Gaulton G N, Triplett E L
J Biol Chem. 1983 Dec 25;258(24):14839-44.
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.
利用高度特异性的兔抗酪氨酸酶抗体和山羊抗兔免疫球蛋白对胚胎多核糖体进行间接免疫沉淀，从第22阶段（尾鳍循环）胚胎中分离出北美豹蛙酪氨酸酶信使核糖核酸（mRNA）。蔗糖梯度分析表明，抗酪氨酸酶抗体特异性结合到300 - 350 S类胚胎多核糖体上，这与新生酪氨酸酶的酶活性位置和酪氨酸酶mRNA的位置一致。加入山羊抗兔免疫球蛋白G后，这些与抗酪氨酸酶结合的多核糖体被完全免疫沉淀。通过在寡聚（dT）纤维素上连续过柱，从经苯酚抽提的抗体-多核糖体复合物中获得聚腺苷酸加尾（Poly(A+)）RNA。酪氨酸酶mRNA的最终纯化通过制备性蔗糖梯度分级分离实现。在5 - 30%蔗糖梯度中，酪氨酸酶mRNA沉降为单一的13 S峰，在十二烷基硫酸钠-聚丙烯酰胺凝胶上显示为一条分子量为4.5×10⁵道尔顿（1275个核苷酸）的单带。在无细胞翻译系统中进行检测时，这种mRNA指导合成一种单一的35000道尔顿蛋白质，该蛋白质在十二烷基硫酸钠-聚丙烯酰胺凝胶上与天然酪氨酸酶共迁移，并且用抗酪氨酸酶免疫球蛋白进行免疫沉淀时，其免疫沉淀率大于98%。最终纯化产物相对于起始多核糖体RNA的纯化倍数为4103倍。
