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Contribution of the Mismatch DNA Repair System to the Generation of Stationary-Phase-Induced Mutants of Bacillus subtilis. Mario Pedraza-Reyes, 2004.A reversion assay system previously implemented to demonstrate the existence of adaptive or stationary-phase-induced mutagenesis in Bacillus subtilis was utilized in this report to study the influence of the mismatch DNA repair (MMR) system on this type of mutagenesis . Results revealed that a strain deficient in MutSL showed a significant propensity to generate increased numbers of stationary-phase-induced revertants . These results suggest that absence or depression of MMR is an important factor in the mutagenesis of nongrowing B . subtilis cells because of the role of MMR in repairing DNA damage . In agreement with this suggestion, a significant decrease in the number of adaptive revertant colonies, for the three markers tested, occurred in B . subtilis cells which overexpressed a component of the MMR system . Interestingly, the single overexpression of mutS, but not of mutL, was sufficient to decrease the level of adaptive mutants in the reversion assay system of B . subtilis . The results presented in this work, as well as in our previous studies, appear to suggest that an MMR deficiency, putatively attributable to inactivation or saturation with DNA damage of MutS, may occur in a subset of B . subtilis cells that differentiate into the hypermutable state . Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells. Chuanwu Xi, 2003.DNA and peptide nucleic acid (PNA) molecular beacons were successfully used to detect rRNA in solution . In addition, PNA molecular beacon hybridizations were found to be useful for the quantification of rRNA: hybridization signals increased in a linear fashion with the 16S rRNA concentrations used in this experiment (between 0.39 and 25 nM) in the presence of 50 nM PNA MB . DNA and PNA molecular beacons were successfully used to detect whole cells in fluorescence in situ hybridization (FISH) experiments without a wash step . The FISH results with the PNA molecular beacons were superior to those with the DNA molecular beacons: the hybridization kinetics were much faster, the signal-to-noise ratio was much higher, and the specificity was much better for the PNA molecular beacons . Finally, it was demonstrated that the combination of the use of PNA molecular beacons in FISH and flow cytometry makes it possible to rapidly collect quantitative FISH data . Thus, PNA molecular beacons might provide a solution for limitations of traditional FISH methods, such as variable target site accessibility, poor sensitivity for target cells with low rRNA content, background fluorescence, and applications of FISH in microfluidic devices .
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