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Proc. Natl. Acad. Sci. USAVol. 92, pp. 4362-4366, May 1995Genetics
Physical mapping, cloning, and identification of genes within a500-kb region containingBRCAlMELISSA 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 forCancer 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-bility gene on human chromosome 17q21. We describe acomplete and detailed physical map of a 500-kb region ofgenomic DNA containing the BRCA1 gene and the partialcloning in phage PI artificial chromosomes. Approximately 70exons were isolated from this region, 11 of which werecomponents of the BRCAI gene. Analysis of the other exonsrevealed a rho-related G protein and the interferon-inducedleucine-zipper protein IFP-35.
Breast cancer is a common disease which exists in bothsporadic and inherited forms. A gene responsible for 45% ofinherited breast cancers and nearly all cases from breast/ovarian cancer families (1) was mapped to chromosome 17q21in 1990 (2). Recently this gene, BRCA1, was identified by usingpositional cloning techniques (3).During the search for the BRCAI gene, our laboratory and
others have carried out fine physical mapping and character-ization of.the 1.0- to 1.5-Mb region known to contain BRCA1(e.g., refs. 4 and 5). This paper describes the detailed charac-terization and partial cloning of a 500-kb region of chromo-some 17q12-21, between the gene 1A1-3B (6) and the poly-morphic marker D17S856 (7). We also describe the isolationand analysis of a number of genes mapping to this region,including BRCA1.11
MATERIALS AND METHODS
Physical Mapping. Single-copy probes across the BRCAIregion (see Fig. 1 legend) were hybridized to pulsed-field gelelectrophoresis (PFGE) Southern filters as previously de-scribed (4).
Isolation of Cosmid and Phage P1 Artificial Chromosome(PAC) Clones. Cosmid clones were isolated from a flow-sortedchromosome 17 cosmid library (8) by using either [y-32p]ATP-labeled primers for the polymorphic markers D17S855 andD17S856 or cDNA fragments from the 5' ends of the genes1A1-3B and EDH-1 7B, generated by PCR using primers basedon the published DNA sequence (6, 9). PAC clones wereisolated from a total human PAC library [generated by P.d.J.,(10)] by using fragments from the above-mentioned cosmids asprobes. Gaps between PAC clones were filled by using theriboprobe II core system kit (Promega).Exon Trapping. Exon sequences were isolated from PAC
clones by following a modification of the procedure describedby Buckler et aL (11). Briefly, PAC DNA was digested witheither Pst I or a combination ofBamHI and Bgl II, inserted intothe exon-trapping vector pSPL3, and then transfected intoCos-7 cells. Total RNA was isolated 48 hr after transfectionand used as a template for reverse transcriptase (RT)-PCR,using pSPL3 sequence-specific primers, and shotgun sub-
The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.
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cloned in the pAMP PCR cloning vector (GIBCO/BRL) togenerate exon libraries.
Analysis of Exon-Trapped Products. pAMP subclones ofexon-trapped products were sequenced in both directions withvector primers, either manually using a Sequenase kit (UnitedStates Biochemical) or automatically using an ABI 373 DNAsequencer. Sequencing results were analyzed by using the GCGprogram. Expression patterns and transcript sizes of new geneswere determined by hybridization of exon-trapped products tocommercially available multiple-tissue Northern blots (Clon-tech), exactly according to the supplier's instructions.
RESULTS
Physical Mapping. A long-range physical map surroundingBRCAI and incorporating markers and genes generated by ourlaboratory and others was constructed by PFGE analysis (Fig.1). Hybridization with EDH and 855RF probes (see Fig. 1legend) revealed a 550-kb Not I fragment to which both probeshybridize, as shown by the arrowed bands. RF18 (probe C) alsomapped to the same Not I, Nru I, and Nru I/Not I fragmentsas 855RF (data not shown). ET-A37 (BRCAI exon 13; seebelow) hybridized to the same 480-kb Nru I fragment as RF18,as indicated by the arrowed bands in autoradiographs C and D,but it hybridized to a larger Not I fragment of 750 kb. ThislargerNot I fragment and theMlu I andEag I fragments to whichthe ET-A37 probe hybridizes are the same ones to which the1A1.3B gene hybridizes (4). Taken together, these resultssuggest that EDH and 855RF reside on the same 550-kb NotI fragment and that the ET-A37 probe resides on the next distalNot I fragment, which also hybridized to the genes 1A1.3B andRNU2 and the markers D17S858/D17S859 (4). ET-A37 alsohybridizes to the same Nru I fragment as the D17S855 probe.Consequently, the long-range restriction maps generated ateach locus could be superimposed, giving the detailed mapshown in A+B+C+D at the bottom of Fig. 1.Genomic Cloning of the Region Between 1A1-3B and
D17S856. Initial efforts to clone the BRCA1 genomic regioninvolved the isolation of yeast artificial chromosome (YAC)clones by using both PCR and hybridization strategies. Screen-ing of four YAC libraries generated several YAC clones;however, all contained only one marker, were chimeric, orcarried deletions (4). Thus we turned to alternate genomiccloning vectors. The recent construction of a high-qualityhuman genomic PAC library (10), which contains stablenonchimeric clones in the range of 100-300 kb, provided anideal solution. Screening the PAC library with a repeat-free(RF) fragment from the centromeric end of the 1A1-3B-positive cosmid A11100 [isolated with the RNU2/1A1-3B YAC
Abbreviations: PFGE, pulsed-field gel electrophoresis; PAC, phage P1artificial chromosome; HIV, human immunodeficiency virus; RF,repeat-free.tTo whom reprint requests should be addressed."The sequences reported in this paper have been deposited in theGenBank data base (accession nos. U21493-U21545).
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A B C DM 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 N N M M N Bs Ea Ea M N Nr N M Bs Nr
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Ea N
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FIG. 1. Construction of a long-range restriction map around BRCA1 by PFGE analysis.A and B correspond to sequential hybridization of one filterand 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 arepeat-free fragment from a cosmid containing marker D17S855 (855RF); probe C was a repeat-free fragment from PAC 22157 (RF18); and probe Dwas 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 ofautoradiographs. Individual restriction mapsA, B, C, andD 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 thelocation of the KiAg gene (5) and ET A38. N, Not I; M, Mlu I; Bs, BssHII; Ea, Eag I; Nr, Nru I; CEN, centromere.
EDH gene
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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 toscale). (B) Cosmid and PAC clones isolated, indicating the presence of various sequences by hybridization (broken lines) or by PCR (rectangularblocks) and the orientation of riboprobe ends (blocks labeled T7 or SP6) if known. (C) Exon-trapped products, showing mapping results of theexon-trapped products described in this paper. Solid lines indicate the PAC to which the exons hybridize. (D) Mapping of exons to PACs by Southernhybridization, showing the ethidium bromide (EtBr)-stained agarose gel of EcoRI-digested PAC DNA and the results of Southern analysis withfour 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.
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12H4 (see ref. 6) and containing the 5' end of the 1A1-3B gene(H. Nicolai, D.B., and E.S., unpublished results)] identified a
single PAC, 103014 (Fig. 2B). Further characterization of thisPAC revealed that it also contained the marker Dl 7S855 (Fig.2 B and D). Using a RF fragment from a D17S855 cosmid, asecond PAC, 44B1, was identified. PAC 44B1 contained onlythe marker D17S855. Hybridization with the EDH cosmid RFfragment generated two PAC clones, 231K9 and 63F12, and atthe marker Dl 7S856 a single PAC, 171Q6, was isolated. Threefurther PAC clones were isolated by riboprobe walking (Fig.2B).
Identification of Previously Unknown Genes BetweenD17S856 and IAI-3B. Genomic clones were analyzed forexonic sequences by using the technique of exon trapping.Several libraries were made (see Materials and Methods), andfrom these approximately 70 cloned exons were isolated andanalyzed.
Analysis of predicted amino acid sequences of exon-trappedproducts revealed several homologies and identities (Table 1).For example, ET-A3 showed 46% amino acid identity and 86%amino acid similarity, over a 77-amino acid stretch, to the yeastrho-1 protein as well as 37% identity and 75% similarity, overa 120-amino acid stretch, to human rho-A and rho-B proteins(Fig. 3A). In addition, ET49, ET53, et al (Table 1) showedidentity to the interferon-induced leucine zipper proteinIFP-35 (12). Other clones displayed high similarity to HIVproteins (Fig. 3 B and F), which is most likely an exon-trappingartifact, as the exon-trapping vector, pSPL3, contains 2.7 kb ofHIV sequence (11). Data base searches of the sequences of theremaining clones showed no homology to genes present in theGenBank or European Molecular Biology Laboratory databases (Table 1).
Subsequent to the cloning of BRCAI (3), translated exon
sequences were compared with the BRCAI amino acid se-
quence. This revealed 100% identity of ET-A12 and ET-A37to BRCAI exons 21 and 13, respectively (Fig. 3 C and D) andof ET18, ET23, and ET29 with a combination ofBRCA1 exons22 and 23 (Fig. 3E). Furthermore, hybridization analysis ofexons from this region, with various probes generated by PCRusing primers based on the nucleotide sequence in and be-tween exons 16 and 20, revealed that ET2, ET14, ET26, ET40,and ET83 corresponded to sequences between exons 16 and 20(Table 1). The fact that ET-A37 (exon 13) maps to PAC103014 only, while ET-A12 (exon 21) maps to both PAC103014 and PAC 44B1 (Fig. 2), orients the BRCA1 gene in atelomeric to centromeric direction.The size and distribution of transcripts corresponding to
several of the exon-trapped products were determined byNorthern analysis. As shown in Fig. 4, ET-B1 hybridized to a2-kb transcript in testis; ET-A3, to a 1.5-kb message, predom-inantly in testis but also in other tissues; and ET-B53, to a2.5-kb mRNA in a wide range of tissues. ET-A37 hybridizes toa transcript of approximately 8 kb present in thymus and testis,consistent with its being an exon from the BRCAI gene (3).
DISCUSSIONThe work presented in this paper confirms the order ofmarkers between and including D1 7S856 and 1A1-3B (e.g., seerefs. 5 and 7) on chromosome 17q12-21 and gives an estima-tion of the physical distances between them. Given that one ofthe probes used in the generation of this map, ET-A37, was a
component of the BRCA1 gene, this information has provideda scaffold for more detailed analysis of the region surroundingthis gene. The physical mapping data generated here were alsocritical for genomic cloning and, indeed, most of the genomicDNA between D17S856 and 1A1-3B has been cloned in fourcosmids and eight PAC clones. Approximately 70 exon sequenceswere isolated from these PAC clones, using the exon-trappingtechnique.
Proc. Natl. Acad Sci. USA 92 (1995)
Table 1. Results of searches for homology ofexon-trapped productsCloneno.
ET-B1ET-B2ET-B31ET-B53ET-A3
ET-A12ET-A38ET-A37ET1ET2ET3ET4ET5ET6ET7ET8ET9ET10ET11ET12ET13ET14ET15ET16ET17ET18ET19ET20ET21ET22ET23ET24ET25ET26ET27ET28ET29ET40ET49ET52ET53ET54ET56ET57ET58ET59ET61ET62ET63ET64ET66ET67ET68ET69ET70ET71ET72ET73ET74ET75ET76ET77ET78ET83ET85ET92ET93
BRCA1exon
21
13
(16-20)*21
(16-20)*
22 + 23
22 + 23
(16-20)*
22 + 23(16-20)*
(16-20)*
Possibly homologousgene or product
HIV vpu
rho-likeG protein
Alu repeat
HIV gp160
IFP-35
IFP-35
IFP-35IFP-35
IFP-35IFP-35IFP-35
IFP-35IFP-35IFP-35
IFP-35
IFP-35IFP-35
IFP-35IFP-35
IFP-35IFP-35
% amino acididentity
92
Yeast: 46Human: 37
78
100
100
100100
100100100
100100100
100
100100
100100
100100
HIV, human immunodeficiency virus; refers to no homology togenes present in the GenBank on European Molecular BiologyOrganization data bases.*(16-20) refers to positive hybridization of the cloned exon-trapped products with a PCR product spanning BRCA1 exons16-20.
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Proc. NatL Acad Sci USA 92 (1995) 4365
YDNVRPLAYPDSDAVLICFDISRPETLDSVLKKWQGETQEFCPNAKVVLVGCKLDMRTDLATLRELSKQRLIPVTHEP
Yeast rho-1:(AN: P06780)
Human rho-a:(AN: P0749)
Human rho-b:(AN: P08134)
BET-B1:
HIV vpu:(AN: P05920)
CET-A12:
BRCA1 exon 21:
DET-A37:
BRCA1 exon 13:
YDRLRPLSYPDSNVVLICFSIDLPDSLENVQEKWIAEVLHFCQGVPIILVGCKVDLRNDPQTIEQLRQEGQQPVTSQE
YDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMQE PVKPEE
YDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRQDEHTRRELAKMKQEPVRSE
MQPIQLAIVALVVAIIIAIVVWSIVIIEYRKILRQRKIDRLIDRLIERVEDSGNESDGEISALVEMGVEMGHHVPXDVDDL
MQPIQLAIVALAIIIAIVVWSIVIIEYRKILRQRKIDRLIDRLIERAEDSGNESEGEISALVEMGVEMGHHAPWDVDDL
IFRGLEICCYGPFTNMPTD
IFRGLEICCYGPFTNMPTD
QRDTMQHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQSTSEK
QRDTMQHNLIKLQQEMAELEAVLEQHGSQPSNSYPSIISDSSALEDLRNPEQSTSEK
EET18, 23 and 29:
BRCA1 exons 22 and 23:
FET22
QLEWMVQLCGASVVKELSSFTLGTGVHPIVVVQPDAWTEDNGFHA
QLEWMVQLCGASVVKELSSFTLGTGVHPIVVVQPDAWTEDNGFHA
LKCTDLRMILIPNSSSGRMIMEKGEIXXCSFNISTSIR
LKCTDLKNDTNTNSSSGRMIMEKGEIKNCSFNISTSIR
GET49,ET53,ET56,ET57,ET59,ET61,ET62,ET67,ET68,ET69,ET72,ET75,ET78,ET85 and ET92:
IFP 35:(AN: P80217)
HET74 and ET77:
IFP 35(AN: P80217)
IRSQPVPRSVLVLNIPDILDGPELHDVLEIH
IRSQPVPRSVLVLNIPDILDGPELHDVLEIH
AQRLCQIGQFTVPLGGQQVPLRVSPYVNGEIQKAEIRSQPVPRSVLVLNIPDILDGPELHDVLEIH
AQRLCQIGQFTVPLGGQQVPLRVSPYVNGEIQKAEIRSQPVPRSVLVLNIPDILDGPELHDVLEIH
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 codesrefer to data base accession numbers (AN).ET-A3 showed highest homology to a number of rho
proteins, which are one of the three members of the RASsuperfamily of G proteins, along with RAS and RAB. RHOproteins are involved in cell morphology, playing a signal-transducing role in the control of actin cytoskeleton organi-zation (13). Interestingly, ET-A3 is the third G protein to beidentified within the 1-Mb region defining BRCA1. In additionto ET-A3, the gene encoding an ADP-ribosylation factor,HAL-64, has been mapped between RNU2 and PPY (D.B., M.Boyd, and E.S., unpublished results), and BC-16, a Rab5c-related gene, has been mapped close to D17S856 (5).Sequence analysis of several exon-trapped products revealed
100% identity with a previously described protein, the inter-feron-induced leucine-zipper protein IFP-35 (12). Interest-ingly, some clones had 30-amino acid stretches of identity whileothers showed identity over 65 amino acids. This suggests that
in the latter case more exons had been trapped, indicating thatthe intervening intron sequences must be quite short. Mappingof these exon-trapped products back to the PACs revealedhybridization solely to PAC 44B1, placing the IFP-35 genecentromeric to BRCA1 (Fig. 2). IFP-35 is a 35-kDa proteinwhich contains a leucine-zipper motif and readily forms ho-modimers in vitro. Unlike many other leucine-zipper-containing proteins, IFP-35 does not contain a DNA-bindingmotif and displays no detectable heterodimerization withother known leucine-zipper proteins, and so the function ofIFP-35, like that of the BRCA1 product, remains unclear.Comparison of the translated sequence of all exons with the
amino acid sequence of the BRCA1 product (3) revealed 100%identity of several products with BRCAI exons. This wasfurther supported by Northern analysis of ET-A37. Detailedmapping of these exons has been helpful in the characteriza-
AET-A3:
HIV GP160(AN: P03375)
Genetics: Brown et al
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28s -
18s
12345678
IET-B1 ET-A3
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
0Al ::,l
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 wereconfirmed previously by hybridization with an actin cDNA probe (4).tion of the genomic DNA housing the BRCA1 gene, inparticular for determining the orientation of BRCA1. Inter-estingly, comparison of these data with similar experimentswith 1A1-3B exons (H. Nicolai, D.B., and E.S., unpublishedresults) indicates that these two genes are transcribed inopposite directions. Intriguingly, we have recently shown thatthe distance between these two genes is less than 300 bp,raising the possibility of coregulation of transcription andpresenting an additional model for BRCAl-mediated onco-genesis (14).The above-mentioned homologies and identities account for
approximately 50% of the exons described in this paper. Theremaining exons displayed no significant homology to genesequences present in the GenBank or European MolecularBiology Laboratory data bases and may therefore representnovel genes. Clearly, some of the sequences may be novelcomponents of previously described genes, includingEDH (9),lAl (6), and the recently described IF-I, BC3-1, BC1-6, andBC1-16 genes (5). Therefore, although the original aim of thisproject was to identify a breast/ovarian cancer gene linked tothis region of 17q12-21, the resulting work has not onlyprovided a detailed physical map about the BRCA1 gene butalso will provide an excellent resource for future work onchromosome 17 as well as contributing to the overall charac-terization of the human genome.The authors are grateful to Fiona Francis for advice on P1 and PAC
libraries and analysis of clones, to Guenther Zehetner and RanjitBhogal for help with picking positive cosmid and PAC clones, and toMike North for advice on the exon-trapping technique. M.A.B. issupported by a European Molecular Biology Organization postdoc-toral research fellowship and M.B. was funded by a Conselho Nacionalde Desenvolvimento Cientifico e Tecnol6gico (Brazil) research grant.
1. Easton, D. F., Bishop, D. T., Ford, D., Crockford, G. P. & TheBreast Cancer Linkage Consortium (1993) Am. J. Hum. Genet.52, 678-701.
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