GENOMICS

11,587-600

(1991)

Characterization

of the Murine

Thrombospondin

Gene

JACK LAWLER, *nl MARK DUQUETTE,” PAULA FERRO,* NEAL G. COPELAND, t DEBRA J. GIL6ERT.t AND NANCY A. JENKlNst *Division

of Vascular Research, Department of Pathology, Brigham and Women‘s Hospital and Harvard Medical Boston, Massachusetts 02175, and the tMammalian Genetics Laboratory, ABL-Basic Research Program, NC/-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Received

May 8, 1991;

revised

Inc.

INTRODUCTION

Thrombospondin is a 420,000-dalton glycoprotein which has recently been identified as a member of the class of adhesive proteins (Lawler and Hynes, 1986; Majack and Bornstein, 1987; Lawler et al., 1988, Kornblihtt and Gutman, 1988). Secreted thrombospondin is incorporated into the extracellular matrices that are produced by endothelial cells, smooth muscle cells, fibroblasts, and keratinocytes in culture

Sequence data from this article have been deposited with EMBL/GenBank Data Libraries under Accession Nos. M62449 M62470. 1 To whom correspondence should be addressed.

July 11, 1991

(for a review see Majack and Bornstein, 1987). Thrombospondin appears to have specialized functions in supporting cell growth. Its biosynthesis by cells in culture is inversely proportional to cell density and is stimulated by platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and interleukin-1 (IL-l) (Donoviel and Bornstein, 1988; Donoviel et al., 1988; Majack et al., 1985, 1986; Mumby et aZ., 1984; Nickoloff et al., 1988). The human thrombospondin promoter has a serum response element that is moderately homologous with the cFOS gene (Donoviel et uZ.,1988; Laherty et al., 1989). The induction of PDGF is concentration dependent and occurs at levels that are suboptimal for a mitogenie response (Majack et uZ., 19851986). Thrombospondin has been shown to act synergistically with epidermal growth factor (EGF) to stimulate mitogenesis of rat vascular smooth muscle cells (Majack et uZ., 1986). In addition, anti-thrombospondin antibodies inhibit the proliferation of smooth muscle cells in culture (Majack et al., 1988). Thrombospondin is composed of three identical subunits that are linked by disulfide bonds (Lawler et uZ., 1985). Like fibronectin, laminin, and tenascin, thrombospondin is composed of multiple copies of several types of internal repeating sequencesthat are homologous to other proteins (Lawler and Hynes, 1986). These data suggest that exon shuffling and endoduplication of exons occurred during the evolution of the thrombospondin gene (Patthy, 1988; Wolf et al., 1990). In this paper we report on the isolation and characterization of murine genomic clones, which include the thrombospondin gene, and on the localization of the thrombospondin gene. Comparison of the mouse and human genes reveals a high level of conservation, in the coding and 3’-untranslated regions. In addition, many potential regulatory elements in the promoter are conserved.

Thrombospondin is an adhesive glycoprotein that supports cell attachment, spreading, and migration. The murine thrombospondin gene is approximately 18 kb in length and includes 22 exons. Interspecific backcross analysis using progeny derived from matings of (C57BLISJ X i’tfus spretua)h’, X C57BL/flJ mice indicates that the thrombospondin gene is tightly linked to the Fehb, Act& Ltk, and B2M loci on murine chromosome 2. The sequence of the murine gene is very similar to that of the human gene in (1) regions of the promoter, (2) the coding region, and (3) the St-untranslated region. The predicted amino acid sequence of the mature murine thrombospondin subunit is 95.1% identical to that of the human. The sequences of these two specie8 are most similar at the regions containing the type 1,2, and 3 repeats as well a8 the COOH-terminal globular domain. The thrombospondin promoter is similar to the 5 flanking region of some housekeeping and growth control genes in that it contains multiple GC-rich regions and lacks a CAAT box. The presence of various consensus sequences suggests that thrombospondin gene expression is regulated Q 18~1 Academic by CAMP, cytokines, and steroid hormones. Preal,

School,

the to

567

OSSS-7543/91$3.90 Copyright 0 1991 by Academic Press, Inc. AI1 rights of reproduction in any form reserved.

588

LAWLER 0

m-0

I

B

BFBB

4

’

,

5I

I

I

L

1

ET 10I

AL.

I,

I

,

15,

,

I

,

I,

20

Kbp

XmgcP

Xmgc7 1

S

E

II LJ 3

uuu 5

S If 7

EBS 9

FE? u uu 11

LIU 13

uu 15

?f UllUrm 17 19

s

7

E

---

21

FIG. 1. Structure of the thromhospondin gene. The restriction maps for the genomic clones Xmgc2 and Xmgc7 for the region containing the thrombospondin gene. Each clone contains additional DNA (dashed line). The positions of sites for BamHI (B), EcoRI(E), and Sac1 (S) are indicated. The open bars below the map of Xmgc7 indicate the position and size of the exons.

MATERIALS

Isolation

AND

METHODS

of the Genomic Clone

An unamplified mouse genomic DNA library was kindly provided by Dr. Doug Gray and Dr. Rudolf Jaenisch. The library was screened with two human endothelial cell cDNA clones, designated Ml and M9, and a cDNA clone containing the entire coding region (Lawler and Hynes, 1986). Hybridization was performed by standard procedures at low strigency (Maniatis et al., 1982). Eight clones were plaque purified and phage DNA was prepared as described previously (Lawler and Hynes, 1986). One of these clones, designed Xmgc7, produced restriction fragments that comigrated with restriction fragments of murine genomic DNA that were observed on Southern blots with the human cDNA probes. The fact that Xmgc7 contains the thrombospondin gene was established by comparison of the nucleotide sequence with that of the human cDNA clones (Lawler and Hynes, 1986). Sequencing of Xmgc7 indicated that the 5’ end of this clone contained approximately 380 bp of promoter sequence. To obtain more promoter sequence a second mouse genomic library, which was kindly provided by Dr. Laurie Jackson-Grusby and Dr. Philip Leder, was screened with a 400-bp probe from the 5 end of the Xmgc7. A single clone, designated Xmgc2, was isolated and validated by sequencing the overlapping region (Fig. 1). Sequencing

Determination

All sequencing was done by the chain termination method of Sanger et al. (1977) with Sequenase reagents (United States Biochemical Corp., Cleveland, OH) and the standard procedures which are recommended by the supplier. EcoRI and EcoRI-Sal1 fragments of Xmgc7 were subcloned into pGEM-4 (Promega Biotec, Madison, WI). BamHl, EcoRI, P&I, and XbaI fragments of Xmgc2 were subcloned with pBluescript KS (Stratagene, LaJolla, CA). The sequence

was completed and the restriction sites that were used for subcloning were crossed with synthetic oligonucleotides. The sequences for the promoter and the 3’-untranslated region were determined on both strands. The sequence of the coding region was determined on one strand, and frame shifts and compression were resolved by sequencing the other strand. Interspecific

Backcross Mapping

Interspecific backcross progeny were generated by mating (C57BL/6J X Mus spretus)F, females and C57BL/6J males as previously described (Buchberg et al., 1988). A total of 205 Nz progeny were obtained, a random subset of these N, mice were used to map the Thbs locus (see text for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer, and hybridization were performed essentially as described (Jenkins et al., 1982). All blots were prepared with Zetabind nylon membrane (AMF-Cuno). The Thbs probe, a 4.5-kb human cDNA probe that represented the entire coding region (Lawler and Hynes, 1986), was labeled with [a-32P]dCTP using a nick translation labeling kit (Boehringer-Mannheim, Indianapolis, IN). Washing was done to a final stringency of 0.5X SSCP, 0.1% SDS, 65°C. A 2.2-kb fragment was detected in XbaIdigested C57BL/6J DNA; a 4.2-kb fragment was detected in XbaI-digested M. spretus DNA. A faintly hybridizing band of approximately 4.9 kb was detected in both parental DNAs. In addition, TaqI digestion of C57BL/6J DNA revealed 4.4-, 1.8-, 1.5-, and 0.9-kb fragments and TaqI digestion of M. spretus DNA revealed 4.4-, 1.3-, and 0.9-kb fragments. The probes and restriction fragment length polymorphisms (RFLPs) for the loci linked to Thbs have been described: follicle-stimulating hormone @ subunit (Fshb) and leukocyte tyrosine kinase (IA) (Siracusa et al., 1990), /3,-microglobulin (B2M) (Siracusa et al., 1989), cardiac a-actin (Actc-1) (Siracusa et al., submitted). Recombination distances were calculated as described (Green, 1981) using the computer program

MURINE

t

FShb

llp13

Actc-I

15qll-qter

Thbs

15q15

Ltk B2m

15qllqter 15q21-q22

THROMBOSPONDIN

FIG. 2. Position of the Thbs locus on mouse chromosome 2. Thbs was placed on mouse chromosome 2 by interspecific backcross analysis. The segregation patterns of Thbs and flanking genes in 108 backcross animals that were typed in common for Thbs are shown at the top of the figure. For individual pairs of loci, more than 108 animals were typed (see text). Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J X i%f. spretus)F, parent. The shaded boxes represent the presence of a C57BL/6J allele and white boxes represent the presence of M. spretus allele. The number of offspring inheriting each type of chromosome is given at the bottom of each column. Transmission ratio distortion, the inheritance of more M. spretus alleles than expected, has previously been reported for this region of chromosome 2 (Siracusa et al., submitted). A partial chromosome 2 linkage map showing the location of Thbs in relation to linked genes is shown at the bottom of the figure. Recombination distances between loci in centimorgans are shown to the left of the chromosome and the positions of all loci in human chromosomes are shown to the right.

SPRETUS MADNESS developed by D. Dave (Data Management Services, Inc., Frederick, MD) and A. M. Buchberg (NCI-FCRDC, ABL-BRP, Frederick, MD). Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. RESULTS

AND

DISCUSSION

Structure of the Murine Thrombospondin Gene The initial genomic clone isolated (Xmgc7) is approximately 20 kb long and includes the majority of

GENE

589

the murine thrombospondin gene at the 5’ end (Fig. 1). The second genomic clone (Xmgc2) includes sequences that correspond to the thrombospondin promoter region at its 3’ end. The gene is approximately 18 kb in length and is composed of 22 exons (Fig. 1). The positions of the intron-exon boundaries have been determined by comparison with consensus sequences for intron-exon splice junctions and are also based on the high level of homology with the human gene, for which the boundaries have been determined (Wolf et al., 1990). This high level of homology includes the nucleotide sequence in (1) portions of the promoter, (2) the coding region, and (3) the 3’-untranslated region. For example, 21 of 26 nucleotides are identical in the region that includes the site for the beginning of transcription of the human gene. Localization of the Thrombospondin Gene The mouse chromosomal location of the Thbs locus was determined by interspecific backcross analysis using progeny derived from matings of (C57BL/6J X M. spretus) F, X C57BL/6J mice. This interspecific backcross mapping panel has been typed for over 675 loci that are well distributed among all the autosomes as well as the X chromosome. C57BL/6J and M. spretus DNAs were digested with several enzymes and analyzed by Southern blot hybridization for informative RFLPs using the Thbs probe. The 4.2-kb M. spretusspecific XbaI RFLP (see Materials and Methods) was used to follow the segregation of the Thbs locus in backcross mice. The mapping results indicated that Thbs is located in the distal region of mouse chromosome 2 tightly linked to Fshb, Actc-1, Ltk, and B2m. Although 108 mice were analyzed for every marker and are shown in the segregation analysis (Fig. 2), up to 194 mice were typed for some markers. Each locus was analyzed in pairwise combinations for recombination frequencies using the additional data. The ratios of the total number of mice exhibiting recombinant chromosomes to the total number of mice analyzed for each pair of loci and the most likely gene order are centromere-Fshb (6/139)-Actc-1 (4/194) Thbs (3/ Ill)-Ltk (l/113)-B2M. The recombination frequencies [expressed as genetic distance in centimorgans (CM) f the standard error] are Fshb (4.3 f 1.7)-A&c-I (2.1 + l.O)-Thbs (2.7 f 1.5)-Ltk (0.9)-B2M. These mapping results were confirmed by following the 1.3kb M. spretus-specific TaqI RFLP (see Materials and Methods) and by using a 5’ mouse genomic clone (data not shown). The placement of Thbs and flanking loci relative to other chromosome 2 markers and a comparison of the interspecific backcross map with the composite intraspecific backcross map have been reported

590

LAWLER

ET

AL.

A AGGCCAACAGAAGGAGAGTAGCAACTTATGTGGGAAGGCTTTTTATATTTAACA

GTGTTGTAT

-3099

TTAACAGTATAGGTGAAGAGGCTATCAAAGGTTTAATCTATGATCTGCCTTTATTTCTGAGCCCTCCAGA

-3029

GGTAAAAAGAGAATCTTGCCAAGTATGAGCAGAAGGGACTAGAAGAGTGAAAGTCATTATGGGAAATGGT HGRE ACCA~~~AGCAACAGAAAACAAGAGCAAATGAGATT~C~TAAGCACGTGTCAT~GACACC LF-Al AGGAGGCAGT~~~TAGGGTTGGTAGGTTTAGTAGCTTG~CTATCCTCTTTAATATCCAAGATA

-2959

TAGAAGCAGGCTAGAAGCCCACCAACTGTCAAATGGAAAAAGGAAATGAAGAAAATATATACAC~GAGT

-2749

TTTATTCAGCCATAAAGAATAATGAAGTTATGACATTTGTGGGAAAATGGATGC~CTAGAGATCATTAT

-2679 GHF-1 -2609

-2889 -2819

GTTAAGTGAGATAAGACAGGCTTAGAAAGACAAATGTCACAGTTTCTCTAGTATGCACAA PEA3 ~GGAGAGGGCACGAGTAGGATTTATGACAGAGGACCATGAGGCCTAACGTGGATGGTGGATGGAA

-2539

GACAAGTGAAAATAATAGACATAAGGTATAGAAGCAGGAGGGGATCCATGCTTGCACTCACTGAGGAGAC

-2469

ACACACACACAAGAAGGGGGTAGCGTACACACAGGCAAACATATACATGCACACATGCTCATATGCACAT

-2399

ACACACACATACACAAACACACACATAAACACATACACATGCACATATACACACACACACATATGAACTC LVC ACACAGACAGACTGTATTCTCCCTACTAAAGATAGTAAC'TTTTCCCTTGCTTCTCTACAACAT

-2329

CCCAAGAGTGTGCTGAATTTCTGTCATCTGCTCACAGCACATGATGATCGGGACTTTGGGTGGTGGTGAT LVC TGGGAATATGGGTGAGACCTCATAGCTGCCTAGGAACCTCATCT~~CCAGGACAGCTGGATTGT

-2189

CATGCTGTGTTTAGGGAAAGACTCTATGTTTGTATTCAACATCTCCTAGGCCACTGCCAGCTTTCAGTAC

-2049

AACAAAGCTCTCCCATAATGCCTTATCTGAAAGGTGTGGAGGAGGTCAAAACTTGTATTGTGTTGTTGAA LF-Al AGGAGCCAAGTTACAAACTGTCCACACCCCCTCAGTCTGGACAGCAC~~~AAGGGGCAGACCCTG

-1979

ATTGCTCCTGATCCATCAGCAAGGTGTAGCTGGGCCTAGGGAAGTTGGTGGCTTTACACAAGGAGAGAAG

-1839

-2259

-2119

-1909

CTCTTGTCTGTGGGCCGCTGACATTCACCTCTGTGGTACAGACC PEA3 AGCCCTCCCAGGGCAGTCCCAAAGGTTCCTG~~~TTAGTGCTGA~~~TTAAAGCAATC LVC GCCTTCCACAAAGCAAAGAGG~~~GAAGGAACTGGAAGCGAAGAGCAA~~~TGATGATCCCG APl AATCTTCAGATTCAGTGGCGAGTCAG~GCCTGTGACTTCAAGGGCAGGAATCT~C~~~C~C

-1769 LVb -1699 LVC -1629 PEA1 -1559

CAGTAGAAACTTTTACTGCTGGCTCTGAGTATCTGAAGTCTAATAAACAAACACATCTTGGGAACGTACT

-1489

TATCTACAAGAGGCACTGGCTGTGTAGCTGTGTACCCGGAGTAACTAGGAAACACTCTGT~GCCCCTCC

-1419

TTGCGTGTACGCACACATACCACGCACACTCACACACCTTGTCACCGAACACTCCCTCCCACACAAGGAA HGRE AGTGAAATTGCTGTGTGATGAGCAT~~~GGTGTTTCCAAGGT~GGGAGTCCAGCC~GTGT~ AP2/Spl CTGATAAGGGGAGGGAGCCCCAGCTGTCCC~CAGCCGCCCCGCCC~ATTCTGCCAAGAT~CTTATTTG~ NFl LVC TGATGCTTTTACCTGGATCCdrGGACTCAGAGCCPIA~~~CACGCAGGGCCACAGCTGGAGAAGCAT ATF CGCCGCCG~~~CTTCCCAAAGAGATGAATGGAATTCCAGGCAGCTGGAGTCATCTTGGCTCCGG

-1349 NFl -1279 CArG -1209 -1139 -1069

FIG. 3. Sequence of the thrombospondin gene. (A) Nucleotide sequence of the promoter region of the thrombospondin gene. The locations of potential regulatory elements are boxed and the identity of each is indicated above the box. For overlapping sequences the total range of sequence is boxed. The nucleotide that aligns with the start site for the human gene is marked by the open triangle and the nucleotides are numbered relative to that start site. Based on the presence of a sequence that matches the consensus site for intron-exon boundaries and on alignment with the human sequence, the last nine bases are in lowercase to indicate they are in the first intron. (B) Nucleotide sequence of the coding region of the murine thrombospondin gene. The amino acid sequence of mouse thrombospondin is indicated below the nucleotide sequence and the amino acids in the human sequence are given below those of the mouSe only in the case where they differ. Sequence motifs for binding proteoglycans and cell surface receptors (VTCG and RGDA) are boxed. Potential sites for N-linked glycosylation are circled. The positions of introns are indicated. Bases that are included in the introns are in lowercase. (C) Nucleotide sequence of exon 22. The mouse and human sequences have been aligned using the Doolittle alignment program which is included in the EuGene sequence analysis software package. Aligned nonidentical bases are in uppercase, unaligned bases are in lowercase, a

MURINE

THROMBOSPONDIN

591

GENE

ACCGATGGCATTGAGACCAGACTTCAGAGACAAAGGATCAGGCAGCAGTCTACTGATGG~GTGTATA

-999

CCCACAGAAATGTAAACCACACCTCGGTTTTGGGAGCCGAGGAGA

-929 GHF-1

AAGATGGGkAATAAAATAATAGGTAGAGAGTAATTTTAT~CTCTACGGGTCCGTTCT~~~GTGT~

-859

GATCCTGGAGCCAGATGGTTCAGCAAATTCTCTCGGG

-789 GHF-1

AATTGTTTTTCAAGACACAATTTCAATTTTTGCCTTGAGGGAGCGGGAT~~~CGCTTCCTT~TG GHF-1 CCGCTAAAAACTCTCCGAAAGGATCCCC~?~~AGAATACA

-719 PEA3 CAAGCGGCCATCTm

-649 -579 -509 -439 -369

ATGGAATTATTCAAGGAGATGTGCTTTAATGAAAAGCCTCCCTA~GGGTCTTAGGTGGTCCCC~AGAAG NFkB CATCGCGTCTCCATGCAGAACGTCTCCAGTTCACATGGCGC~GATCCT~GCGCTAAAGGC SPl TGAGTACGCCAAGGCTGCGlrGGGCGGAGPlCCTATTTTTCTGACAAGTTCCAGGGCTCCTGTGCGGGATCG sp1 GAGTCTCCCCCTTCACTTTCAGCCCGAGAGCTGTGCGCCAAGCAGCAG~GGCGGAG~ Spl/APZ AP2 LVC LVC CGTCCCC@CCCCCGCCCCCGCCC#XAGAACCCTf?$rq=?#T@rtiGGTCCT

-299 -229 -159 LF-Al GAAC & GTC

-89

TATA -19

GGGCTCCTCAGTCAAGCCAGCCACTGCCTGGAGTCAGCCAGCCTCATCGGACTTCTGCAGGC~TCGCGA n AGCTGCTATCCAGTTCTGCCACGGTCTCTCCCGGCGCACCGGCAGTCTCAGCGTCTTCACCGGACTCAGC

52

GTCCTTGTCCTTCACTTCACCTTTGCCACCTCTCCGGGTTACTGAGCCCCGGTGCACACAGgtaaacctc

192

122

dash indicates the bases are identical, and a dot indicates a gap in the sequences. The position of the intron-exon boundary was determined by comparison with consensus sequences for intron-exon splice junctions and based on the high level of homology with the human gene. There are no gaps and only one base that is different when comparing 20 bases of the human and mouse sequences around the 5’ end of exon 22 (Ref. (48)). Note that the intron sequence is included in part B. The TATT and ATTT sequences are boxed. In the case of overlapping sequences, the complete range is boxed. The consensus sequences for polyadenylation are bracketed above the sequence.

previously (Siracusa et al., 1990). These comparative studies indicate that Thbs is located in a region of chromosome 2 in which several mouse mutations have been positioned. A more detailed analysis of the expression patterns of the mouse Thbs locus may reveal that Thbs is a candidate for the structural locus altered in one of these mutations. Finally, our assignment of Thbs to the central region of mouse chromosome 2 is in agreement with the recent results of Jaffe et al. (1990) who placed Thbs on mouse chromosome 2, region F, by in situ hybridization. This region of mouse chromosome 2 also shares a region of homology with the long arm of human chromosome 15 (Fig. 2), suggesting that the human homolog of Thbs resides on 15q. Recent studies have confirmed this expectation; Thbs has been localized to 145qll-qter (Wolf et al., 1990) and, more precisely, to 15q15 (Jaffe et al., 1990). Sequence of the Promoter

Region

Two types of organization within the 5’ flanking sequences of genes have been identified (Dynan,

1986). The majority of eukaryotic genes contain CAAT and TATA boxes, which are used as RNA polymerase II transcription signals. A second group of genes includes the housekeeping and growth control genes (Dynan, 1986). These genes have GC-rich upstream sequences and frequently lack CAAT and TATA boxes. The epidermal growth factor receptor gene and the Harvey ras proto-oncogene are growth control genes that contain multiple copies of GC box sequences (Ishii et al., 1985a,b). The mouse and human thrombospondin genes have sequences that are characteristic of both groups (Donoviel et al., 1988; Laherty et al., 1989; Bornstein et al., 1990) (Fig. 3A). Like the housekeeping and growth control genes, the thrombospondin gene contains multiple copies of GC box sequences and lacks a CAAT box on the sense strand (Fig. 3A). However, the thrombospondin gene contains the TATA box-like sequence TTTAAA at position -26 in both species. This sequence may serve to fix the start site for transcription (Dynan, 1986). C-myc and metallothionein IA are other examples of genes that contain GC boxes and appropriately posi-

592

LAWLER

B mouse:

ET AL.

mouse: human:

CtaCagGCTCCGTGTTGGGCACAAAGGCTCCACCACCATGGAGCTCCTGCGGGGACTAGGTGT ME L L R A W

mouse: mouse: human:

CCTGTTCCTGTTGCATATGTGTGGAAGCAACCGCATTCCAGgtgagt...Intron LFLLHMCGSNRIP M V T

mouse: mouse: human:

..ttacagAGTCTGGGGGAGATAACGGTGTGTGTTTGACATCTTTG~CTCATTGGAGGTGC ESGGDNGVFDI FE S

L

I T

mouse: mouse: human:

ACGAAGGGGCCCCGGTCGCCGACTGGTGAAGGGGCC~GATCTATCCAGCCCCGCCTTCCG RRGPGR LVKGQDLS K S P P

S

PA

mouse: mouse: human:

CATTGAGAATGCCAACCTGATCCCCGCTGTGCCGGATGACAAGTTCC~GACCTACTGGA I EN AN L I PAV P D D K F D P

Q

DLL

mouse: mouse: human:

CGCTGTGTGGGCCGACAAAGGCTTCATCTTCTTGGCTTCCTTGAGGCAGATG~G~GAC AVWA D K G F I F LA S LIR R T E L L

Q

M

K

KT

mouse: mouse: human:

CCGGGGCACACTCCTGGCTGTGGAACGGAAAGACAACACTGGCCAGATCTTCAGTGTGGT ~GTLLAVERKDNTGQ H S L

I V

F

S

V

mouse: mouse: human:

CTCCAACGGCAAAGCTGGCACCCTGGACCTGAGCCTGAGCCTGCCAGGGAAGCAACAAGT SNGKAGTLDLSLS LPGKQQV T V Q

mouse: mouse:

GGTGTCAGTGGAGGAAGCTCTCCTGGCCACTGGCCAGTGG~GAGCATCACGCTGTTTGT VSVEEALLATGQWKS

I

T

L

F

V

mouse: mouse: human:

TCAAGAGGACCGGGCTCAACTCTACATAGACTGTGATAAGATGGAGAGCGCGGAGCTGGA QEDRAQLYI DC D K M E E

S N

A

E

L

D162

mouse: mouse: human:

TGTACCCATCCAGAGCATCTTCACCAGGGATCTGGCCAGCGTTGCCAGGCTCCGAGTTGC F T R D LA S VA V P I Q S I V I

R

LRVA

mouse: mouse: human:

AAAGGGAGATGTCAATGACAATTTTCAGgtaaat...Intron KGDVNDNFQ G

mouse: mouse:

GCTGCAGAATGTGAGGTTTGTCTTTGGAACCACCCCAGAAGG L Q N V R FVFGTTPEDI

mouse: mouse: human:

CTGCTCCAGCTgtgagt...Intron c s s

mouse: mouse:

TGACAACAACGTGGTGAACGGTTCCAGCCCTGCTATCCGCACCAACTACATCGGCCAC~ DNNVV@G SSPAIRTNYI

mouse: mouse: human:

AACAAAGGACCTCCAAGCTATCTGTGGCCTCTCCTGTGATG~CTATCCAGCATGGTCCT TKDLQA I C G LS CD E L I

IV..

FIG.

G

L

G

V -10 II. 4

G

G A

A

22

F

R

42

D

62

V 82

V

102

122 H 142

182 I

III..ccacagGGGGT

L

R

.tagcagCTACCAACGTCCTTCTTACCCT S TN V L S

N

S

V

193

K

G

213

L

224

K

244

L

264

LT

G S

G

MV

H

3-Continued

tioned TATA boxes. The GC box sequences have been shown to bind the transcription factor Spl (Briggs et aZ., 1986; Jones and Tjian, 1985; Mitchell and Tjian, 1989). Briggs and co-workers (1986) have identified a decanucleotide consensus sequence for

the Spl binding site as KRGGCGKRRY. The murine thrombospondin promoter contains two sequences that match this motif in all except the last position (Fig. 3A). In addition, five copies of the inverted complement of this sequence, all of which match in nine of

MURINE

THROMBOSPONDIN

593

GENE

mouse: mouse: human:

GGAACTGAAGGGCCTGCGCACCATCGTGACCACTCTGCAGGACAGCATCCG~GTGgt ELKGLRTIVTTLQDS I R KV R

mouse: mouse: human:

cagt...Intron

mouse: mouse: human:

GCGGCCTCCCCTCTGCTTTCACAATGGAGTCCAGTACAAGCGAGGAGTGGACTGT RPPLCFHNGVQ YKNNEEWTV Y R

mouse: mouse:

AGACAGTTGCACAGAGTGTCACTGCCAGgtaaga...Intron DSCTECHCQ

mouse: mouse:

GGTTACCATCTGCAAAAAGGTGTCCTGTCCCATCATGCCCTGCTCC VTICKKVSCPIMPCSNATVP

mouse: mouse:

TGATGGTGAATGCTGCCCACGGTGCTGGCgtaagt...Intron DGECCPRCW

mouse: mouse: human:

CGACTCTGCTGACGATGGCTGGTCTCCCTGGTCTGAGTGGACCTCCTGCTCTGCCACATG DSADDGWSPWSEWTSCSATC T S

mouse: mouse:

TGGCAATGGAATTCAGCAACGTGGTCGTTCCTGTGACAGCCTCAACAACAGATGCGAGGG GNGIQQR GRSCDSLNNRCEG

397

mouse: mouse:

CTCTTCGGTACAGACGAGGACCTGCCACATTCAGGAGTGTGAC~GATgtaagc..InSSVQTRT CHIQEC D K R

413

mouse: mouse:

tron

mouse: mouse:

CTGTTCTGTGACCTGTGGTGACGGTGTGATCACAAGGATCCGGCTCTGC~CTCCCCCAG C SIV T C G[D G V I TRIRLCNSPS

448

mouse: mouse:

CCCCCAGATGAACGGGAAGCCCTGTGAAGGTGAAGCCCGGGAGACCA~GCCTGC~GAA P Q M N G XPCEGEARETXACKK

468

mouse: mouse:

AGACGCCTGCCCAAgtaaqt...Intron DA C P

479

mouse: mouse: human:

CTGGTCACCATGGGACATCTGCTCTGTCACCTGTGGAGGAGGAGTGCAGAGACGCAGCCG W S P W D ICSIVTCGIGGVQRRSR K

mouse: mouse:

ACTCTGTAACAACCCCACACCCCAGTTTGGAGGCAAAGACTGTGTTGGCGATGTGACAGA LCN@P TPQFGGKDCVGDVTE

mouse: mouse: human:

AAATCAAGTTTGCAACAAGCAGGACTGCCCAATTGgtaagc...Intron NQVCNKQ D C P I I

mouse: mouse: human:

aqATGGATGCCTGTCCAATCCCTGCTTTGCTGGTGCCAAGTGTACTAGCTACCCTGATGG DGCLSNPCFAGAKCTS Y P D V

VIII

V....

283

tttcagACGGAAGAGAACAGAGAGCTGGTCAGTGAGCTGAA TEENRELVSELK K AN

R

315

VI...ttgcagAACTC N

S

CGCCACAGTTCC "Q VII..ccacagCCAG P s

FIG.

326

346

357

377

..cttcagTTAAACAGGATGGTGGCTGGAGTCACTGGTCTCCATGGTCGTC FKQDGGW SHWSPWSS

IX...

295

428

gcacaqTTAATGGAGGCTGGGGTCC INGGWGP

499

519

X....cctc 530

G

550

3-Continued

the ten positions, can be identified. It has been proposed that Spl can interact with other cellular transcription factors including those that bind to APl and AP2 sites (Briggs et al., 1986; Mitchell and Tjian, 1989; Lee et al., 1987; Rauscher et al., 1988). The murine and human thrombospondin genes cantain three potential APl binding sites (Fig. 3A) (Donoviel et al., 1988). The family of transcription factors that bind to APL sites have been proposed to modulate gene tran-

scription in response to the cellular environment (Hai et al., 1988). Many of the genes that contain API sites are upregulated by serum (Mitchell and Tjian, 1989). The. thrombospondin gene has been reported to be induced by platelet-derived growth factor and by interleukin-1 (Donoviel and Bornstein, 1988; Majack et al., 1986). Both the mouse and human promoters contain sequences that conform to the.consensus site for NF-kB binding (Lenardo and Baltimore, 1989). NF-

594

LAWLER mouse:

mouse: human: mouse: mouse:

ET AL.

TAGCTGGAAATGTGGTGCGTGTCCTCCTGGCTACAGTGGAAATGGCATCCAGTGCAAAGA S WKCGACPPGY SGNGIQ

C

CGTCGATGAGqtaqqc...Intron V D E

DA

XI...

ccataqTGCAAAGAAGTGCCTGATGCTTG C K E V P

K T

5

570

C

581

P

601

mouse mouse: human:

: CTTCAATCACAACGGAGAACATCGGTGCAAGAACACAGATCCTGGCTACAACTGCCTGCC F N H N G E H R C KN T D P G E

Y

N

C

mouse: mouse: human:

CTGCCCACCACGATTCACTGG~T~ACAG~CCTTCGGCCGAGGTGT~GAACATG~~ATGG~ C P P R F T G S Q P F G R G V Q

E

H

AMA

mouse: mouse:

CtiCAAACAGqtataq...Intron N K Q

mouse: mouse:

CACGGACGGGACGCATGACTGCAACAAGAACGCTAAGTGCCTACCTGGGTCACTACAG TDGTHDCNKNAKCNYLGHYS

mouse: mouse:

CGACCCCATGTACCGCTGTGAGTGCAAGCCCGGCTATGCAGGC~TGGCATCATCTGCGG DPMYRCECKPGYAGNGII

mouse: mouse:

AGAGGACACAGACCTGGACGGCTGGCCTAATGAAAACCTGCGCAAC EDTDLDGWPNENLVCVAaAT692

mouse: mouse:

CTACCACTGCAAAAAGqtacaq..Intron Y H C K K

mouse: mouse:

TCCCAACTCGGGGCAGGAAGACTATGACAAGGACGGACGGGATTGGCGATGCCTGCGATGATGA P N S G Q EDYDKDG I G DA

mouse: mouse:

CGATGACAACGACAAGATCCCTGATGACAGGqtaqqq...Intron DDNDKIPDDR

mouse: mouse:

CAACTGTCCATTCCATTACAACCCAGCCCAGTATGACTATGACAGAGATGATGTGGGAGA NCPFHYNPAQ Y D Y D R

D

DV

mouse: mouse: humam:

CCGCTGTGACAACTGCCCCTACAAeCACAACCACCAACGG RCDNCPYNHNPDQA

T

D

mouse: mouse: human:

GGAGGGCGATGCCTGTGCTGTGGACATCGATGGAGATGqtaaqq...Intron EGDACAVDIDGD A

mouse: mouse:

CataqGAATCCTCAATGAACGAGACAACTGCCAGTACGTTTAC~CGTGGACCAGAGGGA G I LNERDNCQ YVYNVDQ

R

D

805

mouse: mouse:

CACGGACATGGATGGGGTTGGAGATCAGTGTGACAACTGCCCCCTGGAACAC~TCCAGA TDMDGVGDQCDNCPLEHN

P

D

825

mouse: mouse: human:

CCAGqtaqqt...Intron

G

D

836

mouse: mouse:

CACTTGTGACAACAATCAGGACATCGATGAGGATGGCCATCAG~C~TCTGGAC~CTG TCDNNQDI DE DG HQNN

C

856

Q

XII

P

C

632 652

..ttttaqGACAACTGCCCCAACCT D N C C

C

G

672

P

N

L

703

D

D

D

723

D

734

D

754

G

774

XIV..ttqcaqGA

D

XVI ..ttccaqCTGGACTCTGACTCAGACCTCATAGGGGA L D S D S D

FIG.

621 T

..cctcaqGTGTGCAAACCGCGAAACCCCTG V C K PRN

XIII

L

G

KN N XV...c

786

L R LDN

I

3-Continued

kB binding sites have been reported to be involved in IL-l-induced upregulation of transcription (Lenardo and Baltimore, 1989). The mouse and human promoters also contain two copies of a hexanucleotide (AGTCCT) that has been reported to interact with the glucocorticoid receptor, suggesting that thrombospondin synthesis is regulated by steroid hormones

(Cat0 et al., 1984). The murine promoter contains a potential binding site for polymomavirus enhancer A binding protein 1 (PEAl, TGACTAA), a murine homolog of APl (Martin et al., 1938) (Fig. 3A). Consensus sequences for three nuclear factors (NFl, LVb, and LVc) that have been shown to interact with the Moloney murine leukemia virus enhancer are also

MURINE

THROMBOSPONDIN

595

GENE

mouse :

TCCCTATGTGCCTAACGCCACCAGGCCGACCATGATAAAGGAGATGCCTG PYVPNANQADHDKDGKGDAC

816

mouse: mouse: human:

TGACCATGACGATGACAATGACGGCATCCCTGATGACAGAGACAACTGCAGGCTGGTGCC DHDDDNDGIPDDRDNCRLVP K

896

mouse:

mouse: CAATCCTGACCAGAAGGACTCTGATGgtgagt..Intron mouse : NPDQKDSD

XVII..ccttagGTGATGG G D

G

907

mouse: human:

CCGAGGTGACGCCTGCAAAGACGACTTTGACCATGACAATGTGCCAGATATTGATGACAT (RGDAICKDDFDHDNVPDIDDI S

921

mouse: mouse: human:

CTGTCCTGAGAATTTTGACATCAGTGRAACCGATTTTCCGACGATTCCAGATGATTCCTCT CPENFDISETDFRRFQMIPL V

941

mouse: mouse:

AGATCCCAAAGGAACCTCCCAAAATGACCCTAACTGACCCT~CTGGGTTGTCCGCCATCAGGGC~GA DPKGTSQN DPNWVVRHQGKE 967

mouse: mouse:

ACTCGTCCAGACTGTAAACTGTGACCCTGGACTTGCTGTAGgtaagt..~ntron LVQTVNCDPGLAV

mouse: mouse:

..ttccagGTTATGATGAGTTTAATGCTGTGGACTTCAGCGGTACCTTCTTCATC~CAC GYDEFNAVDFSGTFFINT

998

mouse: mouse:

CGAGAGAGATGATGACTACGCTGGCTTTGTATTCGGCTACCAGTCCAGCAGCCGCTTCTA ERDDDYAGFVFGYQ SSSRFY

1018

mouse: mouse:

CGTTGTGATGTGGAAACAAGTCACCCAGTCCTACTGGGACACC VVMWKQVTQSYWDTNPTRAQ

1038

mouse:

mouse:

mouse:

XVIII 980

CCCCACAAGGGCTCA "g GGGATACTCAGGCCTGTCTGTAAAGGTTGTGAACTCCACCACCGGCCCTGGCGAGCACCT GYSGL~~K~~@STTGPGEHL

mouse:

GCGGAATGCACTGTGGCACACAGGAAACACCCCTGGCCAGgtaaga...Intron RNALWHTGNTPGQ

mouse: mouse:

.cttcagGTGCGCACCCTGTGGCATGACCCTCGCCACATCCTG~ VRTLWHDPRHIGWKDFTA

mouse:

GTACAGATGGCGTCTCAGCCACAGGCCAAAGACCGGTTATATCAGgtaaga...Intron YRWRLSHRPKTGYIR F

mouse:

mouse: human: mouse:

1058

XIX. 1071 1089 1104

XX . ..gtgcagAGTGGTGATGTATGAAGGAAAGAAAATCATGGCTGACTCGGGACCCAT VVMYEGKKIMADSGPI

1120

mouse:

CTATGACAAAACCTACGCCGGCGGTAGACTAGGCCTGTTCGTCTTCTCTCAGG~TGGT YDKTYAGGRLGLFVFSQEMV

1140

mouse: mouse: human:

GTTCTTCTCAGACATGAAATACGAGTGTCGAGgtagga...Intron FFSDMKYECR L

mouse: mouse: human:

TTCCTAA s @ P

mouse: mouse:

FIG.

XXI..taacagA D

1151

3-Continued

present (Speck and Baltimore, 1987). Several elements in the murine thrombospondin promoter suggest that transcription of the gene is upregulated by increases in CAMP. There are five potential AP2 binding sites (Imagawa et al., 1987; Medcaff et al, 1990; Roesler et aZ., 1988) (Fig. 3A). AP2 binding sites have been reported to mediate cellular responses via two

different signal-transduction pathways (Imagawa et aZ., 1987; Medcaff et aZ., 1990). Phorbol ester-induced modulation of protein kinase C and upregulation of CAMP have been reported to increase the activity of AP2. In addition, a consensus sequence for the ATF transcription factor is present (Lin and Green, 1988). ATF is similar or identical to the CAMP regulatory

596

LAWLER

C

ET AL.

mouse human

: ATTCmCATCA....GCTGCC...AA.. .TCATAACCAGCGCTGGCAATGCACCTTC : --C-----------aatt-T--ATtga--gac-G--gac-G--C-TA-A-.....------TGG-AT

50

mouse human

: TAAAAACAAGGGCTAGAGAAACCCCCC...... .ACCCCTGCCGGGATCGCCTTTCCTCG : -G.. ..--CCTT--G--ACT-TGGG-Ttgagaaa-----...-A-----A-T-C----T-

103

mouse human

: CCTTCCTTGCCT.CTCTTCTTGCAT.AGTGTGGACTTGTA~~~~~~G~~CTG~~~~~~ : G-------CTT-~--G-G-------~----------~~--G-A-G--C------------

161

mouse human

: GAAAATGCAGTTTTCGAACCCAGAGTCA.. .GCACTCGGCCTTTAACGAATGAGAATGCA : ---------------A--AA----C---tca---T--A----CC--T----A---...--

218

mouse human

: TCTTCCAAGACCATGAAGAGTTCCTTGGGTTTGCTTTTGGG~GCC~GCGC~~~ : ---------CAT--A--C-A--G---T-----C------~--------.....*.--C-

278

mouse human

: XJZTTCCCAC... TAGGAAGGTGCCCGCTCCACTCTGCCTTACTCACAGAGCCAGAACTTC : ----G-TT-agt-G-----------AT-------------TG---------AG-GTGC-A

335

mouse human

: TTC.GAGGCCACCTCTGAGCAGCACACACAGAAG : --Gt-------T----------TGG--~-Ams

394

mouse human

: AAAATGACTCACTAGAACTCACCGCCAAACAACCTCTGACATAGGTCCTGAG..ATGTGG : G . . . . ------------T-AG-AAA----ACCA-C-------CCTC-T-C--ga-CAC--

452

mouse human

: GGAGGCAGGAGCCAAAGCTCTA..GGGAGGGCATGTACCCAAGAGATGACTGTATGAAGA ----C--T--------: ----.---AG--------A---ag--------GCA-----..

510

mouse human

: AAATGTGGAGGAGCTGTT.......CGGTACTAAAT : ----A-------A-----acatgtt------------G--

mouse

TTT CAGGCATGTCAAAGAAAGGA -----------G----G--$ ----

TT $P ---

CAGGGGACAGACAGACT. -------TT--A-----a

562

:

.TGCTG TT CGCATGCTG.CTGGTGAGAGCTGATTGACCCAATCTTCCACACAGGCA : t - - - - - dT- - - -ATG------a----C-TT--------A-----....-GT-A-T-----

620

human mouse human

: CTTGAGCA..AGCAGGGAAGGGAGGGAGATCATAGCTTCTGGACTTTCTCCCTTTGGTAC --A-------AC-A-GAC-G------------C------GA..... : ---A-AT-gas---

678

mouse human

: TTCTCATCTGCAGTGGCCAGGGT..AGGGGTCAGAAGTGTGTGGGCCATGCTGGCTG...CC : -C-C--C-CTT-C-CAT--CCT-gc--T--C-----T-AG--...-AT-A-~-Caaa--

733

mouse human

: CTTGACTGGTCACGCTGAAACTGTTAAC'TGCATGTACAACATGCATATG..... ---C--GT--CAT--A-AT--gtggc : AG--TAA--CAGT----G..---CC-T.......

788

mouse human

: . . . . . TATAAGGAGAGCTAGAGAACCTTACCGTCTCAGTGAGCTCT....AGCTGCCTCC --AG---GA-AA-AGgaaa-C--A--AT: ttcat-C--GATGT----T-T-C-GA-....

839

mouse human

: GCAG.GAGGGCAGTGCGCCTTTACTTTATGGTTAGAAAGGCACACA......... : T---t---CA-CAGCT----...-CCA-A--......---GG--gccgtgctt

889

mouse human

: TAT.... .CAACCTAACTAAAACATTCCTTTTCTTTCTTTCTTTCTTCCT.......... : ---ggtta---TGGC--A---~................--AA---aactaaaaca

934

mouse human

: TTCTTTTTCTCTCAGTTACCATG~TCCA.AATCTCTTTTGGTTTTTGTTTAACAA : ---C--------.............-------Gt---TA--A..---AG--T-C---TTC

993

mouse human

: ATGCTTTAACAA.TGTAATA&-$ : TCT----TGG--g-A-

mouse human

: GTTCGTCGGAAGGATTGTTACTGAAACAGGAAGCGTGGGACTATCTG~TCATCTTCGTA : .~T-G-A---C.-GG-----GG-~A---A--AGGAA---G-......---

ATTTATT GTTCCCC...... ----AAG-C--TA-Gatgtaaa

FIG.

TT --fzfzk

.eG tatt----T

1040

TT-C. 1100

3-Continued

element binding protein (CREB) and is a member of a family of transcription factors that includes APl (Hai ‘et id., ‘1988). The thrombospondin promoter contains several elements tbat.may be involved in tissue-specific upregulatibn of transcription. A CArG box is present in both

the mouse and human promoters (Donoviel et al., 1988; Laherty et al., 1989). The CArG box has been reported to be involved in the tissue-specific transcription of the cardiac a-actin gene (Miwa and Kedes, 1987). Immunohistochemical stainingofdeveloping mouse embryos reveals that thrombospondin,

MURINE

THROMBOSPONDIN

597

GENE

mouse human

: GTGAGTCTTTATGAC.....TGTAA...G........ATTGTAAATACAGAlT : AA--C-A-CC---T-atctt---TGaga-tcttcgtg-C-----.....----G-~--A

1144

mouse human

: AACCCTGTTCTACCTGGAATTAGA......CTGCATATAGCAAATGTTTGCAA......G : C-G @u-ACtctgtt----C-...-G-+T&--TTcatacg-

1192

mouse human

: CAGGTAGCTGAGGCGGATCAGCAA.........ATCTCTCCAAAACCAGTGTGTGGAAAG .-G-A-A-C-C-T-C--: A-A--GTT--mm . . . . . . . -----gtagttgac--T-A--..

1243

mouse human

: G.CAGCAGA.GGCAGCTGCTTCTCATCTGGGGCTCACAGTGAAT~GACTCCTTTG : Aa-----C-a--A-AA-CAG---A--AA-CT-----....--...,....,..-C----GT

1301

mouse

:

human

GCTTCC.................. : ---CAGagtggatgttatgggatt-----..

1343

mause human

: GGCGGCCGCTTGCATTCACTCCTCCTTGTAAAGG....AA...GCCAGGAGTACTCTAAG ---C---T-tttt--att--A-A--~G-CA-G-: --AATTA-T-G-TTA--CA-.....

mause human

: GCTAGCCCACACCACCCCTTGTGC~~~CAGGG.........AGAAGAAAGAAAACA : -T.... -TT--AT--TGT--TAC--C--CC-TT-T-c~~~~--GGAG---G---GC

mouse human

: : AT--ACA-----..--C---

mouse human mouse human

: dTA....... TT&XCT.C TT TGTGTGACTGAGTAAAGIGAATTAAGAAG : ---AGgtgaaac----A-a--A 5ElACC-C-T-. - . . . . . . . . . -G---TG-G-CT+I : .AAAGAGTTTAAGTGTCAAT.... ,AAIiCTTAAAACTACTGTTGTGTCTAA.......AA : t----~T-GA----Gcggaa-G-G--T--.........--------caaactt--

mouse human

: AGTCGGTGTTGTACATAGCATAAAAATCCTT....TGCCGAGGATGATCCCAAGAAAGAA : --CTAC---A----C--.... ----G--AG-gttg-A-AT--C--......--A--CTCT

mouse human

: :

mouse human

: : -A----T-..

mouse human

: TTGC.CATTGGAATAGAGATCTCAGACTATGTAGATATGCffm@ATA : --'I)-t -----CC--T-GA--...---A---TC----....-G-G--G---tg

mouse human

: CAGGAAATACTGCCTGTAGAGTTA . . . . ..@=fiTGGTTTTTATATGTTGC.ACACT : . . . ..---+T&CAG--AA--ctgcct---GAGT-A-tt-T-A-

1865

mouse human

: GAATTGAA . . ..GAAATGTTGGTTTTTCTTGTTTCGTTTTAGTTTGTTTCTTTGGTTTTG : -T.---C-cact---T--RA-AA--G-TGGT---TTC---TT-------T---TT----T

1921

mouse human

: :

1981

mouse human

: : --T-----------W

2041

mouse human

: AACTTGTATATTACTGTTTCTTATGTACAAGGAAC~C~TCATATGG~GTTT : --G--------------------------------------------------

mouse human

: ATTT 2105 : 1-mm-

CCTTTGTTTTCTCTGTAGTTTCAAGTGGAATTAGGT ---------TT-A--TTT-CA-...--

1396

. ..----------A---AA--..........--

1606 1662

--aaa-C......TCG

. . . . ----CT-T---..

--GG---ACCAT-GCT

FIG. 3-Continued

like cardiac cr-actin, is present in developing heart and skeletal muscle (O’Shea and Dixit, 1988) (unpublished data). There are four copies of the LF-Al element in the mouse promoter (Hardon et ai., 1988). These elements have been reported to be involved in gene transcription in liver cells (Hardon et aZ., 1988). Whereas thrombospondin expression is not detected

in the liver of mouse embryos before Day 135 at Day 14; and later during development the liver does express thrombospondin. Similarly, expression of thrombospondin occurs in the pituitary around terminally differentiated cells (unpublished data). The mouse and human promoters contain sequences that have been reported to bind GHF-1, a transcription

598

LAWLER

factor that regulates synthesis of human growth hormone by the pituitary (Lefevre et al., 1987). Those portions of our sequence that overlap with that of Bornstein and co-workers (1990) are identical with two exceptions between positions -195 and -175. Our sequence starts with five thymidines, whereas theirs contains six and ours ends with three guanines, whereas theirs ends with four. The origin of these differences is unknown; however, since they do not fall in the consensus sequences that constitute our database they do not affect our interpretation of the data.

Sequence of the Coding Region The sequence of the coding region and the positions of the introns for the murine thrombospondin gene are shown in Fig. 3B. Our data agree with the published sequence for exons 2 through 9 (Bornstein et aZ., 1990). Residues that are different in the human sequence as compared with the mouse sequence are indicated below the mouse sequence. There are only 56 differences between the human and mouse sequences over the 1152 residues that constitute the sequence of the mature peptide. The differences are not uniformly distributed. Exon 3 encodes residues 5 through 191 and includes the high-affinity heparinbinding site. In this region there are 29 differences which corresponds to 85% identity when comparing human and mouse. In many cases the replacements are not conservative. For example, in one of the heparin-binding motifs (residues 23-29) a serine residue in the human sequence is replaced by a proline residue in the mouse sequence. Exons 6 and 7 are homologous with exons 1 and 2 of procollagen and may represent an example of exon shuffling (Lawler and Hynes, 1986). There are six substitutions in exon 6 and none in exon 7. This seems to mark a transition point in terms of the degree to which the sequences vary. From exon 7 to the end of molecule the human and mouse sequences have an extremely high level of homology (98% identity over 828 residues). This region includes two copies of the VTCG sequence which has been reported to be involved in hematopoetic cell attachment (Rich et al., 1990). It also includes the RGDA cell binding domain. The attachment of some cells to thrombospondin can be inhibited by RGD-containing peptides (Lawler et aZ., 1988). In addition, the vitronectin receptor is retained on thrombospondin-Sepharose columns and is eluted with RGD-containing peptides (Lawler et al., 1988). Both types of cell binding motifs are conserved in mouse and human. Furthermore, these sequences are also found in the chicken sequence which includes three copies of the VTCG sequence (Lawler et al., 1991).

ET

AL.

Exon 22 encodes the last two residues and the stop codon as well as approximately 2 kb of 3’-untranslated region (Fig. 3C). The 3’ untranslated regions of the human and mouse sequences are very similar. The nucleotide sequence around the AATAAA sequence is highly conserved, with only two differences and no gaps in the last 100 nucleotides. Both the murine and human sequences have multiple copies of the ATTT and TATT sequences which have been reported to decrease message stability (Hennessy et al., 1989). Both sequences end with a TATTT sequence which may result in a decreased stability of the poly(A) tail. The 3’-untranslated portion of the mouse gene includes a second polyadenylation site (AATAAA) that does not exist in the human sequence. The resulting mRNA would be approximately 550 bp shorter than the 6-kb mRNA that has been detected in human and mouse cells. Northern blots for thrombospondin frequently have numerous smaller bands at lower intensity than the principal band, due, at least in part, to the numerous sites for endonuclease cleavage in the 3’-untranslated region (unpublished results). As a result we cannot exclude the possibility that this polyadenylation signal is used. ACKNOWLEDGMENTS We thank D. A. Swing and B. Chor for excellent technical assistance. Dr. Doug Gray, Dr. Rudolf Jaenisch, Dr. Laurie JacksonGrusby, and Dr. Philip Leder kindly provided genomic libraries. We thank Amy Williams and Dr. Tucker Collins for synthesizing the oligonucleotide. We also thank Dr. Temple Smith, Kathleen Klose, and Susan Russo for help with the computer analysis. The manuscript was typed by Alice Callahan and edited by Sami Lawler. This research was supported, in part, by the National Cancer Institute, DHHS, under Contract NOl-CO-74101 with A.B.L. and by National Institutes of Health Grants HL-28749 and HL-42443 from the National Heart, Lung and Blood Institute.

REFERENCES BORNSTEIN, P., ALFI, D., DEVARAYALA, S., FRAIWON, P., AND Lf, P. (1990). Characterization of the mouse thrombospondin gene and evaluation of the role of the first intron in human gene expression. J. Biol. Chem. 265: 16691-16698. BRIGGS, M. R., KADMAGA, J. T., BELL, S. P., AND TJIAN, R. (1986). Purification and biochemical characterization of the promoter-specific transcription factor, SPl. Science 234: 4752. BUCHBERG, A. M., BEDIGJAN, H. G., TAYMR, B. A., BROWNELL, E., IHLE, J. N., NAGATA, S., JENKINS, N. A., AND COPELAND, N. G. (1966). Localization of Eui-2 to chromosome 11: Linkage to other proto-oncogene and growth factor loci using interspecific backcross mice. Oncogene Res. 2: 149-165. CATO, A. C. B., GEISSE, S., WENZ, M., ESTPHAL, H. M., AND BEZATO, M. (1964). The nucleotide sequences recognized by the glucocorticoid receptor in the rabbit uteroglobin gene re-

MURINE gion are located far upstream tion. EMBO J. 3: 2771-2778.

from

the initiation

THROMBOSPONDIN of transcrip-

and sequencing 266: 8039-8043.

5.

DONOVIEL, D. B., AND BORNSTEIN, P. (1988). The thrombospondin gene is inducible by basic fibroblast growth factor (bFGF) and interleukin-1 (IL-l). J. Cell Biol. 107(Part 3): 596a.

22.

6.

DONOVIEL, D. B., FRAMSON, P., ELDRIDGE, C. F., COOKE, M., KOBAYASHI, S., AND BORNSTEIN, P. (1988). Structural analysis and expression of the human thombospondin gene promoter. J. Biol. Chm. 263: 18590-18593.

23.

7.

DYNAN, Trends

8.

GREEN, E. L. (1981). Linkage, recombination, and mapping. In “Genetics and Probability in Animal Breeding Experiments,” pp. 77-113. Macmillan, New York. HAI, T., Lru, F., ALLEGRE?TO, E. A., KARIN, M., AND GREEN, M. R. (1988). A family of immunologically related transcription factors that includes multiple forms of ATF and API. Genes Dev. 2: 1216-1226.

9.

W. S. (1986). Promoters Genet., 2: 196-197.

for

housekeeping

HARDON, E. M., FR.AIN, M., PAONESSA, G., AND CORTESE, R. (1988). Two distinct factors interact with the promoter regions of several liver-specific genes. EMBO J. 7: 1711-1719.

11.

HENNESSY, S. W., FRAZIER, B. A., KIM, D. D., DEZCKWERTH, T. L., BAUMGARTEL, D. M., ROT~EIN, P., AND FRAZIER, W. A. (1989). Complete thrombospondin mRNA sequence includes potential regulatory sites in the 3’ untranplated region. J. Cell Biol. 108: 729-736.

12.

13.

14.

15.

16.

IMAGAWA, M., CHIU, R., AND KARIN, M. (1987). Transcription factor AP-2 mediates induction by two different signaltransduction pathways: Protein kinase C and CAMP. Cell 51: 251-260. ISHII, S., MERLINO, G. T., AND PASTAN, I. (1985a). Promoter region of the human Harvey ras proto-oncogene similarity to the EGF receptor proto-oncogene promoter. Science 230: 1378-1381. ISHII, S., Xv, Y-M, STRATTON, R. G. T., AND PASTAN, I. (1985b). quence of the promoter region growth factor receptor gene. Proc. 4920-4924.

24.

genes.

10.

M., ROE, B. A., MERLINO, Characterization and seof the human epidermal Natl. Ad. Sci. USA 82:

JAFFE, E., BORNSTEIN, P., AND DISTJZCHE, C. M. (1990). Mapping of the thrombospondin gene to human chromosome 15 and mouse chromosome 2 by in situ hybridization. Genomics 7: 123-126. JENKINS, N. A., COPELAND, N. G., TAYLOR, B. A., AND LEE, B. K. (1982). Organization, distribution, and stability of endogenous ecotropic murine leukemia virus DNA sequences in chromosomes of Mus mwculus. J. Virol. 43: 26-36.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

17.

JONES, K. A., AND TJIAN, R. (1985). Spl binds to promoter sequences and activates herpes simplex virus “immediateearly” gene transcription in vitro. Nature 317: 179-182.

18.

KORNBLIH~, A. R., AND GUTMAN, biology of the extracellular matrix 465-507.

19.

LAHERTY, C. D., GERMAN, T. M., ANLI DIXIT, V. M. (1989). Characterization of the promoter region of the human thrombospondin gene. J. Bid. Chem. 264: 11222-11227. LA-R, J., DERICK, L. M., CONNOLLY, J. E., CHEN, J. H., AND CHAO, F. C. (1985). The structure of human platelet thrombospondin. J. Bid. Ckm. 260: 3762-3772.

36.

LAWLER,

37.

20.

21.

J., DUQUFZ~E,

M.,

A. (1988). proteins.

AND FERRO,

The molecular Bid. Rev. 63: 35.

P. (1991).

Cloning

599

GENE of chicken

thrombospondin.

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