Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Shythesis of reovirus oligo adenylic acid in vivo and in vitro. 485 7

The relationship between the in vitro phosphorylation of vesicular stomatitis virus (VSV) proteins and virion uncoating was examined. Activation of the VSV virion kinase with low concentrations of melittin, the active peptide component of bee venom, in the presence of gamma-[32P] ATP resulted in the phosphorylation of virion proteins. Following the in vitro phosphorylation of VSV proteins in the presence of melittin and deoxyadenosine triphosphate, the virion envelope was disrupted based on the accessibility of the internal ribonucleoprotein core (RNP) to the heavy metal stain, uranyl acetate, as determined by electron microscopic observation. The RNP structure was not observed in unphosphorylated virions treated with melittin and uranyl acetate. Phosphorylated virions treated with uranyl acetate subsequently lost the capacity for transcription whereas unphosphorylated virions treated with the stain retained transcriptase activity. These observations suggest that phosphorylation of VSV proteins may contribute to virion uncoating by disrupting the virus envelope.
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PMID:Phosphorylation of vesicular stomatitis virus proteins as a possible contributing factor in virion uncoating. 617 11

Soluble transcriptase containing the L and the NS proteins was isolated from purified vesicular stomatitis virus and its binding with the template ribonucleoprotein containing the N protein-RNA complex was studied with respect to its ability to initiate and synthesize RNA in vitro. By using u.v.-irradiated template reconstituted with soluble transcriptase, it was shown that the synthesis of leader RNA and other small initiated mRNA sequences continued while full-length mRNA synthesis decreased by 90%. In the presence of ATP and CTP, the reconstituted complex synthesized polyphosphorylated oligonucleotides which include AC, AAC and AACA which represent 5'-terminal sequences transcribed from the leader template and genes coding for mRNAs. In the presence of arabinosyl ATP, an inhibitor of RNA synthesis in vitro, the synthesis of leader RNA was found to be inhibited considerably more than other small initiated mRNA sequences. Reconstitution of RNA synthesis with soluble transcriptase and template in the presence of viral matrix (M) protein at low ionic condition resulted in virtual cessation of leader RNA synthesis, although the synthesis of small initiated N mRNA, 11 to 14 bases, continued. These results suggest that transcriptase can bind at multiple sites on the genome template and initiate RNA chains.
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PMID:Interaction of L and NS proteins of vesicular stomatitis virus with its template ribonucleoprotein during RNA synthesis in vitro. 619 59

The basophilic leucaemia cell line RBL-2H3 exhibits a robust inwardly rectifying potassium current, IKIR, which is likely to be modulated by G proteins. We examined the physiological and molecular properties of this KIR conductance to define the nature of the underlying channel species. The macroscopic conductance revealed characteristics typical of classical K+ inward rectifiers of the IRK type. Channel gating was rapid, first order (tau approximately 1 ms at -100 mV) and steeply voltage dependent. Both activation potential and slope conductance were dependent on extracellular K+ concentration ([K+]o) and inward rectification persisted in the absence of internal Mg2+. The current was susceptible to a concentration- and voltage-dependent block by extracellular Na+, Cs+ and Ba2+. Initial IKIR whole-cell amplitudes as well as current rundown were dependent on the presence of 1 mM internal ATP. Perfusion of intracellular guanosine 5'-Q-(3-thiotriphosphate) (GTP[gamma S]) suppressed IKIR with an average half-time of decline of approximately 400 s. It was demonstrated that the dominant IRK-type 25 pS conductance channel was indeed suppressed by 100 microM preloaded GTP[gamma S]. Reverse transcriptase-polymerase chain reactions (RT-PCR) with RBL cell poly(A)+ RNA identified a full length K+ inward rectifier with 94% base pair homology to the recently cloned mouse IRK1 channel. It is concluded that RBL cells express a classical voltage-dependent IRK-type K+ inward rectifier RBL-IRK1 which is negatively controlled by G proteins.
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PMID:Physiological and molecular characterization of an IRK-type inward rectifier K+ channel in a tumour mast cell line. 760 35

To estimate the polyamine distribution in Escherichia coli, the binding constants (K) for DNA, RNA, phospholipids, and ATP were calculated under the condition of 10 mM Tris-HCl, pH 7.5, 150 mM K+, and 10 mM Mg2+. The binding constants of spermidine for E. coli DNA, E. coli 16S rRNA, phospholipids in E. coli membrane, and ATP were 0.015, 0.066, 0.028, and 0.081 mM-1, respectively. Similarly, those of putrescine were 0.010, 0.010, 0.007, and 0.037 mM-1, respectively. The concentrations of putrescine, spermidine, and ATP and phosphates in DNA, RNA, and phospholipids in E. coli harvested at A600 = 0.3 were 32.2, 6.88, and 2.66 and 96.4, 436, and 57.2 mM, respectively. Accordingly, the percentage of spermidine bound to DNA, RNA, phospholipids, and ATP and that of free spermidine were 5.1, 90, 0.7, 0.8, and 3.8%, respectively. The percentage of putrescine bound to DNA, RNA, phospholipids, and ATP and that of free putrescine were 9.3, 48, 1.4, 2.6, and 39%, respectively. The results indicate that most spermidine exists as a spermidine--RNA complex, and about 40% and 50% of putrescine exists as a free form and a putrescine--RNA complex in cells, respectively. Under the conditions that the synthesis of specific proteins such as RNA replicase is stimulated by polyamines in a cell-free system, the amount of spermidine and putrescine bound to RNA was close to the value estimated in cells. Experiments to demonstrate the polyamine stimulation of MS2 RNA-directed RNA replicase synthesis in vivo were thus performed, and the results were confirmed.
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PMID:Estimation of polyamine distribution and polyamine stimulation of protein synthesis in Escherichia coli. 767 29

The 16.7 kbp dsRNA specific to the '447' cytoplasmic male sterility (CMS) line of Vicia faba was labelled in vitro with [alpha-32P]ATP and poly(A) polymerase, and by T4 RNA ligase-mediated addition of [32P]pCp. Analysis of the reaction products under denaturing conditions revealed in both cases extensive labelling of a 4.5 kb ssRNA, already detected in previous experiments in which the RNA-dependent RNA polymerase associated with the dsRNA was allowed to pursue RNA synthesis on preinitiated complexes. Mobility shift analysis of total pCp-labelled dsRNA revealed not two but three different 3' termini. The most prominent sequencing pattern corresponded to the 4.5 kb ssRNA, indicating that this RNA species has a preferentially accessible, free 3' OH extremity. Northern blot analysis of the denatured dsRNA confirmed that the 4.5 kb ssRNA is a subgenomic mRNA and detected its counterpart of about 12 kb. Nearly all 16.7 kbp dsRNA molecules featured an interrupted positive-sense strand, indicating a marked prevalence of transcription over replication complexes. This unusual strategy of transcription by a strand displacement mechanism, following initiation at an internal discontinuity, is compared with that of other dsRNA viruses or defective viruses, and is discussed in relation to the expression of the CMS trait.
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PMID:Unusual structure of the double-stranded RNA associated with the '447' cytoplasmic male sterility in Vicia faba. 768 75

The encephalomyocarditis virus 3C protease has been observed to undergo rapid degradation, both in vivo in mouse cells and in vitro in reticulocyte lysate. Experiments were carried out to characterize the turnover of the 3C protease in reticulocyte lysate. 3C protease prepared in reticulocyte lysate by in vitro translation and processing of a precursor polyprotein could be separated from the proteolytic activity responsible for its degradation. This implies the 3C protease is not directly involved in its own proteolysis. Active 3C protease flanked by only a few amino acids was degraded at a rate identical to that of a similar protein containing an inactivated catalytic site. This indicates that 3C protease activity is not indirectly required for the proteolytic process. Other viral proteins, including the 3D polymerase and capsid proteins, were relatively stable in the lysate. In addition, polyprotein precursors containing 3C protease with an inactive catalytic site and various flanking proteins displayed distinctly different stabilities. These results suggest that the reticulocyte proteolytic system functions in a selective manner toward the viral proteins. The effects of several proteolytic inhibitors on the lysate proteolytic system were evaluated. The results of these experiments indicate that the rapid degradation of the EMC virus 3C protease requires the hydrolysis of ATP.
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PMID:The encephalomyocarditis virus 3C protease is rapidly degraded by an ATP-dependent proteolytic system in reticulocyte lysate. 838 97

The ability of highly purified preparations of poliovirus RNA-dependent RNA polymerase, 3Dpol, to unwind RNA duplex structures was examined during a chain elongation reaction in vitro. Using an antisense RNA prehybridized to an RNA template, we show that poliovirus polymerase can elongate through a highly stable RNA duplex of over 1,000 bp. Radiolabeled antisense RNA was displaced from the template during the reaction, and product RNAs which were equal in length to the template strand were synthesized. Unwinding did not occur in the absence of chain elongation and did not require hydrolysis of the gamma-phosphate of ATP. The rate of elongation through the duplex region was comparable to the rate of elongation on the single-stranded region of the template. Parallel experiments conducted with avian myeloblastosis virus reverse transcriptase showed that this enzyme was not able to unwind the RNA duplex, suggesting that strand displacement by poliovirus 3Dpol is not a property shared by all polymerases.
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PMID:RNA duplex unwinding activity of poliovirus RNA-dependent RNA polymerase 3Dpol. 838 85

The complete amino acid sequence (1961 amino acids) of a vertebrate cellular myosin heavy chain-A was deduced from cDNA clones of a secretory rat mast cell line, the RBL-2H3 cell. The rat, human and chicken cellular myosin heavy chain-A exhibited high similarity in domains that allow binding of ATP and actin. The amino acid sequence of non-muscle myosin heavy chain-A from rat was 96% identical to that in human and 92% identical to that in chicken. Northern blot analysis of mRNA indicated the presence of single message of 7.4 kilobases. Northern blot, reverse-transcriptase polymerase chain reaction, and Western blot with isoform-specific antibodies indicated that RBL-2H3 cells expressed exclusively myosin heavy chain-A. Unlike rat PC12 cells, as well as a wide variety of other cultured cells and tissues, myosin heavy chain-B mRNA and protein were not detectable in RBL-2H3 cells. Because RBL-2H3 cells can be stimulated to release secretory granules as well as newly generated arachidonic acid and cytokines but lack myosin heavy chain-B, this cell line may provide a unique model to study the role of myosin heavy chain-A in cellular responses to antigen and other stimulants.
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PMID:Cloning of the cDNA encoding rat myosin heavy chain-A and evidence for the absence of myosin heavy chain-B in cultured rat mast (RBL-2H3) cells. 874 Apr 33

Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains. Reverse transcriptase-polymerase chain reaction (RT-PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid- and late stages of the chlamydial growth cycle. In addition, C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP-2-keto-3-deoxy-octanoic acid synthetase (CMP-KDO synthetase) as part of a polycistronic mRNA. The co-expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP-KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP-KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co-operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the K(m) of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.
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PMID:Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression. 895 11


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