ISOLATION OF OUTER LAYER OF NUCLEAR ENVELOPE - COMPOSITION OF RNA

A new method is presented for the isolation of the outer layer of the nuclear envelope and its attached ribosomes. Nuclei isolated from rat livers by the method of Chauveau et al. were treated with 2.5% citric acid to separate the outer layer of the nuclear envelope from the remainder of the nucleus. Morphological evidence showed that the precipitate obtained by high speed centrifugation of the citric acid extract contained both membranes and ribosomes. Evidence that the RNA of the outer layer of the nuclear envelope is a mixture of ribosomal and other RNAs was obtained by kinetic studies, UV and 32P nucleotide analyses, and the effects of actinomycin D. The kinetics of labeling of the 18S and 28S RNA of the outer layer of the nuclear envelope with orotic 14C-2-acid were intermediate between those of cytoplasmic ribosomal RNA and nuclear RNA. Maximal labeling was found in 6 h for RNA of the nuclear envelope, 46 h for ribosomal RNA, and 40 min for nuclear RNA. The half-lives of the 18S and 28S RNA of the outer layer of the nuclear envelope were shorter than those of ribosomal RNA and longer than those of nuclear RNA. Although the UV base compositions of nuclear RNA and RNA of the outer layer of the nuclear envelope were similar, the 18S RNA of the latter had a higher content of uridylic acid and a lower content of guanylic acid than nuclear 18S RNA. The 28S RNA of the nuclear envelope had a higher content of adenylic and uridylic acids and a lower content of cytidylic and guanylic acids than nuclear 28S RNA. The distribution of 32P in the nucleotides of 18S RNA of the outer layer of the nuclear envelope was similar to that of nuclear 18S RNA. For the 28S RNA of the nuclear envelope, the differences were more marked. The 28S RNA of the nuclear envelope had a lower 32P content in uridylic acid and a higher 32P content in guanylic and cytidylic acids than nuclear 28S RNA. Actinomycin D inhibited the labeling of nuclear 18S and 28S RNA equally but in the outer layer of the nuclear envelope the labeling of 18S RNA was inhibited by more than 50% with no significant effect on labeling of the 28S RNA. © 1969.