NUCLEOLAR RIBONUCLEASES IN RIBOSOMAL RNA PROCESSING

这项建议的目的是继续并扩大对下列问题的调查： 核仁核糖核酸酶参与核糖体RNA加工。 从埃利希腹水癌细胞中制备的核仁提供了一种 核糖体RNA前体和加工产物的相对纯的来源 以及从中分离酶组分的起始材料。 这些酶组分，特别是核糖核酸酶， 并从核仁系统中纯化，以表征它们的 酶和物理性质。 前核糖体45S RNA及其80S 核糖核蛋白颗粒将被标记并从核仁中纯化， 用作底物，以研究核仁的切割模式 核糖核酸酶 克隆的18S和28S小鼠核糖体DNA杂交探针 将用于区分核糖核酸酶中代表的序列 45S前核糖体RNA的模式。 S1核酸酶图谱研究将在 用特定的核糖体DNA限制性片段进行， 精确定义核糖核酸酶切割位置。 此外还 该提案的目的是开发一种体外系统， 核糖体RNA加工，特别是切割的酶学 反应 为此目的，将在体外处理中进行研究。 可溶性系统与容易获得的前体核糖体RNA作为 基质，并与一个快速和明确的测定处理 产品. 该系统将允许生物化学分离 参与加工反应的组分及其 通过重构实验鉴定。 总的来说，这 信息将用于确定体内功能和重要性 这些降解过程在影响哺乳动物的RNA代谢 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.