Dr. Howard Ochman requests four years of support to investigate biases in the rates and patterns of molecular evolution in the enterobacteria. The specific goals of the proposed research are to determine if leading/lagging or coding/non-coding strands of DNA have unequal mutation rates, thereby explaining an asymmetry in substitution rates. Early work by Wu and Maeda detected inequalities in the rate of certain reciprocal substitutions, which they interpreted as differences in mutation rates between the leading and lagging strands. However, a subsequent analysis of the same data by Bulmer, employing a more restrictive inference of the ancestral sequences, failed to detect such a difference. Recently, Dr. Ochman and his student Ms. Francino have analyzed DNA sequence variation in natural isolates of E. coli and S. enterica, and with respect to detect asymmetries in substitution rates. Although they were unable to detect an asymmetry in base substitution rates between leading and lagging strands, they did detect an asymmetry in substitution rates between coding and non-coding strands. They consider that this difference is best explained by transcription-coupled repair on the non-coding strand. Dr. Ochman now wishes to follow up on this observation, and to examine the conflicting claims of Wu and Maeda, and Bulmer regarding differences in mutation rates between leading and lagging strands. Two complementary lines of investigation are planned. In the first, Dr. Ochman will take an experimental approach. He will measure mutation rates in strains of Salmonella typhimurium that he will construct, containing specific alleles at the lacZ locus integrated into the chromosome at the same position, but in opposite orientations. In this work he will take advantage of sets of lacZ- strains constructed by Jeffrey Miller that harbor a different substitution or frameshift mutations in the same codon position specifying glutamic acid at position 461 in the active site of -galactosidase. He will use a PCR assay to determine the orientation of integration, and use mini-mu cloning and now-standard bacterial genetic procedures to introduce these lacZ alleles into the S. typhimurium chromosome at the same locations. Mutation rate will be measured by a standard Luria-Delbruck fluctuation test. Since the lacZ locus is expressed constitutively in the strains Dr. Ochman will use, any differences in mutation rate can be ascribed to orientation of integration; errors introduced by transcription will be the same in all strains. Dr. Ochman's second approach will be to analyze sequence variation in 11 natural isolates of E. coli. Using procedures that he has already developed and reported on, he will look for mutational asymmetries in regions of the E. coli chromosome that are untranscribed (including two cryptic genes and two 400-bp non-coding segments on opposite sides of the origin of replication located in two so-called """"""""gray holes"""""""") and in regions that are transcribed but untranslated. Each region or loci will be amplified by PCR and then sequenced. The amount of sequencing required is relatively modest, by today's standards. Dr. Ochman will compare the results that he has already obtained for coding regions, with the results that he will obtain for transcribed but untranslated regions, and for untranscribed regions. This should allow him to distinguish between the competing hypotheses of leading/lagging strand mutation rate differences, and transcription-coupled repair, in generating the observed asymmetries.
