Jean Rommelaere

NS-1 and NS-2 proteins may act synergistically in the cytopathogenicity of parvovirus MVMp

The interaction of parvovirus minute virus of mice (prototype strain, MVMp) with simian virus 40 (SV40)-transformed human cells (NB-E) was investigated by means of transfection with MVMp molecular clones derived from the infectious recombinant plasmid (pMM984). pMM984 inhibits stable transformation of NB-E cells to geneticin resistance (G418R) upon cotransfection with the selectable pSV2neo plasmid. We show here that this inhibition is not merely caused by a repression of marker gene expression from the SV40 early region promoter in pSV2neo and rather is likely to reflect the cytotoxic action of the parvovirus. Starting from plasmid pMM984, defined mutations were introduced into the genome of MVMp and more particularly into sequences coding for the NS-1 and/or NS-2 nonstructural proteins. In this way we could show that the NS-1 protein is necessary for the inhibition of transformation to G418R and that the NS-2 protein acts synergistically to enhance this effect. Moreover, results obtained with different viral mutants indicate that the inhibitory action of NS-1 on stable transformation can be dissociated from the ability of this protein both to transactivate the parvoviral p39 promoter of the capsid protein-encoding region and to drive parvoviral DNA amplification. Altogether these data point to a probable direct toxicity of MVMp nonstructural proteins for permissive host cells.

A SENSITIVE METHOD FOR MEASURING PYRIMIDINE DIMERS IN SITU

Abstract. –An immunocytological method for detecting pyrimidine dimers in situ has been developed. The technique is based on the fixation in cell nuclei of radiolabelled antibodies directed against ultraviolet irradiated DNA. The relative amount of bound antibodies was estimated by radioautography. Pyrimidine dimers induced by a dose as low as 2 J m‐2 can easily be detected. With this technique, the dose response of DNA photoproducts and their elimination by excision and photoreactivation were followed in eukaryotic cell cultures.

Chromosome and chromatid exchanges in chinese hamster cells

Chromosomes were studied by autoradiography in a mixed culture of diploid and tetraploid cells, after having induced fusion with Sendai virus between two Chinese hamster cell populations, one labelled with 3H-, the other with 14C-thymidine; sister chromatid exchanges were studied in the 3H diploid cells and exchanges between chromosomes in the 3H-14C tetraploid synkaryons. In both cases, the frequency of exchanges increases after U. V. irradiation.

Detection by density equilibrium centrifugation of recombinant-like DNA molecules in somatic mammalian cells.

When the nuclear DNA of Chinese hamster cells grown in the presence of bromo-deoxyuridine for one or less generation is analysed in an alkaline CsCl density gradient, a small fraction of replicated DNA can be found at densities intermediate between those of the light and heavy strands. The labelling pattern of these displaced molecules and their segregation into fully light and heavy fragments after sonication indicate that they are formed by the covalent joining of light and heavy segments. The duplexes giving rise to displaced DNA upon denaturation are mostly found at the position of light-heavy DNA in a neutral gradient; a minor component, however, bands at densities between light-heavy and heavy-heavy DNA. The high degree of renaturability of the former molecules suggests that they contain cross-links between the complementary strands; the latter, on the contrary, are likely to comprise a segment substituted in both strands, which is interpreted as being a heteroduplex resulting from somatic crossing-over between uninemic sister chromatids. Although the frequency of these recombinant-like molecules is increased several-fold after u.v. irradiation, the data do not support any direct implication of these exchanges in a postreplication repair mechanism of the type found in bacteria.

The genome structure of a new chicken virus identifies it as a parvovirus

The nucleic acid of chicken parvovirus-like particles showed sensitivity to DNase and S1 nuclease treatment and resistance to digestion with RNase. Viral DNA readily served as a template for self-primed conversion in vitro into a double-stranded form of about 5200 base pairs. There was no evidence for encapsidation of strands of opposite polarities. These findings confirm the taxonomic classification of chicken parvovirus-like particles as fowl parvovirus type 1 within the Parvovirus genus of the Parvoviridae.

Isolation of replicating DNA segments from Chinese hamster cells by density equilibrium centrifugation

Abstract When DNA is extracted from Chinese hamster cells grown in the presence of 5-bromodeoxyuridine from the beginning of the S (synthesis) phase until the middle of the first replication round, a significant fraction of total replicated DNA bands at intermediate densities between light-light and light-heavy DNA, in a CsCl gradient. Incomplete bromodeoxyuridine substitution compared with light-heavy DNA justifies the displaced banding of these molecules. Since “intermediate DNA” following alkaline or thermal denaturation gives rise to unsubstituted and fully substituted single strands, its particular density in neutral gradients cannot be ascribed to a uniformly reduced degree of bromodeoxyuridine substitution nor to covalently joined light and heavy strands. The segregation of DNA of intermediate densities into light-light and light-heavy components after shearing suggests that it includes at least one junction between replicated and still unreplicated segments, i. e. one replication fork that may or may not have lost one of its prongs. DNA of intermediate density specifically contains one to two sites sensitive to breakage by Neurospora crassa endonuclease. When a two-minute pulse of tritiated bromodeoxyuridine is given during replication in unlabelled heavy medium, the DNA fragments (mol. wt 35 × 10 6 ) containing labelled segments band essentially at intermediate positions and are progressively converted to light-heavy molecules, with increasing duration of chase. The half-life of this pulse-labelled intermediate DNA (about 25 min) is consistent with the proportion of total replicated DNA found at displaced densities (10 to 15%) and, together with the distribution of the intermediate radioactivity, is compatible with the existence of adjacent growing replicons. If DNA is labelled and extracted during the second replication round in the presence of bromodeoxyuridine, “intermediate DNA” with similar properties is found between the light-heavy and heavy-heavy peaks.

Interaction of caffeine with the DNA of Chinese hamster cells

Incubation of Chinese hamster cells with labelled caffeine leads to transfer of radioactivity to DNA. This association occurs during the S phase of the cell cycle and involves parental as well as newly synthesised strands. The replacement of thymidine by BrdUrd prevents the incorporation radioactivity from caffeine into the DNA strands containing BrdUrd. Thymine is the only base which becomes labelled and data suggesting the participation of methyl groups of caffeine in the biosynthesis of thymine are presented. Ultraviolet irradiation increases the incorporation of radioactivity from caffeine to DNA.

Presence of fowl parvovirus in fibroblast cell cultures prepared from uninoculated white leghorn chicken embryos

Chicken embryo fibroblast cell cultures prepared from commercial uninoculated White Leghorn eggs showed a spontaneous degeneration. Upon staining with haematoxylin-eosin intranuclear inclusion bodies of Cowdry type A were seen. Also, nuclear fluorescence was detected after indirect immunofluorescence staining with anti-serum to fowl parvovirus strain ABU. Electron microscopy of thin sections revealed parvovirus-like particles in both the nucleus and cytoplasm of the affected cells. During purification the viral particles banded in CsCl at a density of 1.42 to 1.43 g/ml and displayed typical parvovirus morphology by negative-staining EM. The viral particles contained single-stranded DNA of one polarity with a natural primer at the 3-end of the molecules. All these properties are characteristic of self-replicating fowl parvovirus strongly suggesting its transovarial transmission.

Atypical nucleoprotein complexes mediate CRE-dependent regulation of the early promoter of minute virus of mice

The P4 promoter of the parvovirus minute virus of mice (MVMp) directs transcription of the genes encoding non-structural proteins. We have previously shown that functional upstream CRE elements contribute to both the ras oncogene-dependent activation of promoter P4 and its down-modulation by known activators of cyclic AMP-dependent protein kinase A (PKA). In the present work, the nucleoprotein complexes formed with the P4 CRE elements were characterized with regard to their polypeptide constituents and the nucleotides taking part in the interaction. Atypical interactions, both at the protein-protein and protein-DNA level, were observed, which may be a reflection of the divergence of the parvoviral CREs from the usual consensus. The CRE-mediated regulation of promoter P4 by PKA and Ras is discussed in light of these findings.

The influence of 5-bromodeoxyuridine on DNA repair in Chinese hamster cells exposed to ultraviolet radiation

Abstract The kinetics and dose dependence were determined both for the repair synthesis and for the induction and repair of single-strand breaks in the 5-bromodeoxyuridine-substituted DNA of ultraviolet-irradiated Chinese hamster cells. A close temporal relationship was observed between repair synthesis and break sealing. The induction of single-strand breaks was linear with dose, whereas a non-linear response was found for repair synthesis. Since almost all breaks were repaired, the calculated number of bases which could be inserted per repaired single-strand break depended on the dose and was close or inferior to a hundred.