GENETICS OF CELL CYCLE & DNA DAMAGE REGULATION IN YEAST

真核细胞周期是一系列高度复杂的过程， 必须以惊人的时间和空间精确度发生。这一程度 复杂性要求存在一个复杂的监管网络 不仅能够协调这些事件，而且能够识别和 纠正在这些复杂过程中发生的错误。这个 这些监管电路的基础设施将在 利用编码核糖核酸酶基因的酿酒酵母 还原酶，RNR1，2和3，作为工具。RNR1和RNR2是细胞周期- 受调控，在S期最高表达，三个基因都是 转录诱导的对损伤药物处理的反应 DNA或阻止DNA复制。RNR1的细胞周期调节将是 通过缺失和亚克隆分析来确定序列 对细胞周期的调节是必要的和充分的。反式作用因素 与这些序列相互作用的序列将进行长期目标的检查 分离参与这一调控的基因。此外，归纳法 细胞周期开始后RNR1基因转录水平的变化 依赖于蛋白质的合成。这种依赖将进一步 利用已知的细胞周期突变和差异基因筛选进行研究 寻找在启动后，但在蛋白质之前诱导的转录本 合成块。 特异性抑制剂对核糖核苷酸还原酶活性的抑制作用 羟基脲(HU)引起细胞周期停滞于S期，其作用不依赖于 RAD9基因。参与对胡的反应的基因将通过以下方式分离 筛选具有“抗CDC”表型的HU敏感突变 关于HU诱导的细胞周期停滞。感兴趣的基因将是 通过这些突变的互补而分离的。对这条路径的分析 将阐明真核细胞的一个关键调控电路 协调DNA复制进入G2和M的周期。 感觉DNA损伤并诱导RNR表达的途径将是 通过分离顺式和反式突变来研究改变 细胞适当调节RNR2和RNR3基因的能力 对DNA损伤的反应。参与这一调控的基因将被分离出来 通过互补。有机体感知和反应的能力 对其遗传物质的破坏是其适应能力的核心 环境压力和生存能力。这一监管电路很可能会 在真核生物中是保守的，并可能揭示更高的 真核生物对DNA损伤的感知和反应。 将构建一种双重用途的伽马基质粒表达载体 它能够调控基因在大肠杆菌和酵母中的表达。 在该载体中构建的文库将用于分离酵母基因 编码感兴趣的序列特异性DNA结合蛋白 在大肠杆菌中进行选择。这些文库还将用于酵母中 通过筛选新的细胞分裂周期(CDC)基因 当表达时，会导致明显的致命性和疾控中心的逮捕 表型。

The eukaryotic cell cycle is a cascade of highly complex processes that must occur with striking temporal and spatial precision. This degree of complexity necessitates the existence of a sophisticated regulatory network capable not only of coordinating these events, but also of recognizing and correcting mistakes that occur during these complex processes. The infrastructured of these regulatory circuits will be probed in Saccharomyces cerevisiae using the genes encoding the enzyme ribonucleotide reductase, RNR1, 2, and 3, as tools. RNR1 and RNR2 are cell cycle- regulated, expressed maximally in S phase, and all three genes are transcriptionally induced in response to treatment with agents that damage DNA or block DNA replication. The cell cycle-regulation of RNR1 will be investigated by deletion and subcloning analysis to determine the sequences necessary and sufficient for cell cycle-regulation. Trans-acting factors that interact with these sequences will be examined with the long-term goal of isolating the genes involved in this regulation. In addition, induction of transcription of the RNR1 gene after passing the start of the cell cycle is dependent on protein synthesis. This dependency will be further investigated using known cell cycle mutants and differential cDNA screens to look for transcripts induced after Start, but prior to the protein synthesis block. Inhabitation of ribonucleotide reductase activity by the specific inhibitor hydroxyurea (HU) causes cell cycle arrest in S-phase that is independent of the RAD9 gene. Genes involved in this response to HU will be isolated by screening for HU-sensitive mutations that have an "anti-CDC" phenotype with respect to HU-induced cell cycle arrest. Genes of interest will be isolated by complementation of these mutations. Analysis of this pathway will shed light on a critical regulatory circuit of the eukaryotic cell cycle that coordinate DNA replication with progression into G2 and M. The pathway that senses DNA damage and induces RNR expression will be investigated by the isolation of cis- and trans-mutations that alter the ability of the cell to properly regulate the RNR2 and RNR3 genes in response to DNA damage. Genes involved in this regulation will be isolated by complementation. The ability of an organism to sense and respond to damage to its genetic material is central to its ability to adapt to environmental stress and to survive. This regulatory circuit is likely to be conserved among eukaryotes and may shed light on the ability of higher eukaryotes to sense and respond to DNA damage. A dual purpose gamma-based plasmid expression vector will be constructed that is capable of regulated expression of genes in E. coli and yeast. Libraries constructed in this vector will be used to isolate yeast genes encoding sequence-specific DNA-binding proteins of interest by genetic selection in E. coli. These libraries will also be used in yeast to identify new cell division cycle (CDC) genes by screening for clones which when expressed, result in dominant lethality and arrest with a CDC phenotype.