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Molecular Biological Study on Rate-limiting Enzyme DNA of de novo Purine Synthesis by Gene Transfer and Controllable Expression

基因转移和可控表达从头合成嘌呤限速酶DNA的分子生物学研究

基本信息

批准号：
63570521
负责人：
ITAKURA Mitsuo
金额：
$ 1.34万
依托单位：
Institute of Clinical Medicine, The University of Tsukuba
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1988
资助国家：
日本
起止时间：
1988 至 1989
项目状态：
已结题

项目摘要

[AIM OF THE STUDY] The regulatory mechanism of amidophosphoribosyltransferase (ATase), the rate-limiting enzyme of de novo purine synthetic pathway, was studied by the molecular biology approach.[METHODS] First, the recombinant plasmid DNA of E.coli ATase and glucocorticoid-dependent mouse mammary tumor virus (MMTV) LTR was transferred to mammalian fibroblasts (CHO ade-A) which are deficient only for ATase. The effect of the modulation of E. coli ATase gene expression bk glucocorticoid on the rate of de novo purine synthesis was studied. Second, the effect of insulin on the rate of de novo purine synthesis and the expression of ATase was studied in primary cultured rat hepatodytes. Third, amalian ATase was purified from rat liver and its native and denatured subunit molecular weights were examined.[RESULTS] The successful transfer and transcription of E. coli ATase gene to a single band of about 3.8kb was shown by Southern and Northern Analysis. The enzyme activity of the extract was increased with glucocorticoid-dependency from 4.4 to 18.3% of the wild type fibroblasts. Western Analysis showed two bands of 57 and 7OkD in E. coli, and a glucocorticoid- dependent 72kD band in the transformants. The rate of DNA or de novo purine synthesis was not increased, nor the purine-independent growth capacity was acquired. Insulin selectively induced ATase enzyme protein in primary cultured rat hepatocytes. ATase purified from rat liver was a heterotetrameric protein with a molecular weight of 240 kD and two subunit molecular weights of 62 and 57 kD.[CONCLUSION AND DISCUSSION] The artificial expression of E. coli ATase in purine-auxotrophic mammalian cells did not complement the purine-dependent growth in spite of the demonstrable enzyme activity in the cell extract, probably because of the limited enzyme activity or of the two molecular species of ATase subunits. The molecular cloning of mammalian ATase is under way.

【研究目的】采用分子生物学方法研究嘌呤从头合成途径的限速酶——氨基磷酸核糖基转移酶（ATase）的调控机制。[方法]首先，将大肠杆菌ATase和糖皮质激素依赖性小鼠乳腺肿瘤病毒（MMTV） LTR重组质粒DNA转移到只缺乏ATase的哺乳动物成纤维细胞（CHO ade-A）中。研究了糖皮质激素调节大肠杆菌ATase基因表达对嘌呤合成速率的影响。其次，在原代培养的大鼠肝细胞中研究胰岛素对嘌呤新生合成速率和ATase表达的影响。第三，从大鼠肝脏中纯化ATase，测定其天然亚基分子量和变性亚基分子量。【结果】通过Southern and Northern分析，大肠杆菌ATase基因成功转移并转录到3.8kb的单条带上。随着糖皮质激素的依赖，提取物的酶活性从野生型成纤维细胞的4.4增加到18.3%。Western分析显示大肠杆菌中存在57和7OkD两条条带，转化体中存在糖皮质激素依赖的72kD条带。DNA或从头合成嘌呤的速率没有增加，也没有获得不依赖嘌呤的生长能力。胰岛素选择性诱导原代培养大鼠肝细胞ATase酶蛋白。ATase是从大鼠肝脏中纯化出来的异四聚体蛋白，分子量为240 kD，两个亚基分子量分别为62和57 kD。【结论与讨论】大肠杆菌ATase在嘌呤型营养不良哺乳动物细胞中的人工表达，虽然在细胞提取物中有明显的酶活性，但并不能补充嘌呤依赖性生长，这可能是由于酶活性有限或ATase亚基的两种分子种类有限。哺乳动物ATase的分子克隆正在进行中。