Mechanisms of Genomic Rearrangement in Yeast

我们已经分离到一个超重组突变体top3，它在酿酒酵母中鉴定了一种新的真核拓扑异构酶。预测的top3基因的开放阅读框与细菌型拓扑异构酶TopA同源，但与真核细胞的I型或II型酶不同源。我们计划研究这种新型酶的生化性质，并确定其底物偏好。零突变表现为生长缓慢、二倍体形成减少和产孢量缺陷等多效性表型。我们将通过检查有丝分裂和减数分裂的核分裂、纺锤体的形成和交配反应来研究这些多向性的后果。基因实验将通过体外诱变top3基因和寻找这些表型的遗传抑制因子来解决这些多向性。一个慢生长抑制因子sgs1已经被分离出来，并将使用我们将要开发的一种改进的Ty突变方法进行克隆和定位。最后，我们将通过使用核酸同源性或蛋白质同源性作为探针，尝试在进化上遥远的裂殖酵母中鉴定同源物，从而开始研究top3基因是否是所有细胞的一般特征。我们的目标是了解这种新的拓扑异构酶在细胞生长和发育中所起的作用。基因重组的过程在所有活着的有机体中都能观察到。尽管这一过程对个体和种群都有重要的生物学后果，其机制的细节多年来一直在多个实验室进行密集研究，但我们仍然没有很好地了解这一过程如何帮助活着的有机体生存。在他之前对酵母中重组机制的研究中，这一建议的作者发现了一个参与这一过程的新基因；该基因编码一种以前在酵母或高等生物中未见过的酶。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提议。

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.