GENETIC AND MOLECULAR ANALYSIS OF GERMLINE DETERMINANTS

种系决定因素的遗传和分子分析

• 批准号: 3329251
• 负责人: RUTH LEHMANN
• 金额: $ 15.93万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-02-01 至 1996-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3329251
• 关键词: Drosophilidae; RNA; alleles; chimeric proteins; chromosome walking; developmental genetics; embryogenesis; fluorescence microscopy; fusion gene; gene expression; genetic regulatory element; genetic transcription; germ cells; immunological substance; in situ hybridization; laboratory rat; molecular cloning; mutant; oogenesis; phenotype; polymerase chain reaction; protein sequence; regulatory gene; transposon /insertion element

The long term goal of this project is to understand the biochemical nature and function of the germ plasm. The posterior pole plasm of Drosophila plays a critical role for the development of somatic and germ line tissues. These functions depend on the normal activity of the maternal effect gene oskar. Mature oocytes derived from homozygous mutant oskar females lack the morphological specializations found at the posterior pole of the normal oocyte. Embryos developing from mutant oocytes do not form pole cells, the germ line precursor cells, and do not develop abdominal segments. Oskar RNA is localized at the posterior pole of the embryo and the oskar protein may thus be a component of the pole plasm. Genetic evidence indicates that the oskar gene product is required for the localization of additional gene products critical for germ cell determination and that it is also required for the localization of the nanos gene product which controls segmentation in the abdominal region. Initially, the molecular nature of the oskar gene product and its distribution during oogenesis and embryogenesis will be characterized. The oskar transcription unit will be defined, the gene will be cloned and the coding region sequenced. The distribution of oskar RNA and protein will be studied during oogenesis and embryogenesis using nonradioactively labelled oskar DNA and/or antisense RNA probes and antibodies directed against the oskar protein. Subsequently, the mechanisms by which oskar products become localized to the posterior pole plasm will be analyzed. The ability of mutants in other pole plasm components to localize oskar RNA and/or protein will be tested. Cis acting sequences in the oskar RNA and protein necessary for proper localization will be determined by analysis of mutant oskar alleles and deletion assays. The sufficiency of these sequences to localize oskar RNA and/or protein will be tested using reporter gene fusions. Finally, to understand the role of oskar for the localization of germ cell and somatic determinants, the effect of oskar mutations on the localization of other components of the posterior pole plasm, e.g. the nanos RNA and the vasa protein, will be studied. Direct interactions between the oskar RNA and protein with other components of the pole plasm will be studied by copurification assays using antibodies directed against pole plasm products.

这个项目的长期目标是了解生物化学的本质 和生殖质的功能。 果蝇后极细胞质 对体细胞和生殖细胞系组织的发育起关键作用。 这些功能依赖于母体效应基因的正常活性 奥斯卡。 来自纯合子突变型Oskar雌性的成熟卵母细胞缺乏 在正常的后极发现的形态学特化 卵母细胞 从突变卵母细胞发育的胚胎不形成极细胞， 生殖系前体细胞，不发育腹节。 Oskar RNA位于胚胎的后极，奥斯卡蛋白 因此可能是极浆的一个组成部分。 遗传学证据表明， Oskar基因产物是定位其它基因所必需的 生殖细胞测定的关键产品， 用于控制分割的纳米基因产物的定位 在腹部 最初，oskar基因产物的分子性质及其 将表征卵子发生和胚胎发生期间的分布。 的 奥斯卡转录单位将被定义，基因将被克隆， 编码区测序。 奥斯卡RNA和蛋白质的分布将是 在卵子发生和胚胎发生过程中使用非放射性标记的 oskar DNA和/或反义RNA探针和针对 奥斯卡蛋白。 随后，奥斯卡产品成为 将分析定位于后极血浆的。 的能力 在其他极细胞质组分中定位oskar RNA和/或蛋白质的突变体 会得到考验 oskar RNA和蛋白质中的顺式作用序列 将通过分析突变体来确定适当定位所必需的蛋白质。 Oskar等位基因和缺失测定。 这些序列足以 将使用报告基因检测定位的oskar RNA和/或蛋白质 融合 最后，了解奥斯卡对于本土化的作用， 生殖细胞和体细胞决定因素，奥斯卡突变对 定位后极浆的其他成分，例如 nanos RNA和vasa蛋白，将被研究。 直接相互作用 奥斯卡RNA和蛋白质与极细胞质的其他成分之间的联系 将通过共纯化试验研究，使用针对 极浆制品。