Replication of mengovirus. II. General properties of the viral-induced ribonucleic acid polymerase

Plagemann, Peter G. W. (Western Reserve University, Cleveland, Ohio), and H. Earle Swim. Replication of mengovirus. II. General properties of the viral-induced ribonucleic acid polymerase. J. Bacteriol. 91:2327-2332. 1966.-Mengovirus induces the appearance of a ribonucleic acid (RNA) polymerase activity in Novikoff hepatoma cells which is readily distinguished from the deoxyribonucleic acid (DNA)-dependent RNA polymerase since it is not inhibited by actinomycin D or deoxyribonuclease, but is inhibited by ammonium sulfate, and is stable at -17 C. The incorporation of uridine into RNA by infected cells in the presence of actinomycin D does not reflect the viral polymerase activity as measured in cell-free preparations. The viral-induced RNA polymerase is produced in a biphasic fashion. Puromycin inhibits the production of viral polymerase, and in its presence the enzyme appears to be unstable between 4 and 6 hr. Puromycin also prevents the secondary rise in polymerase which begins at the end of replicative cycle. Under these conditions, however, the polymerase appears to be stable. The overall data indicated that some unspecified process is responsible for the apparent instability of viral-induced RNA polymerase between 4 and 6 hr and that it becomes inoperative toward the end of the replicative cycle.     

Characterization and expression of a spliced leader RNA in the parasitic nematode Ascaris lumbricoides var. suum.

The parasitic nematode Ascaris spp. contains a 22-nucleotide spliced-leader (SL) sequence identical to the trans-SL previously described in Caenorhabditis elegans and other nematodes. The SL comprises the first 22 nucleotides of a approximately 110-base RNA and is transcribed by RNA polymerase II. The SL RNA contains a trimethylguanosine cap and a consensus Sm binding site. Furthermore, the Ascaris SL RNA has the potential to adopt a secondary structure which is nearly identical to potential secondary structures of similar SL RNAs in C. elegans and Brugia malayi.     

The role of subunits in yeast DNA-dependent ribonucleic acid polymerase A.

The properties of RNA polymerase A, which lacked the subunits of 48 000, 37 000 and 16 000 mol. wt., were compared with those of RNA polymerase A by using native calf thymus DNA as the template. The results showed that: (1) the specific activity of RNA polymerase A was about one-third that of RNA polymerase A; (2) more than 80% of RNA polymerase A, but only about 25% of RNA polymerase A, made RNA; (3) initiation by RNA polymerase A, but not by RNA polymerase A, began after a lag of 2 min; (4) the temperature-dependence for productive binding to DNA was greater for RNA polymerase A; (5) the apparent Km for UTP was greater for RNA polymerase A. These results support the supposition that the subunits missing from RNA polymerase A are involved in DNA binding [Huet, Dezélée, Iborra, Buhler, Sentenac & Fromageot (1976) Biochimie 58, 71-80] and show also that the loss of these subunits affects the elongation reaction.     

Monoclonal antibodies directed against mammalian RNA polymerase I. Identification of the catalytic center

Mouse myeloma cells were fused with splenocytes from a mouse that had been immunized with RNA polymerase I purified from a rat hepatoma. Hybridoma cells were selected and colonies secreting antibodies directed against the enzyme were detected by analysis of cell culture supernatants in a solid phase radioimmunoassay. Two of these cell lines were grown on a larger scale and the interaction between the immunoglobulins obtained from them and RNA polymerase I was studied in detail. Antibodies from both of the hybridoma cell lines were able to inhibit DNA-dependent RNA synthesis catalyzed by RNA polymerases I and III, but not that catalyzed by polymerase II. The antibodies were also capable of reducing the RNA chain elongation reaction catalyzed either by RNA polymerase I associated with isolated nucleoli or by enzyme preinitiated in vitro on calf thymus DNA. Inhibition of RNA polymerase I activity by the monoclonal antibodies was inversely related to the nucleotide concentration. In contrast, the DNA concentration had relatively little effect on inhibition by the antibodies. Analysis of immune complex formation between the antibodies and isolated individual enzyme subunits demonstrated that the monoclonal antibodies were directed against the largest (Mr = 190,000) polypeptide of the polymerase I. These data indicate that the largest subunit of RNA polymerase I contains an immunological determinant in common with RNA polymerase III and suggest that the polymerase I polypeptide of Mr = 190,000 contains a catalytic center involved in RNA chain elongation.