MATERNAL MRNA LOCALIZATIONS IN A DETERMINATE EGG

确定卵子中母体 mRNA 的定位

• 批准号: 3312413
• 负责人: WILLIAM R JEFFERY
• 金额: $ 18.69万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 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 1990-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3312413
• 关键词: Tunicata; complementary DNA; cytoplasm; cytoskeleton; developmental genetics; double stranded RNA; early embryonic stage; egg /ovum; electron microscopy; gene expression; genetic manipulation; genetic recombination; genetic translation; growth /development; in situ hybridization; messenger RNA; molecular cloning; nonmammalian vertebrate embryology; nucleic acid hybridization; oogenesis; pancreatic ribonuclease; radiotracer; ultraviolet radiation

The long term objective of the study is to understand the role of localized maternal mRNA in embryonic development. As a model system, we employ eggs and early embryos of the ascidian Styela, because they exhibit colored cytoplasmic regions which appear to delimit the distribution of localized mRNAs and cytoplasmic determinants. We have already cloned the gene corresponding to a maternal muscle actin mRNA and found that its transcripts are localized primarily in the yellow crescent of the egg and segregated into the embyronic muscle cells. Other evidence indicates that localized maternal mRNAs are bound to the egg cytoskeletal matrix. This proposal is designed to continue this work; it has four specific aims. The first aim is to identify and characterize further examples of localized mRNAs. This will be done by screening an egg cDNA library with probes encoding tissue-specific, cytoskeletal, and yellow crescent mRNAs. The spatial distribution of candidates for localized mRNAs in the egg will then be examined by in situ hybridization. The second aim is to examine the developmental expression of localized mRNAs during oogenesis and embryogenesis. This will be accomplished by Northern blot analysis and in situ hybridization of oocytes, eggs, and developing embryos. The third aim is to determine the mechanism of mRNA localization, specifically whether various localized mRNAs are bound to the egg cytoskeletal matrix and, if so, the nature of the binding sites. This will be accomplished by a combination of Northern blots, in situ hybridization, RNase protection analysis, and UV-crosslinking of mRNA in isolated cytoskeletal matrices. The final aim is to determine the developmental significance of localized mRNAs. This will be accomplished by experiments in which antisense or sense synthetic RNA corresponding to localized mRNAs are microinjected into normal eggs or used to rescue eggs that exhibit developmental defects caused by UV-irradiation. These studies should provide insight into the mechanism of mRNA localization and the function of localized mRNA in eggs and early embryos.

本研究的长期目标是了解 在胚胎发育中定位母体mRNA。 为模特 系统，我们使用海鞘的卵和早期胚胎， 因为它们呈现出有色的细胞质区域， 界定定位的mRNA和细胞质的分布， 决定因素 我们已经克隆了对应于 母亲肌肉肌动蛋白mRNA，并发现其转录本是 主要位于卵的黄色新月形区域， 分离成胚胎肌细胞。 其他证据 表明定位的母体mRNA与卵子结合， 细胞骨架基质 该提案旨在继续这一 工作;它有四个具体目标。 第一个目标是确定和 表征定位mRNA的进一步实例。 这将是 通过用编码以下基因的探针筛选卵子cDNA文库来完成： 组织特异性、细胞骨架和黄新月mRNA。 的 卵中定位mRNA候选物的空间分布 然后通过原位杂交进行检测。 第二个目标是 来检测局部mRNA的发育表达， 在卵子发生和胚胎发生过程中。 这将通过 北方印迹分析和原位杂交卵母细胞，卵子， 和发育中的胚胎 第三个目标是确定 mRNA定位的机制，特别是各种 定位的mRNA与卵细胞骨架基质结合，如果 结合位点的性质。 这将由一个 北方杂交，原位杂交，核糖核酸酶 保护分析和分离的mRNA的UV交联 细胞骨架基质 最终目的是确定 局部mRNA的发育意义。 这将是 通过实验完成，在实验中， 对应于定位mRNA的合成RNA被 显微注射到正常的卵子中，或用于拯救表现出 由紫外线照射引起的发育缺陷。 这些研究 应该提供深入了解mRNA定位的机制， 以及卵和早期胚胎中定位mRNA的功能。