CHARACTERIZATION OF A CELL DIVISION CONTROL-RELATED GENE

细胞分裂控制相关基因的表征

• 批准号: 2182366
• 负责人: VINCENT J. KIDD
• 金额: $ 14.77万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1995-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2182366
• 关键词: autoradiography; cell cycle; cell cycle proteins; cell growth regulation; cell sorting; complementary DNA; enzyme mechanism; galactosyltransferases; genetic transcription; human genetic material tag; human tissue; laboratory rat; messenger RNA; molecular cloning; nucleic acid hybridization; phosphorylation; protein kinase; protein structure function; radionuclides; regulatory gene; site directed mutagenesis

The ability of a cell to reproduce by cell division is one of the most fundamental processes of all living matter. Abnormalities of these processes are often involved in the growth of transformed cells. Therefore, a thorough understanding of the events involved in normal cell division is crucial to understanding transformed cell growth. To this end several groups have identified a cell division control protein kinase, p34cdc2, that is a crucial component of normal mitosis. Removal of this protein kinase activity has recently been shown to lead to a block in mitosis. However, this system is complex and the proteins that activate this kinase, as well as those that are acted upon by this kinase activity, remain unidentified. We have identified a new protein kinase that is highly related (68% homology) to the p34cdc2 kinase. This new kinase is 394 amino acids in size, is apparently regulated by phosphorylation and Ca2+ - calmodulin, and is localized to the cytoplasm and nucleus. Expression of the corresponding mRNA is regulated during the cell cycle, peaking during G(1)-and S-phase. Elevated expression of this CDC-related kinase in eukaryotic cells leads to an apparent block of the cell cycle at the late telophase and early G(1) boundary. We propose to examine, at a molecular and biochemical level, the nature of this CDC-related kinase and its role in cell cycle control. This will be accomplished by examining its regulation during the cell cycle. Careful analysis of steady-state mRNA and protein levels, kinase activity, phosphorylation state, and protein-protein interactions as a function of cell cycle will be performed using cDNA clones and antibodies for this new CDC-related kinase.We will also examine transcriptional and post-transcriptional regulation of the CDC-related mRNA as a function of cell cycle. Hybridization data indicates that sequence-related transcripts exist in humans and mice. These sequences will be isolated and sequenced to determine whether a larger gene family exists. Finally, the structure/function of this new CDC-related kinase will be examined by deletion and site-specific mutagenesis, as well as expression in eukaryotic cells. These studies will ultimately define the role of this CDC-related kinase in both normal and transformed cell growth, and are essential for understanding the molecular basis of cell division control.

细胞通过细胞分裂繁殖的能力是最多的一种 所有生物的基本过程。这些异常 过程通常参与转化细胞的生长。所以， 对正常细胞分裂涉及的事件的透彻理解是 对于理解转化的细胞生长至关重要。为此结束几个 组已经确定了细胞分裂控制蛋白激酶p34cdc2， 这是正常有丝分裂的关键组成部分。去除该蛋白 激酶活性最近已显示导致有丝分裂的障碍。 但是，该系统是复杂的，并且激活该激酶的蛋白质， 以及这种激酶活性作用的 身份不明。我们已经确定了一种高度的新蛋白激酶 与p34CDC2激酶相关（68％同源）。这种新激酶是394氨基 大小的酸显然受磷酸化和Ca2+ - 调节 钙调蛋白，并定位于细胞质和核。表达 在细胞周期中调节相应的mRNA，在 G（1）和S期。该CDC相关激酶在中的表达升高 真核细胞在晚期导致细胞周期的明显块 末期和早期G（1）边界。我们建议在分子上检查 和生化水平，这种与CDC相关激酶的性质及其作用 在细胞周期控制中。这将通过检查其 在细胞周期中调节。仔细分析稳态mRNA和 蛋白质水平，激酶活性，磷酸化态和蛋白质蛋白 将使用cDNA进行相互作用随细胞周期的函数 这种新的与CDC相关的激酶的克隆和抗体。我们还将检查 CDC相关的转录和转录后调节 mRNA与细胞周期的关系。杂交数据表明 序列相关的转录本存在于人类和小鼠中。这些序列会 分离并测序以确定是否存在较大的基因家族。 最后，这种新的与CDC相关激酶的结构/功能将是 通过缺失和位点特异性诱变以及表达进行检查 在真核细胞中。这些研究最终将定义 这种与CDC相关的激酶在正常和转化的细胞生长中，并且是 了解细胞分裂控制的分子基础至关重要。