NUCLEOLAR RIBONUCLEASES IN RIBOSOMAL RNA PROCESSING

这项建议的目的是继续并扩大对 核仁核糖核酸酶在核糖体RNA加工中的作用。 从艾氏腹水癌细胞中制备的核仁提供了一种 相对纯净的核糖体RNA前体和加工产品来源 以及从其分离酶组分的起始材料。 这些酶组分，特别是核糖核酸酶，将被解离。 并从核仁系统中提纯，以表征它们的 酶和物理性质。乳腺体前45S RNA及其80s 核糖核蛋白颗粒将被标记并从核仁中提纯为 作为底物用于研究核仁的切割模式 核糖核酸酶。18S和28S小鼠核糖DNA杂交探针的克隆 将用于区分核糖核酸酶所代表的序列 45S核糖体前核糖核酸的模式。将进行S1核酸酶图谱研究 用特定的核糖体DNA限制性片段进行，以便 精确定义核糖核酸酶的切割位置。此外，它是 这项提议的目的是开发一种体外系统来检查 核糖体RNA的加工，特别是切割的酶学 反应。为此目的，体外处理将在一个 具有易于获得的前体核糖体RNA的可溶性系统 底物，并对加工后的产品进行快速和明确的检测 产品。该系统将允许生化分离 参与加工反应的成分以及它们的 通过重构实验进行鉴定。总而言之，这是 信息将被用来建立体内功能和重要性 这些降解过程对哺乳动物RNA代谢的影响 组织。

The purpose of this proposal is to continue and extend an investigation of the involvement of nucleolar ribonucleases in ribosomal RNA processing. Nucleoli, prepared from Ehrlich ascites carcinoma cells, provide a relatively pure source of ribosomal RNA precursors and processing products as well as the starting material from which to isolate enzymic components. These enzymic components, in particular ribonucleases, will be dissociated and purified from the nucleolar system in order to characterize their enzymic and physical properties. Preribosomal 45S RNA and its 80S ribonucleoprotein particle will be labeled and purified from nucleoli to be used as substrates to study the patterns of cleavage by nucleolar ribonucleases. Cloned 18S and 28S mouse ribosonal DNA hybridization probes will be used to distinguish the sequences represented in the ribonuclease patterns of 45S preribosonal RNA. S1 nuclease mapping studies will be carried out with specific ribosomal DNA restriction fragments in order to precisely define the ribonuclease cleavage positions. In addition, it is the intent of this proposal to develop an in vitro system to examine ribosomal RNA processing and, in particular, the enzymology of the cleavage reaction. To this purpose, in vitro processing will be studied in a soluble system with an easily obtainable precursor ribosomal RNA as substrate, and with a rapid and unambiguous assay for the processed products. This system will allow for the biochemical separation of the components involved in the processing reaction as well as their identification by reconstitution experiments. Taken together, this information will be used to establish in vivo function and the importance of these degradative processes in affecting RNA metabolism in mammalian tissue.