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Physical Mapping, Cloning, and Identification of Genes Within a 500-Kb Region Containing Brcal MELISSA A

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Proc. Natl. Acad. Sci. USA Vol. 92, pp. 4362-4366, May 1995 Genetics Physical mapping, cloning, and identification of genes 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 gene on human chromosome 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 chromosomes. 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 protein 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 chromosome 17 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. A D17S856 EDH gene Dl 7S855 1Al gene -1--3 0746 ........ ... .. A #sAXI coss I) 1116 c(, C:'1)746 c,, B IO4 co~s Al (XX) IAI B 855 PAC 1)3014 prom IO" 85B I'AC 17 IQ6 PAC44BI1 T75 _ PAC 21P119 C PAC 22157 LrF'- AA3 A12 A3? A?X B1 B2 ?31 B53 D ET- to ET-93ET93 trapped t'ron) PAC 10304103()14 and 44144 1 M 2.345 67 23kh- - me ) 0 4 2. _ 0 2 - 0.5 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).
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