WO1995015334A1

WO1995015334A1 - cDNA PROBE FOR BREAST CANCER DIAGNOSIS AND TREATMENT

Info

Publication number: WO1995015334A1
Application number: PCT/US1994/013823
Authority: WO
Inventors: David R. Schott; Helene S. Smith; Julielani Chang
Original assignee: California Pacific Medical Center Research Institute
Priority date: 1993-11-30
Filing date: 1994-11-30
Publication date: 1995-06-08
Also published as: AU1214995A

Abstract

Some cDNA probes cloned from an mRNA coded for by an isolated gene (designated Brush-1) located at 13q12-q13 that is useful in diagnosis and treatment of breast cancer are disclosed. The probe provides a means for detection of premalignant mammary cells and early detection of breast cancer. It is also useful in designing therapeutic treatments for these conditions by traditional pharmaceutical methods or gene therapy.

Description

CDNA PROBE FOR BREAST CANCER DIAGNOSIS AND TREATMENT

FIELD OF THE INVENTION

The invention herein was made with government support under contract 5P01 CA 44768-09 with the National Cancer Institute. The federal government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

Breast cancer is one of the most common malignancies found among women above the age of 35 and results in thousands of deaths in the United States each year. Current treatments, including radiation, chemotherapy and mastectomy, have been successful in halting or slowing the disease's progress, but these known treatments haVe many undesirable side effects of their own.

Early detection of the disease is essential to a positive prognosis for breast cancer treatment. Freguently, by the time a tumor is detected, the disease has already progressed to the point where successful treatment by known methods is difficult or impossible.

Research has produced some new compounds and methods for diagnosis and treatment of certain types of cancerous conditions. U.S. Patent No. 5,262,528, for instance, is directed to a cDNA probe differentiating normal and cancerous tissues and U.S. Patent No. 4,942,123 is directed to a method of 5 diagnosis of retinoblastoma and "involved cancers", said to include breast cancer.

Inactivation of tumor suppressor genes may play an important role in teaaan cancers, including breast cancer. See Ponder „ -. , ϋEatøre (Lond.) 335:

10 400-402 (1988); Sager, R. , Science 246: 14(16-1412

(1989) . This is thought to occur by the inactivation of one allele and the subsequent loss or replacement of the other allele contained on a chromosomal segment. Several localized regions have been

15 implicated by the coincidence of their loss in various breast tumors. These include the short arms of chromosomes 3, 17 and 18 and sites on the long arms of chromosomes 1, 13 and 22. See Sato, T. , et al . , Cancer Res . 50: 7184-7189 (1990); Devilee, p.,

20 et al . , Int . J. Cancer 47: 817-821 (1991); Chen, L-C, et al . , J. Natl . Cancer Inst . 84: 506-510 (1992). Of particular interest is the chromosome 13q region which shows relatively frequent loss of heterozygosity (LOH) in breast tumors suggesting an

25 important role in breast cancer initiation and/or progression. See Lundberg, C . , et al . , Proc . Natl . Acad . Sci . USA 84: 2372-2376 (1987); Devilee, p., et al . , Genomics 5: 554-560 (1989). Most of these studies have focussed on the ql4 region which

30 contains the retinoblastoma (RBI) gene located at 13ql4.2. RBI, the gene involved in heredity and sporadic retinoblastoma, was also the first gene identified and characterized as a tumor suppressor gene. See Stanbridge, E.J. , Functional evidence for

35. human tumour suppressor genes : chromosome and molecular genetic studies, in GENETIC SURVEYS 12: TUMOUR SUPPRESSOR GENES, THE CELL CYCLE AND CANCER 5- 24 (1992)). The relationship between RBI and breast cancer, however, is not clear. Subsequent studies have shown that the LOH for RBI in breast cancer is not correlated with the loss of RBI gene expression. See Borg, A., et al . , Cancer Res . 52: 2991-2994 (1992) . In addition, LOH in the region next to RBI has been found in human breast carcinoma while the RBI gene itself did not show such a genetic change. See Devilee, p., et al . , Genomics 5: 554-560 (1989) . Hence, upon closer re-examination of this region, the finding has been made that RBI expression, at least for mRNA, apparently is not affected by LOH in the region which includes RBI. Instead, a proximal gene demonstrates the expected pattern of a tumor suppressor gene for breast cancer.

The gene cloned and sequenced as described herein, Brush- 1, may represent yet another member of a new class of tumor suppressor genes that function directly as RNA or as the RNA component of a ribonucleoprotein as has been described for the H19 gene (Brannan et al . , 1990, Mol. Cell. Biol. 10:28 Hao et al . , 1993, Nature 365:764). Both Brush-1 and H19 are expressed as a polyadenlyated RNA; are expressed at higher levels in fetal as compared to adult tissues; contain multiple small open reading frames; are both conserved in the monkey genome (shown by zoo blot hybridization) ; are located in regions of frequent LOH; and show loss of RNA expression in tumors demonstrating this LOH.

Known applications of sequenced genes include use of the sequences or of RNA or amino acid sequences derived therefrom for diagnosis or treatment of the corresponding disease. Accordingly, it is useful for the diagnosis and treatment of breast cancer to isolate and further characterize this gene in the region next to RBI.

An object of the present invention is to provide a cDNA probe derived from this gene (designated Brush-1) useful in diagnosis and treatment of breast cancer.

Additional objects and ad-vpaaaifcag/es σ>ff fctte invention will be set forth in the descriptions of the preferred embodiments which follows, and in part will be obvious from the description, or will be learned by practice of the invention.

SUMMARY OF THE INVENTION

The present invention is directed to a novel DNA sequence complementary to an mRNA coded for by an isolated gene (designated Brush-1) located at 13ql2-ql3 that is useful as a probe in diagnosis and treatment of breast cancer. The probe provides a means for detection of premalignant mammary cells and early detection of breast cancer. It is also useful in designing therapeutic treatments for these conditions by traditional pharmaceutical methods or gene therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are incorporated in and constitute a part of the specification.

Figure 1 depicts an autoradiogram of a Northern blot analysis of Brush-1 mRNA. 10 μg per lane of polyadenylated RNA were analyzed by probing with the 1.5 kb Brush-1 cDNA representing the most 3 '-region.

Figure 2 depicts an autoradiogram of a RT- PCR Analysis. Products from RT-PCR run on 1% agarose gel and stained with ethidium bromide. RNA source: lanes 2-4, CAMA1; 5-7, DU4475; 8-10, G94; 11-13, MDA468; Amplimers: lanes 2,5,8 & 11, Brush-1; lanes 3,6,;9,12, RBI; lanes 4,7,10,13, j8-Actin; pGEM marker: lanes 1 and 14.

Figure 3 depicts the position of Brush-1 cDNA clones relative to the 4.7kb mRNA.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following describes the characterization of the gene (designated Brush-1) localized to the 13q region proximal to RBI that is differentially expressed in normal versus tumor mammary epithelial cells and the isolation and derivation of mRNA and cDNA sequences therefrom. This description does not limit the invention and those of ordinary skill in the art will recognize that many variations on this method can be used with equivalent efficacy produce a cDNA probe.

The growth conditions for the various breast cancer cell lines and normal cells isolated from reduction mammoplasties are described in Smith, H.S., In vitro models in human breast cancer in BREAST DISEASES, 2ND EDITION 181-189 (J.R. Harris, et al . , eds., 1991). Human breast primary tumor samples and paired normal skin tissues were collected from 76 individuals. Tumor samples were dissected to remove most of the normal tissue, and stored in liquid nitrogen until use. If the skin tissues are too small to isolate sufficient DNA for analysis, cultured skin fibroblasts from the same patient may be used to extract DNA.

DNA isolation was performed as described by Kallioniemi, A., et al . , Cytogenet . Cell Genet . 60: 190-193 (1962) , incorporated by reference herein. Total RNA was isolated from both tissue culture cells and primary tumors using the Ultraspec^™ RNA method (Biotecx Laboratories, Inc., TX) . Final RNA pellets were resuspended in DEPC water and then stored at - 70°C. Procedures for polyadenylated mRNA selection and subsequent Northern analysis are described in Sa brook, J. , Fritsch, E.F. & Maniatis, T. , MOLECULAR CLONING: A LABORATORY MANUAL (1992).

Single stranded cDNA was synthesized by oligo(dT) priming (0.5 μg) from 3μg of total RNA using 20U of M-MLV Reverse Transcriptase (RT) (Gibco, BRL) in a final volume of 20 μL. The RT enzyme was inactivated by incubation at 70°C for 10 minutes and the product was diluted to 200 μl. A 5μl aliquot of cDNA was used directly for each PCR amplification. Specific amplification for each of three different mRNA species was achieved using sequence specific primers. Amplification primers for the β-Actin gene were obtained from Clontech Laboratories (Palo Alto, CA) and consisted of the following sequences; 5'-

ATGGATGATGATATCGCCGCG-3' and 5•-CTAGAAGCATTTGCGGTGGAC GATGGAGGGGCC-3' . The mRNA from the RBI gene was amplified using the previously described primers: C3-5, 5'-TACTGCAAATGCAGAGACACA-3' and C4-3, 5'-TGTTC CCTCCAGGAATCCGTA-3' (Mori, N. , et al . , Oncogene 5: 1713-1717 (1990)). Both of these primer pairs were chosen to span intron sequences in order to ensure that the resulting products were not due to amplification from genomic DNA. Brush-1 mRNA was amplified using primers homologous to regions within the Brush-1 sequence: 5*-TTAGTGGCACTTTATTC-3 ' and 5'-CATCAGTGTAGCCA AGC-3 • . This primer pair does not span an intron and Reverse Transcription-Polymerase Chains Reaction (RT-PCR) was conducted both with and ^•wifc&αo.: the RT enzyme to assure that results did not reflect DNA contamination. The PCR reaction mixture (50 μL final vol.) consisted of: template cDNA, 1.5 mM MgCl₂, 200 μM dNTPs (each) , 20 p ol of each primer pair and 1 U of Taq DNA polymerase (Promega) . The entire PCR mixture was heated to 95°C for 3 min to assure complete dissociation of the template sequences and then subject to 35 cycles of amplification under the following conditions: 95°C for 30 s, 50°C for 30 s and 72°C for 3 min with a final extension at 72°C for 10 min. The PCR products were separated on a 1% agarose gel and ethidium bromide stained for visualization.

LOH analyses used DNA from both tumors and normal tissues. DNA aliquots (40 ng) were used as templates for PCR amplification of polymorphic markers (Weissenbach, J. , et al . , Nature (Lond . ) , 359: 794-801 (1992)) using primers specific for the D13S219 region at 13ql3 (Research Genetics, Huntsville, AL) . These primers flank a CA repeat polymorphism localized to chromosome 13ql3, proximal to RBI. Primers for the RBI gene (Brandt, B., et al . , Am . J. Hum . Genet . 51: 1450-1451 (1992) flank a variable number terminal repeat (VNTR) which is highly polymorphic. PCR conditions used for the LOH analyses at both the D13S219 and RBI sites are those described for the RT-PCR analyses with the following modifications: Cycle conditions for D13S219 were 35 cycles of amplification under the following conditions: 94°C for 30 s, 56°C for 30 s and 72°C for 30 s with a final extension at 72°C for 10 min. Cycle conditions for RBI VNTR were 35 cycles of amplification under the following conditions: 95°C for 45 s, 53°C for 25 s and 72°C for 2 min. with a final extension at 72°C for 10 min. All PCR products were separated by electrophoresis on a 6% polyacrylamide gel and stained with ethidium bromide for visualization. PCR products from amplification of paired normal and tumor DNA from the same individual were compared to determine first, if the individual was heterozygous at the tested site and second, whether one of the alleles had been lost indicating that there had been a loss of heterozygosity (LOH) .

The 4.UN 1.5 kb cDNA fragment (see Figure 3) was subcloned into the Bluescript plasmid (Stratagene) . Both strands of the cDNA fragment were sequenced using the Sequenase 2.0 system of dideoxynucleotide chain termination (US Biochemicals) . The sequence was analyzed using the Eugene (Baylor College of Medicine) sequence analysis program. The Brush-1 cDNA fragment was labeled with ³²P-dCTP using the Multiprime labeling system (Amersham) . This probe was used to screen a total of 5 X 10⁵ independent clones from an EMBL-3 human placental genomic library (Clontech, Palo Alto, CA) . Two genomic clones corresponding to this cDNA were isolated.

One additional cDNA clone, designated 4.11T, was isolated by using the 4.11N as a probe for screening a cDNA library derived from breast tumor mRNA. Two other clones, designated 4.11K1 and 4.11K2 were isolated, and the relative positions of the clones within the mRNA were determined using the RACE method as described by Frohman, et al . , Proc . Natl . Acad . Sci . USA 85: 8998-9002 (1988) (see Figure 3).

The Northern blots were prepared as described by Sambrook, J. , Fritsch, E.F. & Maniatis, T., MOLECULAR CLONING: A LABORATORY MANUAL (1992) using ϊ.O ug of polγ-A.⁺ selected RNA. These were probed msins the radioac ively labelled cDNA fragment described above. Εfae EMBL-3 genomic DNA clones described above served as Brush-1 templates for the FISH analysis. The RB probe and methods used for the FISH analysis are previously described in Kallioniemi, A., et al . , Cytogenet . Cell Genet . 60: 190-193 (1962).

The Brush-1 mRNA was initially detected on

Northern gels of normal breast epithelium RNA at levels comparable to those seen for RBI (see Figure 1). Brush-1 codes for a single 4.7 kb mRNA. An RT- PCR approach was used for a survey of breast cancer cell lines in order to compare expression for Brush-1 with RBI (which also codes for a 4.7 kb mRNA). An example of this is seen in Figure 2 where RNAs from normal breast epithelium and three breast cancer cell lines were analyzed with this RT-PCR technique. After the initial RT step, three different amplimer sets were used to analyze each of the newly synthesized cDNAs. The first amplimer set was specific to the Brush-1 mRNA, the second was specific to the RBI mRNA and the third, for β-Actin, served as a control. The expected sizes of the amplified DNA products were 592,539 and 1126 base pairs for Brush- 1, RBI and β-Actin, respectively. Two of the breast cancer cell lines (CAMA1 and DU4475) have negligible expression for Brush-1 whereas both the normal breast epithelium (G94) and another breast cancer cell line (MDA468) express high levels of the Brush-1 mRNA. All four types of breast cells have high levels of β- Actin expression and only DU4475 cells do not express RBI.

This survey was extended to additional samples of both normal breast epithelium and breast cancer cell lines. The results are shown in Table 1.

Table 1

Types Expression Expiession LOH at ooff SSppeecciimmeenn ooff BBrruusshh--1l*" ooff RRBBII** 13ql3- σl4^b

A. Primary Breast Tumors

B200 + +

B201 + +

B212 + +

B381 + +

B317 - + +

B349 - + +

B398 - + + B B440066 - — + + +

B. Cells in culture

1. Normal Mammary Epithelium 337EA + +

998E + +

1130E + +^*

G61E + +

G94E + +

2. Breast Cancer Cell Lines

BT20 + *+

MCF7 + +

BT474 + +

MDA157 + +

MDA231 + +

MDA468 + +

MPE600 + +

CAMAl - +

MDA435 - •+ MDA134 - +

SKBR3 - +

UACC812 - +

DU4475 +/-

Detection of specific mRNAs by RT-PCR: (+) = presence; (-) = absence, (+/-) = greatly reduced levels.

Detection of LOH: (+) = LOH for the region; (-)

= no LOH detected for the region.

All five normal keeast ep tfeelial cell cultures expressed high levels of Brusfit-1 mRNA. In contrast, 6 of 13 breast cancer cell lines produced greatly reduced levels of Brush-1 mRNA. It appears that the Brush-1 gene shows no expression in five of these cell lines and only at very low levels for DU4475. These low mRNA levels were consistently observed by both RT-PCR and Northern analyses (data not shown) . Conversely, RBI mRNA is expressed in all normal and breast cancer cell lines except DU4475. The Brush-1 mRNA, therefore, shows much more differential expression in the cancer cell lines than RBI.

The Brush-1 tumor suppressor gene was detected in formalin fixed tissue section using in situ reverse transcriptase polymerase chain reaction fin situ RT PCR) . Brush-1 cDNA was synthesized in situ by reverse transcription using a Brush specific oligonucleotide primer. In situ polymerase chain reaction amplification in the presence of digoxygenin-11-dUTP and subsequent binding with an antidigoxygenin antibody conjugated to alkaline phosphatase allowed direct visualization. Brush-1 is expressed in the luminal layer of epithelial cells of lobules and ductules in tissue sections from normal reduction mammoplasties (5 patients) . In sections of invasive carcinoma, the tumor suppressor gene is expressed in about 10% of the invasive tumor cells (7 patients) . In cases of invasive carcinoma that contain a loss of heterozygosity (LOH) in the 13ql3- 14 region, no tumor cells express Brush-1. The specificity of the in situ reaction described above was demonstrated by performing a reaction without reverse transcriptase and also eluting the amplified fragments from the sections and detection by agarose gel electrophoresis. These results show that in vivo the B sti-l. gene has the expression pattern expected for a fcϊnnor suppressor gene. For immunodetection of Brush-1 message in tissue from reduction mammoplasties, archival formalin/alcohol fixed, paraffin embedded sections were subjected to RT in situ PCR. Lobules and ducts from the same section with no RT step and with the RT step were compared. The results showed that Brush-1 message is expressed at high levels in epithelial cells, and the sections with no RT step are negative.

Immunodetection of Brush-1 message in tumor cells displayed a loss of heterozygosity in the 13ql3-14q region, proximal to the retinoblastoma gene. Archival formalin fixed, paraffin embedded sections were subjected to RT in situ PCR and an area of invasive tumor cells was examined having a section with RT step. The results show tumors bearing an LOH at 13q 13-14 do not express Brush-1 message.

Since in situ RT PCR is applicable to any in vivo system and is capable of detecting low copy mRNAs, it is useful in studying tumor suppressor gene expression in vivo in rare and difficult to obtain cells. Therefore, molecules may be investigated for which there are no antibodies available, as is the case with Brush-1. Since the method is based on the incorporation of digoxygenin-11-dUTP during amplification and then immunodetection, it is rapid, requiring less than two days, and there is essentially no non-specific binding. Moreover, less than 20 copies of mRNA per cell can be detected. See Nuovo, GJ et al. Am J Pathol 1991, 189:847; Heniford, BW et al. NAR 1993, 21:3159. Therefore, this technique is well suited to the study of single cells obtained from nipple aspirates. It is also well suited for the study of heterogen-eous cell populations where only a few cell ess-press tite gene of interest. Such a study was done an peripheral blood cell from patients with HIV where it was shown for the first time by in situ RT-PCR that some leukocytes harbor the HIV virus (Nuovo, GJ et al. J. of acquired Immune Deficiency Syndromes, 1994 7:916).

The Brush-1 cDNA probe is a 4.3 kb sequence assembled from Brush-1 cDNA clone fragments. The correct 5'-3• orientation for each fragment was determined by Northern hybridization of separate single-stranded riboprobes complementary to each of the cDNA strands. Only one orientation hybridized to the 4.7 kb mRNA. SEQ ID N0:1 is the sequence for this cDNA probe. Sequence analysis revealed no significant homology to any known sequences in the Genbank. A longer cDNA sequence (4.3 kb) which hybridizes to the mRNA is SEQ ID NO:2 assembled from fragments used to assemble SEQ ID N0:1 and additional clone fragments. SEQ ID NO:l is contained within SEQ ID NO:2 and begins at position 818 in SEQ ID NO:2.

The relationship of Brush-1 to RBI was first suggested by preliminary mapping of the gene to chromosome 13. The cytological position of Brush-1 relative to RB-1 was done by FISH analysis. Brush-1 is localized to a single position at 13ql2-ql3, proximal to RBI. Previous studies by Lundberg, C. , et al . , Proc . Natl . Acad . Sci . USA 84: 2372-2376 (1987) and Devilee, p., et al . , Genomics 5: 554-560 (1989) found that many breast cancers showed LOH over a large region which included 13ql2-13 and that in one primary breast tumor the LOH on chromosome 13q included 13ql2-ql3 but did not include RBI. Since Brush-1 is proximal to RBI, and Brush-1 is often lost in breast cancer cell lines, it is a useful diagnostic tool for identifying primary breast tumors.

The LOH at D13S219, located at 13ql3, was surveyed for and compared to LOH found at the RBI gene. In one survey of 108 primary breast tumors it was found that LOH at the RBI gene was 45%. Another survey of 76 tumors from the same population gave 42% LOH for D13S219. In all cases where the samples were informative for both RBI and D13S219, the results were identical for LOH. A selection of these tumors were examined for expression of the Brush-1 and RBI mRNA (Table 1) . Four tumors with no LOH in this region demonstrated expression for both Brush-1 and RBI. In contrast, four tumors which clearly demonstrated LOH at both D13S219 and RBI, all showed decreased expression for Brush-1 while maintaining normal levels of expression for RBI. This differential loss of Brush-1 expression, therefore, is manifest in both breast cancer cell lines and primary breast tumors.

The Brush-1 mRNA is thus useful as a diagnostic marker for breast cancer. The Brush-1 cDNA probe, or substantially identical sequences, that is, sequences having 90% or greater homology with the Brush-1 cDNA probe, may be used to detect the presence of this marker in breast tissue or cell samples by generally applied molecular techniques known to those of ordinary skill in the art, such as Northern analysis and in situ hybridization. By comparison of the differential amounts of mRNA in the suspected tumor cells and normal cells, the likelihood of the presence of breast cancer can be ascertained. In addition, the probe may be used in the design and manufacture of new drugs for the treatment of breast cancer. The Brush-1 RNA (or DNA) may be used as gene therapy agents to provide missdbagi tumor suppressor function where it is naturally lacking.

We also noted from a BLAST search that the 5¹ of Brush-1 from the start of the cDNA is 98% homologous (in anti-sense relationship) to the actin binding protein ABP-280 (8360bp; see J.Cell Biol. 111(3) :1089-105(1990) ) . This suggests that Brush-1 could act as antisense RNA to block RNA translation for ABP-280 which is important to cell growth and motility.

In addition, lack of or abnormally low expression of a Brush-1 gene or the presence of a mutant Brush-1 gene in a patient may predispose that individual to breast or other types of cancer. Such a familial breast cancer gene, BRCA1, had been previously localized to chromosome 17 and the target gene has now been identified [Miki et al., 1994, Science 266:66-71; Futreal et al., 1994, Science 266:120-122]. Recently, a second familial breast cancer gene, BRCA2, was localized to chromosome 13ql2-13 [Wooster et al. , 1994, Science 265:2088- 2090] . Furthermore, the BRCA2 gene was most closely linked to the polymorphic microsatellite repeat marker D13S260. Yeast Artificial Chromosomes (YACs) isolated with the D13S260 markers were tested for the presence of the Brush-1 gene. Of eight YACs tested, two were conclusively shown to contain the Brush-1 gene. Therefore, this second familial breast cancer gene is in the same chromosomal location (13ql2-13) as Brush-1 and Brush-1 is contained in the chromosomal segment showing the strongest linkage to BRCA2.

The nucleotide sequences of this invention used for diagnostic applications may be the entire sequence of the gene or may &e fra ments thereof kased on restriction enzyme digestion (-wt -ch fragments may be all or part of the open reading frames) untranslated regions, intermediate coding regions, and fragments and combinations thereof. The minimum size single- stranded fragment will be at least 20 bases and usually at least 50 bases and may be 100 bases or more. The sequence may be obtained as a fragment or be synthesized.

The fragments can be used in a wide variety of ways, depending upon their size, their natural function, the use for which they are desired, and the degree to which they can be manipulated to modify their function. Thus, sequences of at least 20 bases, more usually at least 50 bases, and usually not exceeding about 1000 bases, more usually not exceeding about 500 bases, may serve as probes for detection of the presence of Brush-1 in a host tissue, including the genome, or in a physiological fluid, such as blood, lymph, saliva, spinal fluid, or the like. These sequences may include coding and/or non-coding sequences.

Where the nucleotide sequences are used for duplex formation, hybridization, or annealing, for example, for diagnosis or monitoring of the presence of the Brush-1 in vivo or in vitro, complete base pairing will not be required. One or more mismatches are permissible. To ensure that the presence of one or a few, usually not more than three, mismatches still allows for stable duplexes under the predetermined stringency of hybridizing or annealing conditions, probes will normally be greater than 20 bases, preferably at least about 50 bases or more.

The method of detection will involve duplex formation by annealing or hybridization of an oligonucleotide probe, either labeled or unlabeled, depending upon the nature detection system, with the DNA or RNA of host tissue suspected of harboring Brush-1 . A physiological sample may include tissue, blood, serum, etc. Particularly, blood samples will be taken, more particularly blood samples containing peripheral mononuclear cells, which may be lysed and the DNA or RNA isolated in accordance with known techniques.

The sample polynucleotide mixture obtained from the human host can be bound to a support or may be used in solution depending upon the nature of the protocol. The well-established Southern technique [(1975) J. Mol. Biol. 98:503] may be employed with denatured DNA, by binding the single-stranded fragments to a nitrocellulose filter. Alternatively, RNA can be blotted on nitrocellulose following the procedure described by Thomas, (1980) Proc. Natl. Acad. Sci. (USA) 77:5201. Desirably, the fragments will be electrophoresed prior to binding to a support, so as to be able to select for various sized fractions. Other techniques may also be used such as described in Meinkoth & Wahl, (1984) Anal. Biochem. 138:267-284.

The oligonucleotide probe may be DNA or RNA, usually DNA. The oligonucleotide sequence may be prepared synthetically or in vivo by cloning, where the complementary sequence may then be excised from the cloning vehicle or retained with the cloning vehicle. Various cloning vehicles are available, such as pBR322, M13, Charon 4A, or the like, desirably a single-stranded vehicle, such as M13.

As indicated, the oligonucleotide probe may be labeled or unlabeled. A wide variety of techniques exist for labeling DNA and RNA. As illustrative of such techniques, is radiolabeling using nick translation, tailing with terminal deoxytransferase, or the like, where the bases which are employed carry radioactive <32> P. Alternatively, radioactive nucleotides can be employed where carbon, nitrogen or other radioactive atoms may be part of the nucleoside structure. Other labels which may be used include fluorophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, or the like. Alternatively, instead of having a label which provides for a detectable signal by itself or in conjunction with other reactive agents, ligands can be used to which receptors bind, where the receptors are labeled such as with the above-indicated labels, which labels provide detectable signals by themselves or in conjunction with other reagents See, e.g., Leary et al. (1983) Proc Natl. Acad. Sci. (USA) 80:4045-4049; Cosstick et al. (1984) Nucleic Acids Res. 12:1791-1810.

The oligonucleotide probes are hybridized with the denatured human host nucleic acid, substantially intact or fragmented, or fractions thereof, under conditions of predetermined stringency. The stringency will depend upon the size and composition of the probe, the degree of mismatching, and the like. Usually, an organic solvent such as formamide will be present in from about 30 to 60 vol percent, more usually from about 40 to 50 vol percent, with salt concentration from 0.5 to 1M. Temperatures will generally range from about 30o C to 65o C. , more usually from about 35o C. to 50o C. The times for duplex formation may be varied widely, although minimum times will usually be at least about one hour and not more than about 72 hours, the time being selected in accordance with the amount of DNA or RNA available, the proportion of DNA or RNA as compared to total DNA or RNA, or the like. Stringency may also be modified by ionic strength and temperature. The hybridization and annealing can be carried out in two stages: a first stage in a hybridization medium; and, a second stage, involving washings at a higher stringency, by varying either or both temperature and ionic strength.

As understood in the art, the term "stringent hybridization conditions" as used herein refers to hybridization conditions which allow for closely related nucleic acid sequences to duplex (e.g., greater than about 90% homology) , but not unrelated sequences. The appropriate conditions can be established by routine procedures, such as running Southern hybridization at increasing stringency until only related species are resolved and the background and/or control hybridization has disappeared (i.e., selective hybridization) .

Nucleotide probes may be prepared employing reverse transcriptase using primers, e.g. , random primers or specific primers. The cDNA may be prepared employing a radioactive label, e.g., <32> P, present with one or more of the dNTPs. Reverse transcription will provide various sized fragments depending on the primers, the efficiency of transcription, the integrity of the RNA, and the like. The resulting cDNA sequences may be cloned, separated and used for detection of the presence of Brush-1 in the human genome.

Using specific primers of 10 to 20 bases, or more, Brush-1 may be reverse transcribed and the resulting ss DNA used as a probe specific for the region which hybridized to the primer. By e ploying one or more radionucleotide-labeled bases, tlae probes will be radiolabeled to provide a detectable signal. Alternatively, modified bases may be employed which will be randomly incorporated into the probe and may be used to provide for a detectable signal. For example, biotin-modified bases may be employed. The resulting biotin-containing probe may then be used in conjunction with labeled avidin to provide for a detectable signal upon hybridization and duplex formation.

The Brush-1 sequence may also be used therapeutically through gene therapy on subjects identified as having a genetically aberrant Brush-1 gene. The subjects will then have normal Brush-1 DNA and the ability to utilize its tumor-suppressing activity. For example Brush-1 DNA may be injected into subject after being based to an appropriate vector, such as viral vectors, liposomes and other vectors known in the art. SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: SCHOTT, D.

(ii) TITLE OF INVENTION: CDNA PROBE FOR BREAST CANCER DIAGNOSIS

AND TREATMENT

(iii) NUMBER OF SEQUENCES: 2

(ivj CORRESPONDENCE ADDRESS: (A) ADDRESSEE: REGINALD J. SUYAT, ESQ.

(B) STREET: 333 BUSH STREET

(C) CITY: SAN FRANCISCO

(D) STATE: CALIFORNIA

(E) COUNTRY: USA (F) ZIP: 94104-2878

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentln Release #1.0,

Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE: (C) CLASSIFICATION:

(Vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/160,088

(B) FILING DATE: 30-NOV-1993

(Vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 08/314,598

(B) FILING DATE: 27-SEP-1994

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: SUYAT, REGINALD J.

(B) REGISTRATION NUMBER: 28,172 (C) REFERENCE/DOCKET NUMBER: 11561-0026

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 415-772-6432

(B) TELEFAX: 415-772-6268

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

(A) LENGTH: 3542 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA to mRNA

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

CTGCTGTCTG GGATGGTGCA GCTCCACAGT TTTCTCTAAT GGTGTTCAGG TACACTGAAA 60

TTAGGAATTT TTAATATTTT AACACATTAC TTTGTTACAA AAAAACTTCT CACTTTGAAT 120 GCATGTTTTT TCTCATGAAA CTTTTAATAT TCCCTGAGCT TTTCTCCCCT CAAATTTCTA 180

AAACTTTCTG TCCTTAGTGT CAGTAGAAAA AAAAGTCCAA TAGACATATT TGTTCGTTTA 240

TCTTTAATTT GGAGCCAGCA AAAGGATGTG ATTCTGAACC ACGTGTTGTG TCTGCAGGAA 300

TTCAACTGAA AAAGGTGCAG GAGCAGCGGG AGCAGGAGGC CAAGCGGGAG CCAGTGGGGA 360

ATGACGTGGC CACGATCCTG TCCCGGCGCA TTGCCGTGGA GTACAGCGAC TCTGACGACG 420 ACTCAGAGTT CGACGAGAAC GACTGGTCCG ACTGAGCAAA GGCCGGCGGA GAGGCCGCGT 480

GTGGGAGCGT GTTGAAGATT TTAAGTGGTC TCTACACCCA AATAGTGGTA TTCTAATCCC 540

GTAGCATAGC ACCTTTTGTA TAAACAATGT GATATTGCTT CTGCACATCC AAAAATTCTG 600

GGTCTTTTCA GTATTTACTG TGTAATACTT AAGTGCCACT AAACATAGCA AATTGTGCTG 660

CACATGAGGA AATAGGCTGT CACTATCACA TTGTCCTGAA AACAGCATCT GCTTTCCTCT 720 TGGCCATGAG AGTATTTAGT GCAGTTTGGG TTTACTCTTA CTGATCAATA TAACTCTGCA 780

GACTTGCTGT GTGTTTGTGA AGCTGCCTGG TGTTAGGTCT CTGCAAGACT AATGACTATG 840

TCAGAGTGAT GTCTTCCAAC CAGTAAGTGA TATTGTTTCA CCGCTTTGGT TTTTCCTTTT 900 GTTTTTTTAA AGGATGTGTT TCTGAATAAG TTGGTTTTAG AGGGAAAGGT TCAACAAACA 960

GGGAGAATCC AGTGTTTCTG CTTTCAGTTT CTTGGCTTGG TAGCCTCTGA GTGAATCTGA 1020 TGCTCTGCTG AATAATTTCA TTACCTCTGC ACATGCCTGT CAAATATGAA ATTGGAAGGG 1080

CCCTTTTCAG GCTGGGTTCC CTGTGGGCAT TTGCTTAGTA AATGCCTGTT GATGGTTTTT 1140

(SCaAGaGAAT -ICSfiCAGCTC AACAGTAATG AAAGTGAAGA AC3^GAΘPCCA E CCTTCC 1200

TGGGGCCATT GGGGATGACA CTCAAGATAC TTGCCAGTCT CTCCAGTGTG GGCACCAGCC 1260

GGCCAGAACA GATGCGAGCA GTCCATGACT CTGGGAGCTA CACCGCTGAG CTGGGCAGAG 1320 CTGCGGCACA GGGCCTGGGC TGCAAAGGTG CCCTGCTCCT TTAGTTTCCT GACACCTGTG 1380

TCCTGAGTGA GCCGCAGGAG TTCTTAGCTC CTCAGCGAGC AACAGAGAGC ACTTTAGATG 1440

GCACCTTTCA CCACTTGGTC AGAATTTTAA AAGCTTAGGT TTAGGTGAAA GTAGATATTG 1500

ACAGCTATTC ACCTTTCACG GTGCTGGGGC CAGATTAGGG ATCACTCCCG TGAGGAGGGC 1560

CTTCACCCTG TTCTAGAAGC ACATGGTTGT CCTCCTGTTG TTGGCACATT AAATGATAAA 1620 AAGCACCTCA TGAGATTCCC TTGATCAGCC CTGCAGCTGT AGTACAGTGC TGTGCCCTCA 1680

CCTCCACCCT TCCTGTTGTT CCCACGTGGG CAATACCAGG GACCCATGGG GAAACTCAAG 1740

AATGACAGCT TCTATATTTT GTAATTCTGG ATGAAAGATA ACTGTGTTGA ACAAACAGGT 1800

GCTCCAGGCT TTGATTATAG ATACGACTTC AAAAATATGC TAAGACGCTT GACTTATTAA 1860

GGACTTTAAC CTACTCAACA GTATTTCATA TCCATTGTGG TTAGTTACTC AGTTATGTTG 1920 AGAAGAATCT GGAGCTAAAA GCAGAGATGT TTGAGGTGAC GGTAGGAATG TGAGCAGGAT 1980 GGTGATGGGG GTTTTTGTTA AAATGCATCT GAGCAAGTCA GCCAGCCCCG AAGTCCCCTC 2040

AGTGTGTGTG TCTCGAGTGG CTACCTGTTG GGCTTGTGGG CAGTGATTGT ACAGAGCCTG 2100 TCCATTGGGT GCAGTCATGT AGATCTGAAG CCCTGAAAAG CCTCATGTCT GCATCCCCTT 2160

TCCAAGGCTG CTTCCCTGGT GTGTCTGTTC TCTCCTCTGT CCCAGGTGCT GGGAGCGTCC 2220

TCTTCAGCGT CTTTTCCTAG AGCTGGTCAC CACTAGGCTG TCACTATAAA TTCCTTGAAT 2280

ATAAGTAACA GTTATTAATG AACTTCTAAA TTTCTAATTT CTCTCTCTCT CTCTCTTTTT 2340

TTTTTTTTTG TTGTTGTTAA AAAGGGCCTA CTACATTGGC GCTATTCTTA GGACTTCTGC 2400 AACTTTTAAA GTCTTACTTG TCTTTCTTGT TGCTTTTGTA TTAGGAGTTC CCCGTGTGGG 2460

TCTAGAACTC CCCTTTGGTA ATGCTTCTTT GTTTTTTTAT GGCCCTTCTG TTCTCAGGAT 2520

GGAGAGAACA CAGAAGCTAC TATCCATGTC AGGATTTATT CTATTTATAT CTTATTACAA 2580

TAAAATTAGT GGCACTTTAT TCATAAATAT TCATGAGCCT GTTAATTGTT AGTTGTCTTC 2640

CTGTAGCTGA ATCAACAAGT TATTTTCAAC TCAATTTTAT GACTTGCGAA AAAGCTTTTG 2700 CCCTGTTGTG TACCATAACA TTTAAAAGAA TGGAAAATGA CTGAAATCCA ATTTAGATTA 2760

TTTTTAGAGT ATTTTTCCAG CAAATTCAAT TTATTCTGAA ATTTAAATCC AGATCTTTTC 2820

TAATATGGTA TTACAATGAA AAGAATAAAG AGAAGATTTG AATTTTCAGT TTCATTTTCA 2880

AAAACTATTT ACCAAAACAA ATGGAGAAGA AACATCCAAA AGCACATTTC ATTTCTCCAA 2940

ACTTTGTGTT TTAAATTATA GTTATAAATT GTAAGGTAAT TTTAAATTGT CCCTCGTATT ^' 3000 ATTTCTCCAC GTCTGTTTTA GTTTAATGTC TCCTAAGCTT

TTCTCTCATA GCGTAGACCT 3060 AGGGAAGGGA TGGGAAGATT GCCCAGTCCC CGATGGCTGC GCACACAGGA GGCGGCGGAC 3120

GACAAGGCAA GTGAGTTTGC ACTGTCAGCC CCAGACCGTA AGCTTGGCTA CACTGATGTT 3180 TTTCTTTACT AAGGATACTA TTCAAAAATT AACATTTTCA TCTCAGTAAG TTTTTAGAAC 3240

ATCAAAATGT TTTCTGAGCT CCAAGTGGCT AGGTTGTAAA AGTTTTATAA TAATTTGCAA 3300

TTAAAATACA TGAfEa TAT T&A CQLH!®. AAGACTAGTG GGAATGTATC AGOCAGAGTA 3360

GCAAGTAATT TTTGTTTTAT AAATCATAGT ATCTGTCATC TTGCAGTATT ACCAATGCTG 3420

TTGTAAATTG AATTTAAAGT GGTATTAAAA AAAACTGTTA AACAATTTTT ATCTGTTTGT 3480 ATATCTTACT ATAGATTATG TACAAGTAAC ATCTAAATAA AATTACACTT TTAACCCTAA 3540

AA

3542

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

(A) LENGTH: 4359 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA

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

GGCTGGCACT CCAGCTTCAG CTCTCTCCAG GCCAGGGCCC CCAGCTCGGA CCTTGTGGGC 60

TCCCCCTTCC CCTAGGGGCC CCACGATGAA CTATTTTAGG GGTAAAGGGA CATTATATGC 120

CATTCACTCT CAAATGGTCC AGAGAAAAGT GTCTGTGTCT GTCTGTGTAA AGATGATAGG . 180

GGAAGATTGG GAAATAAATG TGGTCAGATG TTAGCAGTTG GGGAATCCAA GTATATGGGA 240 ATGTTTTTCT GCTGGTCTCA TAGCTGTTCT GTAAATCTGA AATTATTTCA AAACAGTTAA 300 AAAATGAATG GATACCATCT TGGGCAACAT TGCAAGACTC TATCTCTACA AAAAAGAAAA 360

TACAAAATTT AACCGTGCAT GGTGGCGTGC GCCGGTGGTT CCAGCTACTC AGGAGGCTGA 420

5 GGTGGGAGGA TTGCTTGAGC CCAGGAGGTC GAGGCTGCAG TGAGCCATGA TCGTAACACT 480

GCACTCCAGC CTGGGTCACA GAGTGAGACC CAATCTCAAA AAAAAAAAAA ATGGATAAAC 540

G T GTTTCC ACAGTAATTG TTGAAATAAA GGTTTCTCAG TTACTTCTTG GGTTAGCTGA 660

GAGTGAGTAG CATAGAACAC TGTTTCCAAG GCTCTGGATG CTGTTGCTGC CTAGTAGATG 720

15 TCCTGTGGTG ATAGAAATGC TCCAGCCTGC AGCGTCCCAG AGAGTAGCCA CTAGCCACAT 780

GTCAGTTCAT GCTTTTAAGG CTATATGTGC CTAGTGGCTG CTGTCTGGGA TGGTGCAGCT 840

CCACAGTTTT CTCTAATGGT GTTCAGGTAC ACTGAAATTA 20 GGAATTTTTA ATATTTTAAC 900

ACATTACTTT GTTACAAAAA AACTTCTCAC TTTGAATGCA TGTTTTTTCT CATGAAACTT 960

TTAATATTCC CTGAGCTTTT CTCCCCTCAA ATTTCTAAAA CTTTCTGTCC TTAGTGTCAG 1020

25 TAGAAAAAAA AGTCCAATAG ACATATTTGT TCGTTTATCT TTAATTTGGA GCCAGCAAAA 1080

GGATGTGATT CTGAACCACG TGTTGTGTCT GCAGGAATTC AACTGAAAAA GGTGCAGGAG 1140

CAGCGGGAGC AGGAGGCCAA GCGGGAGCCA GTGGGGAATG 30 ACGTGGCCAC GATCCTGTCC . 1200

CGGCGCATTG CCGTGGAGTA CAGCGACTCT GACGACGACT CAGAGTTCGA CGAGAACGAC 1260

TGGTCCGACT GAGCAAAGGC CGGCGGAGAG GCCGCGTGTG GGAGCGTGTT GAAGATTTTA 1320 5 AGTGGTCTCT ACACCCAAAT AGTGGTATTC TAATCCCGTA GCATAGCACC TTTTGTATAA 1380 ACAATGTGAT ATTGCTTCTG CACATCCAAA AATTCTGGGT CTTTTCAGTA TTTACTGTGT 1440

AATACTTAAG TGCCACTAAA CATAGCAAAT TGTGCTGCAC ATGAGGAAAT AGGCTGTCAC 1500 TATCACATTG TCCTGAAAAC AGCATCTGCT TTCCTCTTGG CCATGAGAGT ATTTAGTGCA 1560

GTTTGGGTTT ACTCTTACTG ATCAATATAA CTCTGCAGAC TTGCTGTGTG TTTGTGAAGC 1620

TAAGTGATAT TGTTTCACCG CTTTGGTTT TCCTTTTGTT TTTTTAAAGG ATGTGTTTCT 1740

GAATAAGTTG GTTTTAGAGG GAAAGGTTCA ACAAACAGGG AGAATCCAGT GTTTCTGCTT 1800 TCAGTTTCTT GGCTTGGTAG CCTCTGAGTG AATCTGATGC TCTGCTGAAT AATTTCATTA 1860

CCTCTGCACA TGCCTGTCAA ATATGAAATT GGAAGGGCCC TTTTCAGGCT GGGTTCCCTG 1920

TGGGCATTTG CTTAGTAAAT GCCTGTTGAT GGTTTTTGCA AGAGAATTCA GCAGCTCAAC 1980

AGTAATGAAA GTGAAGAACA GAGTCCATTT TCCTTCCTGG GGCCATTGGG GATGACACTC 2040

AAGATACTTG CCAGTCTCTC CAGTGTGGGC ACCAGCCGGC CAGAACAGAT GCGAGCAGTC 2100 CATGACTCTG GGAGCTACAC CGCTGAGCTG GGCAGAGCTG CGGCACAGGG CCTGGGCTGC 2160

AAAGGTGCCC TGCTCCTTTA GTTTCCTGAC ACCTGTGTCC TGAGTGAGCC GCAGGAGTTC 2220

TTAGCTCCTC AGCGAGCAAC AGAGAGCACT TTAGATGGCA CCTTTCACCA CTTGGTCAGA 2280

ATTTTAAAAG CTTAGGTTTA GGTGAAAGTA GATATTGACA GCTATTCACC TTTCACGGTG 2340

CTGGGGCCAG ATTAGGGATC ACTCCCGTGA GGAGGGCCTT CACCCTGTTC TAGAAGCACA 2400 TGGTTGTCCT CCTGTTGTTG GCACATTAAA TGATAAAAAG CACCTCATGA GATTCCCTTG 2460 ATCAGCCCTG CAGCTGTAGT ACAGTGCTGT GCCCTCACCT CCACCCTTCC TGTTGTTCCC 2520

ACGTGGGCAA TACCAGGGAC CCATGGGGAA ACTCAAGAAT GACAGCTTCT ATATTTTGTA 2580 ATTCTGGATG AAAGATAACT GTGTTGAACA AACAGGTGCT CCAGGCTTTG ATTATAGATA 2640

CGACTTCAAA AATATGCTAA GACGCTTGAC TTATTAAGGA CTTTAACCTA CTCAACAGTA 2700 ECSΓAΓ C A IG GGTTA GTTACTCAGT TATGTTGAGA AGAATCTGGA GCTAAAAGCA 2760

GAGATGTTTG AGGTGACGGT AGGAATGTGA GCAGGATGGT GATGGGGGTT TTTGTTAAAA 2820

TGCATCTGAG CAAGTCAGCC AGCCCCGAAG TCCCCTCAGT GTGTGTGTCT CGAGTGGCTA 2880 CCTGTTGGGC TTGTGGGCAG TGATTGTACA GAGCCTGTCC ATTGGGTGCA GTCATGTAGA 2940

TCTGAAGCCC TGAAAAGCCT CATGTCTGCA TCCCCTTTCC AAGGCTGCTT CCCTGGTGTG 3000

TCTGTTCTCT CCTCTGTCCC AGGTGCTGGG AGCGTCCTCT TCAGCGTCTT TTCCTAGAGC 3060

TGGTCACCAC TAGGCTGTCA CTATAAATTC CTTGAATATA AGTAACAGTT ATTAATGAAC 3120

TTCTAAATTT CTAATTTCTC TCTCTCTCTC TCTTTTTTTT TTTTTTGTTG TTGTTAAAAA 3180 GGGCCTACTA CATTGGCGCT ATTCTTAGGA CTTCTGCAAC TTTTAAAGTC TTACTTGTCT 3240

TTCTTGTTGC TTTTGTATTA GGAGTTCCCC GTGTGGGTCT AGAACTCCCC TTTGGTAATG 3300

CTTCTTTGTT TTTTTATGGC CCTTCTGTTC TCAGGATGGA GAGAACACAG AAGCTACTAT 3360

CCATGTCAGG ATTTATTCTA TTTATATCTT ATTACAATAA AATTAGTGGC ACTTTATTCA 3420

TAAATATTCA TGAGCCTGTT AATTGTTAGT TGTCTTCCTG TAGCTGAATC AACAAGTTAT 3480 TTTCAACTCA ATTTTATGAC TTGCGAAAAA GCTTTTGCCC TGTTGTGTAC CATAACATTT 3540 AAAAGAATGG AAAATGACTG AAATCCAATT TAGATTATTT TTAGAGTATT TTTCCAGCAA 3600

ATTCAATTTA TTCTGAAATT TAAATCCAGA TCTTTTCTAA TATGGTATTA CAATGAAAAG 3660 AATAAAGAGA AGATTTGAAT TTTCAGTTTC ATTTTCAAAA ACTATTTACC AAAACAAATG 3720

GAGAAGAAAC ATCCAAAAGC ACATTTCATT TCTCCAAACT TTGTGTTTTA AATTATAGTT 3780

ATAAATTGTA AGGTAATTTT AAATTGTCCC TCGTAT STF TCTCCACGTC TGTTTTAGTT 3840

TAATGTCTCC TAAGCTTTTC TCTCATAGCG TAGACCTAGG GAAGGGATGG GAAGATTGCC 3900

CAGTCCCCGA TGGCTGCGCA CACAGGAGGC GGCGGACGAC AAGGCAAGTG AGTTTGCACT 3960 GTCAGCCCCA GACCGTAAGC TTGGCTACAC TGATGTTTTT CTTTACTAAG GATACTATTC 4020

AAAAATTAAC ATTTTCATCT CAGTAAGTTT TTAGAACATC AAAATGTTTT CTGAGCTCCA 4080

AGTGGCTAGG TTGTAAAAGT TTTATAATAA TTTGCAATTA AAATACATGA TACATATTAA 4140

TCCATTAAAG ACTAGTGGGA ATGTATCAGC CAGAGTAGCA AGTAATTTTT GTTTTATAAA 4200

TCATAGTATC TGTCATCTTG CAGTATTACC AATGCTGTTG TAAATTGAAT TTAAAGTGGT 4260 ATTAAAAAAA ACTGTTAAAC AATTTTTATC TGTTTGTATA TCTTACTATA GATTATGTAC 4320

AAGTAACATC TAAATAAAAT TACACTTTTA ACCCTAAAA 4359

Claims

WHAT IS CLAIMED IS:

1. Isolated DNA comprising a nucleotide sequence substantially identical to SEQ ID N0:1.

2. Isolated DNA comprising a nucleotide sequence substantially identical to SEQ ID NO:2.

3. A diagnostic agent for detecting tumors and pre¬ malignant cells in host tissue or cells comprising at least one probe selected from the group consisting of a DNA sequence substantially identical to SEQ ID NO:2, its complementary DNA sequence, an RNA sequence substantially identical to the RNA complement of SEQ ID NO:2, the complement of said RNA complement, and fragments of said sequences; and a marker on said probe.

4. A pharmaceutical composition for providing or enhancing the tumor-suppressing ability of a subject comprising a therapeutically effective amount of mRNA, cDNA of said mRNA or DNA sequences of genomic DNA of BRUSH-1, and a pharmaceutically acceptable carrier.

5. A diagnostic agent for detecting tumors and pre¬ malignant cells in host tissue or cells comprising at least one probe selected from the group consisting of a DNA sequence substantially identical to SEQ ID N0:1, its complementary DNA sequence, an RNA sequence substantially identical to the RNA complement of SEQ ID N0:1, the complement of said RNA complement, and fragments of said sequences, and a marker on said probe.

6. A diagnostic method for detecting the presence of breast tumors or pre-malignant breast cells in a subject comprising the steps of

(a) contacting a library containing mRNA derived from tissue or cells from said subject with one or more labelled probes, said probes comprising DNA segments of SEQ N0:1 or SEQ NO:2;

(b) separating the probes which hybridize with mRNA from said library; (c) identifying the mRNA sequences from step

(b) and determining if said sequences define a normal amount of normal BRUSH-1 gene in said subject.

7. A therapeutic method for treating tumors or pre¬ malignant cells in a subject comprising the steps of introducing into a subject known or suspected of having tumors, an agent comprising RNA or DNA or selected from the group consisting of mRNA, cDNA and genomic DNA of BRUSH-1, which enables said subject or augments the ability of said subject to suppress tumor proliferation or growth.

8. A method of providing a subject deficient in a functional BRUSH-1 gene with a functional BRUSH- 1 gene, comprising the step of introducing into said subject an agent comprising RNA or DNA selected from the group consisting of mRNA, cDNA and genomic DNA of BRUSH-1, which enables said subject or augment the ability of said subject to suppress tumor proliferation or growth.

9. A method according to either Claim 7 or 8 wherein said agent comprises a substantially similar variant of said RNA or DNA.

10. A method according to Claim 6 where said probes are labelled.