Studies on processing of rRNA and formation of ribosome in animal cells

为了研究哺乳动物细胞中rRNA加工和核糖体形成的分子机制，开展了以下工作：(1)利用RNA聚合酶I特异性的Ar噬菌体SP6转录系统，将小鼠rRNA和SP6启动子连接到含有小鼠18S rRNA 5‘端的rDNA片段上。(2)用BAL 31核酸酶构建加工位点周围的5’和3‘缺失突变体，确定加工事件的核苷酸序列。(3)用磷酸纤维素柱层析将小鼠S100提取物分成四个组分。在其中一个组分中恢复了处理活动。利用体外处理系统考察了热处理、微球菌核酸酶处理、镁离子、KCL浓度和NTPs对处理活性的影响。

To investigate the molecular mechanism of rRNA processing and formation of Ribosome in mammalian cells, following projects were performed.(1) An in vitro processing system was developed by using RNA polymerase I-specific ar bacteriophage SP6 transcription system, in which mouse rRNA and SP6 promoters were jointed to the rDNA fragments containing the 5' end of mouse 18S rRNA.(2) By constructing the 5'- and 3'- deletion mutants surrounding the processing site with nuclease BAL 31, the nucleotide sequence specified the processing event were ditermined.(3) To identify and characterize the processing factor, mouse S100 extract was fractionated by phosphocellulose column chromatography into four fractions. Processing activity was recovered in one of these fractions. Effects of heat treatment, micrococcal nuclease treatment, Mg^<++> ions, KCL concentrations and NTPs on Processing activity were examined by using an in vitro processing system.