Partial sequence analysis of ribosomal RNA from HeLa cells: I. Oligonucleotide pattern of 28 s and 18 s RNA after pancreatic ribonuclease digestion

Abstract

Highly purified 28 and 18 s ribosomal RNA have been prepared from HeLa cells and subjected to partial sequence analysis. The ratio of 28 to 18 s RNA, determined in material extracted by phenol from total cells or from the ribosome-polysome fraction and fractionated by repeated cycles of sucrose gradient centrifugation, was found to be about 2.5; a very similar figure (about 2.6) was obtained for the ratio of the two RNA components associated with the isolated ribosomal subunits. This finding, and the fact that the observed ratio is substantially equal to that expected on the basis of the molecular weights of the two RNA species (by assuming equimolar amounts of them), argue against any appreciable conversion of one component to the other during extraction. The distribution of nucleotide sequences released by pancreatic RNase digestion has been analyzed in the two rRNA components. In both RNA species, the observed frequency of sequences has shown only a rough agreement with that expected on the basis of random distribution of bases: significant discrepancies from random distribution have been found in several sequences, without any obvious pattern of regularity. Highly significant differences in the frequency of almost all sequences released by RNase digestion have been demonstrated to occur between 28 and 18 s RNA. In both species of ribosomal RNA, the presence of appreciable amounts of pseudouridylic acid has been demonstrated. The analysis of the distribution of this minor component among various RNase digestion products has shown considerable deviations from a constant frequency of sequences containing pseudouridine relative to that of the homologous sequences containing uridine; this finding agrees with the idea of a conversion of uridine to pseudouridine after the assembly of the polynucleotide chain.