GENOMICS 42, 388–392 (1997)ARTICLE NO. GE974769

The Structure and Organization of the Human NPAT Gene

Takashi Imai,*,1 Takehiko Sugawara,* Akiyo Nishiyama,*,† Ryoko Shimada,*,† Reiko Ohki,†Naohiko Seki,‡ Masashi Sagara,* Hiroko Ito,* Masatake Yamauchi,* and Tada-aki Hori*

*Genome Research Group, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage, Chiba 263, Japan;†Department of Molecular Biology, Kyorin University School of Health Sciences, 476 Miyashita, Hachiohji,

Tokyo 192, Japan; and ‡Gene Structure I, Kazusa DNA Research Institute, Kisarazu, Chiba 292, Japan

Received December 26, 1996; accepted April 15, 1997

fects including progressive cerebellar ataxia, oculocuta-Ataxia telangiectasia (AT) is an autosomal recessive neous telangiectasia, immunological defects, and an in-

gene disorder, and ATM, a housekeeping gene, has creased incidence of cancer (for review, see Gatti et al.,been identified as the gene responsible for AT. Re- 1991; Harnden, 1994; Shiloh, 1995). AT heterozygotescently we found that another housekeeping gene, also have an increased risk of developing cancer. It wasNPAT, is located upstream of ATM on human chromo- reported that the estimated risk of cancer of all typessome 11. The two housekeeping genes are transcribed among heterozygotes compared with noncarriers is 3.8in opposite directions and share a 0.5-kb 5* flanking in men and 3.5 in women, and that for breast cancersequence. The structure and organization of NPAT in women is 5.1 (Swift et al., 1991). The gene responsi-were determined by direct sequencing of cosmid ble for AT, ATM, was isolated from human chromosomeclones carrying the gene and by application of the 11q22–q23 (Savitsky et al., 1995a,b) and found to belong and accurate (LA)-PCR method to amplify re- mutated in a large number of AT patients (Savitskygions encompassing the exon/intron boundaries and

et al., 1995a; Gilad et al., 1996; Telatar et al., 1996;all of the exons. The gene spans at least 44 kb andMcConville et al., 1996; Byrd et al., 1996a; Wright etconsists of 18 exons and 17 introns. It has been sug-al., 1996). Recently, Barlow et al. reported the genera-gested that AT heterozygotes have an increased risktion of mice homozygous for the disrupted Atm alleleof developing cancer, especially breast cancer in(Barlow et al., 1996). These mice display symptomswomen. Frequently, loss of heterozygosity at loci onsimilar to those of AT patients and show extreme sensi-11q22–q24 has been observed in DNA isolated fromtivity to ionizing radiation.tumors of the breast, uterine cervix, and colon, per-

The region 11q22–q23 containing the AT locus ex-haps suggesting the location of a tumor suppressorhibits frequent loss of heterozygosity (LOH) in severalgene in 11q22–q24. For investigation of the role of

NPAT in AT and these tumors with allelic loss of types of tumors (Shows et al., 1996). Negrini et al.11q22–q24, appropriate primer sequences and PCR showed that at least two regions at 11q23 are com-conditions for amplification of all the NPAT exons monly deleted in breast carcinomas and that the ATMfrom genomic DNA were determined. We previously locus is contained in one of these regions flanked byreported that no recombinations are found among the markers D11S2000 and D11S897 (Negrini et al.,Atm, Npat, and Acat1 (acetoacetyl-CoA thiolase) loci 1995). Therefore, genes mapped to the commonly de-as determined by fine genetic linkage mapping of the leted region between D11S2000 and D11S897 shouldmouse AT region. The results of the LA-PCR analysis be examined in detail to determine whether theseusing NPAT- and ACAT-specific primers and human genes are involved in the development of tumors.genomic DNA allowed us to map ACAT 12 kb centro- We and other groups independently identified americ to NPAT. q 1997 Academic Press housekeeping gene, NPAT/E14/CAND3, which lies

0.5 kb from the 5* end of ATM and is transcribed inthe direction opposite to that of ATM and speculatedINTRODUCTIONthat each gene could influence the expression of theother (Byrd et al., 1996a; Imai et al., 1996; Chen etAtaxia telangiectasia (AT) is an autosomal recessiveal., 1997). Our nucleotide sequence analysis of NPATgene disorder characterized by a wide spectrum of de-revealed that the gene encodes a 1427-amino-acid pro-tein. This product contains possible nuclear localiza-Sequence data from this article have been deposited with the

DDBJ/EMBL/GenBank Data Libraries under Accession Nos. D89841 tion signals and phosphorylation target sites for sev-to D89854. eral types of protein kinase. To examine this gene fur-1 To whom correspondence should be addressed. Telephone: /81-

ther, we determined the structure and organization of43-251-2111 (Ext. 338). Fax: /81-43-251-9818. E-mail: imait@nirs.go.jp. NPAT. Furthermore, we showed that ACAT, which is

3880888-7543/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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HUMAN NPAT GENE 389

the gene responsible for 3-ketothiolase deficiency (Fu-kao et al., 1990; Kano et al., 1991) and mapped to11q22–q23 as determined by fluorescence in situ hy-bridization (Masuno et al., 1992), is located centromericto NPAT.

MATERIALS AND METHODS

The YACs DA2112B2, 890C12, and 961D6 and cosmid clones de-rived from these YACs were described previously (Imai et al., 1995,1996). DNA sequence analysis was carried out using an automaticsequencer (377; Applied Biosystems) according to the procedure rec-ommended by the manufacturer, and 3 to 5 mg of cosmid DNA and1 mg of plasmid DNA were utilized for the sequencing reactions.Template DNA was purified using Qiagen resin (Qiagen, Hilden) orSephaglas FP (Pharmacia Biotech, Uppsala) before the sequencing

FIG. 1. Structure of NPAT. (A) Gene map of regions flanking thereaction.ATM locus. Arrowheads show transcriptional direction. The struc-Almost all of the genomic region corresponding to NPAT excepttures of ATM and ACAT are described by Uziel et al. (1996) andthe center of intron 1 and a part of the 3 * untranslated region inKano et al. (1991), respectively. PCR primers used for amplificationexon 18 was amplified using the LA-Taq amplification kit (Takara,of the region between NPAT and ACAT were 5*-GACAATTCCATA-Kyoto, Japan), total human DNA and/or the YAC DNAs as templates,TTGTAAATACTGTG-3 * for NPAT and 5*-TGCCATGCTAATTCA-and 11 PCR primer sets, and then the 11 amplified products wereGAAGCTG-3 * for ACAT. // indicates a gap of sequence. (B) Cosmidsubcloned into the pCR2.1 vector (Invitrogen, San Diego). The PCRclones used for sequence analysis. (C) Exon and intron organizationand sequencing primers were designed on the basis of the nucleotideof NPAT. Boxes and vertical lines indicate exons. Horizontal linessequence of the NPAT cDNA and intronic sequences determined byrepresent introns. White box in exon 18 represents the 3* untrans-the first sequence analysis carried out. All exons of NPAT were se-lated region. CDK indicates sequences that may code for possiblequenced.sites for phosphorylation by the CDK/E2F complex, and NLS indi-The long and accurate (LA)-PCRs for amplification of exons (Tablecates sequences that may code for possible nuclear localization sig-2) were performed in a 25-ml volume, containing 11 LA-PCR buffernals.(Takara, Kyoto), 50–100 ng DNA, 0.2 mM each primer, 200 mM dNTP,

0.002 U of Perfect Match Polymerase Enhancer (Stratagene, LaJolla, CA), and 0.012 U of LA-Taq polymerase (Takara, Kyoto). Tem-

rized in Fig. 1 and Table 1. The coding sequence of theperature and time profile was 30 cycles of 947C for 15 s, 60/657C for1 min, and 727C for 2 min. For amplification of all of the introns and NPAT cDNA is composed of 18 exons, ranging in sizeexon 18, the following temperature and time profiles were used: 10 from 41 to 1653 bp, with the average size being 328 bp.cycles of 947C for 10 s, 607C for 1 min, 687C for 10 min, and an The first methionine codon of the open reading frame isadditional 18 cycles of 947C for 10 s, 607C for 1 min, 687C for 10 min.

located in exon 1, whereas the stop codon and theThe duration of the last extension step was increased by 20 s eachpoly(A) addition signal are located in the last exon,cycle. The PCR was carried out with a GeneAmp PCRsystem 9600

(Perkin–Elmer, Norwalk, CT). exon 18. In accordance with the general GT-AG rule,all of the introns begin with the dinucleotide GT andend with AG, except for a variant donor site with aRESULTSGC dinucleotide (Jackson, 1991) present at the start ofintron 17. The junctions at introns 2, 8, 10, 14, andWe had previously constructed the YAC and cosmid17 are type 0 (splicing occurring between codons), thecontigs of the AT region and identified the clones con-junctions at introns 1, 3, 5, 6, 11, 12, 13, and 15 aretaining ATM, NPAT, and ACAT (Imai et al., 1995,type 1 (splicing occurring after the first base of the1996). Almost all of the intron/exon boundaries ofcodon), and the junctions at introns 4, 7, 9, and 16 areNPAT were determined by sequencing of the cosmidtype 2 (splicing occurring after the second base of theclones directly. The nucleotide sequences encom-codon). For amplification of all 18 of the exons frompassing the intron/exon boundaries were also con-genomic DNA, primer sets specific for each intron werefirmed by sequencing of the PCR products that weredesigned and PCR amplification conditions that pro-amplified from human DNA or the YAC DNA as tem-mote amplification of only the desired region were de-plates. The lengths of the introns except intron 1 weretermined (Table 2).determined from the sizes of the LA-PCR products in-

The results of fine linkage mapping for mouse Atm,terposed between two consecutive exons. In the case ofNpat, and Acat1 showed that there was no recombina-the smallest introns, 9, 14, and 16, the length of thetion among these genes in a total of 150 backcross miceintron was determined based on analysis of the nucleo-(Matsuda et al., 1996). Based on previous mapping oftide sequences. The central parts of other introns wereATM, NPAT, and ACAT to the YACs (Imai et al., 1995)not sequenced. The exon sequences are identical toand cosmid contigs of 11q22–q23, we also found thatthose of the corresponding region of the cDNA. TheACAT maps close to the ATM locus. PCR performedstructural organization of NPAT is indicated in Fig. 1.under optimized amplification conditions with LA-TaqBased on these data, the exons of the NPAT cDNA werepolymerase and primers specific for both 5*- and 3 *-concluded to be spread over at least 44 kb.

The results of the sequencing analysis are summa- terminal regions of NPAT and ACAT showed that

AID GENO 4769 / 6r38$$$$61 06-06-97 13:55:52 gnma

IMAI ET AL.390

ACAT is located approximately 12 kb downstream ofNPAT and transcribed from centromere to telomere(Fig. 1A). As reported previously, ATM is also tran-scribed from centromere to telomere (Savitsky et al.,1995a) while NPAT is transcribed from telomere to cen-tromere (Imai et al., 1996).

DISCUSSION

NPAT was identified from the major AT locus usinga positional cloning approach, and it was found thatthis gene is located only 0.5 kb from the 5* end of ATM,the two genes being transcribed in opposite directions.Because both ATM and NPAT are housekeeping genes(Savitsky et al., 1995a; Imai et al., 1996; Byrd et al.,1996b), it was suggested that each gene could influencethe expression of the other (Imai et al., 1996; Byrdet al., 1996b). We previously reported that the NPATproduct contains five possible target sites for phosphor-ylation by cyclin-dependent protein kinases (CDK) as-sociated with E2F. The largest exon of NPAT, exon 13,encodes four of these possible phosphorylation sites.The other possible CDK–E2F target site and two ofthe three possible nuclear localization signals are en-coded by exon 17.

Recently, Byrd et al. reported on the exon/intron or-ganization of E14 (NPAT) and identified 16 exons (Byrdet al., 1996b). However, our sequence and PCR analy-ses using YAC and human DNA indicated that exons14 and 16 of E14 are interrupted by 75-bp- and 1.8-kb-long introns, respectively. Further PCR analysis ofDNAs from 10 Caucasian subjects and one Japanesesubject as templates also revealed the existence ofthese two introns in all of the DNAs examined (datanot shown). Therefore we suggest that NPAT consistsof 18 exons and 17 introns.

Mutations of human chromosome 11 have beenthought to be involved in various types of neoplasm.The results of LOH studies by many laboratories sug-gest that LOH of 11q22–q24 is frequently observed inDNA isolated from, for example, breast (Hampton etal., 1994a; Winqvist et al., 1995; Carter et al., 1994),cervical (Srivatsan et al., 1991; Hampton et al., 1994b;Bethwaite et al., 1995), ovarian (Foulkes et al., 1993),lung (Iizuka et al., 1995; Rasio et al., 1995a), and colo-rectal (Keldysh et al., 1993) carcinomas. In addition,allelic loss of this chromosomal region was observed inmelanomas (Tomlinson et al., 1993; Herbst et al., 1995)and paragangliomas (Devilee et al., 1994). Increasedpredisposition to cancer in AT patients and AT hetero-zygotes suggests that alteration of ATM may lead toaberrations of cells in these tissues (Rasio et al., 1995b).Because no somatic mutations of ATM were identifiedin DNA isolated from breast tumors (Vorechovsky etal., 1996), it is still unclear whether ATM is a suppres-sor oncogene or a modifier gene in these tumors. Therole of NPAT, which might influence or be affected byATM expression, should be investigated by analyzingthe structure of NPAT using DNA isolated from these

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6TAAATGCTTATATGTTTCAG

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AID GENO 4769 / 6r38$$$$61 06-06-97 13:55:52 gnma

HUMAN NPAT GENE 391

TABLE 2

Primer Sets to Amplify NPAT Exons

Approx AnnealExon Primer Primer sequence size (bp) temp (7C)

1 NS29 5*-CATGCGCATCCAGTATCACG-3* 620 60NS30 5*-CTCCGAATGACGAAGAATCAC-3*

2 NS73 5*-AGCAGTGTTGTGAGAACATAG-3* 660 65NS57 5*-GTCTTACCAGTCATGCATGAAT-3*

3 NS64 5*-TCGCTACTCTGCAGTCAGAT-3* 310 60NS59 5*-CTCATTACAACAGGGACGAG-3*

4 NS52 5*-CGCATTATAGCTTTGCAAGCT-3* 370 65NS48 5*-AGTACCAGCAACCTCTAGAG-3*

5 NS36 5*-CCTCATAACTAACTTGAGGTC-3* 230 60NS49 5*-TACTTAGCACATGTGTACAATG-3*

6 NS21 5*-GATGGTCTCGATCTCCTGAC-3* 720 60NS39 5*-CAAATCTAGAAGTCTGTTCCAC-3*

7 NS32 5*-TGGTAGGCACTCAGTACATG-3* 330 60NS62 5*-TAGATCAGAACATAATCTGGTC-3*

8 NS31 5*-GGACAACTGTATCTGTTCTGT-3* 460 60NS72 5*-GGTTGGTCTTGAATTCCTGAG-3*

9, 10 NS33 5*-AATTATGAAGTGTTGTATTCAGG-3* 560 60NS56 5*-ATAACATTTTATGTTGGCAATGG-3*

11 NS55 5*-CAGTGACTCTACCACTGTAC-3* 320 60NS61 5*-TAGATACTCTGGGGAATGTTG-3*

12 NS47 5*-TTTAAGAAACACCTGTCTAGTG-3* 420 60NS40 5*-ATAACAATCTGAACAACTTCTAC-3*

13 NS41 5*-AACACAGCGTATCAGACTC-3* 1870 60NS65 5*-TTCGCAGTCGATAACATCACA-3*

14, 15 NS63 5*-AGTGGATCGTAAGGAAGAGG-3* 560 60NS58 5*-CAAATGTAGGGTGATCACTTG-3*

16, 17 NS68 5*-CTGCTTTGTCAACGTGTTCTAT-3* 1560 65NS35 5*-GCTGTCTTAGCCATAACGTTC-3*

18 NS5a 5*-GAGGAATTAGATGAACGTGAGC-3* 2000 60SR2 5*-ACACCTACTGTCTTATGTGTC-3*

a The primer set NS5/SR2 amplifies intron 17 and only coding region of exon 18, not 3* untranslated region.

tions revealed by sequencing the 5* half of the gene for ataxiatumors. The genomic sequences of NPAT and genera-telangiectasia. Hum. Mol. Genet. 5: 145–149.tion of PCR primer sets to amplify all of the NPAT

Byrd, P. J., Cooper, P. R., Stankovic, T., Kullar, H. S., Watts, G. D. J.,exons using human genomic DNA rather than RNA,Robinson, P. J., and Taylor, A. M. R. (1996b). A gene transcribedwhich is generally difficult to handle, will facilitate from the bidirectional ATM promoter coding for a serine rich pro-

studies to define the function of NPAT. tein: Amino acid sequence, structure and expression studies. Hum.Mol. Genet. 5: 1785–1791.

Carter, S. L., Negrini, M., Baffa, R., Gillum, D. R., Resenberg, A. L.,ACKNOWLEDGMENTSSchwartz, G. F., and Croce, C. M. (1994). Loss of heterozygosity at11q22–q23 in breast cancer. Cancer Res. 54: 6270–6274.We acknowledge with thanks the excellent technical assistance of

Takako Maeda. We also thank Drs. Akira Nakagawara and Hideo Chen, X., Yang, L., Udar, N., Liang, T., Uhrhammer, N., Xu, S., Bay,Tsuji for providing us human DNA. This work was supported in part J.-O., Wang, Z., Dandakar, S., Chiplunkar, S., Klisak, I., Telatar,by grants from Grant-in-Aid for Scientific Research on Priority Areas M., Yang, H., Concannon, P., and Gatti, R. A. (1997). CAND3: Afrom the Ministry of Education, Science and Culture and in part by ubiquitously expressed gene immediately adjacent and in oppositea Research Grant on Aging and Health from the Ministry of Health transcriptional orientation to the ATM gene at 11q23.1. Mamm.and Welfare of Japan. Genome 8: 129–133.

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