RUI: Isolation and Characterization of Cell Cycle Mutants from Kluyveromyces lactis

RUI：乳酸克鲁维酵母细胞周期突变体的分离和表征

• 批准号: 9904900
• 负责人: Linda Silveira
• 金额: $ 12.09万
• 依托单位: University of Redlands
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9904900&HistoricalAwards=false
• 关键词: RUI; Isolation; Characterization; Cell; Cycle

Linda SilveiraMCB- 9904900AbstractGenetic studies in the budding yeast S. cerevisiae and the fission yeast S. pombe have been instrumental in identifying the mechanisms of cell-cycle control. This project leverages the information from these prior studies by extending the genetic analysis of cell division to a third, distantly related yeast species, Kluyveromyces lactis. Temperature-sensitive K. lactis mutants with general defects in cell growth will be isolated after mutagenesis. These mutants will be screened via phase microscopy to identify those with defects in the cell cycle at 36oC. The precise cell-cycle stage that is blocked in each mutant will then be evaluated by a combination of flow cytometry and DAPI-staining of chromosomal DNA. Because regulation of mitosis differs sharply in S. pombe and S. cerevisiae and mutants isolated in previous genetic screens for mitotic regulators have only partially overlapped, K. lactis mutants may help understand the evolution of these differences. Once mutants are identified, classified, and sorted into complementation groups, the corresponding genes will be cloned by complementation of the Ts- growth defect using an existing K. lactis genomic DNA library. The project has the potential to contribute to our understanding of cell division in at least five different ways. First, this study should identify new genes involved in cell cycle control. Second, the phenotypes of K. lactis mutants may vary in informative ways from those of the corresponding S. pombe or S. cerevisiae mutants. Third, comparison of homologous K. lactis, S. cerevisiae, and S. pombe cell cycle genes should help reveal critical features of genes required for this process. Fourth, functional differences between K. lactis and S. cerevisiae homologues can be exploited to identify interacting factors. Fifth, as roles for the same regulator can vary from organism to organism, often for reasons that are as yet unclear, analysis of a second budding yeast should shed light on the evolution of cell cycle regulatory mechanisms and the relationship between these mechanisms and the lifestyle of the organism. The project will also provide numerous opportunities for undergraduate students to conduct independent research. Such research experience fosters improved scientific writing and oral presentation skills, increased technical abilities in genetics and molecular biology, and an in-depth understanding of a central field within cell biology.

Linda SilveiraMCB-9904900摘要酿酒酵母和裂殖酵母的遗传学研究有助于确定细胞周期调控的机制。该项目利用了这些先前研究中的信息，将细胞分裂的遗传分析扩展到第三个远亲酵母物种--乳酸克鲁维酵母。具有一般生长缺陷的温度敏感型乳酸克雷伯氏菌突变株将在诱变后被分离。这些突变体将通过相位显微镜进行筛选，以确定那些在36oC的细胞周期中存在缺陷的突变体。然后将通过结合流式细胞术和染色体DNA的DAPI染色来评估每个突变体中受阻的确切细胞周期阶段。由于金黄色葡萄球菌和酿酒酵母对有丝分裂的调控有很大的不同，而且在以前的有丝分裂调节基因筛选中分离的突变株只有部分重叠，因此乳酸钾突变株可能有助于理解这些差异的进化。一旦突变体被鉴定、分类并归入互补组，相应的基因将通过利用现有的K.lactis基因组DNA文库对ts-生长缺陷进行互补来克隆。该项目有可能通过至少五种不同的方式帮助我们理解细胞分裂。首先，这项研究应该确定与细胞周期控制有关的新基因。其次，乳酸克雷伯氏菌突变体的表型可能在信息方面与相应的庞氏葡萄球菌或酿酒酵母突变体的表型不同。第三，比较乳酸克雷伯菌、酿酒葡萄球菌和庞氏葡萄球菌细胞周期基因的同源，有助于揭示这一过程所需基因的关键特征。第四，乳酸克雷伯氏菌和酿酒酵母同系物之间的功能差异可以用来确定相互作用的因素。第五，由于同一调节器的作用在不同的生物体中可能不同，原因通常尚不清楚，对第二个萌芽酵母的分析应该有助于阐明细胞周期调节机制的进化，以及这些机制与生物体生活方式之间的关系。该项目还将为本科生提供大量进行独立研究的机会。这种研究经验有助于提高科学写作和口头陈述的技能，提高遗传学和分子生物学的技术能力，并深入了解细胞生物学中的一个中心领域。