VIROLOGY

185,

10-17

(1991)

Molecular Cloning and Characterization of a Novel Human Gene Homologous to the Murine Ecotropic Retroviral Receptor TAKAYUKI YOSHIMOTO,

ERIKO YOSHIMOTO,

AND DANIEL MERUELO’

Department of Pathology, New York University Medical Center, 550 First Avenue, New York, New York 10016 Received May 15, 199 1; accepted July 11I 199 1 A novel human cDNA homologous to the murine ecotropic retroviral receptor was cloned from a cDNA library derived from a human T-cell line. The human cDNA is highly homologous to the murine counterpart (87.6% amino acid identity), and its sequence predicts a protein with 629 amino acids (approximately 68 kDa), which is 7 amino acids more than the murine counterpart (622 amino acids). The predicted protein is highly hydrophobic and contains 14 potential transmembrane-spanning domains. No other gene and protein with significant homology to the cloned human gene and the predicted protein were identified by a computer-based search of sequence data banks other than the murine T-cell early activation gene (52.5% amino acid identity) and the murine ecotropic retroviral receptor gene. The human gene is ubiquitously expressed in human tissues and conserved among mammalian species. The genomic gene was also isolated from a cosmid library derived from human lymphocytes, and its organization was elucidated. The gene mapped to human chromosome 13. Q issi ACE&UII~~ PMS, IN.

INTRODUCTION

A-activated T lymphocytes, but whether or not TEA functions as a retroviral receptor remains unknown. In the present study, we molecularly cloned a novel human gene homologous to ERR and also TEA, and characterized the gene. Since we found the existence of a human gene homologous to the ERR cDNA by Southern and Northern blot analyses, we wished to identify the human gene and investigate its function. This gene might regulate retroviral infection and function as a receptor for human retrovirus(es), although its clear role in cells remains to be elucidated.

Viruses infect cells by first attaching to the cellular membrane. This requires specific interactions between molecules on the surface of the virus and receptor molecules on the susceptible cell. Retroviruses utilize diverse cell surface receptors, and human cells are susceptible to infection by a wide variety of retroviruses in vitro. For those retroviruses that can infect human cells, eight receptor interference groups utilizing the same receptor have been identified (Sommerfelt and Weiss, 1990) presumably indicating the existence of at least eight different membrane receptor proteins. To date, only the CD4 antigen receptor for human and simian immunodeficiency viruses (HIV and SIV) (Dalgleish et al., 1984; Sattentau et al., 1988) and the receptor for gibbon ape leukemia virus (GALV) (O’Hara er a/., 1990) have been identified and characterized. The receptors for human T-cell leukemia viruses types 1 and 2 (HTLV-1 and -2) and feline endogenous virus (RDl 14) have been localized to human chromosomes 17 and 19, respectively (Sommerfelt eT al., 1988, 1990). Recently, the murine ecotropic retroviral receptor (ERR) was molecularly cloned by Albritton et a/. (1989). Human cells transfected with ERR cDNA can acquire susceptibility to ecotropic murine leukemia virus (MuLV) infection. A murine gene (T-cell early activation gene, TEA) homologous to ERR was isolated by MacLeod et al. (1990). TEA is expressed on concanavalin’ To whom 0042.6822191

reprint

requests

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$3.00

Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form resewed.

MATERIALS

AND METHODS

Cells The human T-cell lines, H9, Jurkat, and SupTl, and the human B-cell line, Daudi, were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS). Human fibroblast, Hela, and mouse fibroblast, NIH 3T3, were maintained in Dulbecco modified Eagle medium supplemented with 10% FCS, and hamster fibroblast, CHO-Kl , was maintained in Ham’s F-l 0 medium supplemented with 10% FCS. Screening The human T-cell Hut 78 cDNA library (hgtl 1) and the human lymphocyte cosmid library (pWE15) obtained from Clontech (Palo Alto, CA) and Stratagene (La Jolla, CA), respectively, were screened as described by Maniatis et al. (1982). The BarnHI-EcoRI fragment, containing the entire open reading frame, of

be addressed. 10

HUMAN GENE HOMOLOGOUS

ERR cDNA (pJET) which was kindly provided by Drs. Albritton and Cunningham (Harvard Medical School, Boston, MA) was labeled with 32P by nick translation to a specific activity of approximately 2 X 1O* cpm/pg and used as a hybridization probe. DNA sequencing

analysis

cDNA clones from positive phages were recloned into the EcoRl site of plasmid vector pBluescript (Stratagene). Unidirectional deletions of the plasmids were constructed by using exonuclease III and Sl nuclease (Henikoff, 1984) and sequenced by the doublestranded dideoxy-sequencing method @anger et a/., 1977) with Sequenase reagents (United State Biochemical, Cleveland, OH). Restriction maps of positive cosmid inserts were determined using T3 or T7 promoterspecific oligonucleotides to probe partially digested cosmid DNA as described by Evans and Wahl(l987). EcoRI-EcoR1 or EcoRI-HindIll fragments in the cosmids were subcloned into pBluescript or pSport 1 (GIBCO BRL, Gaithersburg, MD). The exons and exonintron junctions were sequenced using synthetic oligonucleotides as primers. Sequences were compiled and analyzed using the Genetics computer group sequence analysis software package (Devereux et a/., 1984). Northern

blot analysis

Total cellular RNA was isolated from cell lines or noncancerous tissues resected with malignancy by the guanidinium isothiocyanate method (Chomczynski and Sacchi, 1987). The RNA sample was electrophoresed through a 1% agarose gel containing formaldehyde, transferred to Nytran filters (Schleicher & Schuell, Keene, NH), and subjected to hybridization as previously described (Amari and Meruelo, 1987). Southern

blot analysis

High molecular weight genomic DNA was prepared from cell lines by the method of Miller et al. (1988). Restriction-enzyme-digested DNA was separated by electrophoresis on 0.8% agarose gels, transferred to nitrocellulose (Schleicher & Schuell), and subjected to hybridization as previously described (Brown et al., 1988; Pampeno and Meruelo, 1986). Chromosomal

mapping

Southern biots of EcoRI-digested DNAs from a commercially available panel of 25 different human-hamster somatic cell hybrid cell lines plus human and hamster control DNA (BIOS, New Haven, CT) were used to

TO MURINE ERR

11

determine the chromosomal location (Kouri et al., 1989). Blots were hybridized with the cloned human cDNA fragment (bp 545-l 638) overnight at 65” in 2x SSPE, 5% Denhardt’s solution, 10% dextran sulfate, 1% SDS, and 0.5 mg/ml denatured salmon sperm DNA, and washed at a final stringency of 0.1 x SSC at 65”. The panels were blindly scored for the presence or absence of the human genes/or chromosomes, and the percentage discordancy was calculated for each of the human chromosomes including the X and Y chromosomes (O’Brien and Nash, 1982).

RESULTS Isolation and DNA sequence

of Ml 3 cD#A clones

Since Southern and Northern blot analyses showed the existence of a human gene homologous to the murine ecotropic retroviral receptor cDNA (data not shown), the human T-cell line (Hut 78) cDNA library was screened using the murine ERR cDNA as a probe. From 5 X 1O5 plaques of the library, 13 positive clones were isolated by cross-hybridization with the murine ERR cDNA. Two clones, designated H13.1-1 and H 13.3-2, containing longer inserts (2.4 and 3.5 kbp, respectively) among them were further anelyzed. Figure 1 shows the nucleotide sequence of the Maul-Pstl fragment of the cloned gene homutogous to the murine ERR cDNA, designated H13, together with the predicted amino acid sequence. The translation initiation site was assigned to base pair 148, since this methionine codon is surrounded by a nucleotide sequence which is reasonably similar to the consensus sequence for eukaryotic initiation (Kozak, 1986). This fragment contains a single long open reading frame of 1887 bp beginning at bp 148 and extending to bp 2034. The cDNA sequence predicts the protein containing 629 amino acids, which has a molecular mass of 67,638 Da. The coding capacity of this open reading frame was confirmed by inserting the HI 3 cDNA (Nrul-Pstl) into a transcription vector (pSP64[poly(A)], Promega, Madison, WI) and using the in V&O transcribed RNA to program a rabbit reticulocyte translation lysate. The H 13 RNA programmed the synthesis of an approximately 68-kDa protein (data not shown), which matches well with the calculated molecular mass as described above. The physical properties of the predicted protein were analyzed by using the computer programs (Devereux et a/., 1984). The predicted protein has four potential N-glycosyletion sites and contains no characteristic signal sequence in the putative N-terminal end. The H 13 protein is highly hydrophobic and contains 14 putative transmembrane-

YOSHIMOTO,

12

YOSHIMOTO,

AND MERUELO

1 CGATCCTGCCGGAGCCCCGCCGCCGCCGGCTTGGATTCTGAAACCTTCCT~GTATCECTCCTGAGACATCTTTGCTGCAAGATCGAGGCT 91 GTCCTCTGGTGAGAAGGTGGTGAGGCTTCCCGTCATATTCCAGCTCTGAACAGCAACATGGGGTGCAAAGTCCTGCTCAACATTGGGCAG MGCKVLLNIGQ 181 CAGATGCTGCGGCGGAAGGTGGTGGACTGTAGCCGGGAGGAGGAGACGCGGCTGTCTCGCTGCCTGAACACTTTTGATCTGGTGGCCCTCGGG QMLRRKVVDCSREETRLSRCLNTFDLVALG 271 GTGGGCAGCACACTGGGTGCTGGTGTCTACGTCCTGGCTGGAGCTGTGGCCCGTGAG~TGCAGGCCCTGCCATTGTCATCTCCTTCCTG YGSTI,GAGVYVLAG$VARENAGpAIVTSFI., 361 ATCGCTGCGCTGGCCTCAGTGCTGGCTGGCCTGTGCTATGGCGAGTTTGGTGCTCGGGTCCCCAAGACGGGCTCAGCTTACCTCTACAGC J A A LA S V LAG LC KG E F G ARV P KT G S AY 451 TATGTCACCGTTGGAGAGCTCTGGGCCTTCATCACCGGCTGG~CTT~TCCTCTCCTACATCATCGGTACTTC~GCGTAGCGAGGGCC Y V T VE F. I, W A F I T G WN L I L S Y I I G T $ S V 541 TGGAGCGCCACCTTCGACGAGCTGATAGGCAGACCCATCGGGGAGTTCTCACGGACACACATGACTCTGAACGCCCCCGCCCCCGGCGTGCTGGCT WSATFDELIGRP I GE F S RT HM T L N A P G 631 GAAAACCCCGACATATTCGCAGTGATCATAATTCTCATCTTGACAGGACTTTTAACTCTTGGTGTGAAAGAGTCGGCCATGGTCAACAAA EN P D 5 F A V 1 I I L I L T G L L T L G V K E SAMYN 721 ATATTCACTTGTATTAACGTCCTGGTCCTGGGCTTCATAATGGTGGTGTCAGGATTTGTGAAAGGATCGGTTAAA~CTGGCAGCTCACGGAG 1 F T C T NV I.!/ I, G F I M V S G EV KG S V K N W Q 811 GAGGATTTTGGGAACACATCAGGCCGTCTCTGTTTGAACAATGACACAAAGAAGGGAAGCCCGGTGTTGGTGGATTCATGCCCTTCGGG ED F G N T S G R LC LN N D T K E G KP GVGG F M 901 TTCTCTGGTGTCCTGTCGGGGGCAGCGACTTGCTTCTATGCCTTCGTGGGCTTTGACTGCATCGCCACCACAGGTGAAGAGGTG~GAAC F S G V T, S G A A T C F Y A F V G F Q C I AT T G E E 991 C~ACAG~GG~~AT~~C~GTGGGGAT~GTGG~GT~~~T~TTGAT~TG~TT~AT~G~~TA~TTTGGGGTGT~GG~TG~~~T~A~GCT~ATG P Q KA I PY G I VA S L L I C F I A Y F GV S A A LT 1081 ATGCCCTACTTCTGCCTGGACAATAACAGCCCCCTGCCCGACGCCTTTAAGCACGTGGGCTGGGAAGGTGCCAAGTACGCAGTGGCCGTG M P Y F C L D N N S P L P DA F K H V G W E G AK Y A 1171 GGCT~~CT~TG~G~T~TTT~~G~~AGT~TT~TAGGTT~~ATGTTT~~~ATG~~T~GGGTTATCTATG~CATGGCTGAGGATGGACTGCTA 5; S LC A L S AS L L G S MEP M P R V I Y A MA E D 1261 TTTAAATTCTTAGCCAACGTCAATGATAGGACCAAAACACC~TAATCGCCACATTAGCCTCGGGTGCCGTTGCTGCTGTGATGGCCTTC F KF LAN VN D RT KTP I'T A T L A S GA V A AV 1351 CTCTTTGACCTGAAGGACTTGGTGGACCTCATGTCCATTGGCACTCTCCTGGCTTACTCGTTGGTGGCTGCCTGTGTGTTGGTCTTACGG UKD LVD LM S I G T L LAY S L V A AC V L 1441 TACCAGCCAGAGCAGCCTAACCTGGTATACCAGATGGCCAGTACTTCCGACGAGTTAGATCCAGCAGACCAA~TGAATTGGCAAGCACC Y Q P E Q P N LV Y Q MA S T SD E L D P AD QN EL 1531 AATGATTCCCAGCTGGGGTTTTTACCAGAGGCAGAGATGTTCTCTTTGAAAACCATACTCTCACCCAAAAACATGGAGCCTTCCAAAATC ND S Q L G F L P EA EM F S LK T I L S P K N M E P 1621 TCTGGGCTAATTGTGAACATTTCAACCAGCCAGCCTTATAGCTGTTCTCATCATCACCTTCTGCATTGTGACCGTGCTTGGAAGGGAGGCT~TC s GL TV N T ST S L T A V I, T T T F C I V IV L G R 1711 ACCAAAGGGGCGCTGTGGGCAGTCTTTCTGCTCGCAGGGTCTGCCCTCCTCTGTGCCGTGGTCACGGGCGTCATCTGGAGGCAGCCCGAG T K GA L WA V F L L AG S A L L C A VV T G y I W R 1801 AGCAAGACCAAGCTCTCATTTAAGGTTCCCTTCCTGCCAGTGCTCCCCATCCTGAGCATCTTCGTGAACGTCTATCTCATGATGCAGCTG s K T K L S FKV P F LP V I, P T LS 1 F VN V Y LMMQ 1891 GACCAGGGCACCTGGGTCCGGTTTGCTGTGTGGATGCTGATAGGCTTCATCATCTACTTTGGCTATGGCCTGTGGCACAGCGAGGAGGCG D Q G T!d V R F A V W M L I G F I I Y F G Y G L w H S 1981 TCCCTGGATGCCGACCAAGCAAGGACTCCTGACGGCAACTTGGACCAGTGCAAGTGACGCACAGCCCCGCCCCCCGGAGGTGGCAGCAGC SLDADQARTPDGNLDQCK' 2071 C~~GAGGGA~G~~~~~AGAGGA~~GGGAGG~A~~~~AC~~T~~~~A~~AGTGC~CAGAAACCACCTGCGTCCACACCCTCACTGCA

11 41 71 LY

S

A

R

V

LA161

101

A131

K191 L

E 221

T

Pm2251 V

K

N

281

LM3ll V

A

V

341

G

L

L

371

MA V

E401 L

R

431

AS

T

461

SK

1491

E

A

L

521

Q

P

E

551

~581 E

E

A

611 629

ho. 1. Nucleotide and deduced amino acid sequences of H13 cDNA. The amino acid sequence is numbered from the presumed initiator methionine, and the termination codon is marked with an asterisk. Potential transmembrane-spanning domains identified by the program of Kyte and Doolittle (1982) are underlined. Potential N-glycosylation sites (NXSIT) occur at amino acids 226, 235, 462, and 497. The EMBUGenBank accession number for this sequence is X591 55.

spanning Fig. 1.

domains

as represented

by underlines

in

Homology of H13 cDNA and amino acid sequences to the murine ERR and TEA Figure 2 shows the comparison of the predicted amino acid sequences between H13 and the murine ERR. The H13 sequences are quite similar to the murine ERR sequences with 87.6% identity at the amino acid level. The total number of amino acids in the H13 predicted protein (629 amino acids) is 7 amino acids more than that of the murine ERR predicted protein

(622 amino acids). These additional amino acids are positioned at amino acids residues 226-238 in the H 13 amino acid sequence by the alignment analysis: 6 amino acids out of them are located at amino acid residues 226-231, and 1 amino acid is located at amino acid residue 238. Recently, the murine gene homologous to the ERR cDNA (T-cell early activation gene, TEA, 453 amino acids), which is expressed on concanavalin-A-activated T lymphocytes, has been identified (Macleod et al., 1990). The comparison between the H13 and the TEA amino acid sequences is also shown in Fig. 2. The H 13 amino acid sequence is similar to the TEA amino

HUMAN GENE HOMOLOGOUSTO MURINE ERR

13

ERR1 MGCKNLLGLGQaMLRRKWDCSREESRLSRCLNTYDLVA;G 70 III1 II.:IIIIIIIIIIIIIII1~11111111:11111111IIIIII1IIIIllllllllIIIIIIII H13

1 MGCKVLLNIGQQMLRRKWDCSREETRLSRCLNTFDLVALGVGSTLGAGV~LAGAV~ENAGPAIVISF 70

IIIIIIIIIIIIIIIIIIIIllllllllllllllllllIllilllllllllllllllIIIIIIIIIIIIl H13

71

ERR

141

LIAALASVLAGLCYGEFGARVPKTGSAYLYSYVTVGELWALILSYIIGTSSVARAWSATFDELI

140

GKPIGEFSRQHMALNAPGVLAQTPDIFAVIIIIILTGLLTLGVKESAMVNKIFTCINVLVLCFIWsGFV

210

l:IIIlIII H13

141

II.IIIIIIII:.

IIIIIIIII:IIIIIIIIillllllllllllllllIII.lI:IIIIl

GRPIGEFSRTHMTLNAPGVLAENPDIFAVIIILILTGLLTLGVKESAMVNKIFTCINVLVLGFIMVSGFV

210

II.II/ TEA ERR

1 211

MVAGFV ..CNNNDT.NVKY.GEGGFMPFGFSGVLSGAATCFYAFVGFDCIATTGEEV

KGSIKNWQLTEKNFS....

III:IIIIIlI.:I: H13

211

ERR

:.I

I 273

Il.:.ll

: 1.1:.

......

280

279

I.IIIII:II.I.I.IIIIIIIIIIIIIIIIIIIIII

KGNVANWKISEEFLKNISASAREPPSENGTSIYGAGGFMPYGFTGTLAGAATCFYAFVGFDCIATTGEEV

76

KNPQKAIPV;;IVASLLICF;AYFGVSAALTLMMPYFC113;DSPLPGAFKHQGWEEAKyAVAIGSLCALST

IIIIlIIIIIIIlIIIIIIlllllllllllllllllllI H13

272

I 11111111111111111111111111111111111

KGSVKNWQLTEEDFGNTSGRLCLNNDTKEGKP.GVGGFMPFGFSGVLSGAATCFYAFVGFDCIATTGEEV

II.1 TEA

I IIII

6

342

:lIIl:IlII

IIl:IIIIll:llllill.

KNPQKAIPVGIVASLLICFIAYFGVSAALTLMMPYFCLDNNSPLPDAFKHVGWEGAKYAVAVGSLCALSA

:IIIIIII:III.III:II:lllllllllllllll:

II:.IIII

349

II..1

I:.lll.ll.lllllll.

TEA

77

RNPQKAIPIGIVTSLLVCFMAYFGVSAALTLMMPYYLLDEKSPLPVAFEWRWGPAKYWAAGSLCALST

i46

ERR

343

SLLGSMFPMPRVIYAMAEDGLLFKFLAKINNRTKTPVIATVTSGAIAAVMAFLFELKDLVDLMSIGTLLA

412

IIIIIllllllllllllllIIIIIIII.:I:IIIII:IIl:. H13

350

III:IIIIIIII:IIIIIIIIIIIIIII

SLLGSMFPMPRVIYAMAEDGLLFKFLANVNDRTKTPIIATLASGAVAAVMAFLFDLKDLVDLMSIGTLLA

IIIII:ll1lIIIlIIIIIIIIII TEA

147

SLLGSIFPMPRVIYAMAEDGLLFKCLAQINSKTKTPVIATLSSGAVAAVMAFLFDLKALVDMMSIGTLMA

ERR

413

YSLVAACVLVLRYQPEQPNLVYQMARTTEELDRVDONEL;

420

IIII

.

.(:......I

I

:[I:.:1

“:

TEA

217

YSLVAACVLILRYQPGLCYEQP~Y~~~~~TLESCTNATLKS..ESQVTML.QGQGFSLRTLFSPSAL.PT

ERR

483

KFSGLIVNISAGLLAALIITVCIVAVLGREALAEGTLWAVFVMTGSVLLCMLVTGIIWRQPESKTKLSFK

l:IIIIIlII 490

ll.llll.l1llll:III

YSLVAACVLVLRYQPEQPNLVYQMASTSDELDPADQNELDP~QNELAST~SQLGFLPEAE~SLKTILSPK~PS

IIIIIIIIl:IIlII:

H13

216 402

IllllllllllllllllllIIIIII.I.:IIl..IIIII.I..:II H13

419

II.:I.:IIII:IIII.IIIIIIIIIIlIIII.III:IIIIII:I

282 552

.:I:I.IIII.III.IIIIIII..I.lIIII::.ll.llI

:lII:lIIIIIIIllllII

I.:

-::I.II:.::I:I..I

:I:

.

I.:

III

559

:IIl.I

TEA

283

~QSRS~VSFLVGFLAFLILGLSILTTYGVQAIARLEAWSLALLALFLVLCAAV;LTIWRQPQNQQKVAFM

ERR

563

VPFVPVLPVLSIFVNIY~QLDQGTWVI;FAVWMLIGFTIYFGYGIWHSEEASL..AAGQAKTPDSNLDQ

IIIII:

I:

620

.I:II:III:I/Il

VPFLPVLPILSIFVNVY~QLDQGTWVRFAVWMLIGFIIYFGYGLWHSEEASL..DADQARTPDGNLDQ

IIIII.II

I 352

III:IIII:IIIIII:IIIIIIIIIIIIIlllllllll.IIIIII:lIIIIIII 560

I.

KISGLIVNISTSLIAVLIITFCIVTVLGREALTKGALWA~L~GSALLCAWTGVI~QPESKTKLSFK

I

H13

489

III:I::II..:

:II:II:lIl:Il..:ll:lI.:ll

:ll:Ill:ll::lI

621

I:.

TEA

353

VPFLPFLPAFSILVNIYLMVQLSADTWIRFSIWMALGFLIYFAYGIRHSLEGNPRDEEDDEDDEDAFSENINV

ERR

621

CK

:.I:.

. .:I:: 422

622

II H13

628

CK

629

TEA

423

ATEEKS~MQANDHHQRNLSLPFILNEKTSEC

453

Ra. 2. Alignment of H 13, ERR, and TEA deduced proteins sequences. The entire amino acid sequences of H t 3, ERR, and TEA deduced proteins are shown on the middle, top, and bottom lines, respectively. The comparison was made by using the Genetics computer group sequence analysis software package (Devereux et a/., 1984) with gaps generated to allow alignment of the highly similar a@~erfr~es Consezvative amino acid differences are indicated by one dot and two dots, and amino acid identities are shown by long dashes between the rows (Doolittle, 1986; Needleman and Wunsch, 1970).

acid sequence with 52.5% identity, although the TEA amino acid sequence is shorter at the N-terminal end, but longer at the C-terminal end. Homology searches with Get-Bank, NBRF, and Swiss data bases revealed no significant similarity between the H13 cDNA or amino acid sequence and other DNA or amino acid sequences previously re-

ported, except for the ERR and TEA. Althou@h the H 13 protein has structural similarity to proteins with multiple transmembrane-spanning domaihs such as ion channels, ion pumps, sugar transport proWns+ P-gfycoprotein, and the gibbon ape leukemia virus receptor, there is no significant sequence similsrity between them either.

14

YOSHIMOTO,YOSHIMOTO,AND MERUELO

H13.7A E 1 ’

H

H

E HBBE

E BHBHBE I ,I II

I,

E I

,

FIG. 3. Genomic organization of the H 13 gene. Two positive cosmid clones (H13.9-1A and H13.7A) are shown on the top, together with their restriction maps with EcoRl (E). BamHl (B), and HindIll (H). The structure of the H13 gene is illustrated schematically with solid (translated regions) and open (untranslated regions) bars, and solid lines (introns) on the middle. The regions which are not determined are drawn with dot lines and boxes surrounded by dot lines. A detailed restriction map of the H13 gene is shown on the bottom. A. Avall; Ap, ApaLl; Bs, BsfXI; Hp. k/pal; K, Kpnl; N, Ncol; and P, Psfl (these do not cover all of the sites present in the gene).

H13 mRNA expression

in various cells

To examine the expression level of the H13 mRNA, Northern blot analyses were carried out using the H 13 cDNA (Nrul-Pstl) as a probe. The H13 cDNA probe detected an approximately 9-kb transcript in human cell lines including T-cell (H9, Jurkat, and SupTl), B-cell (Daudi), and fibroblast (Hela) (data not shown). A similar transcript was easily detected in the hamster cell line, CHO-Kl , and less well in the mouse cell line, NIH 3T3, by cross-hybridization, indicating again that the H13 gene appears conserved in mammalian species. The tissue distribution of the H 13 mRNA was determined by Northern blot analysis using total RNA prepared from various human normal tissues. All tissues examined, including brain, spleen, thymus, kidney, colon, and stomach, are expressing detectable H13 mRNA (data not shown).

and HindIll, were constructed as shown in the top of Fig. 3. To confirm that these clones contain the whole H 13 genomic gene and that there is no rearrangement of the H13 gene during cloning and propagation of these clones, we analyzed restricted genomic DNA by blot hybridization. When genomic DNAs prepared from human cell lines (H9, Jurkat, SupTl, and Hela) were digested with EcoRI, the fragments detected by the H 13 cDNA probe were identical with those seen in the DNA of the H 13.9-l A clone, but not H 13.7A. The 5 end of the H13.7A clone seems to be truncated and rearranged. The sizes of these hybridizing fragments correspond exactly to those expected from the restriction map with EcoRl (Fig. 3). These results indicate that the cosmid clone, H13.9-l A, properly reflects the genomic organization of the human H13 gene. Organization

Isolation of H13 cosmid clones A human lymphocyte cosmid library was screened by using the murine ERR cDNA as a hybridization probe. After screening 5 X 1 O5 recombinant colonies, eight positive clones were isolated. Restriction enzyme digestion analysis with EcoRl revealed that these independent clones actually represented two different groups. The clone, designated H13.9-l A, from one group containing six of the same clones and the clone, designated H 13.7A, from another group containing two of the same clones were further analyzed. From the results of single and double digestions of cosmid DNAs and hybridization using T3 or T7 oligonucleotides to partially digested cosmid DNAs, restriction maps of these clones for the enzymes, EcoRI, BarnHI,

of the H13 gene

To determine the exon-intron junctions, sequence analyses of the H 13.9-l A cosmid and its subclones were performed using synthetic oligonucleotides. Figure 4 shows the exon-intron boundaries sequence of the open reading frame of the H 13 gene. Central parts of the introns were not sequenced. The open reading frame consists of 11 exons and 10 introns. The exon sequences are identical with those of the corresponding region of the cDNA. In accordance with the general GT-AG rule, all of the introns begin with the dinucleotide GT and end with AG. More extensive consensus sequences have been proposed for the RNA splice sites by analyzing a great number of eukaryotic genes that are transcribed by RNA polymerase II (Mount, 1982). The donor [(C or A)-A-G/G-T-(A or G)-A-G-T]

HUMAN GENE HOMOLOGOUS

TO MURINE ERR

15

MetGlyCys (384 bp) IleIleG . . . . . . . . . . TCAGGAAACATCTTTGT'PCTTTTTCAACAG CTCTGAACAGCAACATGGGGTGC..........ATCATCG

GTAGGTCCCAGGGGTCCGGAGCTCGTGCCC........

..ATCTCCTCTGTTGTGTGTGCTTTGTGGCAG ..ACAAAGTTTTGTGTCTATGCACTTTTTCAG

TAAAGCCCACACTTAAGATCAGGT........

1yLeuLeu (175 bp) GACTTTTA..........TT

LeuAsnAs

TGAGTTGCTGCTTCTGACCATTTTTCTTA..........

nAspThr (122 bp) ThrThrG GTGACATTTGTTTGCGGCTCTTTTTTCTAG TGACACA..........ACCACAG

GTA

TGTCCGCCAGCCACTGCGGTCCCGGGT..........

1yGluGlu (223 bp) SerAlaSe CCCTCCTCCGCTGACCCCTTGTGCTTCTAG GTGAAGAG..........TCCGCCAG

GTG

AGAGGCCTTTCTGTGGTGGCTGCTCCT..........

rLeuLeu (140 bp) ValAlaA CACTCACGCGGTTTGCCTCTCCCCATCTAG TCTTCTA..........GTTGCTG

GTGAG

1aValMet (103 bp) ValLeuAr CCACAGATCCTGGTGTCTGTCTCTTCCCAG CTGTGATG..........GTCTTACG

GTATG

TACTAGAACTCATTTCACATGGAAA.......... TATGGCTTTTGGGGGTCTCATGACA........

gTyrGln (218 bp) LeuILe ..GTGGCGGTCACGCAATGCCTTTCTTCCCAG GTACCAG..........CTTAT

CTAGCCCTTCCCTGGGGCCTTGC..........

1aValLeu (167 bp) SerPheLys GCAGCTTGCTGTCTTTTCCTTCCCCCTTAG CTGTTCTC..........TCATTTAAG

GTGAGC

AGCTCGGCCTAGGGAAGGAACCCT........

ValProPhe (109 bp) LeuIleG ..ATGGATTGAGGCCACCTCCTGTCTCTTCAG GTTCCCTTC..........CTGATAG

GTATGT

GATGCTGCTGCCAGCACGTCCAAG.......... FIG. 4. Exon-intron each exon contains

boundaries

sequence

GlnCysLys 1yPheIle TTCACGCCCGTCTCTGCTGTCGGCCCGCAGGCTTCATC..........CAGTGCAAGTGA... of the H 13 gene.

The exons

and the acceptor [(T or C),,-N-(C or T)-A-G/G] consensus sequences are well matched by the exon-intron junctions of the H 13 gene. Since the H 13 cDNA has long untranslated regions (approximately 7 kb), we did not obtain the entire H 13 cDNA equivalent to the H13 mRNA with approximately 9 kb transcript in length. Finally, detailed restriction maps of subclones of the H13.9-1A cosmid were constructed and the precise positions of each exon were assigned as shown in the middle and bottom, respectively, of Fig. 3. Chromosome

location

To identify the chromosome location of the H13 gene, Southern blots of human-hamster somatic cell hybrid DNAs which retain a limited number of different human chromosomes were utilized. Hybrids with the positive hybridization to the H 13 cDNA probe (bp 5451638) were scored, and the percentage discordancy was calculated as shown in Table 1. The concordance was obtained between the presence of the H 13 cDNA sequence and human chromosome 13.

are boxed,

and the numbers

in the box indicate

how

many

* nucleotides

DISCUSSION Retroviruses interact with specific cellular receptors as a first step in the process of infection, and human cells have at least eight different cellular receptors for retroviruses on their surfaces (Sommerfe~t and Weiss, 1990). To date, only the CD4 antigen receptor for human and simian immunodeficiency viruses (Dalgleish et a/., 1984; Sattentau et a/., 1988) and the receptor for gibbon ape leukemia virus (O’Hara et a/., 1990) have been identified and characterized as human retroviral receptors, and the chromosome locations of human T-cell leukemia viruses types 1 and 2 (Sommerfelt et al., 1988) and feline endogenous virus (RDll4) (Sommerfelt et al., 1990) have been assigned to human chromosomes 17 and 19, respectively. The direct comparisons between the H 13 protein and these receptors revealed neither homology in the sequences nor coincidence in the chromosome locations. As human retroviruses, only three retroviruses, HIV and HTLV-1 and -2, have been so far identified and characterized well. The H 13 protein might be a receptor for unknown retroviruses which will be identified in future. Alternatively, the H 13 protein might function to

16

YOSHIMOTO,

YOSHIMOTO.

TABLE1

MAPPINGOFTHE H 13 GENETO HUMANCHROMOSOME13 Number of hybrids (DNA hybridization per chromosome) Chromosome 1 2 3 4 5 6 7 a

9 10 11 12 13

+l+a 2 0 0

1 6

1 2

1 1 0 0 1

14 15 16 17

6 3 2 0 0

ia

1

19

5

20

1

21

3

22

1 1 1

X Y

-Iia ia 15 ia 3 16

19 15 17 16 16 16

19 15 17 16 17 16 17 17 15 16 17 16

+/-

-1-k

4 6 6 5 0 5 4 5 5 6 6 5 0 3 4 6 6 5 2 5 3 5 5 5

1 1 4

1 16 3 0 4 2 3 3 3 0 4 2 3 2 3 1 2 4 3 2 3

Discordancp (%I 20.0 28.0 40.0 24.0 64.0 32.0 16.0 36.0 28.0 36.0 36.0 32.0 0.0 28.0 24.0 36.0 32.0 32.0 12.0 28.0 28.0 32.0 28.0 32.0

a Symbols at the left of the slash indicate the presence (+) or absence (-) of the human H13 restriction fragment as related to (at the right of the slash) the presence (+) or absence (-) of the particular human chromosome indicated, as detected by hybridization with the H13 cDNA probe (bp 545-l 636). b Derived from the sum of the +/- and -/+ observations.

help the infection by viruses in collaboration with other cell surface molecules. Indeed, a number of observations suggest the possibility of the presence of other cell surface molecules, in addition to CD4, which are required for internalization of HIV or development of HIV-induced cytopathic effects such as cell-cell fusion (syncytium formation), although CD4 is a receptor for HIV through interaction with the HIV envelope glycoprotein 120. First, murine cells expressing human CD4 molecules do not internalize HIV following binding to CD4 nor are they capable of cell-cell fusion (Maddon et al., 1986). This may be due to the limited expression of an additional cell surface structure required for virus internalization or their unavailability on the cell surface. Second, fusion of HIV-infected cells to uninfected cells does not correlate with CD4 density on the surface of the uninfected cells (Somasundaran and Robinson, 1987; Kikukawa et a/., 1986). Last, sera from acquired immunodeficiency syndrome patients that are capable of blocking

AND MERUELO

the interaction of HIV with CD4 do not necessarily block fusion and vice versa (Lifson et al., 1986). The sequence of the H 13 gene has been highly conserved across species boundaries, and this mRNA seems to be expressed ubiquitously in various human tissues. This implies a high level of conservation of the normal function which might be indispensable for cell growth. To attempt to identify that function, the computer searches of GenBank, NBRF, and Swiss data bases were made but revealed no significant homologies. Although the lack of any homology of the H 13 protein with known proteins, except ERR and TEA, makes it difficult to speculate on the role of the H13 protein, the cloning and characterization of the H13 gene should permit examination of its possible function as a human viral receptor and elucidation of the normal function of the H13 protein. ACKNOWLEDGMENTS This workwas supported by NIH Grants CA31 346, CA22247, and CA35482 to D. M. The authors thank Drs. Angel Pellicer and Peter D’Eustachio (New York University Medical Center, New York, NY) for critical comments on the manuscript, and Dr. Jone Hill (New York University Medical Center) for helpful comments on the sequence analyses.

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