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)

The RNA genome of hepatitis A virus (HAV) encodes a giant polyprotein that is putatively cleaved proteolytically into four structural and seven non-structural proteins. So far, most of the proposed non-structural proteins and their respective cleavage sites have not been identified. A vaccinia virus recombinant (vRGORF) containing the complete HAV ORF under the control of the bacteriophage T7 promoter was used to express HAV in recombinant animal cells (BT7-H) that constitutively expressed T7 DNA-dependent RNA polymerase. A HAV-specific 27.5 kDa expression product was identified as peptide 2B. The 27.5 kDa 2B antigen was also found in HAV-infected MRC-5 cells. The N-terminal amino acid residues of the new peptide 2B are Ala-Lys-Ile-Ser-Leu-Phe and polyprotein cleavage between 2A and 2B occurred at amino acids 836-837 (Gln-Ala). Furthermore, heterologous expression in the same system of regions P1-P2 and of the protease 3C (3Cpro) gene, showed that P1-P2 polyprotein is not cleaved autocatalytically but by 3Cpro. Hence, 3Cpro is effective in cleaving the polyprotein 2A-2B junction.
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PMID:Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C. 862 28

Gene 1 of the human coronavirus HCV 229E encompasses approximately 20.7 kb and contains two overlapping open reading frames, ORF 1a and ORF 1b. The downstream ORF 1b is expressed by a mechanism involving (-1) ribosomal frameshifting. Translation of mRNA 1, which is thought to be equivalent to the viral genomic RNA, results in the synthesis of two large polyproteins, pp1a and pp1ab. These polyproteins contain motifs characteristic of papain-like and 3C-like proteinases, RNA-dependent RNA polymerases, helicases, and metal-binding proteins. In this study, we have produced pp1ab-specific monoclonal antibodies and have used them to detect an intracellular, 105-kDa viral polypeptide that contains the putative RNA polymerase domain. Furthermore, using trans cleavage assays with bacterially expressed HCV 229E 3C-like proteinase, we have demonstrated that the 105-kDa polypeptide is released from pp1ab by cleavage at the dipeptide bonds Gln-4068/Ser-4069 and Gln-4995/Ala-4996. These data contribute to the characterization of coronavirus 3C-like proteinase-mediated processing of pp1ab and provide the first identification of an HCV 229E ORF 1ab-encoded polypeptide in virus-infected cells.
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PMID:Characterization of a 105-kDa polypeptide encoded in gene 1 of the human coronavirus HCV 229E. 880 2

We recently identified a new cucumovirus-specific gene (2b) which is encoded by RNA 2 of the cucumber mosaic cucumovirus (CMV) tripartite RNA genome and whose coding sequence overlaps the C-terminal 69 codons of ORF 2a encoding the RNA polymerase protein. We have now found that although a CMV mutant lacking ORF 2b accumulated in the inoculated cotyledons of cucumber plants, it was unable to spread systemically, demonstrating involvement of 2b in long distance movement. The same mutant infected tobacco systemically with a much reduced virulence and delayed appearance of symptoms, indicating that 2b may contribute to long distance movement in this host. Deletion of the overlapping C-terminal part of ORF 2a did not change infectivity of the mutant in either host species, ruling out 2a mutation as the reason for the change of phenotype. Further infectivity studies with mutants containing partial deletions in ORF 2b further supported the conclusion that 2b encodes a host-specific long distance movement function. Sequence analysis revealed that 2b may represent a novel naturally occurring hybrid gene important to the evolutionary formation of the cucumovirus group and that it could provide a genetic basis for the wide host range of these viruses.
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PMID:A novel naturally occurring hybrid gene encoded by a plant RNA virus facilitates long distance virus movement. 884 70

Homologous recombination in Saccharomyces cerevisiae and other organisms can be stimulated by transcription. Consistent with this, we find that recombination of a chromosomal ade1 allele with a plasmid-borne ADE1 ORF under the control of the GAL1 promoter increased from 6.1x10(-6) to 1.7x10(-4) when transcription of the plasmid locus was induced by growing the cells in the presence of galactose. Recombination could also be stimulated by over-expressing the Gal4 transcription factor in the presence of the GAL1-ADE1 plasmid, while culturing the cells in dextrose medium. However, when transcription of the same ORF was driven from the highly active promoters of the rDNA (RNA polymerase I), and ADH1 (RNA polymerase II) genes, only background levels of recombination (5-10x10(-6)) were observed, irrespective of the carbon source. Recombination was found to involve integration of the whole plasmid and to depend on RAD51, RAD52 and RAD54. The results indicate that increased accessibility of transcriptionally active chromatin is not sufficient to cause increased rates of this kind of reciprocal exchange.
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PMID:Stimulation of mitotic recombination upon transcription from the yeast GAL1 promoter but not from other RNA polymerase I, II and III promoters. 892 89

We analyzed the location and possible interaction of motifs of functional sites in a DNA sequence of MDG2 (Dm412) revealed by means of computer context analysis. It was shown that motifs of functional sites in the appropriate location can ensure the basic molecular functions of MDG2: expression of its ORF, transcription, induction of transposition, modification of adjacent genes and polygenes, etc. MDG2 ILTR does not contain the NTCAGTYN motif required for initiation of transcription by RNA polymerase II in the absence of the TATA box and located close to the transcription start site in most gypsy-like retrotransposons of Drosophila. Therefore, MDG2 ILTR appears to contain the classic promoter region for RNA polymerase II, contrary to other retrotransposons of the gypsy group. Enhancers of mobile genetic elements are assumed to determine modification of adjacent genes and polygenes. Excisions and transpositions of mobile elements seem to be induced by external stress factors or physiological factors through a heat-shock system.
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PMID:[Analysis of motifs of functional MDG2 sites in assuring its possible molecular functions]. 897 11

A number of Escherichia coli clones were isolated from a Lactobacillus delbrueckii subsp. lactis gene library capable of hydrolysing the chromogenic substrate Gly-Ala-beta-naphthylamide (Gly-Ala-beta NA). Some of the recombinant plasmids carried by these clones have been shown to encode the cysteine aminopeptidase gene pepC. Nucleotide sequence analyses of the plasmid inserts of the remaining clones resulted in the identification of two adjacent ORFs encoding proteins exhibiting a high degree of similarity between themselves (72.6%) and with PepC. One gene, designated pepG, was overexpressed in E. coli and the crude extracts obtained were shown to be peptidolytically active both against chromogenic substrates and peptides, and in a Salmonella typhimurium growth test. PepC and PepG activities were compared using chromogenic beta NA and p-nitroanilide substrates and leucine or proline-containing peptides were applied in growth experiments of recombinant Sal. typhimurium. The results indicate that the enzymes, although structurally related, have different substrate preferences. No enzyme activity could be ascribed to the second ORF (orfW), despite the production of a visible protein using a T7 RNA polymerase system. Primer extension analysis, using mRNA isolated from Lb. delbrueckii subsp. lactis DSM7290 did establish that orfW was transcribed.
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PMID:Lactobacillus delbrueckii subsp. lactis DSM7290 pepG gene encodes a novel cysteine aminopeptidase. 904 29

The 5'-terminal genomic region (8597 nt) of little cherry virus (LChV), a mealybug-borne closterovirus, was cloned from double-stranded RNA, and its sequence determined to complete the 16934 nt sequence of the monopartite LChV RNA genome. In the 5' to 3' direction, the sequence encompasses ORF 1a, encoding the conserved replicative domains of methyltransferase and helicase, and ORF 1b, encoding RNA polymerase. ORFs 1a and 1b partially overlap (in O/+1 configuration), and the LChV replicase is probably expressed by ribosomal frameshifting as a fusion product with a molecular mass of 318 kDa. The N-terminal part of the ORF 1a product contains a papain-like cysteine proteinase (PCP) domain with a predicted cleavage site between Gly-619 and Ser-620. The PCP and the upstream protein domains can be aligned with the equivalent parts of the leader proteins encoded by the whitefly-transmitted lettuce infectious yellows and sweet potato sunken vein closteroviruses. Phylogenetic reconstruction based on the aligned RNA polymerase sequences clearly suggests that the aphid-transmissible and whitefly-transmissible closteroviruses represent two distinct evolutionary lineages, with the mealybug-transmissible LChV being the most remote member of the 'whitefly' lineage.
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PMID:Complete genome structure and phylogenetic analysis of little cherry virus, a mealybug-transmissible closterovirus. 926 8

Recent studies have demonstrated that Rickettsia prowazekii can regulate transcription of selected genes at the level of initiation. However, little information concerning the existence of operons and coordinate gene regulation in this obligate intracellular parasitic bacterium is available. To address these issues, we have focused on the rpoD gene linkage group (greA-open reading frame 23 [ORF23]-dnaG-rpoD), which includes the rickettsial analog (ORF23-dnaG-rpoD) of the major macromolecular synthesis operon (MMSO). The rickettsial MMSO consists of an ORF coding for a protein of unknown function the structural genes for DNA primase (dnaG) and the major sigma factor of RNA polymerase (rpoD). RNase protection assays (RPA) were used to determine if these genes are organized into an operon controlled by multiple promoters and the quantities of transcripts produced by these genes relative to each other. RPA with a probe spanning the 270-base greA-ORF23 intervening region identified a putative transcriptional promoter within the intervening sequence. Multiple RPA probes spanning the next 4,041 bases of the linkage group demonstrated the presence of a continuous transcript and thus the existence of an operon. A probe spanning the dnaG-rpoD region revealed that two additional mRNA fragments were also protected, which enabled us to identify additional putative promoters for rpoD within dnaG. Primer extension determined that the 5' ends of the three transcripts consist separately of adenine (located 227 bases upstream of ORF23) and uracil and adenine (located 336 and 250 bases upstream of rpoD, respectively). Quantitation of transcripts produced by the three ORFs determined the relative amounts of transcripts (ORF23 to dnaG to rpoD) to be 1:2.7:5.1.
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PMID:Transcriptional characterization of the Rickettsia prowazekii major macromolecular synthesis operon. 933 95

The intronic mat-r ORF encodes a protein with significant homology to retroviral reverse transcriptases. Here, we describe the nucleotide sequence of potato mat-r and study the editing status of mat-r transcripts in two systems, potato and wheat, where the mat-r ORF is part of the trans-introns but in two different configurations relative to nad1 exons d and e. In potato and wheat, 13 and 15 C-to-U transitions respectively were observed. Most transcripts were partially edited, but potato transcripts were edited more efficiently than wheat transcripts. As in functional mitochondrial genes, RNA editing increased the similarity between plant mat-r proteins and their homologous non-plant counterparts. Interestingly, editing of mat-r was clustered in the reverse-transcriptase (RT) and the maturase (X) domains, two well defined regions having known functions in other systems. These results, together with the integrity and sequence conservation of mat-r, strongly suggest that the encoded protein plays a functional role in plant mitochondria.
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PMID:Editing status of mat-r transcripts in mitochondria from two plant species: C-to-U changes occur in putative functional RT and maturase domains. 964 5

Three transmissible gastroenteritis coronavirus (TGEV) defective interfering RNAs of 21, 10.6 and 9.7 kb (DI-A, DI-B and DI-C, respectively) were isolated. Dilution experiments showed that the largest DI RNA, DI-A, is a self-replicating RNA (replicon), and thus codes for a functional RNA polymerase and all the necessary replication signals. In order to engineer a cDNA encoding the RNA replicon a strategy based on the cloning of DI-C cDNA, followed by the insertion of the sequences required to complete the DI-A sequence has been developed. A cDNA complementary to DI-C RNA was cloned under the control of the CMV promoter (pDI-C-CMV) and rescued with a helper virus. In the ORF 1a of polymerase gene pDI-C-CMV contained a 10 kb deletion and in ORF 1b a 1.1 kb deletion. The consensus sequence corresponding to the deleted regions was cloned, and the deletions in pDI-C-CMV were replaced to yield a complete cDNA clone of DI-A, pDI-A-21-CMV, containing a full-length TGEV polymerase, driven by a CMV promoter. Expression of a functional TGEV polymerase is being investigated.
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PMID:Progress towards the construction of a transmissible gastroenteritis coronavirus self-replicating RNA using a two-layer expression system. 978 99


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