CELL CYCLE REGULATION OF THE YEAST HO GENE

酵母 HO 基因的细胞周期调控

• 批准号: 3467367
• 负责人: LINDA L. BREEDEN
• 金额: $ 9.75万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-12-01 至 1993-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3467367
• 关键词: DNA binding protein; DNA footprinting; Saccharomyces cerevisiae; cell cycle; fungal genetics; gene expression; genetic enhancer element; genetic transcription; immunochemistry; molecular cloning; nucleic acid sequence; temperature sensitive mutant

The transcription of the S. cerevisiae HO gene occurs immediately after and is dependent upon the start of the cell cycle. The long term goal of this project is to determine how this cell cycle regulation is exerted upon the HO promoter. A repeated sequence has been identified (CACGA4) that is necessary and sufficient for the start-dependent, and transient activation of HO transcription. The specific aims of this proposal are to: 1) determine what forms of regulation are exerted upon the CACGA4 sequence, 2) identify and characterize the trans-acting factors that are responsible for that regulation, 30 determine which factor displays transient activity through the cell cycle, and 4) identify the start- specific signal that is responsible for that transient activation. This work should lead to a better understanding of the early events in the cell cycle and the mechanisms of start. The CACGA4 element acts as a cell cycle regulated upstream activation sequence (UAS). Two proteins that are specifically required for CACGA4- driven transcription (SW14 and SW16) have been cloned and sequenced. IN order to investigate their role in the transient activation of CACGA4, deletion mutant and over-producing strains have been made. Footprint and band-shift assays will be carried out with crude extracts from these mutant strains to see if SW14 or SW16 are required for binding to the CACGA4 sequence. Antibodies will be raised to both proteins and use to see if SW14 or SW16 can be detected in the protein-DNA complex at CACGA4. Antibodies will also be used for observing any qualitative or quantitative changes in these proteins through the cell cycle which might affect their activity. To identify other components of the CACGA4-regulatory system, genetic screens have been devised to look for mutations in negative regulators or positive regulators that result in constitutive CACGA4-driven transcription. Particular attention will be paid to those displaying is lethality, or other defects in growth control. these regulators will be characterized as described above for SW14 and SW16. If cell cycle- specific changes in the CACGA4- protein complex or in the physical state of any of these regulators is observed, the molecules responsible for that cyclic change will be purified and characterized. Such a molecule would be expected to be directly involved in or closely associated with the start of the cell cycle.

酿酒酵母HO基因的转录发生在 并依赖于细胞周期的开始。的长期目标是 这个项目是为了确定这种细胞周期调节是如何实施的。 在HO启动者身上。发现了一个重复序列(CACGA4) 这对于依赖于启动的和瞬变的 HO转录的激活。这项提议的具体目的是 以：1)确定对CACGA4实施哪些形式的监管 序列，2)识别和表征以下反式作用因子 负责该规则，30确定显示哪个因素 细胞周期中的瞬时活动，以及4)识别开始- 负责瞬时激活特定信号。这 工作应该导致对早期事件的更好理解 细胞周期和启动机制。 CACGA4元件作为细胞周期调控的上游激活元件 序列(UAS)。CACGA4特别需要的两种蛋白质- 驱动转录(SW14和SW16)已被克隆和测序。在……里面 为了研究它们在CACGA4瞬时激活中的作用， 获得了缺失突变株和高产菌株。占地面积和 从这些植物中提取的粗提物将进行带移分析。 以查看是否需要SW14或SW16才能与 CACGA4序列。抗体将被提升到两种蛋白质上，并用于 看看在CACGA4的蛋白质-DNA复合体中是否能检测到SW14或SW16。 抗体也将被用来观察任何定性或 这些蛋白质在细胞周期中的数量变化可能 影响他们的活动。 为了确定CACGA4-调控系统的其他组件，基因 已经设计了筛查来寻找负性调节者或 导致结构性CACGA4驱动的正向调节器 抄写。将特别注意那些展示IS的人 致命性，或生长控制方面的其他缺陷。这些监管机构将是 其特征如上所述用于SW14和SW16。如果细胞周期- CACGA4-蛋白质复合体或物理状态的特定变化 这些调节剂中的任何一个都是被观察到的，负责 这种循环变化将被提纯和表征。这样一种分子 预计将直接参与或密切关联于 细胞周期的开始。