Recognition of ribosomal RNA sites in DNA. II. The HeLa cell system.

Abstract

This analysis has shown that a heterogeneous population of RNase -resistant segments can be recovered from hybrids between HeLa 28S and homologous DNA. In this population a fraction corresponding to 3-5 x10-5 of HeLa DNA has sedimentation properties and base composition similar to those of the bulk of the input RNA. This points to a regular and complete H bonding of these chains with DNA. This fact, together with the specificity pattern shown by this portion of the complexes, suggests that their formation occurs at the level of specific ribosomal RNA sites. Based on sedimentation properties of the hybridized RNA, a similar fraction (3-8 x 10- 5) of other DNA''s of human origin appears to be involved in specific hybridization with 28S RNA. A figure of about 5 x 10-5 for the fraction of HeLa DNA complementary to 28S RNA, determined by measuring the over- level of hybrid retained on Millipore filters after a disaggregation step at 60[degree]C [was reported]. Although these results suggest a similar order of magnitude of the specific complexes, they are difficult to compare to these for the difference in the conditions employed and because the nature of the hybrids involved was not analyzed. It can be calculated that the DNA fraction involved in specific hybridization with 28S RNA would be equivalent to the sum of 200-400 stretches of the length of 28S molecules in the HeLa genome and 150-350 stretches in other human genomes. These figures are only minimum estimates, because the absolute efficiency of hybridization is probably significantly lower than 100% and because 1 fraction of the shorter regions of base pairing may actually involve ribosomal RNA sites. The fraction of DNA which appears to be capable of specific hybridization with 18S RNA is of the order of 2-3 x 10-5 in HeLa cells. This would correspond to the sum of 400-600 stretches of the length of the 18S molecule/cell. The cross-hybridization observed between the 2 RNA subclasses introduces an uncertainty as to the fraction of DNA corresponding to 28S and, respectively, 18S sites. In spite of this, the number of sites appears to be very large for both RNA species. This multiplicity, which has also been observed in bacteria, raises the problem of the variability and control of ribosomal RNA genes.