Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences (original) (raw)
1988, The Journal of Antibiotics
Wehave developed a method for gene replacement in Streptomyces hygroscopicus which permits introduction of an in vitro derived mutation carried on a plasmid into the chromosome. Weconstructed the plasmid pMSB212which can replicate in S. hygroscopicus and contains the step5 gene of the bialaphos biosynthetic pathway which was inactivated by a frame-shift mutation caused by filling in the cohesive ends of the EcoKI site in the structural gene. pMSB212 was introduced into a bialaphos producer strain and by protoplast regeneration of the primary thiostrepton-resistant transformants, non-producing mutants, were obtained. Biochemical and genetical analyses indicated that these mutants were specifically blocked by introduction of the frame-shift mutation in the step5 gene on the chromosome. This method will enable us to obtain isogenic mutants of knowngenes and to identify new genes encoded on a cloned fragment. Streptomycetes are very important microorganisms whichproduce manyantibiotics and enzymes of commercial value. Recent developments in Streptomyces gene cloning have resulted in cloning of the resistance, biosynthetic, and regulatory genes of antibiotics, and elucidation of Streptomyces gene organization and regulation1~7). Mutant analysis gives us muchinformation about gene function and regulation. Mutants usually have been obtained by using chemical or physical mutagens, but there have been reports recently about another mutagenic approach, gene replacement techniques in Escherichia coli8), Bacillus subtilis9~12:> or Saccharomyces cerevisiae13 > 14\
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Trends in Biotechnology, 1983
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