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度发生。 这种程度的 复杂性要求有一个复杂的监管网络 不仅能够协调这些事件，而且能够认识和 纠正在这些复杂过程中发生的错误。 的 这些监管电路的基础设施将在 使用编码酶核糖核苷酸的基因的酿酒酵母 还原酶，RNR 1，2和3作为工具。 RNR 1和RNR 2是细胞周期- 调节，在S期表达最多，并且所有三个基因都是 转录诱导的，以响应用损伤 DNA或阻断DNA复制。 RNR 1的细胞周期调节将是 通过缺失和亚克隆分析进行研究，以确定序列 这是细胞周期调节的必要和充分条件。 反式作用因子 与这些序列相互作用的基因将被长期研究， 分离出参与调控的基因。 此外，归纳 在细胞周期开始后， 依赖于蛋白质的合成。 这种依赖性将进一步 使用已知的细胞周期突变体和差异cDNA筛选进行研究 寻找启动后诱导的转录本，但在蛋白质 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）导致细胞周期停滞在S期，这是独立的 RAD 9基因 参与对HU的这种反应的基因将被分离， 筛选具有“抗CDC”表型的HU敏感性突变， 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分离。 分析这一途径 将揭示真核细胞的一个关键调控回路 协调DNA复制与G2和M期进展的周期。 感知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， 细胞适当调节RNR 2和RNR 3基因的能力， 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。 生物体感知和反应的能力 对它的遗传物质的损害是它适应环境的能力的核心。 环境压力和生存。 这条监管电路可能会 在真核生物中是保守的，并且可能揭示高等生物的能力。 真核生物对DNA损伤的感知和反应。 将构建一个双重目的的基于γ的质粒表达载体 能够调控E.大肠杆菌和酵母菌。 在此载体中构建的文库将用于分离酵母基因 编码序列特异性DNA结合蛋白的基因 E.杆菌 这些文库也将用于酵母， 通过筛选 当表达时，导致显性致死性和CDC抑制 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.