Elsevier

Methods

Volume 21, Issue 2, June 2000, Pages 111-124
Methods

Regular Article
Expression and Regulation of the Streptokinase Gene

https://doi.org/10.1006/meth.2000.0982Get rights and content

Abstract

Recent research in various areas has appreciably expanded our knowledge of streptokinase, a plasminogen activator produced by all human group A (GAS), group C (GCS), and group G (GGS) streptococci. Several molecular genetic approaches are described here to study the expression of the streptokinase gene, skn. Southern hybridization analysis demonstrated homology of synteny of ska, skc, and skg in the genomes of the above serogroups. S1 nuclease mapping, the use of transcriptional fusions to β-galactosidase and luciferase reporter genes, in conjunction with site-directed mutagenesis, led to the localization of the core promoter region of skc and the identification of a cis-active upstream region required for full promoter activity. Circular permutation analysis of the promoter upstream region identified an intrinsic DNA bending locus as the pivotal DNA element stimulating the activity of the core promoter. The detection of skn allele-specific expression phenotypes, which proved not to be due to different skn mRNA half-lives, prompted allele swap experiments, showing that promoter activity is dictated by the host genetic background, rather than the sequence of the regulatory region. These findings suggest the involvement in skn expression of an as yet unidentified transcriptional activator that contacts the bent DNA region. Transcription termination of skc is directed by a bidirectional terminator whose structural requirements for termination efficiency were determined with base substitution mutants fused to a chloramphenicol acetyl transferase reporter. Finally, mutagenic plasmids are described for insertion–duplication and allele replacement mutagenesis of the skn locus.

References (33)

  • H Malke et al.

    FEMS Microbiol. Lett.

    (1994)
  • K.C Young et al.

    J. Biol. Chem.

    (1998)
  • H Malke et al.

    Gene

    (1985)
  • H Malke

    Zbl. Bakteriol.

    (1993)
  • W Tewodros et al.

    Microbial Pathogen.

    (1995)
  • L Rao et al.

    J. Mol. Biol.

    (1994)
  • Z Eichenbaum et al.

    Gene

    (1997)
  • Z Li et al.

    J. Microbiol. Methods

    (1997)
  • L.R Christensen

    J. Gen. Physiol.

    (1945)
  • X Wang et al.

    Science

    (1998)
  • H Malke et al.

    Proc. Natl. Acad. Sci. USA

    (1984)
  • H Malke et al.
  • T.T Huang et al.

    Infect. Immun.

    (1989)
  • V Kapur et al.

    Mol. Microbiol.

    (1995)
  • K Okada et al.

    Clin. Nephrol.

    (1995)
  • C Frank et al.

    Med. Microbiol. Immunol.

    (1995)
  • Cited by (23)

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