MITOSIS IN NORMAL AND NEOPLASTIC CELLS

正常细胞和肿瘤细胞的有丝分裂

• 批准号: 6137436
• 负责人: BILL R BRINKLEY
• 金额: $ 30.4万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-06-01 至 2001-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6137436
• 关键词: DNA binding protein; aneuploidy; antinuclear autoantibody; antisense nucleic acid; cell cycle; centromere; chromosome movement; genetic manipulation; human genetic material tag; human tissue; laboratory mouse; laboratory rabbit; microinjections; neoplasm /cancer genetics; nucleic acid sequence; protein biosynthesis; protein structure function; yeast two hybrid system

DESCRIPTION: This is a first revision of a application for renewed support from a senior investigator, Dr. Bill Brinkley, to continue the investigation of kintechore assembly during mitosis in mammalian cells. Previous investigations have demonstrated that underlying mammalian centromeres are large blocks of repetitive DNA sequences, including one called satellite DNA. To dissect the complex DNA elements found in mammalian centromeres, the proposal will focus on the discovery in the previous granting period that centromeres can be fragmented by inducing cells with unreplicated genomes to enter mitosis (a process producing MUGS). With these, the present proposal will identify functional centromere sequences following chromosomal fragmentation by combining in situ hybridization and immunocytochemistry to follow specific DNA sequences and centromere proteins, particularly CENP-B and CENP-C. Three aims are now proposed. Using probes specific for alphoid DNA subfamilies known to localize within the centromere-kintechore complex of human chromosomes, the functional and molecular organization of these sequences will be followed using in situ hybridization. Second, to test the function of centromere protein B (CENP-B), the gene will be disrupted in embryonic stem cells to test whether elimination of this protein has a consequence in mice. Further, proteins that interact with CENP-B will be examined using the yeast two hybrid system and the properties of any proteins so identified will be further characterized. This will focus on using strategies to suppress protein function by overexpression of protein fragments, microinjection of antibodies, and suppression of protein expression using antisense oligonucleotides. Lastly, the properties of newly identified centromere protein-F (CENP-F) will be examined during kinetochore maturation as cells progress from interphase to mitosis, as well as using methods to interfere with CENP-F function to test its role in mitotic chromosome segregation.

描述：这是对更新支持申请的首次修订 来自高级调查员比尔·布林克利（Bill Brinkley）博士继续调查 哺乳动物细胞有丝分裂过程中的kintechore组装。 以前的 调查表明，潜在的哺乳动物centromeres是 重复的DNA序列的大块，包括一个称为卫星的序列 脱氧核糖核酸。 为了剖析在哺乳动物centromeres中发现的复杂DNA元素， 该提案将重点介绍上一个授予期的发现 通过诱导无重复的细胞，可以将这些centromeres分散 输入有丝分裂的基因组（一种产生杯子的过程）。 有了这些 目前的建议将确定以下功能性的中心粒序列 通过原位杂交和 免疫细胞化学遵循特定的DNA序列和丝粒 蛋白质，特别是CENP-B和CENP-C。 现在提出了三个目标。 使用针对已知本地化的字段DNA亚家族特异的探针 人类染色体的Centromere-Kintechore复合物，功能和 这些序列的分子组织将遵循原位 杂交。 第二，测试中心粒蛋白B的功能 （CENP-B），该基因将在胚胎干细胞中被破坏，以测试是否是否 消除该蛋白质在小鼠中具有后果。 此外，蛋白质 与CENP-B相互作用将使用酵母两个混合系统进行检查 而且，因此所鉴定的任何蛋白质的特性将进一步 特征。 这将着重于使用策略抑制蛋白质 通过过表达蛋白质片段的功能，对 抗体，并使用反义抑制蛋白质表达 寡核苷酸。 最后，新确定的中心粒的特性 蛋白-F（CENP-F）将在动力学成熟过程中检查为细胞 从相间到有丝分裂的进展，以及使用方法进行干扰 具有CENP-F功能来测试其在有丝分裂染色体分离中的作用。