Genes for catecholamine biosynthesis: cloning by expression and identification of the cDNA for rat dopamine beta-hydroxylase.
mRNA for dopamine beta-hydroxylase [3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1] has been partially purified from poly(A)+ mRNA isolated from a rat pheochromocytoma cell line. Shared antigenic determinants between tyrosine hydroxylase and dopamine beta-hydroxylase allowed us to obtain enriched fractions of dopamine beta-hydroxylase mRNA by immunoprecipitating translated mRNA products with tyrosine hydroxylase antisera. The enriched dopamine beta-hydroxylase mRNA was used to synthesize the corresponding cDNAs, which were then cloned in the Pst I site of pBR322. Recombinant colonies were characterized by an in situ colony immunoassay and hybrid-selected translation. In vitro translation of the mRNA selected from one recombinant clone produced a protein of 75,000 daltons that comigrated with authentic dopamine beta-hydroxylase. Partial proteolysis of both authentic dopamine beta-hydroxylase and the protein encoded by the recombinant clone produced identical peptide patterns.
D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004299:Dopamine beta-Hydroxylase / Q000235:genetics*; D005786:Gene Expression Regulation; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000032:analysis; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats
D010446:Peptide Fragments; D012333:RNA, Messenger; D004247:DNA; D004299:Dopamine beta-Hydroxylase