CONTROL OF THE MAMMALIAN CELL DIVISION CYCLE

哺乳动物细胞分裂周期的控制

• 批准号: 3305375
• 负责人: EDWIN M BRADBURY
• 金额: $ 18.41万
• 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/3305375
• 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.

我们已经证明了小鼠乳腺肿瘤细胞系FT210中的TS损伤 位于pombe链霉菌cdc2基因的哺乳动物同源物cdc2基因中。 已知是酵母细胞分裂周期中的关键调控基因。二 已经在CDC2基因中发现了导致氨基的点突变 P34蛋白激酶保守区的酸替换。这其中的一个 替换位于严格保守的PSTAIR区域。其他实验室 已经表明，注射含有PSTAIR的多肽就能诱导 细胞周期事件的主要变化。我们已经拯救了FT2-10细胞 人CDC2基因，获得了6株稳定转化的FT2Hs细胞系。四 已经分离出H1激酶活性，其中三个含有p34和 不耐热。我们建议研究保守的PSTAIR多肽的作用 在细胞周期进程中，特别是通过G1/S和G2/M。 庞氏链霉菌CDC2Mm基因突变的其他研究 仅通过G2/M影响进展cdc2 S.pombe基因将 经过修改以反映FT210中发现的相同氨基酸替代 CDC2Mmts基因，通过直接替换内源酵母基因， 将测试PSTAIR区域的突变对细胞的影响 在酵母细胞周期中的进展。四种hl激酶的活性 将BC充分表征为其分子的鉴定 它们的H1底物的组成和磷酸化的位置。 亲和纯化技术，特别是适用于p34的技术 激酶，将被用来获得高纯度的H1激酶组分。 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的FT210中进行研究 细胞。尽管HL的G2/M过度磷酸化与 染色体凝聚的过程、因果关系尚待研究 演示了。DNA超螺旋分析现在已经建立起来了，我们 关于确定HI过度磷酸化对DNA影响的建议 拓扑学。HI激酶活性将进行测试，以确定它们是否能够 诱导染色质在均一的和高度清晰的 超螺旋试验、使用天然染色质的体外试验和原位试验 从温度滞留的G2期FT210细胞中分离出细胞核。这个 H1磷酸化对染色质结构的影响将通过 监测染色质、电子的溶解度的变化 染色质的显微镜、低角x射线和中子散射以及DNA 细胞核底物染色。