Homologous chromosomes display associations in many organisms. Drosophila melanogaster (here after, Drosophila) serves as an excellent model to study pairing interactions since chromosomes are paired in all somatic cells throughout development. For many genes, the degree of homolog association influences gene expression. These effects, collectively referred to as transvection, can promote gene activation or silencing. Requirements for transvection are poorly understood. Chapter One reviews what is known about transvection in Drosophila and chromosome interactions in mammals. Recent cell culture studies implicated a requirement for Topoisomerase 2 (Top2) in chromosome pairing. Top2 encodes an ATP dependent homodimeric enzyme that generates double stranded breaks to change DNA topology. This enzyme is a common target of anticancer drugs due to its role in DNA metabolism. To understand the in vivo role of Drosophila Top2, an EMS screen was completed. Chapter Two describes the identification and characterization of fifteen new EMS generated Top2 mutations. Fifteen null and hypomorphic alleles were obtained, including one that displays temperature sensitivity. Molecular analyses of these alleles uncovered single or multiple base pair substitutions within the coding region of each mutant gene. Even though flies carrying individual missense alleles in trans to a deficiency are inviable, heteroallelic combinations of several missense alleles produced viable flies, including two lines carrying mutations that display resistance to anti-cancer drugs. These data indicate that Top2 activity can be restored by dimerization of defective subunits. Our new Top2 alleles establish a novel allelic series and provide a platform for understanding drug resistance. In Chapter Three, the role of Top2 in chromosome associations was tested to determine whether mutations in Top2 disrupted transvection. Viable heteroallelic combinations of Top2 mutations were used to test transvection at three classically studied loci. For each gene, homologous interactions were analyzed by screening for alterations in pairing-dependent changes in phenotype involving transvecting alleles. Only one of the three genes tested displayed phenotypic changes in Top2 complementing adults that were consistent with an alteration in pairing dependent changes in expression. Transcript levels were assessed at the three genes studied that display transvection. Our studies indicate that changes in the phenotype, due to altered Top2, are likely gene specific transcriptional changes. Further investigation of gene associations in Top2 mutants employed fluorescence in situ hybridization (FISH). These studies showed that all loci examined were paired near wild type levels, suggesting that Top2 does not globally disrupt homolog associations in vivo. The differences observed in Top2 function in vivo and in vitro may be explained by two possibilities. First, the probes studied differ from those used in vitro, indicating that different genetic loci may have different sensitivities to unpairing. Second, Top2 plays a role in the segregation of sister chromatids during anaphase and loss of Top2 causes improper resolution of chromosomes resulting in aneuploidy. In cell culture, cells were allowed to go through one division and then were subsequently fixed, permitting analyses on all cells. It is possible that nuclei exhibiting aneuploidy have undergone cell death in vivo, explaining why we do not see increased amounts of unpairing. In conclusion, Top2 contributions to nuclear functions are complex. Loss of Top2 may result in subtle changes in pairing that may affect transcription and transvection.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(医学) / 甲第23090号 / 医博第4717号 / 新制||医||1050(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 篠原 隆司, 教授 小川 修, 教授 溝脇 尚志 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM