Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4362-4366, May 1995 Genetics

Physical mapping, cloning, and identification of within a 500-kb region containing BRCAl MELISSA A. BROWN*t, KAREN A. JONES*, HANS NICOLAI*, MARISA BONJARDIM*, DONALD BLACKt, ROBERT MCFARLANEt, PIETER DE JONG§, JEREMY P. QUIRK¶, HANS LEHRACH¶, AND ELLEN SOLOMON* *Somatic Cell Genetics and lGenome Analysis Laboratories, Imperial Cancer Research Fund, London, WC2A 3PX, United Kingdom; DBeatson Institute for Cancer Research, Bearsden, Glasgow, G61 1BD, United Kingdom; and §Roswell Park Cancer Institute, Buffalo, NY Communicated by Walter Bodmer, Imperial Cancer Research Fund, London, United Kingdom, December 21, 1994 ABSTRACT BRCA1 is a breast/ovarian cancer suscepti- cloned in the pAMP PCR cloning vector (GIBCO/BRL) to bility on human 17q21. We describe a generate exon libraries. complete and detailed physical map of a 500-kb region of Analysis of Exon-Trapped Products. pAMP subclones of genomic DNA containing the BRCA1 gene and the partial exon-trapped products were sequenced in both directions with cloning in phage PI artificial . Approximately 70 vector primers, either manually using a Sequenase kit (United exons were isolated from this region, 11 of which were States Biochemical) or automatically using an ABI 373 DNA components of the BRCAI gene. Analysis of the other exons sequencer. Sequencing results were analyzedbyusing the GCG revealed a rho-related G and the interferon-induced program. Expression patterns and transcript sizes of new genes leucine-zipper protein IFP-35. were determined by hybridization of exon-trapped products to commercially available multiple-tissue Northern blots (Clon- Breast cancer is a common disease which exists in both tech), exactly according to the supplier's instructions. sporadic and inherited forms. A gene responsible for 45% of inherited breast cancers and nearly all cases from breast/ RESULTS ovarian cancer families (1) was mapped to chromosome 17q21 in 1990 (2). Recently this gene, BRCA1, was identified by using Physical Mapping. A long-range physical map surrounding positional cloning techniques (3). BRCAI and incorporating markers and genes generated by our During the search for the BRCAI gene, our laboratory and laboratory and others was constructed by PFGE analysis (Fig. others have carried out fine physical mapping and character- 1). Hybridization with EDH and 855RF probes (see Fig. 1 ization of.the 1.0- to 1.5-Mb region known to contain BRCA1 legend) revealed a 550-kb Not I fragment to which both probes (e.g., refs. 4 and 5). This paper describes the detailed charac- hybridize, as shown by the arrowed bands. RF18 (probe C) also terization and partial cloning of a 500-kb region of chromo- mapped to the same Not I, Nru I, and Nru I/Not I fragments some 17q12-21, between the gene 1A1-3B (6) and the poly- as 855RF (data not shown). ET-A37 (BRCAI exon 13; see morphic marker D17S856 (7). We also describe the isolation below) hybridized to the same 480-kb Nru I fragment as RF18, and analysis of a number of genes mapping to this region, as indicated by the arrowed bands in autoradiographs C and D, including BRCA1.11 but it hybridized to a larger Not I fragment of 750 kb. This largerNot I fragment and theMlu I andEag I fragments to which the ET-A37 probe hybridizes are the same ones to which the MATERIALS AND METHODS 1A1.3B gene hybridizes (4). Taken together, these results Physical Mapping. Single-copy probes across the BRCAI suggest that EDH and 855RF reside on the same 550-kb Not region (see Fig. 1 legend) were hybridized to pulsed-field gel I fragment and that the ET-A37 probe resides on the next distal electrophoresis (PFGE) Southern filters as previously de- Not I fragment, which also hybridized to the genes 1A1.3B and scribed (4). RNU2 and the markers D17S858/D17S859 (4). ET-A37 also Isolation of Cosmid and Phage P1 Artificial Chromosome hybridizes to the same Nru I fragment as the D17S855 probe. (PAC) Clones. Cosmid clones were isolated from a flow-sorted Consequently, the long-range restriction maps generated at cosmid library (8) by using either [y-32p]ATP- each locus could be superimposed, giving the detailed map labeled primers for the polymorphic markers D17S855 and shown in A+B+C+D at the bottom of Fig. 1. D17S856 or cDNA fragments from the 5' ends of the genes Genomic Cloning of the Region Between 1A1-3B and 1A1-3B and EDH-1 7B, generated by PCR using primers based D17S856. Initial efforts to clone the BRCA1 genomic region on the published DNA sequence (6, 9). PAC clones were involved the isolation of yeast artificial chromosome (YAC) isolated from a total human PAC library [generated by P.d.J., clones by using both PCR and hybridization strategies. Screen- (10)] by using fragments from the above-mentioned cosmids as ing of four YAC libraries generated several YAC clones; probes. Gaps between PAC clones were filled by using the however, all contained only one marker, were chimeric, or riboprobe II core system kit (Promega). carried deletions (4). Thus we turned to alternate genomic Exon Trapping. Exon sequences were isolated from PAC cloning vectors. The recent construction of a high-quality clones by following a modification of the procedure described human genomic PAC library (10), which contains stable by Buckler et aL (11). Briefly, PAC DNA was digested with nonchimeric clones in the range of 100-300 kb, provided an either Pst I or a combination ofBamHI and Bgl II, inserted into ideal solution. Screening the PAC library with a repeat-free the exon-trapping vector pSPL3, and then transfected into (RF) fragment from the centromeric end of the 1A1-3B- Cos-7 cells. Total RNA was isolated 48 hr after transfection positive cosmid A11100 [isolated with the RNU2/1A1-3B YAC and used as a template for reverse transcriptase (RT)-PCR, using pSPL3 sequence-specific primers, and shotgun sub- Abbreviations: PFGE, pulsed-fieldgel electrophoresis; PAC, phage P1 artificial chromosome; HIV, human immunodeficiency virus; RF, repeat-free. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in "The sequences reported in this paper have been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession nos. U21493-U21545). 4362 Downloaded by guest on October 2, 2021 Genetics: Brown et at Proc. Natl. Acad. Sci USA 92 (1995) 4363 A B C D M BssBs B;sbia Nr Nr Nr Ea M sBsBs Bs Ea Ea Nr Nr Nr Ea M Bs Bs Bs Ea Ea Nr Nr Nr Ea M Bs Bs Bs Ea Ea Nr Nr Nr Ea N N M M N Bs .aaEa M N Nr N M Bs Nr N N M M N Bs Ea Ea M N Nr N M Bs Nr N N M M N Bs Ea Ea M N Nr N M Bs Nr NN M M N Bs Ea Ea M N Nr N M Bs Nr -Ar * A 530- ~' _ " * *::^o 300- f --*j 6*,; *« -0 *a«e'*I.& a . 50-

a -" i * * J ..*c ^ --- CEN -- Bs It' (Fal; A Nr lM) Nr i N I;"1.1 Bs Bs B N rl Nr M Ea Ea MN Nr I.1 11 .Wl 855RF C Bs Bs N Nr IHa Ea MN Nr I I I- 750kb RF18 Bs D PAC 22157 MNEa Nr Bs Ea N II . I. ~ ~ ~ ~ ~ ~ ~ ~ II .. //-", -j Hs M ET A37 A+B+C+D ls (Eal Bs Bs Bs Bs N Nr l-:; (M) Nr Ea Ea Ea MNEa Nr Bs EaBsNr MBs N _ _ _ I l1-1l11 _ I EDHI RF18 KiAg 855RF ET A37 IAI.3B RNU2N PAC 22157 ET A38 FIG. 1. Construction of a long-range restriction map around BRCA1 by PFGE analysis. A and B correspond to sequential hybridization of one filter and C and D to the sequential hybridization of a second filter. Probe A was a PCR product from the 3' untranslated region of EDH; probe B was a repeat-free fragment from a cosmid containing marker D17S855 (855RF); probe C was a repeat-free fragment from PAC 22157 (RF18); and probe D was ET-A37 (BRCAI exon 13, see text). Size markers (kb) corresponding to each of the two PFGE filters used are indicated to the left of each pair of autoradiographs. Individual restriction mapsA, B, C, and D were constructed by using the data provided by each of the corresponding autoradiographs. The maps could be superimposed at the regions of the shared restriction fragments (A+B+C+D). Subsequent PFGE Southern analysis indicated the location of the KiAg gene (5) and ET A38. N, Not I; M, Mlu I; Bs, BssHII; Ea, Eag I; Nr, Nru I; CEN, centromere.

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EtBr ET- BI ET-B2 ET-A3 ET-A37 FIG. 2. Cloning and exon trapping of the D17S856 to 1AI-3B region of 17q12-21. (A) Map showing the order of genes and markers (not to scale). (B) Cosmid and PAC clones isolated, indicating the presence of various sequences by hybridization (broken lines) or by PCR (rectangular blocks) and the orientation of riboprobe ends (blocks labeled T7 or SP6) if known. (C) Exon-trapped products, showing mapping results of the exon-trapped products described in this paper. Solid lines indicate the PAC to which the exons hybridize. (D) Mapping of exons to PACs by Southern hybridization, showing the ethidium bromide (EtBr)-stained agarose gel of EcoRI-digested PAC DNA and the results of Southern analysis with four of the exons (as indicated). Lanes: M, A phage DNA HindIII-digested markers; 1, PAC 171Q6; 2, PAC 231K9; 3, PAC 63F12; 4, PAC 024217; 5, PAC 12257; 6, PAC P21119; 7, PAC 44B1; and 8, PAC 103014. Downloaded by guest on October 2, 2021 4364 Genetics: Brown et al Proc. Natl. Acad Sci. USA 92 (1995) 12H4 (see ref. 6) and containing the 5' end of the 1A1-3B gene Table 1. Results of searches for homology of (H. Nicolai, D.B., and E.S., unpublished results)] identified a exon-trapped products single PAC, 103014 (Fig. 2B). Further characterization of this Clone BRCA1 % amino acid PAC revealed that it also contained the marker Dl 7S855 Possibly homologous (Fig. no. exon gene or product identity 2 B and D). Using a RF fragment from a D17S855 cosmid, a second was identified. PAC 44B1 contained ET-B1 HIV vpu 92 PAC, 44B1, only ET-B2 the marker D17S855. Hybridization with the EDH cosmid RF ET-B31 fragment generated two PAC clones, 231K9 and 63F12, and at ET-B53 the marker Dl 7S856 a single PAC, 171Q6, was isolated. Three ET-A3 rho-like Yeast: 46 further PAC clones were isolated by riboprobe walking (Fig. G protein Human: 37 2B). ET-A12 21 Identification of Previously Unknown Genes Between ET-A38 D17S856 and Genomic clones were ET-A37 13 IAI-3B. analyzed for ET1 exonic sequences by using the technique of exon trapping. ET2 (16-20)* Several libraries were made (see Materials and Methods), and ET3 21 from these approximately 70 cloned exons were isolated and ET4 analyzed. ET5 Analysis of predicted amino acid sequences of exon-trapped ET6 products revealed several homologies and identities (Table 1). ET7 For ET-A3 showed amino acid and ET8 example, 46% identity 86% ET9 amino acid similarity, over a 77-amino acid stretch, to the yeast ET10 rho-1 protein as well as 37% identity and 75% similarity, over ET11 a 120-amino acid stretch, to human rho-A and rho-B ET12 (Fig. 3A). In addition, ET49, ET53, et al (Table 1) showed ET13 identity to the interferon-induced leucine zipper protein ET14 (16-20)* IFP-35 (12). Other clones displayed high similarity to HIV ET15 3 B and which is most an ET16 proteins (Fig. F), likely exon-trapping ET17 artifact, as the exon-trapping vector, pSPL3, contains 2.7 kb of ET18 22 + 23 HIV sequence (11). Data base searches of the sequences of the ET19 Alu repeat remaining clones showed no homology to genes present in the ET20 GenBank or European Molecular Biology Laboratory data ET21 bases (Table 1). ET22 HIV gp160 78 Subsequent to the cloning of BRCAI (3), translated exon ET23 22 + 23 were with the amino acid se- ET24 sequences compared BRCAI ET25 quence. This revealed 100% identity of ET-A12 and ET-A37 ET26 (16-20)* to BRCAI exons 21 and 13, respectively (Fig. 3 C and D) and ET27 of ET18, ET23, and ET29 with a combination ofBRCA1 exons ET28 22 and 23 (Fig. 3E). Furthermore, hybridization analysis of ET29 22 + 23 exons from this region, with various probes generated by PCR ET40 (16-20)* using primers based on the nucleotide sequence in and be- ET49 IFP-35 100 tween exons 16 and revealed that ET52 20, ET2, ET14, ET26, ET40, ET53 IFP-35 100 and ET83 corresponded to sequences between exons 16 and 20 ET54 (Table 1). The fact that ET-A37 (exon 13) maps to PAC ET56 IFP-35 100 103014 only, while ET-A12 (exon 21) maps to both PAC ET57 IFP-35 100 103014 and PAC 44B1 (Fig. 2), orients the BRCA1 gene in a ET58 telomeric to centromeric direction. ET59 IFP-35 100 The size and distribution of transcripts corresponding to ET61 IFP-35 100 several of the were ET62 IFP-35 100 exon-trapped products determined by ET63 Northern analysis. As shown in Fig. 4, ET-B1 hybridized to a ET64 2-kb transcript in testis; ET-A3, to a 1.5-kb message, predom- ET66 inantly in testis but also in other tissues; and ET-B53, to a ET67 IFP-35 100 2.5-kb mRNA in a wide range of tissues. ET-A37 hybridizes to ET68 IFP-35 100 a transcript of approximately 8 kb present in thymus and testis, ET69 IFP-35 100 consistent with its an exon from the ET70 being BRCAI gene (3). ET71 ET72 IFP-35 100 DISCUSSION ET73 ET74 IFP-35 100 The work presented in this paper confirms the order of ET75 IFP-35 100 markers between and including D17S856 and 1A1-3B (e.g., see ET76 refs. 5 and 7) on chromosome 17q12-21 and gives an estima- ET77 IFP-35 100 tion of the physical distances between them. Given that one of ET78 IFP-35 100 the used in the of this was a ET83 (16-20)* probes generation map, ET-A37, ET85 IFP-35 100 component of the BRCA1 gene, this information has provided ET92 IFP-35 100 a scaffold for more detailed analysis of the region surrounding ET93 this gene. The physical mapping data generated here were also critical for most of the HIV, human immunodeficiency virus; refers to no homology to genomic cloning and, indeed, genomic genes present in the GenBank on European Molecular Biology DNA between D17S856 and 1A1-3B has been cloned in four Organization data bases. cosmids and eight PAC clones. Approximately 70 exon sequences *(16-20) refers to positive hybridization of the cloned exon- were isolated from these PAC clones, using the exon-trapping trapped products with a PCR product spanning BRCA1 exons technique. 16-20. Downloaded by guest on October 2, 2021 Genetics: Brown et al Proc. NatL Acad Sci USA 92 (1995) 4365 A ET-A3: YDNVRPLAYPDSDAVLICFDISRPETLDSVLKKWQGETQEFCPNAKVVLVGCKLDMRTDLATLRELSKQRLIPVTHEP

Yeast rho-1: YDRLRPLSYPDSNVVLICFSIDLPDSLENVQEKWIAEVLHFCQGVPIILVGCKVDLRNDPQTIEQLRQEGQQPVTSQE (AN: P06780) Human rho-a: YDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMQE PVKPEE (AN: P0749) Human rho-b: YDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRQDEHTRRELAKMKQEPVRSE (AN: P08134) B ET-B1: MQPIQLAIVALVVAIIIAIVVWSIVIIEYRKILRQRKIDRLIDRLIERVEDSGNESDGEISALVEMGVEMGHHVPXDVDDL HIV vpu: MQPIQLAIVALAIIIAIVVWSIVIIEYRKILRQRKIDRLIDRLIERAEDSGNESEGEISALVEMGVEMGHHAPWDVDDL (AN: P05920) C ET-A12: IFRGLEICCYGPFTNMPTD BRCA1 exon 21: IFRGLEICCYGPFTNMPTD D ET-A37: QRDTMQHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQSTSEK BRCA1 exon 13: QRDTMQHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQSTSEK E ET18, 23 and 29: QLEWMVQLCGASVVKELSSFTLGTGVHPIVVVQPDAWTEDNGFHA BRCA1 exons 22 and 23: QLEWMVQLCGASVVKELSSFTLGTGVHPIVVVQPDAWTEDNGFHA F ET22 LKCTDLRMILIPNSSSGRMIMEKGEIXXCSFNISTSIR HIV GP160 LKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIR (AN: P03375) G ET49,ET53,ET56,ET57,ET59,ET61,ET62,ET67, ET68,ET69,ET72,ET75,ET78,ET85 and ET92: IRSQPVPRSVLVLNIPDILDGPELHDVLEIH IFP 35: IRSQPVPRSVLVLNIPDILDGPELHDVLEIH (AN: P80217) H ET74 and ET77: AQRLCQIGQFTVPLGGQQVPLRVSPYVNGEIQKAEIRSQPVPRSVLVLNIPDILDGPELHDVLEIH IFP 35 AQRLCQIGQFTVPLGGQQVPLRVSPYVNGEIQKAEIRSQPVPRSVLVLNIPDILDGPELHDVLEIH (AN: P80217)

FIG. 3. Homologies and amino acid identities of exon-trapped products with known genes. Figure shows only the minimal area of homology. Letters in boldface correspond to amino acid identity. Amino acid similarity is not shown, but percentages are given in the text. Parenthetic codes refer to data base accession numbers (AN). ET-A3 showed highest homology to a number of rho in the latter case more exons had been trapped, indicating that proteins, which are one of the three members of the RAS the intervening intron sequences must be quite short. Mapping superfamily of G proteins, along with RAS and RAB. RHO of these exon-trapped products back to the PACs revealed proteins are involved in cell morphology, playing a signal- hybridization solely to PAC 44B1, placing the IFP-35 gene transducing role in the control of actin cytoskeleton organi- centromeric to BRCA1 (Fig. 2). IFP-35 is a 35-kDa protein zation (13). Interestingly, ET-A3 is the third G protein to be which contains a leucine-zipper motif and readily forms ho- identified within the 1-Mb region defining BRCA1. In addition modimers in vitro. Unlike many other leucine-zipper- to ET-A3, the gene encoding an ADP-ribosylation factor, containing proteins, IFP-35 does not contain a DNA-binding HAL-64, has been mapped between RNU2 and PPY (D.B., M. motif and displays no detectable heterodimerization with Boyd, and E.S., unpublished results), and BC-16, a Rab5c- other known leucine-zipper proteins, and so the function of related gene, has been mapped close to D17S856 (5). IFP-35, like that of the BRCA1 product, remains unclear. Sequence analysis of several exon-trapped products revealed Comparison of the translated sequence of all exons with the 100% identity with a previously described protein, the inter- amino acid sequence of the BRCA1 product (3) revealed 100% feron-induced leucine-zipper protein IFP-35 (12). Interest- identity of several products with BRCAI exons. This was ingly, some clones had 30-amino acid stretches of identity while further supported by Northern analysis of ET-A37. Detailed others showed identity over 65 amino acids. This suggests that mapping of these exons has been helpful in the characteriza- Downloaded by guest on October 2, 2021 4366 Genetics: Brown et at. Proc. Natl. Acad. Sci. USA 92 (1995)

12345678 12345678 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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ET-B1 ET-A3 ET-A37 ET-B53 FIG. 4. Northern analysis of exon-trapped products. Results from hybridization of four of the exons to Clontech multiple-tissue (human) Northern blots. Lanes: 1, spleen; 2, thymus; 3, prostate; 4, testis; 5, ovary; 6, small intestine; 7, colon; 8, peripheral blood leukocyte; 9, heart; 10, brain; 11, placenta; 12, lung; 13, liver; 14, skeletal muscle; 15, kidney; and 16, pancreas. Approximately even loadings on these Northern blots were confirmed previously by hybridization with an actin cDNA probe (4). tion of the genomic DNA housing the BRCA1 gene, in 2. Hall, J. M., Lee, M. K., Newman, B., Morrow, J. E., Anderson, particular for determining the orientation of BRCA1. Inter- L. A., Huey, B. & King, M.-C. (1990) Science 250, 1684-1689. estingly, comparison of these data with similar experiments 3. Miki, Y., Swensen, J., Shattuck-Eidens, D., Futreal, P. A. & with 1A1-3B exons (H. Nicolai, D.B., and E.S., unpublished Harshman, K. et al. (1994) Science 266, 66-71. results) indicates that these two genes are transcribed in 4. Jones, K. A., Black, D. M., Brown, M. A., Griffiths, B. L., Nicolai, opposite directions. Intriguingly, we have recently shown that H. M., Chambers, J. A., Bonjardim, M., Xu, C.-F., Boyd, M., the distance between these two is less than 300 McFarlane, R., Korn, B., Poustka, A., North, M. A., Schalkwyk, genes bp, L., Lehrach, H. & Solomon, E. (1994) Hum. Mol. Genet. 3, raising the possibility of coregulation of transcription and 1927-1934. presenting an additional model for BRCAl-mediated onco- 5. Albertsen, H. M., Smith, S.A., Mazoyer, S., Fujimoto, E., genesis (14). Stevens, J., Williams, B., Rodriguez, P., Cropp, C. S., Slijepcevic, The above-mentioned homologies and identities account for P., Carlson, M., Robertson, M., Bradley, P., Lawrence, E., approximately 50% of the exons described in this paper. The Harrington, T., Mei Sheng, Z., Hoopes, R., Sternberg, N., remaining exons displayed no significant homology to gene Brothman, A., Callahan, R., Ponder, B. A. J. & White, R. (1994) sequences present in the GenBank or European Molecular Nat. Genet. 7, 472-479. Biology Laboratory data bases and may therefore represent 6. Campbell, I. G., Nicolai, H. M., Foulkes, W. D., Senger, G., novel genes. Clearly, some of the sequences may be novel Stamp, G. W., Allan, G., Boyer, C., Jones, K., Bast, R. C., components of previously described genes, including EDH (9), Solomon, E., Trowsdale, J. & Black, D. M. (1994) Hum. Mol. lAl (6), and the recently described IF-I, BC3-1, BC1-6, and Genet. 3, 589-594. BC1-16 the aim of this 7. Anderson, L. A., Friedman, L., Osborne-Lawrence, S., Lynch, E., genes (5). Therefore, although original Weissenbach, J., Bowcock, A. & King, M.-C. (1993) Genomics 17, project was to identify a breast/ovarian cancer gene linked to 618-623. this region of 17q12-21, the resulting work has not only 8. Lehrach, H. (1990) in Genome Analysis: Genetic and Physical provided a detailed physical map about the BRCA1 gene but Mapping, eds. Davies, K. E. & Tilghman, S. M. (Cold Spring also will provide an excellent resource for future work on Harbor Lab. Press, Plainview, NY), Vol. 1, pp. 39-81. chromosome 17 as well as contributing to the overall charac- 9. Peltoketo, H., Isomaa, V. & Vihko, R. (1992) Eur. J. Biochem. terization of the . 209, 459-466. 10. Ioannou, P.A., Amemiya, C.T., Games, J., Kroisel, P.M., The authors are grateful to Fiona Francis for advice on P1 and PAC Shizuya, H., Chen, C., Batzer, M. A. & de Jong, P. J. (1994) Nat. libraries and analysis of clones, to Guenther Zehetner and Ranjit Genet. 6, 84-89. Bhogal for help with picking positive cosmid and PAC clones, and to 11. Buckler, A. J., Chang, D. D., Graw, S. L., Brook, J. D., Haber, Mike North for advice on the exon-trapping technique. M.A.B. is D. A., Sharp, P. A. & Housman, D. E. (1991) Proc. Natl. Acad. supported by a European Molecular Biology Organization postdoc- Sci. USA 88, 4005-4009. toral research fellowship and M.B. was funded by a Conselho Nacional 12. Bange, F.-C., Vogel, U., Flohr, T., Kiekenbeck, M., Denecke, B. de Desenvolvimento Cientifico e Tecnol6gico (Brazil) research grant. & Bottger, E. C. (1994) J. Biol. Chem. 269, 1091-1098. 13. Nobes, C. & Hall, A. (1994) Curr. Opin. Genet. Dev. 4, 77-81. 1. Easton, D. F., Bishop, D. T., Ford, D., Crockford, G. P. & The 14. Brown, M. A., Nicolai, H., Xu, C.-F., Griffiths, B. L., Jones, Breast Cancer Linkage Consortium (1993) Am. J. Hum. Genet. K. A., Solomon, E., Hosking, L., Trowsdale, J., Black, D. M. & 52, 678-701. McFarlane, R. (1994) Nature (London) 372, 733. Downloaded by guest on October 2, 2021