POST-TRANSCRIPTIONAL REGULATION OF R-PROTEIN SYNTHESIS

这些实验旨在描述 作为翻译调控决定因素的mRNAs和 信使核糖核酸稳定性。我们将研究R蛋白的mRNAs。 我们已经证明了有选择地 在发育开始时排除在多倍体之外，并到 在发育后期经历明显的稳定性下降。1) 翻译规则：翻译的相对效率 R-蛋白mRNAs将在体外编程系统中进行评估 随着Poly(A)+RNA总量的增加， 开始或伸长：通过添加 抑制剂。用于识别以下序列的实验 对体内的翻译歧视负责的人将利用 编码哪些基因区域的重组质粒 发育中具有不同翻译效率的mRNAs 已经被交换了。嵌合mRNA的翻译 从这些质粒中表达出来的基因将在转化后进行检测， 发育中的细胞。此外，r-蛋白mRNAs的表达 一个活跃在发展中的推动者将允许我们问 转录后修饰可能会随着mRNA的变化而改变 “年龄”在决定发展中的可译性方面很重要 细胞。未翻译但稳定的r-蛋白的细胞定位 MRNAs将通过原位杂交进行检测，以形成 细胞。2)信使核糖核酸的稳定性： R-蛋白mRNA稳定性的发育变化将是 由mRNA转录测量确定(以核为单位 径流分析)和信使核糖核酸半衰期(在脉冲追逐实验中)。 已知的影响Dictyostelialm RNA稳定性的事件将是 与r-蛋白相对丰度的下降有关 MRNAs。特定顺序和有效率的重要性 开发过程中的翻译将使用嵌合体进行检查 通过转化从质粒中导入转录本。我们会 通过原位杂交检查稳定性的变化是否 与亚细胞位置的变化相关。最后，我们会 尝试确定与特定阶段相关的核酸酶活性 发育细胞裂解物中r蛋白mrna的降解。

These experiments are designed to characterize features of mRNAs which are determinants of translational regulation and mRNA stability. We will study the r-protein mRNAs of Dictyostelium which we have already shown to be selectively excluded from polysomes at the start of development and to undergo an apparent decrease in stability later in development. 1) Translational regulation: The relative translational efficiency of r-protein mRNAs will be assessed in in vitro systems programmed with increasing amounts of total poly(A)+ RNA, with either initiation or elongation made rate limiting by the addition of inhibitors. Experiments to identify sequences which are responsible for translational discrimination in vivo will utilize recombinant plasmids in which regions of genes which encode mRNAs with different translational efficiencies in development have been exchanged. Translation of the chimeric mRNAs expressed from these plasmids will be examined in transformed, developing cells. In addition, expression of r-protein mRNAs from a promoter active in development will allow us to ask whether post-transcriptional modifications which may change with mRNA "age" are important in determining translatability in developing cells. The cellular location of untranslated, but stable, r-protein mRNAS will be examined by in situ hybridization to developing cells. 2) mRNA stability: The time and magnitude of the developmental change in r-protein mRNA stability will be determined by measurement of mRNA transcription (in nuclear run-off assays) and mRNA half-life (in pulse-chase experiments). Events known to affect mRNA stability in Dictyostelium will be correlated with the decrease in relative abundance of r-protein mRNAs. The importance of specific sequences and of efficient translation during development will be examined using chimeric transcripts from plasmids introduced by transformation. We will examine by in situ hybridization whether changes in stability are correlated with changes in subcellular location. Finally, we will try to identify a nuclease activity responsible for stage specific degradation of r-protein mRNAS in developmental cell lysates.