Ribonuclease H levels in herpes simplex virus-infected cells.

Two forms of ribonuclease H (RNase H) have been identified both in uninfected and Herpes Simplex virus (HSV-)infected BHK cells. Identical RNase H species were detected in control- as well as in infected cells. RNase H I and II have not been found to be associated both with host cell DNA polymerase alpha and beta and HSV-induced DNA polymerase. Infection of BHK cells with HSV type 1 does not lead to a pronounced alteration of RNase H II activity but to an increase (3-fold) of the extractable RNase H I activity. RNase H I activity increases to a maximum between 8-10 hours p.i.; the bulk of HSV-DNA synthesis occurs between 6-8 hours p.i. From these experiments we draw the preliminary conclusi…

LEVELS OF RIBONUCLEASE H IN CELLS INFECTED WITH HERPES SIMPLEX VIRUS TYPE1

Biochemistry and Molecular Biology of DNA Replication in Yeast

For the past two decades, the study of the mechanism of DNA replication has been focused mainly on the chromosomes of the simple prokaryotes and their viruses (1). The complexity of the eukaryotic genome and multiple levels of control during the replication of eukaryotic chromosomes have until recently prevented similar studies. In recent years, a lower eukaryote, the yeast Saccharomyces cerevisiae, has become a major focus of efforts in molecular biology. In this chapter, I will briefly review accomplishments in this area. Yeast is an ideal model system for studies on the structure and replication of the eukaryotic chromosome. Yeast cells are easy to grow and study biochemically. Genetic a…

Control of Enzymic Hydrolysis of Polyadenylate Segment of Messenger RNA: Role of Polyadenylate-Associated Proteins

The role of poly(A)-associated proteins in the breakdown of poly(A) sequences in both mammalian polyribosomes and in isolated poly(A) · protein complexes has been studied on an enzymic level. Two nucleases (alkaline exoribonuclease and endoribonuclease IV; both isolated from eukaryotic tissue), which preferentially hydrolyze poly(A) sequences, have been applied to determine the susceptibility of poly(A) in dependence on the presence of poly(A) · protein(s). Polysomes, isolated from L5178y mouse lymphoma cells, do not contain endogenous poly(A) nuclease activity. The poly(A) segment in polysomes is hydrolyzed by the exoribonuclease, irrespective of the preincubation conditions used. Pretreat…

Virazole (Ribavirin) a Cytostatic Agent

Virazole (1-β-D-ribofuranosyl-1,2,4-triazole-3carboxamide,ribavirin) is a synthetic triazol nucleoside with a broad spectrum of antiviral activity. But virazole affects the metabolism not only of virus infected cells. Virazole strongly inhibits the cell proliferation of mouse lymphoma cells (L 5178y), which were not infected with DNA- or RNA-viruses. Starting with 3 × 103 cells/ml and an incubation period of 72 hr, the drug reduces the cell proliferation to 50% (= ED50 concentration) in a concentration of 4,7 μM.

Cell cycle-dependent alterations of the two types of ribonucleases H in L5178y cells.

Alterations of Activities of Ribonucleases and Polyadenylate Polymerase in Synchronized Mouse L Cells

The activities of the three known catabolic and the one anabolic polyadenylate enzymes have been determined in synchronized L5178y cells: endoribonuclease, exoribonuclease, 5'-nucleotidase and poly(A) polymerase (Mg2+-dependent). These four enzymes were found primarily in the nuclear fraction. The activity of poly(A) polymerase remains essentially constant during the transition from G1 to S phase. However, the poly(A) catabolic enzyme activities increase parallel with DNA synthesis; the endoribonuclease activity increases 4-fold during G1 to S phase, the exoribonuclease and the nucleotidase activities increasing 30-fold and 16-fold. During the S phase the poly(A)-degrading enzymes are far m…

Filter paper disk techniques for assay of nucleotidase

A DE filter disk technique for assaying the activity of nucleotidase is described. This method is based on the observation that nucleotides bind to the filters at 5 mM Tris-HCl (pH 7.8) while nucleosides do not. As parameter for the nucleotidase activity the decrease of bound nucleotides is determined. In parallel experiments the amount of the product (nucleoside) formed can be measured by DEAE Sephadex column chromatography. The filter disk technique can be applied for the determination of vmax and Km of a nucleotidase by using different ribonucleosidase monophosphate substrates.

Differential mode of inhibition of terminal deoxynucleotidyl transferase by 3′-dATP, ATP, βaraATP and αaraATP

DNA-replication complex from cells infected with herpes virus.

Herpes simplex virus (HSV) DNA synthesis is initiated in an intact cell system by a 36-residue ribonucleotide stretch [W.E.G. Müller, R.K. Zahn, J. Arendes, and D. Falke (1979) Virology, 98, 200-210]. In the present study a nucleoplasmic fraction was isolated from rabbit kidney cells infected with HSV (type 1), which catalyzes DNA synthesis. By means of specific assays, containing single-stranded deoxyribopolymers, it was elucidated that the replication complex contains both an RNA-synthesizing and a DNA-synthesizing enzyme. These enzymes were characterized as host cell RNA polymerase II and HSV-induced DNA polymerase. The RNA polymerase II synthesizes an RNA initiator with an average chain…