Analysis of the 5'-terminal nucleotide sequences of ribonucleic acids. II. Comparison of the 5'-terminal nucleotide sequences of ribosomal RNA's from different organisms.

The use of end-group analysis is valuable for determining the homogeneity and molecular weight of macromolecules. The synthesis of RNA upon a DNA template proceeds from the 5'to the 3'-end of the RNA sequence, and the identification of the 5'-termini of RNA molecules is therefore of particular importance in characterizing them. With this in view, a procedure for analyzing 5'-terminal nucleotide sequences of large RNA molecules was developed by one of us.' By this technique, it was found that two ribosomal RNA's present in Escherichia coli had unique sequences at their 5'-termini. A logical next step was to compare these sequences with those of ribosomal RNA's from other organisms. According to Midgley2 and Miura,3 the ribosomal RNA of bacteria is quite constant in nucleotide composition, in contrast to DNA, which varies widely in composition from species to species. When tested with cell-free amino acid-incorporating systems, ribosomes from various bacterial species have been found to be interchangeable.4' Accordingly, ribosomal RNA's were prepared from four species of bacteria with GC/AT ratios in DNA ranging from 0.53 (Bacillus cereus) to 2.33 (Sarcina lutea), and from one species of yeast, and their 5'-termini were analyzed. The results obtained are described in this communication. Materials and Methods.-Organisms: The bacterial and yeast species with GC/AT ratios of their DNA and the nucleotide composition of their RNA's are listed in Table 1. Culture media and harvesting cells: Culture media were as follows: S. lutea and B. stearothermophilus-10 gm polypeptone, 2 gm yeast extract, and 8 gm NaCl in 1 liter of water, pH 7.4; B. cereus-4 gm glucose, 1 gm NH4Cl, 120 mg MgSO4, 160 jg FeCi3, and 2 gm casamino acids in 1 liter of 0.05 M Tris-HCl, pH 7.4; B. subtilis-1 gm Na-citrate, 2 gm (NH4)2SO4, 5 gm casamino acids, 120 mg MgSO4, 5 gm sucrose in 1 liter of 0.05 M phosphate buffer, pH 7.4; yeast-5 gm yeast extract, 4 gm (NH14)2SO4, 0.25 gm MgSO4, 0.25 gm CaCl2, 5 gm casamino acids, 5 gm peptone, and 40 gm glucose in 1 liter of water, pH 5.0. All bacteria were grown with aeration at 37°C, except B. stearothermophilus, which was grown at 60°C. Yeast was cultured at 30°C. Cells were harvested during the logarithmic phase (ODeso = 0.6-0.8) and stored in a freezer until use.