Characterizing Sperm Chromatic Assembly in C. elegans

秀丽隐杆线虫精子染色组装的特征

• 批准号: 7106843
• 负责人: Diana S. Chu
• 金额: $ 1.7万
• 依托单位: SAN FRANCISCO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2007-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7106843
• 关键词: Caenorhabditis elegans; chromatin; chromosomes; developmental genetics; mass spectrometry; nucleoproteins; protein localization; protein structure function; proteomics; sperm; spermatogenesis

DESCRIPTION (provided by applicant): Successful reproduction requires faithful transmission of DNA during gamete formation and union. The DNA of the paternal gamete is packaged uniquely from that of other cell types. Sperm chromatin packaging consists of both a uniquely tight compaction of DNA by sperm-specific scaffolding molecules and the association of factors required for spermatogenesis or functions after fertilization. Sperm-specific chromatin packaging is conserved from worms to humans; however, the mechanisms used to package sperm DNA are largely unknown. Systematic dissection of factors required for sperm chromatin assembly in a genetically amenable system, such as Caenorhabditis elegans may identify vital components required for spermatogenesis in all organisms. The goal of this project is to develop methodology to identify and characterize sperm chromatin assembly factors in C. elegans. A proteomic approach is used to identify candidate sperm chromatin assembly factors. Chromatin is biochemically purified from C. elegans sperm and oocyte cells. Each sample is then subjected to specialized mass spectrometric analysis using the multidimensional protein identification technique (MudPIT). Results are analyzed against the sequenced C. elegans genome allowing identification of associated proteins. Protein profiles from sperm and oocyte samples are compared to subtract general factors and to identify proteins that are specific to highly condensed sperm DNA. The role of identified proteins in sperm chromatin assembly will be assessed by reducing function of candidate proteins. Requirements for these proteins in spermatogenesis, chromosome organization, and chromosome dynamics will be examined. Localization of candidate proteins will be used to define how and when these proteins function in chromosome assembly. The methodology using C. elegans developed in this proposal will allow rapid identification of sperm-specific factors that condense and organize chromatin. These factors may be conserved and play vital roles in fertility and reproduction in all organisms.

描述（由申请人提供）：成功的繁殖需要在配子形成和结合过程中忠实地传递 DNA。父本配子的 DNA 的包装与其他细胞类型的 DNA 不同。精子染色质包装由精子特异性支架分子对 DNA 的独特紧密压缩以及精子发生或受精后功能所需因素的关联组成。从蠕虫到人类，精子特异性染色质包装都是保守的；然而，用于包装精子 DNA 的机制在很大程度上尚不清楚。对遗传顺从的系统（例如秀丽隐杆线虫）中精子染色质组装所需的因素进行系统分析，可以识别所有生物体中精子发生所需的重要成分。该项目的目标是开发识别和表征线虫精子染色质组装因子的方法。蛋白质组学方法用于识别候选精子染色质组装因子。染色质是从秀丽隐杆线虫精子和卵母细胞中生化纯化的。然后使用多维蛋白质鉴定技术 (MudPIT) 对每个样品进行专门的质谱分析。根据已测序的秀丽隐杆线虫基因组对结果进行分析，从而鉴定相关蛋白质。对精子和卵母细胞样本的蛋白质谱进行比较，减去一般因素，并鉴定高度浓缩的精子 DNA 特有的蛋白质。将通过降低候选蛋白质的功能来评估已鉴定蛋白质在精子染色质组装中的作用。将检查这些蛋白质在精子发生、染色体组织和染色体动力学中的需求。候选蛋白质的定位将用于定义这些蛋白质如何以及何时 蛋白质在染色体组装中发挥作用。本提案中开发的使用秀丽隐杆线虫的方法将允许快速识别浓缩和组织染色质的精子特异性因子。这些因素可能是保守的，并且在所有生物体的生育和繁殖中发挥着至关重要的作用。