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H J Raskas

Publications and source records attributed to H J Raskas.

At least 55 records · Page 3Linked to original sources
PubMed1975

Sequence relationships between adenovirus 2 early RNA and viral RNA size classes synthesized at 18 hours after infection.

Synthesis of cytoplasmic viral RNA was studied during infection of cultured human (KB) cells with adenovirus 2. At 6 h, before viral DNA synthesis began 5% of the poly(A)-containing RNA hybridized to viral DNA; by 12 h and at later times more than 80% was virus specified. At 18 h after infection, four major size classes of cytoplasmic viral RNA were identified among the poly(A)-containing molecules. These size classes migrated as 27S, 24S, 19S, and 12 to 15S in polyacrylamide gels. The three larger size classes could also be identified in denaturing formamide gels. Hybridization of the 27S, 24S, and 19S viral RNAs was not inhibited by RNA harvested from cells at early times in infection. Therefore, these three major RNAs must code for late viral proteins. Hybridization of the 12 to 15S RNA was partially inhibited by RNA from cultures harvested at early times, suggesting that in this size class some of the RNA labeled at 18 h codes for early viral proteins.

Adenine Nucleotides↗
PubMed1975

Identification of early adenovirus type 2 RNA species transcribed from the left-hand end of the genome.

Unique fragments of adenovirus type 2 DNA generated by cleavage with endonuclease R-Eco RI or endonuclease R-Hsu I (Hin dIII) were used to map cytoplasmic viral RNAs transcribed early in productive infection. Radioactive early viral RNA was first fractionated by polyacrylamide gel electrophoresis. Eluted viral RNAs were then tested for hybrid formation with DNA fragments. The Eco RI DNA fragment (Eco RI-A) which contains the left-hand 58% of the genome hybridized 13S and 11S RNAs. More detailed mapping of these RNAs was achieved by hybridization to the seven Hsu I fragments of Eco RI-A. The early RNA annealed only to Hsu I-G and C, two fragments which comprise the extreme left-hand 17% of the genome. Viral RNA migrating as 13S and 11S annealed to Hsu I-G, and 13S RNA annealed to Hsu I-C. A 13S RNA is transcribed from Eco RI-A late in infection (18 h). Hybridization-inhibition studies with Eco RI-A DNA, early cytoplasmic RNA, and 3H-labeled 13S late RNA demonstrated that this RNA synthesized at late times is an early RNA species which continues to be synthesized in large amounts at 18 h. This 13S RNA synthesized at 18 h hybridized to Hsu I-C but not to Hsu I-G DNA. These results establish that the 13S RNAs transcribed from Hsu I-G and C at early times must be different species.

Adenoviridae↗
PubMed1975

Viral RNA synthesis and levels of DNA-dependent RNA polymerases during replication of adenovirus 2.

The rates of RNA synthesis in cultured human KB cells infected by adenovirus 2 were estimated by measuring the endogenous RNA polymerase activities in isolated nuclei. The fungal toxin alpha-amanitin was used to determine the relative and absolute levels of RNA polymerases I, II, and III in nuclei isolated during the course of infection. Whereas the level of endogenous RNA polymerase I activity in nuclei from infected cells remained constant relative to the level in nuclei from mock-infected cells, the endogenous RNA polymerase II and III activities each increased about 10-fold. These increases in endogenous RNA polymerase activities were accompanied by concomitant increases in the rates of synthesis in isolated nuclei of viral mRNA precursor, which was quantitated by electrophoretic analysis on polyacrylamide gels. The cellular RNA polymerase levels were measured with exogenous templates after solubilization and chromatographic resolution of the enzymes on DEAE-Sephadex, using procedures in which no losses of activity were apparent. In contrast to the endogenous RNA polymerase activities in isolated nuclei, the cellular levels of the solubilized class I, II, and III RNA polymerases remained constant throughout the course of the infection. Furthermore, no differences were detected in the chromatographic properties of the RNA polymerases obtained from infected or control mock-infected cells. These observations suggest that the increases in endogenous RNA polymerase activities in isolated nuclei are not due to variations in the cellular concentrations of the enzymes. Instead, it is likely that the increased endogenous enzyme activities result from either the large amounts of viral DNA template available as a consequence of viral replication of from replication or from functional modifications of the RNA polymerases or from a combination of these effects.

Adenoviridae↗
PubMed1974

Localization of adenovirus 2 messenger RNA's to segments of the viral genome defined by endonuclease R-R1.

Adenovirus 2 mRNAs synthesized in productive infection were assigned to specific regions of the genome by hybridization to unique fragments of viral DNA. Radioactive viral RNA synthesized early or late in infection was first fractionated by polyacrylamide gel electrophoresis. Eluted RNAs were then tested for complementary hybrid formation with each of the six fragments of adenovirus 2 DNA generated by cleavage with endonuclease R.R1. Early RNA species migrating as 13S, 19S, and 20S RNAs were identified as transcription products of fragments A, B, and D, respectively. In addition to the 13S RNA transcribed from A fragment DNA, 13S RNA also hybridized to the D and E fragment DNAs; 11S RNA annealed to both A and B fragments. The RNA that hybridized to fragment C DNA was heterogeneous in size. Viral RNA synthesized late in infection included 27S, 24S, 19S, and 11S size classes, all of which annealed to A fragment DNA. Additional RNA migrating as 24S hybridized to E and C fragment DNA, and 23S RNA annealed to F fragment DNA. The results of the hybridizations of size fractionated RNAs with viral DNA fragments enabled formation of a partial map of viral mRNAs with respect to the adenovirus 2 genome. Some of the viral RNAs may represent transcripts which overlap R1 cleavage sites, because in at least three instances hybridization revealed viral RNAs which have the same electrophoretic mobility and anneal to fragments that are contiguous on the genome.

Adenoviridae↗
PubMed1974

Role of DNA-dependent RNA polymerases II and III in transcription of the adenovirus genome late in productive infection.

DNA-dependent RNA polymerases I, II, and III were isolated and partially purified from KB (human) cells 18 hr after infection with adenovirus 2. As reported previously for the enzymes from other animal cells, RNA polymerase II was completely sensitive to low concentrations of alpha-amanitin (50% inhibition at 0.02 mug/ml), RNA polymerase III was completely sensitive to high concentrations of alpha-amanitin (50% inhibition at 20 mug/ml) and RNA polymerase I was totally resistant to concentrations of alpha-amanitin less than or equal to 200 mug/ml. RNA synthesis by the endogenous RNA polymerase activities in nuclei isolated from infected cells was completely sensitive to alpha-amanitin, thus suggesting that RNA polymerase I is not involved in viral DNA transcription even though it is present in these cells. The alpha-amanitin inhibition curve was biphasic and showed inflection points at about 0.02 and 20 mug/ml, suggesting the participation of both RNA polymerases II and III in the synthesis of RNA in these nuclei. Furthermore, at least a large fraction of the synthesis of the nuclear precursors to viral mRNA, monitored by hybridization to viral DNA, showed the same sensitivity to alpha-amanitin as did RNA polymerase II; and the synthesis of both viral 5.5S RNA and (presumably cellular) 5S RNA in the isolated nuclei exhibited the same sensitivity to alpha-amanitin as did purified RNA polymerase III. Thus, these data provide strong supporting evidence for previous studies which suggested the involvement of an RNA polymerase II in transcription of the adenovirus genome and demonstrate the role of an RNA polymerase III activity in the synthesis of viral 5.5S RNA and cellular 5S RNA.

Adenoviridae↗
PubMed1974

Effect of cycloheximide on RNA metabolism early in productive infection with adenovirus 2.

The presence of cycloheximide during the early phase of adenovirus 2 replication causes an increase in the virus-specific content of newly synthesized mRNA. The total cytoplasmic RNA from control cultures labeled 2 to 5 h after infection hybridized to viral DNA 0.8%, whereas RNA synthesized in the presence of cycloheximide annealed 6%. Cytosine arabinoside, an inhibitor of DNA synthesis, did not affect the percent hybridization to viral DNA. Oligo(dT)-cellulose chromatography was used to purify the portion of cytoplasmic RNA containing poly(A). The poly(A)-containing RNA from cultures labeled in the presence of cycloheximide hybridized to viral DNA 32% as compared to 2.2% for RNA from control cultures. Hybridization-inhibition experiments between RNAs from control- and cycloheximide-treated cultures demonstrated that the cultures treated with cycloheximide did not have an increased content of viral RNA or a new class of viral RNA sequences. Therefore, the increased hybridization appears to be caused by a reduction in synthesis of cellular cytoplasmic mRNA. Nucleoplasmic RNAs lacking and containing poly(A) were annealed to viral DNA. For both classes, RNA from cultures treated with cycloheximide hybridized 5- to 10-fold more than RNA from control-infected cultures. Therefore, the increased hybridization of cytoplasmic RNA synthesized in the presence of cycloheximide is caused either by reduced transcription of the cellular genome or by greatly increased instability of cellular heterogeneous nuclear RNA.

Adenoviridae↗
PubMed1974

Two classes of cytoplasmic viral RNA synthesized early in productive infection with adenovirus 2.

The RNA sequences and RNA size classes transcribed early in productive infection with adenovirus 2 were analyzed by RNA-DNA hybridization. Two independent procedures demonstrated that early cytoplasmic viral RNA is composed of two sequence classes, class I which is absent or present in greatly reduced quantities at 18 h, and class II which persists throughout the infection. When the sequences in early viral RNA were analyzed by hybridization-inhibition studies, the hybridization of early [(3)H]RNA was inhibited only 50% by RNA from cultures harvested late (18 h) in infection. Liquid hybridizations with radioactive viral DNA confirmed that early RNA includes two classes. Duplex formation of RNA with (32)P-labeled viral DNA was assayed by hydroxylapatite chromatography and resistance to S(1) nuclease digestion. Both methods showed that the cytoplasmic RNA present early in infection annealed 12 to 15% of the viral DNA; late cytoplasmic RNA hybridized 21 to 25% of the DNA. Mixtures of early plus late cytoplasmic RNAs hybridized 30 to 34% of the viral DNA, demonstrating the reduced concentration of early class I RNA in the late RNA preparations. Experiments were performed to correlate class I and class II early RNA with size-fractionated cytoplasmic RNA synthesized early in infection. Fractionation of RNA by gel electrophoresis or sucrose gradient centrifugation confirmed three major size classes, 12 to 15S, 19 to 20S, and 26S. Total cytoplasmic RNA and RNA selected on the basis of poly(A) content contained the same size classes of viral RNA. In standard electrophoresis conditions, the 19 to 20S viral RNA could be resolved into two size classes, and the distribution of 12 to 15S RNA also indicated the presence of more than one size component. Hybridization-inhibition studies under nonsaturating conditions were performed with 26S, 19 to 20S, and 12 to 15S viral RNAs fractionated by gel electrophoresis. Late RNA inhibited the hybridization of 26S RNA only 20%, 19 to 20S RNA was inhibited 45%, and 12 to 15S RNA was inhibited 50%. When 18 to 19S and 12 to 15S viral RNAs purified by sucrose gradient centrifugation were similarly analyzed, late RNA inhibited hybridization of 18 to 19S RNA 50%, and the annealing of 12 to 15S RNA was inhibited 70%.

Adenoviridae↗
PubMed1972

Processing of adenovirus RNA before release from isolated nuclei.

Nuclei isolated from cultured human cells (KB) infected with adenovirus type-2 were used to study events occurring during the adenosine triphosphate-dependent release of viral RNA. When cells were labeled with [(3)H]uridine for 50 min beginning 18 hr after infection, most nuclear viral messenger RNA was in the form of high molecular weight precursors with sedimentation coefficients greater than 28 S. When nuclei were incubated in the presence of ATP and an ATP-generating system, labeled RNA the size of viral mRNA (10-29 S) was released. Cleavage of viral RNA precursor molecules of high molecular weight occurred during incubation of nuclei. The change in size of viral RNA did not require exogenous ATP and occurred before release of RNA from nuclei. This RNA cleavage seems comparable to processing of mRNA in vivo, for discrete viral species were produced. Cleavage was completed after 10 min of incubation but was not the rate-limiting step in the release reaction, which required about 20 min for completion.

Adenosine Triphosphate↗
PubMed1972

Potentiation of the antifungal effects of antibiotics by amphotericin B.

Amphotericin B was found to potentiate the antifungal effects of mycophenolic acid glucuronide, tetracycline, and actinomycin D by increasing the penetration of these antibiotics through the fungal cytoplasmic membrane. Because the fungi were over 100 times more susceptible than animal cells to these effects of amphotericin B, these observations might have clinical application.

Amphotericin B↗
PubMed1972

Procedure for the preparation of milligram quantities of adenovirus messenger ribonucleic acid.

Late after adenovirus 2 infection (18 hr), nearly all newly synthesized polysomal messenger ribonucleic acid (mRNA) is viral specified. Large amounts of adenovirus mRNA have been purified by utilizing membrane filtration at high ionic strength. With this procedure, molecules that contain polyadenylic acid [poly (A)] tracts are bound selectively, and ribosomal RNA can be separated from the viral mRNA which contains poly(A). Polysomal RNA synthesized 18 hr after infection was labeled in the presence of 0.02 mug of actinomycin D per ml and extracted at pH 9.0. This RNA annealed 40% to 3 mug of adenovirus 2 deoxyribonucleic acid; the RNA selected by membrane filtration bound 80% under the same conditions. The RNA eluted from membrane filters was 80 to 90% greater than 18S and contained species migrating as 31, 27, and 24S. Binding of polysomal RNA to individual membrane filters was linear, using as much as 300 mug of RNA per membrane. A 1.1-mg amount of viral RNA was prepared from 17.7 mg of polysomal RNA that had been purified by extraction at pH 9.0.

Adenoviridae↗