Amplification and cloning of the Chinese hamster glutamine synthetase gene.
A Chinese hamster ovary cell line, KG1MS which is resistant to 5 mM methionine sulphoximine overproduces glutamine synthetase. Overproduction of this 42 000 mol. wt. polypeptide is not seen in either parental KG1 or revertant KG1MSC4-0 cell lines which are resistant to 3 microM and 8 microM methionine sulphoximine, respectively. Restriction endonuclease analysis of DNA from KG1MS cells produces a pattern of amplified DNA fragments not seen in parental KG1 or revertant KG1MSC4-0 digests. Hybridization of cDNA probes complementary to KG1MS poly(A) mRNA against Southern blots of KG1MS restriction digests identifies a specific subset within these amplified sequences which is not detected by cDNA probes made from parental KG1 poly(A) mRNA. One 8.2-kb BglII fragment of KG1MS DNA identified by cDNA hybridization has been cloned to produce recombinant pGS-1. mRNA hybrid selected by pGS-1 translates to a 42 000 mol. wt. polypeptide which co-migrates in polyacrylamide gels with the over-produced protein in KG1MS cells and purified glutamine synthetase. pGS-1 also hybridizes to several mRNA species abundant in KG1MSC4-M, but not KG1, poly(A) mRNA extracts. The high level of resistance to methionine sulphoximine shown by KG1MS cells is due to amplification of a DNA sequence of at least 50 kb which contains the coding region for the enzyme glutamine synthetase.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D004351:Drug Resistance; D005260:Female; D005784:Gene Amplification; D005796:Genes; D005974:Glutamate-Ammonia Ligase / Q000235:genetics* / Q000302:isolation & purification; D008717:Methionine Sulfoximine / Q000633:toxicity; D008970:Molecular Weight; D010053:Ovary; D014176:Protein Biosynthesis
D008717:Methionine Sulfoximine; D005974:Glutamate-Ammonia Ligase