Isolation and characterization of trout testis protamine mRNAs lacking poly (A)

Abstract

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.