POST-TRANSCRIPTIONAL REGULATION OF R-PROTEIN SYNTHESIS

这些实验旨在表征 作为翻译调控决定因素的mRNA， mRNA稳定性 我们将研究 我们已经证明， 在发育开始时从多聚核糖体中排除， 在发育后期稳定性明显下降。 第一章 翻译调节：翻译的相对效率 将在体外系统中评估r蛋白mRNA， 随着总poly（A）+ RNA量的增加， 通过添加下列物质限制引发或伸长速率 抑制剂的 实验来识别序列， 负责体内翻译辨别的基因将利用 重组质粒，其中编码 在发育过程中具有不同翻译效率的mRNA 被交换了。 嵌合mRNA的翻译 将在转化的， 发育细胞 此外，从细胞中表达的r-蛋白mRNA， 一个积极参与发展的推动者将使我们能够问， 可能随mRNA变化的转录后修饰 “年龄”在确定可译性方面是重要的， 细胞 未翻译但稳定的r蛋白的细胞定位 将通过原位杂交检测mRNAS，以显色 细胞 2)mRNA稳定性：mRNA表达的时间和幅度。 R蛋白mRNA稳定性的发育变化将是 通过测量mRNA转录（在细胞核中）来确定 径流测定）和mRNA半衰期（在脉冲追踪实验中）。 已知影响网骨藻中mRNA稳定性的事件将 与r-蛋白相对丰度的降低相关 mRNA。 特定序列和有效的 在发育过程中的翻译将使用嵌合 通过转化引入的质粒的转录物。 我们将 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。 最后我们将 试图确定负责阶段特异性的核酸酶活性， 发育细胞裂解物中的r-蛋白mRNAS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.