Functional Analysis of Codon Bias in the Adh Gene of Drosophila melanogaster

American University, Washington DC

Investigators

Abstract

There are two types of single nucleotide polymorphisms (SNPs) in protein-coding genes: "replacement" or non-synonymous polymorphisms, which result in amino acid substitutions in the encoded polypeptide, and "silent" or synonymous polymorphisms, which do not result in amino acid substitutions. Due to their effects on the primary structures of encoded polypeptides, the functional effects of non-synonymous SNPs have been studied extensively. The functional effects of synonymous SNPs, on the other hand, have received comparatively little attention from researchers. Synonymous SNPs can potentially affect the level of protein synthesis; consequently, silent variation can also affect fitness. This is evidenced by the fact that synonymous codons are not used with equal frequency, a phenomenon known as codon bias. Several lines of evidence suggest that codon bias is due to natural selection for translational accuracy and/or efficiency at synonymous sites, but few experimental studies have been conducted to confirm the importance of codon bias in multicellular eukaryotes as a means of regulating protein expression. The goal of this project is to determine the functional effects of synonymous substitutions by experimentally altering codon bias in the Drosophila alcohol dehydrogenase (Adh) gene. In particular, this research will accomplish the following three objectives: (1) The translational selection hypothesis will be vigorously tested through the in vivo introduction of a number of optimal and sub-optimal codons into the Drosophila Adh gene. Transgenic flies will be constructed using P-element mediated germline transformation, and the translational effects of synonymous mutations will be assessed through measurements of protein expression. (2) The position dependence of synonymous mutations will be investigated by comparing the expression and fitness of constructs containing ten unpreferred mutations at the 5' end of the Adh gene with constructs containing 10 unpreferred mutations at the 3' end of the gene. (3) The fitness effects of preferred and unpreferred synonymous substitutions will be investigated directly by performing ethanol tolerance and population cage experiments. Regression analysis of the fitness and protein expression data will determine if the effects of synonymous mutations are additive or epistatic. Synonymous codons, those that encode the same amino acid, are not used with equal frequency in most organisms. This pattern of variation is known as codon bias. The functional significance of synonymous variation will be investigated by experimentally altering levels of codon bias in the fruit fly Drosophila melanogaster. The effects of changes in codon bias will be examined by measuring protein expression and fitness in transgenic flies. This research will contribute to our understanding of the significance and evolutionary dynamics of synonymous variation in natural populations.