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Biomedical subjects

G Acs

Publications and source records attributed to G Acs.

At least 109 records · Page 6Linked to original sources
PubMed1974

Shythesis of reovirus oligo adenylic acid in vivo and in vitro.

The formation of reovirus double-stranded (ds) RNA and of oligo adenylic acid (oligo A) is inhibited by 5 mug of actinomycin D per ml added at the time of viral infection. Viral proteins are synthesized and assembled into dsRNA-deficient particles under these conditions. The addition of cycloheximide to infected cells during the mid-logarithmic phase of viral replication terminates protein and dsRNA synthesis, but allows continued oligo A synthesis for about 1 h. The (3)H-labeled oligo A formed in the presence of cycloheximide is incorporated into particles whose density in CsCl is identical to that of reovirions. Using the large particulate or virus factory-containing cytoplasmic fraction of infected L-cells, we have established an in vitro system for the synthesis of oligo A. The in vitro product migrates slightly faster in sodium dodecyl sulfate acrylamide gels than marker oligo A. Oligo A synthesis in vitro continues for about 1 h, requires, the presence of only one ribonucleoside triphosphate (ATP), is not inhibited by DNase or RNase, but is abruptly terminated by the addition of chymotrypsin to the reaction mixture. Oligo A formed both in vivo and in vitro is released from the factory fraction by chymotrypsin digestion. The enzymes which catalyze the synthesis of oligo A, dsRNA, and single-stranded RNA all exhibit a similar temperature dependence with an optimum of approximately 45 C. These results indicate that oligo A is formed within the core of the nascent virion after the completion of dsRNA synthesis; they suggest that the oligo A polymerase is an alternative activity of the virion-bound transcriptase and that it is regulated by outer capsomere proteins.

Adenosine Monophosphate↗
PubMed1973

Protein initiation in eukaryotes: formation and function of a ternary complex composed of a partially purified ribosomal factor, methionyl transfer RNA, and guanosine triphosphate.

A protein factor contained in a 1 M KCl extract of L-cell ribosomes and partially purified by chromatography on DEAE-cellulose forms a specific ternary complex with rat-liver Met-tRNA(f) and GTP. The complex is measured by its quantitative retention on nitrocellulose membranes. Complex assembly is optimal at 100 mM KCl and 0.2 mM MgCl(2), and is independent of mRNA and of ribosomes. The GTP requirement can be replaced over 65% by its methylene analogue GDPCH(2)P, indicating that GTP hydrolysis is not involved. Complex formation is inhibited by 10 muM aurintricarboxylic acid, but is unaffected by 100 muM pactamycin, 100 muM fusidic acid, or by excess uncharged methionine tRNA(f). The ternary complex is relatively stable and appears at the void volume during filtration on Sephadex G-100. At 1-3 mM MgCl(2) and in the presence of other factors, the ternary complex is implicated in protein initiation by (i) its capacity to bind to the 40S ribosomal subunit to form a 48S complex; and (ii) the subsequent association of the 48S complex with a 60S subunit to form a functional "80S complex."

Adenosine Triphosphate↗
PubMed1972

Hydrocortisone induction of rat-liver leucyl-transfer RNA and its synthetases.

Within 3 hr after the intraperitoneal administration of hydrocortisone to female rats, a new leucine-accepting tRNA and a new leucyl-tRNA synthetase activity appear in the liver cytosol. The new isoaccepting tRNA can be acylated only with the synthetase derived from livers of hormone-treated animals. Both components are transient; by 12 hr after hydrocortisone administration, the isoaccepting tRNA and its synthetase disappear from livers of treated animals.

Amino Acyl-tRNA Synthetases↗
PubMed1972

Formation of a mammalian initiation complex with reovirus messenger RNA, methionyl-tRNA F , and ribosomal subunits.

Previous data demonstrated that reovirus mRNA, synthesized in vitro with the particulate RNA transcriptase of reovirus cores, efficiently directs the synthesis of polypeptides in vitro. The present studies indicate that all of the three size classes of reovirus mRNA produced in vitro can form protein initiation complexes with rat liver [(36)S]Met-tRNA(F) and incubated 40S and 60S ribosomal subunits, which had been washed in 0.5 M KCl of mouse fibroblast L-929 cells. Mild prior treatment of the mRNA with HCHO was required to expose the initiator region. The initiation complex reacted quantitatively with puromycin to form a puromycin peptide, whose electrophoretic properties were identical to methionyl-puromycin formed in response to poly(A,G,U) or the initiator codon AUG. The complex was relatively stable and specific for [(35)S]Met-tRNA(F); rat liver [(35)S]Met-tRNA(M) was unreactive unless the supernatant factors EF T(1) and EF T(2) were also present. However, the addition of fusidic acid, at a concentration that did not affect complex formation with [(35)S]Met-tRNA(F), completely inhibited Met-tRNA(M) utilization. Exogenous ribosomal factors and GTP were not required unless the separated 40S and 60S subunits were further treated with 1 M KCl. The data suggest that reovirus mRNA contains AUG initiator codons that form a complex with Met-tRNA(F) at a puromycin-reactive site on ribosomes.

Animals↗
PubMed1971

Asynchronous synthesis of the complementary strands of the reovirus genome.

The mechanism of replication of the double-stranded RNA genome of reovirus has been analyzed by tracing the fate of the parental double-stranded RNA genome and by determining whether the complementary strands, which comprise the progeny double-stranded RNA, are synthesized simultaneously or sequentially. The results indicate that the parental double-stranded RNA is conserved as the original duplex molecule within a subviral particle throughout the viral replicative cycle. The complementary strands, which form the progeny double-stranded RNA, are produced asynchronously. Minus strands are synthesized on preformed plus-strand templates, whereas plus strands appear to be synthesized on double-stranded RNA templates.

Centrifugation↗
PubMed1971

Mechanism of reovirus double-stranded ribonucleic acid synthesis in vivo and in vitro.

The complementary strands of reovirus double-stranded ribonucleic acid (ds RNA) are synthesized sequentially in vivo and in vitro. In both cases, preformed plus strands serve as templates for the synthesis of the complementary minus strands. The in vitro synthesis of dsRNA is catalyzed by a large particulate fraction from reovirus-infected cells. Treatment of this fraction with chymotrypsin or with detergents which solubilize cellular membranes does not alter its capacity to synthesize dsRNA. The enzyme or enzymes responsible for dsRNA synthesis remain sedimentable at 10,000 x g after these enzyme or detergent treatments, indicating their particulate nature. Pretreatment of this fraction with ribonuclease, however, abolishes its ability to catalyze dsRNA synthesis, emphasizing the single-stranded nature of the template and its location in a structure permeable to ribonuclease. In contrast, the newly formed dsRNA is resistant to ribonuclease digestion at low salt concentrations and hence is thought to reside within a ribonuclease-impermeable structure.

Animals↗
PubMed1970

Properties of RNA transcriptase in reovirus subviral particles.

Subviral particles containing reovirus RNA transcriptase have been isolated from extracts of virus-infected mouse fibroblast cells. The purified particles which lacked the outer protein capsomeres of the mature virion had a buoyant density of 1.43-1.44 g/ml in CsCl and contained all of the double-stranded RNA genome of the intact virus. The particles were free of nuclease activity. RNA synthesis required all four ribonucleoside triphosphates and was dependent on magnesium or manganese; optimal activity required potassium or ammonium ions. In the presence of a ribonucleoside triphosphate regenerating system, reaction rates were linear for 20 hr. RNA yields of 40-fold in excess of input template could be obtained. Completed RNA chains were released from the subviral particles. In the course of RNA synthesis, the double-stranded RNA template was fully conserved. The RNA products formed in vitro displayed profiles in sucrose gradients similar to those found for in vitro reovirus mRNA. The RNA products were single-stranded and did not self-anneal. Over 90 percent of the transcriptase products could be annealed with template double-stranded RNA. The annealed products migrated in acrylamide gels as double-stranded RNA, indicating efficient in vitro transcription.

Centrifugation, Density Gradient↗
PubMed1970

The reovirus replicative cycle: conservation of parental RNA and protein.

The fate of parental reovirions in the viral replicative cycle has been analyzed using CsCl density centrifugation. After penetration of L-cells, reovirus is converted from a particle of density 1.39 g/cm(3) to a subviral particle of density 1.41 g/cm(3). This alteration in density is temporally correlated with the hydrolysis of viral coat proteins and is qualitatively similar when particles are labeled in their RNA or protein. Ten hours after infection, when synthesis of progeny virus is underway, the parental RNA and protein are again found at density 1.39 g/cm(3). These data demonstrate conservation of the parental RNA and protein in the subviral particle throughout the replicative cycle.

Centrifugation, Density Gradient↗