Yamauchi M, Yamauchi N, Meuth M
Imperial Cancer Reserach Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
EMBO J. 1990 Jul;9(7):2095-9. doi: 10.1002/j.1460-2075.1990.tb07377.x.
Successive rounds of chromosome-mediated gene transfer were used to complement a hamster cytidine auxotroph deficient in CTP synthetase activity and eventually to clone human genomic and cDNA fragments coding for the structural gene. Our approach was to isolate human Alu+ fragments from a tertiary transfectant and to utilize these fragments to screen a panel of primary transfectants. In this manner two DNA fragments, both mapping within the structural gene, were identified and used to clone a partial length cDNA. The remaining portion of the open reading frame was obtained through the RACE polymerase chain reaction technique. The open reading frame encodes 591 amino acids having a striking degree of similarity to the Escherichia coli structural gene (48% identical amino acids with 76% overall similarity including conservative substitutions) with the glutamine amide transfer domain being particularly conserved. As regulatory mutations of CTP synthetase confer both multi-drug resistance to agents widely used in cancer chemotherapy and a mutator phenotype, the cloning of the structural gene will be important in assessing the relevance of such phenotypes to the development of cellular drug resistance.
连续几轮的染色体介导基因转移被用于补充仓鼠胞苷营养缺陷型（缺乏CTP合成酶活性），并最终克隆编码该结构基因的人类基因组和cDNA片段。我们的方法是从第三代转染子中分离人Alu +片段，并利用这些片段筛选一组第一代转染子。通过这种方式，鉴定出了两个均位于结构基因内的DNA片段，并用于克隆一个部分长度的cDNA。开放阅读框的其余部分通过RACE聚合酶链反应技术获得。该开放阅读框编码591个氨基酸，与大肠杆菌结构基因具有显著程度的相似性（48%的氨基酸相同，总体相似性为76%，包括保守替换），其中谷氨酰胺转移结构域特别保守。由于CTP合成酶的调节突变赋予了对癌症化疗中广泛使用的药物的多药耐药性和突变体表型，该结构基因的克隆对于评估此类表型与细胞耐药性发展的相关性将具有重要意义。
