Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008. / Title from title screen (site viewed Mar. 5, 2009). PDF text: 78 p. : ill. (some col.) ; 8.43 Mb. UMI publication number: AAT 3330302. Includes bibliographical references. Also available in microfilm and microfiche formats.

Meiosis is the process by which sexually-reproducing organisms ensure that precisely half a chromosome set is passed from each parent to the following generation; this circumvents the doubling of the genome that would otherwise occur upon fertilisation. Meiosis occurs via a single round of DNA replication followed by two successive chromosome segregation events. In the first segregation, homologous chromosomes align and become physically linked through the process of meiotic recombination, which is crucial for the accurate segregation of homologous chromosomes. During the second round of segregation, sister chromatids are segregated to produce four haploid daughter cells. Failure to physically tether homologous chromosomes to each other through meiotic recombination can result in the aberrant segregation of homologous chromosomes, which can cause hereditary diseases (aneuploidies) and miscarriages in humans. Meiotic recombination also shuffles alleles of the parental chromosomes, which is crucial for evolution. The study of meiotic recombination, and its regulation, is thus paramount for our understanding of how genetic diversity is generated within populations. The work in this thesis has helped characterise factors, both genetic and environmental, that modulate meiotic recombination in the fission yeast, Schizosaccharomyces pombe. Here, I identify temperature as a major determinant of meiotic recombination outcome; when meiosis is performed at 16°C, significant reductions in meiotic recombination outcome are observed relative to meiosis performed at higher temperatures. Additionally, I present genetic and cytological evidence that the strand resection and strand invasion steps of meiotic recombination are impaired at 16°C relative to higher temperatures, but that double strand break levels appear not to be influenced by temperature. I have also characterised several novel genes predicted to be involved in meiotic recombination, and explored the genetic relationship between several genes already known to be crucial in modulating meiotic recombination. Finally, I have laid the foundations for a future project aiming to map the meiotic recombination landscape across the entire S. pombe genome.

Abstract The evolution of sociality is often associated with close relatedness and genetic similarity of interacting individuals. However, colonies of advanced social insects (e.g. ants, bees and wasps) characterized by large colony size and division of tasks, are also shaped by acquisition of genetic diversity by polyandry, polygyny, recombination and even by hybridization. The balance between forces selecting for high relatedness on one hand and for improved colony performance though increased genetic diversity on the other hand forms an intriguing area of research. My study has produced the first genetic linkage maps for ants (Acromyrmex echinatior and Pogonomyrmex rugosus) and social wasps (Vespula vulgaris). Together with the findings of earlier honeybee research, it is shown that advanced eusocial insects have higher recombination rates than any other insect (or animal) studied so far. The estimates obtained here were 14 cM/Mb for P. rugosus, 9.7 cM/Mb for V. vulgaris and 6.2 cM/Mb for A. echinatior. Pogonomyrmex harvester ants have a genetic caste determination system in which workers arise from mating between two hybridizing lineages whereas sexuals are produced by within-lineage mating. I evaluated the origin of the lineages and the caste determination system by using 751 variable nuclear genetic markers. Fertile hybrids would lead to introgression, particularly in genomic regions characterized by a high recombination rate and lack of strongly selected loci. The hybridizing lineages (lineage pairs J1/J2 and H1/H2) showed many fixed differences. Nineteen of them were in the constructed linkage map, scattered in different linkage groups. The results suggest that there has been no recent introgression. As the hybrids are viable (as workers), caste differentiation can be affected by many loci scattered throughout the ant genome or by a small number of very strongly selected loci. Genetic diversity in colonies of the ant Formica cinerea is affected by varying levels of polygyny. I tested the hypotheses that the prevalence of endosymbiotic bacteria can vary in polygynous colonies but be either very low or very high in monogynous colonies. However, I found no association between the level of polygyny and endosymbiont prevalence. In addition to Wolbachia, I found two other endosymbiotic bacteria Cardinium and Candidatus Serratia symbiotica which have not been earlier reported from ants. Genetic diversity in insect colonies is affected by polyandry and polygyny. My results indicate that high a recombination rate is also an important factor influencing diversity. Genotypically diverse progenies can enhance colony success, e.g. through effects on division of labour or defence against pathogens. Recombination differs from the other factors in its effects on genetic relatedness among colony members.

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The role of the T7-induced exonuclease (gene 6) in recombination was studied using both molecular and genetic techniques. In the molecular method the fate of parental DNA during parent-to-progeny recombination was examined. A comparison of infections with T7⁺, T7am6 (amber gene 6), or T7ts6 (temperature sensitive gene 6) under permissive and nonpermissive conditions was made. CsCl density gradient analysis of replicative DNA indicated that the T7 exonuclease is necessary for recombination to occur, i.e., in the absence of the exonuclease the parental DNA replicated continuously as a hybrid molecule and did not recombine. Analysis of denatured replicative DNA by CsCl density gradient centrifugation indicated that the exonuclease also may be needed for a limited amount of covalent repair of recombinants. Further confirmation of the essential role which the exonuclease plays in recombination came from genetic analysis. The T7 exonuclease was shown to be necessary for intragenic and intergenic recombination in several areas of the T7 genetic map; genetic recombination frequencies were found to be decreased from 3 to 18-fold under conditions nonpermissive for the exonuclease. The role of the T7-induced endonuclease (gene 3) in molecular recombination was studied by examining the fate of parental DNA during parent-to-progeny recombination using a shear technique. The T7 endonuclease was found to be necessary for the dispersion of parental DNA in the newly replicated DNA. Concatemers synthesized by either T7⁺ or T7am3 (amber gene 3) phage containing the newly replicated DNA were sheared to the size of mature phage DNA and also to quarter size molecules. In the presence of gene 3 protein, parental DNA and newly replicated DNA were interspersed, i.e., the 32P-label from the sheared DNA was found to sediment at the density of recombined DNA. In the absence of gene 3 protein, the parental strand of each sheared DNA molecule was usually found intact, i.e., the ³²P-label from the sheared DNA was found to sediment at the density of hybrid DNA. These results support the previous genetic data (52, 83) that the gene 3 protein is essential for T7 recombination. The role of T7 recombination enzymes in the formation of concatemers was studied by examining selected gene 3 and gene 6 mutants. Results of sucrose gradient analysis showed that DNA concatemers were formed when both the T7 exonuclease (gene 6) and the T7 endonuclease (gene 3) were absent. Further results showed that concatemers cannot be maintained in the absence of the exonuclease unless the endonuclease was eliminated. In a T7am6 infection DNA concatemers formed early were prematurely broken down and accumulated as fragments smaller than mature size phage DNA. In a T7am3am6 (amber in both genes 3 and 6) infection concatemers accumulated and were not matured. These results indicate that concatemers are formed by a process other than normal phage recombination. However, selective defects in the recombination system do interfere with the stability of concatemers. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18897号 / 医博第4008号 / 新制||医||1009(附属図書館) / 31848 / 京都大学大学院医学研究科医学専攻 / (主査)教授 松岡 雅雄, 教授 朝長 啓造, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Dissociative recombination of molecular ions by collisions with electrons is a reactive collision, in which the electronic kinetic energy is transferred to the excitation of the molecule that, then, dissociates. The goals of this dissertation was (1) to improve existing approaches in theory of DR of triatomic ions in the time-independent framework developed in recent years by Kokoouline and Greene, and (2) to develop a time-dependent theoretical framework for DR treatment based on quantum defect theory. The theoretical method developed by Kokoouline and Greene is based on multichannel quantum defect theory and accounts for the major non-Born-Oppenheimer Jahn-Teller interaction between electronic and vibrational motions of the molecule. The study of this dissertation is partially based on this method but improved, extended, and systematically applied in the framework of my thesis. This dissertation presents the calculated DR rate coefficient for H3+ within the time-independent framework and the description of the method used to obtain the (qualitative) flux with time-dependent method. The time-independent results show good agreement with experimental data from storage ring experiments. The DR rate coefficients for ortho- and para-H3+ were calculated separately and show a significant difference at very low electronic energies; a result that agrees with recent storage ring experiments. Also, it is discussed results for other isotopologues of H3+ (H2D+, D2H+, and D3+) and the results for vibrationally-excited initial states of H3+. It was found that the DR rate coefficients for vibrationally-excited initial states are larger than the rates for the ion initially in the ground vibrational state. At the end, this dissertation discuss the time-dependent calculations done with a diatomic model system.

Meiosis is a specialized form of cell division in which haploid gametes are produced from diploid progenitors. This reduction in ploidy results from proper meiotic chromosome segregation and is ensured by crossover recombination events. Given their importance, it is no surprise that crossover formation is regulated in most eukaryotes. Crossover assurance is a regulatory mechanism that helps to ensure that each pair of chromosomes gets at least one crossover during meiosis. We seek to better understand how crossover assurance works. To do so, we have developed a system in which crossover formation between a pair of chromosomes is restricted to a defined region. If crossover assurance functions in this context, then crossovers should frequently form in this defined region. Our experiments involve three yeast strains: Homolog: diploid Saccharomyces cerevisiae. Homeolog: Diploid S. cerevisiae, but with one copy of III derived from S. paradoxus and one from S. cerevisiae. Homo-meolog: The homeolog strain, but with the HIS4 region of the S. paradoxus III replaced with the corresponding S. cerevisiae sequence. S. cerevisiae and S. paradoxus are largely syntenic and have 80-90% sequence homology. This level of sequence divergence greatly reduces the incidence of meiotic crossing over. Thus, in the Homeolog strain chromosomes III will frequently fail to form crossovers. In the Homo-meolog strain, a defined region of homology surrounding HIS4 (a hotspot for meiotic recombination) exists in a chromosomal context of homeology. In the Homo-meolog strain, crossover assurance should result in a high incidence of crossover formation in the HIS4 region. By comparing the spectrum of meiotic recombination events in the HIS4 region in the three strains, we will gain insight into the means through which crossover assurance is enforced. These experiments are in the preliminary stage. Strain construction and data collection are ongoing, but our preliminary results demonstrate an elevated incidence of crossing over in the HIS4 region in the homo-meolog strain relative to both the homolog and homeolog strains. Spore viability patterns in the homo-meolog strain are not statistically distinguishable from that of the homolog strain, but are different from that of the homeolog strain. Taken together, these results suggest that the crossovers are targeted to the HIS4 region in the homo-meolog strain, possible through the action of a crossover assurance mechanism. Further analysis of the patterns of recombination in these strains may provide insight into the means through which this regulation is exerted.

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No description available.