RELATIONSHIP BETWEEN HELA CELL RIBOSOMAL RNA AND ITS PRECURSORS STUDIED BY HIGH RESOLUTION RNA-DNA HYBRIDIZATION

The relationship between ribosomal RNA and its precursors in HeLa cells has been investigated by RNA-DNA hybridization. Highly purified 28 and 18 s RNA have been prepared from isolated ribosomal subunits, and 45 and 32 s ribosomal RNA precursors from isolated nucleoli. Analysis of size and base composition of the RNA recovered from RNase-treated RNA-DNA hybrids has been used to monitor the specificity of the hybrids investigated. The values obtained for the fraction of HeLa cell DNA complementary to 18, 28 and 45 s RNA were found to be proportional to the molecular weights of these species taken as 0.6, 1.6 and 4.6 × 106, respectively. No competition for sites in DNA was detected between 28 and 18 s RNA. Under the stringent specificity conditions defined in the present work, 45 s RNA was found to compete with 28 and 18 s RNA to the extent expected if about 35 and 13%, respectively, of the precursor molecule were involved. The saturation and hybridization-competition results described above provide strong support for the model, suggested by previous structural studies, according to which the 45 s molecule contains the sequences of one 28 s molecule and one 18s molecule, with the remaining 50% of its length being represented by non-ribosomal sequences. 32 s RNA competed with 28 s RNA for sites in DNA to the extent expected if about 70% of the precursor molecule were involved, whereas it did not show any capacity to compete with 18 s RNA: these results indicate, in agreement with the previous conclusion of the partial sequence analysis, that in the 32 s molecule (mol. wt about 2.2 × 106) approximately 70% is represented by the sequences of 28 S RNA and the rest by sequences of non-ribosomal type. Both in saturation experiments and in tests of hybridization-competition with 45 s RNA, 32 s RNA showed abnormal behavior, which suggested that the non-ribosomal portion of this half of the 45 s molecule can hybridize, although with a lower efficiency, also with the DNA segment homologous to the nonribosomal portion of the other half of the 45 s precursor: these observations are in agreement with the remarkable similarity in partial sequence distribution between the two non-ribosomal stretches. The high resolution obtained in the hybridization experiments described in the present work provides a further confirmation of the specificity exhibited, under appropriate conditions, by RNA-DNA hybrids involving ribosomal RNA in eukaryotic cells. © 1969.