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The way in which herpes virus of a well adapted strain penetrates susceptible HeLa cells has been investigated using thin sectioning techniques for electron microscopy. Mature virus particles and cells were mixed together in suspension cultures for 15, 30, 60, or 120 minutes so that the stages in virus uptake could be followed in sequence. The ingestion of particles of colloidal gold by HeLa cells under similar conditions was studied for comparison in parallel experiments. After 15 minutes' contact, the mature virus was found adsorbed on the surface of the cells but separated from them by a narrow gap in which phosphotungstic acid staining was sometimes able to reveal an extraneous coat which appeared as an amorphous layer on the outer aspect of the plasma membrane. When mixing continued for longer the particles were present in deep invaginations or actual cytoplasmic vacuoles, with their outer layers in various stages of stripping and digestion. The stripped, naked, central portion of the virus was occasionally found in these vacuoles but was more commonly free in the cytoplasmic matrix; the mode of transition between these sites could not be determined. Where contact continued for 2 hours these phenomena were much less frequently observed. The larger particles of colloidal gold were ingested in the same way as the virus, but smaller ones were taken up in micropinocytosis vesicles. The gold passed through membrane-bounded cytoplasmic spaces to accumulate in vacuoles from which, in contrast to herpes particles, it did not escape. These findings are discussed, and considered with particular reference to their bearing on the initiation of infection, the uptake and disposal of particles by cells, and the influence on the latter of virus morphology.

Agrobacterium rhizogenes induces root formation at the wound site of inoculation in plants and inserts a fragment of its plasmid (Ri) into the plant nuclear DNA. Parts of the transferred region (T-region) of the Ri plasmid of A. rhizogenes strain A4 or 8196 are cloned in Escherichia coli. Insertions of the E. coli lacZ coding region into the hybrid plasmids were made in vivo using transduction by miniMu. Twenty insertions localized in the TL-DNA of pRiA4 (or pRi1855) and 2 inserts in the T-DNA of pRi8196 were obtained in E. coli. One of the TL-DNA insertions is saved up because it is linked to an internal T-DNA deletion; the others because they confer a lactose plus phenotype on E. coli; this indicates that the T-DNA harbours sequences that are expressed in E. coli. Fifteen of these T-DNA insertions were transfered to Agrobacterium where they substitute the corresponding wild-type T-DNA of the Ri plasmid by homologous recombination. These strains corresponding to insertion-directed mutagenesis were used to inoculate Daucus carota slices and stems and leaves of Kalanchoe daigremontiana. The two insertions strains obtained in the T-DNA of pRi8196 are avirulent on K. daigremontiana; but their phenotypes differ on D. carota slices, suggesting that insertions affect distinct loci on the T-DNA involved in hairy root formation. Only one insertion out of the twenty obtained in the TL-DNA of pRiA4 (or 1855) induces a loss of virulence on leaves of K. daigremontiana. However the TL-DNA deletion harbouring strain induces a loss of virulence on D. carota and K. daigremontiana (stems and leaves), confirming the importance of the TL-DNA for hairy root induction. re]19850711 rv]19851230 ac]19860114.

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