Mechanisms of Genomic Rearrangement in Yeast

我们已经分离出一个超重组突变体，TOP3，在酵母中鉴定出一种新的真核拓扑异构酶，酿酒酵母。预测的TOP3基因开放阅读框与细菌I型拓扑异构酶TopA同源，而与真核生物I型或II型酶不同源。我们计划研究这种新酶的生化特性，并确定其底物偏好。零突变表现出多效性表型，如生长缓慢，二倍体形成减少和孢子形成缺陷。我们将通过检查有丝分裂和减数分裂核分裂，纺锤体形成和交配反应来研究这些多效性的后果。遗传实验将通过TOP3基因的体外诱变和寻找这些表型的遗传抑制因子来解决这些多效性。一种生长缓慢的抑制因子sgs1已经被分离出来，并将使用我们将开发的改良的Ty诱变方法进行克隆和定位。最后，我们将开始研究TOP3基因是否是所有细胞的普遍特征，通过使用核酸同源性或蛋白质同源性作为探针，试图在进化上遥远的酵母裂糖酵母中鉴定同源物。我们的目标是了解这种新型拓扑异构酶在细胞生长和发育中的作用。基因重组的过程在所有生物体中都存在。尽管这一过程对个体和种群都有重要的生物学影响，并且其机制的细节已经在许多实验室中进行了多年的深入研究，但我们仍然没有很好地了解这一过程如何帮助生物体生存。在他之前对酵母重组机制的研究中，本提案的作者发现了参与该过程的新基因；这种基因编码了一种酵素，这种酵素以前在酵母菌或更高级的生物体中从未见过。该基因缺陷的突变体在其生命周期的许多方面也存在缺陷。这是一项研究该基因如何参与这些其他生命过程的建议。

We have isolated a hyper-recombination mutants, TOP3 that identifies a novel eukaryotic topoisomerase in the yeast, Saccharomyces cerevisiae. The predicted open reading frame of the TOP3 gene is homologous to the bacterial type, I topoisomerase, TopA, but not homologous to eukaryotic type I or II enzymes. We plan to investigate the biochemical properties of this novel enzyme and to define its substrate preferences. Null mutations exhibit pleiotropic phenotypes such as slow growth, reduced diploid formation and a sporulation defect. We will investigate the consequences of these pleiotropies by examining mitotic and meiotic nuclear division, spindle formation and the mating reaction. Genetic experiments will address these pleiotropies by in vitro mutagenesis of the TOP3 gene and by searching for genetic suppressors of these phenotypes. A slow growth suppressor, sgs1, has already been isolated and will be cloned and mapped using a modified Ty mutagenesis method that we will develop. Lastly, we will begin to investigate whether the TOP3 gene is a general feature of all cell by attempting to identify a homologue in the evolutionarily distant yeast Schizosaccharomyces pombe by using either nucleic acid homology or protein homology as a probe. It is our goal to understand the role that this novel topoisomerase plays in cells growth and development. The process of genetic recombination is observed in all living organisms. Although this process has important biological consequences both for individuals and for populations, and the details of its mechanism has been under intense study in a number of laboratories for years, we still do not have a good understanding of how this process helps living organisms survive. During his previous investigations into the mechanism of recombination in yeast, the author of this proposal discovered anew gene involved in this process; this gene encoded an enzyme of a type not previously seen in yeast or higher organisms. Mutants defective in this gene also had defects in many aspects of their life cycle. This is a proposal to investigate how this gene is involved with these other life processes.