Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes.
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.
Yamauchi
M
M
Imperial Cancer Reserach Fund, Clare Hall Laboratories, South Mimms, Herts, UK.
Yamauchi
N
N
Meuth
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000878:Anthranilate Synthase / Q000235:genetics; D001483:Base Sequence; D019731:Carbon-Nitrogen Ligases; D002460:Cell Line; D002877:Chromosomes, Human / Q000648:ultrastructure*; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005796:Genes; D005816:Genetic Complementation Test; D006801:Humans; D008025:Ligases / Q000235:genetics*; D008677:Metaphase; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection
D015345:Oligonucleotide Probes; D004247:DNA; D000878:Anthranilate Synthase; D008025:Ligases; D019731:Carbon-Nitrogen Ligases; C019951:CTP synthetase