BIOCHEMISTRY AND GENETICS OF MAMMALIAN/APRT

我们将继续调查突变的机制， 中国仓鼠、小鼠和人细胞系中的APRT基因座。 突变体的分析将通过DNA测序进行， 与突变蛋白质的比较研究。 之间 致突变作用，我们有兴趣研究的影响， 非整倍体化剂，据报道， 这个基因座的突变频率。 体内诱变将是 采用体外诱变进行的重复性研究。 调控 “管家”基因的转录和翻译将是 以APRT为模型进行研究。 这将由 启动子区缺失作图法 用APRT基因的启动子片段连接到 CAT基因，并通过分析该基因上的DNA结合位点。 的 增强子元件和病毒LTR对APRT表达影响 将被审查。 我们将分析放置 APRT基因在激素控制下的转录、翻译， 和核苷酸库。 E.大肠杆菌， 黑腹果蝇和哺乳动物的资料显示， 氨基酸同源性 我们将测序酵母APRT基因， 比较核苷酸和氨基酸序列 使用 比较序列分析方法，体外诱变， 和单克隆抗体相互作用，我们将定义 蛋白质的功能结构域。

We shall continue invistigating the mechanism of mutation at the APRT locus in Chinese hamster, mouse, and human cell lines. Analysis of mutants will be done by DNA sequencing, and comparative studies with the mutated proteins. Among mutagenic effects, we are interested in studying the effect of aneuploidizing agents, which have been reported to cause a high frequency of mutations at this locus. In vivo mutagenesis will be paralleled by studies with in vitro mutagenesis. Regulation of transcription and translation of 'house keeping' genes will be studied using APRT as a model. This will be done by a combination of deletion mapping in the promoter region, CAT assays with promoter segments of the APRT gene linked to the CAT gene, and by analysis of DNA binding sites on the gene. The effect of enhancer elements and viral LTRs on APRT expression will be examined. We shall analyze the effect of placing the APRT gene under hormonal control on transcription, translation, and nucleotide pools. Comparative analysis of phosphoribosyltransferases from E. coli, Drosophila melanogaster, and mammalian sources show extensive aminoacid homology. We shall sequence the yeast APRT gene, and compare the nucleotide and aminoacid sequences. Using the approach of comparative sequence analysis, in vitro mutagenesis, and monoclonal antibody interaction, we shall define the functional domains of the protein.