Download PDF
Thromb Haemost 1997; 78(03): 1104-1110
DOI: 10.1055/s-0038-1657694
Rapid Communication
Schattauer GmbH Stuttgart

Characterization and Targeting of the Murine α2-Antiplasmin Gene

K Okada
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
2   The Department of Physiology, Kinki University School of Medicine, Osaka, Japan
,
H R Lijnen
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
M Dewerchin
3   The Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium
,
A Belayew
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
,
O Matsuo
2   The Department of Physiology, Kinki University School of Medicine, Osaka, Japan
,
D Collen
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
3   The Center for Transgene Technology and Gene Therapy, Flanders Interuniversity Institute for Biotechnology, Leuven, Belgium
,
R Bernaerts
1   The Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
› Author Affiliations
Further Information

Publication History

Received 19 1996

Accepted after revision 08 April 1997

Publication Date:
12 July 2018 (online)

  PDF Download  Permissions and Reprints

Summary

α2-Antiplasmin (α2-AP) is the main physiological plasmin inhibitor in mammalian plasma. As a first step toward the generation of α2-AP deficient mice, the murine α2-AP 1 gene was characterized and a targeting vector for homologous recombination in embryonic stem (ES) cells constructed. Alignment of nucleotide sequences obtained from genomic subclones allowed location of exons 2 through 10 of the α2-AP 1gene, but failed to identify the 5’ boundary of exon 1. Compared to the human gene, exons 2 through 9 in the murine gene have identical size and intron-exon boundaries obeying the GT/AG rule. The 5’ boundary of exon 10 is identical in both genes while the 3’ non-coding region is 64 bp longer in the human gene. Introns 2,3,6 and 8 have similar sizes in the mouse and human genes; intron 1 is 6-fold smaller, introns 5, 7 and 9 are 2- to 3-fold smaller, whereas intron 4 is about 2-fold larger in the mouse gene. Compared to the human 5’ flanking sequence, an insertion of a simple repeat region with sequence (TGG)n has occurred. The open reading frame of the mouse α2-AP gene encodes a 491-amino acid protein comprising the experimentally determined NH2-terminus of the mature protein Val-Asp-Leu-Pro-Gly-.

A targeting vector, ppPNT.α2-AP, was constructed by introducing a homologous sequence of 8.3 kb in total in the parental pPNT vector. In pPNT.α2-AP, the neomycin resistance expression cassette replaces a 7 kb genomic fragment comprising exon 2 through part of exon 10 (including the stop codon), which represents the entire sequence encoding the mature protein, including the fibrin-binding domain, the reactive site peptide bond and the plasmin(ogen)-binding region. Electroporation of 129R1 embryonic stem (ES) cells with the linearized vector pPNT.α2-AP yielded three targeted clones with correct homologous recombination at the 5’- and 3’-ends, as confirmed by Southern blot analysis of purified genomic DNA with appropriate restriction enzymes and probes. These targeted clones will be used to generate α2-AP deficient mice.

 

  • References

  • 1 Collen D, Lijnen HR. Basic and clinical aspects of fibrinolysis and throm bolysis. Blood 1991; 78: 3114-3124
    PubMedGoogle Scholar
  • 2 Carmeliet P, Collen D. Gene targeting and gene transfer studies of the biological role of the plasminogen/plasmin system. Thromb Haemost 1995; 74: 429-436
    Article in Thieme ConnectPubMedGoogle Scholar
  • 3 Collen D. Identification and some properties of a new fast-reacting plas min inhibitor in human plasma. Eur J Biochem 1976; 69: 209-216
    CrossrefPubMedGoogle Scholar
  • 4 Moroi M, Aoki N. Isolation and characterization of alpha2-plasmin inhibi tor from human plasma. A novel proteinase inhibitor which inhibits acti vator-induced clot lysis. J Biol Chem 1976; 251: 5956-5965
    PubMedGoogle Scholar
  • 5 Mullertz S, Clemmensen I. The primary inhibitor of plasmin in human plas ma. Biochem J 1976; 159: 545-553
    CrossrefPubMedGoogle Scholar
  • 6 Wiman B, Collen D. On the mechanism of the reactions between human a2-antiplasmin and plasmin. J Biol Chem 1979; 254: 9291-927
    PubMedGoogle Scholar
  • 7 Holmes WE, Nelles L, Lijnen HR, Collen D. Primary structure of human a2-antiplasmin, a serine protease inhibitor (serpin). J Biol Chem 1987; 262: 1659-1664
    PubMedGoogle Scholar
  • 8 Lijnen HR, Wiman B, Collen D. Partial primary structure of human α2-antiplasmin. Homology with other plasma protease inhibitors. Thromb Haemost 1982; 48: 311-314
    Article in Thieme ConnectPubMedGoogle Scholar
  • 9 Lijnen HR, Holmes WE, Van HoefB, Wiman B, Rodriguez H, Collen D. Amino acid sequence of human a2-antiplasmin. Eur J Biochem 1987; 166: 565-5674
    CrossrefPubMedGoogle Scholar
  • 10 Bangert K, Johnsen AH, Christensen U, Thorsen S. Different N-terminal forms of α2-plasmin inhibitor in human plasma. Biochem J 1993; 291: 623-625
    CrossrefPubMedGoogle Scholar
  • 11 Sugiyama N, Sasaki T, Iwamoto M, Abiko Y. Binding site of α2-plasmin inhibitor to plasminogen. Biochim Biophys Acta 1988; 952: 1-7
    CrossrefPubMedGoogle Scholar
  • 12 Ichinose A, Tamaki T, Aoki N. Factor XHI-mediated cross-linking of NH2-terminal peptide of α2-plasmin inhibitor to fibrin. FEBS Lett 1983; 153: 369-371
    CrossrefPubMedGoogle Scholar
  • 13 Lijnen HR, Collen D. Congenital and acquired deficiencies of components of the fibrinolytic system and their relation to bleeding or thrombosis. Fibrinolysis 1989; 03: 67-77
    PubMedGoogle Scholar
  • 14 Mui PTK, James HI, Ganguly P. Isolation and properties of a low molecu lar weight antiplasmin of human blood platelets and serum. Br J Haematol 1975; 29: 627-637
    CrossrefPubMedGoogle Scholar
  • 15 Kato A, Nakamura Y, Miura O, Hirosawa S, Sumi Y, Aoki N. Assignment of the human alpha2-plasmin inhibitor gene (PLI) to chromosome region 18pl 1.1-ql 1.2 by in situ hybridization. Cytogenet Cell Genet 1988; 47: 209-211
    CrossrefPubMedGoogle Scholar
  • 16 Hirosawa S, Nakamura Y, Miura O, Sumi Y, Aoki N. Organization of the human α2-plasmin inhibitor gene. Proc. Natl. Acad. Sci. USA 1988; 85: 6836-640
    CrossrefPubMedGoogle Scholar
  • 17 Menoud PA, Sappino N, Boudal-Khoshbeen M, Vassalli JD, Sappino AP. The kidney is a major site of α2-antiplasmin production. J Clin Invest 1996; 97: 2478-2484
    CrossrefPubMedGoogle Scholar
  • 18 Lijnen HR, Van HoefB, Beelen V, Collen D. Characterization of the murine plasma fibrinolytic system. Eur J Biochem 1994; 224: 863-871
    CrossrefPubMedGoogle Scholar
  • 19 Pierce JC, Sternberg N. Using bacteriophage PI system to clone high molecular weight genomic DNA. Methods Enzymol 1992; 216: 549-574
    PubMedGoogle Scholar
  • 20 Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, N.Y; 1989
    Google Scholar
  • 21 Güssow D, Clackson TP. Direct clone characterization from plaques and colonies by the polymerase chain reaction. Nucl Acids Res 1989; 17: 4000
    CrossrefPubMedGoogle Scholar
  • 22 Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 1984; 12: 387-395
    CrossrefPubMedGoogle Scholar
  • 23 Quandt K, Freeh K, Karas H, Wingender E, Werner T. Matlnd and Matlnspector – New fast and versatile tools for the detection of consensus matches in nucleotide sequence data. Nucleic Acids Res 1995; 23: 4878-4884
    CrossrefPubMedGoogle Scholar
  • 24 Tybulewicz VL, Crawford CE, Jackson PK, Bronson RT, Mulligan RC. Neonatal lethality and lymphopenia in mice with a homozygous disruption of the c-abl proto-oncogene. Cell 1991; 65: 1153-1163
    CrossrefPubMedGoogle Scholar
  • 25 Carmeliet P, Kieckens L, Schoonjans L, Ream B, Van NuffelenA, Prendergast G, Cole M, Bronson R, Collen D, Mulligan RC. Plasminogen activator inhibitor 1 gene-deficient mice. I. Generation by homologous recombination and characterization. J Clin Invest 1973; 92: 2746-2755
    PubMedGoogle Scholar
  • 26 Breathnach R, Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem 1981; 50: 349-383
    CrossrefPubMedGoogle Scholar
  • 27 Krayev AS, Markusheva DA, Kramerov DA, Ryskov AP, Skryabin KG, Bayev AA, Georgiev GP. Ubiquitous transposon-like repeats bl and b2 of the mouse genome. Nucleic Acids Res 1982; 10: 7461-7475
    CrossrefPubMedGoogle Scholar
  • 28 Klug J, Beato M. Binding of YY1 to a site overlapping a weak TATA box is essential for transcription from the uteroglobin promoter in endometrialcells. Mol Cell Biol 1996; 16: 6398-6407
    CrossrefPubMedGoogle Scholar
  • 29 Carmeliet P, Collen D. Targeted gene manipulation and transfer of the plasminogen and coagulation systems in mice. Fibrinolysis 1996; 10: 195-213
    PubMedGoogle Scholar
  • 30 Guigo R, Knudsen S, Drake N, Smith T. Prediction of gene structure. J Mol Biol 1992; 245: 45-56
    PubMedGoogle Scholar
  • 31 Vilalta A, Kickhoefer VA, Rome LH, Johnson DL. The rat vault RNA gene contains a unique RNA polymerase III promoter composed of both external and internal elements that function synergistically. J Biol Chem 1994; 269: 29752-29759
    PubMedGoogle Scholar
  • 32 Willis IM. RNA polymerase III: genes, factors and transcriptional specifi city. Eur J Biochem 1993; 212: 1-12
    CrossrefPubMedGoogle Scholar