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The Tuberin (TSC2), Autosomal Dominant Polycystic Kidney Disease(PKD1), and Somatostatin Type V Receptor (SSTR5) Genes Form

a Synteny Group in the Fugu Genome

RICHARD SANDFORD,1 BARBARA SGOTTO, TIMOTHY BURN,* AND SYDNEY BRENNER

Molecular Genetics, Department of Medicine, Addenbrookes Hospital, Hills Road, Cambridge, CB2 2QQ, England, United Kingdom;and *Genzyme Genetics, Genzyme Corporation, Framingham, Massachusetts 01701-9322

Received July 10, 1996; accepted September 12, 1996

genes (9). The widely expressed 14-kb PKD1 transcriptThe tuberous sclerosis 2 (TSC2) and polycystic kid- encodes a novel predicted protein containing a leucine-

ney disease 1 (PKD1) genes are adjacent on human rich repeat motif, multiple internally repeated do-chromosome 16p13.3 and form part of a conserved syn- mains, and a transmembrane region (8). Although theteny group with mouse chromosome 17. We have deter- 3 * end is single copy, the duplicated 5* region has mademined that the PKD1 gene is evolutionarily conserved, full characterization of the gene and mutation screen-single copy, and linked to TSC2 in the Fugu genome. ing very difficult. No data on its evolutionary conserva-A short cosmid contig has been identified containing tion exist.both genes based on hybridization, exon trapping, and We reasoned that a cross-species comparison wouldrandom sequence data. In addition sequences homolo- aid the full characterization of the PKD1 gene includ-gous to the somatostatin type V receptor (SSTR5) were

ing functional domains and regulatory elements. Theidentified 5* to PKD1, defining a larger syntenic region,Fugu genome was therefore used to screen for evolu-as this gene has also been mapped to human chromo-tionary conservation, as teleosts form the largest andsome 16p13.3. As in mammalian genomes, the Fuguoldest group of vertebrates. The 400-Mb genome of theTSC2 and PKD1 genes are adjacent in a tail-to-tail ori-Japanese pufferfish, Fugu rubripes, has been shown toentation. q 1996 Academic Press, Inc.

be relatively free of repetitive DNA and to containgenes with small introns, at high density (3). Assuming70,000 genes in 400 Mb, this represents an averageThe majority of cases of autosomal dominant polycys-gene density of one every 6 kb, suggesting that a singletic kidney disease (ADPKD) are caused by mutationscosmid clone may contain an average of 6 genes. Genesat the PKD1 locus on human chromosome 16p13.3 (7).are therefore easily amenable to genomic sequenceMutations in the PKD2 gene on chromosome 4q21–q23analysis and may span considerably less DNA thanaccount for most of the remainder, and these two genestheir mammalian homologues (2). This gives access tohave recently been shown to have some sequence simi-information about a gene that is not available fromlarity (10). Several PKD1-like sequences have also beencDNA sequencing alone. Several well-characterizedmapped to 16p13.1 that are highly conserved but repre-and evolutionarily conserved genes have been de-sent a recent duplication, as they are absent from thescribed in the Fugu genome and confirm that not onlymouse genome (11). PKD1 has also been shown to besequence but also exon–intron organization and regu-contiguous with the TSC2 gene in a tail-to-tail orienta-latory elements are conserved (1, 2). The greater evolu-tion in both human and rodent genomes, identifying ationary distance between Fugu and human than be-conserved synteny group between mouse chromosometween mouse and human can also be exploited to deter-17 and human chromosome 16p13.3 (7, 11).mine functional domains, as these regions are likely toThe 5.5-kb TSC2 gene (6) has homology to theshow a higher degree of sequence conservation.GTPase-activating protein GAP3 and is highly con-

This initial study was designed to investigateserved during vertebrate evolution (9). It is likely towhether the PKD1 gene is conserved across vertebratefunction as a classical tumor suppressor gene, and itsevolution and linked to TSC2 in the Fugu genome. A41 exons span about 43 kb of human genomic DNA.previously described genomic library cloned in theThe Fugu homologue of this gene has 42 exons, spansBamHI site of l2001 (3) was screened using the TSC217.3 kb, and demonstrates a pattern of alternative4.9 and 4B2 cDNAs (6) and the PKD1 3A3 cDNA (7).splicing identical to that of the human and rodentUnder conditions of low stringency using the Church–Gilbert protocol (5), 105 clones were screened, and du-1 To whom correspondence should be addressed. Telephone:/1223

402436. Fax: /1223 210136. E-mail: rsandfor@med.cam.ac.uk. plicate positives were obtained with both the TSC2 and

84GENOMICS 38, 84–86 (1996)ARTICLE NO. 05960888-7543/96 $18.00Copyright q 1996 by Academic Press, Inc.All rights of reproduction in any form reserved.

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FIG. 1. Restriction map of lambda and cosmid clones spanning the Fugu PKD1 locus. The regions producing hybridization signals withthe TSC2 and PKD1 probes are shown by shaded arrows, and the orientation of genes are shown by filled arrows. The maximum distancebetween TSC2 and PKD1 predicted by restriction mapping is therefore 2 kb. Data derived from sequencing are shown as the amino acidpositions of the human gene (aa). Fragments derived from SSTR5 by exon trapping were mapped back to cosmid 48D10 by hybridization.

the PKD1 probes. Two clones were positive with both sequence analysis identified 61% with homologies toknown genes: 6 were vector derived (1 from pSPL3B,probes, suggesting that these genes are part of a con-

served linkage group. To define this region further, 5 from cosmid), 6 had homology to four different re-gions of TSC2, 14 had homology to three differentfragments from one clone (l10) were used to screen a

gridded Fugu cosmid library. A single positive clone, regions of PKD1, 2 had homology to a single regionof SSTR5, and 8 produced no significant hits.295C6, was identified. To determine the extent and

orientation of the TSC2 and PKD1 genes, both lambda Thus data from both sequencing and hybridizationplaced almost the entire TSC2 gene and a region ho-and cosmid clones were used to construct a physical

map of this region (Fig. 1). Further cosmid clones, mologous to 31 of the 46 exons of the PKD1 gene oncosmid 295C6. This region of PKD1 spans approxi-52G18 and 48D10, were identified using end probes

from 295C6. mately 25 kb of human genomic DNA and 20 kb ofFugu genomic DNA, suggesting that PKD1 is of equiva-Probes for the 3 * ends of TSC2 (4.9) and PKD1 (3A3)

were used to determine the orientation of these genes. lent size in both genomes. The rest of PKD1 thereforeresides on cosmid 48D10, with SSTR5 defining an ex-Additional data were provided by random, end-clone,

and ordered sequencing and by exon trapping. The tended syntenic region.The sequence of the Fugu TSC2 gene is highly con-complete sequence of the Fugu TSC2 gene has already

been reported (9). Sequences homologous to TSC2, served, with 32% of its genomic region composed ofcoding sequence and 70% of its introns less than 200PKD1, and SSTR5 were found using these techniques

(Fig. 1). End-clone sequences corresponding to amino bp (range 62–1928 bp). Therefore the total predictedexon density of 295C6 is approximately 72 in 37 kb,acids 36–89 (exons 1 and 2) of the TSC2 gene and

1596–1638 (repeat domain PKD R8, exon 15) of the covering 13.6 kb of coding sequence (37% of the insert).A single random sequence of 300 bp from 295C6 wouldPKD1 gene were obtained from 295C6. The PKD1 3A3

probe hybridized to fragments of the cosmid clone that therefore be expected to include both coding and non-coding sequence. With exon trapping identifying onlyindicated that the Fugu PKD1 homologue was adjacent

to TSC2 in a tail-to-tail orientation, consistent with 4 from 41 exons of TSC2 and random sequencing 13from 41, the study of an equivalent number of se-these sequence data. Southern hybridization and li-

brary screening also suggested that TSC2 and PKD1 quences favors random sequence analysis or cosmidsequence scanning as the method of choice for identi-were single copy in the Fugu genome.

Further confirmation of the presence of a Fugu fying coding sequence in the Fugu genome.Cosmid clones 295C6 and 48D10, spanning 65 kb,PKD1 homologue adjacent to TSC2 was obtained

with random sequences from 295C6 and exon trap- therefore define the PKD1 locus in the Fugu genomeand demonstrate a region of conserved synteny includ-ping. Of 34 random sequences obtained from 295C6,

13 (38%) were homologous to different regions of ing TSC2 and SSTR5. They currently form the startingpoint for assembling a large cosmid contig of this regionTSC2 and 7 (21%) to different regions of PKD1. Exon

trapping analysis was carried out to compare its util- to investigate synteny around the PKD1 locus further.Obtaining the complete sequence of this region willity in isolating coding sequence from the Fugu ge-

nome with random sequencing of cosmid clone frag- yield important information on the structure of PKD1including comparisons of exon–intron boundaries, con-ments. The modified pSPL3 vector described by Burn

et al. (4) was used to analyse 295C6 and 48D10. Of 36 served domains, and regulatory elements. The elucida-tion of the extent of this and other syntenies will haveexon trapping products obtained from these cosmids,

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(Fugu) genome as a compact model vertebrate genome. Natureimportant implications in the use of the Fugu genome366: 265–268.in positional cloning, gene hunting, and transcriptional

4. Burn, T. C., Connors, T. D., Klinger, K. W., and Landes, G. M.mapping strategies. (1995). Increased exon-trapping efficiency through modifica-tions to the pSPL3 splicing vector. Gene 161: 183–187.

5. Church, G. M., and Gilbert, W. (1984). Genomic sequencing.ACKNOWLEDGMENTSProc. Natl. Acad. Sci. USA 81: 1991–1995.

6. The European Chromosome 16 Tuberous Sclerosis Consortium.R.S. and B.S. are funded by a Medical Research Council Clinician(1993). Identification and characterisation of the tuberous scle-Scientist Fellowship awarded to R.S. B.S. is also in receipt of fundingrosis gene on chromosome 16. Cell 75: 1305–1315.from the Tuberous Sclerosis Association. The TSC2 probe was sup-

7. The European Polycystic Kidney Disease Consortium. (1994).plied by The European Chromosome 16 Tuberous Sclerosis Consor-The polycystic kidney disease 1 gene encodes a 14 kb transcripttium and the PKD1 3A3 probe by The European Polycystic Kidneyand lies within a duplicated region on chromosome 16. Cell 77:Disease Consortium. The Fugu cosmid library is available by inquiry881–894.to the UK HGMP Resource Centre.

8. Hughes, J., Ward, C. J., Peral, B., Aspinwall, R., Clark, K., SanMillan, J. L., Gamble, V., and Harris, P. (1995). The polycystic

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