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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gaucher disease results from impaired activity of the lysosomal enzyme glucocerebrosidase. Aiming at overexpressing the human glucocerebrosidase and testing the efficiency of the two in-frame ATGs of its gene in directing synthesis of an active enzyme, it was coupled to the T7 RNA polymerase promoter in a vaccinia virus-derived expression vector (pTM-1). cDNAs containing either one or both ATGs of the glucocerebrosidase mRNA were linked to the T7 polymerase promoter. Recombinant viruses were produced and used for infecting human cells in tissue culture. The results demonstrated that both ATGs directed translation of active glucocerebrosidase, resulting in a 10-fold increase in enzymic activity. Most of the protein remained sensitive to endoglycosidase H. The active enzyme represented a small fraction of the expressed glucocerebrosidase. The recombinant enzyme had the same Km and optimal pH towards the artificial substrate 4-methylumbelliferyl glucopyranoside as the authentic endogenous human enzyme. Measurements of intracellular enzymic activity directed by the cDNAs with either one or both ATGs in cells loaded with a fluorescent glucosylceramide demonstrated a 30% increase in activity directed by the cDNAs containing the first ATG over that containing the second ATG. This indicates that the protein synthesized from the first ATG, with a 38 amino acid leader, is translocated through the endoplasmic reticulum more readily than its counterpart directed by the second ATG, with a 19 amino acid leader. The elevation in glucocerebrosidase activity and the reproducibility of the data leads us to propose the use of the vaccinia virus-derived expression system as a tool for studying glucocerebrosidase mutants in Gaucher disease.
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PMID:Overexpression of human glucocerebrosidase containing different-sized leaders. 869 90

The role of ongoing RNA synthesis in chromatin organization in Chinese hamster ovary cells was examined upon exposure to the transcription inhibitor alpha-amanitin. Treatment with alpha-amanitin led to pleomorphic nuclei with chromatin heavily condensed and with the remaining ribonucleoprotein aggregated in large compact granular masses around the margins of the nuclear periphery. Concommitant with the changes in nuclei morphology transient focal dilatation of the rough endoplasmic reticulum was observed while other cytoplasmic organelles appeared structurally unaffected. The morphological changes occurred after complete inhibition of RNA polymerase II mediated transcription. The molecular integrity of isolated DNA was monitored in parallel with the structural analysis. Fragmentation of cellular DNA occurred in a time-dependent fashion and well after the complete inhibition of RNA synthesis. Characteristic oligonucleosomesized DNA fragments of about 187 base pairs in length was produced in a cotemporal time-dependent fashion. Our findings indicate that ongoing transcription and the structural state of chromatin are very closely integrated, and provide further evidence that RNA is a structural component of the nuclear matrix, which in turn is involved in keeping chromatin physically dispersed and decondensed.
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PMID:Ongoing activity of RNA polymerase II precludes chromatin collapse and DNA fragmentation in Chinese hamster ovary cells. 888 93

Lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (hex A) requires the presence of the GM2 activator protein (GM2AP) as an essential cofactor. A deficiency of the GM2 activator causes the AB variant of GM2 gangliosidosis, a recessively inherited disorder characterized by excessive neuronal accumulation of GM2 and related glycolipids. Two novel mutations in the GM2 activator gene (GM2A) have been identified by the reverse-transcriptase-PCR method--a three-base deletion, AAG262-264, resulting in a deletion of Lys88, and a single-base deletion, A410, that causes a frameshift. The latter results in substitution of 33 amino acids and the loss of another 24 amino acid residues. Both patients are homoallelic for their respective mutations inherited from their parents, who are heteroallelic at the GM2A locus. Although the cultured fibroblasts of both patients produce normal levels of activator mRNA, they lack a lysosomal form of GM2AP. Pulse/chase labeling of cultured fibroblasts of the patients, in presence and absence of brefeldin A, indicates a premature degradation of both--mutant and truncated--GM2APs in the endoplasmic reticulum or Golgi. These results were supported by in vitro translation experiments and expression of the mutated proteins. When the mutated GM2APs were expressed in Escherichia coli, both mature GM2AP forms turned proved to exhibit only residual activities in an in vitro assay.
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PMID:Molecular analysis of a GM2-activator deficiency in two patients with GM2-gangliosidosis AB variant. 890 Feb 33

Peripheral primitive neuroectodermal tumors (PNETs) consistently demonstrate a reciprocal translocation, t(11;22)(q24;q12). This translocation has not been found in PNETs of the central nervous system including the cerebellar medulloblastoma. We report an unusual cerebellar PNET in a 4-year-old boy in which tumor cells were surrounded by pools of Alcian blue-positive material. Tumor cells were immunoreactive for neuron-specific enolase and synaptophysin. Electron microscopy revealed well-developed rough endoplasmic reticulum, cell processes with intermediate filaments, microtubules, and dense core granules, and extracellular material reminiscent of mucopolysaccharide. Reverse transcriptase polymerase chain reaction (PCR) revealed an 11;22 translocation-specific PCR product. Clinically the tumor was a cerebellar PNET with leptomeningeal dissemination and there was no evidence to suggest that it was metastatic. Histopathology, however, was indicative of an unusual PNET that also manifested t(11;22) and was associated with an aggressive clinical course.
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PMID:An unusual cerebellar primitive neuroectodermal tumor with t(11;22) translocation: pathological and molecular analysis. 896 22

The helicase-like 1a and polymerase-like 2a proteins of brome mosaic virus (BMV) are required for viral RNA replication in vivo, are present in membrane-bound viral RNA polymerase extracts, and share conservation with the many other members of the alphavirus-like superfamily. To better understand BMV RNA replication and BMV-host interactions, we used confocal microscopy and double-label immunofluorescence to determine and compare the sites of 1a, 2a, and nascent viral RNA accumulation in BMV-infected barley protoplasts. 1a and 2a showed nearly complete colocalization throughout infection, accumulating in defined cytoplasmic spots usually adjacent to or surrounding the nucleus. These spots grew throughout infection and by 16 h postinoculation often assumed a vesicle-like appearance. The BMV RNA replication complex incorporated 5-bromouridine 5'-triphosphate into RNA in vitro and in vivo, allowing immunofluorescent detection of nascent RNA. The cytoplasmic sites of BMV-specific RNA synthesis coincided with the sites of 1a and 2a accumulation, and at the resolution of confocal microscopy, all sites of 1a and 2a accumulation were sites of BMV RNA synthesis. Double-label immunofluorescence detection of selected subcellular markers and 1a or 2a showed that BMV replication complexes were tightly associated with markers for the endoplasmic reticulum but not the medial Golgi or later compartments of the cellular secretory pathway. Defining this association of BMV RNA replication complexes with endoplasmic reticulum markers should assist in identifying and characterizing host factors involved in BMV RNA replication.
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PMID:Brome mosaic virus helicase- and polymerase-like proteins colocalize on the endoplasmic reticulum at sites of viral RNA synthesis. 897 Oct 20

Hepatitis C virus NS5B protein is an RNA-dependent RNA polymerase. To investigate the properties and function of this protein, we have expressed the NS5B protein in insect and mammalian cells. NS5B was found to be present as fine speckles in the cytoplasm, particularly concentrated in the perinuclear region, suggesting its association with the nuclear membrane, the endoplasmic reticulum, or the Golgi complex. This conclusion was supported by the biochemical demonstration that NS5B was associated with the membranes in the cells. Furthermore, it was shown that NS5B protein is a phosphoprotein. These properties may be related to its function as an RNA polymerase.
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PMID:Hepatitis C virus NS5B protein is a membrane-associated phosphoprotein with a predominantly perinuclear localization. 901 43

The cellular and molecular mechanisms underlying the postsynaptic aggregation of ionotropic receptors in the central nervous system are not understood. The glycine receptor (GlyR) and its cytoplasmic domain-associated protein, gephyrin, are clustered at the postsynaptic membrane and constitute a good model for addressing these questions. The glycine receptor is inhibited by strychnine. The effects of chronic strychnine treatment on the expression and cellular distribution of gephyrin and glycine receptor were therefore tested using primary cultures of spinal cord neurons. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the glycine receptor alpha1, alpha2, beta subunits and gephyrin mRNAs were expressed at comparable levels in strychnine-treated and untreated cultures. The number of immunoreactive cells and the subcellular distribution of gephyrin and GlyR subunits was determined with standard and confocal immunofluorescence. The proportion of gephyrin and glycine receptor-immunoreactive (IR) cells was unaffected by strychnine treatment. Confocal microscopy revealed that the glycine receptor was mainly localized intracellularly near the nucleus. This cytoplasmic glycine receptor was not associated with the Golgi apparatus nor with the rough endoplasmic reticulum and therefore is not likely to correspond to neosynthesized proteins. The number of GlyR clusters on the somato-dendritic membrane was dramatically reduced on neurons displaying intracellular staining. In contrast, the subcellular distribution and the number of gephyrin clusters was not modified by the treatment. The fact that gephyrin postsynaptic localization was not modified by strychnine suggests that the aggregation of glycine receptor and gephyrin is governed by different mechanisms. The distribution of other cell surface molecules such as NCAM or GABAA receptor beta2/3 subunits was not modified by strychnine treatment. Chronic exposure of the cultures to tetrodotoxin did not affect gephyrin or glycine receptor cluster formation. Taken together, these results indicate that functional glycine receptor, but not electrical synaptic activity, is required for the formation of glycine receptor clusters.
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PMID:Strychnine-sensitive stabilization of postsynaptic glycine receptor clusters. 942 82

The brain is an immunoprivileged organ isolated from the peripheral immune system. However, it has been shown that resident cells, notably astrocytes and microglia, can express numerous innate immune molecules, providing the capacity to generate a local antipathogen system. Perforin is a cytolytic protein present in the granules of cytotoxic T lymphocytes and natural killer cells. Expression in cells other than those of the hemopoetic lineage has not been described. We report here that fetal astrocytes in culture (passages 2 to 15), astrocytoma, and adult astrocytes expressed perforin. Reverse transcriptase polymerase chain reaction followed by Southern blot was carried out using multiple specific primers and all cDNAs were cloned and sequenced. Human fetal astrocyte perforin cDNA sequence was approximately 100% identical to the reported perforin cDNA cloned from T cells. Western blot analysis using monoclonal and polyclonal antiperforin peptide antibodies revealed a protein of 65 kD in both human fetal astrocyte and rat natural killer cell lysates (n = 4). Immunostaining followed by FACS(R) and confocal and electron microscopy analysis revealed that perforin was expressed by 40-50% of glial fibrillary acidic protein positive cells present in the fetal brain culture (n = 11). Perforin was not localized to granules in astrocytes but was present throughout the cytoplasm, probably in association with the endoplasmic reticulum. Perforin was not detected in normal adult brain tissue but was present in and around areas of inflammation (white and grey matter) in multiple sclerosis and neurodegenerative brains. Perforin-positive cells were identified as reactive astrocytes. These findings demonstrate that perforin expression is not unique to lymphoid cells and suggest that perforin produced by a subpopulation of astrocytes plays a role in inflammation in the brain.
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PMID:Identification of an astrocyte cell population from human brain that expresses perforin, a cytotoxic protein implicated in immune defense. 946 95

Among the functions of the replicase of equine arteritis virus (EAV; family Arteriviridae, order Nidovirales) are important viral enzyme activities such as proteases and the putative RNA polymerase and RNA helicase functions. The replicase is expressed in the form of two polyproteins (open reading frame 1a [ORF1a] and ORF1ab), which are processed into 12 nonstructural proteins by three viral proteases. In immunofluorescence assays, the majority of these cleavage products localized to the perinuclear region of the cell. A dense granular and vesicular staining was observed, which strongly suggested membrane association. By using confocal microscopy and double-label immunofluorescence, the distribution of the EAV replicase was shown to overlap with that of PDI, a resident protein of the endoplasmic reticulum and intermediate compartment. An in situ labeling of nascent viral RNA with bromo-UTP demonstrated that the membrane-bound complex in which the replicase subunits accumulate is indeed the site of viral RNA synthesis. A number of ORF1a-encoded hydrophobic domains were postulated to be involved in the membrane association of the arterivirus replication complex. By using various biochemical methods (Triton X-114 extraction, membrane purification, and sodium carbonate treatment), replicase subunits containing these domains were shown to behave as integral membrane proteins and to be membrane associated in infected cells. Thus, contribution to the formation of a membrane-bound scaffold for the viral replication-transcription complex appears to be an important novel function for the arterivirus ORF1a replicase polyprotein.
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PMID:ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex. 965 16

The replication of tobacco mosaic virus (TMV) RNA involves synthesis of a negative-strand RNA using the genomic positive-strand RNA as a template, followed by the synthesis of positive-strand RNA on the negative-strand RNA templates. Intermediates of replication isolated from infected cells include completely double-stranded RNA (replicative form) and partly double-stranded and partly single-stranded RNA (replicative intermediate), but it is not known whether these structures are double-stranded or largely single-stranded in vivo. The synthesis of negative strands ceases before that of positive strands, and positive and negative strands may be synthesized by two different polymerases. The genomic-length negative strand also serves as a template for the synthesis of subgenomic mRNAs for the virus movement and coat proteins. Both the virus-encoded 126-kDa protein, which has amino-acid sequence motifs typical of methyltransferases and helicases, and the 183-kDa protein, which has additional motifs characteristic of RNA-dependent RNA polymerases, are required for efficient TMV RNA replication. Purified TMV RNA polymerase also contains a host protein serologically related to the RNA-binding subunit of the yeast translational initiation factor, eIF3. Study of Arabidopsis mutants defective in RNA replication indicates that at least two host proteins are needed for TMV RNA replication. The tomato resistance gene Tm-1 may also encode a mutant form of a host protein component of the TMV replicase. TMV replicase complexes are located on the endoplasmic reticulum in close association with the cytoskeleton in cytoplasmic bodies called viroplasms, which mature to produce 'X bodies'. Viroplasms are sites of both RNA replication and protein synthesis, and may provide compartments in which the various stages of the virus mutiplication cycle (protein synthesis, RNA replication, virus movement, encapsidation) are localized and coordinated. Membranes may also be important for the configuration of the replicase with respect to initiation of RNA synthesis, and synthesis and release of progeny single-stranded RNA.
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PMID:Replication of tobacco mosaic virus RNA. 1021 41


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