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Isolation of the Mouse Homologue of BRCA1 and Genetic Mapping to Mouse Chromosome 11
GENOMICS
29, 576–581 (1995)
Isolation of the Mouse Homologue of BRCA1 and Genetic Mapping to Mouse Chromosome 11 L. MICHELLE BENNETT,*,1 ASTRID HAUGEN-STRANO,* CHARLES COCHRAN,* HEATHER A. BROWNLEE,* FRED T. FIEDOREK, JR.,† AND ROGER W. WISEMAN* *Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and †Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received March 8, 1995; accepted July 27, 1995
The BRCA1 gene is in large part responsible for hereditary human breast and ovarian cancer. Here we report the isolation of the murine Brca1 homologue cDNA clones. In addition, we identified genomic P1 clones that contain most, if not all, of the mouse Brca1 locus. DNA sequence analysis revealed that the mouse and human coding regions are 75% identical at the nucleotide level while the predicted amino acid identity is only 58%. A DNA sequence variant in the Brca1 locus was identified and used to map this gene on a (Mus m. musculus Czech II 1 C57BL/KsJ)F1 1 C57BL/KsJ intersubspecific backcross to distal mouse chromosome 11. The mapping of this gene to a region highly syntenic with human chromosome 17, coupled with Southern and Northern analyses, confirms that we isolated the murine Brca1 homologue rather than a related RING finger gene. The isolation of the mouse Brca1 homologue will facilitate the creation of mouse models for germline BRCA1 defects. q 1995 Academic Press, Inc.
involved in DNA binding (Freemont, 1993). The presence of this conserved structural motif suggests that the BRCA1 protein may regulate gene expression (Futreal et al., 1994; Miki et al., 1994). As an initial step toward understanding BRCA1 functions, we have isolated the murine homologue. The mouse provides a good system in which to study human disease syndromes because approximately 37% of the murine genome contains segments in which both synteny and gene order are conserved with humans (Nadeau, 1989). One of the most extensively characterized syntenic regions between the mouse and the human is the distal half of mouse chromosome 11, which contains at least 54 human chromosome 17 loci (Lossie et al., 1994). In this report we describe the sequence of the coding region for the mouse Brca1 homologue, isolation of mouse genomic P1 clones, and mapping of Brca1 to a 1.9-cM interval on mouse chromosome 11 that also contains the Mox1 and keratin gene cluster loci (Candia et al., 1992; Lossie et al., 1994). MATERIALS AND METHODS
INTRODUCTION
The human BRCA1 locus, mapped to chromosome 17q in 1990 (Hall et al., 1990), was recently identified by positional cloning (Miki et al., 1994). Germline defects in this gene confer a profound predisposition to the development of hereditary, early-onset breast and ovarian cancers (Futreal et al., 1994; Miki et al., 1994). Since the wildtype allele is almost always lost in hereditary breast and ovarian tumors, BRCA1 appears to function as a tumor suppressor gene (Wooster and Stratton, 1995). The human BRCA1 gene contains 24 exons, 22 of which encode an acidic protein 1863 amino acids in length. BRCA1 contains a Cys3-His-Cys4 (C3HC4) RING finger domain near the amino terminus that has also been observed in proteins predicted to be 1 To whom correspondence should be addressed at the National Institute of Environmental Health Sciences, MD C4-06, 111 Alexander Dr., Research Triangle Park, NC 27709. Telephone: (919) 5413229. Fax: (919) 541-3720. E-mail: [email protected].
0888-7543/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Isolation of the murine Brca1 homologue. A human polymerase chain reaction (PCR) product (nucleotides 74 to 517 of Accession No. U14680) was used as a hybridization probe to isolate the initial Brca1 cDNAs from a mouse mixed germ cell library. Since Northern analysis of multiple mouse tissues with the human probe indicated that Brca1 transcript levels were highest in testis (data not shown), a mixed germ cell cDNA library from CD-1 mice was screened to yield a series of clones. Genomic clones from Lambda FIX II library of 129SV/J mice (Stratagene) were used to obtain Brca1 sequences from exon 11. In addition, cDNA was generated from mouse testis and spleen RNA using the reverse transcription system (Promega). These cDNAs were used as templates to amplify mouse sequences with combinations of mouse and human oligonucleotides to obtain Brca1 coding sequences between exons 12 and 24. The PCR conditions for mouse primer combinations were as follows: 947C for 3 min followed by 35 cycles of 947C/1 min, 557C/1 min, 727C/1 min. These conditions were modified when human/mouse primer combinations were used to include 5 cycles of 947C/1 min, 377C/1 min, 727C/1 min following the initial denaturation step and before the 35 cycles with an annealing temperature of 557C. Isolated clones and PCR products were sequenced using a PRISM dye terminator kit on an ABI 373 automated fluorescent sequencer (Applied Biosystems). Sequence comparison of the mouse Brca1 and
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ISOLATION AND MAPPING OF MURINE Brca1 the human BRCA1 gene was done using GAP and BESTFIT software (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group). Southern and Northern analysis. Multiple-species Southern and multiple mouse tissue Northern filters (Clontech) were probed with human- or mouse-specific sequences containing exon 2. Southern and Northern blots were hybridized in Hybrisol I (Oncor) at 427C overnight and washed twice in 21 SSC (11 SSC: 1.5 M NaCl/0.15 M sodium citrate, pH 7.0) for 5 min at room temperature, twice in 21 SSC/1% SDS for 30 min at 657C, and twice in 0.11 SSC/0.1% SDS at room temperature for 30 min. The membranes were exposed to X-Omat AR (Kodak) overnight to 1 week. The mouse exon 2 probe was generated using the primers MBF2, 5*-ACT GGA ACT GGA AGA AAT GGA, and MBR2, 5*-TYA CCA GAT CGG ACA CTC TAA. Isolation of P1 clones containing Brca1. PCR primers were designed based on the Brca1 cDNA sequence (MBF3, 5*-GCA CAT TTA TTA CAG GAC CAC A; MBR4, 5*-ACT TCC ACC TCA GCC TAT TTT T) that amplified a 390-bp fragment from the 5* end of Brca1 exon 11 from mouse genomic DNA. Using these primers, three Brca1containing clones were obtained by screening a commercially available 129 mouse P1 library (Genome Systems). Southern analysis of the P1 clones after restriction enzyme digestion with PvuII, EcoRI, and BamHI was performed using BRCA1 probes containing mouse exon 2 or human exons 1 through 6. The digested P1 DNA was electrophoresed on a 0.7% agarose gel in 11 TBE (0.9 M Tris–borate/ 0.002 M EDTA, pH 8.0) for 15 h at 35 V. The DNA was transferred to GeneScreen membrane (DuPont) in 101 SSC after denaturation (1.5 M NaCl/0.5 M NaOH) and neutralization (1.5 M NaCl/0.5 M Tris, pH 7.5). Following transfer, DNA was cross-linked to the membrane with a Stratalinker (Stratagene) and hybridized as described above. Identification of sequence variants and mapping. The intersubspecific backcross (Czech II 1 C57BL/KsJ)F1 1 C57BL/KsJ was used to determine the order of genes in the murine Brca1 region. One hundred fifty-eight N2 progeny of a previously characterized (Mus m. musculus Czech II 1 C57BL/KsJ m/m)F1 1 C57BL/KsJ m/m backcross (Fiedorek and Kay, 1994) were initially genotyped using the mouse chromosome 11 simple sequence repeat (SSR) markers D11Mit32, -52, -58, and -10 (MIT Database of SSLP Markers: [email protected]). Animals identified as being recombinant in this region were subsequently typed with the SSR markers D11Mit10, -14, -32, -52, -58, -59, -70, -98, -99, -145, -160, and -197 (Research Genetics). The PCR products were electrophoresed on a 7% native acrylamide gel at 200 V for 2–4 h with subsequent staining by ethidium bromide and visualization with UV light. A (CA)n sequence variant between C57BL/KsJ and Czech II mice was identified in intron 3 of the Brca1 gene (Accession No. U32585). Oligonucleotides were designed based on sequences flanking the CA repeat to amplify a 370-bp fragment in the 129SV/J mouse strain. Forward and reverse primers were MBF43, 5*-CAG AGT TTT TGT TTA CTT GGT CC, and MBR42, 5*-TGC ACT TAC CTG CAT ATG ACT T. The PCR conditions for this amplification were as described above except that the number of cycles was limited to 27. Comparison of the PCR templates from the two inbred strains revealed that the Czech II-specific PCR product was larger than that of C57BL/KsJ. A Mox1 gene sequence varient was identified to distinguish between the C57BL/KsJ and the Czech II alleles. Primers for the Mox1 gene were selected (MMOX1F, 5*-CTG GGG CCC TCT TTT CTG, and MMOX1R, 5*-CTG TCT TGA TGG GGT GGG) to amplify a 660-bp fragment from the 3* untranslated region (Accession No. Z15103). Sequence analysis of these PCR products revealed restriction fragment length polymorphism between the two strains. Restriction enzyme digest of this PCR product with Bbs1 yields two fragments of 403 and 257 bp from the C57BL/KsJ allele, while the product from the Czech II allele remains intact. The genetic order and recombination intervals expressed in centimorgans were determined using Map Manager software (Manley, 1993).
RESULTS
Isolation of the Mouse Brca1 Homologue Screening of the murine mixed germ cell cDNA library with a human probe containing the RING finger
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domain yielded three clones. DNA sequence analysis revealed that these cDNAs contained the putative murine Brca1 homologue and extended from exon 1, which is untranslated, to exon 11. A composite sequence for the entire murine Brca1 coding region was assembled using additional mixed germ cell cDNAs, genomic clones containing exon 11, and cDNA PCR products for exons 12 to 24 (Accession No. U32446). Partial genomic sequencing revealed that exon 11 comprises about 60% of the total coding region and the murine Brca1 gene contains intron–exon boundaries similar to that of the human. While the overall homology between human and mouse nucleotide sequences is relatively high (75%), the amino acid identity is only 58%. Figure 1 shows the alignment of the predicted mouse and human gene products. The C3HC4 RING finger domain (residues 20 through 70) is highly conserved in mice relative to humans with only a single conservative substitution of aspartic acid for glutamic acid. As in the human the mouse Brca1 gene product is very acidic, with a net excess of 53 acidic residues. Hybridization Analysis of the Mouse Brca1 Homologue To confirm that we had isolated the murine Brca1 homologue rather than a related RING finger gene, additional studies were performed. Northern analysis of multiple-tissue mRNAs with a mouse exon 2 probe revealed a 7.5-kb transcript that was most abundant in mouse testis and spleen (Fig. 2), consistent with previous observations for human tissues (Miki et al., 1994). Likewise, Southern analysis with this mouse probe detected the expected human EcoRI fragment as well as single-copy sequences in all mammalian DNAs examined (Fig. 2). Southern analysis was also performed on the mouse P1 clones. Duplicate filters containing P1 DNA digested with various enzymes were probed with mouseand human-specific sequences from the RING finger domain (Fig. 3). Common hybridization patterns between the two filters indicate that two P1 clones (P14020 and P14022) include the 5* half of the mouse Brca1 gene homologue. The third clone (P14021) contains a smaller portion of 5* Brca1 sequence that does not extend to the second exon. Finally, PCR analysis with mouse exon 24 primers revealed that all three P1 clones contain at least part of the 3* untranslated region of the Brca1 gene (data not shown). Linkage Analysis The MOX1 and BRCA1 loci are tightly linked on human chromosome 17q21 (Neuhausen et al., 1994). Based on known human–mouse synteny, we predicted that the murine Brca1 gene would be tightly linked to the Mox1 locus, previously mapped to chromosome 11 (Candia et al., 1992). To map the murine Brca1 homologue, DNA sequence variants of the mouse Brca1 and the Mox1 genes were identified and used to localize
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FIG. 1. Alignment of the predicted murine Brca1 (upper, U32446) and human BRCA1 (lower, U14680) amino acid sequences. Vertical lines between amino acids indicate identity, while colons and dots indicate greater and lesser degrees of amino acid similarity, respectively, as determined by the GAP program. The C3HC4 RING finger residues are indicated by asterisks.
these two genes relative to known SSR markers in the Brca1 region. Segregation analysis of the Brca1 and Mox1 se-
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quence variants and chromosome 11 SSR markers was carried out on 158 (Mus m. musculus Czech II 1 C57BL/KsJ m/m)F1 1 C57BL/KsJ m/m backcross prog-
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ISOLATION AND MAPPING OF MURINE Brca1
D11Mit52, which is approximately 2.6 cM distal to its placement by the MIT group. The second difference lies in the recombination distance between D11Mit32 and D11Mit98. We calculate this distance to be 7.6 cM from the data obtained in our backcross population, while these two anchor loci had been previously assigned a recombination distance of 17.9 cM. DISCUSSION
Contrary to expectations, sequence comparisons between the mouse and the human homologues indicated that BRCA1 is more highly conserved at the nucleotide than at the amino acid level. The coding region of the mouse and human genes are 75% identical at the nucleic acid level, while the predicted amino acid identity is only 58% overall. This result is in contrast to what has been typically observed for other cancer susceptibility genes. For example, the retinoblastoma and adenomatous polyposis coli tumor suppressor gene products are 90% identical between mice and humans (Bernards et al., 1989; Su et al., 1992). Despite the low evolutionary conservation between the two species, several lines of evidence support the conclusion that we have isolated the mouse Brca1 homologue rather than a related gene. Hybridization analyses demonstrated single-copy sequences in all mammalian species examined and transcripts of the expected size and a tissue-specific pattern of expression (Miki et al., 1994). Likewise, we localized the Brca1 gene to a 1.9cM region of mouse chromosome 11 that contains the Mox1 gene and the keratin gene cluster. This region of mouse chromosome 11 was predicted to harbor the
FIG. 2. Northern and Southern analysis of the mouse Brca1 gene. The upper panel illustrates Northern analysis of Brca1 transcripts in mRNA isolated from the mouse tissues indicated. The lower panel contains a Southern blot of EcoRI-digested DNA from various species. Both filters were hybridized with a mouse exon 2 probe.
eny (Fiedorek and Kay, 1994). Four chromosome 11 SSR markers were typed that spanned the syntenic region predicted to harbor the Brca1 and Mox1 genes. This analysis identified 19 recombinant progeny that were then used to type the Brca1 CA repeat, the Mox1 BbsI restriction fragment length variant, and a dozen additional SSR markers by PCR. The haplotypes from this analysis are shown in Fig. 4A. The Brca1 and Mox1 loci cosegregate with the keratin gene cluster (D11Mit57) in a 1.9-cM interval defined by D11Mit99 and D11Mit52 (Fig. 4B). The order of the markers for this subchromosomal segment on distal mouse chromosome 11 is consistent with the human physical map in the BRCA1 region (Albertsen et al., 1994) and corresponds to the order and approximate cM distances defined for the murine composite map (Lossie et al., 1994). Only two discrepancies between our results and those obtained from the MIT genome center were detected. We observed the cosegregation of D11Mit58 with
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FIG. 3. Southern analysis of mouse P1 clones. (A) Hybridization with a mouse exon 2 probe reveals identical DNA fragments for P14020 (lane 1) and P14022 (lane 2); P14021 (lane 3) does not contain exon 2 of Brca1. (B) Hybridization of a duplicate filter with a human cDNA probe that extends from exon 1 to exon 6 shows cross-hybridization to all three P1 clones. P14021 has fewer hybridizing fragments consistent with its lack of the 5* most exons.
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FIG. 4. Meiotic mapping of the Brca1 region. (A) Distribution of the haplotypes for 158 progeny of a (Czech II 1 C57BL/KsJ)F1 1 C57BL/KsJ backcross. The black squares represent the presence of the Czech II allele, and the white squares signify progeny homozygous for the C57BL/KsJ allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. (B) A genetic linkage map of distal mouse chromosome 11 including the locations of the Brca1 and Mox1 loci is shown. Recombination distances between loci in cM are shown to the left of the chromosome.
Brca1 locus based on synteny with human chromosome 17q21 (Lossie et al., 1994). The only structural motif revealed by computer analysis of the predicted mouse and human Brca1 gene products, which has been highly conserved, is a C3HC4 RING finger near the amino terminus. RING finger domains have been observed in more than 40 other proteins from a wide range of species (Freemont, 1993). Many of these RING finger proteins are involved in transcriptional regulation, and several are thought to bind DNA. The presence of this conserved structural motif suggests that at least one function of Brca1 may be to control the expression of other genes. In contrast to that observed for the RING domain only short stretches of amino acid identity are evident over the remainder of the two gene products. Additional functional domains of BRCA1 may be revealed by future comparisons of the mouse and human gene products. While providing strong support for the functional significance of the RING finger domain, these observations of amino acid conservation suggest that the evolutionary constraints on much of the remainder of the BRCA1 gene product are unexpectedly weak. Despite the low degree of homology between the two species at the protein level, several apparently important amino acids have been conserved. In particular, we have determined that the amino acid positions at which missense mutations in BRCA1 families have been reported are identical between mice and humans in six of seven cases (Shattuck-Eidens et al., 1995). In contrast the residue representing the common human allele is conserved in the mouse for only one of five published human polymorphisms that result in amino acid substitutions (Freidman et al., 1994; Miki et al., 1994). Hence, comparison of the human and mouse genes may provide insights into whether sequence variants represent predisposing mutations or rare polymorphisms that are biologically neutral. Based on the likely significance of the RING finger domain in BRCA1 protein function, we are creating
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mice with a germline BRCA1 alteration using a targeting vector designed to disrupt this structural motif. As in humans, we predict that Brca1 defects may predispose mice to neoplastic development in the mammary gland, ovary, and perhaps other tissues. Generation of these mice will also allow us to ask whether Brca1 alterations, either in the heterozygous and homozygous null state, result in any developmental or reproductive abnormalities. Given that a patient homozygous for a BRCA1 defect has recently been identified (Boyd et al., 1995), it seems likely that Brca1-null mice will be viable. Finally, these Brca1-deficient mice will provide an important new model system for the analysis of interactions between this important cancer predisposing gene and a variety of environmental factors. ACKNOWLEDGMENTS We thank Eric Kay for technical assistance with mapping analysis. The CD-1 mixed germ cell cDNA library and total testis RNA were kindly provided by Dr. Jeff Welch. This work was supported in part by Grant DK44074 from the National Institutes of Health (F.T.F.).
REFERENCES Albertsen, H. M., Smith, S. A., Mazoyer, S., Fujimoto, E., Stevens, J., Williams, B., Rodriguez, P., Cropp, C. S., Slijepcevic, P., Carlson, M., Robertson, M., Bradley, P., Lawrence, E., Harrington, T., Mei Sheng, Z., Hoppes, R., Sternberg, N., Brothman, A., Callahan, R., Ponder, B. A. J., and White, R. (1994). A physical map and candidate genes in the BRCA1 region on chromosome 17q12–21. Nature Genet. 7: 472–479. Bernards, R., Schackleford, G. M., Gerber, M. R., Horowitz, J. M., Friend, S. H., Schartl, M., Bogenmann, E., Rapaport, J. M., McGee, T., Dryja, T. P., and Weinberg, R. A. (1989). Structure and expression of the murine retinoblastoma gene and characterization of its encoded protein. Proc. Natl. Acad. Sci. USA 86: 6474–6478. Boyd, M., Harris, F., McFarlane, R., Davidson, H. R., and Black, D. M. (1995). A human BRCA1 gene knockout. Nature 375: 542– 543. Candia, A. F., Hu, J., Crosby, J., Lalley, P. A., Noden, D., Nadeau, J. H., and Wright, C. V. E. (1992). Mox-1 and Mox-2 define a novel homeobox gene subfamily and are differentially expressed during
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ISOLATION AND MAPPING OF MURINE Brca1 early mesodermal patterning in mouse embryos. Development 116: 1123–1136. Fiedorek, F. T., Jr., and Kay, E. S. (1994). Mapping of PCR-based markers for mouse Chromosome 4 on a backcross penetrant for the misty (m) mutation. Mamm. Genome 5: 479–485. Freemont, P. S. (1993). The RING finger: A novel protein sequence motif related to the zinc finger. Ann. N. Y. Acad. Sci. 684: 174– 192. Freidman, L. S., Ostermeyer, E. A., Szabo, C. I., Dowd, P., Lynch, E. D., Rowell, S. E., and King, M.-C. (1994). Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nature Genet. 8: 399–404. Futreal, P. A., Liu, Q., Shattuck-Eidens, D., Cochran, C., Harshman, K., Tavtigian, S., Bennett, L. M., Haugen-Strano, A., Swensen, J., Miki, Y., Eddington, K., McClure, M., Frye, C., Weaver-Feldhaus, J., Ding, W., Gholami, Z., Soderkvist, P., Terry, L., Jhanwar, S., Berchuck, A., Iglehart, J. D., Marks, J., Ballinger, D. G., Barrett, J. C., Skolnick, M. H., Kamb, A., and Wiseman, R. (1994). BRCA1 mutations in primary breast and ovarian carcinomas. Science 266: 120–122. Hall, J. M., Lee, M. K., Newman, B., Morrow, J. E., Anderson, L. A., Huey, B., and King, M.-C. (1990). Linkage of early-onset familial breast cancer to chromosome 17q21. Science 250: 1684–1689. Lossie, A. C., MacPhee, M., Burchberg, A. M., and Camper, S. A. (1994). Mouse Chromosome 11. Mamm. Genome 5: S164–S180. Manley, K. F. (1993). A Macintosh program for storage and analysis of experimental genetic mapping data. Mamm. Genome 4: 303– 313. Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P. A., Harshman, K., Tavtigian, S., Cochran, C., Bennett, L. M., Ding, W., Bell, R., Rosenthal, J., Hussey, C., Tran, T., McClure, M., Frye, C.,
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Hattier, T., Phelps, R., Haugen-Strano, A., Katcher, H., Yakumo, K., Gholami, Z., Shaffer, D., Stone, S., Bayer, S., Wray, C., Bogden, R., Dayananth, P., Ward, J., Tonin, P., Narod, S., Bristow, P. K., Norris, F. H., Helvering, L., Morrison, P., Rosteck, P., Lai, M., Barrett, C. J., Lewis, C., Neuhausen, S., Cannon-Albright, L., Goldgar, D., Wiseman, R., Kamb, A., and Skolnick, M. H. (1994). A strong candidate for the breast and ovarian cancer susceptibility gene BRCA1. Science 266: 66–71. Nadeau, J. H. (1989). Maps of linkage and synteny homologies between mouse and man. Trends Genet. 5: 82–86. Neuhausen, S. L., Swensen, J., Miki, Y., Liu, Q., Tavtigian, S., Shattuck-Eidens, D., Kamb, A., Hobbs, M. R., Gingrich, J., Shizuya, H., Kim, U.-J., Cochran, C., Futreal, P. A., Wiseman, R. W., Lynch, H. T., Tonin, P., Narod, S., Cannon-Albright, L., Skolnick, M. H., and Golgar, D. E. (1994). A P1-based physical map of the region from D17S776 to D17S78 containing the breast cancer susceptibility gene BRCA1. Hum. Mol. Genet. 3: 1919–1926. Shattuck-Eidens, D., McClure, M., Simard, J., Labrie, F., Narod, S., Couch, F., Hoskins, K., Weber, B., Castilla, L., Erdos, M., Brody, L., Friedman, L., Ostermeyer, E., Szabo, C., King, M.-C., Jhanwar, S., Offit, K., Steel, M., Ingles, S., Haile, R., Lindblom, A., Olsson, H., Borg, A., Bishop, D. T., Solomon, E., Radice, P., Spatti, G., Gayther, S., Ponder, B., Warren, W., Stratton, M., Liu, Q., Fujimura, F., Lewis, C., Skolnick, M., and Goldgar, D. E. (1995). A collaborative survey of 80 mutations in the BRCA1 breast and ovarian cancer susceptibility gene. J. Am. Med. Assoc. 273: 535– 541. Su, L.-K., Kinzler, K. W., Vogelstein, B., Preisinger, A. C., Moser, A. R., Luongo, C., Gould, K. A., and Dove, W. F. (1992). Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science 256: 668–670. Wooster, R., and Stratton, M. R. (1995). Breast cancer susceptibility: A complex disease unravels. Trends Genet. 11: 3–5.
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Isolation of the Mouse Homologue of BRCA1 and Genetic Mapping to Mouse Chromosome 11 L. MICHELLE BENNETT,*,1 ASTRID HAUGEN-STRANO,* CHARLES COCHRAN,* HEATHER A. BROWNLEE,* FRED T. FIEDOREK, JR.,† AND ROGER W. WISEMAN* *Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709; and †Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received March 8, 1995; accepted July 27, 1995
The BRCA1 gene is in large part responsible for hereditary human breast and ovarian cancer. Here we report the isolation of the murine Brca1 homologue cDNA clones. In addition, we identified genomic P1 clones that contain most, if not all, of the mouse Brca1 locus. DNA sequence analysis revealed that the mouse and human coding regions are 75% identical at the nucleotide level while the predicted amino acid identity is only 58%. A DNA sequence variant in the Brca1 locus was identified and used to map this gene on a (Mus m. musculus Czech II 1 C57BL/KsJ)F1 1 C57BL/KsJ intersubspecific backcross to distal mouse chromosome 11. The mapping of this gene to a region highly syntenic with human chromosome 17, coupled with Southern and Northern analyses, confirms that we isolated the murine Brca1 homologue rather than a related RING finger gene. The isolation of the mouse Brca1 homologue will facilitate the creation of mouse models for germline BRCA1 defects. q 1995 Academic Press, Inc.
involved in DNA binding (Freemont, 1993). The presence of this conserved structural motif suggests that the BRCA1 protein may regulate gene expression (Futreal et al., 1994; Miki et al., 1994). As an initial step toward understanding BRCA1 functions, we have isolated the murine homologue. The mouse provides a good system in which to study human disease syndromes because approximately 37% of the murine genome contains segments in which both synteny and gene order are conserved with humans (Nadeau, 1989). One of the most extensively characterized syntenic regions between the mouse and the human is the distal half of mouse chromosome 11, which contains at least 54 human chromosome 17 loci (Lossie et al., 1994). In this report we describe the sequence of the coding region for the mouse Brca1 homologue, isolation of mouse genomic P1 clones, and mapping of Brca1 to a 1.9-cM interval on mouse chromosome 11 that also contains the Mox1 and keratin gene cluster loci (Candia et al., 1992; Lossie et al., 1994). MATERIALS AND METHODS
INTRODUCTION
The human BRCA1 locus, mapped to chromosome 17q in 1990 (Hall et al., 1990), was recently identified by positional cloning (Miki et al., 1994). Germline defects in this gene confer a profound predisposition to the development of hereditary, early-onset breast and ovarian cancers (Futreal et al., 1994; Miki et al., 1994). Since the wildtype allele is almost always lost in hereditary breast and ovarian tumors, BRCA1 appears to function as a tumor suppressor gene (Wooster and Stratton, 1995). The human BRCA1 gene contains 24 exons, 22 of which encode an acidic protein 1863 amino acids in length. BRCA1 contains a Cys3-His-Cys4 (C3HC4) RING finger domain near the amino terminus that has also been observed in proteins predicted to be 1 To whom correspondence should be addressed at the National Institute of Environmental Health Sciences, MD C4-06, 111 Alexander Dr., Research Triangle Park, NC 27709. Telephone: (919) 5413229. Fax: (919) 541-3720. E-mail: [email protected].
0888-7543/95 $12.00 Copyright q 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Isolation of the murine Brca1 homologue. A human polymerase chain reaction (PCR) product (nucleotides 74 to 517 of Accession No. U14680) was used as a hybridization probe to isolate the initial Brca1 cDNAs from a mouse mixed germ cell library. Since Northern analysis of multiple mouse tissues with the human probe indicated that Brca1 transcript levels were highest in testis (data not shown), a mixed germ cell cDNA library from CD-1 mice was screened to yield a series of clones. Genomic clones from Lambda FIX II library of 129SV/J mice (Stratagene) were used to obtain Brca1 sequences from exon 11. In addition, cDNA was generated from mouse testis and spleen RNA using the reverse transcription system (Promega). These cDNAs were used as templates to amplify mouse sequences with combinations of mouse and human oligonucleotides to obtain Brca1 coding sequences between exons 12 and 24. The PCR conditions for mouse primer combinations were as follows: 947C for 3 min followed by 35 cycles of 947C/1 min, 557C/1 min, 727C/1 min. These conditions were modified when human/mouse primer combinations were used to include 5 cycles of 947C/1 min, 377C/1 min, 727C/1 min following the initial denaturation step and before the 35 cycles with an annealing temperature of 557C. Isolated clones and PCR products were sequenced using a PRISM dye terminator kit on an ABI 373 automated fluorescent sequencer (Applied Biosystems). Sequence comparison of the mouse Brca1 and
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ISOLATION AND MAPPING OF MURINE Brca1 the human BRCA1 gene was done using GAP and BESTFIT software (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group). Southern and Northern analysis. Multiple-species Southern and multiple mouse tissue Northern filters (Clontech) were probed with human- or mouse-specific sequences containing exon 2. Southern and Northern blots were hybridized in Hybrisol I (Oncor) at 427C overnight and washed twice in 21 SSC (11 SSC: 1.5 M NaCl/0.15 M sodium citrate, pH 7.0) for 5 min at room temperature, twice in 21 SSC/1% SDS for 30 min at 657C, and twice in 0.11 SSC/0.1% SDS at room temperature for 30 min. The membranes were exposed to X-Omat AR (Kodak) overnight to 1 week. The mouse exon 2 probe was generated using the primers MBF2, 5*-ACT GGA ACT GGA AGA AAT GGA, and MBR2, 5*-TYA CCA GAT CGG ACA CTC TAA. Isolation of P1 clones containing Brca1. PCR primers were designed based on the Brca1 cDNA sequence (MBF3, 5*-GCA CAT TTA TTA CAG GAC CAC A; MBR4, 5*-ACT TCC ACC TCA GCC TAT TTT T) that amplified a 390-bp fragment from the 5* end of Brca1 exon 11 from mouse genomic DNA. Using these primers, three Brca1containing clones were obtained by screening a commercially available 129 mouse P1 library (Genome Systems). Southern analysis of the P1 clones after restriction enzyme digestion with PvuII, EcoRI, and BamHI was performed using BRCA1 probes containing mouse exon 2 or human exons 1 through 6. The digested P1 DNA was electrophoresed on a 0.7% agarose gel in 11 TBE (0.9 M Tris–borate/ 0.002 M EDTA, pH 8.0) for 15 h at 35 V. The DNA was transferred to GeneScreen membrane (DuPont) in 101 SSC after denaturation (1.5 M NaCl/0.5 M NaOH) and neutralization (1.5 M NaCl/0.5 M Tris, pH 7.5). Following transfer, DNA was cross-linked to the membrane with a Stratalinker (Stratagene) and hybridized as described above. Identification of sequence variants and mapping. The intersubspecific backcross (Czech II 1 C57BL/KsJ)F1 1 C57BL/KsJ was used to determine the order of genes in the murine Brca1 region. One hundred fifty-eight N2 progeny of a previously characterized (Mus m. musculus Czech II 1 C57BL/KsJ m/m)F1 1 C57BL/KsJ m/m backcross (Fiedorek and Kay, 1994) were initially genotyped using the mouse chromosome 11 simple sequence repeat (SSR) markers D11Mit32, -52, -58, and -10 (MIT Database of SSLP Markers: [email protected]). Animals identified as being recombinant in this region were subsequently typed with the SSR markers D11Mit10, -14, -32, -52, -58, -59, -70, -98, -99, -145, -160, and -197 (Research Genetics). The PCR products were electrophoresed on a 7% native acrylamide gel at 200 V for 2–4 h with subsequent staining by ethidium bromide and visualization with UV light. A (CA)n sequence variant between C57BL/KsJ and Czech II mice was identified in intron 3 of the Brca1 gene (Accession No. U32585). Oligonucleotides were designed based on sequences flanking the CA repeat to amplify a 370-bp fragment in the 129SV/J mouse strain. Forward and reverse primers were MBF43, 5*-CAG AGT TTT TGT TTA CTT GGT CC, and MBR42, 5*-TGC ACT TAC CTG CAT ATG ACT T. The PCR conditions for this amplification were as described above except that the number of cycles was limited to 27. Comparison of the PCR templates from the two inbred strains revealed that the Czech II-specific PCR product was larger than that of C57BL/KsJ. A Mox1 gene sequence varient was identified to distinguish between the C57BL/KsJ and the Czech II alleles. Primers for the Mox1 gene were selected (MMOX1F, 5*-CTG GGG CCC TCT TTT CTG, and MMOX1R, 5*-CTG TCT TGA TGG GGT GGG) to amplify a 660-bp fragment from the 3* untranslated region (Accession No. Z15103). Sequence analysis of these PCR products revealed restriction fragment length polymorphism between the two strains. Restriction enzyme digest of this PCR product with Bbs1 yields two fragments of 403 and 257 bp from the C57BL/KsJ allele, while the product from the Czech II allele remains intact. The genetic order and recombination intervals expressed in centimorgans were determined using Map Manager software (Manley, 1993).
RESULTS
Isolation of the Mouse Brca1 Homologue Screening of the murine mixed germ cell cDNA library with a human probe containing the RING finger
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domain yielded three clones. DNA sequence analysis revealed that these cDNAs contained the putative murine Brca1 homologue and extended from exon 1, which is untranslated, to exon 11. A composite sequence for the entire murine Brca1 coding region was assembled using additional mixed germ cell cDNAs, genomic clones containing exon 11, and cDNA PCR products for exons 12 to 24 (Accession No. U32446). Partial genomic sequencing revealed that exon 11 comprises about 60% of the total coding region and the murine Brca1 gene contains intron–exon boundaries similar to that of the human. While the overall homology between human and mouse nucleotide sequences is relatively high (75%), the amino acid identity is only 58%. Figure 1 shows the alignment of the predicted mouse and human gene products. The C3HC4 RING finger domain (residues 20 through 70) is highly conserved in mice relative to humans with only a single conservative substitution of aspartic acid for glutamic acid. As in the human the mouse Brca1 gene product is very acidic, with a net excess of 53 acidic residues. Hybridization Analysis of the Mouse Brca1 Homologue To confirm that we had isolated the murine Brca1 homologue rather than a related RING finger gene, additional studies were performed. Northern analysis of multiple-tissue mRNAs with a mouse exon 2 probe revealed a 7.5-kb transcript that was most abundant in mouse testis and spleen (Fig. 2), consistent with previous observations for human tissues (Miki et al., 1994). Likewise, Southern analysis with this mouse probe detected the expected human EcoRI fragment as well as single-copy sequences in all mammalian DNAs examined (Fig. 2). Southern analysis was also performed on the mouse P1 clones. Duplicate filters containing P1 DNA digested with various enzymes were probed with mouseand human-specific sequences from the RING finger domain (Fig. 3). Common hybridization patterns between the two filters indicate that two P1 clones (P14020 and P14022) include the 5* half of the mouse Brca1 gene homologue. The third clone (P14021) contains a smaller portion of 5* Brca1 sequence that does not extend to the second exon. Finally, PCR analysis with mouse exon 24 primers revealed that all three P1 clones contain at least part of the 3* untranslated region of the Brca1 gene (data not shown). Linkage Analysis The MOX1 and BRCA1 loci are tightly linked on human chromosome 17q21 (Neuhausen et al., 1994). Based on known human–mouse synteny, we predicted that the murine Brca1 gene would be tightly linked to the Mox1 locus, previously mapped to chromosome 11 (Candia et al., 1992). To map the murine Brca1 homologue, DNA sequence variants of the mouse Brca1 and the Mox1 genes were identified and used to localize
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FIG. 1. Alignment of the predicted murine Brca1 (upper, U32446) and human BRCA1 (lower, U14680) amino acid sequences. Vertical lines between amino acids indicate identity, while colons and dots indicate greater and lesser degrees of amino acid similarity, respectively, as determined by the GAP program. The C3HC4 RING finger residues are indicated by asterisks.
these two genes relative to known SSR markers in the Brca1 region. Segregation analysis of the Brca1 and Mox1 se-
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quence variants and chromosome 11 SSR markers was carried out on 158 (Mus m. musculus Czech II 1 C57BL/KsJ m/m)F1 1 C57BL/KsJ m/m backcross prog-
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D11Mit52, which is approximately 2.6 cM distal to its placement by the MIT group. The second difference lies in the recombination distance between D11Mit32 and D11Mit98. We calculate this distance to be 7.6 cM from the data obtained in our backcross population, while these two anchor loci had been previously assigned a recombination distance of 17.9 cM. DISCUSSION
Contrary to expectations, sequence comparisons between the mouse and the human homologues indicated that BRCA1 is more highly conserved at the nucleotide than at the amino acid level. The coding region of the mouse and human genes are 75% identical at the nucleic acid level, while the predicted amino acid identity is only 58% overall. This result is in contrast to what has been typically observed for other cancer susceptibility genes. For example, the retinoblastoma and adenomatous polyposis coli tumor suppressor gene products are 90% identical between mice and humans (Bernards et al., 1989; Su et al., 1992). Despite the low evolutionary conservation between the two species, several lines of evidence support the conclusion that we have isolated the mouse Brca1 homologue rather than a related gene. Hybridization analyses demonstrated single-copy sequences in all mammalian species examined and transcripts of the expected size and a tissue-specific pattern of expression (Miki et al., 1994). Likewise, we localized the Brca1 gene to a 1.9cM region of mouse chromosome 11 that contains the Mox1 gene and the keratin gene cluster. This region of mouse chromosome 11 was predicted to harbor the
FIG. 2. Northern and Southern analysis of the mouse Brca1 gene. The upper panel illustrates Northern analysis of Brca1 transcripts in mRNA isolated from the mouse tissues indicated. The lower panel contains a Southern blot of EcoRI-digested DNA from various species. Both filters were hybridized with a mouse exon 2 probe.
eny (Fiedorek and Kay, 1994). Four chromosome 11 SSR markers were typed that spanned the syntenic region predicted to harbor the Brca1 and Mox1 genes. This analysis identified 19 recombinant progeny that were then used to type the Brca1 CA repeat, the Mox1 BbsI restriction fragment length variant, and a dozen additional SSR markers by PCR. The haplotypes from this analysis are shown in Fig. 4A. The Brca1 and Mox1 loci cosegregate with the keratin gene cluster (D11Mit57) in a 1.9-cM interval defined by D11Mit99 and D11Mit52 (Fig. 4B). The order of the markers for this subchromosomal segment on distal mouse chromosome 11 is consistent with the human physical map in the BRCA1 region (Albertsen et al., 1994) and corresponds to the order and approximate cM distances defined for the murine composite map (Lossie et al., 1994). Only two discrepancies between our results and those obtained from the MIT genome center were detected. We observed the cosegregation of D11Mit58 with
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FIG. 3. Southern analysis of mouse P1 clones. (A) Hybridization with a mouse exon 2 probe reveals identical DNA fragments for P14020 (lane 1) and P14022 (lane 2); P14021 (lane 3) does not contain exon 2 of Brca1. (B) Hybridization of a duplicate filter with a human cDNA probe that extends from exon 1 to exon 6 shows cross-hybridization to all three P1 clones. P14021 has fewer hybridizing fragments consistent with its lack of the 5* most exons.
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FIG. 4. Meiotic mapping of the Brca1 region. (A) Distribution of the haplotypes for 158 progeny of a (Czech II 1 C57BL/KsJ)F1 1 C57BL/KsJ backcross. The black squares represent the presence of the Czech II allele, and the white squares signify progeny homozygous for the C57BL/KsJ allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. (B) A genetic linkage map of distal mouse chromosome 11 including the locations of the Brca1 and Mox1 loci is shown. Recombination distances between loci in cM are shown to the left of the chromosome.
Brca1 locus based on synteny with human chromosome 17q21 (Lossie et al., 1994). The only structural motif revealed by computer analysis of the predicted mouse and human Brca1 gene products, which has been highly conserved, is a C3HC4 RING finger near the amino terminus. RING finger domains have been observed in more than 40 other proteins from a wide range of species (Freemont, 1993). Many of these RING finger proteins are involved in transcriptional regulation, and several are thought to bind DNA. The presence of this conserved structural motif suggests that at least one function of Brca1 may be to control the expression of other genes. In contrast to that observed for the RING domain only short stretches of amino acid identity are evident over the remainder of the two gene products. Additional functional domains of BRCA1 may be revealed by future comparisons of the mouse and human gene products. While providing strong support for the functional significance of the RING finger domain, these observations of amino acid conservation suggest that the evolutionary constraints on much of the remainder of the BRCA1 gene product are unexpectedly weak. Despite the low degree of homology between the two species at the protein level, several apparently important amino acids have been conserved. In particular, we have determined that the amino acid positions at which missense mutations in BRCA1 families have been reported are identical between mice and humans in six of seven cases (Shattuck-Eidens et al., 1995). In contrast the residue representing the common human allele is conserved in the mouse for only one of five published human polymorphisms that result in amino acid substitutions (Freidman et al., 1994; Miki et al., 1994). Hence, comparison of the human and mouse genes may provide insights into whether sequence variants represent predisposing mutations or rare polymorphisms that are biologically neutral. Based on the likely significance of the RING finger domain in BRCA1 protein function, we are creating
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mice with a germline BRCA1 alteration using a targeting vector designed to disrupt this structural motif. As in humans, we predict that Brca1 defects may predispose mice to neoplastic development in the mammary gland, ovary, and perhaps other tissues. Generation of these mice will also allow us to ask whether Brca1 alterations, either in the heterozygous and homozygous null state, result in any developmental or reproductive abnormalities. Given that a patient homozygous for a BRCA1 defect has recently been identified (Boyd et al., 1995), it seems likely that Brca1-null mice will be viable. Finally, these Brca1-deficient mice will provide an important new model system for the analysis of interactions between this important cancer predisposing gene and a variety of environmental factors. ACKNOWLEDGMENTS We thank Eric Kay for technical assistance with mapping analysis. The CD-1 mixed germ cell cDNA library and total testis RNA were kindly provided by Dr. Jeff Welch. This work was supported in part by Grant DK44074 from the National Institutes of Health (F.T.F.).
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ISOLATION AND MAPPING OF MURINE Brca1 early mesodermal patterning in mouse embryos. Development 116: 1123–1136. Fiedorek, F. T., Jr., and Kay, E. S. (1994). Mapping of PCR-based markers for mouse Chromosome 4 on a backcross penetrant for the misty (m) mutation. Mamm. Genome 5: 479–485. Freemont, P. S. (1993). The RING finger: A novel protein sequence motif related to the zinc finger. Ann. N. Y. Acad. Sci. 684: 174– 192. Freidman, L. S., Ostermeyer, E. A., Szabo, C. I., Dowd, P., Lynch, E. D., Rowell, S. E., and King, M.-C. (1994). Confirmation of BRCA1 by analysis of germline mutations linked to breast and ovarian cancer in ten families. Nature Genet. 8: 399–404. Futreal, P. A., Liu, Q., Shattuck-Eidens, D., Cochran, C., Harshman, K., Tavtigian, S., Bennett, L. M., Haugen-Strano, A., Swensen, J., Miki, Y., Eddington, K., McClure, M., Frye, C., Weaver-Feldhaus, J., Ding, W., Gholami, Z., Soderkvist, P., Terry, L., Jhanwar, S., Berchuck, A., Iglehart, J. D., Marks, J., Ballinger, D. G., Barrett, J. C., Skolnick, M. H., Kamb, A., and Wiseman, R. (1994). BRCA1 mutations in primary breast and ovarian carcinomas. Science 266: 120–122. Hall, J. M., Lee, M. K., Newman, B., Morrow, J. E., Anderson, L. A., Huey, B., and King, M.-C. (1990). Linkage of early-onset familial breast cancer to chromosome 17q21. Science 250: 1684–1689. Lossie, A. C., MacPhee, M., Burchberg, A. M., and Camper, S. A. (1994). Mouse Chromosome 11. Mamm. Genome 5: S164–S180. Manley, K. F. (1993). A Macintosh program for storage and analysis of experimental genetic mapping data. Mamm. Genome 4: 303– 313. Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P. A., Harshman, K., Tavtigian, S., Cochran, C., Bennett, L. M., Ding, W., Bell, R., Rosenthal, J., Hussey, C., Tran, T., McClure, M., Frye, C.,
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