Characterizing Sperm Chromatic Assembly in C. elegans

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。父系配子的DNA与其他细胞类型的DNA包装独特。精子染色质包装既包括精子特异性支架分子对DNA的独特紧密压实，也包括精子发生或受精后功能所需的相关因素。精子特异性染色质包装从蠕虫到人类都是保守的；然而，用于包装精子DNA的机制在很大程度上是未知的。在遗传可调节的系统中，如秀丽隐杆线虫，系统地解剖精子染色质组装所需的因素，可以识别所有生物中精子发生所需的重要成分。本项目的目标是开发方法来鉴定和表征秀丽隐杆线虫的精子染色质组装因子。蛋白质组学方法用于鉴定候选精子染色质组装因子。染色质是从秀丽隐杆线虫的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。结果分析与测序秀丽隐杆线虫基因组允许鉴定相关蛋白。从精子和卵母细胞样品的蛋白质谱进行比较，减去一般因素，并确定蛋白质是特异性的高度浓缩的精子DNA。鉴定的蛋白质在精子染色质组装中的作用将通过降低候选蛋白质的功能来评估。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将被检查。候选蛋白的定位将用于确定这些蛋白如何以及何时

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.