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PMID: 6142821 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Amplification and cloning of the Chinese hamster glutamine synthetase gene.

The EMBO journal ·Vol. 3 ·No. 1 ·1984-01-00 ·Pages 65-71

Sanders PG, Wilson RH

Abstract

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.

MeSH Terms
Animals Cell Line Cloning, Molecular Cricetinae Cricetulus Drug Resistance Female Gene Amplification Genes Glutamate-Ammonia Ligase/genetics,isolation & purification Methionine Sulfoximine/toxicity Molecular Weight Ovary Protein Biosynthesis
Chemicals
Methionine Sulfoximine Glutamate-Ammonia Ligase
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Sanders P G
Wilson R H
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33 references, click to expand
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1984-01-00
Pages
65-71
Language
English
Region
England
NLM ID
8208664
PMCID
PMC557298
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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