Cloning of the E. coli O-acetylserine sulfhydrylase gene: ability of the clone to produce a mutagenic product from azide and O-acetylserine.
The gene coding for O-acetylserine sulfhydrylase (OASS) from E. coli K12 was cloned into the vector pBR322 plasmid and expressed in a cysk mutant strain of E. coli that is deficient in O-acetylserine sulfhydrylase (OASS-). The clone containing the OASS gene was selected by using tetracycline-ammonium bismuth citrate medium. Retransformation of the hybrid plasmid into competent cysk mutant cells resulted in the recovery of a clone containing normal levels of O-acetylserine sulfhydrylase. Negative selection of retransformed cysk cells on 1,2,4-triazole plates resulted in the complete inhibition of growth indicating the presence of a functional OASS gene. The ability of the new clone to convert azide to its mutagenic metabolite was tested. Cultures of the clone cells containing significant levels of OASS activity were able to produce a mutagenic product from azide and O-acetylserine as tested on Salmonella typhimurium TA1530. This cloning method could be applied also to clone the same gene from eukaryotic sources.
Owais
W M
WM
Zoology Department, Kuwait University, Safat.
Gharaibeh
R
R
D001386:Azides / Q000378:metabolism*; D003001:Cloning, Molecular; D003547:Cysteine Synthase / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D012694:Serine / Q000031:analogs & derivatives* / Q000378:metabolism; D019810:Sodium Azide; D014170:Transformation, Genetic
D001386:Azides; D012694:Serine; D019810:Sodium Azide; D003547:Cysteine Synthase; C043943:O-acetylserine