Isolation of newly synthesised RNA can be achieved by treatment of cells in culture with 6-thioguanosine or 4-thiouridine followed by separation of thiol-containing RNA by affinity chromatography on mercurated cellulose columns. After short periods of treatment with 6-thioguanosine the proportion of RNA retained on mercurated cellulose is the same for both poly(A)-containing and poly(A)-free RNA, indicating similar incorporation of the drug into mRNA and rRNA. However, after longer periods of exposure, the cytotoxic effect of 6-thioguanosine results in diminished incorporation of radioactive uridine into RNA and of radioactive leucine into protein ; this suggests that synthesis of both RNA and protein are impaired. On the other hand, even after long exposure to high concentrations of 4-thiouridine, the syntheses of RNA and protein are not significantly affected. Proteins synthesised after treatment of cells with 6-thioguanosine are less stable than proteins synthesised after treatment of cells with 4-thiouridine.Following treatment of cells with thiol-containing analogues of nucleic acid precursors, RNA containing thiol groups can be physically separated from nonthiol RNA by affinity chromatography on columns of mercurated cellulose or agarose [I, 21. The possible application of this technique to studies of RNA metabolism and gene expression has been pointed out An alternative technique involving the use of mercury-substituted UTP and CTP as precursors for RNA biosynthesis in isolated chromatin preparations and subsequent isolation of mercurated RNA on thiol-Sepharose columns has already been applied to studies such as the fidelity of transcription of chromatin by Escherichiu coli RNA polymerase [3,4]. However, it appears that mercurated RNA may be subject to aggregation [5,6] and, in addition, the technique is limited to studies on isolated nuclei or chromatin [3]. Aggregation of mercurated RNA molecules with normal RNA leads to contamination of the RNA newly synthesised in vitvo with endogenous RNA carried over in the nuclear preparation Thiol-containing RNA can be isolated from intact cells and does not appear to be subject to aggregation. Physical isolation of RNA newly synthesised in vivo is therefore possible and provides a technique with which such questions as the extent of reutilisation of RNA precursors and the real half-lives of nuclear RNA and mRNA molecules can be tackled. In this paper we present further information on the use of [I, 21. PI. different precursors as regards incorporation into thiol-containing RNA, and their influence on macromolecular metabolism. MATERIALS AND METHODS Mutevials6-Thioguanosine, 6-mercaptopurine ribonucleoside, 8-thioguanosine, 2-mercaptopyrimidine, 2-thiocytosine and 4-thiouridine were obtained from Sigma

SUMMARYThree hybridoma antibodies, prepared against the RSN-2 strain of human respiratory syncytial (RS) virus, have been used to identify antigenic variation between 41 isolates of RS virus collected from widely separated geographical regions over a period of 29 years. One antibody was directed against an antigenic site on the virus fusion protein, VP70. This site was shared by 21 virus isolates tested and its recognition by the antibody was sensitive to the presence of 2-mercaptoethanol. The remaining two antibodies used react against the virus phosphoprotein, VPP32. Two independent sites were recognized on VPP32 by these antibodies. One antibody reacted with all of the virus isolates screened while the second reacted with only 21 out of the 41 virus isolates. On the basis of the variable epitope, two antigenic types of human RS virus were identified. The distribution of each antigenic group among 28 RS virus isolates from the Grampian Region, north-east Scotland, collected between 1982 and 1984 was determined. The reactivity of these antibodies was examined using immunofluorescence staining and by immunoblotting; the latter technique also revealed that the electrophoretic mobility of VPP32 varied in parallel with the variable antigenic site.

SUMMARYInfection of BHK 2I (C 13) or HEp-2 cells with herpes virus was followed by a marked increase in the activity of alkaline DNase which was prevented by actinomycin D and puromycin. Activation of a latent enzyme was not responsible for the increase. The properties of the DNase appearing after virus infection in both cell types were the same, but they differed from those of the enzyme from uninfected cells in specificity towards the secondary structure of the DNA substrate, heat-stability, requirement for thiol groups, inhibition by K + and Na + ions and response to various concentrations of Mg ~+ and Mn ~+. The activities of acid DNase and alkaline phosphomonoesterase were not significantly altered after herpes infection. Further, the activity of alkaline RNase was not altered by infection, and this implies that the induced DNase was specific for DNA. The new DNase could be separated from the DNase present in uninfected cells and from alkaline phosphomonoesterase by chromatography on columns of DEAE-cellulose; its enzymic properties were the same as those observed with soluble extracts of cells infected with herpes virus.

No abstract

SUMMARYHuman sera containing respiratory syncytial (RS) virus-specific antibodies enhance RS virus infection of the U937 macrophage cell line. There was an increase in the number of cells expressing virus antigen when U937 cells were infected with RS virus in the presence of human serum compared to cells infected in the absence of human serum. Human sera enhanced virus yield, as measured by the cell-released infectious virus, by an average of 50-fold compared to virus infection in the absence of human serum. The comparison of the enhancing activities of paired acute and convalescent human sera showed that the titre of enhancing antibody increased in parallel with the titre of RS virus-specific antibody measured by complement fixation and virus neutralization. An RS virus-specific neutralizing monoclonal antibody directed to the virus F protein enhanced virus infection of U937 cells. A non-neutralizing monoclonal antibody directed to the virus nucleoprotein did not enhance virus infection. The possible role of enhancing antibodies in vivo is discussed. INTRODUCTIONRespiratory syncytial (RS) virus is a major cause of acute respiratory infections in young children. The use of a formalin-inactivated RS virus vaccine predisposed children to severe illness when subsequently infected with RS virus (Kim et al., 1969). Following immunization, children developed lower neutralizing antibody titres than those of a comparable age who had natural RS virus infections and had not been immunized (Murphy et al., 1986). Chin et al. (1969) found higher virus shedding and virus isolation rates in vaccinees compared to unvaccinated children when both groups were naturally infected with RS virus. These data led to the speculation that low antibody titres contributed to the severity of the RS virus infections. A possible explanation for these data is antibody-dependent enhancement of virus infection, as proposed by Porterfield (1982) and Halstead (1982), although no experimental data were presented at that time.The in vitro enhancement of virus infection of macrophage cell lines by virus-specific antibodies at sub-neutralizing concentrations has been reported for several viruses (reviewed by Halstead, 1982). The initial stages of virus enhancement require the formation of a virusantibody complex which then attaches, via the Fc portion of the antibody, to the macrophage Fc receptor; this facilitates the entry of the virus as compared to virus infection in the absence of antibody (Gollins & Porterfield, 1984). The sites of replication of RS virus in the respiratory tract are poorly understood and the role of antibody-dependent enhancement in RS virus pathogenesis remains to be determined. Recently, RS virus has been shown to replicate in vitro in human peripheral blood mononuclear leukocytes (Krilov et al., 1987). In addition, RS virus antigen has been found in circulating mononuclear leukocytes of infants with RS virus infections (Dumorat et al., 1985). The present report describes the infection of the U937 human macrophage cell line by...

Two DNA polymerases of high molecular weight, pol A (mol.wt. 190 000) and pol B (mol.wt. 240 ooo), have been purified 6300-fold and 1600-fold respectively from an extramitochondrial supernatant of a bleached strain of Euglena gracilis. They have very similar requirements when assayed with an 'activated'-DNA primer-template [the optimum conditions of pH and ionic (K+ and Mn2+) composition being 7.2, 25 mM and 0.2 mM respectively]. 0.2 mM-Mn2+ was about 1.5-2-fold as effective as 2 mM-Mg2+, owing to substrate activation by deoxyribonucleoside 5'-triphosphates in the presence of Mn2+. Km values for the triphosphates in the absence of activation were about 10(-6)M with Mn2+ and 8 X 10(-6) M with Mg2+ for both enzymes. They were inhibited to the same extent by N-ethylmaleimide, novobiocin and o-phenanthroline, but differed in their chromatographic behaviour on DEAE-cellulose and in their electrophoretic mobilities on polyacrylamide gel. No evidence was found for the existence in these cells of a DNA polymerase of low molecular weight, but there were indications that a third enzyme of high molecular weight might exist.

No abstract

No abstract