Centromere Structure and Function

着丝粒的结构和功能

• 批准号: 6571335
• 负责人: John A Carbon
• 金额: $ 36.88万
• 依托单位: UNIVERSITY OF CALIFORNIA SANTA BARBARA
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6571335
• 关键词: Candida albicans; DNA binding protein; affinity chromatography; cell cycle; cell morphology; centromere; chromosome movement; fungal genetics; gel mobility shift assay; gene deletion mutation; gene expression; immunoprecipitation; mass spectrometry; microarray technology; molecular genetics; polymerase chain reaction; protein structure function; yeast two hybrid system

DESCRIPTION (provided by applicant): The long-range objective of this research program is to understand in molecular terms how the centromere-kinetochore functions in eukaryotic cell division. Our previous research in this area emphasized centromere structure/function studies in the budding yeast Saccharomyces cerevisiae and fission yeast Schizo-saccharomyces pombe. Building on the valuable information gained in these groundwork studies, we will now extend the work to investigate centromere-kinetochores in the two most common and clinically significant pathogenic budding yeasts, Candida albicans and Candida glabrata. All budding yeast species examined to date contain relatively small point centromeres (CEN less than 400 bp in length), whereas other organisms contain large regional centromeres characterized by the presence of long stretches of heterochromatic repeated DNA sequences. It is postulated that this fundamental difference in centromere structure could be exploited eventually to develop agents selectively toxic to the budding yeasts. Specific aims are: (1) combined molecular and genetic strategies will be used to isolate and characterize CEN DNAs of C. albicans; (2) stable CEN-based plasmid and artificial chromosome vector systems will be constructed to facilitate molecular genetic research in C. albicans; and (3) inner kinetochore-associated proteins from both C. albicans and C. glabrata will be identified and characterized as potential targets for specific antifungal drug therapy. We have identified, epitope-tagged, and expressed an evolutionarily conserved, centromere-specific histone H3 protein (CaCse4p) in C. albicans. Chromatin immunoprecipitation (CHIP) with antibodies directed against this protein will be used to isolate and clone CEN DNAs. Putative CEN DNAs will be identified and characterized in terms of their nucleotide sequence and ability to mitotically stabilize plasmid and artificial chromosome vectors in actively dividing cells. Inner kinetochore proteins of Candida species will be isolated by CEN DNA affinity chromatography and the corresponding genes will be cloned and characterized. One-hybrid and two-hybrid screens currently under development for use with C. glabrata will be applied to a search for additional genes specifying CEN-associated proteins. Kinetochore association of candidate proteins will be confirmed by ChIP analysis and by studying the effects of gene deletion and/or mutagenesis on cell division and chromosome segregation.

描述（由申请人提供）：该研究计划的远程目标是用分子术语来理解丝粒 - 金属孔如何在真核细胞分裂中的功能。我们先前在这一领域的研究强调了酿酒酵母和裂变酵母酵母菌精神分裂症pombe中的丝粒结构/功能研究。在这些基础研究中获得的有价值的信息的基础上，我们现在将扩展工作，以研究两种最常见且具有临床意义的致病性酵母，白色念珠菌和念珠菌glabrata中的丝粒 - 金属粒。迄今为止研究的所有萌芽的酵母菌物种都包含相对较小的点丝粒（长度小于400 bp的cen），而其他生物体则包含大型的区域丝粒，其特征是存在长长的异质性重复DNA序列。据推测，最终可以利用中心粒结构的这种基本差异，以使剂对萌芽的酵母有选择性毒性。具体目的是：（1）分子和遗传策略的组合将用于分离和表征白色念珠菌的CEN DNA； （2）将构建稳定的基于CEN的质粒和人工染色体载体系统，以促进白色念珠菌的分子遗传研究； （3）将鉴定出来自白色念珠菌和glabrata的内部动力学相关蛋白，并将其描述为特定抗真菌药物治疗的潜在靶标。我们已经在白色念珠菌中鉴定出，标记并表达了一种进化保守的，丝粒特异性的组蛋白H3蛋白（CACSE4P）。用针对该蛋白质的抗体的染色质免疫沉淀（CHIP）将用于分离和克隆CEN DNA。假定的CEN DNA将以其核苷酸序列以及在主动分裂细胞中稳定质粒和人造染色体载体的能力来识别和表征。 CEN DNA亲和力色谱法将分离念珠菌的内部动力学蛋白，并将相应的基因克隆和表征。目前正在开发的杂菌晶状体蛋白酶的一杂交和两个杂交筛网将用于搜索指定CEN相关蛋白的其他基因。候选蛋白的动力学结合将通过CHIP分析以及研究基因缺失和/或诱变对细胞分裂和染色体分离的影响来确认。