Expression and regulation of the streptokinase gene

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 a pproaches are described here to study the expression of the streptokinase g ene, skn. Southern hybridization analysis demonstrated homology of synteny of ska, skc, and skg in the genomes of the above serogroups. S1 nuclease ma pping, the use of transcriptional fusions to beta-galactosidase and lucifer ase reporter genes, in conjunction with site-directed mutagenesis, led to t he localization of the core promoter region of skc and the identification o f a cis-active upstream region required for full promoter activity. Circula r permutation analysis of the promoter upstream region identified an Intrin sic DNA bending locus as the pivotal DNA element stimulating the activity o f the core promoter. The detection of skn allele-specific expression phenot ypes, 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 uni dentified transcriptional activator that contacts the bent DNA region. Tran scription termination of skc is directed by a bidirectional terminator whos e structural requirements for termination efficiency were determined with b ase substitution mutants fused to a chloramphenicol acetyl transferase repo rter. Finally, mutagenic plasmids are described for insertion-duplication a nd allele replacement mutagenesis of the skn locus. (C) 2000 Academic Press .