Thesis (Ph. D.)--University of Texas at Austin, 2004. / Supervisors: David M. Hillis and James J. Bull. Vita. Includes bibliographical references.

Thesis (Ph.D.)--Bowling Green State University, 2006. / Document formatted into pages; contains viii, 97 p. Includes bibliographical references.

Big Blue® lacI transgenic mice and rats carry the E. coli lacI gene integrated as a tandem array of approximately 40 copies integrated at a single site in chromosome 4. This mutationally well-characterized gene is highly sensitive to base substitution mutations and is readily recovered from virtually any tissue of the transgenic host, facilitating the in vivo study of mutation. The Big Blue® assay was used to investigate spontaneous and induced mutation, with an emphasis on dietary influences on mutational frequency and specificity. The effects of ageing and dietary restriction on spontaneous mutation in the lacI transgene were determined in mice, permitting evaluation of several well established theories of ageing. Mutation frequencies were found to increase with age in tissues that proliferate (bladder and liver, but not brain), validating a principle tenet of the somatic ageing theory. However, the unexpected lack of a change in mutational specificity in ageing mice suggests that theories of ageing based on oxidative damage, or a reduction in DNA repair efficiency, may not be seminal to the ageing process, at least until more advanced age. Similarly, dietary restriction, which is known to extend lifespan in rodents and was predicted to decrease oxidative DNA-damage, had no appreciable effect on either the frequency or the specificity of spontaneous mutation in liver of younger (6 month old) and older (12 month old) mice. Dietary influences on induced mutation were examined following treatment with aflatoxin B₁ (AFB₁) and 2-amino-1-methyl-6-phenylimidazo[4,5- b]pyridine (PhIP) powerful animal carcinogens which demonstrate tissue, species and sex-linked differences in rats and mice. As expected, AFB₁ was found to be potently mutagenic in rat but not mouse liver, in agreement with rodent carcinogenicity studies that found F344 rats to be highly susceptible to AFB₁-induced hepatocarcinogenesis, while C57BL/6 mice are highly resistant. PhIP was found to be potently and equally mutagenic in colon of both male and female rats. The result in female colon was surprising since PhIP predominantly induces colon cancer in male rats but mammary tumors in female rats. Therefore, the progression of PhIP-induced colon cancer in the rat colon is likely due to factors acting at a later stage in the tumorigenic process, following the damage and mutation of DNA. Rat prostate tissue, another tumor target tissue in PhIP-treated rats, was also found to be highly susceptible to PhIP-induced mutagenesis. Lastly, the PhIP studies were extended to an additional transgene target located in the shuttle vector construct from Big Blue® rodents, the bacteriophage λ-derived cII gene. These studies validated the use of the λ cII gene as an alternative mutational target for use in the Big Blue® assay, while the analyses of mutation in the lacI and the λcII transgenes serves as a paradigm for mutational studies which compare mutational responses in different genes. Collectively, these studies demonstrate the utility of the lacI (λ cII) transgenic mutagenicity assay for the in vivo investigation of mutational processes as a function of age, diet, sex, species, and target tissue specificity with respect to sites of mutation and cancer. / Graduate

Mutations are generally thought to be targeted events. The distribution of mutations is based upon the initial original deposition of DNA damage and the fidelity and efficiency of repair of this damage. These factors are dependent on the primary site of DNA modification and the surrounding nucleotides (i.e., mutation is “context sensitive"). To better understand mutagenesis, I measured DNA damage and/or mutation at the DNA sequence level, then considered the impact of mutation location and the surrounding nucleotide environment. The selected mutagens, ultraviolet light (UV) and benzo(a)pyrene [B(a)P], were chosen because they produce well-characterized lesions and are environmentally relevant. UVC (254nm) light-induced DNA damage is well documented. UVC produces a characteristic spectrum of mutations. The predominant UV-induced mutations are C → T transitions occurring at TC or CC sites, as well as CC → TT tandem transitions. The latter class of mutation is considered the hallmark of UV mutagenesis. Quantitatively speaking, the primary types of UV-induced DNA lesions are cyclobutane pyrimidine dimers (CPDs) and the 6-4 pyrimidine/pyrimidone (64PyPy). These are also the suspected predominant pre-mutagenic lesions. Each lesion was independently measured at the DNA sequence level in a defined region of DNA. The pattern of UVC-induced DNA damage revealed a complex induction pattern. The flanking DNA nucleotides partially influenced the pattern of damage deposition. Sites where C → T transitions and CC → TT transitions were recovered at high frequencies were also frequently damaged. Thus, at these sites, mutation fixation was potentially more influenced by initial DNA damage than the rate of DNA repair. Two other components of the UV spectrum [UVB (290-320 nm) and UVA (320-400 nm)] are more environmentally relevant than UVC since UVB and UVA reach the surface of the earth.The results of UVC experiments were used as a guide to interpret the results obtained using UVB since direct light absorption by DNA has been shown to be one of the main biological effect at both wavelengths. The model that was chosen for the studies was an in vivo transgenic rodent mutagenesis assay. The research presented in the thesis represents one of the first studies to characterize UV-induced mutation at the DNA sequence level in rodent skin. The backs of female C57B1/6 lacl transgenic mice were shaved and exposed to either UVB or UVA light. UVB was found to be significantly more mutagenic than UVA. The UVB-induced mutation spectrum was characterized by C → T transitions at dipyrimidine sites, implicating CPD and/or 64PyPy lesions as premutational DNA lesions. The majority of UVA-induced mutations was C → T transitions at dipyrimidines sites and hence, as with UVB-induced mutation, attributed to CPDs and 64PyPy. In the UVA-dose response experiments, the induced mutant frequency was lower than expected at higher doses. A statistically significant increase in putative clonal expansion suggested that skin cells might have undergone cell killing followed by repopulation. In a final study, C57Bl/6 lacI transgenic male mice were intraperitoneally injected with the mutagen B(a)P at doses of 0, 62.5, 125, 250, and 500 mg/kg. This resulted in a linear increase in mutation frequency (4.8 to 53 × 10⁻⁵). All mutations increased at GC basepairs and not AT basepairs following B(a)P treatment. This was consistent with models suggesting guanosine adducts to be mutagenic lesions. In conclusion, the transgenic lacI mouse mutagenesis model was a sensitive target for in vivo mutagenesis from UVB, UVA and benzo(a)pyrene exposures. The system detected class-specific mutation frequency differences and increases in cell proliferation after mutagen exposure. With a further refinement of techniques, the correlation of DNA damage and mutation will allow even more exquisite studies. / Graduate

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published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

At least some areas of the embryonic blastoderm in Drosophila melanogaster appear to be determined by substances produced under control of the maternal genome without involvement of the embryonic genome. Study of mutant genes controlling the production of such morphogenic substances would provide valuable information about these substances and the nature of determination itself. In this thesis, the adult ovaries in offspring of females containing sex chromosomes originating from a wild-type strain of Drosophila melanogaster collected on Margarita Island, Venezuela have been examined. After selection on such chromosomes, over 50% of the offspring produced by homozygous females contained either one or two agametic gonads. The delayed expression is due to temperature-sensitive gene expression duringoogenesis. Two micron sections of normal and abnormal ovaries of this strain indicate the mesodermal components of the abnormal ovaries including the ovarian sheaths and the ovariole epithelial sheaths, were normal. In addition, attachment of the ovaries to the oviducts are normal. The abnormal ovarioles contain cells which appear to be mesodermally-derived follicle cells, but no normal egg chambers are seen. The germaria also contain follicle cells but no cells resembling stem cells or cystocytes are seen. It is concluded: (1) the development of ovarian germinal and mesodermal components are completely independent and (2) the mutant gene(s) prevent normal differentiation of pole cells into germ cells.

Mutation is a fundamental process that drives evolutionary change; however, most new mutations are deleterious for organismal fitness and can readily propagate within populations under a broad range of conditions. Mutational processes able to counteract deleterious mutation accumulation include: 1) reversion mutation back to wildtype, 2) acquisition of generally beneficial mutations, and 3) compensatory mutations that specifically mitigate the effects of previously-acquired deleterious mutations through epistasis. The potential for any of these mutation types alters our expectations for the impact of deleterious mutation in populations, but since the fitness effects of individual mutations are rarely characterized, the relative importance of beneficial and compensatory epistatic mutations is unknown. In this thesis, I characterized the nuclear mutations that arose in a previous mutation accumulation (MA) experiment using Caenorhabditis elegans nematodes, in which mutations were allowed to accumulate under extreme drift conditions in replicate, independently evolving lines initiated from a low-fitness mitochondrial electron transport chain (ETC) mutant, gas-1. In contrast to the results of typical MA experiments, gas-1 MA lines improved fitness slightly compared to their ancestor. Here, I find that the gas-1 MA lines demonstrate little increase in among-line variance and that the gas-1 MA nuclear mutations are more narrowly functionally defined than wildtype MA nuclear mutations. When combined with evidence for zygotic or post zygotic selection these data suggest that selection--both purifying and positive--can be an extremely powerful force even in conditions of extreme genetic drift. Furthermore, functional characterization of a four-mutation set isolated from one of the gas-1 MA lines on gas-1 and wildtype backgrounds shows fitness improvements on both backgrounds. This beneficial four-mutation set is associated with a decrease in steady-state endogenous ROS on the gas-1 background while exhibiting no effect on wildtype. I also find that steady-state ATP levels associated with the beneficial four-mutation set decreased compared to wildtype suggesting that fermentation may be metabolic strategy to cope with increase oxidative stress. These findings suggest that we can detect and characterize specific genetic changes that lead to a partial recovery of fitness and phenotype in a low-fitness ETC-deficient mutant strain of C. elegans. I extended my thesis to include analyses of fitness and phenotype of 24 replicate lineages of the gas-1 ETC mutant evolved in large population (n = 1000) sizes for 60 generations--conditions optimal for selection and fitness recovery (RC). I find that two distinct gas-1 RC fitness groups emerged: one group with significantly higher average fitness than the ancestor and containing two lines that exceeded wildtype fitness levels, and another group with more modest and non-significant fitness gains. Interestingly, many lines in the first group were observed to generate appreciable numbers of males during experimental evolution--consistent with evolution of outcrossing either accompanying or driving rapid fitness recovery. Bioinformatic functional analyses of the nuclear mutations that arose in the gas-1 RC lines show the availability of potentially more paths to fitness recovery for large populations than small ones. Combined, these data allow us to identify patterns in selection and drift in gas-1 recovery under MA and RC (recovery) conditions. My research advances our understanding of the genetic bases of adaptive evolution under extremely unfavorable population genetic conditions and how mitochondrial dysfunction affects evolutionary dynamics.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 290-314). Also available in print.