MESSENGER RNA METABOLISM IN DICTYOSTELIUM DISCOIDEUM

该方案利用细胞黏菌盘基网柄菌， 关于基因转录后调控的两个问题 表达：1)聚(A)的功能是什么？和2)什么是 信使核糖核酸稳定性的决定因素？我们假设Poly(A)尾巴不会 调节信使核糖核酸的稳定性，而不是它们在蛋白质中起作用 由细胞质Poly(A)结合蛋白介导的合成。 本提案的前半部分通过以下方式解决了这一假设：a) 翻译效率和Poly(A)尾长的测定 生长和发育细胞中的单个mRNA，b)特征 Poly(A)结合蛋白的特异性结合特性及c)检测 识别聚(A)结合蛋白的抗体是否能抑制 蛋白质的体外合成。这后半部分的实验 提案将考虑对信使核糖核酸调节的替代解释 稳定性。编码稳定或不稳定信使核糖核酸的两组DNA， 分别被克隆，并将被用于比较属性 这两类信使核糖核酸。我们将确定mRNAs的稳定性 与翻译效率或是否存在特定的 5‘或3’未翻译的序列，转录的重复序列，或 Poly(A)。此外，我们将确定是否有特定的半衰期 在网柄网柄菌生命周期中，mRNAs受到不同程度的调控。

This proposal utilizes the cellular slime mold, Dictyostelium discoideum, to consider two problems of post-transcriptional regulation of gene expression: 1) What is the function of poly(A)? and 2) What are the determinants of mRNA stability? We postulate that poly(A) tails do not regulate mRNA stability, but rather that they have a role in protein synthesis that is mediated by the cytoplasmic poly(A)-binding proteins. The first half of this proposal addresses this hypothesis by: a) determining the translational efficiency and poly(A) tail length of individual mRNAs in growing and developing cells, b) characterizing specific binding properties of the poly(A)-binding proteins and c) testing whether antibodies which recognize the poly(A)-binding proteins can inhibit protein synthesis in vitro. The experiments of the second half of this proposal will consider alternate explanations for the regulation of mRNA stability. Two sets of cDNAs which encode stable or unstable mRNAs, respectively, have been cloned and will be used to compare the properties of these two classes of mRNA. We will determine whether mRNA stability correlates with translational efficiency or with the presence of specific 5' or 3' untranslated sequences, transcribed repetitive sequences, or poly(A). In addition, we will determine whether the half-lives of specific mRNAs are differentially regulated during the Dictyostelium life cycle.