The scope of this research incorporates isolation of new E. coli mutants, development of a new target DNA system for explicit mutation assays and a substantial enquiry into the nature of the antimutation mechanism mutation frequency decline (MFD). The study is focused around measurements of mutagenesis at glutamine tRNA genes (class 2 suppressor mutation). Nonsense defective E. coli B/r are exposed to ultraviolet radiation (UV) and this particular type of revertant is distinguished from other types by a simple T4 phage test. Class 2 suppressor mutation results from G:C to A:T transitions in the genes glnl and gln2, apparently targeted at T=C pyrimidine dimers in the transcribed strand (for de novo) or the non-transcribed strand (for conveted suppressor mutation). Transposon insertions (TN5) will be used to produce the new mutants defective in specific mutation processes. The screening will exploit our experience with different types of reversion and present day microtiter procedures. The target sequence for mutation assays consists of an E. coli DNA segment with the seven tRNA operon that includes adjacent redundant copies of glnl and gln2 (sites for class 2 suppressor mutation). This sequence is in a temperature sensitive lambda hybrid so that it may be replicated in the E. colin genome or amplified as free phage convenient for sequence analysis. A series of forward mutations inactivating expression of the operon will be determined in the target sequence, and reversion mutation at these different sites will detect any pattern in mutation sensitivity as a function of sequence context. MFD, an unexplained mechanism that can rapidly eliminate de novo class 2 suppressor mutation potentiated by UV, will be investigated in four ways. Studies with endogenous photoenzymatic monomerization will consider possible DNA conformation changes and whether the two strands in this DNA region are similar. A probe fragment of glnl-DNA in a plasmid will measure repair of different photoproducts with and without MFD conditions. MFD of reversion at the sites affecting expression in the target sequence and of class 1 and 3 suppressor mutation will be considered, and also MFD of de novo class 2 suppressor mutation as affected by endogenous polyamines. The results in total should increase understanding of the molecular genetics for information maintenance and/or reorganization.
Showing the most recent 10 out of 22 publications
