MATERNAL MRNA LOCALIZATIONS IN A DETERMINATE EGG

确定卵子中母体 mRNA 的定位

• 批准号: 3312415
• 负责人: WILLIAM R JEFFERY
• 金额: $ 15.27万
• 依托单位: UNIVERSITY OF CALIFORNIA AT DAVIS
• 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 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3312415
• 关键词: RNA biosynthesis; Tunicata; alternatives to animals in research; cell type; cytoplasm; cytoskeleton; developmental genetics; early embryonic stage; egg /ovum; gene expression; genetic manipulation; genetic transcription; growth /development; laboratory rabbit; larva; messenger RNA; microinjections; molecular cloning; muscle cells; muscle proteins; mutant; myogenesis; nonmammalian vertebrate embryology; northern blottings; nucleic acid hybridization; nucleic acid sequence; oogenesis; radiation genetics; radiotracer

Embryonic development is initiated by the activity of ooplasmic determinants. Some of these determinants are localized mRNA molecules, yet few such mRNAs have been identified and the mechanisms involved in their localization and function are poorly understood. The long term objective of this study is to understand the role of maternal mRNAs in the initiation of embryonic development. Eggs and embryos of ascidians, which contain cytoplasmic regions with distinct developmental fates, are used as a model system. This proposal exploits unique attributes of ascidians, including the ability to isolate an egg cytoplasmic region containing localized mRNAs, the autonomous and precocious differentiation of muscle cells during embryogenesis, and the existence of sibling species with radically different modes of development, to identify and characterize developmentallysignificant maternal mRNAs. I have already cloned several mRNAs that are localized in the myoplasm, a egg cytoplasmic region that specifies embryonic axis and muscle cell development, and one mRNA that is localized in the ectoplasm, a region inherited by ectodermal cells. At least one of the mRNAs localized in the myoplasm is associated with the egg cytoskeletal matrix. This proposal of six specific aims is designed to continue and extend this work. The first specific aim is to identify localized and other maternal mRNAs that are candidates for ooplasmic determinants. This will be done by screening cDNA libraries for (1) clones encoding mRNAs localized in the myoplasm, (2) clones encoding an mRNA localized in the ectoplasm, and (3) clones homologous to the vertebrate myogenic genes, MyoD and myogenin, which may be involved in muscle development; and (4) screening a subtracted cDNA library for clones with messages expressed in eggs of an ascidian species exhibiting larval development but not in eggs of a sibling species lacking larval development The second specific aim is to characterize the mRNAs by sequencing and determining their temporal and spatial expression during development. The third specific aim is to determine whether the expression of these mRNAs is changed in the species lacking larval development. The fourth specific aim is to determine whether these mRNAs are associated with the egg cytoskeletal matrix and whether this association is altered in the species lacking larval development. The fifth specific aim is to identify and characterize proteins encoded by these mRNAs with respect to their temporal and spatial expression during development and whether changes in expression patterns occur in the species lacking larval development. The sixth specific aim is to test the function of each mRNA by microinjecting synthetic transcripts into LTV irradiated eggs, which lack functional axial determinants, and eggs of the species lacking larval development, which are defective in muscle determinants.

胚胎发育是由卵质的活性启动的 决定因素。 其中一些决定因素是局部 mRNA 分子， 然而，很少有这样的 mRNA 被鉴定出来，并且涉及的机制 人们对它们的定位和功能知之甚少。 长期来看 本研究的目的是了解母体 mRNA 在 胚胎发育的开始。 海鞘的卵和胚胎， 包含具有不同发育命运的细胞质区域，是 用作模型系统。 该提案利用了独特的属性 海鞘，包括分离卵细胞质区域的能力 含有局部 mRNA，自主且早熟的分化 胚胎发生过程中肌肉细胞的形成，以及兄弟姐妹的存在 具有完全不同的发展模式的物种，以识别和 表征具有发育意义的母体 mRNA。 我已经 克隆了几个位于肌质（卵子）中的 mRNA 指定胚胎轴和肌细胞的细胞质区域 发育过程中，一种 mRNA 位于外质（一个区域） 由外胚层细胞遗传。 至少一种 mRNA 位于 肌质与卵细胞骨架基质相关。 这 提出六个具体目标的提议旨在继续并扩展这一目标 工作。 第一个具体目标是确定本地和其他孕产妇 作为卵质决定簇候选者的 mRNA。 这将完成 通过筛选 cDNA 文库中的 (1) 编码 mRNA 的克隆，该 mRNA 位于 肌质，（2）编码位于外质中的 mRNA 的克隆，以及 (3)与脊椎动物生肌基因MyoD同源的克隆 肌生成素，可能参与肌肉发育； (4)筛选 一个扣除 cDNA 文库，用于克隆，其信息在卵中表达 一种海鞘物种，表现出幼虫发育，但在卵中不表现出 缺乏幼体发育的兄弟物种 第二个具体目标是 通过测序和确定 mRNA 的时间和特性来表征 mRNA 发育过程中的空间表达。 第三个具体目标是 确定这些 mRNA 的表达在物种中是否发生变化 缺乏幼虫发育。 第四个具体目标是确定 这些 mRNA 是否与卵细胞骨架基质相关，以及 在缺乏幼虫的物种中这种关联是否会改变 发展。 第五个具体目标是识别和表征 这些 mRNA 编码的蛋白质在时间和 发育过程中的空间表达以及表达是否发生变化 模式发生在缺乏幼虫发育的物种中。 第六个 具体目的是通过显微注射测试每个mRNA的功能 将合成转录物放入 LTV 辐照的鸡蛋中，这些鸡蛋缺乏功能 轴向决定因素，以及缺乏幼虫发育的物种的卵， 肌肉决定因素有缺陷。