YEAST CDC6 GENE CELL CYCLE PROGRESSION

酵母 CDC6 基因细胞周期进展

• 批准号: 2185960
• 负责人: AMBROSE Y JONG
• 金额: $ 16.41万
• 依托单位: CHILDREN'S HOSPITAL OF LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1997-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2185960
• 关键词: DNA replication; adenosinetriphosphatase; alleles; binding proteins; cell cycle; chemical binding; enzyme activity; fungal genetics; fusion gene; gene interaction; guanosinetriphosphatases; laboratory rabbit; methane sulfonate; nucleic acid sequence; phenotype; polymerase chain reaction; protein structure function; suppressor mutations; temperature sensitive mutant; yeasts

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.

该资助的总体目标是研究CDC6基因功能及其 细胞周期调节。 早期遗传学研究表明，酵母 温度敏感突变体 cdc6 在 G1 和 S 晚期有缺陷 相边界，其功能可以是直接或 间接参与DNA复制。 我们之前已经隔离并 CDC6 基因的特征。 我们还证明了 CDC6 mRNA CDC6蛋白的入核和入核是细胞周期依赖性的。 在这个 投稿期间，我们发现GTS::CDC6融合蛋白具有 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化方法来研究 CDC6 的生物学功能及其细胞周期调节。 首先，我们将识别与相互作用的细胞成分 CDC6基因产物。 为了实现这一目标，我们将隔离并 表征抑制因子以在 CDC6 之间建立关联途径 以及细胞周期进程中的其他相关基因。 我们还将使用 分子遗传学方法，2-杂交系统，来识别 在体内与 CDC6 相互作用的蛋白质成分。 结果将 补充我们的基因研究，提供额外的信息 建立细胞周期进展的途径。 其次，我们将通过以下方式探索 CDC6 的基因功能： 生化方法。 我们将通过以下方式表征纯化的 CDC6 蛋白： 研究其核苷酸结合、ATP 酶和/或 GTP 酶活性。 根据序列基序，我们还将检测可能的解旋酶 活动。 点突变的 cdc6 蛋白也将被纯化为 补充上述生化研究。 预计 结果将揭示 CDC6 的生物学功能。 我们期待对酵母CDC6基因及其相关性的研究 抑制器将导致对功能的理解 其他真核生物中的相应基因，可能为我们提供有用的信息 揭示细胞深层调控机制的信息 分裂周期。