IN SITU LOCALIZATION OF INFORMATIONAL EMBRYO RNAS

信息胚胎 RNAS 的原位定位

• 批准号: 2174486
• 负责人: ROBERT C ANGERER
• 金额: $ 32.47万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 1978
• 资助国家: 美国
• 起止时间: 1978-07-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2174486
• 关键词: DNA footprinting; SDS polyacrylamide gel electrophoresis; affinity chromatography; alternatives to animals in research; antisense nucleic acid; developmental genetics; early embryonic stage; gene expression; genetic promoter element; genetic regulatory element; genetic transcription; immunochemistry; in situ hybridization; invertebrate embryology; messenger RNA; microinjections; molecular cloning; nucleic acid probes; polymerase chain reaction; protein structure function; regulatory gene; sea urchins; site directed mutagenesis

A set of messenger RNAs has been identified which are coordinately expressed in a temporally and spatially restricted pattern during development of the sea urchin (Strongylocentrotus purpuratus) embryo. The messages accumulate in the very early blastula (VEB) and then decay rapidly. The have a non-uniform concentration along the maternally specified animal-vegetal axis, being absent from cells at the vegetal pole, but are uniformly distributed along the oral-aboral axis that is specified after fertilization. Several lines of evidence suggest that the genes encoding these messages (VEB genes) are regulated by maternal factors asymmetrically arrayed along the animal-vegetal axis. The VEB genes are activated as early as 4-cell stage, are first transcribed in specific blastomeres of the 16-cell embryo, and accumulate autonomously in cells separated beginning at 2-cell stage. The promoters of two of the VEB genes will be analyzed to identify sites of interaction with regulatory proteins, especially those factors responsible for spatial regulation. These analyses will use standard in vitro biochemical assays of protein-DNA interaction, and in vivo tests of reporter gene constructs driven by intact and altered promoters. Recombinant clones representing the mRNAs encoding the factors responsible for spatial regulation of the VEB genes will be identified, and antibodies will be raised that are specific for the corresponding proteins. These probes will be used to distinguish among possible mechanisms of localized activity of the spatial regulators-localized maternal mRNA, localized maternal protein, or localized activation of a uniformly distributed protein. Antisense methods and/or antibody inhibition will be tested for their efficacy in blocking the function of the VEB regulators, in order to investigate their role in determination and differentiation along the animal-vegetal axis.

已经鉴定出一组信使RNA， 以时间和空间受限的模式表达， 海胆（Strongylocentrotus purpuratus）胚胎的发育。 这些信息在极早期囊胚（VEB）中积累， 迅速腐烂。 它们在沿着 母体特有的动物-植物轴，在细胞中不存在， 植物极，但均匀分布沿着口-背口 受精后指定的轴。 几行 有证据表明，编码这些信息的基因（VEB基因） 受母亲因素的调节，这些因素沿着 动植物轴 VEB基因早在4细胞期就被激活， 阶段，首先在16细胞的特定卵裂球中转录 胚胎，并在分离的细胞中自主积累， 2-细胞期 将分析两个VEB基因的启动子 确定与调节蛋白相互作用的位点，特别是 这些因素负责空间调节。 这些分析 将使用标准的蛋白质-DNA体外生化分析 相互作用，并在体内测试的报告基因构建体驱动 完整和改变的启动子。 重组克隆代表 编码负责空间调节的因子的mRNA VEB基因将被识别，抗体将被提出， 对相应的蛋白质具有特异性。 这些探针将用于 为了区分可能的局部活动机制， 空间调节子-定位于母体mRNA，定位于母体 蛋白质，或均匀分布的蛋白质的局部活化。 将测试反义方法和/或抗体抑制， 有效阻断VEB调节因子的功能， 研究它们在决定和分化沿着 动植物轴