WO1999002682A1

WO1999002682A1 - p53 MUTANTS FOUND IN TUMORS

Info

Publication number: WO1999002682A1
Application number: PCT/US1998/013949
Authority: WO
Inventors: David H. Mack
Original assignee: Affymetrix, Inc.
Priority date: 1997-07-09
Filing date: 1998-07-09
Publication date: 1999-01-21

Abstract

Mutations in tumor suppressor p53 found in human cancers are disclosed which are useful for diagnosis, monitoring process of chemotherapy and detecting metastasis. Antibodies, polypeptides, primers, probes, specific for these mutations can be used for detection and monitoring.

Description

p53 MUTANTS FOUND IN TUMORS

This application claims the benefit of application U.S. Serial No. 60/052,805, filed July 9, 1997, as well as of application PCT/US98/01206 filed January 12, 1998, as well as of application U.S. Serial No. 60/035,327, filed January 13, 1997, as well as of application U.S. Serial No. 60/049,627, filed June 13, 1997. The disclosures of all applications whose benefit is claimed are expressly incorporated herein by reference. BACKGROUND OF THE INVENTION p53 is a commonly mutated gene associated with neoplasia; mutations in p53 are found in 50% or more of all human cancers. The p53 gene protein product is a nuclear phosphoprotein that functions in cell-cycle regulation and the preservation of genetic integrity (reviewed in Levine, A.J. p53, "The Cellular Gatekeeper for Growth and Division", Cell 3, 323-331 (1997)). The protein possesses numerous biochemical properties necessary to carry out these functions, including sequence-specific DNA binding activity, transcriptional activation, and transcriptional repression. The observation that p53 mutations found in human cancers overwhelmingly select for p53 gene products that have lost the ability to bind DNA and transcriptionally regulate target genes, strongly suggests that these properties are crucial in p53's regulation of cell proliferation and apoptosis.

Wild-type p53 protein transcriptionally activates a number of known genes that are linked with its tumor-suppressor activity and are responsible in part for p53-dependent functions in a cell. The biological and clinical importance of the p53 tumor suppressor gene is discussed in Velculescu, V.E. & El-Deiry, W.S., CLINICAL CHEMISTRY 6 Pt 1, 858-68 (1996). See also,

Soussi T, et al, ONCOGENE 5(7): 945-952 (July 1990); Levine, Arnold, J., et al, Cancer Prevention, Vol. 768 of the ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, 111-125 (September 30, 1995); Chumakov, P.M., Englehardt Institute of Molecular Biology, ACADEMY OF SCIENCE OF THE U.S.S.R., (August 2, 1990); Futreal, P.A., et al, NUCLEIC ACIDS RES. 19 (24), 6977 (1991); MOL. CELL. BlOL. 6:1379-1385 (1986); MOL. CELL BlOL. 7:961-963

(1987); Crawford, L.V., (1983). INT. REV. EXP. PATH., 25, 1-50; Oren, M. (1985). BlOCHIM. BlOPHYS. ACTA, 823, 67-68; and Jenkins, J.R. and Sturzbecher, H.W. (1988), In: THE ONCOGENE HANDBOOK, Reddy, E.P., Skalka, A.M. and Currant, T. (eds.) Elsevier, 403-423. See also U.S. Patent Nos. 5,532,220; 5,527,676; 4,871,838; 4,725,550; 5,085,983; 5,382,510;

5,411 ,860; 5,571,905; 5,604,113; 5,552,283; 5,569,824; and 5,620,848. See also PCT Published Application Nos. WO 95/32223; WO 94/08049; WO 94/08241; WO 91/03489; WO 95/19448; and WO 94/00603.

The cDNA sequence for wild-type p53 is set forth in SEQ ID NO: 1 (see Matlashewski et al., "Isolation and Characterization of a human p53 cDNA clone : expression of the p53 gene". EMBO J. 3:3257-3262 (1984)).

The amino acid sequence for wild- type p53 is set forth in SEQ ID NO: 2 .

(see Lamb,P. et al., "Characterization of the human p53 gene", MOL. CELL.

BIOL. 6 (5), 1379-1385 (1986)). Because of the apparent centrality of p53 to cancer, it is widely used as a diagnostic indicator. However, to make detection of mutations in p53 more sensitive, there is a need in the art for identification of additional mutations which occur in human cancers and for correlation of particular mutations with particular types of cancer.

SUMMARY OF THE INVENTION

The invention relates to p53 mutations. The invention also involves a method for detecting and distinguishing neoplastic tissues. This invention further rdates to the detection of mutations at the protein level which result from the altered p53 gene. This invention also rdates to the detection of p53 mutations by raising antibodies spedfic to the protein product of the expressed gene.

It is an object of the invention to provide isolated and purified nudeic acid molecules which contain a portion of a p53 coding sequence which contains a mutation found in a human tumor.

It is another object of the invention to provide isolated and purified p53 polypeptides which contain a mutation found in a tumor.

It is still another object of the invention to provide preparations of antibodies which spedfically bind to a p53 protein containing a mutation found in a tumor.

It is even another object of the invention to provide methods of identifying a cell or tissue with a defective p53, particularly in colorectal cancers.

These and other objects of the invention are provided by one or more of the emobdiments shown below. In one embodiment of the invention an isolated and purified nucleic add molecule is provided. The nucleic add molecule comprises at least 10 nudeotides of a mutant p53 coding sequence.

The nudeic add molecule comprises a contiguous sequence of nudeotides which indudes codon 163, and codon 163 spedfies an aspartic add residue. The nucleic add molecule is selected from the group consisting of: a single stranded coding strand, a single stranded anti-sense strand, a double stranded molecule.

According to another embodiment of the invention an isolated and purified nudeic add molecule is provided which comprises at least a 10 nudeotides of a mutant p53 coding sequence. The nucleic add molecule comprises a contiguous sequence of nudeotides which includes codon 427 or 179. The codon 427 or 179 in the nucleic add molecule spedfies a lysine residue or a glutamine residue, respectively. The nudeic add molecule is selected from the group consisting of: a single stranded coding strand, a single stranded anti-sense strand, a double stranded molecule.

In another embodiment of the invention an isolated and purified p53 polypeptide is provided which comprises at least six contiguous amino adds of p53. The p53 polypeptide has an aspartic add residue for p53 residue no. 163.

In another aspect of the invention an isolated and purified p53 polypeptide is provided which comprises at least six contiguous amino adds of p53. The p53 polypeptide has a lysine residue for p53 residue no. 427 or a glutamine residue at p53 residue no. 179. According to another aspect of the invention a preparation of antibodies is provided which spedfically bind to a p53 protein which has an aspartic add residue at position 163. The preparation does not spedfically bind to a p53 protein which has a tyrosine at position 163.

In another aspect of the invention a preparation of antibodies is provided which spedfically bind to a p53 protein which has a lysine residue at position 427 or a glutamine at position 179. The preprataion does not spedfically bind to a p53 protein which has a glutamate at position 427 or a histidine at position 179, respectively.

Another embodiment of the invention provides a method of identifying a cell or tissue with a defective p53. The method comprises the step of determining whether the cell or tissue harbors a p53 protein with an aspartic acid residue at position 163 or a lysine at position 427 or a glutamine at position 179. The presence of said amino adds indicates a defective p53.

According to another aspect of the invention a method is provided of identifying a colorectal cell or colorectal tissue with a defective p53. The method comprises the step of determining whether the colorectal cell or colorectal tissue harbors a p53 protein with a glutamine residue at position

179. The presence of said amino add indicates a defective p53.

Another aspect of the invention is a method of identifying a cell or tissue with a defective p53. The method comprises the step of determining whether the cell or tissue harbors a p53 gene with a G (guanine) at nucleotide

489 or an A (adenine) at nudeotide 1279 or an A (adenine) at nucleotide

537. The presence of any of said codons indicates a defective p53.

Yet another embodiment of the invention is a method of identifying a colorectal cell or colorectal tissue with a defertive p53. The method comprises the step of determining whether the colorectal cell or colorectal tissue harbors a p53 gene with an A (adenine) at nudeotide 537. The presence of said codon indicates a defective p53.

These and other embodiments of the invention which will become dear upon reading the more detailed description below, provide the art with additional tools for identifying and treating cancers.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a portion of the wild type p53 coding and amino add sequences. Fig. 1 also shows the sequence obtained from a Dukes D staged adenocardnoma of the colon. As shown, the tumor sequence contains a T to G base substitution resulting in a Y to D amino acid change at position designated 163.

Fig. 2 shows a portion of the wild type p53 coding and amino add sequences. Fig. 2 also shows the sequence obtained from a Dukes D staged adenocardnoma of the colon. As shown, the tumor sequence contains a T to A mutation at the nudeotide position designated 537 resulting in a his to gin mutation at amino acid position designated 179.

Fig. 3 shows a portion of the wild type p53 coding and amino add sequences. Fig. 3 also shows the sequence obtained from a cancer. As shown, the tumor sequence contains a G to A mutation at the nucleotide position designated 1279 resulting in a glu to lys mutation at amino add position designated 427. Figure 3 shows the result of GeneChip^® sequence analysis of p53 genes in normal and malignant breast epithelium cells. GeneChip® data analysis output is shown (bottom) that unambiguously identifies a G-A base change at nudeotide 1,279 of p53 in BT-474 resulting in a glutamic add to lysine amino add change in exon 8 (DNA binding domain).

DEFINITIONS An "oligonudeotide" or "nudeic add molecule" can be DNA or RNA, and single- or double-stranded, naturally or unnnaturally occurring as well as analogs thereof. Preferred oligonudeotides of the invention indude segments of DNA, or their complements induding any one of the variant sites shown in, for example, Figures 1 or 2 or 3. The segments are often between 5 and 100 bases, and often indude between 5-10, 5-20, 10-20, 10-50, 20-50 or 20- 100 bases. Typically the segments are at least about 10, 15, 18, or 20 nudeotides in length. Moreover, the segments may be limited to less than

100, 50, 30, 26, or 20 nudeotides in length. The variant site can occur within any position of the segment, although preferably it will occur approximatdy equidistant from the 5' and the 3' ends, or close to the 3' end. These positions fadlitate certain assays which rely on distinguishing between perfectly and imperfectly matched nucleic acid hybridizing partners. An oligonudeotide as used in the context of the present invention generally means a nudeic add or analog thereof without limitation on source or length. Thus, an oligonudeotide can indude as a nonlimiting example the full length cDNA or gDNA of the p53 gene.

"Hybridization probes" are oligonucleotides capable of binding in a base-spedfic manner to a complementary strand of nucleic add. Such probes include peptide nucleic adds, as described in Nielsen et al., SCIENCE 254, 1497-1500 (1991). Hybridizations are usually performed under stringent conditions, for example, at a salt concentration of no more than 1 M and a temperature of at least 25 ^CC. For example, conditions of 5X SSPE (750 mM NaCl, 50 mM NaPhosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-

30 °C are suitable for allele-spedfic probe hybridizations.

The term "primer" refers to a single-stranded oligonudeotide capable of acting as a point of initiation of template-direrted nucleic add synthesis under appropriate conditions {e.g., in the presence of four different nucleoside triphosphates and an agent for polymerization, such as, DNA or RNA polymerase or reverse transcriptase) in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer but typically ranges from 15 to 30 nudeotides. Short primer molecules generally require cooler temperatures to form suffidently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template but must be sufficiently complementary to hybridize with a template. The term primer site refers to the area of the target DNA to which a primer hybridizes. The term primer pair means a set of primers including a 5' upstream primer that hybridizes with the 5' end of a DNA sequence to be amplified and a 3', downstream primer that hybridizes with the complement of the 3' end of a sequence to be amplified. A "mutation" is a permanent alteration in the nucleotide sequence of an organism. A mutation may arise inter alia due to substitution of one nudeotide for another at the variant site. A transition is the replacement of one purine by another purine or one pyrimidine by another pyrimidine. A transversion is the replacement of a purine by a pyrimidine or vice versa. Single nudeotide mutations can also arise from a deletion of a nucleotide or an insertion of a nucleotide relative to a reference allele.

Alleles in the present application refers to nucleic adds containing different sequences including for example sequences that are wild-type at a locus and sequences containing a mutation or polymorphism of interest at the same locus.

"Isolated and purified" according to the present invention means that the substance is not present in the same milieu as it is in nature. Thus isolated and purified nudeic acids are present in the absence of other sequences with which they are assodated in the cell, including other genes on the same chromosome or other genes on different chromosomes. The substance need not be isolated and purified from a cell directly, but may be syntherized in vitro, in a recombinant organism, or in a transgenic manmal.

Intron-free sequences are typically derived from reverse transcription of an mRNA molecule. These can be susbsequently propagated as cDNA dones, without using an mRNA intermediate. Thus they need not be made directly by reverse transcription. DETAILED DESCRIPTION OF THE INVENTION

This invention provides useful nudeic adds and polypeptides, fragments and analogs thereof, encoding the p53 gene or pep tide product induding at least one mutation shown in Figures 1 or 2 or 3. Complements of the nucleic acid segments are also included. The nucleic acid segments can be DNA, RNA, and analogues thereof and can be double- or single- stranded. Some segments are 6 or more, 10 or more or 20 or more bases long. In some embodiments the bases are less than 100 bases long, less than 50 bases long, or less than 10 bases long. Those skilled in the art will appredate that the source and length of the polypeptides and nudeic adds of the present invention is any source and length suitable for the assay of choice. Accordingly, no restriction on source and length is contemplated by the present invention.

The invention further provides allele-spedfic oligonudeotides that hybridize to nudeic adds containing at least one of the mutations shown in

Figures 1 , 2 and 3 thdr complements. These oligonudeotides can be probes or primers. Also provided are isolated polypeptides and nudeic adds comprising the amino add or nuddc add sequences (induding complements) shown in Figures 1, 2 and 3. The invention further provides a method of analyzing a nudeic add or polypeptide from an individual. One such method determines which base or amino add is present at any one of the variant sites shown in Figures 1, 2, or 3. Optionally, a set of bases occupying a set of the variant sites shown in Table 1 is deteπr ned. This type of analysis can be performed on a plurality of individuals who are tested for the presence of a disease phenotype. The presence or absence of disease phenotype can then be correlated with a base or set of bases present (or amino add or sets of amino adds) at the variant sites in the individuals tested.

Methods which can be used to detect a mutant p53 gene sequence indude any analytical techniques which are known in the art. These include the use of nucleic add sequendng, hybridization to allele-spedfic probes, analysis of proteins produced from a gene in an in vitro transcription and translation system, etc.

Similarly, when mutations are being detected at the protein level, any technique known in the art can be used. These include the myriad immunological methods, such as ELISA, immunopredpitation, Western blots, immunohistochemistry, etc.

Any tissue known in the art to experience p53 mutations can be screened for the presence of mutations, as can tissues that are hitherto unknown to experience p53 mutations. These include leukemic cells, colorectal cancer cells, breast cancer cells, brain tumor cells, liver cells, kidney cells, lung cells, etc. Techniques for obtaining cancer cells relatively free of other cells to fadlitate the analysis are well known in the art and can be used here as well. Techniques for isolating nucleic adds and proteins from test samples are also well known, and any can be used as is deemed appropriate by the skilled artisan.

DESCRIPTION OF THE PRESENT INVENTION

I. Analysis of Mutations

A. Preparation of Samples

In general, mutations are detected in a target nudeic add from an individual being analyzed. For assay of genomic DNA, virtually any biological sample (other than pure red blood cells) is suitable. For example, convenient tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair. For assay of cDNA or mRNA, the tissue sample must be obtained from an organ in which the target nucleic acid is expressed. For example, if the target nucleic acid were a cytochrome P450 gene, the liver would be a suitable source. Many of the methods described below require amplification of DNA from target samples. This can be accomplished by e.g., PCR. See generalyl PCR TECHNOLOGY: PRINCIPLES AND APPLICATIONS FOR DNA AMPLIFICATION (ed. HA Erlich, Freeman Press, NY, NY, 1992); PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS (eds. Innis, et al., Academic Press, San Diego, CA, 1990); Mattila et al., NUCLEIC ACIDS RES. 19, 4967 (1991);

Eckert et al., PCR METHODS AND APPLICATIONS 1 , 17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Patent 4,683,202 (each of which is incorporated by reference for all purposes).

Other suitable amplification methods indude the ligase chain reaction (LCR) (see Wu and Wallace, Genomics 4, 560 (1989), Landegren et al.,

SCIENCE 241, 1077 (1988), transcription amplification (Kwoh et al., PROC. NATL. ACAD. SCI. USA 86, 1173 (1989)), and self-sustained sequence replication (Guatelli et al., PROC. NAT. ACAD. SCI. USA, 87, 1874 (1990)) and nucleic add based sequence amplification (NASBA). The latter two amplification methods involve isothermal reactions based on isothermal transcription, which produce both single stranded RNA (ssRNA) and double stranded DNA (dsDNA) as the amplification products in a ratio of about 30 or 100 to 1 , respertively.

B. Detection of Mutations in Target Nucleic Adds There are a variety of suitable procedures, some of which are described in turn. 1. Allele-Spedfic Probes

The design and use of allele-spedfic probes for analyzing polymorphisms (or mutations) is described by e.g., Saiki et al., NATURE 324, 163-166 (1986); Dattagupta, EP 235,726, Saiki, WO 89/11548. Allele- specific probes can be designed that hybridize to a segment of target DNA from one individual but do not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms in the respective segments from the two individuals. The same procedure applies to mutation detection. Probes are designed that hybridize to a target nucleic add containing the mutation but do not hybridize to target nucleic adds that do not contain the mutation.

Hybridization conditions should be suffidently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Some probes are designed to hybridize to a segment of target DNA such that the polymorphic site aligns with a central position (e.g., in a 15 mer at the 7 position; in a 16 mer, at either the 8 or 9 position) of the probe. This design of probe achieves good discrimination in hybridization between different allelic forms. Allele-spedfic probes are often used in pairs, one member of a pair showing a perfect match to a reference form of a target sequence and the other member showing a perfect match to a variant form. Several pairs of probes can then be immobilized on the same support for simultaneous analysis of multiple polymorphisms within the same target sequence. 2. Tiling Arrays

The mutations can also be identified by hybridization to nucleic add arrays, some example of which are described by WO 95/11995 (incorporated by reference in its entirety for all purposes). The same array or a different array can be used for analysis of characterized mutations or polymorphisms. WO 95/11995 also describes subarrays that are optimized for detection of a variant forms of a precharacterized polymorphism or mutation. Such a subarray contains probes designed to be complementary to a second reference sequence, which is an allelic variant of the first reference sequence. The second group of probes is designed by the same prindples as described in the Examples except that the probes exhibit complementarily to the second reference sequence. The inclusion of a second group (or further groups) can be particularly useful for analyzing short subsequences of the primary reference sequence in which multiple mutations are expected to occur within a short distance commensurate with the length of the probes (i.e., two or more mutations within 9 to 21 bases).

The existence of a variant site is also manifested by differences in normalized hybridization intensities of probes flanking a mutation or polymorphic site when the probes hybridized to corresponding targets from wildtype or different individuals, respectively. For example, relative loss of hybridization intensity in a "footprint" of probes flanking a polymorphism or mutation site signals a difference between the target and reference (i.e., a polymorphism) (see EP 717,113, incorporated by reference in its entirety for all purposes). Additionally, hybridization intensities for corresponding targets from different individuals can be dassified into groups or clusters suggested by the data, not defined a priori, such that isolates in a give cluster tend to be similar and isolates in different dusters tend to be dissimilar. (See US 08/797,812 incorporated by reference in its entirety for all purposes).

3. Allele-Spedfic Primers An allele-spedfic primer hybridizes to a site on target DNA overlapping a variant gene site and only primes amplification of an allelic form to which the primer exhibits perfect complementarily. See Gibbs, NUCLEIC ACID RES. 17, 2427-2448 (1989). This primer is used in conjunction with a second primer which hybridizes at a distal site. Amplification proceeds from the two primers leading to a detectable product signifying the particular allelic form is present. A control is usually performed with a second pair of primers, one of which shows a single base mismatch at the polymorphic or mutation site and the other of which exhibits perfect complementarily to a distal site. The single-base mismatch prevents amplification and no detectable product is formed. The method works best when the mismatch is induded in the 3'-most position of the oligonudeotide aligned with the polymorphism or mutation because this position is most destabilizing to elongation from the primer. See, e.g., WO 93/22456.

4. Direct-Sequendng The direct analysis of the sequence of the gene mutation of the present invention can be accomplished using either the dideoxy chain termination method or the Maxam Gilbert method (see Sambrook et al., MOLECULAR CLONLNG, A LABORATORY MANUAL (2nd Ed., CSHP, New York 1989); Zyskind et al, RECOMBINANT DNA LABORATORY MANUAL, (Acad. Press, 1988)).

5. Denaturing Gradient Gel Electrophoresis Amplification products generated using the polymerase chain reaction can be analyzed by the use of denaturing gradient gel electrophoresis. Different alleles can be identified based on the different sequence-dependent melting properties and electrophoretic migration of

DNA in solution. Erlich, ed., PCR TECHNOLOGY, PRINCIPLES AND APPLICAΉONS FOR DNAAMPLIFICAΉON, (W.H. Freeman and Co, New York, 1992), Chapter 7.

6. Single-Strand Conformation Polymorphism or Mutation Analysis

Alleles of target sequences can be differentiated using single- strand conformation polymorphism (or mutation) analysis, which identifies base differences by alteration in electrophoretic migration of single stranded PCR products, as described in Orita et al., PROC. NAT. ACAD. SCI. 86, 2766- 2770 (1989). Amplified PCR products can be generated as described above, and heated or otherwise denatured, to form single stranded amplification products. Single-stranded nudeic adds may refold or form secondary structures which are partially dependent on the base sequence. The different electrophoretic mobilities of single-stranded amplification products can be related to base-sequence difference between alleles of target sequences.

III. Methods of Use After determining a mutation is present in an individual, this information can be used in a number of methods induding without limitation those taught below.

A. Correlation of Mutation with Phenotypic Traits

In general, a genetic mutation may result in variant gene products which, in turn, can contribute to the phenotype of an organism in different ways. Some mutations occur within a protein coding sequence and contribute to phenotype by affecting protein structure. The effect may be neutral, beneficial or detrimental, or both benefidal and detrimental, depending on the drcumstances. Other mutations may occur in noncoding regions but may exert phenotypic effects indirectly via influence on replication, transcription, and translation. A single mutation may affect more than one phenotypic trait. Likewise, a single phenotypic trait may be affected by a mutation in different genes. It is believed that the mutations identified herein are selected in neoplasms because those mutations confer an advantage to neoplastic tissues. Hence, the mutations of the present invention are particularly useful in the identification and differentiation of abnormal tissue types.

B. Immunohistochemical Methods

Polyclonal and/or monoclonal antibodies that spedfically bind to variant gene products but not to corresponding prototypical (wild-type) gene products are also provided. Antibodies can be made by injecting mice or other animals with the variant gene product or synthetic peptide fragments thereof. Monoclonal antibodies are screened as are described, for example, in Hariow SL Lane, ANTIBODIES, A LABORATORY MANUAL, Cold Spring Harbor Press, New York (1988); Goding, MONOCLONAL ANTIBODIES, PRINCIPLES AND PRACTICE (2d ed.) Academic Press, New York (1986). Monodonal antibodies are tested for spedfic immunoreactivity with a variant gene product and lack of immunoreactivity to the corresponding prototypical gene produd. These antibodies are useful in diagnostic assays for detection of the variant form, or as an active ingredient in a pharmaceutical composition.

IV. Modified Polypeptides and Gene Sequences

The invention further provides variant forms of nucleic adds and corresponding proteins. The nucleic adds comprise at least a portion of one of the sequences described in the Figures in which the mutation position is occupied by one of the alternative bases for that position. Some of the useful nuddc adds of the present invention also indude nucleic acids encoding full- length variant forms of p53. Variant genes can be expressed in an expression vector in which a variant gene is operably linked to a native or other promoter. Usually, the promoter is a eukaryotic promoter for expression in a mammalian cell. The transcription regulation sequences typically indude a heterologous promoter and optionally an enhancer which is recognized by the host. The selertion of an appropriate promoter, for example trp, lac, phage promoters, glycolytic enzyme promoters and tRNA promoters, depends on the host selected. Commerdally available expression vectors can be used. Vedors can indude host-recognized replication systems, amplifiable genes, sdectable markers, host sequences useful for insertion into the host genome, and the like. The means of introdudng the expression construct into a host cell varies depending upon the particular construction and the target host. Suitable means indude fusion, conjugation, transfedion, transduction, dectroporation or injection, as described in Sambrook, supra. A wide variety of host cells can be employed for expression of the variant gene, both prokaryotic and eukaryotic. Suitable host cells include baderia such as E. ωli, yeast, filamentous fungi, insect cells, mammalian cells, typically immortalized, e.g., mouse, CHO, human and monkey cell lines and derivatives thereof. Preferred host cells are able to process the variant gene produd to produce an appropriate mature polypeptide. Processing includes glycosylation, ubiquitination, disulfide bond formation, general post- translational modification, and the like.

The protein may be isolated by conventional means of protein biochemistry and purification to obtain a substantially pure product, i.e., 80, 95 or 99% free of cell component contaminants, as described in Jacoby, METHODS IN ENZYMOLOGY Volume 104, Academic Press, New York (1984); Scopes, PROTEIN PURIFICATION, PRINCIPLES AND PRACTICE, 2nd Edition, Springer- Verlag, New York (1987); and Deutscher (ed) , GUIDE TO PROTEIN

PURIFICATION, METHODS IN ENZYMOLOGY, Vol. 182 (1990). If the protein is secreted, it can be isolated from the supernatant in which the host cell is grown. If not secreted, the protein can be isolated from a lysate of the host cells. The invention further provides transgenic nonhuman animals capable of expressing an exogenous variant gene. Expression of an exogenous variant gene is usually achieved by operably linking the gene to a promoter and optionally an enhancer, and microinjecting the construct into a zygote. See Hogan et al., MANIPULATING THE MOUSE EMBRYO, A LABORATORY MANUAL, Cold Spring Harbor Laboratory. Inactivation of an endogenous variant genes can be achieved by forming a transgene in which a cloned variant gene is inactivated by insertion of a positive sdection marker. See Capecchi, SCIENCE 244, 1288-1292 (1989). The transgene is then introduced into an embryonic stem cell, where it undergoes homologous recombination with an endogenous variant gene. Mice and other rodents are preferred animals.

Such animals provide useful drug screening systems.

In addition to substantially full-length polypeptides expressed by variant genes, the present invention includes biologically active fragments of the polypeptides, or analogs thereof, induding organic molecules which simulate the interactions of the peptides. Biologically active fragments indude any portion of the full-length polypeptide which confers a biological function on the variant gene product, induding ligand binding, and antibody binding. Ligand binding indudes binding by nucleic adds, proteins or polypeptides, small biologically active molecules, or large cellular structures. Substances that recognize the mutations identified herein can be queried using peptide arrays. Particularly useful peptide arrays are high density peptide arrays. See U.S. Patent. No. 5,143,854, 5,405,783; and

5,324,633.

Protdns and polypeptides which comprise the mutations can be used, inter alia, to immunize animals to raise antibodies.

V. Kits The invention further provides kits comprising at least one allele- specific oligonudeotide as described above. Often, the kits contain one or more pairs of allele-spedfic oligonudeotides useful in the practice of the present invention. In some kits, the allele-spedfic oligonudeotides are provided immobilized to a substrate. For example, the same substrate can comprise allele-spedfic oligonudeotide probes for detecting the mutations shown in the Figures. Optional additional components of the kit indude, for example, restriction enzymes, reverse-transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions. Usually, the kit also contains instrudions for carrying out the methods as described herein.

Similarly, the kits may contain the instructions and all or some of the reagents necessary to perform antibody assays. Preferably, such kits contain polyclonal or monoclonal antibodies useful in detecting the polypeptide product of at least one of the p53 mutations identified herein.

The kits of the present invention can also include peptide arrays. EXAMPLES Example 1

The mutations shown in Figures 1 and 2 were identified by resequendng target sequences from normal (control) and neoplastic colon tissue samples both taken from patients with a Dukes D staged colon adenocardnoma. The resequendng was accomplished by hybridization to probes immobilized to microfabricated arrays: the GeneChip® p53 probe array (Affymetrix, Inc., Santa Clara, CA, USA). Analysis of the target sequences isolated from those tissues revealed two separate mutations of the p53 gene, whereas the target sequences from the normal colon tissue from each patient was wild-type at the relevant p53 locus. In general, P53 mutation detection can be performed using the GeneChip® p53 assay and reagents available from, through, or as suggested by Affymetrix, Inc. See also WO 95/11995.

Example 2

The analysis of BT-474 versus HT-125 p53 genomic DNA using the p53 genotyping array revealed a single base substitution of G to A in exon 8 (DNA binding domain), resulting in an amino add change at position 285 from E to K (Fig. 3). The hybridization signal difference centered about the mutation identified by the footprint analysis (see Fig. 3), and subsequent base calling of a single genotype (see Fig. 3) in BT-474 by the GeneChip software indicated that this cardnoma had a loss of heterozygosity at the p53 locus (confirmed by dideoxy sequence analysis). These data unambiguously show that the loss of wild-type p53 transcriptional function in these cells was due to the absence of wild-type p53 protein. We have applied this analysis to other breast cardnomas with similar outcomes correlating altered gene expression patterns of targets of p53 transcriptional modulation with mutant p53 gene status.

p53 PCR and labeling for re-sequence analysis by array hybridization. The p53 gene was genotyped by amplifying coding exons

2-11 in a 100 μl multiplex PCR reaction using 100 ng of genomic DNA extracted from cells using a QIAmp Blood Kit (Qiagen). PCR Buffer II (Perkin-Elmer) was used at IX along with 2.5mM MgCh, 200 μM of each dNTP and 10 units of Taq Polymerase Gold (Perkin-Elmer). The multiplex PCR was performed using 10 exon-spedfic primers (Table 4) with the following cycling conditions: 1 cycle at 94 °C (5 min), 50 cydes of 94°C (30 sec), 60°C (30 sec) and 72°C (30 sec), followed by 1 cyde at 72°C (7 min). 45 μl of the PCR reaction was then fragmented and dephosphorylated by incubation at 25° C for 15 min with 0.25 units of Amp Grade DNAse I (Gibco/BRL) and 2.5 units of Calf Alkaline

Phosphatase (Gibco/BRL), followed by heat-inactivation at 99 °C for 10 min. The fragmented PCR products were then labeled in a 100 μl reaction using 10 μM fiourecein-N6-ddATP (Dupont-NEN) and 25 units of terminal transferase (Boehringer Mannheim) in 200 μM K-Cacodylate, 25 mM Tris-HCl (pH 6.6), 0.25 mg/ml BSA and 2.5 mM CoC The labeling reaction was incubated at 37 °C for 45 min and heat-inactivated at 99°C for 5 min. p53 re-sequence analysis array hybridization and scanning. The fragmented, labeled PCR readion was hybridized to the p53 re- sequence analysis array in 6xSSPE-T containing 2mg/ml BSA and 1.67 nM fluorescein-labeled control oligo (5'-CTGAACGGTAGCATCTTGAC-3') at 45 °C for 30 min. The array was then washed with 3X SSPE-T at 35 °C for 4 cycles (10 drains-fiUs/cyde) in the GeneChip Fluidics Station (RELA). The hybridized p53 array was scanned using an argon-ion laser scanner (Hewlett-Packard) with a resolution setting of 6.0 μm/pixel (—70 pixels/probe cell) and wavelength detection setting of 530 nm. A fluorescence image was created, intensity information analyzed and nucleotide determination made by GeneChip Analysis Software (Affymetrix). Footprint analysis was done using Ulysses Analysis Software (Affymetrix) essentially as described. All publications and patent applications dted above are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication or patent application were spedfically and individually indicated to be so incorporated by reference. Although the present invention has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be apparent that certain changes and modifications may be practiced within the scope of the invention. The scope of the invention should, therefore, be considered with reference to the appended daims along with their full scope of equivalents.

Claims

I claim:

1. An isolated and purified nucleic acid molecule comprising at least 10 nudeotides of a mutant p53 coding sequence, wherein the fragment comprises a contiguous sequence of nudeotides which includes codon 163, wherein codon 163 specifies an amino add residue which is not tyrosine, wherein the polynudeotide molecule is seleded from the group consisting of: a single stranded coding strand, a single stranded anti-sense strand, a double stranded molecule.

2. An isolated and purified nucleic add molecule comprising at least a

10 nudeotides of a mutant p53 coding sequence, wherein the fragment comprises a contiguous sequence of nudeotides which includes codon 427, wherein codon 427 spedfies an amino add residue which is not glutamate, wherein the polynudeotide molecule is selected from the group consisting of: a single stranded coding strand, a single stranded anti-sense strand, a double stranded molecule.

3. An isolated and purified nucleic add molecule comprising at least 10 nudeotides of a mutant p53 coding sequence, wherein the fragment comprises a contiguous sequence of nudeotides which includes codon 179, wherein codon 179 spedfies an amino add residue which is not histidine, wherein the polynudeotide molecule is selected from the group consisting of: a single stranded coding strand, a single stranded anti-sense strand, a double stranded molecule.

4. The nucleic add molecule of claim 1 wherein the amino add residue is aspartic add.

5. The nucleic add molecule of claim 2 wherein the amino add residue is lysine.

6. The nucleic acid molecule of claim 3 wherein the amino add residue is glutamine.

7. The nudeic add molecule of claim 1, 2, or 3 which is intron-free.

8. The nudeic add molecule of claim 1, 2, or 3 which comprises an intron.

9. The nucleic add molecule of claim 1 wherein codon 163 comprises a T ΓåÆG substitution at nudeotide 487.

10. The nucleic add molecule of claim 2 wherein codon 427 comprises a G ->A substitution at nucleotide 1279.

11. The nucleic add molecule of claim 3 wherein codon 179 comprises a T ΓåÆA substitution at nucleotide 537.

12. The nudeic add molecule of daim 1 , 2, or 3 which is single stranded.

13. The nudeic add molecule of daim 1, 2, or 3 which is RNA.

14. The nucleic add molecule of claim 1 , 2, or 3 which is less than 50 nudeotides in length.

15. The nudeic add molecule of daim 1 , 2, or 3 which is less than 30 nudeotides in length.

16. The nucleic add molecule of claim 1 , 2, or 3 which is less than 26 nudeotides in length.

17. The nudeic add molecule of claim 1 , 2, or 3 which is greater than

15 nudeotides in length. 18. The nucleic add molecule of daim 1 , 2, or 3 which is greater than

18 nudeotides in length.

19. The nucleic add molecule of claim 1, 2, or 3 which is greater than 20 nudeotides in length.

20. The nudeic add molecule of daim 1 wherein codon 163 is approximately equidistant from both ends of the nucleic add molecule.

21. The nudeic add molecule of daim 2 wherein codon 427 is approximately equidistant from both ends of the nucleic add molecule.

22. The nucleic add molecule of claim 3 wherein codon 179 is approximately equidistant from both ends of the nucleic acid molecule.

23. An isolated and purified p53 polypeptide which comprises at least six contiguous amino adds of p53 wherein the p53 polypeptide has an amino add residue for p53 residue no. 163 which is not tyrosine.

24. An isolated and purified p53 polypeptide which comprises at least six contiguous amino adds of p53 wherein the p53 polypeptide has an amino add residue for p53 residue no. 427 which is not glutamate.

25. An isolated and purified p53 polypeptide which comprises at least six contiguous amino adds of p53 wherein the p53 polypeptide has an amino add residue for p53 residue no. 179 which is not histidine.

26. The p53 polypeptide of claim23 wherein the amino add residue is aspartic add.

27. The p53 polypeptide of claim 24 wherein the amino add residue is lysine.

28. The p53 polypeptide of claim23 wherein the amino add residue is glutamine.

29. The isolated and purified p53 polypeptide of claim 23, 24, or 25 which comprises at least 10 contiguous amino adds of p53.

30. The isolated and purified p53 polypeptide of claim 23, 24, or 25 which comprises at least 12 contiguous amino adds of p53.

31. The isolated and purified p53 polypeptide of daim 23, 24, or 25 which comprises at least 15 contiguous amino adds of p53.

32. The isolated and purified p53 polypeptide of claim 23, 24, or 25 which comprises at least 20 contiguous amino adds of p53.

33. A preparation of antibodies which spedfically bind to a p53 protein which has an aspartic add residue at position 163 and does not spedfically bind to a p53 protein which has a tyrosine at position 163.

34. A preparation of antibodies which spedfically bind to a p53 protein which has a lysine residue at position 427 and does not spedfically bind to a p53 protein which has a glutamate at position 427.

35. A preparation of antibodies which spedfically bind to a p53 protein which has an glutamine residue at position 179 and does not spedfically bind to a p53 protein which has a histidine at position 179.

36. The preparation of claim 34, 35, or 36 wherein the antibodies are monoclonal.

37. The preparation of claim 34, 35, or 36 wherein the antibodies are affinity-purified.

38. A method of identifying a cell or tissue with a defective p53, comprising the step of: determining whether the cell or tissue harbors a p53 protein with a glutamine at position 179, an aspartic acid residue at position 163 or a lysine at position 427, wherein the presence of either of said amino acids indicates a defective p53.

39. The method of claim 38 wherein the cell or tissue is a colorectal cell or colorectal tissue.

40. The method of daim 38 wherein the step of determining utilizes an antibody which spedfically binds to a p53 protein as redted, but which does not bind to a wild-type p53 protein.

41. A method of identifying a cell or tissue with a defective p53, comprising the step of: determining whether the cell or tissue harbors a p53 gene with a A (adenine) at nudeotide 537, a G (guanine) at nucleotide 489, or an A (adenine) at nucleotide 1279, wherein the presence of either of said codons indicates a defective p53.

42. The method of claim 41 wherein the cell or tissue is a colorectal cell or colorectal tissue.

43. The method of claim 41 wherein the step of determining utilizes a single-stranded oligonudeotide whidi hybridizes to a p53 gene.

44. The method of claim 41 wherein the oligonudeotide is attached to a solid support.

45. The method of claim 44 wherein the solid support is an array of oligonudeotide probes.