The identification and characterization of the mammalian mutation mei4

哺乳动物突变 mei4 的鉴定和表征

• 批准号: 7128397
• 负责人: Jeremy Ogden Ward
• 金额: $ 19.31万
• 依托单位: MIDDLEBURY COLLEGE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7128397
• 关键词: DNA repair; animal genetic material tag; antibody; apoptosis; chimeric proteins; cyclins; developmental genetics; gene expression; gene expression profiling; gene mutation; immunocytochemistry; laboratory mouse; male; meiosis; messenger RNA; polymerase chain reaction; protein protein interaction; spermatogenesis; testis

DESCRIPTION (provided by applicant): Human infertility and complications of reproductive processes have devastating social and monetary costs. We are attempting to identify genes necessary for fertility by studying mammalian meiosis using chemical mutagenesis, expression profiling, and immunocytochemistry in the mouse. For meiosis to succeed a complicated series of processes must occur including cell cycle progression, recombination, chromosome segregation and cell division. Phenotype-driven mutagenic screens have generated the mutation mei4. mei4 causes male and female sterility by disrupting gametogenesis during meiosis. Meiosis in mei4/mei4 homozygotes fails to complete due to a lack of recombination. While other DNA repair proteins such as Rad51 localize normally to meiotic chromosomes, the mismatch repair protein Mlh1 fails to load onto meiotic chromosomes during prophase I of meiosis in mei4/mei4 animals. We have identified the causative mutation behind the mei4 phenotype in a putative E3 ligase that functions to regulate cyclin degradation. This application proposes to characterize the mutant mei4 allele in the context of meiotic DNA repair and cell cycle biology. The role of mei4 in DNA mismatch repair and cell cycle progression will be elucidated via the following specific aims and strategies: (1) mRNA and protein expression profiling, (2) in vitro, in situ, and in vivo protein-protein interaction studies, (3) immunocytochemistry of protein constituents at recombination sites. This work has significant health relatedness as several of the mismatch repair proteins found to function in meiosis are also associated with hereditary non-polyposis colorectal cancer (HNPCC). E3 ligases and their substrates have been implicated in several cancers, including inherited breast cancer, and also in neurodegenerative disorders. Lastly, aneuploidy is a major source of birth defects resulting from non- disjunction or premature separation of meiotic chromosomes. mei4 has a severe meiotic aneuploid phenotype and thus its characterization will address potential causes of clinical aneuploidy and birth defects. The characterization of mei4 will not only contribute to our understanding of the basic molecular biology of meiosis but also increase our knowledge of important human health issues.

描述（由申请人提供）：人类不孕症和生殖过程的并发症具有毁灭性的社会和货币成本。我们试图通过使用小鼠中的化学诱变，表达分析和免疫细胞化学研究哺乳动物减数分裂来鉴定生育所必需的基因。为了减数分裂，必须发生一系列复杂的过程，包括细胞周期进程，重组，染色体分离和细胞分裂。表型驱动的诱变筛选产生了突变MEI4。 MEI4通过破坏减数分裂过程中的配子发生引起男性和女性不育。由于缺乏重组，MEI4/MEI4纯合子中的减数分裂未能完成。尽管其他DNA修复蛋白（例如RAD51）正常定位于减数分裂染色体，但不匹配修复蛋白MLH1在MEI4/MEI4动物中减数分裂的预言I期间未能加载到减数分裂染色体上。我们已经在推定的E3连接酶中确定了MEI4表型背后的因果突变，该连接酶起作用可调节细胞周期蛋白降解。该应用建议在减数分裂DNA修复和细胞周期生物学的背景下表征突变体MEI4等位基因。 MEI4在DNA失配修复和细胞周期进程中的作用将通过以下特定目的和策略阐明：（1）mRNA和蛋白质表达谱分析，（2）体外，原位，体内蛋白质 - 蛋白质相互作用研究，（3）重新组合蛋白质成分的不可分裂的蛋白质成分。这项工作具有重要的健康相关性，因为发现在减数分裂中起作用的几种不匹配修复蛋白也与遗传性非型型结直肠癌（HNPCC）有关。 E3连接酶及其底物与几种癌症有关，包括遗传性乳腺癌以及神经退行性疾病。最后，非整倍性是由于非分离或减数分裂染色体的过早分离而导致的先天缺陷的主要来源。 MEI4具有严重的减数分裂性非整倍表型，因此其表征将解决临床非整倍性和先天缺陷的潜在原因。 MEI4的特征不仅有助于我们对减数分裂的基本分子生物学的理解，而且还增加了我们对重要人类健康问题的了解。