J. Huppert

Examen au microscope electronique du RNA du virus de la myeloblastose aviaire

Single-stranded RNA, with a molecular weight of 107, has been obtained by phenol extraction of avian myeloblastosis virus treated with pronase. The RNA was prepared for electron microscopy according to Kleinschmidt. The usual tangling of single-stranded RNA was avoided by diluting the purified RNA in 8 M -urea before spreading. Very long molecules, with a modal length of 8·7 μ and a mean length of 8·3 μ can be seen. Shorter pieces of variable lengths, which may be broken molecules, are also present. From the base composition, the molecular weight and the length of the intact molecules, it can be calculated that two successive bases are separated by 2·96 A

The interaction of basic dyes with ribonucleic acid

Abstract Ribonucleic acid reacts in vitro with basic dyes such as toluidine blue, acridine orange, and pyronine to provoke a shift in the absorption maximum of the dye, either toward a longer (negative metachromasy) or shorter wavelength (positive metachromasy). The direction of the metachromatic shift is dependent on both the relative concentration of the polymer to the dye and on the secondary structure and the chainlength of the polymer. In studies with synthetic polymers, RNA, and RNA-fractions, at known polymer-dye ratios, it was found that the length of the polymer chain determined the extent of positive metachromasy. On the other hand, negative metachromasy appears dependent on density of double-stranded regions in RNA molecules.

Ribonuclease activity associated with mammalian cell walls.

Abstract An enzymatically active component, capable of degrading RNA, has been found associated with cell wall preparations obtained from Krebs ascites cells. The active material has been partially purified and characterized, and appears to be an endonuclease, failing to degrade RNA to nucleotides. It is thought that this activity is responsible for the destruction of infectious RNA by a mechanism different from that of pancreatic ribonuclease.

Polyadénylate synthétase des cellules d'ascite de souris: I. Purification et caractérisation de l'enzyme

Abstract Poly (A) synthetase from mouse ascites cells. I. Purification and characterization of the enzyme 1. 1. The poly(A) synthetase from mouse ascites cells which incorporates [14C]AMP into a preexisting polyribonucleotide chain is a weakly acidic protein ( isoelectric point = 6 ± 0.05 ) with a molecular weight, determined by sucrose gradient centrifugation, close to 80 000. Its exclusion from Sephadex G-200 would indicate that it can exist in a complex form. Incubation of the enzyme in a medium of high ionic strength (0.5 M KCl) results in a reversible dissociation. However, centrifugation in a high gravitational field ( 284 000 × g ) in presence of 0.5 M KCl leads to an irreversible dissociation of the enzyme. 2. 2. The enzyme is heat labile and loses 50 % of its activity in 4 min at 45 °C. The polyribonucleic acid primer and ATP have distinct roles in the stabilisation of the enzyme. 3. 3. The enzyme was 150-fold purified. Nevertheless, it still contains trace amounts of nucleic acids and has slight nuclease activity. Purification of the enzyme results in the appearance of new properties, which are discussed in the second paper.

Endonucleolytic DNase in oncorna viruses: role of mycoplasma.

Abstract The DNA-directed DNA polymerase activity of avian myeloblastosis virus and Rauscher leukemia virus was not enhanced by SV40 DNA, whereas that of murine sarcoma virus preparations was. The latter stimulation is ascribed to an endonucleolytic DNase which occurs in a mycoplasma contaminant. This can be removed by differential velocity centrifugation. Deoxyribonuclease activity is not a property of oncorna viruses, and the possibility is raised that other nonpolymerase enzymatic activities associated with these viruses may be contaminants.

Host-range mutant of fowl plague virus (FPV): Comparison of the genome and virus proteins

Abstract A host-range mutant of fowl plague virus, which has the ability to grow in mammalian cells, has been investigated. It was found that one or two of the structural proteins were apparently smaller than the corresponding proteins of the wild-type virus which only multiplied in chicken cells. There was also a corresponding reduction in the size of one of the genome fragments.

Newcastle Disease Virus: Virus Particle and RNA Synthesis in Different Host Cells and at Different Temperatures*

Summary When incubated in vitro at 40 °C, chorioallantoic membranes produce 80 to 200 p.f.u./cell of Newcastle disease virus (NDV), whereas chick embryo cells and mouse L cells produce only 2 to 10 and 0.1 p.f.u./cell, respectively. Under such conditions NDV ‘minus’ strand RNA accumulates in chick embryo cells (as well as in mouse L cells) but not in chorioallantoic membranes. Lowering the incubation temperature from 40 to 36 °C resulted in delays in virus release by 5 h from chick embryo cells, but by 1 h from chorioallantoic membranes. The maximum rate of virus RNA synthesis was observed at times when the release of progeny virus had been terminated. When protein synthesis is inhibited by early addition of cycloheximide, some NDV ‘minus’ strand RNA is found in the chick embryo cells but not in the chorioallantoic membranes. When the inhibitor is added later (2 h after virus infection), no virus RNA is labelled in the chick embryo cells, although some ‘plus’ strand RNAs are found labelled in the chorioallantoic membranes. It is suggested that the observed difference in the permissiveness of various cell types for NDV depends on the amount of some cellular component involved in NDV ‘plus’ strand RNA synthesis.

Heterogeneity of chick embryo cells with regard to Newcastle disease virus multiplication.

Summary Newcastle disease virus (NDV) production by individual chick embryo cells was determined after infection of monolayer cultures, trypsinization and distribution of the cells into a large number of test tubes so that each tube would contain an average of one cell (or in some experiments 10 cells). The content of each tube was assayed for p.f.u. 18 h later. The statistical analysis of the results indicates the presence of at least three distinct populations of cells with regard to NDV production: one non-producing population (50 to 90% of the cells); one low-producing population (80% of the producing cells) and one high-producing (20% of the producing cells). The relative proportions of these cell populations was the same whether primary, secondary or tertiary cultures were used, and also when the cell-cultures were obtained from different tissues of the chick embryo.

Polyadenylate synthetase des cellules d'ascite de souris. II. Etude de la reaction enzymatique.

Poly(A) synthetase from mouse ascites cells. II. Study of the enzyme reaction 1. 1. The poly(A) synthetase of mouse ascites cells has an absolute specificity for ATP as substrate and requires the presence of a primer (natural RNA or synthetic polyribonucleotide more than two nucleotides long) for its activity. The secondary structure of the primer does not appear to be critical but a free 3′-OH group, preferably that of adenosine, is an obligatory requirement. The product of the reaction is a poly(A) (30–40 nucleotides long) which remains attached to the primer. 2. 2. The pH-activity curves reveal two pH values for optimum enzymatic activity, one between 8.1 and 8.5 and the other above 9. Depending on the pH, two optimum concentrations for Mn2+ are also found. Mg2+ is not required for enzyme activity, although it stimulates the reaction rates under some conditions. An initial lag period of the reaction, abolished by increased enzyme concentrations, is observed. Further, the incorporation of [14C]AMP is not linear with enzyme concentration. 3. 3. The properties of the enzyme depend on its degree of purification and aging, as well as the source from which it is derived. 4. 4. The results suggest that the poly(A) synthetase of mouse ascites cells may exist in the form of a complex with other proteins and/or nucleic acids.

The Effect of Ethidium Bromide on Lcells and Encephalomyocarditis Virus Replication

Summary Ethidium bromide inhibits multiplication of encephalomyocarditis virus and synthesis of infectious single-stranded virus RNA. Infectious double-stranded virus RNA and cellular RNA synthesis are inhibited to a lesser degree. It is suggested that the effect is due mainly to binding of the drug to double-stranded replicative virus RNA.