title: Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences.
abstract: We have 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. We constructed the plasmid pMSB212 which 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 EcoR I 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 known genes and to identify new genes encoded on a cloned fragment.
authors.affiliation: Pharmaceutical Research Laboratories, Meiji Seika Kaisha, Ltd., Yokohama, Japan.
authors.forename: H
authors.initials: H
authors.lastname: Anzai
authors.forename: Y
authors.initials: Y
authors.lastname: Kumada
authors.forename: O
authors.initials: O
authors.lastname: Hara
authors.forename: T
authors.initials: T
authors.lastname: Murakami
authors.forename: R
authors.initials: R
authors.lastname: Itoh
authors.forename: E
authors.initials: E
authors.lastname: Takano
authors.forename: S
authors.initials: S
authors.lastname: Imai
authors.forename: A
authors.initials: A
authors.lastname: Satoh
authors.forename: K
authors.initials: K
authors.lastname: Nagaoka
mesh_terms: D002876:Chromosomes, Bacterial; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D009943:Organophosphorus Compounds / Q000096:biosynthesis*; D010957:Plasmids; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
chemical_list: D004269:DNA, Bacterial; D009943:Organophosphorus Compounds; C003122:bialaphos