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The cloning of the Escherichia coli K-12 deoxyribonucleoside operon
Authors:M Fischer  S A Short
Institution:Department of Microbiology, Wellcome Research Laboratories, Research Triangle Park, NC 27709 U.S.A.
Abstract:A 6.1-kb EcoRI DNA fragment containing the four structural genes (deoC, deoA, deoB, deoD) of the deoxyribonucleoside operon has been cloned into the plasmid pMFS53. By use of a unique, asymmetrically positioned HindIII site on the 6.1 kb insert, plasmids containing the deoC,deoA genes (pMFS50) or the deoB,deoD genes (pMFS55) have been constructed. Enzyme assays performed on extracts prepared from clones harboring pMFS53, pMFS50 or pMFS55 revealed that each clone possessed amplified deo enzyme levels and that the spectrum of enzyme amplification corresponded to the genetic composition of the plasmids carried by each clone. A plasmid (pMFS50l) having functional deoA, deoB and deoD genes but devoid of the deo regulatory region and a portion of the deoC structural gene has been isolated following treatment of BamHI cleaved pMFS53 and BAL31 nuclease. Comparison of the deo enzyme levels for clones harboring pMFS53 and pMFS501 suggest that plasmid pMFS53 possesses a functional deo regulatory region in addition to the four structural genes of the operon.
Keywords:Thymidine phosphorylase  purine nucleoside phosphorylase  recombinant DNA  pBR325 derivative vector  ampicillin resistance  chloramphenicol resistance  DRA  deoxyriboaldolase  DRM  deoxyribomutase  EtBr  ethidium bromide  kb  kilobase pairs  PNP  purine nucleoside phosphorylase  tetracycline resistance  TPP  thymidine phosphorylase  [ ]  indicates plasmid-carrier state  ( )  indicates prophage in lysogen
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