleotide 192-498 of the nucleotide 891-1016 of SEQ ID NO:3 and SEQ ID NO:4.

46. method as claimed in claim 36 is characterized in that, the change of wild type mts gene is to detect by the hybridization with MTS allele-specific probe.

47. method as claimed in claim 36 is characterized in that, the detection of the change of wild type mts gene is then extension increasing sequence to be checked order to produce extension increasing sequence by amplification total length or part mts gene in this tissue.

48. method as claimed in claim 36 is characterized in that, the detection of the change of wild type mts gene is by at specificity MTS allelomorph, amplification total length or part mts gene.

49. method as claimed in claim 36 is characterized in that, the detection of the change of wild type mts gene is by total length in this tissue or part mts gene are carried out molecular cloning, to produce clone's sequence, and the sequence order-checking to cloning then.

50. method as claimed in claim 36, it is characterized in that, the detection of the change of wild type mts gene is by when molecule (1) and (2) when mutual cross forms two strands mutually, differentiates the mispairing between the nucleic acid probe of molecule (1)---genomic DNA of isolated mts gene or MTSmRNA and molecule (2) from this tissue---and the complementation of people's wild type MTS regional DNA.

51. method as claimed in claim 36 is characterized in that, the detection of the change of wild type mts gene is by the mts gene sequence in this tissue that increases, then with sequence that increases and the nucleic acid probe hybridization that contains wild type mts gene sequence.

52. method as claimed in claim 36 is characterized in that, the detection of the change of wild type mts gene is by the mts gene sequence in this tissue that increases, then with sequence that increases and the nucleic acid probe hybridization that contains non-wild type mts gene sequence.

53. method as claimed in claim 36 is characterized in that, the detection of the change of wild type mts gene is by the screening deletion mutation.

54. method as claimed in claim 36 is characterized in that, the detection of the change of wild type mts gene is by the screening point mutation.

55. method as claimed in claim 36 is characterized in that, the detection of the change of wild type mts gene is to insert sudden change by screening.

56. method as claimed in claim 36 is characterized in that, the detection of the change of wild type mts gene is by carrying out in situ hybridization with the nucleic acid probe and the mts gene that contain mts gene.

57. method as claimed in claim 36 is characterized in that, expression product is a protein molecule.

58. method as claimed in claim 57 is characterized in that, the change of wild type mts gene protein detects by Western blot.

59. method as claimed in claim 57 is characterized in that, the change of wild type mts gene protein detects with monoclone antibody.

60. method as claimed in claim 57 is characterized in that, the change of wild type mts gene protein detects by immunocytochemistry.

61. method as claimed in claim 57 is characterized in that, the detection of the change of wild type mts gene protein is by analyzing the effect of mutually combining of isolated mts gene protein and Cdk from this tissue.

62. method as claimed in claim 61 is characterized in that, this Cdk is Cdk4.

63. method as claimed in claim 58 is characterized in that, the change of wild type mts gene protein is by analyzing the inhibitory action of Cdk chemical-biological activities.

64., it is characterized in that this Cdk is Cdk4 as the described method of claim 63.

65. one kind confirms that the mts gene seat in the mammal does not have neoplastic method, it is characterized in that, comprise: in the mammal sample, detect wild type mts gene or its expression product, exist wild type gene or its expression product promptly to represent the neoplasia that does not cause by the mts gene seat.

66., it is characterized in that this mts gene is MTS1 as the described method of claim 65.

67., it is characterized in that this mts gene is MTS2 as the described method of claim 65.

68., it is characterized in that this expression product is the mRNA of MTS1 as the described method of claim 65.

69., it is characterized in that this expression product is the mRNA of MTS2 as the described method of claim 65.

70., it is characterized in that wild type mts gene mRNA is by detecting from the mRNA and the mts gene probe hybridization of this tissue sample as the described method of claim 68.

71., it is characterized in that wild type mts gene mRNA detects by hybridizing from the mRNA of this tissue sample and mts gene probe as the described method of claim 69.

72., it is characterized in that the wild type mts gene is by detecting from genomic DNA and the mts gene probe hybridization that this separate tissue goes out as the described method of claim 65.

73., it is characterized in that mts gene probe and the exon hybridization that is selected from down group: the nucleotide 192-498 of the nucleotide 891-1016 of SEQ ID NO:3 and SEQ ID NO:4 as the described method of claim 72.

74. as the described method of claim 65, it is characterized in that, the detection of wild type mts gene be by total length or part mts gene in this tissue that increases to produce extension increasing sequence, then extension increasing sequence is checked order.

75., it is characterized in that the detection of wild type mts gene is by total length in this tissue or part mts gene are carried out molecular cloning as the described method of claim 65, to produce clone's sequence, the sequence order-checking to cloning then.

76., it is characterized in that the detection of wild type mts gene is by carrying out in situ hybridization with the nucleic acid probe and the mts gene that contain mts gene as the described method of claim 65.

77., it is characterized in that expression product is a protein molecule as the described method of claim 65.

78., it is characterized in that the change of wild type mts gene protein detects by Western blot as the described method of claim 77.

79., it is characterized in that the change of wild type mts gene protein detects by immunocytochemistry as the described method of claim 77.

80., it is characterized in that the detection of the change of wild type mts gene protein is by analyzing the effect of mutually combining of isolated mts gene protein and Cdk from this tissue as the described method of claim 77.

81., it is characterized in that this Cdk is Cdk4 as the described method of claim 80.

82., it is characterized in that the change of wild type mts gene protein is by analyzing the inhibitory action of Cdk chemical-biological activities as the described method of claim 77.

83., it is characterized in that this Cdk is Cdk4 as the described method of claim 82.

84. one kind to the cell that lacks wild type mts gene function or cause gene function to change because of the sudden change of mts gene provide wild type mts gene function or basically with the method for the similar MTS function of wild type, it is characterized in that, comprise to this cell introduce wild type mts gene nucleic acid or with basic homology of wild type mts gene nucleic acid and nucleic acid that can cell cycle regulation, this nucleic acid is expressed in cell.

85., it is characterized in that this nucleic acid is the part of wild type mts gene as the described method of claim 84, this part coding makes this cell carry out the part of the required wild type mts gene polypeptide of non-neoplasia growth.

86., it is characterized in that this nucleic acid is the part of wild type mts gene as the described method of claim 84, this part coding makes this cell carry out the required total length wild type mts gene polypeptide of non-neoplasia growth.

87. as the described method of claim 84, it is characterized in that, this nucleic acid and the basic homology of part wild type mts gene, this part encoded polypeptides and the basic homology of part wild type MTS polypeptide, and have to making this cell and carry out the required similar substantially function of part wild type MTS polypeptide of non-neoplasia growth.

88. as the described method of claim 84, it is characterized in that, this nucleic acid and the basic homology of part wild type mts gene, this part encoded polypeptides and the basic homology of total length wild type MTS polypeptide, and have to making this cell and carry out the required similar substantially function of total length wild type MTS polypeptide of non-neoplasia growth.

89., it is characterized in that this nucleic acid contains the mts gene regulating and controlling sequence as the described method of claim 84-88.

90., it is characterized in that this nucleic acid is integrated into the genome of cell as the described method of one of claim 84-89.

91. one kind to the cell that lacks wild type mts gene function or cause gene function to change because of the sudden change of mts gene provide wild type mts gene function or basically with the method for the similar MTS function of wild type, it is characterized in that, comprise to this cell introducing making cell carry out required total length or the part wild type MTS polypeptide of non-neoplasia growth.

92. one kind to the cell that lacks wild type mts gene function or cause gene function to change because of the sudden change of mts gene provide wild type mts gene function or basically with the method for the similar MTS function of wild type, it is characterized in that, comprise molecule from simulation wild type MTS polypeptide function to this cell that introduce.

93., it is characterized in that this molecule and cell carry out the required basic homology of complete wild type MTS polypeptide of non-neoplasia growth and have and the basic similarly function of wild type MTS polypeptide as the described method of claim 92.

94., it is characterized in that this molecule and cell carry out the required basic homology of part wild type MTS polypeptide of non-neoplasia growth and have and the basic similarly function of part wild type MTS polypeptide as the described method of claim 92.

95. a method of screening potential cancerous swelling therapeutic agent is characterized in that, comprising:

(a) measure the ATP that mixes compound, Cdk, cyclin, Cdk substrate and the mark that may be the cancerous swelling therapeutic agent in the compatibility solution at cell cycle protein dependent kinase (Cdk);

(b) quantity of the substrate of mensuration phosphorylation; With

(c) quantity with the phosphorylated substrate in the step (b) compares with the control sample for preparing according to step (a), in this control sample, replaces this therapeutic agent with the MTS polypeptide.

96., it is characterized in that this Cdk is Cdk4 as the described method of claim 95.

97. the method for the potential cancerous swelling therapeutic agent of screening is characterized in that, comprising: cell mixing cyclin-dependent kinase (Cdk), MTS polypeptide and may be the compound of cancerous swelling therapeutic agent, measure the quantity of MTS polypeptide and Cdk combination then.

98., it is characterized in that this Cdk is Cdk4 as the described method of claim 97.

99. the method for the potential cancerous swelling therapeutic agent of screening is characterized in that, comprising: cell mixing cyclin-dependent kinase (Cdk) and may be the compound of cancerous swelling therapeutic agent, measure the biologic activity of Cdk then.

100., it is characterized in that this Cdk is Cdk4 as the described method of claim 99.

101. method of screening potential cancerous swelling therapeutic agent, it is characterized in that, comprise: under the condition that the compound that may be the cancerous swelling therapeutic agent exists, make the conversion eukaryotic host cell growth that contains the mts gene that suddenlys change, measure the growth rate of this host cell then.

102., it is characterized in that it is by measuring the biologic activity of cell cycle protein dependent kinase (Cdk) that the growth rate of this host cell is measured as the described method of claim 102.

103. transgenic animal is characterized in that, it carries the MTS allelomorph from the sudden change of another animal in genome.

104., it is characterized in that the MTS allelomorph of this sudden change is the allelomorph that contains deletion mutation as the described transgenic animal of claim 103.

105., it is characterized in that the MTS allelomorph of this sudden change is interrupted allelomorph as the described transgenic animal of claim 103.

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