An empirical test of the concomitantly variable codon hypothesi

Core models of molecular evolution assume that each amino acid site in a protein evolves independently and that the rate of evolution at a single site does not affect the rate at other sites. An alternative hypothesis proposes that amino acid replacements at one site constrain replacements that can occur at interacting sites. This concomitantly variable codon (covarion) hypothesis specifically suggests that only a few sites in a protein are free to vary at any one time, and that as molecular changes accumulate, this set of sites changes. While this hypothesis is assumed to be biochemically realistic, it has never been evaluated empirically. To identify constraints on protein evolution imposed by interactions between sites, we created hybrid proteins of the enzyme triosephosphate isomerase from different lineages and determined if their activities fell outside the range of wild-type proteins. Results show that covarions develop only over large evolutionary distances. Covarions of lethal effect are extremely rare whereas covarions of small effect are common. In most cases, large numbers of amino acid changes had little effect on the ability to complement a triosephosphate isomerase deficient strain. However, more sensitive kinetic assays showed that many hybrids have decreased performance compared to wild-type enzymes.;This thesis also investigates genetic variability in natural populations of Escherichia coli. Examination of triosephosphate isomerase sequences from 82 E. coli strains shows no evidence for selection at this locus, though there is evidence for recombination. Analysis of the three amino acid polymorphisms uncovered demonstrates that replacement polymorphisms do not significantly affect enzyme performance, indicating that the amino acid changes are neutral.;A single nucleotide polymorphism between E. coli strains K12 and B is known to alter the mechanism by which the arginine repressor regulates arginine biosynthesis. Laboratory experiments have demonstrated that the different regulatory strategies are selectively favored under different environmental conditions. In this study we show that the deregulated strategy is rare in isolates obtained from natural sources. Moreover, sequence analysis reveals no evidence of selection at the arginine repressor locus, suggesting that analysis of sequence data is insufficient to detect selection of uncommon alleles in rare environments.