Expression of viral early functions in rat 3Y1 cells infected with human papovavirus BK.

A plaque morphology mutant (pm-522) of BK virus (BKV) with a small deletion at map unit 0.72 can readily transform rat 3Y1 cells, but wild-type BKV (wt-501) cannot. We examined the expression of the viral early functions in BKV (wt-501 or pm-522)-infected 3Y1 cells within a 2-week period after infection, before foci of transformed cells became detectable, to know how the difference between the two BKVs occurs. After a high-multiplicity infection, comparable amounts of free viral DNA (forms I and II) were found by Southern blotting analyses to persist in the nuclei of the cells infected with wt and pm BKVs. Whereas the proportion of T antigen-positive cells, as revealed by the indirect immunofluorescence method with complement, remained at a level of 60% in pm BKV infection, the level of T antigen-positive cells in wt BKV infection decreased from the initial 45% to 1% on day 9. The results obtained by the immunoprecipitation analyses of radiolabeled proteins from the infected cells were consistent with the immunofluorescence data. Viral early mRNA was detectable on day 2 and increased on day 9 in pm BKV infection, but in wt BKV infection, the low level of early mRNA detected on day 2 disappeared on day 9. Cell DNA synthesis and cell growth were enhanced more in pm BKV infection than in wt BKV infection. The low level of viral DNA synthesis that occurred in the infected rat cells was more prominent in pm BKV infection than in wt BKV infection. These data indicate that the expression of viral early functions continued much longer in pm BKV-infected rat cells than in wt BKV-infected rat cells, where the expression was probably repressed soon after infection. Continued T antigen production directed by the unintegrated viral genomes appears to be required for efficient transformation of rat cells by BKV.     

Genetic basis of the neurovirulence of pseudorabies virus.

Lomniczi et al. (J. Virol. 49:970-979, 1984) have shown previously that two attenuated vaccine strains of pseudorabies virus have a similar deletion in the short unique (US) region of the genome. The region which is deleted normally codes for several translationally competent mRNAs. As expected, these mRNAs are not formed in the cells infected with the vaccine strains. The function specified by these mRNAs is thus not necessary for growth in cell culture. Using intracerebral inoculation of 1-day-old chicks as a test system, we have attempted to determine whether a gene within the region that is missing from the attenuated strains specifies functions that are required for the expression of virulence. An analysis of recombinants between the Bartha vaccine strain and a virulent pseudorabies virus strain (having or lacking a thymidine kinase gene [TK+ or TK-]) revealed the following. None of the recombinant plaque isolates that were either TK- or which had a deletion in the US was virulent. Not all recombinant plaque isolates which were both TK+ and had an intact US were virulent. These results indicate that both thymidine kinase activity and an intact US were necessary but not sufficient for the expression of virulence. Marker rescue experiments involving cotransfection of the Bartha strain DNA and a restriction fragment spanning the region of the genome that was missing from the Bartha strain resulted in the isolation of virions to which an intact US had been restored. These virions were not virulent but had an improved ability to replicate in the brains of chicks compared with that of the parental nonrescued Bartha strain. Our results show that genes in the US region, which are missing from the Bartha strain, were necessary for virulence but that this strain was also defective in other genes required for the expression of virulence. Thus, the virulence of pseudorabies virus, as measured by intracerebral inoculation into chicks, appears to be controlled multigenically.     

A variant in the restriction endonuclease cleavage pattern of mitochondrial DNA in the domestic fowl, Gallus gallus domesticus

The cleavage patterns of mitochondrial DNAs (mtDNAs) were investigated from 15 lines of domestic fowls, Gallus gallus domesticus, using 11 restriction endonucleases. The cleavage patterns with 10 restriction endonucleases were identical in all the lines. A variant was found in a line of White Leghorn in the pattern with MspI digestions. Cleavage patterns of the red jungle fowl, Gallus gallus gallus, were identical to the common patterns shown by the 14 lines of domestic fowls.     

Replication timing: histone genes replicate during early S phase in cleavage-stage embryos of sea urchin.

Newly synthesized DNA was separated from the bulk of the DNA by pulse-labeling with BUdR and centrifugation in an alkaline CsCl buoyant density gradient. The content of histone gene in the newly synthesized DNA was determined by DNA dot hybridization. The gene contents in DNA replicated during the early half of S phase and during the whole S phase were compared. Results showed that histone genes were replicated during the first half of the S phase in embryos in the early cleavage stage.     

Investigation of actin in Tetrahymena cells. A comparison with skeletal muscle actin by a devised two-dimensional gel electrophoresis method

Total protein constituents of Tetrahymena thermophila strain B1868 III were studied by two-dimensional agarose-polyacrylamide gel electrophoresis to detect actin among the constituents. In the attempts to prepare a whole-cell extract of Tetrahymena, it was found that protease activity in the extract was so high that high molecular components were quickly digested with the endogenous protease into small peptides unless the homogenization and heat-treatment in a sodium dodecylsulfate solution were performed within 5 s. It was eventually found that employment of 8 M guanidine hydrochloride (HCl) in the homogenization of cells perfectly prevented the degradation of protein components, even through a long preparation procedure. A devised two-dimensional agarose-polyacrylamide gel electrophoresis of the guanidine HCl extract gave a 'protein map' on which most proteins were located in their respective positions, including proteins with more than 200,000 mol. wt. Addition of rabbit skeletal muscle actin on the protein map revealed that no protein with isoelectric point and molecular weight identical with those of the actin was contained in the whole Tetrahymena extract, suggesting that Tetrahymena actin may have characteristics far different from those of skeletal muscle actin.     

Identification of Cytokinins in Root Exudate of the Rice Plant

Cytokinins, cis-zeatin and cis- and (trans-ribosylzeatin, wereidentified in the root exudate of the rice plant (Oryza sativa,indica cultivar IR-24) after several chromatographic separationsand combined gas-liquid chromatography-selected ion monitoring(GC-SIM) analysis. The presence of trans-zeatin ribotide wassuggested by enzyme hydrolysis, subsequent chromatographic separationand GC-SIM. The comparatively high content of the ribotide inthe root exudate suggests the form of cytokinins to be transportedfrom roots to other parts in the rice plant. (Received July 22, 1982; Accepted November 25, 1982)     

Statistical measurement of signal transmission in the central nervous system of the crayfish

Summary Transmission of nerve signals in the crayfish brain was studied by means of the transfer function derived from input-output analysis with random stimulation. The transfer function was measured in the form of frequency-response-function and represented by Bode plot. Two classes of the frequency-response-functions were discriminated, corresponding to two types of response patterns evoked by the constant frequency stimuli. The first type had the characteristics of a band pass filter similar to an underdamped resonant circuit. The second one had the characteristics of a phase lag circuit, occasionally, with additional small positive or negative peak at almost the same frequency as the resonance of the first type. A few possibilities for the resonance and the phase lag mechanisms were discussed.     

Superinfection with R Factors by Transduction in Escherichia coli and Salmonella typhimurium

Superinfection immunity is found in the conjugal transfer of R factors between two fi(+) R factors and between two fi(-) R factors (fi = fertility inhibition), as we reported previously. In contrast, no reduction in the frequencies of transduction of an fi(+) R factor 222 was caused by the presence of fi(+) R factors in the recipients in transduction systems with phage P1kc in Escherichia coli K-12 and with phage P22 in Salmonella typhimurium LT-2. The absence of superinfection immunity in transduction may be due to the difference in the route of entry of the R factor. The frequencies of transduction of an fi(+) R factor were reduced, although slightly, by the presence of fi(-) R factors in the recipients. This reduction is probably due to host-controlled restriction of the entering fi(+) R factor by the fi(-) R factors in the recipients, since transduction of an fi(+) R factor by the transducing phage propagated on the strain carrying both fi(+) and fi(-) R factors was not reduced by the presence of homologous fi(-) R factors in the recipients. The fi(+) R factor 222, when transduced to the recipient strains carrying other R factors, recombined genetically at high frequencies with these resident R factors, regardless of their fi type.