Isolation of a High‐Molecular‐Weight Hybrid between Ribosomal RNA and DNA

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Abstract
The genes for rRNA of Bacillus subtilis have been purified as high molecular weight DNA: RNA hybrids with a DNA/RNA ratio of 1. Two different methods were used. (1) High molecular weight denatured DNA (Mr= 2.2 × 107) was hybridized with rRNA and the excess of single‐stranded DNA was digested with Neurospora crassa endonuclease and Escherichia coli exonuclease I. The hybrid was then purified on Sepharose 4B and two subsequent CsCl gradients. This is a marked improvement on the previously published procedure of Sgaramella, Spadari and Falaschi (1968) insofar as it gives high molecular weight products (8 × 105 for the DNA moiety of the hybrid). (2) A simplified non‐enzymic procedure that used relatively short single‐stranded DNA molecules (Mr= 4 × 106) and several subsequent CsCl gradients led to a hybrid with a DNA: RNA ratio of 1.1 and a molecular weight higher than the previous procedure (1.7 × 106 for the DNA moiety). The DNA of the isolated hybrid, after removal of the RNA moiety, rehybridizes completely with rRNA.After hybridization of rRNA with DNA having a single‐stranded molecular weight of 2.2 × 107, the hybrid bands mainly at a density corresponding to that of denatured DNA; hybrid with a density corresponding to that of 1:1 DNA/RNA ratio can be obtained only if the DNA has been reduced by enzymic digestion or shearing to a single‐stranded molecular weight close to 2 × 106; this indicates that between each set of 16‐S + 23‐S rRNA genes, there must be regions of DNA non‐complementary to rRNA; these regions have a single‐stranded molecular weight that can be estimated as greater than 1.8 × 107.When the isolated DNA is treated with N. crassa endonuclease, in conditions that cause 95% hydrolysis of denatured DNA and 5% digestion of native DNA, it is digested only up to 50%. This suggests the presence of self‐annealing sequences in the rRNA genes, in agreement with the secondary structure of rRNA.