Gedamu L, Iatrou K, Dixon G H
Cell. 1977 Mar;10(3):443-51. doi: 10.1016/0092-8674(77)90031-9.
Poly(A)+ protamine mRNA was isolated from trout testis cells in a very pure form, and artificial poly(A)- protamine mRNA molecules were derived from it by enzymatic deadenylation with RNAase H from calf thymus after hybridization with oligo(dT). The deadenylated protamine mRNA was found to be active in a wheat germ cell-free system and yielded a labeled product which co-migrated with authentic protamine. These deadenylated mRNA molecules were subsequently used as markers on denaturing polyacrylamide gels to identify and allow the purification of the poly(A)- protamine components known to exist in vivo in the total cellular poly(A)- RNA. RNA species of molecular weights similar to the enzymatically deadenylated subcomponents of protamine mRNA were observed in the natural poly(A)-RNA population of the testis cells. These naturally occurring poly(A)- protamine mRNAs were isolated by preparative gel electrophoresis and further characterized by 3H-poly(U) hybridization assay, by hybridization to complementary DNA made against highly purified poly(A)+ protamine mRNA, and by their ability to direct protamine synthesis in a cell-free system.
从鳟鱼睾丸细胞中以非常纯的形式分离出聚腺苷酸（Poly(A)+）鱼精蛋白mRNA，在与寡聚（dT）杂交后，通过用来自小牛胸腺的RNA酶H进行酶促去腺苷酸化，从其衍生出人工聚腺苷酸（Poly(A)-）鱼精蛋白mRNA分子。发现去腺苷酸化的鱼精蛋白mRNA在小麦胚芽无细胞系统中具有活性，并产生了一种与 authentic鱼精蛋白共迁移的标记产物。这些去腺苷酸化的mRNA分子随后被用作变性聚丙烯酰胺凝胶上的标记物，以鉴定并允许纯化已知存在于总细胞聚腺苷酸（Poly(A)-）RNA中的体内聚腺苷酸（Poly(A)-）鱼精蛋白成分。在睾丸细胞的天然聚腺苷酸（Poly(A)-）RNA群体中观察到分子量与鱼精蛋白mRNA的酶促去腺苷酸化亚成分相似的RNA种类。通过制备性凝胶电泳分离这些天然存在的聚腺苷酸（Poly(A)-）鱼精蛋白mRNA，并通过3H-聚尿苷酸（3H-poly(U)）杂交试验、与针对高度纯化的聚腺苷酸（Poly(A)+）鱼精蛋白mRNA制备的互补DNA杂交以及它们在无细胞系统中指导鱼精蛋白合成的能力进行进一步表征。
