Cloning and expression of a Streptomyces fradiae neomycin resistance gene in Escherichia coli |
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Authors: | W H Rodgers W Springer F E Young |
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Institution: | 1. Department of Microbiology, University of Rochester, School of Medicine and Dentistry, Rochester, NY 14642 U.S.A.;1. Institut für Biochemie, Bayer AC, Pharma Forschungszentrum, Postfach D5600, Wuppertal 1 F.R.G. |
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Abstract: | A Streptomyces fradiae DNA sequence, which codes for a neomycin phosphotransferase, has been subcloned from the Streptomyces recombinant plasmid pIJ2 a chimera between the Streptomyces plasmid SLP1.2 and chromosomal DNA containing a neomycin (Nm) resistance gene] into the BamHI restriction enzyme site of pHV14. Three different recombinant plasmids (pWHR1, pWHR2, pWHR3) have been isolated which transform Escherichia coli to Nm resistance. Southern transfer hybridization experiments show that the recombinant plasmids contain the cloned Streptomyces Nm resistance gene, and lysates of E. coli containing the recombinant plasmids were shown to have Nm phosphotransferase activity, demonstrating that a gene from Streptomyces can be expressed in E. coli. |
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Keywords: | Recombinant DNA Southern hybridization cloned neomycin phosphotransferase gene Ap ampicillin bp base pairs Cm chlorarnphenicol EtBr ethidium bromide kb kilobase pairs LB Luria broth (1% Difco Uyptone 0 5% yeast extract 0 5% Nad pH 7 0) MIC minimal inhibitory concentration Nm neomycin SL66 TBAB tryptone blood agar base (Difco) Tc tetracycline [ ] indicates plasmid-carrier state |
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