IN SITU LOCALIZATION OF INFORMATIONAL EMBRYO RNAS

信息胚胎 RNAS 的原位定位

• 批准号: 2174487
• 负责人: ROBERT C ANGERER
• 金额: $ 33.13万
• 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/2174487
• 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.

已经确定了一组相互协调的信使RNA 以在时间和空间上受限的模式在 紫球海胆胚胎发育的初步研究 这些信息在极早期的囊胚(VEB)中积累，然后 腐烂得很快。它们的浓度不均匀地沿 特定于母体的动植物轴，不存在于 植物杆，但沿口流产均匀分布 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) 是由母性因素不对称地沿 动植物轴线。VEB基因早在4-细胞就被激活了 阶段，首先在16-细胞的特定卵裂球中转录 胚胎，并自主地积累在从 2-细胞期。我们将分析两个VEB基因的启动子 识别与调节蛋白相互作用的部位，尤其是 那些负责空间调控的因素。这些分析 将使用标准的体外蛋白质-DNA生化分析方法 相互作用，和体内测试的报告基因结构驱动 原封不动和更改过的启动子。重组克隆体 编码负责空间调控的因子的mRNAs VEB基因将被鉴定，并将产生符合以下条件的抗体 对于相应的蛋白质是特异的。这些探头将用于 为了区分可能的局部活动机制， 空间调节因子-定位的母体mRNA，定位的母体 蛋白质，或均匀分布的蛋白质的局部激活。 将对反义方法和/或抗体抑制进行测试 阻断VEB调节器功能的有效性，以便 研究它们在决定和分化过程中的作用 动植物轴线。