CONTROL OF THE CELL DIVISION CYCLE

细胞分裂周期的控制

• 批准号: 2183496
• 负责人: EDWIN M BRADBURY
• 金额: $ 19.15万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183496
• 关键词: DNA replication; Saccharomyces cerevisiae; Schizosaccharomyces pombe; affinity chromatography; cell cycle; chromatin; circular DNA; fungal genetics; high performance liquid chromatography; histones; molecular cloning; phosphorylation; point mutation; protein kinase; protein reconstitution; protein structure function; radiotracer; regulatory gene; synchronous cell division; temperature sensitive mutant; tissue /cell culture; transfection

We have shown that the ts lesion in the mouse mammary tumor cell line FT210 is located in CDC2 gene, the mammalian homologue of the S. pombe cdc2 gene known to be a key regulatory gene in the yeast cell division cycle. Two point mutations have been identified in the CDC2 gene resulting in amino acid replacements in conserved regions of the p34 kinase. One of these replacements is in the rigidly conserved PSTAIR region. Other laboratories have shown that injection of just the PSTAIR containing peptide induces major changes in cell cycle events. We have rescued FT2 10 cells with the human CDC2 gene and isolated 6 stably transformed FT2Hs cells lines. Four H1 kinase activities have been isolated, three of which contain p34 and are thermolabile. We propose to study the role of the conserved PSTAIR peptide in cell cycle progression, particularly through G1/S and G2/M. Unlike all other cdc2 mutations studies in S. pombe, the mutation in the CDC2Mm gene affects progression through only G2/M. The cdc2 S. pombe gene will be modified to reflect the same amino acid substitution found in the FT210 CDC2Mmts gene and, by direct replacement of the endogenous yeast gene, the mutation in the PSTAIR region will be tested for its effect on cellular progression through the yeast cell cycle. The four Hl kinase activities will bc fully characterized by the identification of their molecular components and the sites of phosphorylation of their H1 substrates. Affinity purification techniques, particularly those applicable to p34 kinases, will be employed to obtain highly purified H1 kinase fractions. The cell cycle behavior of the H1 kinases as related to G1, S, and G2/M phosphorylation of the H1 histones will be studies in synchronized FT210 cells. Although G2/M hyperphosphorylation of Hl has been correlated with the process of chromosome condensation, cause and effect have yet to be demonstrated. DNA supercoiling assays have now been established and we propose to determine the effects of HI hyperphosphorylation on DNA topology. The HI kinase activities will be tested for their ability to induce compaction of chromatin in the homogenous and highly defined supercoiling assay, in in vitro assays using native chromatin, and in situ with nuclei isolated from temperature arrested G2-phase FT210 cells. The effects of H1 phosphorylation on chromatin structure will be assessed by monitoring alterations in the solubility of the chromatin, electron microscopy, low-angle x-ray and neutron scatter of chromatin, and by DNA staining of nuclei substrates.

我们已经证明，小鼠乳腺肿瘤细胞系FT 210中的ts病变 CDC 2基因是S.粟酒酵母cdc 2基因 已知是酵母细胞分裂周期中的关键调控基因。 两 已经在CDC 2基因中鉴定了点突变， p34激酶保守区的酸置换。 其中一 置换位于严格保守的PSTAIR区域。 其他实验室 已经表明，仅注射含有PSTAIR的肽诱导 细胞周期事件的重大变化。 我们已经拯救了FT 2 10细胞， 人CDC 2基因，并分离了6个稳定转化的FT 2 H细胞系。 四 H1激酶活性已被分离，其中三个含有p34， 不耐热的 我们建议研究保守的PSTAIR肽的作用 在细胞周期进程中，特别是通过G1/S和G2/M。 不像所有 其他在S.粟酒裂殖酵母，CDC 2 Mm基因突变 仅影响G2/M期进展。 CDC2 S.粟酒基因将是 经修饰以反映FT 210中发现的相同氨基酸取代 CDC 2 Mmts基因，并通过直接替换内源酵母基因， 将测试PSTAIR区域中的突变对细胞的影响。 通过酵母细胞周期的进展。 四种Hl激酶活性 将通过鉴定它们的分子来充分表征 组分及其H1底物的磷酸化位点。 亲和纯化技术，特别是适用于p34的那些 激酶，将用于获得高度纯化的H1激酶级分。 与G1、S和G2/M相关的H1激酶的细胞周期行为 H1组蛋白的磷酸化将在同步FT 210中进行研究。 细胞 尽管H1的G2/M过度磷酸化与H2/M过度磷酸化相关，但H1的G2/M过度磷酸化与H2/M过度磷酸化相关。 染色体浓缩过程、原因和影响还有待于 演示。 DNA超螺旋分析现在已经建立，我们 建议确定HI过度磷酸化对DNA的影响 topology. 将测试HI激酶活性的能力， 诱导染色质致密化， 超螺旋测定、使用天然染色质的体外测定和原位测定 从温度阻滞的G2期FT 210细胞中分离细胞核。 的 H1磷酸化对染色质结构的影响将通过 监测染色质溶解度的变化，电子 显微镜，染色质的低角X射线和中子散射，以及DNA 核基质染色。