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Biochemical and Biophysical Research Communications 271, 693–698 (2000)

doi:10.1006/bbrc.2000.2685, available online at http://www.idealibrary.com on

solation of Two Novel Genes, DSCR5 and DSCR6,rom Down Syndrome Critical Regionn Human Chromosome 21q22.21

azunori Shibuya, Jun Kudoh, Shinsei Minoshima, Kazuhiko Kawasaki,huichi Asakawa, and Nobuyoshi Shimizu2

epartment of Molecular Biology, Keio University School of Medicine, 35 Shinanomachi,hinjuku-ku, Tokyo 160-8582, Japan

eceived April 11, 2000

DSCR6 genes are candidates for the pathogenesis ofDr

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We have isolated two novel genes, designatedSCR5 and DSCR6, from the Down syndrome critical

egion (DSCR) on chromosome 21q22.2 which has beenefined as minimal overlapping region of partial tri-omy 21 patients and located between t(4;21) breakoint and ERG (approximately 1.6 Mb). DSCR5 andSCR6 genes consist of 6 and 5 exons, respectively.lternative use of transcription start sites and alter-ative splicing events produce different RNA speciesnd proteins from both genes. Three different tran-cripts of DSCR5 gene encode three putative trans-embrane proteins of 158, 134, and 108 amino acids,hile 4 different transcripts of DSCR6 gene encode

wo forms of proteins with 190 and 106 amino acids.he DSCR5 gene is expressed in various human tis-ues examined, whereas the DSCR6 gene is expressednly in limited tissues at low level. Both DSCR5 and

Abbreviations used: DSCR, Down syndrome critical region; ERG,-ets avian erythroblastosis virus E26 oncogene related; BAC, bac-erial artificial chromosome; HLCS, holocarbyoxylase synthetase;TC3, tetratricopeptide repeat domain 3; DCRA, Down syndromeritical region A; DYRK1A, dual-specificity tyrosine-(Y)-phosphor-lation regulated kinase 1A; KCNJ6, potassium inwardly-rectifyinghannel, subfamily J, member 6; DCRB, Down syndrome criticalegion B; KCNJ15, potassium inwardly-rectifying channel, subfam-ly J, member-15; RACE, rapid amplification of cDNA ends; ORF,pen reading frame; UTR, untranslational region; PCR, polymerasehain reaction; EST, expressed sequence tag; G3PDH, glyceralde-yde-3-phosphate dehydrogenase.

1 The HUGO Nomenclature Committee approved symbols for theown syndrome critical region gene 5 and the Down syndrome

ritical region gene 6 are DSCR5 and DSCR6, respectively. Theucleotide sequence data reported in this paper will appear in theDBJ, EMBL, and GenBank nucleotide sequence databases underccession Nos. AP000704 (KB318C2), AB037158 (DSCR6a), AB037159

DSCR6b), AB037160 (DSCR6c), AB037161 (DSCR6d), AB037162DSCR5a), AB037163 (DSCR5b), and AB037164 (DSCR5c).

2 To whom correspondence should be addressed. Fax: 81-3-3351-370. E-mail: shimizu@dmb.med.keio.ac.jp.

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own syndrome, although the function of these genesemains to be elucidated. © 2000 Academic Press

Key Words: chromosome 21; trisomy 21; Down syn-rome; cDNA; genome sequencing; DSCR5; DSCR6.

Down syndrome is the most frequent birth defect,ccurring in approximately 1/1000 births and is asso-iated with mental retardation, congenital heart dis-ase, facial and physical features, and defects of themmune and endocrine systems. It is caused by fullhree copies or partial trisomy of human chromosome1. Thus, it was expected that particular regionshould be profoundly related with the pathogenesis ofown syndrome. This minimal region is called Down

yndrome critical region (DSCR) and was defined be-ween D21S17 and ETS2 (1, 2), then limited to 2.5 Mbegion between CBR and ERG (3) and finally definedp to approximately 1.6 Mb between t(4;21) and ERG4). The proposition to elucidate the pathogenesis ofown syndrome is to completely identify every gene onSCR. Six genes have been found out from DSCR,

ncluding TTC3 (5, 6), DCRA (7), DYRK1A (8–12),CNJ6 (12–15), DCRB (16) and KCNJ15 (12, 17). Be-

ides these, it has recently been reported that SIM218) located outside the DSCR may be important forhe pathogenesis of Down syndrome, especially mentaletardation (19). In order to find out further novelenes, we have attempted to completely determine theequence of a BAC clone, KB318C2, which located inistal region of LA68 (D21S396), and analyzed its se-uence by computer-aided exon prediction and homol-gy search through the BLAST server at NCBI. Con-equently, we could successfully isolate two novelenes from the DSCR, which were named DSCR5 andSCR6.

0006-291X/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

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ATERIALS AND METHODS

BAC DNA sequencing of KB318C2. Genomic sequencing of BAClone, KB318C2 was performed using the shot-gun sequencingethod as described previously (20).

Computational analysis of DNA and amino acid sequences. Theasking of the repetitive elements in the genomic sequence andLASTN-homology searches of the nonredundant database at Gen-ank were performed using our Xmsv system based on nsm program

21). The exon-predictions were carried out using Xgrail version1.3c22), GENSCAN (23) and MZEF (24), respectively. Further detailedomologies of nucleotide and amino acid sequences were analyzedhrough the BLAST server at NCBI using BLASTN, BLASTP andLASTX (25). A motif analysis of gene product was performedy SMART v3.1 (26). Protein sorting signal and localization sitesn amino acid sequences were predicted by PSORT II (http://sort.nibb.ac.jp:8800/).

cDNA cloning of the putative genes, DSCR5 and DSCR6. cDNAloning was performed by means of internal amplification of cDNAsnd 59- and 39-Rapid Amplification of cDNA Ends (RACE) method27) according to the manufacturer’s protocol. PCRs for internalmplification and 59 and 39 RACE were executed as illustrated inig. 2 and Fig. 3 using primers summarized in Table 1. 59 and 39ACE fragments and internal fragments were amplified by Expandigh Fidelity PCR System containing thermostable Taq and PwoNA polymerase (Boehringer mannheim) from testis cDNA pools

Marathon-Ready cDNA, Clontech) against DSCR5 and fetal kidney,etal brain, testis and retina cDNA pools against DSCR6, respec-ively, under the condition of 94°C for 30 sec, 65°C for 1 min, and2°C for 2 min for 35 cycles in an automated thermal cycler, TRIOhermoblock 48 (Biometra) and then subcloned into pBluescriptI(SK1) (Stratagene). Sequencing of those fragments was performedith DNA sequencer (Models 377, Applied Biosystems) by theideoxy Terminator Cycle Sequencing method using a combinationf BigDye Terminator and AmpliTaq/FS DNA polymerase (Perkinlmer Corporation).

Multiple tissue cDNA panel analysis. Expression analyses of theSCR5 and DSCR6 genes were performed using the Human Multi-le Tissue cDNA (MTC) panels (Clontech; I, II, fetal, and immuneystem panels) containing cDNAs from 27 human tissues. The am-lification of the DSCR5 cDNA were carried out with a primerair 59-CGGAGAACTCAGCGCTGAGATTGTCTA-39/59-CGTGCTT-GTGTTACTATGGTTACACA-39 (DSCR5a), 59-CGCTGACGCTGA-TGTGTTCC-39/59-GCTGGAATGGCCTCCTCTTGGT-39 (DSCR5b)nd 59-GTCGTGGTCTTGTGGAGGAGACAGAT-39/59-CGTGCTTG-TGTTACTATGGTTACACA-39 (DSCR5c) using approximately 2g, 20 pg and 2 pg each of MTC panel cDNA as template, respec-ively. Also, the amplification of the DSCR6 cDNA were carried out

Primer Sequences Used in This Study

Name Sequence

A1F-primer 59-TGTCACTGCCCCGGGGACGCT-39A1R-primer 59-AGCGTCCCCGGGGCAGTGACA-39A2F-primer 59-GGAGTTCCGGGGAGCAAGTACTG-39A2R-primer 59-GGGTCCTTCCTCTGGCTCTTCA-39A3R-primer 59-CTTGGTTGATGCCCTGGTCTCTG-39A3F-primer 59-ACTCCACCTCCTACTTAACCTGA-39A4F-primer 59-CCCTACCCTTAACCCATGAACAC-39A4R-primer 59-ACAGGAAGACACCACATGTAAGA-39I1F-primer 59-GCTGGAATGGCCTCCTCTTGGT-39I1R-primer 59-GTGGGCCTTTATTCCTGAATCTTGGCT-39P2-primer 59-ACTCACTATAGGGCTCGAGCGGC-39

694

GTTGATGCCCTGGTCTCTG-39 (DSCR6a) and 59-ACATCCCA-ATCCGGCAGGGGAA-39/59-CTTGGTTGATGCCCTGGTCTCTG-39

DSCR6b) using approximately 0.2 ng each of MTC panel cDNA asemplate. As a control, PCRs were performed with the glycer-ldehyde-3-phosphate dehydrogenase (G3PDH) control amplimer setsing 2 pg each of MTC panel cDNA as template.

ESULTS AND DISCUSSION

enomic Sequencing and Computational Analysisof KB318C2

In order to elucidate the pathogenesis of Down syn-rome, it is very important to identify all genes whichxist in DSCR. For this purpose, we completely deter-ined a contiguous 122,672 nucleotide sequence of aAC clone, KB318C2, which located in distal region ofA68 (D21S396), using shot-gun sequencing method

20). We have analyzed this sequence data byomputer-aided exon prediction (Xgrail, GENSCAN,nd MZEF) and homology search through the BLASTerver at NCBI. As a result, 23 exons by Xgrail, 22xons by MZEF and 5 gene models by GENSCAN werendependently predicted on a BAC clone KB318C2Fig. 1). Since the results of each prediction werecarcely consistent each other, we did not regard theseredictions as a significant clue for gene-finding. Ho-ology search analysis toward the nucleotide sequence

atabase revealed that 2 mRNAs, 9 ESTs, 5 trappedxons and 4 CpG islands matched with a BAC cloneB318C2 in the high degree as shown in Fig. 1. TwoRNAs were different type of transcripts from holo-

arbyoxylase synthetase (HLCS) gene (12, 28). Six outf 9 ESTs continuously matched with the genomic se-uence, whereas 3 ESTs (GenBank accession numberA025925, AA944415, and AI131153) were found out

o be split on genomic sequence. AI131153 matchedith only a part of KB318C2, remaining parts matchedith the genomic sequence (Accession No. AP000150)hich was located in distal region of KB318C2.Though AA025925 discontinuously matched with 4

arts of KB318C2, the end of this EST was immedi-tely followed by 17 bp poly-A on the genomic sequencef KB318C2 and then it was deduced that this ESTight be derived from cDNA to be incompletely syn-

hesized by reverse transcriptase. Thus, it is unclearhether this EST is a part of true gene on this region.

haracterization of DSCR5

AI131153 is a human EST and a partial segment ofhis EST only matched with the sequence of KB318C2,nd extra segment of this EST discontinuouslyatched with 3 parts of the genomic sequence (Acces-

ion No. AP000150). We designed PCR-primers for 59nd 39 RACE, and attempted to amplify fragmentsrom testis cDNA pools. Accordingly, we could deter-

ine the sequence and structure of 2 kinds of tran-

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cripts, DSCR5a and DSCR5b (Fig. 2), and it was re-ealed that there was a putative polyadenylationignal in 24-bp upstream of poly(A) sequence. Besides,here was an EST, R11132, as shown in Fig. 2, but suchype of mRNA could not be obtained from testis cDNAools. Thereafter, we have determined the sequence ofhe full insert of IMAGE:129210 clone and we couldetermine the sequence and structure of another tran-cript, DSCR5c (Fig. 2).In DSCR5, 3 transcripts existed due to alternative

plicing, and it is noted that DSCR5b held a transitionutation (C to T) and DSCR5c held both transitionutation (A to G) and transversion mutation (A to C).hese mutations could be regarded as SNP, however,ne mutation (A to G) on DSCR5c was observed inording region and could substitute an amino acid, i.e.,soleucine to valine. As isoleucine and valine are anal-gous amino acid, this substitution may not influencehe function of DSCR5c protein. It was deduced that

FIG. 1. The relationship between Down syndrome critical regionnd human chromosome 21, and schematic representation of com-utational analysis of KB318C2. (A) The scheme of the location ofSCR toward whole human chromosome 21. DSCR has been defineds interval (approximately 1.6 Mb) between t(4;21) breakpoint andRG. Arrows indicate positions and orientation of known genesround KB318C2. (B) Summarized scheme of computational analy-is of KB318C2. Total length of KB318C2 is 122,672 bp. Three DNAarkers, ESTs and cDNA which were found by homology search

hrough the BLAST server at NCBI, and analysis for protein codingotential using Xgrail, MZEF, and GENSCAN toward the genomicequence of KB318C2, were represented. Predictions of Xgrail areategorized by three phases; i.e., symbol “*” indicates “excellent,” noymbol indicates “good,” and symbol “1” indicates “marginal.”

695

rames corresponding to 134, 158 and 108 amino acids,espectively (Fig. 2). Although significant homologousrotein could not be detected by BLASTP, signal pep-ides and transmembrane domains were predicted byMART (Fig. 2). Thus, it was deduced that DSCR5ight be transmembrane protein. It was predicted thatSCR5a and DSCR5b would be likely to be localized inndoplasmic reticulum with the probability of 66.7%,nd DSCR5c would be likely to be localized in endo-lasmic reticulum, Golgi and cytoplasm with the prob-bility of 44.4%, 22.2% and 22.2% by PSORT II, respec-ively. As it was predicted that DSCR5c does not holdny signal peptide sequence, it is not determinedhether DSCR5c is really localized in endoplasmic

eticulum.Expression analysis of DSCR5 was carried out byorthern blotting, and the band with adequate size

FIG. 2. Genomic, mRNA, and protein structure of DSCR5. (A)chematic representation of genomic structures of three transcripts

n DSCR5. Each exon of DSCR5a–DSCR5c, segments of EST (Acces-ion No. AI131153), which was the trigger for gene isolation, andegments of EST (Accession No. R11132), which became DSCR5ctself, are indicated. (B) Schematic representation of mRNA struc-ures of three transcripts, and protein structures and motifs ofSCR5. mRNA structures of DSCR5a and b were determined byCR as illustrated, and mRNA structures of DSCR5c were deter-ined by sequencing the IMAGE clone, 129210. Abbreviations TM

nd SP indicate transmembrane domain and signal peptide se-uence, respectively. A transmembrane domain was predicted byMART, and signal peptide cleavage sites in amino acid sequencesere predicted by signal version 2.0 (29) linked with SMART.

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approximately 800 bp) could be detected (data nothown). For more detailed analysis, we practiced ex-ression analysis of DSCR5 using MTC panels. It wasbserved that DSCR5a and DSCR5b are expressed inany tissues, although DSCR5b were detected onlyhen ten-fold of template MTC panel cDNA was useds compared with DSCR5a (Fig. 4). DSCR5c was ex-ressed in fetal liver at the highest level among allissues examined (Fig. 4). This expression profile isonsistent with the origin of EST, R11132, that is de-ived from a fetal liver/spleen library. Since expressionf the DSCR5c was not detected in any adult tissuesxcept for testis, it may be postulated that this geneill be functionally active in early embryogenesis.

haracterization of DSCR6

AA944415 is a rat EST and spans as 4 exons on theenomic sequence of KB318C2 as shown in Figs. 1 and. We designed PCR-primers (AA1F/AA3R) and at-empted to amplify from fetal kidney, testis and retinaDNA pools. Amplified PCR products hold partial

FIG. 3. Genomic, mRNA, and protein structure of DSCR6. (A)chematic representation of genomic structures of four transcripts inSCR6. Each exon of DSCR6a–DSCR6d and segments of EST (Ac-

ession No. AA944415), which was the trigger for gene isolation, arendicated. (B) Schematic representation of mRNA structures of fourranscripts. All mRNA structures were determined by PCR as illus-rated. (C) Schematic representation of protein structures of DSCR6.

significant motif toward these proteins could not be predicted.

696

uently performed 59 RACE using fetal kidney cDNAool (Fig. 3). In addition, we designed PCR-primers fornother 59 and 39 RACE based on the human genomicequence matched with AA944415. As a result of themplification from fetal kidney, testis and retina cDNAools, it was found that poly(A) sequence of 39 RACEragment corresponded with genomic sequence ofB318C2 (nt. 74,601–74616), suggesting that cDNA

ynthesis by reverse transcriptase might run from thealfway of mRNA. Therefore, this 39 RACE fragmentay not reflect on the full length of an actual mRNA.

n order to determine true 39-end of this gene, we newlyesigned PCR-primers and attempted to amplify frag-ent from fetal kidney, testis and retina cDNA pools.s PCR product of approximately 1kb was amplified,e subsequently performed 39-RACE using fetal kid-ey, fetal brain and retina cDNA pool. As there was autative polyadenylation signal 13-bp upstream ofoly(A) sequence, we could estimate that this 39 endas real. As shown in Fig. 3, we have named these

ranscripts DSCR6a, DSCR6b, DSCR6c and DSCR6d,nd finally determined the sequence and structure of 4inds of transcripts (Fig. 3).For DSCR6, 5 transition mutations (A to G) and an

nsertion mutation (2 bp) of adenine were observed inhe 39 UTR of mRNA when compared with the genomicequence of KB318C2. The 39 UTR of mRNA containedAlu repetitive elements and all the above-mentionedutations were found out in Alu repetitive regions.ence, these mutations may be apparent polymor-hisms containing single nucleotide polymorphismSNP).

It was deduced that there are 4 transcripts by alter-ative splicing; DSCR6a contains an open readingrame of 190 amino acids and DSCR6b, DSCR6c andSCR6d contain open reading frame of 106 amino acids.either the sequence-similarity search by BLASTP nor

he motif analysis by SMART detected significant ho-ologous protein and motif, whereas PSORTII pre-

icted that two products of DSCR6 would be likely to beocalized in nucleus with the probability of 65.2%, al-hough actual nuclear localization signals could not bedentified. DSCR6 is the gene which has been found outo far in most centromeric side of Down syndromeritical region (Fig. 1).Expression analysis of DSCR6 was carried out byorthern blotting, but the clearly positive band couldot be detected (data not shown). For more detailednalysis, we practiced expression analysis of DSCR6sing MTC panels. DSCR6a and DSCR6b were ex-ressed at very low level, but PCR fragments weremplified from 0.2 ng of template MTC panel cDNA. Itas confirmed that DSCR6a is expressed in fetal kid-ey and DSCR6b is expressed in fetal kidney and fetalrain at higher level than other tissues examined, al-hough both DSCR6a and DSCR6b were scarcely ex-

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Vol. 271, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ressed in adult kidney and brain (Fig. 4). Human ESTor this gene is not found in GenBank database butnly rat ESTs, which are derived from normalized ratmbryo, are found. Therefore, it may be able to specu-ate that this gene as well as DSCR5c will be function-lly active in early embryogenesis.DSCR5 and DSCR6 isolated in this study remain un-

nown in biological functions. However, in respect ofheir localization in Down syndrome critical region andossible function in early embryogenesis, these can beonsidered as candidate genes for Down syndrome. Wexpect that these two novel genes may greatly contributeo the elucidation of the pathogenesis of Down syndrome.

CKNOWLEDGMENTS

The authors thank Miss Mio Takahashi for technical assistancend all the members of genomic sequencing team in the Laboratoryf Genomic Medicine for their contribution to this work. This workas supported in part by Fund for Human Genome Sequencingroject from the Japan Science and Technology Corporation (JST);rant in Aid for Scientific Research on Priority Areas from theinistry of Education, Science, Sports and Culture of Japan; andrant in Aid for Scientific Research and Fund for “Research for theuture” Program from the Japan Society for the Promotion of Sci-nce (JSPS).

EFERENCES

1. McCormick, M. K., Schinzel, A., Petersen, M. B., Stetten, G.,Driscoll, D. J., Cantu, E. S., Tranebjaerg, L., Mikkelsen, M.,

FIG. 4. Expression pattern of DSCR5 and DSCR6 mRNA analyrom 27 different cDNAs in MTC panels (Clontech) using a primer paithout template cDNA. Lanes 2–28 display PCR products using 20s template MTC panel cDNA, respectively. Control PCR of the G3Pnd no template (lane 1).

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Watkins, P. C., and Antonarakis, S. E. (1989) Genomics 5, 325–331.

2. Delabar, J. M., Theophile, D., Rahmani, Z., Chettouh, Z., Blouin,J. L., Prieur, M., Noel, B., and Sinet, P. M. (1993) Eur. J. Hum.Genet. 1, 114–124.

3. Lucente, D., Chen, H. M., Shea, D., Samec, S. N., Rutter, M.,Chrast, R., Rossier, C., Buckler, A., Antonarakis, S. E., andMccormick, M. K. (1995) Hum. Mol. Genet. 4, 1305–1311.

4. Ohira, M., Ichikawa, H., Suzuki, E., Iwaki, M., Suzuki, K., Saito-Ohara, F., Ikeuchi, T., Chumakov, I., Tanahashi, H., Tashiro, K.,Sakaki, Y., and Ohki, M. (1996) Genomics 33, 65–74.

5. Ohira, M., Ootsuyama, A., Suzuki, E., Ichikawa, H., Seki, N.,Nagase, T., Monura, N., and Ohki, M. (1996) DNA Res. 3, 9–16.

6. Tsukahara, F., Hattori, M., Muraki, T., and Sakaki, Y. (1996)J. Biochem. 120, 820–827.

7. Nakamura, A., Hattori, M., and Sakaki, Y. (1997) J. Biochem.122, 872–877.

8. Song, W.-J., Sternberg, L. R., Kasten-Sportes, C., Van Keuren,M. L., Chung, S.-H., Slack, A. C., Miller, D. E., Glover, T. W.,Chiang, P.-W., Lou, L., and Kurnit, D. M. (1996) Genomics 38,331–339.

9. Shindoh, N., Kudoh, J., Maeda, H., Yamaki, A., Minoshima, S.,Shimizu, Y., and Shimizu, N. (1996) Biochem. Biophys. Res.Commun. 225, 92–99.

0. Guimera, J., Casas, C., Pucharcos, C., Solans, A., Domenech, A.,Planas, A. M., Ashley, J., Lovett, M., Estivill, X., and Pritchard,M. A. (1996) Hum. Mol. Genet. 5, 1305–1310.

1. Wang, J., Kudoh, J., Shintani, A., Minoshima, S., and Shimizu,N. (1998) Biochem. Biophys. Res. Commun. 250, 704–710.

2. Ohira, M., Seki, N., Nagase, T., Suzuki, E., Nomura, N., Ohara,O., Hattori, M., Sakaki, Y., Eki, T., Murakami, Y., Saito, T.,Ichikawa, H., and Ohki, M. (1997) Genome Res. 7, 47–58.

by PCR. The DSCR5 and DSCR6 cDNA fragments were amplifiedescribed under Materials and Methods. Lane 1 is a negative controlfor DSCR5b, 2 pg for DSCR5a and DSCR5c, and 0.2 ng for DSCR6cDNA using 2 pg each of template MTC panel cDNA (lanes 2–28)

zedir dpgDH

13. Lesage, F., Duprat, F., Fink, M., Guillemare, E., Coppola, T.,

1

1

1

1

1

1

20. Kawasaki, K., Minoshima, S., Nakato, E., Shibuya, K., Shintani,

22

222

2

2

2

2

Vol. 271, No. 3, 2000 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Lazdunski, M., and Hugnot, J.-P. (1994) FEBS Lett. 353, 37–42.4. Sakura, H., Bond, C., Warren-Perry, M., Horsley, S., Kearney,

L., Tucker, S., Adelman, J., Turner, R., and Ashcroft, F. M.(1995) FEBS Lett. 367, 193–197.

5. Tsaur, M.-L., Menzel, S., Lai, F.-P., Espinosa, R., III, Concannon,P., Spielman, R. S., Hanis, C. L., Cox, N. J., Le Beau, M. M.,German, M. S., Jan, L. Y., Bell, G. I., and Stoffel, M. (1995)Diabetes 44, 592–596.

6. Nakamura, A., Hattori, M., and Sakaki, Y. (1997) DNA Res. 4,321–324.

7. Gosset, P., Ghezala, G. A., Korn, B., Yaspo, M.-L., Poutska, A.,Lehrach, H., Sinet, P.-M., Creau, N. (1997) Genomics 44, 237–241.

8. Chen, H., Chrast, R., Rossier, C., Gos, A., Antonarakis, S. E.,Kudoh, J., Yamaki, A., Shindoh, N., Maeda, H., Minoshima, S.,and Shimizu, N. (1995) Nature Genet. 10, 9–10.

9. Ema, M., Ikegami, S., Hosoya, T., Mimura, J., Ohtani, H., Na-kao, K., Inokuchi, K., Katsuki, M., and Fujii-Kuriyama, Y. (1999)Hum. Mol. Genet. 8, 1409–1415.

698

A., Schmeits, J. L., Wang, J., and Shimizu, N. (1997) GenomeRes. 7, 250–261.

1. Gotoh, O. (1987). Comput. Appl. Biosci. 3, 17–20.2. Uberbacher, E. C., Xu, Y., and Mural, R. J. (1996) Methods

Enzymol. 266, 259–281.3. Burge, C., and Karlin, S. (1997) J. Mol. Biol. 268, 78–94.4. Zhang, M. Q. (1997) Proc. Natl. Acad. Sci. USA 94, 565–568.5. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman,

D. J. (1990) J. Mol. Biol. 215, 403–410.6. Schultz, J., Milpetz, F., Bork, P., and Ponting, C. P. (1998) Proc.

Natl. Acad. Sci. USA 95, 5857–5864.7. Frohman, M. A., Dush, M. K., and Martin, G. R. (1988) Proc.

Natl. Acad. Sci. USA 85, 8998–9002.8. Suzuki, Y., Aoki, Y., Ishida, Y., Chiba, Y., Iwamatsu, A., Kishino,

T., Niikawa, N., Matsubara, Y., and Narisawa, K. (1994) Nat.Genet. 8, 122–128.

9. Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G.(1997) Protein Eng. 10, 1–6.