Browsing by Subject "Mutation (Biology)"

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Analysis of mutations in the kinesin motor that decouple ATPase activity and microtubule interaction
(2004) Auerbach, Scott David, 1965-; Johnson, Kenneth A. (Kenneth Allen)
Conventional kinesin is a dimeric, microtubule-dependent motor whose activity is tightly coupled to ATP hydrolysis. Mutations that might affect the coupling between ATPase and motor activities of kinesin were predicted to fall within the gamma- phosphate sensor apparatus, a set of domains in the protein believed to detect the phosphorylation state of the bound nucleotide and mechanically transmit the information via conformational change to the microtubule-binding domain. An additional element, the relay helix, has been postulated to undergo axial translation, rotation, and/or elongation, in response to the loss of the gamma phosphate from the bound nucleotide, and serve as intermediary between nucleotide- and microtubule-binding sites. An N-terminal truncation of rat conventional kinesin was examined using steady- and transient-state kinetic methods. Rate constants for ATP and microtubule binding were determined, as well as those for microtubule-dependent ADP and phosphate release. The dimeric state of the motor in solution was confirmed using analytical ultracentrifugation. Conserved residues within the gamma phosphate sensor were selected for mutagenesis. The residue E237 is believed to form a transient salt bridge with R204 when the motor is in the ATP state, based on crystal structure analysis, and the mutations E237A and E237D were examined using transient state kinetic methods. Both mutants showed > 10-fold reduction in steady-state ATPase activity, although rate constants for ATP and microtubule binding, as well as ADP release were little affected. These results suggested a disruption in the catalysis step caused by the mutations. An electrostatic interaction between E200 and R204 may also form in response to changes in nucleotide phosphorylation state, however, E200D and E200A mutants were scarcely compromised in steady-state ATPase activity, and this was attributed to a reduction in the rate constants governing product release. Finally, the N256K mutation caused a >1000- fold reduction in the rate of ADP release and a ~100-fold reduction in the steady-state ATPase rate. N256 falls within the relay helix, although the mechanism by which the N256K defect arises cannot yet be determined.
An analysis of the effect of varying the duration of X-ray treatment upon the frequency of mutations
(1931) Oliver, Clarence P. (Clarence Paul), 1898-; Not available
The effects of the aging of X-rayed male germ cells upon the frequency of sex-linked lethals in Drosophila melanogaster
(1929) Harris, Benjamin Bee; Muller, H. J. (Hermann Joseph), 1890-1967
Mutagenic analysis of the decarboxylases and hydratases in parallel meta-fission pathways
(2008-08) Miller, Scott Garrett; Whitman, Christian P.
The catechol meta-fission pathway, a degradation pathway for simple aromatic compounds, is rich in enzyme chemistry and replete with structural and evolutionary diversity. Vinyl pyruvate hydratase (VPH) and MhpD catalyze the same reaction in this pathway, but in different bacterial species. These metal ion-dependent enzymes reportedly catalyze a 1,5-keto-enol tautomerization reaction followed by a Michael addition of water. MhpD, and most likely VPH, are members of the fumarylacetoacetate hydrolase (FAH) superfamily. The crystal structure of MhpD and the sequence of VPH identified four potential active site residues, Lys-60, Leu-72, Asp-78, and Ser-160 (Ser-161 in VPH). The K60A and D78N mutants of VPH and MhpD had the most damaging effects on catalysis. Moreover, the K60A mutant seemingly uncoupled tautomerization from hydration and provided evidence for an [alpha, beta]-unsaturated ketone in the reaction. The effects of the L72A and S160A (S161A in VPH) mutants were smaller, suggesting less important roles in the mechanism. 5-(carboxymethyl)-2-Oxo-3-hexene-1,6-dioate decarboxylase (COHED) is a metal ion-dependent enzyme in the homoprotocatechuate (HPC) pathway, a chromosomally encoded meta-fission pathway from Escherichia coli C that parallels the catechol meta-fission pathway. COHED is also a member of the FAH superfamily. It is a monomeric protein with two domains. It is postulated that the C-terminal domain catalyzes the decarboxylation reaction and the N-terminal domain carries out the 1,3-keto-enol tautomerization reaction. Site-directed mutagenesis, NMR, and kinetic analysis with different substrates and inhibitors have identified three potential active-site residues Glu-276, Glu-278 (in the C-terminal domain), and Lys-110 (in the N-terminal domain). Replacement of either glutamate with a glutamine eliminated both the decarboxylase and tautomerase activities. The K110A mutant also diminished both activities, but more importantly eliminated the C-3 proton/deuteron exchange reaction observed for substrate analogs. The enzymes of the catechol and homoprotocatechuate pathways provide examples of enzyme optimization toward a specific substrate even among related compounds, as reflected by the FAH superfamily. Hence, the results of these studies add to the growing body of information about how enzymes evolve and how pathways are assembled.
Mutation: lessons from RNA models
(2008-05) Cowperthwaite, Matthew Cranston, 1973-; Meyers, Lauren Ancel
Mutation is a fundamental process in evolution because affects the amount of genetic variation in evolving populations. Molecular-structure models offer significant advantages over traditional population-genetics models for studying mutation, mainly because such models incorporate simple, tractable genotype-to-phenotype maps. Here, I use RNA secondary structure models to study four basic properties of mutation. The first section of this thesis studies the statistical properties of beneficial mutations. According to population genetics theory, the fitness effects of new beneficial mutations will be exponentially distributed. I show that in RNA there is sufficient correlation between a genotype and its point mutant neighbors to produce non-exponential distributions of fitness effects of beneficial mutations. These results suggest that more sophisticated statistical models may be necessary to adequately describe the distribution of fitness effects of new beneficial mutations. The second section of this thesis addresses the dynamics of deleterious mutations in evolving populations. There is a vast body of theoretical work addressing deleterious mutations that almost universally assumes that the fitness effects of deleterious mutations are static. I use an RNA simulation model to show that, at moderately high mutation rates, initially deleterious mutations may ultimately confer beneficial effects to the individuals harboring them. This result suggests that deleterious mutations may play a more important role in evolution than previously thought. The third section of this thesis studies the global patterns of mutations connecting phenotypes in fitness landscapes. I developed a network model to describe global characteristics of the relationship between sequence and structure in RNA fitness landscapes. I show that phenotype abundance varies in a predictable manner and critically influences evolutionary dynamics. A study of naturally occurring functional RNA molecules using a new structural statistic suggests that these molecules are biased towards abundant phenotypes. These results are consistent with an "ascent of the abundant" hypothesis, in which evolution yields abundant phenotypes even when they are not the most fit. The final section of this thesis addresses the evolution of mutation rates infinite asexual populations. I developed an RNA-based simulation model in which each individual's mutation rate is controlled by a neutral modifier locus. Using this model, I show that smaller populations maintain higher mutation rates than larger populations. I also show that genome length and shape of the fitness function do not significantly determine the evolved mutation rate. Lastly, I show that intermediate rates of environmental change favor evolution of the largest mutation rates.
Polyploid origins, experimental evolution of gene duplicates, and duplication and divergence of reproductive genes
(2004) Holloway, Alisha Kay; Hillis, David M.; Bull, James J.
Gene and genome duplication are major sources of material for evolutionary innovations in eukaryotes that provide opportunities for novel function, increased complexity, and rapid speciation. Single gene duplications are prevalent even in eukaryotes with small genomes. Whole genome duplication plays an important role in the evolution of plants and some vertebrate groups. I studied the origins of tetraploid gray tree frogs using molecular sequence data and advertisement calls. Molecular sequence data support multiple allopolyploid origins of tetraploids with a surprising twist. Apparently, tetraploids are composed of a single interbreeding lineage that was created from at least three distinct diploid species, a paradigm of polyploid formation that has never been seen before. The evolutionary fate of gene duplicates has been debated since the 1970s. Duplicate genes are maintained by purifying selection or evolve novel function. Drift, adaptive evolution of one duplicate, or subfunctionalization (the parsing of multiple ancestral functions between duplicates) leads to the diversification duplicates. Empirical evidence for all four of these models exists in nature, but the relative importance of each remains to be determined. I developed an experimental model system in which the gene β-lactamase was duplicated. The system utilized the antibiotic resistance properties of the gene to test factors involved in maintenance and divergence of duplicates. Native β-lactamase confers resistance to ampicillin and to very low levels of cefotaxime. Mutations in β- lactamase allow it to confer increased resistance to cefotaxime, but decrease resistance to ampicillin, creating a tradeoff. I found that the mere existence of a tradeoff between old and new functions is insufficient for retention of the second copy; the environment must be such that the copy evolving novel function is no longer able to serve the original function. Third, I examined the relative roles of novel function and subfunctionalization in evolution of duplicated male reproductive genes in Drosophila. Polymorphism and divergence data from these duplicated genes suggest that protein divergence between D. melanogaster and D. simulans is a result of adaptive evolution leading to novel function. These data strengthen the conclusion that male reproductive genes may often be under directional selection in Drosophila.