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Tyrosine aminotransferase mRNA turnover in CHO cells

Conference · · FASEB Journal (Federation of American Societies for Experimental Biology); (United States)
OSTI ID:5148380
; ; ;  [1]
  1. Oak Ridge National Lab., TN (United States) Univ. of Tennessee, Oak Ridge (United States)
An expression vector containing a minigene designed for precise expression of the mRNA and the protein of rat tyrosine aminotransferase (TAT), ordinarily only expressed in liver, was constructed. This minigene was placed under control of the LTR promoter of murine leukemia virus and was cotransfected into CHO cells deficient in dihydrofolate reductase (DHFR) with another vector (pSV2dhfr) encoding DHFR. Upon methotrexate selection and further amplification, cell lines which produce large amounts of TAT mRNA and the enzyme have been isolated. Northern blots exhibited the expected 2.4 kb band of TAT mRNA, and S1-nuclease protection assay established that the mRNA was correctly initiated and terminated. When transcription was blocked with actinomycin, the 2.4 kb TAT mRNA in CHO cell lines was degraded with half-life ranging 1.5 to 2.5 hr, a rate similar to that found in liver or hepatoma cells. Furthermore, the rapid turnover of TAT mRNA in CHO cells could be completely stabilized by cyclohexamide as observed previously in rat liver. With their high content of TAT mRNA exhibiting normal turnover properties, the CHO cell lines established should be useful for analysis of the molecular mechanisms of intracellular turnover of this labile message.
DOE Contract Number:
AC05-84OR21400
OSTI ID:
5148380
Report Number(s):
CONF-9104107--
Conference Information:
Journal Name: FASEB Journal (Federation of American Societies for Experimental Biology); (United States) Journal Volume: 5:5
Country of Publication:
United States
Language:
English