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.

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.

Flow cytometric analysis of hepatoma tissue and HeLa cells grown on various types of microbeads using hydroxyurea, nocodazole and aphidicolin in succession.

Applying Hydroxyurea, Nocodazole and Aphidicolin in succession to obtain parasynchronous growth, the progression of HTC and HeLa cells through the cell cycle has been monitored by laser flow cytometry. The experimental results show that HTC cells behave identically whether grown in monolayer or attached to dextran-based microbeads but that the chemical nature of the micro-support itself plays an important role especially on the speed with which the cells pass from mitosis into G1, polyacrylamide-based microbeads being superior in this respect.

Laser flow cytofluorometric analysis of HTC cells synchronized with hydroxyurea, nocodazole and aphidicolin.

The technique of laser flow cytofluorometry has been used to monitor the arrival in G1 and the subsequent progression through the cell cycle of HTC cells accumulated in metaphase with colcemid alone or after treatment with hydroxyurea and Nocodazole. Under the experimental conditions used in this study, the latter procedure gives much better results, avoiding in particular the extensive formation of micronucleated cells. Aphidicolin, an inhibitor of DNA polymerase, in combination with Nocodazole, provides a useful method to tightly synchronize these cells at the G1/S border.

The eucaryotic cell-lipochromosome as a new host-vector system in gene amplification and expression.

Although procaryotes such as E. Coli are generally considered to be ideal hosts, able to amplify eucaryotic gene sequences contained within hybrid DNA plasmid molecules (3, 14), recent experimental evidence such as the decreased bacterial viability observed during the cloning of mouse mitochondrial DNA, clearly shows the limitations of this type of approach (6). In addition, major technical difficulties are encountered during the isolation and purification of the specific messenger RNA (mRNA) needed to synthetize the complementary DNA (cDNA) molecule (1, 4). Last but not least, after having screened many different bacterial clones -- provided that the foreign gene product is not toxic for its host -- it is still a question of good luck to get one clone producing adequate quantities of the biologically active protein of interest, free of contaminants. On the other hand, the low but significant frequency with which eucaryotic cells exposed to fragmented DNA molecules or metaphasic chromosomes phenotypically express a particular marker suggests that this approach might offer an alternative to the bacteria-plasmid system used in "classic" genetic engineering (10, 17, 18, 19). It is the purpose of this work to briefly discuss some of the difficulties involved in this approach and to propose solutions.

Synchronization of HeLa cell cultures by inhibition of DNA polymerase alpha with aphidicolin.

Both the inhibitory effect of aphidicolin on the replicative alpha-polymerase and the reversibility of its action in vivo (Pedrali-Noy & Spadari, 1979, Biochem. Biophys. Res. Commun. 88, 1194-2002) allow the synchronization of cells in culture. Aphidicolin prevents G1 cells from entering the DNA synthetic period, blocks cells in "S" phase, allows G2, M and G1 cells to continue the cell cycle and to accumulate at the G1/S border. Aphidicolin is a more useful reagent than hydroxyurea and thymidine because it does not affect cell viability or "S" phase duration and does not interfere with the synthesis of dNTPs or DNA polymerases. In fact cells exposed to the drug continue to synthesize all three DNA polymerases alpha, beta and gamma as well as all dNTPs which, when the block is removed, are present at levels optimal for DNA initiation and replication. The technique is simple and can be applied to cells growing in suspension or monolayers and allows one to harvest large quantities of synchronized cells.

Study of messenger-like RNA extracted from HeLa cells polysomes. II. Identification of the types of RNA eluted from methylated albumin kieselguhr columns.

Together with the elution pattern of pure messenger RNA molecules of various origin, the labelling kinetics of rapidly labelled heterogeneously sedimenting RNA (HSRNA) extracted from polysomes of HeLa cells have been studied by chromatogrphy on columns made of methylated bovine serum albumin adsorbed on kieselguhr. HSRNA is eluted within three peaks-IP, Q2P and TDP-following in that order the increase of NaC1 concentration in the eluting buffer. Besides peak TDP which results from an experimental artefact, our data suggest that the appearance of peaks IP and Q2P reflects the absence and presence respectively of polyadenylic acid stretches in these molecules. Within peak Q2P, the critical factor affecting the order of elution is the size of the polyadenylic acid stretch.