The research plan deals with the chemical asymmetry found in Bacillus subtilis DNA as it effects "strand switching" during transcription, strand bias in integration of donor DNA, repair and mutagenesis during transformation. The experimental approach involves the following: 1) Physical isolation of L-specific sporulation and H-specific germination 32P-labeled RNAs obtained either by characterizing discrete-size species as displayed by electrophoresis on polyacrylamide or by hybridization-competitions. 2) Utilization of these RNAs as hybridization probes to survey and recover in an EcoRI digest, DNA fragments which specifically encode for L or H transcripts. 3) Restriction endonucleases cleavage site evaluations of the B. subtilis genome through the electrophoretic separation of prototrophic transforming fragments before and after marker "rescue" analyses involving ligation with recipient DNA that has been similarly restricted. 4) Molecular cloning in B. subtilis of transforming fragments using the plasmids pUB110 and pC194 as vectors, or amplifying DNA fragments which encode for sporulation or germination RNAs in E. coli by insertion into the plasmid pMB9 followed by sequence analyses of their promoter regions. 5) Studies on strand bias (H/L), cotransfer frequencies and mutations (selfing") in rec positive and rec negative recipients transformed with various mismatched heteroduplex DNAs, including in vitro repair of endonuclease S1 treated hybrids with lysates from W.T. and po1A5 cells. These studies will elucidate the mechanisms which control the asymmetric transcription during morphogenesis and whether mismatch repair is an asymmetric process and error-prone during the gap-filling step by DNA polymerase I.
NIH ExPorter
NIH ExPorter