GENOMICS 40, 267–276 (1997)ARTICLE NO. GE964595

Construction of a Transcription Map around the Gene for AtaxiaTelangiectasia: Identification of at Least Four Novel Genes

TATJANA STANKOVIC, PHILIP J. BYRD, PAUL R. COOPER, CARMEL M. MCCONVILLE, DAVID J. MUNROE,*JOHN H. RILEY,† GILES D. J. WATTS, HELEN AMBROSE, GERMAINE MCGUIRE, ALEXANDRA D. SMITH,

ANDREW SUTCLIFFE, TRACY MILLS, AND A. MALCOLM R. TAYLOR1

CRC Institute for Cancer Studies, The Medical School, University of Birmingham, Birmingham, B15 2TA, United Kingdom;*Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139;

and †Zeneca, Alderley Park, Nr Macclesfield, Cheshire, SK10 4TG, United Kingdom

Received August 21, 1996; accepted December 16, 1996

task (Collins, 1995). High-density transcription mapsWe have constructed YAC, PAC, and cosmid contigs are currently being constructed in two ways: first,

in the ataxia–telangiectasia gene region and used the through systematic identification of transcribed se-assembled clones to isolate expressed sequences by quences across the whole genome and second, throughexon trapping and hybridization selection. In the in- various positional cloning projects related to the identi-terval between D11S1819 and D11S2029, exons and fication of specific disease genes. While systematiccDNAs for potentially 13 different genes were identi- searches for transcribed sequences (expressed se-fied. Three of these genes, F37, K28, and 6.82, are large quence tag maps) offer the partial sequence of a largenovel genes expressed in a variety of different tissues. number of genes, transcription maps derived by indi-K28 shows sequence homology to the Rab GTP binding vidual positional cloning projects are much more de-protein family and gene 6.82 homology to the rabbit tailed, as they usually provide information regardingvasopressin activated calcium mobilizing receptor, not only the number of genes in a particular region,while gene F37 has no homology to any known se-

but also their orientation, order, distribution, and evo-quence in the database. Three further clones, exon 6.41lutionary conservation as well as their pattern of ex-and cDNAs K22 and E74, from the interval betweenpression in different tissues.D11S1819 and D11S2029, appear to be expressed en-

The gene for the recessively inherited disorder ataxia–dogenous retrovirus sequences. The fourth large noveltelangiectasia (A-T) was cloned recently (Savitsky et al.,gene, E14, together with two further possible novel1995a). The identification of this gene followed severalgenes, E13 and E3, was identified from exons andyears of genetic and physical mapping studies in differentcDNAs in the more telomeric 300-kb interval betweenlaboratories. A YAC contig of the A-T gene region (Rot-markers D11S2029 and D11S2179. These are in addi-man et al., 1994; Arai et al., 1996) and also a genomiction to the genes for mitochondrial acetoacetyl-CoA-

acetyltransferase (ACAT) and the ATM gene in the map (Ambrose et al., 1994) have been previously pub-same region. Genes E3, E13, and E14 do not show ho- lished. We have developed a refined physical map of 800mology to any known genes. K28, 6.82, ACAT, and ATM kb–1 Mb of the A-T region and have derived a numberall appear to have the same transcriptional orienta- of regional transcripts. Our map contains contigs builttion toward the telomere. q 1997 Academic Press up by using three alternative cloning systems: YACs, cos-

mids, and PACs. Various methods have been developedand described in detail for deriving transcripts. We used

INTRODUCTION mainly exon amplification (Buckler et al., 1991; Churchet al., 1994; Burfoot and Campbell, 1994), direct selection(Lovett et al., 1991), and screening of arrayed librariesThe isolation and accurate mapping of genes is the(Munroe et al., 1995), but in addition we used evolution-major goal of the human genome mapping initiative.ary conservation and CpG island identification (LarsenThe aim and expectation of this initiative is to developet al., 1992; Rouleau et al., 1993). As a consequence ofa transcription map of such density that positionalthese combined approaches, we assembled a transcrip-cloning of different disease genes will become an easytion map of a region that includes the ATM gene andseveral novel widely expressed genes.Sequence data from this article have been deposited with the

EMBL/GenBank Data Libraries under Accession Nos. X81882, MATERIALS AND METHODSX99961, X99962, and Y10269–Y10274.1 To whom correspondence should be addressed. Telephone: 0121- Construction of YAC, cosmid, and PAC contigs. YACs from the

region of the A-T gene were mostly obtained by screening the ICI414-4488. Fax: 0121-414-4486.

2670888-7543/97 $25.00

Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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YAC library (Anand et al., 1990) using regional STSs (Ambrose et al., (5* AGC AAG TTC AGC CTG GTT AAG) and purified using a Qia-Quick PCR purification kit (Qiagen).1994) developed from the ends of YACs (Riley et al., 1990). Additional

Selector DNA was generated from YAC 6E9 and from three cosmidYACs were identified by screening the chromosome 11 YAC librarycontigs containing 35 cosmids from the centromeric part of the A-T(Qin et al., 1993) with Alu-vectorette PCR products from the regionalregion. One hundred nanograms of DNA from each cosmid was par-YACs, and mega-YACs were obtained by screening a mega-YAC li-tially digested with NdeII or Sau3A, and the digest was pooled intobrary (Bellanne-Chantelot et al., 1992) with the same products. Thecontig groups, purified, ligated to a linker containing a MboI sitedegree of overlap between the individual YACs was estimated by(linker 2) (Morgan et al., 1992), and column purified again. SelectorSTS mapping and by comparison with YAC restriction maps. Orien-DNA was generated by PCR amplification of 6 ml of the purifiedtation of the YAC contigs was assessed by pulse-field gel electropho-ligation, using a biotin-labeled primer complementary to the linkerresis (PFGE) separation.sequence 5898 (5* biotinTGG TCT CAC GAA TTC GTC GA) andThe two YAC clones 20FE12, and 6E9, which are 720 and 450 kb,purified using a QiaQuick PCR purification kit.respectively, were used to generate human cosmid libraries. High-

Hybridization selection of contig-specific cDNAs was carried outmolecular-weight DNA was prepared from these YACs, partially di-by capture of selected cDNAs on streptavidin-coated magnetic beadsgested with Sau3A, and ligated to the arms of the cosmid vector(Promega), essentially as described by Futreal et al. (1994), exceptSuperCos 1 (Stratagene). The ligated DNA was packaged with lthat hybridization was carried out for 24 h and the hybridizationphage packaging extracts (Stratagene) and propagated in the hostmix contained 1 mg of cDNA and 200 ng cosmid DNA. Following twoNM554 (Stratagene), and the library was plated out. Colonies ob-cycles of selection, eluted cDNAs were reamplified and blunt endtained were lifted onto filters, which were hybridized with total hu-cloned into a Bluescript vector.man DNA to identify human clones. Additional cosmid clones were

Characterization of amplified exons and selected cDNA clones.obtained by screening the chromosome 11 cosmid library (LoscDNA clones were PCR amplified using vector primers and used asAlamos) with total Alu-PCR products of the YACs from the A-Thybridization probes on chromosome 11q22–q23-specific YACs andregion.somatic cell hybrids to confirm their localization. Amplified exonsCosmid walking was performed by two methods. First, cosmidsand cDNA clones mapping to the A-T region were hybridized to thewere screened for the presence of regional STSs, and initial minicon-cosmid contig across the A-T region to establish their exact positiontigs were obtained. The end clones from each of these minicontigsand relation to each other. To obtain the patterns of expression forwere then used as hybridization probes to screen the remainder ofindividual exons the exon-specific primers were used to amplify Clon-the cosmids not previously assigned by PCR assay. Typically, severaltech cDNA libraries from different tissues. cDNAs and exons wererounds of hybridization were necessary to join the initial minicontigs.also used as hybridization probes on multiple tissue Northern blotsThe PAC library containing 120,000 clones with an average insert(Clontech) and Zoo blots (Clontech).size of 120 kb (Ioannou et al., 1994) was screened with eight cosmids

Rapid amplification of cDNA ends (RACE). Placental poly(A)/that had been assigned to contigs from the A-T region, and PAC sizemRNA (Clontech) was reverse transcribed using gene-specific prim-was determined by PFGE. The PAC contig was built up by STSers. An adaptor (Clontech Marathon cDNA kit) was ligated to themapping and by the establishment of the NotI restriction map ofcDNA following second-strand synthesis. Long-range PCR was thenindividual PACs.performed using adaptor-specific primer (AP1; Clontech Marathon

Exon amplification. DNA from six groups of two overlapping cos- cDNA kit) and primers specific for the individual genes: ATM (Byrdmids and from the two YACs (6E9 and 13G9) of the A-T region et al., 1996a), E14 (Byrd et al., 1996b), K28 (primer 9953: 5* TAAwas completely digested with enzymes BglII, BamHI, and PstI. Fifty CCC CTT CCC AGC CAT CCT GGA TAC), and 6.82 (Byrd, in press).nanograms of the digested DNA was subcloned into the splicing vec- The major amplification products were digested with a series of re-tor pSPL3. The ligated products were transformed into Escherichia striction enzymes and ligated to vectorette for sequencing.coli DH5a, and plasmid DNAs were purified from a mixture of the Sequencing and analysis of cDNA and exon sequences. DNA se-transformants. Transfection of these plasmid DNAs into COS7 cells, quencing was performed using the Applied Biosystems Prism Readyisolation of total cytoplasmic RNA, synthesis of cDNA, digestion of Reaction Dye Terminator Cycle sequencing kit and the ABI 373AcDNA with BstXI, and amplification of spliced fragments were per- DNA sequencer. Sequenced exons and cDNA clones were investi-formed according to the methods described by Church et al. (1994). gated for similarities to known genes by searching DNA and proteinAmplified fragments were subcloned into pBluescript, and DNA se- databases. DNA searches were performed on the combined GenBankquences were determined. Southern blot hybridization of individual 85 and EMBL 40 DNA databases using FASTA (Pearson et al., 1988).exons with a probe corresponding to the intron sequence of the vector The protein database search was performed using the Blastx proce-was undertaken to eliminate the artifacts of cryptic splicing. dure of BLAST (Basic Local Alignment Search Tool).

Screening of arrayed thymus and frontal cortex cDNA libraries.Two libraries, a human frontal cortex cDNA library and a thymus

RESULTSlibrary (Stratagene), were arrayed by Munroe et al. (1995) and our-selves. PCR assays were used to identify wells that were positive forthe exon or cDNA sequence. The PCR-positive well for each of cDNAs Contigs across the A-T Regionor exons was plated out at a density of 1000 plaques per plate, andplaques were lifted on Hybond-N/ filters and hybridized with the Contigs of three types of cloned DNA (YAC, PAC,probes generated from the relevant exon or cDNA. The ExAssist and cosmid) were constructed across 1 Mb of chrom-Interference-Resistance helper Phage kit from Stratagene was used osome 11q22–q23 between markers D11S1819 andfor the in vivo excision of the positive phage DNA. The cultures were

D11S1294, a region that linkage analysis had showntested by PCR for the presence of the exon or cDNA sequence, andto be the most likely location for the A-T gene (GattiDNA was prepared from positive clones.et al., 1994). Figure 1 shows a minimal coverage of theDirect cDNA selection. mRNA was prepared from lymphoblastoidA-T gene region with YAC, PAC, and cosmid clones.cell lines (LCLs) using a Pharmacia QuickPrep mRNA purification

kit. Poly(A)/ RNA (1.5 mg) from LCLs of five individuals was used as Twenty-five YACs were identified by screening threea template for oligo(dT)12–18 and pd(N)6 random hexamer primed YAC libraries and mapped to the region of the A-TcDNA synthesis (Pharmacia TimeSaver cDNA synthesis kit). A gene. Several of these YAC clones were shown to belinker (linker 1), prepared as described by Futreal et al. (1994), was

chimeric or contain repetitive insert terminal se-ligated to the cDNA, and the ligated product was column purified.quences and, therefore, extension of the complete YACTarget cDNA was generated by PCR amplification of 6 ml of the

ligated cDNA using a primer complementary to the linker sequence contig required screening of more than one YAC li-

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STANKOVIC ET AL.270

brary. Eventually, the whole A-T region was covered maining 8 clones appeared to represent six indepen-dent fragments (Table 1).by three nonchimeric YACs: 6E9, 13G9, and 20FE12

(Fig. 1). Restriction mapping of the YAC and corre-sponding PAC clones within the A-T region identified Assembly of a Transcription Map in the Region of thetwo NotI sites, about 60 kb apart, between markers A-T GeneD11S2025 and D11S2026 (Fig. 1). Three CpG islands

A transcription map of the A-T region was establishedwere identified, two associated with each of the NotIby alignment of identified exons and cDNAs to the cosmidrestriction sites and the third one in the region 300and PAC contig backbone. The assembly allowed a clus-kb distal to the telomeric NotI site. The 60-kb regiontering of the identified transcripts with subsequent isola-between the two NotI sites appeared to be both repeti-tion of several genes (Fig. 1). In addition, amplified frag-tive and unstable, resulting in rearrangements and de-ments from the 5* and 3* ends of the ATM gene (Savitskyletions in many YAC and cosmid clones containing thiset al., 1995a,b; Byrd et al., 1996) were used to place thisregion. In contrast, the PAC clones seemed to be unaf-gene within the cosmid/PAC contig.fected by this genomic instability.

The interval between D11S1819 and D11S2029.The cosmids that map to the 60-kb NotI fragment be-Identification of Exons and cDNAs from the Region oftween markers D11S2025 and D11S2026 were found tothe A-T Genecontain repetitive sequences. Two exons were trappedfrom this fragment. The 354-bp exon 6.41 was found toTranscripts from the A-T region were identified by

two complementary methods: exon amplification and be homologous (75% identical) to expressed retrovirusLTR sequences while the 136-bp exon 1.89 was founddirect cDNA selection.

Fifty-two potentially unique exons were derived from to be unique.A number of exons, 6.15, 6.32, and 6.84, togetherthe A-T region using exon amplification from the YAC

and cosmid clones: 35 from the region of the YAC with hybridization selected cDNAs F37, M1, M17, M22,M43, M64, M72, M83, M93, M96 and cDNA D4, specific20FE12 and 17 from the region of the YAC 6E9. The

average length of the identified exons varied between to the exon 6.84, were mapped centromeric to the 60-kb NotI fragment (Fig. 1, Table 1). These form a 1.5-29 and 420 bp. A data search of their sequences indi-

cated that four exons (three clones) were part of the kb cDNA contig representing a unique anonymous geneF37 that extends in a telomeric direction to the nearestACAT gene (acetoacetyl-coenzyme-A-acetyltransfer-

ase) (Masuno et al., 1992), previously mapped to the CpG island associated with the centromeric NotI site oncosmid 104H4. We formed a contig of F37 overlappingA-T region. One exon was found to be homologous to

the sequence of an endogenous retrovirus and 6 ap- clones on the basis of their sequence and their hybrid-ization patterns. Analysis of the aligned sequences ofpeared to show homology to repeat sequences (3 to Alu

and another 3 to LINE-1 repetitive sequences). One the overlapping clones enabled us to establish the orderof exons 6.84, 6.15, and 6.32 from the telomere towardexon, 6.82, showed homology to an EST from a testis

cDNA library and another one, exon A8, was subse- the centromere. The alignment of the sequence of theexons and cDNAs together with their hybridizationquently found to be a part of the ATM gene (Table 1).

The remaining 40 clones–exons represented poten- patterns to the cosmid contig allowed us to establishthe transcription orientation of the F37 gene as beingtially unique sequences.

Exon-specific primers were designed for the purpose telomere to centromere (Fig. 1). This gene appearedto be widely expressed, producing a 3-kb message onof screening arrayed cDNA libraries and extending ex-

ons in the 5* and 3 * direction by RACE. Exons E3, E10, Northern blots in a variety of tissues including spleen,thymus, prostate, testis, ovary, small intestine, colon,and E14 were used to screen an arrayed human frontal

cortex cDNA library (Munroe et al., 1995), and three and peripheral blood leukocyte (Fig. 2). FASTA andBLAST search, however, identified no homology tocorresponding cDNA clones were isolated. In addition

two exons, 6.45 and 6.82, from the centromeric part of known sequence in the DNA or protein sequence data-base (Table 1). One further hybridization selectedthe A-T region were also used to screen the arrayed

cDNA library, and three cDNA clones were identified. cDNA, M27, and the related exon 6.45 (Table 1) ap-peared not to be part of the F37 gene (Fig. 1). TheNo cDNA clones were isolated following screening of

the human frontal cortex and the thymus cDNA librar- coding sequences for F37 were found on the nonover-lapping cosmids 131H11 and 104H4, which were linkedies, with exons E12 and E13. RACE was used to extend

the transcript from exon 6.82 and also to obtain the 5* through cosmid 138. Exon 6.45 and cDNA M27 weremapped to cosmid 138, which was placed between cos-end of the ATM gene (Byrd et al., 1996a).

YACs and cosmids from the A-T region were also mids 131H11 and 104H4, possibly identifying a genein an intron of the F37 gene or a gene transcribed fromused to generate cDNA clones by direct selection.

Twenty-three clones identified by direct selection were the antisense strand relative to F37.On the telomeric side of the 60-kb NotI fragment ananalyzed and 18 mapped back to the A-T region. How-

ever, 10/18 clones were either identical or different exon, 6.83, and two hybridization selected cDNAs, E74and K28, were mapped to cosmids that were close tofragments of the same gene, F37 (Table 1). The re-

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EXPRESSION MAP AROUND THE ATM GENE 271

TABLE 1

Characteristics of Genes and Individual Transcripts in the A-T Region

Initial CloneGene/ isolation size Transcript Accession

transcript method (kb) Sibling clones Cosmids or PACs Expression size (kb) Sequence homologies No.

F37 S 1.5 6.32, 6.15, 6.84 33 6E9, 131H11, Wide 3 No homology X99961M1, M17, M22, 104H4

M43, M64,M72, M83,M93, M96, D4

M27 S 1.1 6.45 138 6E9 No homology6.41 E 0.354 94G8 Expressed retrovirus

LTR sequences1.89 E 0.136 143 6E9, 43B2 LCL No homology Y10274E74 S 0.160 143 6E9, 31G7, HERV-K class of

59 6E9 endogenousretrovirus sequence

6.83 E 0.176 31G7 No homologyK28 S 1.3 143 6E9, 43B2, Wide 2

59 6E9, 31G7, Rab family gene X9996299 6E9

K22 S 1.2 31G7, 99 6E9, Integrase and dUTPase114 6E9 domain of the

HERV-L4.14 E 0.113 114 6E9 LCL No homology Y10273M25 S 2.1 K44 186B7 No homology6.82 E 0.097 6.82, K35 114 6E9, Wide 7.5 Rabbit vasopressin

186B7, 64, activated calcium X818826E9, 179C1, mobilizing receptor63E11, 65B10 gene (VACM-1)

K47 S 1.3 179C1, 63E11 No homologyE23 0.144E15 E 0.210 2B1, 59D5 Wide 1.5 ACAT geneE2 0.065E3 E 0.140 CJ52.193 Wide No homology Y10272E13 E 0.249 X25b, 32a No homology Y10271E14 E 0.370 CJ52.193, Wide 5.3 No homology Byrd et al.,

X26b, X25b, 6.25 199632a

A8 E 0.090 PAC 22P10 Wide ATM geneE34 E 0.093 89F5, Y4b No homology Y10269E36 E 0.149 35a, 16a No homology Y10270

Note. Designations of the individual clones are given in centromeric/telomeric order from the top to the bottom on the left hand side. Thecharacterization of the individual transcripts includes the method of isolation (cDNA selection, S; and exon trapping, E), size presence ofoverlapping clones, position within the cosmid and PAC contig, pattern of expression, size of mRNA message, and sequence homology toother known sequences. The sequences of genes F37, K28, 6.82 and other transcripts have been submitted to the EMBL Nucleotide SequenceData base and the accession numbers of these genes are given.

or overlapped the telomeric NotI site. The 176-bp exon genes including 47.9% identity with Hu-Rab 2 over 190amino acids. This includes four domains involved in6.83 appeared to be unique, while the small 160-bp

cDNA E74 was found to be ú85% homologous to the GTP/GDP binding (residues 15–22, 68–74, 124–131,and 155–161) that are common to both Ras and RabHERV-K class of endogenous retrovirus sequences (Ta-

ble 1). Despite the fact that they map to the same geno- gene families as well as an additional three domainscorresponding to residues 44–49, 62–67, and 74–96mic region, exon 6.83 and cDNA clone E74 do not ap-

pear to be part of the K28 cDNA, by either sequence that are highly conserved among the Rab protein fam-ily but diverge from sequences found in the p21Rascomparison or exon connection.

The 5* end of the 1.3-kb K28 cDNA was obtained by (Fig. 3) (Zahraoui et al., 1989). One of these highlyconserved regions among the Rab family, the effectorRACE, and a database search with the complete coding

sequence was performed. Conceptual protein transla- region, (residues 44 to 49), is thought to be involved inthe interaction of the protein with its putative effector.tion and BLAST and FASTA searches confirmed that

K28 represents a new member of the Rab gene family. K28 shows only limited similarity to other Rab sub-classes with respect to another region (residues 110–Identities of 42–49% were obtained with various Rab

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STANKOVIC ET AL.272

4.40, was found to be the exon preceding the 6.82 exon,and the cDNA clone K35 (0.9 kb) was found to represent3 * sequences of the same gene, which is transcribedtoward the telomere of the long arm of chromosome 11.Northern blotting identified a widely expressed 7.5-kbmRNA in tissues such as heart, brain, placenta, lung,liver, skeletal muscle, kidney, pancreas, spleen, thy-mus, prostate, testis, ovary, small intestine, colon, andperipheral blood leukocytes, and BLAST search identi-fied homology with rabbit vasopressin-activated cal-cium mobilizing receptor gene (Byrd, in press).

The interval between D11S2029 and D11S1294.The gene for mitochondrial ACAT was mapped to11q22–q23 by Masuno et al. (1992), and we subse-quently localized it centromeric to the region of the A-T gene. Our previous radiation hybrid mapping (Am-brose et al., 1994) indicated that the ACAT gene waslocated proximal to D11S384 and within 50 kb of theend of the YAC 20FE12 (D11S2029). This order wasconfirmed by PCR analysis of cosmids that spannedD11S2029. In addition, PCR analysis on cosmids usingACAT primers from the promoter and different partsFIG. 2. Northern blot analysis of genes K28 and F37 in multipleof the body of the gene showed that this gene is tran-human tissues. (Top) Hybridization of PCR products corresponding

to the body of genes K28 and F37 on multiple human tissue mRNA scribed toward the telomere of chromosome 11.filters. (Bottom) The hybridization signal obtained with a b-actin Between the gene for ACAT and the ATM gene weprobe on the same filter, for comparison of the amount of mRNA have evidence for potentially three novel genes. Fiveloaded in each track.

exons were trapped within this interval: E3, E10, E12,E13, and E14. The exon E3 (140 bp), which was trapped

120) that is thought to interact with GEFs (guanine from the cosmid pair CJ52.193 (containing the markernucleotide exchange factor) (Moore et al., 1995), sug- D11S384) and X26b, was extended by screening cDNAgesting that this gene is distinct from previously identi- libraries and by 5* and 3 * RACE. Differentially splicedfied Rab family members (Fig. 3). All members of the E3-specific cDNAs of up to 2.4 kb have been obtainedRas superfamily possess at least one cysteine near the from brain, thymus, and placenta. However, NorthernCOOH-terminus, which has been shown to be required blotting analysis failed to identify a correspondingfor fatty acylation, membrane association, and biologi- mRNA in 16 analyzed human tissues. It is possible,cal activity. K28 contains a terminal Cys-X-Cys motif therefore, that the E3 exon is part of a low abundancethat is also found in Rabs 3a, 3b, 4, and 6 (Fig. 3) gene or it represents an exon that is differentially(Zahraoui et al., 1989). The K28 gene seems to be spliced in the majority of tissues or functions in eitherwidely expressed, producing a message of about 2 kb on a tissue or developmentally restricted way. Despite theNorthern blots in a variety of tissues, including spleen, fact that E3 maps close to exons E10, E12, and E14,thymus, prostate, testis, ovary, small intestine, colon, which are part of the E14 gene (see below), it is unlikelyand peripheral blood leukocytes (Fig. 2). The transcrip- that this exon belongs to the E14 gene, which is ex-tional orientation of the K28 Rab gene on chromosome pressed ubiquitously.11 is from the centromere toward the telomere (Fig. 1). The exon, E13 (249 bp), from this region, which was

Immediately telomeric to the K28 RAB gene we have amplified from the cosmid pair X25b and 32a, had amapped a 1.2-kb hybridization selected cDNA K22 and high G / C composition and a CpG content suggestivea 113-bp exon 4.14. No homologies have been found for of the presence of a CpG island (Fig. 1); it also containedthe exon but K22 shows ú80% homology to the integ- sites for the rare-cutting enzymes AscI (invariablyrase and dUTPase domains of the HERV-L class of found in CpG islands), BssHII, and NgoMI. The 9.5-kbendogenous retrovirus sequences (Cordonnier et al., EcoRI restriction fragment of E13 was seen in genomic1995). DNA from human and monkey, and possibly a smaller

Four hybridization selected cDNAs, M25, K44, K47, 1-kb fragment was seen in cow DNA (data not shown).and K35, have been mapped telomeric to K22. Two No hybridizing restriction fragments were detected inclones, M25 (2.1 kb) and K47 (1.3 kb), are unique and genomic DNA from any of the other species. This exondo not appear to be part of another large gene, desig- was, however, not amplified from any of the tissuesnated 6.82, which extends from cosmid 114 to cosmid (liver, pancreas, heart, lung, skeletal muscle, kidney,2B1 (Fig. 1). The original 97-bp exon 6.82 that was used brain, and placenta) included in the Clontech cDNAto identify this gene was expanded by cDNA library library panel and, like exon E3, may have restricted

tissue or developmental expression.screening and 5* and 3 * RACE. A second 112-bp exon,

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EXPRESSION MAP AROUND THE ATM GENE 273

FIG. 3. Alignment of the amino acid sequence of K28 and other members of the Rab/Ras family of proteins. Sets of identical or conservedresidues are boxed. The reference numbering is that of the K28 gene.

Exon E14 has identified a new gene. Extension of has been reported (Byrd et al., 1996; Imai et al., 1996)but no homologous sequences in analyzed DNA or pro-the 5* end of the ATM cDNA (Savitsky et al., 1995a)

on genomic DNA showed that the first exon was part tein databases have been identified (Imai et al., 1996;Byrd et al., 1996b).of a CpG island (Byrd et al., 1996a). More than 2 kb of

genomic sequence was determined upstream to the 5* Two exons, E34 (93 bp) from cosmid pair 89F5/Y4band E36 (149 bp) from cosmid pair 35a/16a, were iso-end of the A-T cDNA. Within this sequence and 681 bp

5* to the start of the ATM gene we found the first exon lated telomeric to the ATM gene. These exons showedno homology to known sequence in the database andof a new gene, E14, a cDNA that had been identified

with trapped exon E14 (370 bp). The ATM and E14 may potentially be part of two novel genes.genes were found to be oriented in opposite directions,suggesting that they were transcribed from a bidirec- DISCUSSIONtional promoter (Byrd et al., 1996a). This was con-firmed recently and also shown to be the case in the We have constructed YAC, PAC, and cosmid contigs

covering a 1-Mb region on chromosome 11q22–q23 thatmouse (Byrd et al., 1996b). The E14 gene is, therefore,transcribed in a telomere to centromere direction, giv- includes the gene for ataxia telangiectasia (ATM) and

used exon trapping and hybridization selection to iden-ing transcripts of Ç6.25 and Ç5.3 kb in a variety oftissues (Byrd et al., 1996b). The complete amino acid tify genes within this region. The exons and cDNAs

that we isolated have been mapped back to the cosmidssequence of E14 (also called NPAT; Imai et al., 1996)

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STANKOVIC ET AL.274

and PAC clones, and a transcription map has been de- exon trapping and hybridization selection truly repre-sented genomic DNA. The third region, telomeric to theveloped.

The A-T region appears to be a gene-rich region. Ex- ATM gene, contains only two potential genes repre-sented by two exons, E34 and E36. Again, without fur-ons and cDNAs for potentially 19 genes were isolated

across the 1 Mb of genomic DNA, of which 4 have so far ther characterization of these genes, including ob-taining the full-length cDNA and mapping of the full-been identified as new human genes. This represents a

density of 1 gene every 50 kb and approximates the length cDNA to the cosmid and PAC backbone, it isdifficult to gauge the gene density in this part of theexpected frequency for the presence of 70,000 genes

distributed throughout the human genome. There are, A-T region.The A-T region contains three endogenous retrovirushowever, segments of the A-T region with a gene den-

sity lower than this, probably due to the presence of insertion sites (6.41, K22, and E74) clustered within60 kb, each in proximity to one of the two CpG islandsthe large genes F37, 6.82, E14, and ATM. The genes

ACAT, K28, and E3 as well as many other exons and associated with the NotI sites in the centromeric partof the A-T region. The roles of such transcripts gener-cDNAs that represent only fragments of larger tran-

scripts not fully identified seem to correspond to a ally are unclear but they are a recurring theme of manytranscription maps recently developed across the hu-smaller genomic region. Many of these smaller frag-

ments do not appear to be connected to each other and man genome (Sedlacek et al., 1993). It has been sug-gested that endogenous retroviruses, if expressed, mayare included in the transcription map as separate

units. For some, such as exons 1.89 and 4.14, PCR be involved in autoimmunity (Query and Keene, 1987)and leukemias (Brodsky et al., 1993) and that theirscreening of representative libraries of different tissues

showed that apart from cDNA made from lymphoblas- reinsertion at new sites in the genome could activateor inactivate genes (Kongsuwan et al., 1989).toid cell mRNA, they were not expressed in any other

tissue and therefore may well represent parts of tissue- Expression patterns of the individual transcripts andgenes within the A-T region indicate a common, widespecific genes. cDNA fragments K28, K22, and K47 and

exon 1.83 seem to be parts of separate genes since inter- distribution for the majority of genes analyzed. Novelgenes such as F37, 6.82, E14, and K28 as well as pre-fragment amplification from the cDNA libraries or di-

rectly from cDNA made from lymphoblastoid cell viously reported genes, ACAT and ATM, show expres-sion in a wide variety of tissues. On the other hand,mRNA was not possible. In addition, the K28 cDNA

gene shows homology to the human family of the Rab information regarding restricted expression or lack ofexpression of isolated exons and fragments of tran-genes while the other three cDNA fragments show no

homology to any other human sequence. In the same scripts has to be read with caution, as it is well knownthat individual exons could be affected by alternativemanner, exons E34 and E36 seem to be part of novel

genes with no homologous sequences in the GenBank splicing in specific tissues.The existence of ‘‘transcriptional domains’’ has beendatabase.

Several regions poor in transcripts were identified reported throughout the genome (Yeom et al., 1991;Bione et al., 1993). This situation is where clusters ofduring construction of the transcription map. From

centromere to telomere these regions are the region genes show the same orientation and expression in thesame tissues and it is believed that genes involved incentromeric to the derived cosmid 33 6E9 and gene

F37, the region between the two NotI sites, and the these domains may have related functions. The cloningof a single ATM gene ruled out the possibility of theregion telomeric to the ATM gene. The first region,

centromeric to cosmid 33 6E9, appeared to be genuinely existence of several A-T genes, which had been postu-lated following the demonstration of different geneticgene poor as the generation of transcripts from two

sources, the cosmid contig and YAC 6E9, using both complementation groups. Although transcriptional do-mains usually involve genes clustered within a smallexon trapping and direct selection of cDNAs, failed to

identify transcripts other than ones that were part of genomic region (up to 100 kb), it is interesting that fiveneighboring genes within 400 kb of the A-T region,gene F37. However, the complete coding sequence of

gene F37 has not been obtained and therefore the cen- distal to the telomeric NotI site, appear to have a re-lated transcriptional orientation. Genes K28, 6.82,tromeric boundary of this gene is not known. The sec-

ond region, between the two NotI sites, is associated ACAT, and ATM are all transcribed from the centro-mere toward the telomere, and the E14 gene, althoughwith remarkable genomic instability. The cosmid con-

tig could not be extended across this region and we and transcribed in the opposite direction, is transcribedfrom the same promoter as the ATM gene (Byrd et al.,others (Arai et al., 1996) identified deletions in many

YAC clones across this region. The YAC 6E9 colony, 1996a). In addition, the recently identified, ubiqui-tously expressed, putative DEAD-Box RNA helicase,which was used for exon trapping and direct selection,

contained a NotI fragment of Ç50 kb, the largest NotI which maps to a region telomeric to marker D11S1294,is also orientated 5* r 3 * centromere to telomere (Savit-fragment seen in 10 colonies. Physical mapping of hu-

man genomic DNA indicated that one of these two NotI sky et al., 1996).Prior to the identification of the ATM gene, whichsites was methylated, so it is not known whether the

NotI fragment identified in the 6E9 colony used for was found to be mutated in various A-T patients, any

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EXPRESSION MAP AROUND THE ATM GENE 275

human VACM-1, a cullin gene family member, located on chromo-of the widely expressed genes in the A-T region couldsome 11q22–23. Genome Res., in press.have been considered as a candidate for various aspects

Collins, F. S. (1995). Positional cloning moves from perditorial toof the A-T phenotype. However, at present, there is notraditional. Nature Genet. 9: 347–350.evidence that any of these genes has a role in the A-T

Cordonnier, A., Casella, J-F., and Heidmann, T. (1995). Isolation ofphenotype and, indeed, one gene, E14, was found not novel human endogenous retrovirus-like elements with foamy vi-to be mutated in A-T patients (Byrd et al., 1996b; Imai rus-related pol sequence. J. Virol. 69: 5890–5897.et al., 1996). The widely expressed genes from the A-T Church, D. M., Stoler, C. J., Rutter, J. L., Murrell, J. R., Trofatter,

J. A., and Buckler, A. J. (1994). Isolation of genes from complexregion are likely to be involved in vital cellular func-sources of mammalian genomic DNA using exon amplification. Na-tions. The interest in these genes is maintained furtherture Genet. 6: 98–105.by the fact that the A-T region appears to be involved in

Futreal, P. A., Cochran, C., Rosenthal, J., Miki, Y., Swenson, J.,loss of heterozygosity in some tumors, including breast Hobbs, M., Bennett, L. M., Haugen-Strano, A., Marks, J., Barett,cancer (Vorechovsky et al., 1996). J. C., Tavtigan, S. V., Shattuck-Eidens, D., Kamb, A., Skolnick,

M., and Weiseman, R. (1994). Isolation of Diverged homeobox gene,MOX from BRCA1 region on 17q21 by solution hybrid capture.ACKNOWLEDGMENTSHum. Mol. Genet. 3: 1359–1364.

Gatti, R. A., Lange, E., Rotman, G., Chen, X., Uhrhammer, N., Liang,We thank the Wellcome Trust, the Cancer Research Campaign,T., Chiplunkar, S., Yang, L., Udar, N., Dandekar, S., Sheikha-the U.K. A-T Society, the A-T Research and Support Trust, and thevandi, S., Wang, Z., Yang, H.-M., Polikow, J., Elashoff, M., Teletar,Medical Research Council for their continued support.M., Sanal, O., Chessa, L., McConville, C., Taylor, M., Shiloh, Y.,Porras, O., Borresen, A.-L., Wegner, R.-D., Curry, C., Gerken, S.,

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