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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058405-02
Application #
2883070
Study Section
Genetics Study Section (GEN)
Project Start
1998-03-01
Project End
2001-02-28
Budget Start
1999-03-01
Budget End
2000-02-29
Support Year
2
Fiscal Year
1999
Total Cost
Indirect Cost
Name
University of Rochester
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
208469486
City
Rochester
State
NY
Country
United States
Zip Code
14627
Baines, John F; Chen, Ying; Das, Aparup et al. (2002) DNA sequence variation at a duplicated gene: excess of replacement polymorphism and extensive haplotype structure in the Drosophila melanogaster bicoid region. Mol Biol Evol 19:989-98
Carlini, D B; Chen, Y; Stephan, W (2001) The relationship between third-codon position nucleotide content, codon bias, mRNA secondary structure and gene expression in the drosophilid alcohol dehydrogenase genes Adh and Adhr. Genetics 159:623-33
Innan, H; Stephan, W (2001) Selection intensity against deleterious mutations in RNA secondary structures and rate of compensatory nucleotide substitutions. Genetics 159:389-99
Parsch, J; Braverman, J M; Stephan, W (2000) Comparative sequence analysis and patterns of covariation in RNA secondary structures. Genetics 154:909-21
Parsch, J; Russell, J A; Beerman, I et al. (2000) Deletion of a conserved regulatory element in the Drosophila Adh gene leads to increased alcohol dehydrogenase activity but also delays development. Genetics 156:219-27
Chen, Y; Carlini, D B; Baines, J F et al. (1999) RNA secondary structure and compensatory evolution. Genes Genet Syst 74:271-86
Begun, D J; Betancourt, A J; Langley, C H et al. (1999) Is the fast/slow allozyme variation at the Adh locus of Drosophila melanogaster an ancient balanced polymorphism? Mol Biol Evol 16:1816-9
Parsch, J; Stephan, W; Tanda, S (1999) A highly conserved sequence in the 3'-untranslated region of the drosophila Adh gene plays a functional role in Adh expression. Genetics 151:667-74
Stephan, W; Kim, Y (1998) Persistence of microsatellite arrays in finite populations. Mol Biol Evol 15:1332-6
Parsch, J; Stephan, W; Tanda, S (1998) Long-range base pairing in Drosophila and human mRNA sequences. Mol Biol Evol 15:820-6