Structural Analysis of Nucleolar Precursors of Ribosomal Ribonucleic Acid: POLYPURINE SEQUENCES IN NUCLEOLAR 28 S RIBONUCLEIC ACID OF NOVIKOFF HEPATOMA ASCITES CELLS

Abstract

Complete pancreatic RNase digestion was carried out on twice purified nucleolar 28 S RNA of Novikoff hepatoma ascites cells which were highly labeled in vitro with 32P-orthophosphate. The resultant oligonucleotides of the digests were separated into well defined peaks by chromatography on DEAE-Sephadex A-25 at pH 7.6. Peak 8 containing octanucleotides and Peak 7 containing heptanucleotides were rechromatographed on DEAE-Sephadex A-25 at pH 3.3. By this procedure, octanucleotides and heptanucleotides were resolved into seven and eight major peaks, respectively. The hexanucleotides were separated into 12 spots by two-dimensional paper electrophoresis. On the basis of an estimated chain length of 4500 nucleotides for nucleolar 28 S, the calculated numbers of isopliths were in close agreement with the numbers of components isolated at acid pH. Sequence studies were carried out on the octa-, hepta-, and hexanucleotides by means of nucleotide analysis, complete digestion with T1 RNase and partial digestion with spleen phosphodiesterase. The polypurine sequences in nucleolar 28 S RNA defined thus far are the octanucleotides: G-A-A-A-A-G-A-Cp, G-A-A-G-A-A-G-Cp, G-A-G-G-A-A-G-Cp, and G-A-A-A-G-A-G-Up; the heptanucleotides A-A-G-A-A-G-Cp and G-G-G-G-G-G-Up; and the hexanucleotides G-G-G-G-G-Up, A-A-A-A-G-Cp and A-A-G-A-A-Cp. The octanucleotide G-A-G-G-A-A-G-Cp appears three times in the molecule. Interestingly, none of the sequences found are identical with those reported to be present in bacterial or viral RNA.