Regulation of tyrosine hydroxylase mRNA by glucocorticoid and cyclic AMP in a rat pheochromocytoma cell line. Isolation of a cDNA clone for tyrosine hydroxylase mRNA

Abstract

Treatment of a subclone of the PC12 pheochromocytoma cell line, PC8b, with either dexamethasone or 8-bromo cyclic AMP resulted in increased translational activity of tyrosine hydroxylase mRNA (mRNATH). Poly(A+)-containing RNA from cells treated with both inducers was used to construct a cDNA library. Double-stranded cDNA was inserted into the PstI site of pBR322 using GC tailing, and plasmids were used to transform Escherichia coli HB101. Colonies containing plasmids with inserted sequences were initially screened by DNA dot hybridization, and those positive colonies were then screened by hybrid selected translation. One plasmid, pTH.4, was identified as containing a 400-base pair sequence complementary to mRNATH. Nick-translated pTH.4 DNA was used to identify mRNATH as containing approximately 1800 nucleotides by Northern blot analysis. PC8b cells treated with either dexamethasone or 8-bromo cyclic AMP yielded greater mRNATH hybridization on Northern blot analysis and accumulated higher molecular weight tyrosine hydroxylase RNA species. Following treatment of cells with inducers, the temporal increase in tyrosine hydroxylase enzyme activity was associated in all cases with an increase in the translational activity and relative amount of mRNATH, and the fold increase in the latter two parameters was equal to or greater than the increase in enzyme activity.