Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.

Abstract

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.