h111111lllO-

Immunogenetics 36: 175-181, 1992

genetics

© Springer-Verlag 1992

Genomic organization and chromosomal location of the human gene encoding the B-lymphocyte activation antigen B7 Annamalai Selvakumar 1, Bhaskara K. Mohanraj 1, Roger L. Eddy z, Thomas B. Shows z, Perrin C. White 3, and Bo Dupont ~ 1 Human Immunogenetics Laboratory, Sloan-Kettering Institute for Cancer Research, New York, NY 10021, USA 2 Department of Human Genetics, Rosewell Park Memorial Institute, Buffalo, New York, NY 14263, USA 3 Division of Pediatric Endocrinology, Cornell University Medical College, New York, NY 10021, USA Received November 7, 1991

Abstract. The human B lymphocyte activation antigen B7 provides regulatory signals for T lymphocytes as a consequence of binding to its ligands CD28 and CTLA-4. The cDNA for B7 has previously been isolated and predicted to encode a type I membrane protein. The predicted polypeptide has a secretory signal peptide followed by two contiguous Ig-like domains, a hydrophobic transmembrane region and a short cytoplasmic tail. Here we report the exon-intron genomic organization of human B7 and the chromosomal location. The gene has six exons that span approximately 32 kilobases of DNA. Exon 1 is not translated and the second exon contains the initiation ATG codon and encodes a predicted signal peptide. This gene structure is characteristic for several eukaryotic genes with tissue-specific expression. The third and fourth exons correspond to two Ig-like domains whereas the fifth and sixth exons encode respectively the trans-membrane portion and the cytoplasmic tail. This close relationship between exons and functional domains is a characteristic feature of genes of the Ig superfamily. Cell surface expression of the B7 gene product has previously been mapped to human chromosome 12 by antibody reactivity with the B7-specific monoclonal antibody BB-1. We here demonstrate that the B7 gene is located to the q21-qter region of chromosome 3 by DNA blot analysis of human x rodent somatic cell hybrids.

Introduction T-cell receptor (Tcr) occupancy by antigen major histocompatibility complex (MHC) is the first and manThe nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M83071-M83075, M83077. Address correspondence and offprint requests to: B. Dupont, Sloan-Kettering Institute for Cancer Research, 1275 York Avenue (Room $709), New York, NY 10021, USA.

datory signal for antigen-specific activation of T-lymphocytes. Several co-stimulatory signals are, however, required for induction of interleukin 2 (IL-2) production and clonal T-cell expansion (Mueller et al. 1989). One of these co-stimulatory signals is provided by receptor ligand interaction between the Tcr CD28 and the B-lymphocyte restricted activation antigen B7 (Linsley et al. 1990; Linsley et al. 1991a; Koulova et al. 1991; Gimmi et al. 1991). Recently it has been shown that B7 also binds to the receptor CTLA-4 (Linsley et al. 1991b) which is expressed in cytotoxic T cells (Dariavach et al. 1988). CTLA-4 and CD28 are genetically closely linked and are structurally similar (Lafage-Pochitaloff et al. 1990; Harper et al. 1991). B7 is a cell surface molecule expressed on activated B-lymphocytes, some B-cell lymphomas, Epstein-Barr virus (EBV) transformed Blymphoblasts (Yokochi et al. 1982; Freedman et al. 1987) and interferon 3' (INF-3,) treated monocytes (Feeman et al. 1990). cDNA clones encoding human (Freeman et al. 1989) and mouse (Freeman et al. 1991) B7 have been isolated. The mature human B7 predicted from the cDNA sequence is a type I membrane protein (i. e., it is synthesized with a signal peptide that is cleaved upon translocation across the endoplasmic reticulum) with 262 amino acids. The protein is predicted to contain two extracellular domains structurally similar to those of Ig, a hydrophobic transmembrane region, and a short cytoplasmic domain. It has eight potential glycocylation sites (Freeman et al. 1989). Human and mouse B7 protein have 44% amino acid identity (Freeman et al. 1991). The B7 molecule is detected by two mouse monoclonal antibodies (mAbs) B7 (Freedman et al. 1987) and BB-1 (Yokochi et al. 1982; Clark et al. 1986) which both immunoprecipitate a M r 44-54 000 molecular complex from B7 transfected COS cells (Freeman et al. 1989). The B7 mAb, however, immunoprecipates a M r 60 000 molecule from B-lymphoblasts (Freedman et al. 1987) whereas BB-1 detects a Mr 37 000 molecule in western blots of B-cells

176

A. Selvakumar et al. : Genomic organization of human B7

Human B7probe isolation. Oligonucleotides 311-350 (5') and 750-794 (3') were synthesized based on the published human B7 cDNA sequence (Freeman et al. 1989) and end-labeled. These oligonucleotides were used to screen a Daudi (B-lymphoblast) library kindly provided by J.P. DiSanto (Memorial Sloan-kettering Cancer Center, NY). Screening and hybridization was performed by standard procedures (Sambrook et al. 1989). Five positive clones were identified in the primary screening. The 1.5 kilobase (kb) insert of one clone was subcloned into pBluescript II KS (Stratagene, La Jolla, CA). Denatured plasmid DNA was sequenced with oligonucleotide primers (selected at 150 base pair (bp) intervals from the published B7 cDNA sequence) by the chain termination method (Tabor and Richardson 1987) using T7 DNA polymerase (US Biochemicals, Cleveland, OH). The sequence of the clone was identical to the published sequence (Freeman et al. 1989).

(Clark et al. 1986). These differences in biochemical characterization of the molecular species identified by the two B7-specific mAbs are currently unexplained. The gene for B7 has previously been mapped to human chromosome 12 using antibody reactivity with BB-1 tested on human-mouse somatic cell hybrids (Katz et al. 1985). Here we report that B7 is encoded by a single copy gene which has a genomic organization characteristic of genes of the Ig superfamily. We also demonstrate by DNA blot analysis of human x rodent somatic cell hybrids that this gene is located on human chromosome 3.

Genomic library screening and characterization of clones. A human genomic library in the Lambda Dash bacteriophage vector (Stratagene, La Jolla, CA) was screened (Sambrook et al. 1989) with a full-lenght human B7 cDNA probe that was labeled with 32p using a random priming kit (Boehringer Mannheim Biochemicals, Indianapolis, IN; Feinberg and Vogelstein 1984). The final washes of the filters were in 0.1 x Standard sodium citrate (SSC) and 0.1% sodium dodecyl sulfate (SDS) at 55 °C. Ten positive clones were selected and plaque purified. Restriction digests of these clones with Eco RI and subsequent hybridizations

Materials and methods Oligonucleotide synthesis and labeling. Oligonucleotides were synthesized using a 381A DNA synthesizer (Applied Biosystems, Foster City, CA) and end-labeled with 32p (NEN, Boston, MA) using T4 polynucleotide kinase (New England Biolabs, Beverly, MA) essentially as described (Sambrook et al. 1989),

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Fig. 1. Exon-intron organization of the human BTgene. The BTgene has six axons shown in soEd boxes; introns are shown as a sofid line. Recognition sites in genontic clones (X3.5, X3.8, ~3.4, and X5.4) for Eco RI, Sac I, and Xba I are shown below. All exons and intron-exon boundaries were sequenced on both strands, 4.2 kb, 4.1 kb, 2.6 kb, 2.3 kb. Unordered Eco RI and Xba I fragments are shown in parentheses. The dotted line in clone 3.8 indicates an extraneous segment that is not present in B7 gene.

Exon 1

•

~08 AAGTGATTTGT~ATTGCTTTATAGACTGTAAGAAGAGAA~ATCT~AGAAGTGGAGTCTTACCCTGAAATCAAAGGATTTAAAGAA/ULAGTGGAATTTTTCTTCAGCAA ~16 ccctgtcc~ctcct~gc~ctg~c~agtactg~gt~actcaaaccctctgta~g~a~caga~gttaga~ggggaaatgtcgcctctctg~ga~t~/U~AGA/~

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587

Exon 2 GcTGTGAAAcTAAATCCACAA~TTTGGAGA~CCAGGAACAC~CTC~AATCT~TGTGTGTTTT~TAAACAT~A~TGGAGGGT~TT~TACGTGAGCAATTGGATTGT~A 495 TCAGC~CTGCCTGTTTTGCACCTGGGAAGTGCCCTGGT~TTACTT~GGTCCAAATTGTTGGCTTTCACTTTTGACCCTAAGCATCTGAAG~C ATG GGC CAC ACA Her Gty His Thr

599

+1

CGG AGG CAG GGA ACA TCA CCA TCC /LAG TGT CCA TAC CTC AAT TTC TTT CAG CTC TTG GTG CTG GCT GGT CTT TCT CAC TTC A r g A r g Gln GLy Thr Ser Pro Set Lys Cys Pro Tyr Leu Asn Phe Pha Gln Leu Leu Vat Leu Ata Gly Leu Set His Phe

680 (5)

TGT TCA G Cys Ser G

6~T (7)

~gcB~ctttcaggncttaactgagt~ctg~t~cegg..~|ntr~n.B(-12~5Kb)°~gacacaacatt~taagttctgttactgccgacttt~gattcctgm ccatcgaggeatctgtatttaateaetetecaatcttgttctgtttgcctctccatsg

795 853

A. Selvakumar et el. : Genomie organization of human B7

177

Exon 3 GT GTT ATC CAC GTG ACC AAG GAA GTG AAA GAP.GTG GCA ACG CTG TCC TGT GGT CAC AAT GTT TCT GTT GAA GAG CTG GCA CAA t y V e t I t e His Vet Thr Lys GtuVet Lys GluVat ALe Thr Leu Set Cys GLy His ^snVat Set Vet GLu GLu LeuAla Gin

936 (35)

ACT CGC ATC TAC TGG CAA AAG GAG A.a,GAk.AATG GTG CTG ACT ATG ATG TCT GGGGAC ATG AAT ATA TGG CCC GAG TAC AAG AAC Thr Arg Ire Tyr Trp Gln Lys Gtu Lys Lys Her Vet Leu Thr Her Her Set GLy Asp Hat Asn l i e Trp Pro GLu Tyr Lye Asn

1020 (63)

CGG ACC ATC TTT GAT ATC ACT AAT AAC CTC TCC ATT GTG ATC CTG GCT CTG CGC CCA TCT GAC GAG GGC ACA TAC GAG TGT GTT Arg Thr Ire Phe Asp l l e Thr Asn Asn Leu Set I l e Vet I t e Leu ALe Leu Arg Pro sap Asp Gtu GLy Thr Tyr Gtu Cys Vet

1104 (91)

GTT CTG AAG TAT GAA ~ GAC GCT TTC AAG CGG GAA CAC CTG GCT GA.AGTG ACG TTA TCA GTC AAA G Vet Leu Lys Tyr Gtu Lye Asp ALe Phe Lye ^rg Gtu H|s Leu Ate Gtu Vet Thr Leu Set Vet Lye A

1171

gttg~tgggattttctg~tttt~ctcte~gccggcag~tt..'Intr~n.~(-6.6Kb)...ccttt~tc~ttcttatt~ct~g~ttattteee~t~e~c

1279

ctctaceeecataggtttcteegtatecgtaattetatettcttgttctttctttag

1336

Exon 4 CT GAC TTC CCT ACA CCT AGT ATA TCT GAC TTT GAAATT CCA ACT TCT AAT ATT AGA AGG ATA ATT TGC TCA ACC TCT GGA GGT le Asp Phe Pro Thr Pro Ser l t e Ser Asp Phe Gtu Ire Pro Thr SerAsn l i e f rg Arg Zte l i e Cys Set Thr Set GLy GIy

1419 (141)

TTT CCA GAG CCT CAC CTC TCC TGG TTG GAAAAT GGA GAA GAA TTA /U~T GCC ATC AAC ACA ACA GTT TCC CAA GAT CCT GAA ACT Phe Pro GLu Pro His Leu Set Trp Leu Gtu Asn Gty GLU Gtu Leu Ash Ate l i e Asn Thr Thr Vat Set Gin Asp Pro Gtu Thr

150] (169)

GAG CTC TAT GCT GTT AGC AGC AAA CTG GAT TTCAAT ATG ACA ACC AAC CAC AGC TTC ATG TGT CTC ATCAAG TAT GGA CAT TTA Gtu Leu Tyr Ate Vat Set SaP Lys Leu Asp Phe Asn Net lhr Thr Asn His Set Phe Hat Cye Leu ire Lye Tyr Gty His Leu

1587 (197)

AGA GTG AAT CAG ACC TTC AAC TGG AAT ACA A Arg Vet Asn Gtn Thr Phe ASh Trp Asn Thr T

1618 (207)

gtgec~tt9ttctgg~g~gttt~t~tcg~etcte~'-.~ntr~n-~(-6~8Kb)~-~c~t~cca~g~eeg~t~ctc~ec~t¢~etc~teg~e~ gecegeetgegtetceggcegtaggaeetecattecegctgecettettcttt.tcccccceteg

1726 1790

(113)

Exon 5 CC AAG CAA GAG CAT TTT CCT GAT AAC CTG CTC CCA TCC TGG GCC ATT ACC TTA ATC TCA GTA AAT GGAATT TTT GTG ATA TGC

1873

hr Lys Gtn Gtu H|s Phe Pro Asp Asn Leu Leu Pro Set Trp Ate l i e Thr Leu Ire Ser Vet Asn Gty Ire Phe Vat l i e Cye

(235)

tGC CTG ACC TACT Cys Leu Thr Tyr C

1886 (239)

gt~agtactatc~t~ccatcat~ctgatc~ttctgge.~.~ntr~n.E(-1~9Kb)...tataegea~eact~ct~c~ttcta~c~gtct~ng~cttgcta eegtaeetettaacatatgggcecaattceagggaeecttacttatcatttttattgtttgttcttgtcteceag

1994 2069

Exon 6 GC TTT GCC CCA AGA TGC AG& GAGAGA AGG AGG AAT GAG AGA TTG AGA AGG GAA AGT GTA CGC CCT GTA TAACAGTGTCCGCAGAAG ,2155

ys Phe ALe Pro Arg Cys krg Glu kng Arg Arg Asn Glu Ar9 Leu Arg Arg Glu Set Vet ^tO Pro Vat * (262) CAAGGGGCTGAA~GATCTG~G~AGCCTCCGTCATCTCT~cTGGGATACATG~ATCGTGGG~AT~ATG&GGCATTCTTCCCTT~C/~TTT~GCTGTTTTA~C~ 2263

ACT^CCTCACCTTCTTAA~/~CCTCTTTCAGATTAAGCTGAACAGTTACAAGATGGCTGGCATCCCTcTccTTTCTCCCCATATGCAATTTGCTTAATGTAACCTcTT .2371 ~TT~GccATGTT~ccATTcTG~cA~cT~GAATTGTcTTGT~AGcc~TTcATTA~cTATTAAAcA~TAATTTGAGtt~ctt~aat~tt~t~ggta~tt~tta~479 ttctgatcettcetttttccctctatctctcegtettgetggacatgggttggggtcactttgtatteecacggteace

2558

Fig. 2. Nucleotide sequence of the human B7 gene. Nucleotides are numbered at right. The translated amino acids of the coding exons are shown below the nucleotide sequence and the introns (A to E) are shown in lower case letters. The triangle indicates the transcription initiation site and the stop codon is marked by an asterisk. Position + 1 is the first amino acid of the mature B7 protein. with cDNA and oligonucleotides under high stringency conditions identiffed four clones that contained between them the entire cDNA sequence. Restriction mapping of these clones was performed with different combinations of restriction enzymes in the polylinker region (Sambrook et al. 1989). All Eco RI fragments containing exons were subcloned into pBluescript II KS and both strands of exons and intronexon boundaries were sequenced with oligonucleotide primers (Tabor and Richardson 1987).

Primer extension. Total RNA was isolated from the RAJI (Burkitt's lymphoma) cell line by guanidium is0thiocyanate/CsC1 gradient centrifugation (Asubel et al. 1989). Primer extension was perfomed as described (Asubel et al. 1989) using an antisense oligonucleotide corresponding to nt. +51 to + 8 6 of exon 1 and 40 or 50 pg of total RNA. Chromosomal localization. The human B 7 c D N A probe was hybridized to Southern blots containing Eco RI-digested DNA from the human-

178 mouse hybrids as described (Shows 1983, 1982, 1984), Final washing were in 0.1 x SSC and 0.1% SDS at 50 °C. Blots were scored for the presence of human-specific bands in each hybrid and compared with the presence or absence of particular human chromosomes in each hybrid.

A. Selvakumar et al.: Genomic organization of human B7 1

bp

2

3

90-

Results and Discussion

Genomic organization of human B7. Overlapping B7 genomic clones (),3.5, X3.8, X3.4, and X5.4) were isolated from a human genomic library after screening with full-length human B7cDNA. The clones were aligned by comparisons of their restriction maps and by hybridization with various oligonucleotide probes. Clone )x3.8 contained an extraneous stretch of approximately 5 kb of DNA as determined by discrepancies between its restriction map and those of other clones and of uncloned genomic DNA (not shown). A restriction map of the human B7gene is shown in Figure 1. The gene spans approximately 32 kb and contains six exons with five intervening (A-E) introns. The sizes of the introns range from approximately 2-12.5 kb. All exon-containing fragments were identified by Southern blots with oligonucleotide probes. The sizes of cloned Eco RI fragments containing exons (4.2 kb, 4.1 kb, 2.6 kb, 2.3 kb) were consistent with the fragments observed in Southern blots of uncloned genomic DNA (not shown). The nucleotide sequence of the B7 gene is shown in Figure 2. All intron-exon junctions have conventional splice signal sequences (Shapiro and Senapathy 1987). In order to determine the transcription initiation start site, primer extension analysis was performed on mRNA from the B7-positive B cell line RAJI (Freeman et al. 1989). The transcription start site was located 58 bases upstream from the 5' end of the cDNA clone (Fig. 3). No consensus transcriptional regulatory sequences, such as TATA and CCAAT elements (Shapiro and Senapathy 1987), were observed in the 5' flanking region. A number of other genes encoding lymphocyte-specific proteins also lack these elements (Breathnach and Chambon 1981; Yoshikawa et al. 1991; Schanberg et al. 1991; Hall et al. 1988; Hogarth et al. 1991). Sequence analysis of B7cDNA (Freeman et al. 1989) predicts a polypeptide with a signal peptide of 26 amino acids. The mature polypeptide is predicted to be a typical type I membrane protein consisting of a 216 amino acid extracellular domain, a hydrophobic transmembrane region (27 amino acids), and a short cytoplasmic domain (19 amino acids). The extracellular domain consists of two contiguous Ig-like domains (Freeman et al. 1989). The predicted domains of the protein correspond well to the organization of the B7 gene. The first exon does not encode a peptide, whereas the second exon contains the initiation ATG codon and encodes the predicted signal peptide. A similar gene structure was also observed for the

65-

Fig. 3. Primer extension analysis of transcriptional start site of the human B7 gene. A synthetic 36 met oligonucleotide complementary to position +51 to + 86 of B7 cDNA was 5' end labeled and hybridized to total RNA (40 and 50 Ixg) isolated from human B cell line RAJI and yeast tRNA (40 Ixg) was used as control. Hybridized primer was extended by reverse transcriptase and the extension products were analyzed on a 6% sequencing gel (Asubel et al. 1989). The same primer was used in a sequencing reaction of a genomic clone which is shown as a size marker. Numbers indicate the nucleotide length of DNA fragment. Lanes 1 and 2 represent RAJI RNA (40 and 50 pg) and lane 3 yeast tRNA (40 pg). The major primer extension product was located at 58th base upstream to the 5' end of the cDNA.

4"2

I

Kb 2-8-

1

2

3

4

Fig. 4. Mapping of the human B7 gene. The human B7 cDNA probe was hybridized to a southern blot ofEco RI digested DNA from humanmouse hybrid cell lines and controls. Lane 1, hybrid containing chromosome 3; lane 2, hybrid lacking chromosome 3; lane 3, mouse control; lane 4, human control.

leucocyte common antigen CD45 (Schanberg et al. 1991) and for the low affinity IgG receptors /3Fc3,RII in the mouse (Hogarth et al. 1991). Exon 2 also contains the start of the first Ig-like domain. Exon 3 encodes most of the region resembling an IgV domain whereas exon 4 corresponds closely to an IgC-like domain. Exon 5 corresponds to the predicted transmembrane region while exon 6 contains the short cytoplasmic domain. An alternate polyadenylation signal ATTAAA (Freeman et al. 1989) was present in exon 6. This close relationship between exons and functional domains is a characteristic feature of genes in the lg gene superfamily (Hood et al. 1985).

A. S e l v a k u m a r et al.: G e n o m i c o r g a n i z a t i o n o f h u m a n B7 T a b l e 1. S e g r e g a t i o n o f Hybrid

B7 D N A

B7

179

p r o b e with h u m a n c h r o m o s o m e s in

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Chromosomal mapping of human B7.

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Concortdant # of Hybrids

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31L-39 38L-39 55R-16 55R-33 ATR-13 DUA-5BSAGA DUA-6 DUM-13 EXR-5CSAZ GAR- 1 ICL-15CSBF JSR-2 JSR-14 JSR-17S JWR-22H NSL-5 NSL-9 P E W - 11 REX-11BSAgB REX-11BSHF RSR-1 SIR- 11 TSL-1 TSL-2 VTL-6 VTL-19 VTL-23 W12 WlL-2 WlL-6 WlL-8X W l L - 14 WlL-15 WTR-15 XER-7 XER-11 XOL-6 XTR-2 XTR-3BSAgB XTR-22 Chromosome

line is shown

digested h u m a n - m o u s e cell h y b r i d D N A .

Human Chromosomes 1

probe

Eco RI

human hybrid

Three not

cells.

and negative

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cell

to human

hybrids

carry

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an

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were

41

3BSAgB

and XTR-2

both contained

3pter-3q21:Xq28-Xqter, translocation, localize

the

58

and

a 3/X

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17 qter-17p21:3p21-3pter.

B7

chromosome

gene

to the

3 (Table

A previous

intact

the mouse

mAb

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encoding

BB-1

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These region

a

17/3

findings of human

1).

study has mapped BB-1 to human reactivity

translocation,

contained

serological

chromosome

has been

previously

reactivity

to

12. The gene designated

180

MIC17. The study utilized mouse-human hybrid cell lines formed by fusion of either human acute lymphoblastic or chronic lymphocytic leukemia cells and the mouse myeloma p3.X63.Ag8/653 (Katz et al. 1985). The evidence that the two antibodies B7 (Freedman et al. 1987) and BB1 (Yokochi et al. 1982; Clark et al. 1986) detect the same molecule is based on similar immunoprecipitation patterns obtained with B7-transfected COS cells (Freeman et al. 1989). Prior studies with the two antibodies did not reveal this similarity in patterns of reactivity on human B-cell lines (Yokochi et al. 1982; Freedman et al. 1987; Clark et al. 1986). Furthermore, a recent study has used binding assays with fusion proteins CTLA-4/Ig and B7/Ig and demonstrated insufficient inhibition with mAb BB-1 (Linsley et al. 1991). It is, therefore, currently unknown if the antibodies B7 and BB-1 detect the same molecule. The present study using DNA blot analysis, however, revealed that the B7 gene is located on human chromosome 3, whereas chromosome 12 has been excluded. Acknowledgments. This work was supported by National Institutes of

Health grants CA-22507, CA-08748, CA-49096, CA-23766, HG00333, and HD-05196. PCW is a scholar of the Irma Hirschl Trust. Mr. Andre Ambron is thanked for the preparation of the manuscript. The genomic clones k3.5, X3.8, )~3.4, and X5.4 are available on request.

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