The synthesis of polyribonucleotides by cytoplasmic enzymes

Abstract

1. The possibility that the cell cytoplasm contains enzymes catalysing the biosynthesis of RNA was investigated in fractions obtained by differential centrifugation of homogenates of Landschutz ascites-tumour cells. 2. The microsomal fraction was shown to be most active in incorporating UMP residues from [alpha-(32)P]UTP into polyribonucleotide material. 3. The same fraction also incorporated [(3)H]CTP, [(3)H]ATP and [(3)H]GTP separately and independently of the presence of complementary ribonucleoside 5'-triphosphates. 4. The reaction was promoted by the addition of RNA and showed an absolute requirement for Mg(2+) ions. 5. Analysis of alkaline hydrolysates of the reaction products after the incorporation of [alpha-(32)P]UTP showed that most of the radioactivity was recovered in (2',3')-UMP residues irrespective of whether CTP, ATP and GTP were present in the reaction mixture. 6. Extraction of RNA from the reaction mixtures after the incorporation of [(3)H]ATP, [(3)H]GTP or [(3)H]CTP and analysis by sucrosedensity-gradient centrifugation showed no labelling of the ribosomal RNA. Radioactive material appeared between the 4s region and the meniscus of the sucrose gradient. In agreement with this observation, determinations of the chain length of the product showed that only short sequences of polynucleotides were synthesized. It is concluded that only homopolyribonucleotide synthesis is catalysed by the microsomal fractions and that there is little or no synthesis of RNA-like heteropolymers.