MATERNAL MRNA LOCALIZATIONS IN A DETERMINATE EGG

确定卵子中母体 mRNA 的定位

• 批准号: 6079324
• 负责人: WILLIAM R JEFFERY
• 金额: $ 25.93万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-08-01 至 2002-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6079324
• 关键词: RNase protection assay; Tunicata; antisense nucleic acid; developmental genetics; egg /ovum; gene expression; genetic mapping; genetic translation; immunoprecipitation; in situ hybridization; intermolecular interaction; laboratory rabbit; messenger RNA; microinjections; molecular cloning; myogenesis; nonmammalian vertebrate embryology; nucleic acid sequence; proliferating cell nuclear antigen; protein localization; protein tyrosine kinase; yeast two hybrid system

DESCRIPTION (Adapted from applicant s abstract): Embryonic development involves cell fate specification, cell division, and cell differentiation. These events begin with the localization of regulatory molecules, including RNAs, in the egg. The PI has used ascidian eggs as a model system to investigate localized RNAs. Ascidians are simple chordates with advantages for studying developmental processes, including eggs with colored regions of specific developmental fate (e.g., the yellow crescent or myoplasm) and closely related species with different modes of development (e.g., tailed and tailless larva). Using these attributes, the PI has identified the yellow crescent (YC) and Uro-1 RNAs, which are localized in the myoplsam (muscle-forming region), and proliferating cell nuclear antigen (PCNA) mRNA, which is localized in the ectoplasm (epidermal and neural-forming region) of ascidian eggs. The YC and PCNA RNAs have complementary 3 UTRs and may represent a novel means for controlling the extent of embryonic cell division by antisense RNA. Uro-1 is an ankrin repeat-containing (SHARK family) protein tyrosine kinase (PTK), which is localized in the myoplsam and required for muscle differentiation. The objective is to determine the functions of these localized RNAs in development. The first specific aim is to map the YC/PCNA locus by sequencing genomic clones and to determine whether YC RNA codes for a protein. The second specific aim is to determine the expression patterns of YC and PCNA RNA during development by blot and in situ hybridization. The third specific aim is to investigate the role of YC/PCNA RNA interactions in PCNA mRNA stability and translation using in vivo assays and by 3 UTR swapping experiments. The fourth specific aim is to investigate the function of YC and PCNA RNA by gain- and loss-of-function studies using synthetic mRNAs and antisense oligos. The fifth specific aim is to determine the subcellular localization of Uro-1 PTK using antibodies. The sixth specific aim is to identify proteins associated with the Uro-1 PTK by immunoprecipitation and two-hybrid system cloning. The seventh specific aim is to determine the developmental function of Uro-1 using antisense oligos and synthetic mRNAs. This investigation will provide new information on the regulation of cell division and cell fate determination during embryogenesis.

描述（改编自申请人的摘要）：胚胎发育 涉及细胞命运规范、细胞分裂和细胞分化。 这些事件始于调节分子的定位，包括 RNA，在鸡蛋中。 PI 使用海鞘卵作为模型系统 研究局部RNA。 海鞘是简单的脊索动物，具有优点 用于研究发育过程，包括带有彩色区域的鸡蛋 特定的发育命运（例如黄新月体或肌质）和 具有不同发育模式的密切相关物种（例如，尾 和无尾幼虫）。 使用这些属性，PI 已确定 黄新月体 (YC) 和 Uro-1 RNA，位于肌体中 （肌肉形成区）和增殖细胞核抗原（PCNA）mRNA， 它位于外质（表皮和神经形成区域） 海鞘卵。 YC 和 PCNA RNA 具有互补的 3 个 UTR，并且可能 代表了控制胚胎细胞范围的新方法 被反义RNA分裂。 Uro-1 是一种含有锚蛋白重复序列​​（SHARK 家族）蛋白酪氨酸激酶（PTK），位于肌质体中 并且是肌肉分化所必需的。 目标是确定 这些局部RNA在发育过程中的功能。 第一个具体目标是 通过对基因组克隆进行测序来绘制 YC/PCNA 基因座图并确定 YC RNA 是否编码蛋白质。 第二个具体目标是确定 通过 blot 和 in 观察发育过程中 YC 和 PCNA RNA 的表达模式 原位杂交。 第三个具体目标是研究 YC/PCNA RNA 相互作用在 PCNA mRNA 稳定性和翻译中的应用 体内测定和 3 个 UTR 交换实验。 第四个具体目标是 通过功能获得和丧失来研究 YC 和 PCNA RNA 的功能 使用合成 mRNA 和反义寡核苷酸的研究。 五、具体目标 的目的是使用抗体确定 Uro-1 PTK 的亚细胞定位。 第六个具体目标是鉴定与 Uro-1 PTK 相关的蛋白质 通过免疫沉淀和双杂交系统克隆。 第七具体 目的是使用反义确定 Uro-1 的发育功能 寡核苷酸和合成 mRNA。 这项调查将提供新信息 细胞分裂的调控和细胞命运的决定 胚胎发生。