Currently, the most widely used in vivo mutation monitoring system in humans is the hypoxanthine-guanine phosphoribosyltransferase (hprt) T-cell clonal assay. This dissertation examines the current state of the hprt monitoring system and the usefulness of hprt mutational spectra in revealing environmental exposures. The nature of spontaneous mutational spectra recovered through the implementation of this system is detailed. An examination of hprt mutation frequencies obtained from a set of monozygotic twins revealed a striking influence of genetic factors. As age increases, the influence of genetic factors controlling mutation frequency appears to be modified by environmental factors. Mutational spectra obtained from Russian individuals living in Moscow were distinct from the spectrum of mutation observed in age-matched Western controls. Analysis of the relationship between mutation frequency and subject age clearly demonstrated the lack of any relationship for subjects after the age of 55. This finding contradicts many previously published reports on the relationship between mutation frequency and age. Finally, the influence of tobacco smoking on mutational frequency is clear, however, no change in the mutational spectrum of smokers was revealed. Changes in mutational spectrum are analyzed in the context of the T-cell biology and reveal that the dynamics of this tissue are likely responsible for the observations made in this dissertation. Although the hprt gene is a highly robust and suitable target for the analysis of mutation, the target has not yet been saturated, and new single base-pair substitutions are still being characterized. The data clearly suggest that the T-cell clonal assay in its current state may not be a suitable mutational monitoring system for human populations. This dissertation concludes that new mutational assays need to be developed for monitoring mutations in human populations. / Graduate

The subject of this study is a new dominant suppressor mutation Su(pr) which acts on the purple eye-colour mutant (pr) of Drosophila a melanogaster. The Induction of Su (pr) was originally associated with the synthesis of a compound-2R chromosome In SD72/cn bw females. The suppression of p_ was first observed in combination with a homologous pr bearing compound-2L chromosome. Suppressed-pr flies appeared to have a fully wild eye phenotype. The intention of this study was to determine the chromosomal constitution necessary for Su(pr) induction, and to map the suppressor site. To do this, many compound-2R chromosomes were synthesized from several combinations of standard seconds. It was found that SD72 must be present to produce a suppressing compound-2R. The SD72 second carries a pericentric inversion that results in a duplication of 2L heterochromatIn, and an associated deficiency of 2R heterochromatin in the compound-2R S u (p r) chromosome. Suppression, therefore, Is associated with the pericentric inversion found only on SD72. The role of this segmental aneuploidy was studied by detaching several C(2L)pr: C(2R)SD72/,cn bw suppressed strains such that both arms of the Su(pr) compound autosome were recovered independently and established in standard strains. Suppressing and non-suppressing detachment products were recovered with a frequency that varied according to the compound-2R Su(pr) strain from which they were derived. The chromosome mechanics involved in the process of C(2R)SD72/cn bw formation and subsequent detachment implicates alterations to a segment of proxlmial 2R heterochromatin from SD72 in Su(pr) Induction. Loss of Su(pr) in the detachment process correlates predominantly with deletions generated In 2R heterochromatIn. Recombination mapping relative to the two visible heterochromatic markers, Iight and rolled, revealed that Su(pr) Iies to the left of rolIed. SpectrophotometrIc measurements of eye pigments revealed that suppressed-pr and suppressed-pru bw flies had pigment levels that exceeded the wild type. The lethal allele prc4. was not found to be suppressible. / Science, Faculty of / Zoology, Department of / Graduate

The sex-linked vermilion (v) locus is probably the structural gene for the enzyme tryptophan pyrrolase. Mutations at the locus invariably are recessive and result in a bright-red eye colour phenotype accompanied by a loss of tryptophan pyrrolase activity. Extensive genetic, biochemical and developmental studies of v mutations have shown that the gene is a relatively small cistron controlling the catalytic activity of tryptophan pyrrolase which gives rise to kynure-nine, a brown eye pigment precursor, in the larval fat body during a defined developmental period. Alleles of the locus can be broadly grouped into two classes: 1) spontaneous v mutations, the majority of which are suppressible by mutation at the non-allelic suppressor of sable [su(s)] locus, 2) induced v mutations which are all unsuppressible by su(s) alleles. Alleles of both classes behave nonautonomously during development and all map within the definable limits of the v cistron. This investigation was initiated to recover conditional (temperature-sensitive) v alleles which could be used to study further the regulation of the activity of the v gene during development, and to extend our knowledge of the genetic functioning of the locus. A temperature-sensitive (ts) allele of a known structural gene, affecting the catalytic activity of an assayable enzyme, could also enable a determination of the factors responsible for temperature-sensitivity in Drosophila in terms of changes in the gene product. The temperature-sensitive period (TSP) of a ts mutant in Drosophila is defined as that period during development when exposure to the restrictive temperature commits the organism to a mutant phenotype. With a ts v allele, a correlation can be made between the TSP determined phenotypically and the variation in tryptophan pyrrolase activity during development, and thus contribute to a molecular understanding of the TSP. This study has consisted mainly of the following approaches: 1) mutagenesis and genetic screening to recover ts v alleles, 2) an examination of the phenogenetics of one ts v allele, including fine structure mapping, complementation properties, nonautonomous expression in gynandromorphs, and suppressibility, and a comparison of these properties with those exhibited by some non-ts v mutations, 3) a biochemical analysis of the effect of a ts v mutation on the properties of tryptophan pyrrolase, a deter-mination of the TSP of a ts v allele based on the eye phenotype. Both ts v alleles, v[sup ts1] and v[sup ts2], recovered in this investigation cause a vermilion phenotype if v flies are raised at the restrictive temperature (29°C), whereas v[sup ts] flies raised at the permissive temperature (17° or 22°C) have almost normal eye colour. The activity of tryptophan pyrrolase, extracted from [sup ts1] flies raised at 29°G and 22°C respectively, parallels the temperature-dependent phenotypic properties; enzyme activity is markedly reduced in flies raised at 29°C but is almost normal in flies raised at the permissive temperature. The v[sup ts1] mutation behaves like non-ts,induced v alleles at 29°C in its complementation, suppressibility and nonautonomy. Thus, it fails to complement any other v point mutant, is unsuppressible by su(s)² and is developmentally nonautonomous when present with v* tissue in gynandromorphs raised at 29°C. Since the v[sup ts1] allele is viable when heterozygous with deletions removing the v locus and maps within the v cistron as a point, it is assumed to be a point mutation in the v structural gene. Furthermore, the tryptophan pyrrolase controlled by the v[sup ts1] mutant has different in vitro kinetic and temperature-dependent properties when v[sup ts1] flies are raised at 29 C compared to either wild type or tryptophan pyrrolase extracted from v[sup ts1] flies raised at 22°C. The v[sup ts1] mutant demonstrates different phenotypic and enzyme properties between males and females raised at 29°C; hemizygous males are more mutant in phenotype and have lower tryptophan pyrrolase activity than their homozygous sibs. This result apparently is the reverse of the dosage compensation nor-mally demonstrated by wild type tryptophan pyrrolase in which males with one dose of the v⁺ gene have at least the enzyme activity obtained from females with two doses of the v⁺ gene. How-ever, the TSP for the v[sup ts1] mutant is the same for males and females and falls between the early third instar larva and early pupa stages of development. This period corresponds to the maximum pre-adult activity of tryptophan pyrrolase and also correlates with the formation of kynurenine in the cells of the fat bffidy. These results are discussed in relation to a molecular model explaining the genetic and molecular functioning of the v locus during development. The results are consistent with the hypothesis that v[sup ts] and nonconditional v mutations affect different aspects of active tryptophan pyrrolase structure rather than regulation of the rate of synthesis of the enzyme. Thus, suppressible v mutations affect allosteric or regulatory sites of the enzyme which interact with metabolic and develop-mental cofactors, whereas the nonconditional, unsuppressible, induced v mutations probably affect the catalytic sites of t tryptophan pyrrolase. The ts v mutation, v[sup ts1] , has genetic and biochemical properties which are compatible with an effect on the aggregation of enzyme subunits due to conformational changes during enzyme synthesis at the restrictive temperature. / Science, Faculty of / Zoology, Department of / Graduate

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Early events occurring during induction of the SOS response in Escherichia coli are poorly understood. In order to understand the early steps in SOS induction more fully, we have isolated several mutations which constitutively express the SOS regulon. Using a Mud(Apᴿ,lac) fusion to the SOS regulated gene sulA, we isolated Lac⁺ colonies as mutants in which RecA protein is constitutively activated for repressor cleavage. The mutations map to four loci: dam, lig, uvrD and recA. The extent of constitutive SOS induction in these mutants varied greatly, indicating different levels or types of signal in the cell. The mutations isolated demonstrate two early steps in SOS induction. The first step in SOS induction is signal generation and includes mutations found in dam, lig and uvrD genes. The mutant gene products presumably alter DNA metabolism to produce an inducing signal. These non-lethal mutations lead to sub-induction and probably generate very specific signals, such as abnormally unwound DNA in the case of DNA helicase II mutants or unsealed DNA nicks that result from deficient ligation in lig mutants. Greater induction may require quantitatively more signal or different types of signal generated by severe defects leading to cell death. These mutations also show that signal is a variable quantity, allowing the cell to fine tune the levels of SOS repair activity according to the amount or type of signal (damage) perceived. In some cases (such as dam mutations), blocking the SOS response by lexA(Ind⁻) alleles leads to cell death. In this type of constitutively activated strain, the increased level of repair from SOS induction is required to allow the cell to tolerate potentially lethal DNA structures generated by the mutant gene product. The second step in induction is the interaction of signal with RecA protein and is shown by isolating 8 recA mutants. Mutant recA alleles caused the strongest SOS induction in any mutants obtained, similar to the level found in strains lacking repressor (lexA(Def) mutants). This full induction in the absence of lethal DNA damage underscores the pivotal role of RecA protein in regulating the SOS response.

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / Biological Sciences / Master / Master of Philosophy

The lexA41 (formerly tsl-l) mutant was previously isolated as a UV-resistant, temperature-sensitive derivative of its UV-sensitive lexA3(Ind⁻) parent. Cells exhibit a so-called "split-phenotype", a phenomenon in which only a subset of the SOS responses can be detected physiologically following inducing treatments. In this work, lexA41 has been cloned and sequenced; the mutant gene retains the lexA3 mutation (Gly to Asp at position 85) and has a second mutation, lexA41 (Ala to Thr at position 132). LexA41 protein is not cleaved by the RecA protein-catalyzed pathway in vivo, but the mutant protein is degraded by the Lon protease at both 32° and 42°C. β-galactosidase activities of lac fusions to thirteen different SOS promoters were measured at 30° and 42° to determine levels of expression and were found to vary considerably. LexA41 protein is deficient in repressor function. The temperature sensitive phenotype is due to increased expression of sulA, which encodes a division inhibitor, at 42°. Excision repair genes, including uvrA, uvrB and uvrD, are constitutively expressed at 30° accounting for the UV resistance of the lexA41 mutant, but the SOS mutagenesis operon, umuDC, is not adequately derepressed explaining the failure to induce mutagenesis in this background. This differential expression of SOS genes gives a plausible explanation of the "split-phenotype" associated with lexA41. In another set of experiments, I have examined the level of expression of the SOS regulon in cells lacking DNA adenine methylase activity (dam⁻). Mud (Ap, lac) fusions to several SOS operons (recA, lexA, uvrA, uvrB, uvrD, sulA, dinD, and dinF) were found to express higher levels of (beta)-galactosidase in dam⁻ strains than in isogenic dam⁺ strains. The attempted construction of dam⁻ strains that were also mutant in one of several SOS genes indicated that viability of methylase-deficient strains correlates with the inactivation of the SOS repressor (LexA protein). Consistent with this, the wild-type functions of two LexA-repressed genes (recA and ruv) appear to be required for viability of dam⁻ strains.

The past fifteen years have seen a revolution in human population genetics. We have gone from anecdotal genetic data from a few individuals at a few genetic loci to an avalanche of genome-wide sequencing data, from many individuals in many different human populations. These new data have opened up many new directions of research in human population genetics. In this work, I explore two such directions. Genomic data have uncovered that recent changes in human population size have had dramatic effects of on the genomes of different human populations. These effects have raised the question of whether historic changes in population size have led to differences in the burden of deleterious mutations, or mutation load, between different human populations. In Chapter 1 of this thesis, I show that despite earlier arguments to the contrary only minor differences in load are expected and indeed observed between Africans and Europeans. Over the past decade, genome-wide association studies (GWAS) have begun to systematically identify the genetic variants underlying heritable variation in quantitative traits. The number, frequencies and effect sizes of these variants reflect the selection, and other evolutionary processes, acting on traits. In Chapter 2, I develop a model for traits under pleiotropic, stabilizing selection, relate the model’s predictions to GWAS findings, and show that GWAS findings for height and BMI indeed follow model predictions. In Chapter 3, I develop a method to infer the distribution of selection coefficients acting on genome-wide significant associations made by GWAS.