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H J Raskas

Publications and source records attributed to H J Raskas.

At least 37 records · Page 2Linked to original sources
PubMed1979

In vitro translation products specified by the transforming region of adenovirus type 2.

Region 1 DNA sequences (map positions 0 to 11% on the linear adenovirus 2 genome) are expressed both early and late in lytic infection and are required for transformation by the virus. During productive infection six distinct cytoplasmic RNAs are synthesized from this region. These RNAs comprise two families, each consisting of three size classes that share 3' sequences. Region 1 RNA's were purified by hybridization selection, using restriction fragments bound to nitrocellulose membranes, and by size fractionation. The isolated RNAs were then translated in cell-free systems derived from wheat germ and rabbit reticulocytes. The family of RNAs specified by 0 to 4.4 sequences includes two RNAs, which are 12S and 13S in size. These RNAs were partially separated by molecular weight and translated. The 13S RNA produced 53,000-dalton (53K) and 41K peptides, and the 12S RNA synthesized 47K and 35K products. The family of RNAs mapping from 4.4 to 11.0 encodes three separate polypeptides, each of which can be assigned to a specific RNA. A 12K product that comigrates with structural polypeptide IX is synthesized from the 9S RNA as previously reported (U. Pettersson and M. B. Mathews, Cell 12:741-750, 1977). The 13S RNA encodes a 15K polypeptide that corresponds to a 15K polypeptide in infected cell extracts. The 22s RNA encodes a 52K protein distinct from the 0 to 4.4 polypeptides.

Adenoviruses, Human↗
PubMed1978

Two regions of the adenovirus 2 genome specify families of late polysomal RNAs containing common sequences.

Late cytoplasmic RNAs specified by two regions of the adenovirus 2 genome (39.3--51.8 and 70.7--83.4 map units) were analyzed by size fractionation of poly(A)-[3H]RNA and subsequent hybridization to DNA fragments. Both regions encode RNAs whose sequence content exceeds the coding capacity of the region. These multiple transcripts are likely to function as mRNAs, because they are present on polyribosomes. The DNA segment 39.3--51.8 specifies 27S, 22S, and 18S RNAs. The genome sites specifying these three size classes were determined by hybridizations with seven different DNA fragments from this region of the genome. The 3' termini of all three size classes are specified by sequences near a common site, position 50.1. The 27S RNA includes sequences beginning near 39.3, the 22S RNA contains sequences from 41.0, and the 18S RNA includes sequences from approximately 45.3 on the unit genome. A second family of four RNAs is transcribed from 70.7--83.4. These 28S, 22S, 18S, and 16S RNAs have a relationship similar to the RNAs transcribed from 39.3--51.8. Sequences near the 5' ends of these four size classes are specified by different genome sites. However, the 3' termini of all four size classes were localized near map position 80.4. The synthesis of families of RNA may allow the translation of multiple polypeptides from a genome segment that has only one terminator site for mRNA synthesis.

Adenoviruses, Human↗
PubMed1978

Effect of protein synthesis inhibitors on viral mRNA's synthesized early in adenovirus type 2 infection.

Viral mRNA species synthesized early in adenovirus type 2 infection in the presence of cycloheximide were compared with those synthesized in the absence of drug or in the presence of the DNA synthesis inhibitor 1-beta-D-arabinofuranosylcytosine. Cycloheximide caused approximately a 10-fold stimulation in the accumulation of [3H]uridine into early viral mRNA species. The only exception was a 24s mRNA transcribed from the transforming end of the genome; in the presence of cycloheximide, accumulation of this mRNA species was stimulated no more than 2-fold. Treatment with cycloheximide also resulted in the accumulation of polyadenylated RNAs transcribed from EcoRI-C that are heterogeneous and smaller than the 20S mRNA. Other translation inhibitors were shown to have similar effects, suggesting that inhibition of protein synthesis early after infection induces alterations in the metabolism of specific RNA sequences.

Adenoviruses, Human↗
PubMed1977

Species identification and genome mapping of cytoplasmic adenovirus type 2 RNAs synthesized late in infection.

Adenovirus type 2 cytoplasmic RNAs synthesized late in productive infection were resolved by electrophoresis on formamide gels. Regions of the adenovirus 2 genome specifying RNAs of distinct size were determined by hybridization to specific DNA fragments generated by cleavage with endo R.EcoRI and endo R.SmaI. From these studies 13 distinct viral RNA species were identified. A 26S to 28S size class and a 21S to 23S size class were each found to consist of four distinct RNA species. Three RNA species were identified in a 16S to 18S size class, and a fourth size class, 11S to 13S, was resolved into two components. The SmaI-D region (0.38 to 0.51 on the unit genome) and the EcoRI-F, D region (0.70 to 0.83) of the genome were found to code for multiple transcripts. Three RNAs (28S, 22S, and 18S) are specified by SmaI-D, and four components, 28S, 22S, 18S, and 16S, are encoded by EcoRI-F,D. The RNA represented by each set of multiple transcripts exceeds the coding capacity of the respective region, and the species within each set of RNAs appear to contain common sequences. The relationship between the cytoplasmic RNA species synthesized at late times and early cytoplasmic RNAs was determined by hybridization-inhibition experiments. The multiple transcripts encoded by the EcoRI-D fragment were found to contain sequences that are present in early cytoplasmic RNA. These studies enabled preparation of a map which accounts for transcription of approximately 67% of the r strand of the adenovirus 2 genome.

Adenoviruses, Human↗
PubMed1976

Nuclear transcripts larger than the cytoplasmic mRNAs are specified by segments of the adenovirus genome coding for early functions.

Nuclear viral RNAs synthesized early in productive infection with adenovirus 2 were analyzed by hybridization to specific viral DNA fragments. Radioactive RNAs were subjected to electrophoresis in polyacrylamide gels containing 98% formamide, and the fractionated RNA was hybridized to specific DNA fragments generated by cleavage with endo R-Eco Ri or endo R-Sma l. The viral genes expressed early in infection are located in four different segments of the genome. When nuclear RNA was hybridized to DNA fragments representing each of these segments, discrete RNA size classes were detected. For each of these four regions of the genome, some of the discrete nuclear RNAs were larger than the cytoplasmic mRNAs. As calculated from electrophoretic mobility, the molecular weight of these nuclear RNA size classes ranged from 15% to several fold greater than that of the corresponding cytoplasmic mRNAs. Hybridization-inhibition experiments were performed to analyze the sequences present in one of these larger nuclear transcripts, a 22S RNA transcribed from the left-hand end of the genome. This 22S nuclear RNA was transcribed from the same strand as the viral mRNA, for it contained cytoplasmic sequences; it also contained sequences restricted to the nucleus. The larger nuclear RNAs may serve as precursors to the cytoplasmic viral mRNAs synthesized early in infection.

Adenoviridae↗
PubMed1976

Low molecular weight viral RNAs transcribed by RNA polymerase III during adenovirus 2 infection.

Nuclei isolated from human cells productively infected with adenovirus 2 have been shown to synthesize four low molecular weight RNA species which hybridize efficiently to viral DNA. One species corresponds to the 5.5S or VA RNA (Ohe, Weissman, and Cooke, 1969), and is designated V156. The other three species are novel and have been designated V200, V140, V130, since they are approximately 200, 140, and 130 nucleotides in length, respectively. These viral RNAs retain their distinct electrophoretic properties after denaturation with formamide. RNA species with electrophoretic mobilities similar to those of the V200, V156, and V140 RNAs have been found in the cytoplasmic fraction of cells at late times after adenovirus infection. In isolated nuclei, the V200, V156, V140, and V130 RNAs are all synthesized by DNA-dependent RNA polymerase III, since synthesis is sensitive to high but not to low concentrations of alpha-amanitin. The synthesis of these low molecular weight RNAs continues for a prolonged period of time in isolated nuclei, suggesting that reinitiation occurs. Adenovirus 2 DNA fragments obtained by digestion with restriction endonucleases Eco RI and Sma I were used to map the location of the DNA sequences which encode the RNAs. All the low molecular weight RNAs hybridized to a region of the genome between o.18 and 0.38 fractional lengths from the left end of the adenovirus genome, suggesting that the respective DNA sequences are clustered. Other nonviral low molecular weight RNAs are synthesized in nuclei isolated from infected cells. These include the cellular 5S rRNA species which was minitored by its hybridization to purified 5S DNA from Xenopus laevis.

Adenoviridae↗
PubMed1975

Temperature-sensitive cell mutations that inhibit adenovirus 2 replication.

Five temperature-sensitive growth mutants of the hamster cell line BHK-21 were tested for the ability to support adenovirus 2 multiplication at 39 degrees and 33 degrees. Wild-type BHK-21 and mutants ts 422E and ts BCH yielded comparable amounts of virus at 33 degrees and 39 degrees, whereas in three other mutants, ts T22, ts T23, and ts AF8, virus production at 39 degrees was reduced to about 1% of that at 33 degrees. Virus yield in the three mutants was not reduced because of a delay in virus production; for all cells tested maximal virus yield at 39 degrees was obtained by 40-50 hr after infection. Normal yields of infectious virus were not obtained from ts AF8 even with a very high multiplicity of infection. In contrast, the virus yield from ts T22 and ts T23 was multiplicity-dependent. Shiftup experiments demonstrated that in ts AF8, a cell cycle mutant which at 39 degrees becomes arrested in G1, virus multiplication was thermosensitive for the first 40 hr of infection. In ts T22 and ts T23, the thermosensitivity was only for the first 3-4 hr of the infection. In all three mutants viral DNA synthesis was reduced by at least 95% at the higher temperature. The cell function specified by the ts AF8 mutation seems to be required for the early period of adenovirus 2 replication, after virus entry into the cell but before the onset of viral DNA replication.

Adenoviridae↗
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↗