YEAST CDC6 GENE CELL CYCLE PROGRESSION

该基金的总体目标是研究CDC 6基因的功能及其对人类的影响。 细胞周期调控 早期的遗传学研究表明， 一个温度敏感突变体cdc 6在G1和S期晚期有缺陷 相边界，其功能可以是，直接或 间接参与DNA复制。 我们之前已经分离出 鉴定了CDC 6基因。 我们还证明了CDC 6 mRNA CDC 6蛋白进入细胞核与细胞周期有关。 在这 在提交期间，我们发现GTS：：CDC 6融合蛋白具有 a DNA依赖性ATP酶活性。 在本提案中，我们将使用 结合分子遗传学和生物化学方法来研究 CDC 6的生物学功能及其细胞周期调控。 首先，我们将确定与细胞相互作用的细胞成分。 CDC 6基因产物。 为了做到这一点，我们将隔离和 表征抑制子，以建立CDC 6与 以及其他与细胞周期进程相关的基因。 我们还将使用 分子遗传学方法，2杂交系统，以确定 在体内与CDC 6相互作用的蛋白组分。 结果将 补充我们的基因研究，提供更多的信息， 建立细胞周期进展途径。 其次，我们将通过基因组学研究CDC 6的基因功能。 生物化学方法 我们将通过以下方法表征纯化的CDC 6蛋白： 研究其核苷酸结合、ATP酶和/或GTP酶活性。 基于序列基序，我们还将检测可能的解旋酶 活动 点突变的cdc 6蛋白也将被纯化， 补充上述生物化学研究。 预计该 结果将阐明CDC 6的生物学功能。 我们希望对酵母CDC 6基因及其表达的研究能为酵母CDC 6基因的研究提供新的思路。 抑制剂将导致对功能的理解， 其他真核生物中的相应基因，并可能为我们提供有用的 揭示细胞深层控制机制的信息 分裂周期

The overall goal of this grant is to study CDC6 gene function and its cell cycle regulation. Early genetic studies revealed that yeast temperature-sensitive mutant, cdc6, is defective in the late G1 and S phase boundary, and that its function may be, either directly or indirectly, involved in DNA replication. We have previously isolated and characterized the CDC6 gene. We have also demonstrated that CDC6 mRNA and nuclear entry of CDC6 protein are cell cycle dependent. In this submission period, we have found that the GTS::CDC6 fusion protein has a DNA-dependent ATPase activity. In this proposal, we will use a combined molecular genetic and biochemical approach to investigate the biological function(s) of CDC6 and its cell cycle regulation. Initially, we will identify the cellular components which interact with CDC6 gene products. In order to accomplish this, we will isolate and characterize the suppressor(s) to establish a linked pathway between CDC6 and other related genes in the cell cycle progression. We will also use a molecular genetic approach, the 2-hybrid system, to identify the protein component(s) which interact with CDC6 in vivo. The results will complement our genetic studies, providing additional information to establish a pathway of cell cycle progression. Secondly, we will explore the gene function(s) of CDC6 via the biochemical approach. We will characterize the purified CDC6 protein by investigating its nucleotide-binding, ATPase, and/or GTPase activities. Based on the sequence motif, we will also detect possible helicase activity. Point-mutated cdc6 proteins will also be purified to complement the above biochemical studies. It is anticipated that the results will shed light on the biological function(s) of CDC6. We expect that the investigation of the yeast CDC6 gene and its suppressors will lead to an understanding of the functions of corresponding genes in other eukaryotes, and may provide us with useful information to disclose the profound control mechanism of the cell division cycle.