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Organization of the gene encoding mouse vitronectin
Gene, 134(1993)303-304 ElsevierScience
Publishers
303
B.V.
GENE 07457
Organization (Complement
Dietmar
of the gene errcoding mouse vitronectin*
S-protein;
cell adhesion;
Seiffert, Judith
Lahorutory,jiwCIinicul
Exprrirnental
Received by J.A. Gorman:
fibrinolysis;
Poenninger Physiology.
blood coagulation;
gene cloning;
murine)
and Bernd R. Binder
Unicersify
4 June 1993; Revis~d~Accepted:
of’I/irnnu,
13 July/l4
Vienncc, Ausfritr.
Tel. ( 45 222)
July 1993; Received at publishers:
404X023(): F
4087500
27 July 1993
SUMMARY
The gene (Vn) encoding the mouse vitronectin was isolated and its nucleotide sequence determined. approximately 3 kb of genomic DNA. Alignment of the genomic sequence with that of the cDNA consists of eight exons, interrupted by seven introns ranging in size from 78 to 723 bp.
Vitronectin (Vn) belongs to a group of cell adhesion molecules which mediate adhesion through a common RGD-dependent mechanism (for a review, see Tomasini and Mosher, 1990; Preissner, 1991). Vn is identical to the S-protein of the complement system, and also appears to exert several regulatory functions in the coagulation and fibrinolytic system. The primary structure of mouse, rabbit and human Vn (i.e., the protein) has been determined by deduction from cDNA sequences. Comparison of the deduced aa sequences revealed a high degree of conservation (approximately 80%) between all three species (Jenne and Stanley, 1985; Suzuki et al., 1985; Sato et al., 1990; Seiffert et al., 1991). The functional similarity of human and murine Vn in respect to its binding to type-l plasminogen activator inhibitor, cell, and heparin suggests that the mouse is an ideal model system to elucidate the role(s) of Vn (Seiffert et al., 1991). To further characterize the gene encoding murine Vn, a mouse (BALB/c) genomic library in hEMBL3 SP6/T7 was screened with a full-length mouse Vn cDNA probe Correspondencr
Research
(Seiffert et al., 1991). Two non-overlapping, independent phage clones were isolated and partially sequenced. The intervening sequence between the two non-overlapping clones (i.e., a small fragment of exon III) was obtained by standard PCR techniques. The transcribed nt sequence for the Vn protein covers about 3 kb of genomic DNA. The position of exons and introns was determined by alignment of the genomic sequence with that of the cDNA. The gene consists of eight exons interrupted by seven introns ranging in size from 78 to 725 bp. There is only one polyadenylation site (AATAAA) located 55 bp downstream from the stop codon. Comparison of the gene organization of the mouse gene with that of the human gene (Jenne and Stanley, 1987) revealed a similar size and identical localization and phase class of introns, indicating that the structure of the gene was highly conserved during mammalian phylogeny. This work was supported by grants to D.S. and B.R.B. from the ‘Fond zur Foerderung der wissenschaftlichen Forschung Oesterreichs’.
CO: Dr. D. Seiffert
Institute,
CVB-3,
at his present address: The Scripps 10666 North Torrey Pines Road, La Jolla,
CA 92037, IJSA. Tel. (1-619) 554-7153; Fax (1-619) 554-6404. *On request, the authors will supply detailed experimental evidence the conclusions
The gene covers revealed that I/n
reached
for
REFERENCES
in this Brief Note.
Abbreviations: aa, amino acid(s): bp, base pair(s); cDNA, DNA compiementary to RNA; kb, kilobase or 1000 bp: nt, nucleotide(s); PCR, polymerase chain reaction: Vn, vitronectin; Vn, gene encoding Vn.
Jenne, D. and Staniey, K.K.: Mo)ecuFdr cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion. EMBO J. 4 (1985) 3153-3157. Jenne, D. and Stanley, K.K.: Nucleotide sequence and organization of
120 3 240 20 360 43 480 74 600 114 720 154 CTTCCGAGGTGACTCTATGCAGGTCTGGTGGCGGTCG~TCT~CTTAGGCTCATCTGCTTCTCACACAGTTTCCCCTGTTCTAGGGCAGTACTGCTAT~GCTA~T~~CGG~G 1yGlnTyrCysTyrGluLeuAspGluThrAlaV aPheArgG
640 168 960 203
GTATGTCCTCACCCTTCTGATCTCTGTCTTTCAGGGTAGTCAGTACTGGCGCTTT~G~TGGGGTCCTG~CCCTGGTTATCCCC~CATCTCC~GGCTTCAGTGGCATACCA~ GlySerGlnTyrTrpArgPheGluAspGlyValLeuAspProGlyTyrProArgAsnIleSerGluGlyPheSerGlyIleProAs
1200 232
CAATGTTGATGCAGCGTTCGCCCTTCCTGCCCACCGTTACAGTGGCCGG~GGGTCTACTTCTTCMGGGTACTCAGGGTG~G~~GGGGGCAGGT~TG~GTCCCTGTTCTAT pAsnValAspAlPAlaPheAlaLeuProAlaHisArgTyrSerGlyArgGluArgValTyrPhePheLysG
1320 256 1440 276 1560 307 1680 1600 1920 2040 2160
‘3:: AAAAAACAGMGTCTMGCGTAG
CATCAGCCG~CTGGCATGGTGTGCCAGGGAAAGTGGACGCTGCTATGGCCGGCCGCATCTACGTCACTGGCTCCTTATCCCACTCTGCCCMGCC eIleSerArgA5nTrpHisGlyValProGlyLysValAspAl~laMetAlaGlyArgIleTyrValThrGlySerLeuSerHisSerAlaGlnAlaLysLysGlnLysSerLysArgAr
2400 354
MGCCOAAAGCGCTATCGTTCACGCCOAGGGCGTGGCCACA~CGCAGCCA~GCTC~CTCCCGTCGTTCATCACGTTCMTCTGGTTCTCTTTGTTCTCCAGC~G~~GTGGGCT gSerArgLysArgTyrArgSerArgArgGlyArgGlyHisArgArgSerGlnSerSerAsnSerArgArgSerSerArgSerIleTrpPheSerLeuPheSerSerGluGluSerGlyLe
2520 394
AGGAACCTACAACAACTATGTTATGATATGCACTGGCTTGTACCTGCCACCTGC~GCCCATTCA~GCGTCTATTTCTTCTCTGGAGGTGGGAGCCCCTTTCTACTCCTTAWICTGGT uGlyThrTyrAsnAsnTyrAspTyrAspMetAspTrpLeuValProAlaThrCysGluProIleGlnSerValTyrPhePheSerGlyA
2640 424
GAAAMGCCTTTATCCTTCT
TTTGGCTAGGCCTTGCCTGCAGGTCTTGGTCCCTGGGTGCT~CACAGCCTGTA~TGGT~CATTATCTGCTTGGTG~TGTTCACTTTCCCTTCTGA
2760
TGGGACCAGTGOAW\GGATCAGGCAWV\TCTCACATMCCCTTTTTCCATTCTTTCTTTCA~C~TACTACC~GTC~CCTTA~CCCGGC~GTG~CTCTGT~TCCTCCCTA spLysTyrTyrArpValAsnLeuArgThrArgArgValAspSerValA5nProProTy
2880 443
CCCACGCTCCATTOCTCAGTATTGGCTGGGCTGCCCOACCTCT~~GTAG~TCA~GCCCACTCGGCT~GCTTCAG~GCCTCATCTCTTTCTCCCAGCCC~T~GTCTGT rProArgSerIleAlaGlnTyrTrpLeuGlyCysProThrSerGluLys l
3000 459
TGGCTACOAGTTTMOACTACTGTCCTGAATAGGGTGGCCTCTCACTGGGCTCA
3120
GGTGCTCTCCCTAAATCAGAGACACTGGGGGCTTGTGG~GCACCCTTCCTTCTGTGGCACCACACCCTCCCCGGGT~GGCTTCTTT~CCAT~GTT~CTAG~GGCCGGG~CG
3240
TGAGAAACAGGTGGGTGGA
Fig. I. The nt sequence
3259
of the mouse
I/n gene and 3’ flanking
region,
and the deduced
aa sequence.
The aa sequence
for the mature
protein
is
numbered l-459, and the aa sequence of the inferred signal peptide is represented by negative numbers. Underlined sequences are from 5’ to 3’: the PCR primer used for the amplification of parts of exon 111 from genomic DNA, and the polyadenylation signal. Asterisk marks the stop codon. Methods used for molecular cloning were based on Sambrook et al. ( 1989). The GenBank accession number is X72091. the human S-protein gene: repeating peptide motifs in the ‘pexin’ family and a model for their evolution, Biochemistry 26 (1987) 673556742. Preissner, K.T.: Structure and biological role of vitronectin. Ann”. Rev. Cell. Biol. 7 (1991) 2755310.
Seiffert, D.. Keeton, M., Eguchi, Y., Sawdey. M. and Loskutoff, D.J.: Detection of vitronectin mRNA in tissues and cells of the mouse. Proc. Natl. Acad. Sci. USA 88 (1991) 9402-9406. E.G., Pierschbacher. M.D. and Suzuki. S.. Oldberg, A.. Hayman. Ruoslahti. E.: Complete amino acid sequence of human vitronectin
Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY, 1989. Sato, R., Komine, Y., Imanaka, T. and Takano, T.: Monoclonal antibody EMR la,I212D recognizing site of deposition of extracellular lipid in atherosclerosis: isolation and characterization of a cDNA clone for the antigen. J. Biol. Chem. 265 ( 1990) 21232 21236.
deduced from cDNA. Similarity of cell attachment sites in v,itronectin and tibronectin. EMBO J. 4 (1985) 2519-2524. Tomasini, B.R. and Mosher, D.F.: Vitronectin. Prog. Hemostasrs Thromb. IO ( 1990) 269- 305.
Publishers
303
B.V.
GENE 07457
Organization (Complement
Dietmar
of the gene errcoding mouse vitronectin*
S-protein;
cell adhesion;
Seiffert, Judith
Lahorutory,jiwCIinicul
Exprrirnental
Received by J.A. Gorman:
fibrinolysis;
Poenninger Physiology.
blood coagulation;
gene cloning;
murine)
and Bernd R. Binder
Unicersify
4 June 1993; Revis~d~Accepted:
of’I/irnnu,
13 July/l4
Vienncc, Ausfritr.
Tel. ( 45 222)
July 1993; Received at publishers:
404X023(): F
4087500
27 July 1993
SUMMARY
The gene (Vn) encoding the mouse vitronectin was isolated and its nucleotide sequence determined. approximately 3 kb of genomic DNA. Alignment of the genomic sequence with that of the cDNA consists of eight exons, interrupted by seven introns ranging in size from 78 to 723 bp.
Vitronectin (Vn) belongs to a group of cell adhesion molecules which mediate adhesion through a common RGD-dependent mechanism (for a review, see Tomasini and Mosher, 1990; Preissner, 1991). Vn is identical to the S-protein of the complement system, and also appears to exert several regulatory functions in the coagulation and fibrinolytic system. The primary structure of mouse, rabbit and human Vn (i.e., the protein) has been determined by deduction from cDNA sequences. Comparison of the deduced aa sequences revealed a high degree of conservation (approximately 80%) between all three species (Jenne and Stanley, 1985; Suzuki et al., 1985; Sato et al., 1990; Seiffert et al., 1991). The functional similarity of human and murine Vn in respect to its binding to type-l plasminogen activator inhibitor, cell, and heparin suggests that the mouse is an ideal model system to elucidate the role(s) of Vn (Seiffert et al., 1991). To further characterize the gene encoding murine Vn, a mouse (BALB/c) genomic library in hEMBL3 SP6/T7 was screened with a full-length mouse Vn cDNA probe Correspondencr
Research
(Seiffert et al., 1991). Two non-overlapping, independent phage clones were isolated and partially sequenced. The intervening sequence between the two non-overlapping clones (i.e., a small fragment of exon III) was obtained by standard PCR techniques. The transcribed nt sequence for the Vn protein covers about 3 kb of genomic DNA. The position of exons and introns was determined by alignment of the genomic sequence with that of the cDNA. The gene consists of eight exons interrupted by seven introns ranging in size from 78 to 725 bp. There is only one polyadenylation site (AATAAA) located 55 bp downstream from the stop codon. Comparison of the gene organization of the mouse gene with that of the human gene (Jenne and Stanley, 1987) revealed a similar size and identical localization and phase class of introns, indicating that the structure of the gene was highly conserved during mammalian phylogeny. This work was supported by grants to D.S. and B.R.B. from the ‘Fond zur Foerderung der wissenschaftlichen Forschung Oesterreichs’.
CO: Dr. D. Seiffert
Institute,
CVB-3,
at his present address: The Scripps 10666 North Torrey Pines Road, La Jolla,
CA 92037, IJSA. Tel. (1-619) 554-7153; Fax (1-619) 554-6404. *On request, the authors will supply detailed experimental evidence the conclusions
The gene covers revealed that I/n
reached
for
REFERENCES
in this Brief Note.
Abbreviations: aa, amino acid(s): bp, base pair(s); cDNA, DNA compiementary to RNA; kb, kilobase or 1000 bp: nt, nucleotide(s); PCR, polymerase chain reaction: Vn, vitronectin; Vn, gene encoding Vn.
Jenne, D. and Staniey, K.K.: Mo)ecuFdr cloning of S-protein, a link between complement, coagulation and cell-substrate adhesion. EMBO J. 4 (1985) 3153-3157. Jenne, D. and Stanley, K.K.: Nucleotide sequence and organization of
120 3 240 20 360 43 480 74 600 114 720 154 CTTCCGAGGTGACTCTATGCAGGTCTGGTGGCGGTCG~TCT~CTTAGGCTCATCTGCTTCTCACACAGTTTCCCCTGTTCTAGGGCAGTACTGCTAT~GCTA~T~~CGG~G 1yGlnTyrCysTyrGluLeuAspGluThrAlaV aPheArgG
640 168 960 203
GTATGTCCTCACCCTTCTGATCTCTGTCTTTCAGGGTAGTCAGTACTGGCGCTTT~G~TGGGGTCCTG~CCCTGGTTATCCCC~CATCTCC~GGCTTCAGTGGCATACCA~ GlySerGlnTyrTrpArgPheGluAspGlyValLeuAspProGlyTyrProArgAsnIleSerGluGlyPheSerGlyIleProAs
1200 232
CAATGTTGATGCAGCGTTCGCCCTTCCTGCCCACCGTTACAGTGGCCGG~GGGTCTACTTCTTCMGGGTACTCAGGGTG~G~~GGGGGCAGGT~TG~GTCCCTGTTCTAT pAsnValAspAlPAlaPheAlaLeuProAlaHisArgTyrSerGlyArgGluArgValTyrPhePheLysG
1320 256 1440 276 1560 307 1680 1600 1920 2040 2160
‘3:: AAAAAACAGMGTCTMGCGTAG
CATCAGCCG~CTGGCATGGTGTGCCAGGGAAAGTGGACGCTGCTATGGCCGGCCGCATCTACGTCACTGGCTCCTTATCCCACTCTGCCCMGCC eIleSerArgA5nTrpHisGlyValProGlyLysValAspAl~laMetAlaGlyArgIleTyrValThrGlySerLeuSerHisSerAlaGlnAlaLysLysGlnLysSerLysArgAr
2400 354
MGCCOAAAGCGCTATCGTTCACGCCOAGGGCGTGGCCACA~CGCAGCCA~GCTC~CTCCCGTCGTTCATCACGTTCMTCTGGTTCTCTTTGTTCTCCAGC~G~~GTGGGCT gSerArgLysArgTyrArgSerArgArgGlyArgGlyHisArgArgSerGlnSerSerAsnSerArgArgSerSerArgSerIleTrpPheSerLeuPheSerSerGluGluSerGlyLe
2520 394
AGGAACCTACAACAACTATGTTATGATATGCACTGGCTTGTACCTGCCACCTGC~GCCCATTCA~GCGTCTATTTCTTCTCTGGAGGTGGGAGCCCCTTTCTACTCCTTAWICTGGT uGlyThrTyrAsnAsnTyrAspTyrAspMetAspTrpLeuValProAlaThrCysGluProIleGlnSerValTyrPhePheSerGlyA
2640 424
GAAAMGCCTTTATCCTTCT
TTTGGCTAGGCCTTGCCTGCAGGTCTTGGTCCCTGGGTGCT~CACAGCCTGTA~TGGT~CATTATCTGCTTGGTG~TGTTCACTTTCCCTTCTGA
2760
TGGGACCAGTGOAW\GGATCAGGCAWV\TCTCACATMCCCTTTTTCCATTCTTTCTTTCA~C~TACTACC~GTC~CCTTA~CCCGGC~GTG~CTCTGT~TCCTCCCTA spLysTyrTyrArpValAsnLeuArgThrArgArgValAspSerValA5nProProTy
2880 443
CCCACGCTCCATTOCTCAGTATTGGCTGGGCTGCCCOACCTCT~~GTAG~TCA~GCCCACTCGGCT~GCTTCAG~GCCTCATCTCTTTCTCCCAGCCC~T~GTCTGT rProArgSerIleAlaGlnTyrTrpLeuGlyCysProThrSerGluLys l
3000 459
TGGCTACOAGTTTMOACTACTGTCCTGAATAGGGTGGCCTCTCACTGGGCTCA
3120
GGTGCTCTCCCTAAATCAGAGACACTGGGGGCTTGTGG~GCACCCTTCCTTCTGTGGCACCACACCCTCCCCGGGT~GGCTTCTTT~CCAT~GTT~CTAG~GGCCGGG~CG
3240
TGAGAAACAGGTGGGTGGA
Fig. I. The nt sequence
3259
of the mouse
I/n gene and 3’ flanking
region,
and the deduced
aa sequence.
The aa sequence
for the mature
protein
is
numbered l-459, and the aa sequence of the inferred signal peptide is represented by negative numbers. Underlined sequences are from 5’ to 3’: the PCR primer used for the amplification of parts of exon 111 from genomic DNA, and the polyadenylation signal. Asterisk marks the stop codon. Methods used for molecular cloning were based on Sambrook et al. ( 1989). The GenBank accession number is X72091. the human S-protein gene: repeating peptide motifs in the ‘pexin’ family and a model for their evolution, Biochemistry 26 (1987) 673556742. Preissner, K.T.: Structure and biological role of vitronectin. Ann”. Rev. Cell. Biol. 7 (1991) 2755310.
Seiffert, D.. Keeton, M., Eguchi, Y., Sawdey. M. and Loskutoff, D.J.: Detection of vitronectin mRNA in tissues and cells of the mouse. Proc. Natl. Acad. Sci. USA 88 (1991) 9402-9406. E.G., Pierschbacher. M.D. and Suzuki. S.. Oldberg, A.. Hayman. Ruoslahti. E.: Complete amino acid sequence of human vitronectin
Sambrook, J., Fritsch, E.F. and Maniatis, T.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor. NY, 1989. Sato, R., Komine, Y., Imanaka, T. and Takano, T.: Monoclonal antibody EMR la,I212D recognizing site of deposition of extracellular lipid in atherosclerosis: isolation and characterization of a cDNA clone for the antigen. J. Biol. Chem. 265 ( 1990) 21232 21236.
deduced from cDNA. Similarity of cell attachment sites in v,itronectin and tibronectin. EMBO J. 4 (1985) 2519-2524. Tomasini, B.R. and Mosher, D.F.: Vitronectin. Prog. Hemostasrs Thromb. IO ( 1990) 269- 305.