Bovine Spleen Phosphodiesterase Research Articles

Inactivation of E. coli alkaline phosphatase prior to phosphodiesterase digestion of oligodeoxyribonucleotides

Experiments are described that demonstrate the inability of chloroform-isoamyl alcohol deproteinization to remove E. coli alkaline phosphatase from aqueous reaction mixtures. The inconstant inactivation of alkaline phosphatase with the use of ethylenediaminetetraacetic acid alone is also noted. Total inactivation of alkaline phosphatase was obtained by incubation of the enzyme-containing reaction mixture for 3 hr at pH 2.1 and 25°C in the presence of 0.005 M EDTA; following restoration of the pH to 8.0 and addition of Mg ++ to 0.05 M, no reactivation of the enzyme was noted. The presence of inorganic phosphate in the reaction mixture during the pH 2.1 incubation did not protect the enzyme against inactivation. Both bovine spleen and snake venom phosphodiesterase, when placed in reaction mixtures that had first been subjected to inactivation conditions followed by pH restoration and addition of Mg ++, were found to be quite active. The procedure described is thus useful for removal of terminal phosphate groups from oligonucleotides prior to complete phosphodiesterase digestions.

The Incorporation of Sangivamycin 5′-Triphosphate into Polyribonucleotide by Ribonucleic Acid Polymerase from Micrococcus lysodeikticus

Abstract Sangivamycin is incorporated into RNA in the reaction catalyzed by the RNA polymerase from Micrococcus lysodeikticus with the use of calf thymus DNA or the copolymer of deoxyadenosine and deoxythymidine (poly d(A-T)). The incorporation of sangivamycin 5'-triphosphate into RNA is dependent on primer, time, protein concentration, and Mn++ or Mg++. When calf thymus DNA is primer, GTP, CTP, and UTP are necessary for the incorporation of sangivamycin 5'-triphosphate into RNA. In similar experiments when sangivamycin 5'-triphosphate is the only nucleotide in the assay mixture, little or no homopolymer is formed. However, an acid-insoluble product forms when ATP is added. When poly d(A-T) is the primer, UTP is necessary for the incorporation of sangivamycin 5'-triphosphate to form polyribonucleotide. The copolymer formed with poly d(A-T) primer consists of regularly alternating residues of sangivamycin 5'-monophosphate and UMP. Bovine spleen phosphodiesterase can hydrolyze this copolymer. These results indicate that the phosphodiester bonds between sangivamycin and the adjacent uridine residue are susceptible to phosphodiesterase. Sangivamycin 5'-monophosphate and sangivamycin 5'-diphosphate are not inhibitors of RNA polymerase, nor are these compounds incorporated into RNA. It has also been found that 3'-deoxyadenosine 5'-triphosphate inhibits the incorporation of sangivamycin 5'-triphosphate. The data show that sangivamycin 5'-triphosphate competes with ATP in the various polymerization reactions. Nitrogen atom 7 of the purine ring of ATP is not essential for binding to RNA polymerase, as evidenced by the incorporation into RNA of the nucleotide antibiotic, sangivamycin 5'-triphosphate, which has a pyrrolopyrimidine ring.