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)

We used direct RNA sequencing to determine the genomic organization of the region downstream from the G gene of viral hemorrhagic septicemia virus (VHSV), a fish rhabdovirus. This region contains a gene coding for a protein, identified as nonvirion protein (NV), and the gene coding for the RNA polymerase (L). Thus, VHSV genome organization was confirmed to be 3'-N-P-M-G-NV-L-5'. In both a virulent European (07-71) and an avirulent North American (Makah) strain, the NV gene is transcribed into a small mRNA that codes for a protein of 122 amino acids. It has no significant sequence similarity with the infectious hematopoietic necrosis virus NV protein nor with any other known protein. We expressed the NV protein as a fusion protein with the glutathione S-transferase of Schistosoma japonicum and used the purified fusion protein to immunize rabbits. The rabbit antiserum precipitated from infected cell extracts--and not from noninfected cells or purified virions--a protein of 14 kDa, well in accordance with the expected NV gene product size. The prediction that the NV protein is a nonstructural protein is supported by its absence from mature virions although it is present in infected cells.
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PMID:Distant strains of the fish rhabdovirus VHSV maintain a sixth functional cistron which codes for a nonstructural protein of unknown function. 757 46

The lipase gene from Pseudomonas aeruginosa TE3285 is followed by another gene, lipB. The lipase gene was expressed in Escherichia coli BL21(DE3)pLysS using the T7 RNA polymerase expression system. The mature lipase was accumulated as inclusion bodies at 42% of the total cell proteins. The inclusion bodies were solubilized with 8 M urea, but lipase activity was not detected in the solubilized preparation containing 85% lipase protein even after removing urea by dialysis. The lipB gene, positioned downstream of the lipase gene and thought to be necessary for the expression of the lipase gene, was expressed in Escherichia coli JM109 as a fusion with the glutathione transferase gene from Schistosoma japonicum. The fusion protein was partially purified on glutathione-agarose beads to 36% purity. Incubated with the fusion protein at a molar ratio of 1:1 at 4 degrees C for 24 h, the solubilized lipase showed lipase activity of about a tenth that of the purified lipase prepared from Pseudomonas aeruginosa TE3285. Magnesium ions and ATP were not essential but increased the activation. When the fusion protein was treated with thrombin to release the glutathione transferase part, it retained its activity. The lipase activation with lipB protein probably proceeds to form a 1:1 complex with the inactive, solubilized lipase protein but by a different mode from known chaperones.
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PMID:Lipase from Pseudomonas aeruginosa. Production in Escherichia coli and activation in vitro with a protein from the downstream gene. 834 92

We cloned and sequenced the second open reading frame of the RNA polymerase gene, ORF1b, of bovine coronavirus. In the region representing nucleotide positions 4919-5677 upstream from the initiation codon of the 32K non-structural protein gene, we identified two putative functional domains. One of these domains contained four leucine residues repeated exactly in every seventh position, and the other domain represented a cluster of cysteine and histidine residues. The DNA sequence representing these domains was cloned and expressed in Escherichia coli as fusion proteins with glutathione S-transferase from Schistosoma japonicum. A high level expression of the cysteine-rich domain was achieved as a fusion protein when the bacterial culture was induced with IPTG. In a solid phase zinc binding assay using the recombinant fusion protein, we found that the protein containing the cysteine-rich domain was able to bind to radioactive zinc in vitro, demonstrating that the polypeptide encoded by the ORF1b of coronavirus is a zinc-binding protein.
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PMID:Zinc-binding of the cysteine-rich domain encoded in the open reading frame of 1B of the RNA polymerase gene of coronavirus. 883 May 21

Schistosomes utilise haemoglobin from ingested host erythrocytes as their main source of amino acids. Using reverse-transcriptase PCR, we detected transcripts encoding cathepsin D in eggs, miracidia, and adult male, female and mixed-sex Schistosoma japonicum. Using the synthetic fluorogenic peptide, o-aminobenzoyl-isoleucyl-glutamyl-phenylalanyl-p-nitro-phenylalanyl-a rgi nyl-leucine-NH2, and human haemoglobin as substrates, we detected cathepsin D-like aspartic protease activity at pH 3.6 in extracts of these developmental stages which was completely inhibited by the addition of l0 microM pepstatin. Using immunoblotting with rabbit antibodies raised against recombinant S. japonicum cathepsin D, we detected the aspartic protease in extracts of all developmental stages examined, although it appeared to be expressed at higher levels in the adult female schistosome. These results indicate that (almost) all stages of S. japonicum, express an aspartic protease. Moreover, they are consistent with the hypothesis that this enzyme plays a key role in maturing and adult schistosomes in the proteolysis of host haemoglobin from ingested erythrocytes.
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PMID:Developmental expression of cathepsin D aspartic protease in Schistosoma japonicum. 1061 28

Smalpha is a short interspersed element (SINE)-like retroposon that occurs in high copy number of the genome of the human blood fluke Schistosoma mansoni. The sequence of the consensus Smalpha element includes the hallmark features of SINE-like elements including a promoter region for RNA polymerase III, an AT-rich stretch at its 3'-terminus, a short length of 500 bp or less, and short direct repeat sequences flanking the insertion site. Interestingly, the sequence of Smalpha also encodes an active ribozyme bearing a hammerhead domain. Contrary to the recent findings of Ferbeyre et al. (Mol. Cell. Biol. 18 (1998) 3880-8) that indicated that Smalpha-like elements were absent from the genome of the Oriental blood fluke Schistosoma japonicum, we report here that the genome of S. japonicum does contain a family of Smalpha-like retroposons, elements that we have named the Sjalpha family. Like Smalpha, Sjalpha elements are SINE-like in structure and sequence, are present at high copy number interspersed throughout the S. japonicum genome, and contain an ostensibly functional, hammerhead ribozyme motif. The presence of these elements in all species of Schistosoma so far examined suggests that the hammerhead domain was acquired by vertical transmission from a common schistosome ancestor.
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PMID:Sjalpha elements, short interspersed element-like retroposons bearing a hammerhead ribozyme motif from the genome of the oriental blood fluke Schistosoma japonicum. 1100 17

We characterized the consensus sequence and structure of a long terminal repeat (LTR) retrotransposon from the genome of the human blood fluke, Schistosoma japonicum, and have named this element, Gulliver. The full length, consensus Gulliver LTR retrotransposon was 4788 bp, and it was flanked at its 5'- and 3'-ends by LTRs of 259 bp. Each LTR included RNA polymerase II promoter sequences, a CAAT signal and a TATA box. Gulliver exhibited features characteristic of a functional LTR retrotransposon including two read through (termination) ORFs encoding retroviral gag and pol proteins of 312 and 1071 amino acid residues, respectively. The gag ORF encoded motifs conserved in nucleic acid binding proteins, while the pol ORF encoded conserved domains of aspartic protease, reverse transcriptase (RT), RNaseH and integrase, in that order, a pol pattern conserved in the gypsy lineage of LTR retrotransposons. Whereas the sequence and structure of Gulliver was similar to that of gypsy, phylogenetic analysis revealed that Gulliver did not group particularly closely with the gypsy family. Rather, its closest relatives were a LTR retrotransposon from Caenorhabditis elegans, mag from Bombyx mori and, to a lesser extent, easel from the salmon Oncorhynchus keta. Dot blot hybridizations indicated that Gulliver was present at between 100 and several thousand copies in the S. japonicum genome, and Southern hybridization analysis suggested its probable presence in the genome of Schistosoma mansoni. Transcripts encoding the RT domain of Gulliver were detected by RT-PCR in larval and adult stages of S. japonicum, indicating that (at least) the RT domain of Gulliver is transcribed. This is the first report of the sequence and structure of an LTR retrotransposon from any schistosome or indeed from any species belonging to the phylum Platyhelminthes.
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PMID:Gulliver, a long terminal repeat retrotransposon from the genome of the oriental blood fluke Schistosoma japonicum. 1124 79

Saliva has been suggested as an easily accessible and a noninvasive diagnostic alternative for detection of antibodies. To identify and characterize Schistosoma japonicum (S. japonicum) antigens that are recognized by saliva of infected host, we have used a pool of saliva from infected patients to immunoscreen an egg cDNA library of S. japonicum. The open reading frame of the isolated two clones encodes same protein of 116 amino acids exhibiting 100% identity to an amino acid sequence (AY222893) of S. japonicum in NCBInr database. The protein encoded is inferred a secretory protein with a molecular mass of 13 kDa (Sj13) and shares no homology to any entries in the NCBInr database, demonstrating that Sj13 might be a schistosome-specific protein. Reverse transcriptase polymerase chain reaction, Western blotting, and immunolocalization analysis revealed Sj13 could be detected in cercaria, adult, and egg and was localized to forehead and tegument of cercaria, cell body ("cytons") of adult worm, egg shell, and epidermal plate of miracidium. Furthermore, Sj13 showed a good antigenicity when reacted with saliva or serum from schistosomiasis patients. The recombinant Sj13 (rSj13) expressed and purified from Escherichia coli was applied to detect its specific salivary antibody for schistosomiasis diagnosis by an indirect enzyme linked immunosorbent assay (ELISA). Preliminary laboratory test of 116 subjects, 40 with parasitologically proven S. japonicumm infection, 46 with other infectious diseases, and 30 negative controls exhibited 92.50% sensitivity with saliva/rSj13 and 95.00% with serum/SWAP (P > 0.05). The specificity of the ELISA using saliva/rSj13 was 92.11% versus 85.53% with serum/SWAP (P < 0.05). No direct correlations of anti-Sj13 IgG levels with egg counts in stool were observed in saliva detection. These results suggest that Sj13 specific salivary antibody detection may be useful as an antigen for the salivary diagnosis of schistosomiasis japonica and contribute to epidemiological study of schistosomiasis infection in endemic areas.
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PMID:Cloning, molecular characterization of a 13-kDa antigen from Schistosoma japonicum, Sj13, a putative salivary diagnosis candidate for Schistosomiasis japonica. 1963 40

Schistosomes are the causative agent of schistosomiasis. The 70-kDa heat-shock proteins (HSP70) are considered the predominant HSP family and play a key regulatory role in parasite development and pathogenesis. Based on the published sequences in Genbank/EMBL, an open-reading frame (ORF) encoding 653 amino acids (XP_002581385.1) and belonging to the Schistosoma HSP70 protein family with a molecular weight of 71.49 kDa was identified by bioinformatic analysis. Since the sequence shared 77% identity with the published full-length Homo sapiens HSP70 protein, it was named Schistosoma mortalin-like protein (MLP/Hsp70). Here, we report the molecular and functional characterization of the Schistosoma japonicum SjMLP/hsp70 as a member of the HSP70 family. The complete SjMLP/hsp70 coding sequence was amplified from a S. japonicum adult worm cDNA library by polymerase chain reaction (PCR) and subcloned into the pET28a expression vector. The purified recombinant protein, rSjMLP/hsp70, was identified as a member of 70-kDa HSP family by mass spectrometry and could be recognized by the S. japonicum-infected mouse serum. Reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting analysis revealed that SjMLP/hsp70 was widely expressed in the eggs, cercariae, schistosomula, and adult worms of S. japonicum. A thermotolerance assay showed that rSjMLP/hsp70 could protect Escherichia coli cells from heat damage. This chaperone-like activity was demonstrated by full-length SjMLP/hsp70. The detection of specific antibody levels by indirect enzyme-linked immunosorbent assay and IFN-gamma secretion of splenocytes by ELISpot assay suggested that mice immunized with SjMLP/hsp70 were able to elicit Th1-type bias immune response. The challenge-protective experiment showed that DNA vaccine of SjGST combined with SjMLP/hsp70 could induce a 31.31% reduction of worm burden and 58.59% reduction of egg burden in intestinal tissue of immunized mice. Our results imply that SjMLP/hsp70 has a potential adjuvant function and might be a vaccine candidate for schistosomiaisis, which is the first report of the expression and preliminary characterization analysis of this molecule.
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PMID:Molecular and functional characterization of a mortalin-like protein from Schistosoma japonicum (SjMLP/hsp70) as a member of the HSP70 family. 2060 14

Schistosoma japonicum is the pathogen responsible for schistosomiasis japonica, one of the major infectious diseases targeted for prevention nationally in China. Expression of heat shock proteins (HSPs) following stress plays a very important biological role in many organisms including S. japonicum. Among the HSP family, the 70-kDa HSPs are most responsible for intracellular chaperone and extracellular immunoregulatory functions. Based on the published sequences in GenBank/EMBL (AF044412.1), open reading frame belonging to HSP70 protein corresponds to a full-length cDNA containing an open reading frame of 1,947 bp encoded of 648 amino acids was identified as HSP70 from schistosome. In this study, the coding region that we named rSj648/hsp70 was amplified from S. japonicum adult worm cDNA library, and the recombinant protein was expressed in vector pET32a(+) and purified using a Ni-NTA purification system. The target protein rSj648/hsp70 was determined by matrix-assisted laser desorption/ionization mass spectrometer after thrombin digestion and dialysis. Reverse transcriptase polymerase chain reaction and Western blotting analysis confirmed that Sj648/hsp70 could be expressed in the eggs, normal cercariae, ultraviolet-attenuated cercariae (UVAC), and adult worms of S. japonicum. Real-time quantitative PCR analysis indicated that Sj648/hsp70 was expressed significantly higher in eggs than that in cercariae and adult worms, and the expression in UVAC was higher than that in normal cercariae. A thermotolerance assay showed that rSj648/hsp70 could protect Escherichia coli cells from heat damage. The detection of specific antibody levels by indirect enzyme-linked immunosorbent assay demonstrated that mice immunized with rSj648/hsp70 induced higher level of specific anti-rSj648/hsp70 IgG1 compared with those vaccinated with adjuvant alone, indicating that rSj648/hsp70 was able to elicit Th2-type bias immune response. Our results suggest that Sj648/hsp70 might be an important molecule in parasite-host interaction and display potential roles in mice immunoregulation system.
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PMID:Molecular cloning and characterization of a HSP70 gene from Schistosoma japonicum. 2207 51

Sj7TR is a 13 kDa repetitive region of a 31 kDa protein in Schistosoma japonicum known as Sjp_0110390 that showed high sensitivity and specificity in antibody detection against schistosomiasis patients. However, the current database for S. japonicum genes characterized it only as an expressed protein. A more thorough understanding of this antigenic protein is therefore necessary to possibly give more information about the nature of this protein and its role in the parasite. In this study, immunolocalization and expression profiling were done for Sjp_0110390 on the different stages of the parasite. Immunofluorescent assay showed that Sjp_0110390 was expressed in the young stages of the parasites including the schistosomula, eggs, aquatic and intra-molluscan stages. This was supported by the reverse-transcriptase PCR which confirmed the stage-specific expression of Sjp_0110390 and Western blot test which detected the protein in the extracted eggs proteins, but not in the adults. Furthermore, it was also highly expressed in infected Oncomelania hupensis nosophora snails suggesting that Sjp_0110390 might have a role in the development of the parasite inside the intermediate host. This result also suggests that Sj7TR might be used not only for human diagnosis but to detect snail infection as well.
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PMID:Localization and expression profiling of a 31 kDa antigenic repetitive protein Sjp_0110390 in Schistosoma japonicum life stages. 2325 1


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