The proposed research would be a detailed investigation of the prevalence and functional significance of compensatory mutations within genes. Primary transcripts are linear single stranded RNA molecules that take on secondary and tertiary structures as a function of intra- molecular patterns of well studied feature of ribosomal and transfer RNAs. In general, a point mutation in a region of secondary structure (i.e. that forms base pairs with another region of the molecule) will disrupt the function of that molecule and be deleterious. However, if a secondary mutation occurs in the complementary position of the gene, it is possible for the secondary structure of the RNA to be restored. Thus, pairs of mutations may not alter the function of the molecule, even though either mutation individually is deleterious. Dr. Stephan and colleagues have recently shown that compensatory mutations also occur in the RNA of the Alcohol dehydrogenase (Adh) gene in species of Drosophila. The presence of secondary structure was inferred with a phylogenetic approach, an excess of paired mutations in positions consistent with a model of a stem-loop structure. These apparent regions of conserved secondary structure lie in introns, where it is reasonable to think that secondary structure may be important for proper intron splicing. The proposal details two main areas of investigation. The first is a phylogenetic investigation of DNA sequences, to try and identify regions of genes that correspond to RNA secondary structure. The major insight comes from pairs of sites that covary evolutionarily to maintain Watson- Crick pair rules. By looking at Adh across Diptera, including new sequences to be obtained as part of the proposed research, as well as rRNA and tRNA genes that exist in the database, Dr. Stephan and colleagues will use this phylogenetic criterion to identify candidate regions of genes as sites of secondary structure. Such regions will also then be checked by three additional criteria expected of secondary structure compensatory pairs: the overall rate of substitution in pair loop stem regions is expected to be lower than in unpaired regions; the single mutation step intermediate between double mutant steps that preserve secondary structure is expected to go through the more stable GU intermediate more often than through less stable intermediates; and the substitution rate of double mutants is expected to be less as recombination between the sites goes up, so that paired regions that are farther apart on a gene should experience fewer substitutions that those that are close. The second major area of investigation is on the functional effects of changes in regions that appear to be involved in secondary structure. This work entails extensive molecular biology benchwork, and will be conducted by Dr. Stephan's main collaborator on this project, Dr. Soichia Tanda. Plasmid constructs containing a D. melanogaster Adh sequence will be subjected to site directed mutagenesis in regions hypothesized to support secondary structure in primary transcripts. These constructs will also be used to transform flies for the measurement of Adh enzyme activity. Also, Adh RNA processing efficiency will be assessed using Schneider cell lines (reportedly Adh null) cotransfected with wild type and mutated Adh genes. RNA will be isolated and assessed, using a quantitative PCR assay, for the relative presence of differently processed RNAs.
