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At least 451 records · Page 25Linked to original sources
PubMed1979

High molecular weight virus DNA in KB cells infected with ts mutants of adenovirus type 2 under permissive and non-permissive conditions.

Temperature-sensitive (ts) mutants of adenovirus type 2 (Ad2), which are deficient in virus DNA synthesis at the non-permissive temperature, have been used to investigate whether virus DNA replication is required for the occurrence of high mol. wt. Ad2 DNA (greater than 100S, 50 to 90S) in human cells productively infected with Ad2. The high mol. wt. virus DNA has been previously shown to consist of virus and cellular DNA molecules covalently linked. The present data indicate that after infection with DNA-ts mutants, the production of high mol.wt. virus DNA is much less sensitive to restrictive conditions than the synthesis of unit length (34S) Ad2 DNA. This finding lends further support to the idea that the occurrence of high mol. wt. virus DNA is independent of the synthesis of unit length virus DNA.

Adenoviruses, Human↗
PubMed1975

The transcriptional role of host DNA-dependent RNA polymerases in adenovirus-infected KB cells.

From the present experiments, we can conclude that late in infection two classes of DNA-dependent RNA polymerase transcribe the viral genome. Although we cannot definitively exclude the possibility that one or both of these enzymes are coded for by the virus, it appears more probable that these are hos enzymes, because of the coincidence of the alpha-amanitin sensitivities for synthesis of specific viral RNAs with the toxin sensitivities of the respective host RNA polymerases. Furthermore, the chromatographic properties of the enzymes, the alpha-amanitin sensitivities of the purified RNA polymerases and the levels of solubilized activities are the same for uninfected and late infected cells. It seems probable that some virus-coded or virus-induced factor(s) modifies either the selectivity or the activity of these host RNA polymerases. As an example, the "inactivation" of RNA polymerase I activity in vivo or the increased endogenous activity of isolated infected cell nuclei, without changes in the solubilized level of the respective RNA polymerases, could be mediated by such factor(s). The effect of such factors may not be detected by activity measurements on exogenous DNA, because it acts as a nonspecific template. Analysis of such factors will require reconstitution of appropriate in vitro systems, which retain some transcriptional specificity. Since several viral mRNAs synthesized at late times (Tal et al. 1974) and the 5.5S RNA (J. Pan, pers, comm.) are transcribed from the same region (R-R1 restriction enzyme fragment A), initiation and termination signals for both RNA polymerase II and III are contained in this portion of the genome. Further studies of the interaction of these two enzymes with the adenovirus 2 genome should contribute to understanding the control of transcription in eukaryotic cells, in particular in the case of virus-infected or -transformed cells.

Adenoviridae↗
PubMed1976

Susceptibility of Haemophilus influenza to ampicillin as determined by use of a modified, one-minute beta-lactamase test.

Fifty-seven cultures of Haemophilus influenzae were tested for production of beta-lactamase by use of a modified, 1-min test. Only the 24 cultures that were ampicillin resistant, whether type b or not, yielded positive results for the enzyme; all ampicillin-susceptible isolates were uniformly negative. The beta-lactamase procedure is simple to perform and requires only commonly available reagents.

Ampicillin↗
PubMed1976

Viral transcription in KB cells infected by temperature-sensitive "early" mutants of adenovirus type 5.

RNA:DNA hybridization was used to study the synthesis of viral RNA in two DNA-minus, temperature-sensitive mutants of type 5 adenovirus (H5ts125 and H5ts149) belonging to two different, non-overlapping complementation groups. Hybridization competition analysis showed that both mutants transcribed all early gene sequences at the restrictive temperature (41 C). In mutant-infected cells at 41 C, the rate of viral transcription was similar to the rate of early RNA synthesis in wild-type virus infection and was dependent on the multiplicity of infection; little or no late transcription was detected. The shutoff of class I early RNA transcription was shown to be a late function during wild-type virus infection and did not occur at 41 C in mutant-infected cells. When mutant-infected cells were incubated at the permissive temperature (32 C) for 25 h and then shifted to 41 C, the rate of viral DNA synthesis decreased rapidly for H5ts125 and slowly for H5ts149. However, the rate of viral transcription remained unchanged in H5ts125-infected cells for at least 3 h after the temperature shift; although the synthesis of viral DNA had stopped by this time, the synthesis of late viral RNA sequences continued.

Adenoviridae↗
PubMed1976

Adenovirus type 12-specific RNA sequences during productive infection of KB cells.

The complementary strands of adenovirus type 12 DNA were separated, and virus-specific RNA was analyzed by saturation hybridization in solution. Late during infection whole cell RNA hybridized to 75% of the light (1) strand and 15% of the heavy (H) strand, whereas cytoplasmic RNA hybridized to 65% of the 1 strand and 15% of the h strand. Late nuclear RNA hybridized to about 90% of the 1 strand and at least 36% of the h strand. Double-stranded RNA was isolated from infected cells late after infection, which annealed to greater than 30% of each of the two complementary DNA strands. Early whole cell RNA hybridized to 45 to 50% of the 1 strand and 15% of the h strand, whereas early cytoplasmic RNA hybridized to about 15% of each of the complementary strands. All early cytoplasmic sequences were present in the cytoplasm at late times.

Adenoviridae↗
PubMed1979

DNase induced after infection of KB cells by herpes simplex virus type 1 or type 2. II. Characterization of an associated endonuclease activity.

Purified preparations of the "exonuclease" specified by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) possess an endonuclease activity. The exonuclease and endonuclease activities copurify and cosediment in a sucrose density gradient. Endonuclease activity is only observed in the presence of a divalent cation, and Mg(2+) or Mn(2+) is equally effective as a cofactor with an optimal concentration of 2 mM. A slight amount of endonuclease activity is observed in the presence of Ca(2+), whereas no activity occurs in the presence of Zn(2+). In the presence of Mg(2+), Ca(2+) and Zn(2+) are inhibitory. Comparison of exonuclease and endonuclease activity in the presence of various divalent cations revealed that, at concentrations of Mn(2+) greater than 1 mM, only endonuclease activity occurs whereas endonuclease and exonuclease activity occur at all concentrations of Mg(2+). The endonuclease was affected by putrescine and spermidine to the same extent as the exonuclease activity, but in marked contrast the endonuclease was inhibited by a 10-fold-lower concentration of spermine compared to the exonuclease. The activity specified by HSV-1 and HSV-2 has very similar properties. HSV-1 and HSV-2 endonuclease cleave covalently closed circular DNA to yield, firstly, nicked circles and then linear DNA which is subsequently hydrolyzed to small oligonucleotides. Cleavage does not appear to be base sequence specific. Conversion of nicked circles to linear DNA and subsequent degradation of linear DNA occurs more rapidly in the presence of Mg(2+) than Mn(2+) presumably by virtue of the presence of the exonuclease activity. Nonsuperhelical covalently closed circular duplex DNA is cleaved by the endonucleases at a rate 60 times slower than the rate observed on the supercoiled form. These data indicate that the HSV-1 and HSV-2 endonuclease preferentially recognize single-stranded DNA regions.

Cations, Divalent↗
PubMed1971

Synthesis of virus-specific ribonucleic acid in KB cells infected with type 2 adenovirus.

By using the technique of deoxyribonucleic acid (DNA)-ribonucleic acid (RNA) hybridization, virus-specific RNA (cRNA) was detected 6 hr after infection in preparations of total RNA from cells infected with type 2 adenovirus in the presence of 2 mum 5-fluorodeoxyuridine. In the absence of 5-fluorodeoxyuridine, there was a continuous increase in the incorporation of (3)H-uridine into viral cRNA until 20 hr after infection, at which time approximately 40% of the (3)H-uridine entering RNA was found in virus-specific RNA. When RNA was prepared from polyribosome fractions obtained from cytoplasmic extracts of infected cells, virus-directed transcription was detected at 3 hr after infection (i.e., 3 to 4 hr before the initiation of viral DNA synthesis). Viral cRNA species synthesized at different times after infection were compared by the technique of DNA-RNA hybridization-inhibition ("presaturation" hybridization-competition). Three hybridization-inhibition techniques were compared. The techniques differed in the manner in which the DNA-RNA complex was isolated after the first hybridization reaction. Depending on the procedure employed, various degrees of inhibition were measured. The variation could be essentially eliminated if prior to hybridization the inhibitory RNA species were alkali-degraded to a uniform size of about 4S. Undegraded RNA could be used if the DNA-RNA complex was isolated by using a procedure involving rigorous washing (preferably including ribonuclease treatment) before the second hybridization with labeled RNA. When a rigorous hybridization-inhibition procedure was used, three classes of virus-specific RNA species could be distinguished: (i) early RNA class I whose synthesis began prior to viral DNA replication and stopped at some time after the initiation of viral DNA replication-it comprised about 70% of the early RNA species and was apparently degraded by 18 hr after infection; (ii) early RNA class II whose synthesis began prior to viral DNA replication and apparently continued at an enhanced rate late in infection; and (iii) late RNA whose synthesis began after the initiation of viral DNA synthesis.

Adenoviridae↗