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An Expression Map from Human Chromosome 14q24.3

Vikram Sharma,*,†,1 Parvoneh Poorkaj,*,† Fuki Hisama,‡ Lori Bonnycastle,*,†,2

Chang-En Yu,*,† Hillary Massa,§ Barbara Trask,§ Kevin P. Clancy,Ø David Patterson,ØSherman M. Weissman,‡ and Gerard D. Schellenberg,*,†

*Geriatric Research Education Clinical Center, Seattle Veterans’ Affairs Medical Center, Seattle, Washington, 98108; †Division ofGerontology and Geriatric Medicine and Division of Neurology, Department of Medicine and §Department of Biotechnology,University of Washington, Seattle, Washington 98195; ‡Boyer Center for Molecular Medicine, Yale University, New Haven,

Connecticut 06510; and ØEleanor Roosevelt Institute, Denver, Colorado 80206

Received July 30, 1997; accepted October 23, 1997

resolution of this genomic region for future human dis-We have constructed an expression map of chromo- ease studies (9).

some 14q24.3 between markers D14S42 and D14S63. We constructed a YAC contig by screening the Wash-cDNA selection with YACs from 14q24.3 was used to ington University YAC libraries and early CEPH andgenerate expressed sequence tags (ESTs). The localiza- CEPH Mega-C YAC libraries with short tandem repeattion of ESTs was confirmed on a YAC contig. PCR prod- polymorphic markers (STRP) and genes previously lo-ucts of ESTs were used as probes to screen cDNA li- calized to chromosome 14q24 (2, 4, 5, 7, 12). P1 clonesbraries leading to the isolation of transcripts for for genetic and EST markers were isolated by PCR-known and unknown genes. In total, the expression based screening services provided by Genome Systems,map contains 7 known genes previously mapped to Inc. (St. Louis, MO) or by screening an arrayed P1 clone14q24.3, 6 cDNA transcripts, and 15 anonymous ESTs. library on nylon membranes (Genome Systems, Inc.)The addition of 21 unique transcribed loci from anÉ5- with radiolabeled probes. A flow-sorted chromosome 14to 7-Mb region of chromosome 14q24.3 will facilitate cosmid library (LA14NC01, Larry Deaven, Los Alamosfuture efforts to identify human disease genes from National Laboratory) was pooled and arrayed in twothis region. q 1998 Academic Press

dimensions for screening by PCR. New STRP markerswere developed by subcloning YAC and cosmids intopBluescript (Stratagene) as described (22).A goal of the Human Genome Project is the construc-

Fluorescein-labeled inter Alu-PCR products ampli-tion of a physical and transcriptional map of the humanfied from total yeast DNA of 30 YACs were mappedgenome (8). The construction of transcriptional mapsrelative to fluorescent R-bands in metaphase chromo-is underway with the submission of anonymous ex-somes as described (11). Twelve YACs were found topressed sequence tags (ESTs) from a variety of cell linesbe chimeric (Fig. 1). Cosmids for 4 genetic markersand tissues (1, 13, 18). Another approach is the creationwere unambiguously ordered as cen–D14S57–D14S289of transcription maps as part of positional cloning to–D14S77–D14S43–tel, using two-color FISH on atidentify disease-causing genes. We employed cDNA se-least 50 chromatids per cosmid pair. Cosmids repre-lection on YACs from chromosome 14q24.3 to generatesenting D14S43 and D14S273 could not be resolved,region-specific ESTs as a step toward identifying thesuggesting that they lie õ500 kb apart (20) (Fig. 1).chromosome 14 Alzheimer disease gene. With the clon-The cytogenetic location of the contig as determined bying of this gene, presenilin-1 (19), several chromosomeFISH, extends from 14q24.1 to 14q24.3.14q24 contigs consisting of nontranscribed loci have The order of common markers, contig size, and place-been published (6, 10, 14). The transcription map pre- ment of YACs are consistent with those of other contigssented here contains 28 expressed sequences, including from this region (6, 10, 14, 19). New additions include5 genes not previously reported, thus increasing the the development of 5 STRP markers from YACs or cos-mids (D14S1449–D14S1454) and the placement of 12

Sequence data from this article have been deposited with theP1 clones and 4 cosmids to the contig. We noted consid-EMBL/GenBank Data Libraries under Accession Nos. G20215–erable YAC instability as independent clones of theG20218, N64901–N64914, N65920–N65937, N81199–N81206,

U25972, and W33209. same YAC varied in size or STS content. Inconsisten-1 To whom correspondence should be addressed at present address: cies in the map regarding the localization of ESTs or

PPD Discovery Inc., 1505 O’Brien Drive, Suite B, Menlo Park, CA STSs to overlapping YAC’s appear to result from cryp-94025. Telephone: (415) 617-9570. Fax: (415) 617-9574. tic deletions in YACs. However, the redundancy of2 Present address: Institute of Molecular Biology and Biochemis-

YACs for given markers strongly suggests that theytry, Department of Chemistry, Simon Fraser University, BurnabyBC, Canada V5A156. derive from the appropriate genomic region.

314GENOMICS 47, 314–318 (1998)ARTICLE NO. GE9751060888-7543/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

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FIG. 1. The expression map and contig from 14q24.3. The STS content of the genomic clones is shown. Genomic clones and markersare listed in centromeric to telomeric order. PCR primers and conditions for new markers have been deposited with the Genome Data Baseand GenBank. P1 and cosmid clones are noted after the clone address as (P) and (C), respectively. Genomic clones tested for only a singleSTS are indicated by a plus sign; genomic clones not tested for an STS are indicated by a minus sign. Boldface underline of markersindicates that the relative order of these to each other could not be determined. Types of markers are distinguished by P (STRP), G (knowngene), C (cDNA), E (EST), and S (STS). DNA preparations for the eight most telomeric YACs did not originate from single colonies. Onesignificant gap (denoted by the slash) exists on the contig between D14S258 and D14S57. Superscripts C and NC denote chimeric by FISHand not chimeric by FISH, respectively.

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cDNA selections were performed as described (15, 2H), though a number of these were represented byunmapped, anonymous ESTs (Table 1). The cDNA16), on CEPH YACs 852A2, 788H12, 964E2, 746B4,

948B6, and 932C7. The average insert size of selected transcript for 788IG9 shares a conserved domain at theamino acid level for human bile acid-CoA amino acidclones was 125–250 bp. Approximately 10–15% of

clones contained human repetitive sequences. DNA se- N-acyl transferase (L34081; 67% identity over 49 resi-dues) and rat kan-1 (D43964; 60% identity over 50 resi-quences were obtained for ú200 human repeat-free

clones, identifying 75 unique sequences. Identities or dues).The cDNA transcripts recovered for ESTs 788IIH5closely related homologs to 21 sequences deposited in

GenBank/EMBL were found. These included 10 genes and 4F-AD4 from the 788H12 selection library are thePS-1 Alzheimer disease gene (19). We cloned by PCR[PS-1 (L40401), TGFB3 (M60556), PGF (X54936),

DLST (D17297), rat neuro-D (X66022), yeast riboprot- 0.9- and 1.2-kb gene fragments from a spinal cordcDNA library (ATCC LMG4). Additional transcriptsein L-41 (X76487), rat riboprotein L-36 (X68284), pro-

tein phosphatase 2A (X12656), activating transcription were isolated by screening cDNA libraries with ESTprobes. One cDNA transcript, 4F-2A, had a unique 5*factor 3 (L19871), 7 SL-RNA], 10 ESTS (T07673,

Z40498, Z28781, Z31231, Z44006, H40373, H85607, UTR sequence (Accession No. W33209), which was al-ternatively spliced to nucleotide 112 of the PS-1 geneH41018, R02189 and N54188), and 1 anonymous

mRNA transcript (L40400). The majority of the cDNA (L42110). Characterization of a 10-kb EcoRI genomicfragment from a P1 clone (662A10) confirms the pres-selected clones had no identity to sequences in the data-

bases. cDNA sequences were checked for the presence ence of traditional consensus splice sites for the unique5* UTR and PS-1 (data not shown). The presenilin-1of potential exons by the program GRAIL-1 (21).

PCR primers were designed for 37 cDNA selected gene is represented on our map by 3 ESTs (Table 1,Fig. 1).clones, 24 of which were mapped to the contig. The

chromosome 14 origin of new EST and STRP loci was cDNA selection proved to be a robust method for rap-idly isolating gene sequences from a targeted genomicconfirmed by PCR of total human genomic DNA, a

human chromosome 14-only somatic cell hybrid region. In addition to clones derived from genes knownto be present on the genomic target, cDNA selected(GM10479A), the YAC used in the cDNA selection, and

the remaining contig YACs. All localizations of markers clones provided the initial localization of PGF and PS-1 to our map. Of 24 ESTs mapped to the contig, 16 hadto the contig were performed by PCR. DNA and PCR

primer sequences for new EST and STRP markers have no homology to sequences in GenBank and are expectedto represent novel genes. Some ESTs were successfullybeen deposited with the GenBank/EMBL Data Librar-

ies (NCBI/NIH) and the Human Genome Database used as probes on cDNA libraries to isolate the repre-sentative cDNA transcript (Table 1). For ESTs with no(Johns Hopkins University). Although we chose to map

ESTs by PCR, 13 primer pairs failed to amplify genomic associated cDNA transcript, we note that only a singletranscript was recovered for each of 3 ESTs (Table 1)DNA. We were able to show for a number of ESTs

that PCR failure was attributable to primers spanning after screening 4.5 1 106 PFU from cDNA libraries.By screening additional clones or cDNA libraries fromintronic splice sites that disrupted primer annealing to

genomic DNA or that spanned introns that were too different tissue sources, rare transcripts or those withlimited expression profiles may be recovered for manylarge to amplify by PCR (data not shown). The appro-

priate sized fragments were recoverable when total of the remaining mapped ESTs.A comparison of our expression map with the othercDNA was amplified.

A total of 3 1 106 PFU from a fetal brain cDNA transcription map of this region (19) shows that neithermap has all the genes identified by the other, althoughlibrary (Clontech HL3003b) and 1.5 1 106 PFU from

a lymphocyte cDNA library (Clontech HL5007b) were both were developed by cDNA selection from the sameregion of chromosome 14. For example, we had cDNAplated at 150,000 PFU/150-mm plate and screened by

traditional methods (17) using labeled PCR products transcripts or ESTs for 4 of 14 novel transcripts thatSherrington et al. (19) mapped to 14q24.3, while we(T7 Labeling Kit, Pharmacia). cDNA transcripts were

also cloned into TA cloning vectors (Invitrogen) by PCR isolated PGF and cDNA transcripts for 5 genes thatthey did not identify. In addition, in our selection exper-using EST and vector primers from 100 ng of total

cDNA from basal ganglia (LMG3) and spinal cord iments, we did not isolate clones for the RAP1A pseu-dogene and EST D14S101E, which have been mapped(LMG4) cDNA libraries (American Type Culture Col-

lection, Rockville, MD). Screening cDNA libraries for to YACs 964E2 and 746B4, respectively (6, 10). Thesedifferences between maps regarding which transcripts17 of the mapped ESTs resulted in the isolation of tran-

scripts for 8 ESTs (Table 1). The cDNA transcripts were identified will reflect how extensively the selectedlibraries were screened or the YAC used for selectionsranged in size from 0.6 to 2.9 kb. Partial sequencing of

the transcripts confirmed the presence of the originat- or result from differences in the tissue source for thecDNAs used for selection. cDNA selection quickly iden-ing EST. GenBank searches by BLAST (3) found no

significant homology to known genes for 5 cDNA tran- tifies a set of genes from a target genomic region, butprobably does not select all the genes within a YACscripts (788IE1, 788IIH2, 964IH2, 788IID1, and AD3-

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TABLE 1

EST Characteristics and GenBank Homologies

EST GDB Accession Selection No. of EST GRAIL GenBank No. of cDNAname amplimer No. library homologsa EST score identities Description (kb)

788IIH2 D14S1353 N81199 788H12 4 Good 2.2788IG9 D14S1367 788H12 0 Excellent Z28781, Anonymous EST 1.3 and 1.0

L40401 Partial mRNA transcript788IID1 D14S1371 N81201 788H12 0 0 2.8788IE1 D14S1373 N81203 788H12 3 0 Z40498 Anonymous EST 2.9AD3-2H D14S1381 N81205 964E2 1 Marginal T07673 Anonymous EST 0.7 and 0.6964IH2 D14S1385 N81206 964E2 1 0 2.0 and 0.7788IIH5 788H12 3 Good L42110 Presenilin 1 gene 2.2, 1.2, and 1.04F-AD4 788H12 2 Excellent L42110 Presenilin 1 gene; cDNA 4F-2A has a unique 2.0, 1.4, and 1.2

alternatively spliced 5*UTR (W33209)788IIA8 788H12 1 0 L42110 Presenilin 1 gene746CDNA5 746B4 5 nd M60556 Transforming growth factor b-3964IC3 964E2 0 0 X54936 Placenta growth factor964IIB6 964E2 2 0 D17297 Dihydrolipoamide succinyl transferaseAD6-2E D14S1355 N64914 852A2 0 0 Z31231 Anonymous mouse EST788IG4 D14S1357 N64909 788H12 0 0788IIA4 D14S1359 N64910 788H12 6 GoodAD3-4B D14S1361 N64905 964E2 2 0788IIA7 D14S1363 N64911 788H12 0 0788IID8 D14S1365 N64912 788H12 0 0788IG3 D14S1369 N64908 788H12 5 02H-AD4 D14S1375 N64907 788H12 3 0AD4-2 D14S1377 N64902 788H12 3 0AD3-5 D14S1379 N64901 964E2 1 0964ID1 D14S1383 N64904 964E2 2 04E-AD4 D14S1387 N64906 788H12 1 0AD4-5 D14S1389 N64903 788H12 4 0788IIE3 D14S1391 N64913 788H12 2 06C-AD4b 788H12 4 Excellent X66022 Rat neuro-d homologcDNA6c 746B4 1 nd L19871 Activating transcription factor 3 homolog964IIA4 964E2 2 nd L40400 Partial mRNA transcript mapped to 14q24.3d

788IIB7 788H12 3 Good Z44006, Anonymous ESTL40393 Partial mRNA transcript mapped to 14q24.3d

a Number of independent clones with overlapping sequences found in the selection library.b Maps to contig YACs 788H12, 885B5, 852A2, and 834G11 by hybridization.c Maps to contig YACs 746B4 and 797D11 by hybridization.d Sherrington et al. (19).

93ER61553) (B.T.); and the Yale-L. P. Markey Trust Physician-Sci-clone with equal efficiency. Additionally, it will missentist Training Fellowship (F.H.). We thank Elaine Loomis for expertidentifying genes expressed in a tissue-specific mannertechnical assistance.or in a developmentally narrow window. The construc-

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