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[UV-mutagenesis in Bacillus subtilis. VI. Mutagenesis under conditions of experimental delay of post-radiation DNA replication].

To test a suggestion that mutation frequency decline (MFD) in UV-irradiated bacteria starved by nitrogen sources is caused by long delay of the first postradiation cycle of DNA replication, we used a strain 1201-05 of Bacillus subtilis 168 (ade6 met5tsdnaC) which does not synthesize DNA at 42 degrees C. In a suspension of UV-irradiated cells of this strain which is incubated at 42 degrees in liquid deprived by nitrogen sources, a decrease of frequencies of Ade+ and Met+ revertants is observed. This shows that the mechanism responsible for MFD normally operates at higher temperature. Such a level of decline is not observed in a suspension incubated in nutritional medium when the DNA replication is blocked by non-permissive temperature for the same period as in starved cells. On the basis of these facts it is concluded that MFD in suspension of bacteria starved by nitrogen sources can not be caused only by delay of postradiation replication of DNA.

Bacillus subtilis↗

Targeted Forward Genetics: Saturating Mutational Analyses of Specific Target Loci Within the Genome.

Precise allele replacement by homologous recombination (also known as "gene targeting" or "genome editing") allows scientists to engineer altered DNA sequences, insertions, or deletions at specific locations in the genome. Such reverse genetics provides powerful tools to elucidate the structure and function of regulatory DNA elements, genes, RNAs, and proteins within their natural, endogenous context. Here, we describe in detail the methodology for Targeted Forward Genetics (TFG), which supports population-scale, saturating screens of allele replacements spanning thousands of base pairs at a specific target locus in the genome. The overall approach and detailed protocols, developed for the fission yeast Schizosaccharomyces pombe, are extensible to other organisms in which gene targeting is feasible.

Schizosaccharomyces↗