CELL CYCLE REGULATION OF THE YEAST HO GENE

S.酿酒酵母HO基因发生后立即 并且依赖于细胞周期的开始。的长期目标 这个项目是为了确定这种细胞周期调节是如何发挥作用的， HO启动子。已确定重复序列（CACGA 4） 这是必要的和充分的启动依赖，和瞬态 HO转录的激活。这项建议的具体目标是 1）确定对CACGA 4施加何种形式的监管 序列，2）识别和表征反式作用因子， 负责该调节30确定显示哪些因子 通过细胞周期的瞬时活性，和4）识别起始- 负责瞬时激活特定信号。这 这项工作应有助于更好地了解联合国系统内的早期事件， 细胞周期和启动机制。 CACGA 4元件作为细胞周期调节的上游激活 序列（UAS）。CACGA 4特异性需要的两种蛋白质- 驱动转录（SW 14和SW 16）的基因组序列进行了克隆和测序。在 为了研究它们在CACGA 4瞬时激活中的作用， 已经制备了缺失突变体和高产菌株。足迹和 将用这些植物的粗提物进行带移测定， 突变菌株，以确定SW 14或SW 16是否是结合所需的 CACGA 4序列。将针对这两种蛋白质产生抗体， 看看SW 14或SW 16是否可以在CACGA 4的蛋白质-DNA复合物中检测到。 抗体也将用于观察任何定性或定量的结果。 这些蛋白质在细胞周期中的定量变化， 影响他们的活动。 为了鉴定CACGA 4调节系统的其他成分，遗传学 已经设计了筛选来寻找负调节因子中的突变， 导致组成型CACGA 4驱动的正调控因子 转录。将特别注意那些显示是 致死性或生长控制中的其他缺陷。这些监管机构将 其特征如上文对SW 14和SW 16所述。如果细胞周期- CACGA 4-蛋白质复合物或物理状态的特定变化 任何这些监管机构的观察，分子负责 这种循环变化将被净化和表征。这种分子 将直接参与或密切相关， 细胞周期的开始。

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.