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Creation of a test plasmid for detecting G-C-to-T-A transversions by changing serine to arginine in the active site of beta-lactamase

Foster, Patricia L. and Dalbadie-McFarland, Gloria and Davis, Elaine F. and Schultz, Steve C. and Richards, John H. (1987) Creation of a test plasmid for detecting G-C-to-T-A transversions by changing serine to arginine in the active site of beta-lactamase. Journal of Bacteriology, 169 (6). pp. 2476-2481. ISSN 0021-9193. PMCID PMC212096.

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Oligonucleotide-directed mutagenesis of the beta-lactamase gene, bla, on pBR322 was used to change the codon for the active-site serine 70, AGC, to CGC, coding for arginine. Escherichia coli cells carrying the mutant plasmid, pGD104, were sensitive to ampicillin, indicating that the arginine-containing enzyme is inactive. We characterized the reversion of the mutant bla gene by a number of mutagens and in different genetic backgrounds and demonstrated that full ampicillin resistance can be restored only by a G-C-to-T-A transversion occurring at the first base of the codon. Thus, reversion of the mutant bla gene is diagnostic for G-C-to-T-A transversions, and bacteria carrying pGD104 can be used as test strains to detect the occurrence of this mutation.

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Additional Information:© 1987 American Society for Microbiology. Received 9 January 1987/Accepted i9 March 1987. We gratefully acknowledge the contributions of David Botstein to the conception, support, and completion of this project. We thank Eric Eisenstadt for helpful discussions and for critically reading this manuscript. We also thank the people mentioned in Materials and Methods for strains and plasmids, especially Steven Elledge and Graham Walker, and Jeffrey Miller for discussion. This work was supported by grant 85-16 from the Whitaker Health Sciences Fund to P.L.F. and David Botstein and by Public Health Service grants CA37880 from the National Cancer Institute to P.L.F. and GM16424 from the National Institute of General Medical Sciences to J.H.R. G.D.-M. was supported by NRSA grant GM07626.
Funding AgencyGrant Number
Whitaker Health Science Fund85-16
NIH Predoctoral FellowshipGM07626
Issue or Number:6
PubMed Central ID:PMC212096
Record Number:CaltechAUTHORS:FOSjbact87
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:11830
Deposited By: Archive Administrator
Deposited On:02 Oct 2008 20:50
Last Modified:03 Oct 2019 00:22

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