Analysis of Frameshifting Frequencies Due to Homopolymeric Nucleotide Tracts in Neisseria gonorrhoeae
Analysis of Frameshifting Frequencies Due to Homopolymeric Nucleotide Tracts in Neisseria gonorrhoeae
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Date
2006-03-10
Authors
Holder, Robert Christopher
Advisor
Stein, Daniel C
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Abstract
Organisms that are sensitive to the antimicrobial agent nitrofurantoin express nitroreductases, which reduce nitrofuran containing compounds to highly reactive and damaging intermediates. A single nitroreductase, nfsB, was identified in N. gonorrhoeae FA1090. The sequence of nfsB was modified to contain polyguanine tracts of varying lengths. Analysis indicated that mutations yielding nitrofurantoin resistance occurred at a higher rate in strains containing modified nfsB genes when compared to wildtype FA1090. The frequency of mutation also increased as polyguanine tract length increased. A polyguanine tract length of 5 residues did not produce elevated mutation frequencies.
The use of PCR and polyacrylamide gel electrophoresis proved to be a reliable method for quickly identifying nitrofurantoin resistant mutants that contained nfsB frameshift mutations. When nfsB fragments amplified from nitrofurantoin resistant mutants were electrophoresed, the presence of insertions or deletions in the nfsB coding sequence was easily detected.