title: Control of tyrosinase gene expression and its relationship to neural crest induction in Rana pipiens. I. Isolation and characterization of amphibian tyrosinase mRNA.
abstract: Rana pipiens tyrosinase mRNA was isolated from Stage 22 (tailfin circulation) embryos by indirect immunoprecipitation of embryonic polysomes using highly specific rabbit anti-tyrosinase and goat-(anti-rabbit) immunoglobulins. Analysis on sucrose gradients indicated that anti-tyrosinase bound specifically to embryonic polysomes of the 300-350 S class coincident with the location of nascent tyrosinase enzyme activity and tyrosinase mRNA. These same anti-tyrosinase-bound polysomes were fully immunoprecipitated by the addition of goat-(anti-rabbit) IgG. Poly(A+) RNA was obtained from phenol-extracted antibody. polysome complexes by sequential passage over oligo(dT)-cellulose. The final purification of tyrosinase mRNA was achieved by preparative sucrose gradient fractionation. Tyrosinase mRNA sedimented as a single 13 S peak in 5-30% sucrose gradients and tracked on sodium dodecyl sulfate-polyacrylamide gels as a single band of 4.5 X 10(5) Da (1275 nucleotides). When assayed in a cell-free translation system, this mRNA directed the synthesis of a single 35,000-Da protein which co-migrated with native tyrosinase on sodium dodecyl sulfate-polyacrylamide gels and which was greater than 98% immunoprecipitable by anti-tyrosinase immunoglobulin. Final purification was 4103-fold over the starting polysomal RNA.
mesh_terms: D000818:Animals; D004156:Catechol Oxidase / Q000235:genetics*; D004627:Embryonic Induction; D005786:Gene Expression Regulation; D008970:Molecular Weight; D014442:Monophenol Monooxygenase / Q000235:genetics*; D009432:Neural Crest / Q000201:enzymology*; D011061:Poly A / Q000302:isolation & purification; D011132:Polyribosomes / Q000201:enzymology; D012333:RNA, Messenger / Q000302:isolation & purification*; D011894:Rana pipiens / Q000196:embryology*
chemical_list: D012333:RNA, Messenger; D011061:Poly A; D004156:Catechol Oxidase; D014442:Monophenol Monooxygenase