Selection By [3H] Amino Acids Of CHO-cell Mutants With Altered Leucyl

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Abstract

Efficient selection procedures, using [3H]amino acids as the selecting agent, were developed for isolating temperature-sensitive (TS) mutations in CHO cells affecting protein synthesis. After chemical mutagenesis, leucyl-tRNA synthetase mutants were obtained when [3H]leucine was used as the selecting agent in two independent experiments. These mutations seem to involve the same genetic locus as the TSH1 mutant described previously (1). A selection with [3H]valine, in which all amino acids except leucine were at low concentration in the selective medium, resulted in a new class of mutants with reduced asparagyl-tRNA synthetase activity. These results were consistent with the finding that all mutants were phenotypically dependent on the concentration of amino acid, specific to the altered synthetase, in the medium. Our observations suggest that although leucyl synthetase mutations are a relatively common class of TS mutations in CHO cells, the spectrum of mutants obtained can be at least partially manipulated through concentrations of amino acids in selective media. The asparagyl-synthetase mutation was shown to be recessive and to complement the leucyl-synthetase mutation in cell-cell hybrids.

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• Mutations in the structural genes of CHO cell histidyl-, valyl-, and leucyl-tRNA synthetases. Ashman CR. Ashman CR. Somatic Cell Genet. 1978 May;4(3):299-312. doi: 10.1007/BF01542844. Somatic Cell Genet. 1978. PMID: 694722
• Six complementation classes of conditionally lethal protein synthesis mutants of CHO cells selected by 3H-amino acid. Adair GM, Thompson LH, Lindl PA. Adair GM, et al. Somatic Cell Genet. 1978 Jan;4(1):27-44. doi: 10.1007/BF01546491. Somatic Cell Genet. 1978. PMID: 628883
• The effect of amino acids on the temperature sensitive phenotype of the mammalian leucyl-tRNA synthetase mutant tsHl and its revertants. Molnar SJ, Rauth AM. Molnar SJ, et al. J Cell Physiol. 1979 Feb;98(2):315-26. doi: 10.1002/jcp.1040980208. J Cell Physiol. 1979. PMID: 422660
• A mammalian cell mutant with a temperature-sensitive leucyl-transfer RNA synthetase. Thompson LH, Harkins JL, Stanners CP. Thompson LH, et al. Proc Natl Acad Sci U S A. 1973 Nov;70(11):3094-8. doi: 10.1073/pnas.70.11.3094. Proc Natl Acad Sci U S A. 1973. PMID: 4361675 Free PMC article.
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• Isolation of a Chinese hamster cell mutant with low intracellular phosphoribosylpyrophosphate concentration. Crawford CR, Bryant RE. Crawford CR, et al. Mol Cell Biol. 1982 Dec;2(12):1619-27. doi: 10.1128/mcb.2.12.1619-1627.1982. Mol Cell Biol. 1982. PMID: 14582202 Free PMC article.
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• Efficient procedure for transferring specific human genes into Chinese hamster cell mutants: interspecific transfer of the human genes encoding leucyl- and asparaginyl-tRNA synthetases. Cirullo RE, Dana S, Wasmuth JJ. Cirullo RE, et al. Mol Cell Biol. 1983 May;3(5):892-902. doi: 10.1128/mcb.3.5.892-902.1983. Mol Cell Biol. 1983. PMID: 6346061 Free PMC article.
• Purification and characterization of DNA polymerase alpha of Chinese hamster ovary cells. Khan NN, Brown NC. Khan NN, et al. Mol Cell Biochem. 1985 Oct;68(2):169-79. doi: 10.1007/BF00219381. Mol Cell Biochem. 1985. PMID: 3001502

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