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)

The unicellular eukaryote Trypanosoma brucei evades the immune defence of its mammalian host by antigenic variation. The genes for variant-specific surface glycoproteins (VSGs) are expressed within large multicistronic transcription units. Mature messenger RNAs are produced by trans-splicing and polyadenylation. A remarkable feature of the transcription of VSG genes is its insensitivity to the RNA polymerase II inhibitor alpha-amanitin. This has led to the speculation that RNA polymerase I, normally only involved in the transcription of ribosomal RNA genes, also mediates expression of these surface antigen genes. In higher eukaryotes, however, transcripts produced by RNA polymerase I were found to be poor substrates for processing into mature mRNAs. In contrast, we show here that the RNA polymerase I of T. brucei can mediate the efficient production of functional mRNA for neomycin phosphotransferase. This exceptional ability may be related to the unusual way in which pre-mRNAs are capped in trypanosomes. In most eukaryotes, mRNAs are modified at their 5' end by a capping activity associated with RNA polymerase II; in trypanosomes, mRNAs acquire their 5'-cap from capped mini-exon donor RNA by trans-splicing, a process that could be independent of the RNA polymerase producing the pre-mRNA.
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PMID:Efficient production of functional mRNA mediated by RNA polymerase I in Trypanosoma brucei. 165 58

Transcription of the predominant surface antigen genes in Trypanosoma brucei is unusual in its resistance to the RNA polymerase inhibitor alpha-amanitin, a property typical for rDNA transcription in eukaryotes. Transcription of most other protein-coding genes in trypanosomes is sensitive to alpha-amanitin. To investigate whether RNA polymerase I, the polymerase that transcribes rRNA genes, can give rise to functional mRNAs in trypanosomes, we have fused the putative promoter of the T.brucei rRNA genes to the chloramphenicol acetyl transferase (CAT) gene and determined CAT activity after transient expression of chimeric constructs in procyclic trypanosomes. We show here that the rRNA promoter yields the same high CAT activity as the promoters for the two predominant surface antigen genes of trypanosomes, the Variant-specific Surface Glycoprotein (VSG) gene of bloodstream trypanosomes and the procyclin gene of insect-form trypanosomes, both of which are also transcribed by an alpha-amanitin-insensitive RNA polymerase. RNA polymerase I of trypanosomes seems therefore able to synthesize pre-mRNAs that are effectively processed into translatable mRNAs. Dissection of the promoter segments showed the minimal elements for a VSG gene expression site promoter to be confined to a segment of -60 to +77 bp, overlapping the most 5' putative transcription start sites as determined in vivo by RNase protection experiments. For the ribosomal promoter region a segment of -258 to +200 bp relative to the putative transcription start site was sufficient for maximal CAT activity. There is a precise requirement for specific nucleotides at the rRNA transcription start site. We detect no homology between the sequences required for promoter function of the three alpha-amanitin-resistant transcription units, rRNA, VSG and procyclin (parp) genes. This suggests that the sequence-specific recognition of these promoters either occurs by common factors detecting sequence homologies that escape us, or by separate factors that bind to different DNA sequences but interact with a common alpha-amanitin-resistant RNA polymerase.
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PMID:Alpha-amanitin-resistant transcription units in trypanosomes: a comparison of promoter sequences for a VSG gene expression site and for the ribosomal RNA genes. 192 1

A novel expression system based on coinfection of cells with two recombinant vaccinia viruses has been developed. One recombinant vaccinia virus contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second recombinant vaccinia virus contained a target gene of choice flanked by bacteriophage T7 promoter and termination sequences. Maximum expression of the target gene occurred when cells were infected with 10 PFU of each recombinant virus. Although T7 RNA polymerase synthesis began shortly after infection, the target gene was not expressed until late times and was largely inhibited when DNA replication was blocked. Target gene transcripts were analyzed by agarose gel electrophoresis and had the predicted size. With this system, Escherichia coli beta-galactosidase, hepatitis B virus surface antigen, and human immunodeficiency virus envelope proteins were made. In each case, the level of synthesis was greater than had previously been obtained with the more conventional recombinant vaccinia virus expression system.
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PMID:Use of a hybrid vaccinia virus-T7 RNA polymerase system for expression of target genes. 311 59

DNA fragments preceding open reading frames in a conserved segment of the vaccinia virus genome (Plucienniczak A., et al. (1985) Nucleic Acids Res. 13, 985-998) were cloned into plasmids upstream of the S gene of the hepatitis B virus encoding the surface antigen (HBsAg). Recombinant vaccinia virus obtained after insertion of these constructs into the thymidine kinase gene were used to infect mouse 1D cells. HBsAg was assayed in cellular supernatants. A strong promoter was thus identified in a 295 bp fragment preceding the coding region of the 147 kDa subunit of the vaccinia RNA polymerase.
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PMID:Identification of vaccinia promoters by heterologous expression of hepatitis B surface antigen in mouse cells infected by recombinant vaccinia viruses. 367 23

We employed an in vitro cell-free transcription system to locate RNA polymerase II promoters on the hepatitis B virus genome. The strongest promoter precedes the surface antigen (HBsAg) gene, which is comprised of a long (500 base pairs) presurface region as well as the mature HBsAg coding sequence. The origin of this transcript was localized by using truncated templates and S1 endonuclease mapping. The activity of the promoter was confirmed in transfection experiments in which the complete HBsAg gene was introduced into monkey kidney cells via a simian virus 40 expression vector. A second RNA polymerase II promoter preceding the HBcAg gene was also active in the cell-free system. The presence of multiple promoters in the hepatitis B virus genome suggests that the relative levels of viral-specific proteins detected in liver and serum may reflect differential or regulated promoter efficiency.
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PMID:Transcription of hepatitis B virus by RNA polymerase II. 664 22

The chromosome of Yersinia enterocolitica encodes a heat-stable enterotoxin called Yst and a surface antigen called Myf, which closely resembles enterotoxin-associated fimbriae. Both factors could act in conjunction to produce diarrhea. Production of the enterotoxin is regulated by temperature, osmolarity, and pH and occurs only when bacteria reach the stationary phase. Myf production is regulated by temperature and pH and, as we show in this work, also occurs after the exponential growth phase. In an attempt to understand the late-phase expression of yst and myf, we cloned, sequenced, and mutagenized the gene encoding RpoS, an alternative sigma factor of the RNA polymerase involved in expression of stationary-phase genes in other enterobacteria. An intact rpoS gene was necessary for full expression of yst in the stationary phase but not for the expression of myf and of pYV-encoded virulence determinants.
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PMID:The rpoS gene from Yersinia enterocolitica and its influence on expression of virulence factors. 772 93

The Yersinia enterocolitica surface antigen Myf is a fibrillar structure that resembles CS3 fimbriae. Gene myfA encodes the 21-kDa major subunit of the antigen, while genes myfB and myfC are required for the transport and assembly of pilin subunits at the bacterial cell surface. Here we show that the expression of Myf is regulated at the transcriptional level by temperature and pH. Gene myfA is transcribed at 37 degrees C and in acidic medium. The transcription start is preceded by a putative -10 box for the vegetative RNA polymerase as well as by sequences resembling the consensus sequence recognized by sigma 28. Thus, myfA could be transcribed either from a classical sigma 70 promoter or from a sigma 28 promoter. Transcription of myfA requires at least two genes, myfF and myfE, situated immediately upstream from myfA. The myfF product does not show similarity to any known regulatory protein. It is an 18.5-kDa protein with no typical helix-turn-helix motif and a unique hydrophobic domain in the NH2-terminal part. T7 expression, osmotic shock, fractionation experiments, and TnphoA fusion analyses carried out in Escherichia coli suggest that MyfF is associated with the inner membrane by means of its hydrophobic domain whereas the hydrophilic part protrudes in the periplasm. These features strikingly evoke ToxS, a protein involved in regulation of Tcp pilus production in Vibrio cholerae. MyfE resembles PsaE, a protein involved in regulation of pH6 antigen in Yersinia pestis. Genes myfF and myfE are presumably part of a whole regulatory network. MyfF could be an element of the signal transducing system.
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PMID:MyfF, an element of the network regulating the synthesis of fibrillae in Yersinia enterocolitica. 783 9

Toxoplasma gondii isolates can be classified into mouse-virulent and -avirulent strains. Since the major surface antigen of Toxoplasma gondii, SAG1, has been shown to be important for the invasion process and might thus be involved in mouse virulence as well, we analyzed the corresponding gene of mouse-virulent and -avirulent strains. In addition to eight mismatches, mouse-virulent strains harbored five copies of an up-stream 27-bp repeat in the promotor region of SAG1 as compared with four copies in avirulent strains. Reverse-transcriptase polymerase chain reaction revealed that SAG1 expression levels in the mouse-virulent T. gondii strains were at least 4-fold those in the avirulent strains. Since SAG1 seems to mediate invasion, it is suggested that the possibly higher steady-state expression of SAG1 in mouse-virulent strains of T. gondii is associated with virulence and facilitates faster invasion by these strains in comparison with avirulent strains.
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PMID:Toxoplasma gondii strain-specific transcript levels of SAG1 and their association with virulence. 889 6

Within its intermediate host, Toxoplasma gondii switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterless HXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasma family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.
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PMID:Isolation of developmentally regulated genes from Toxoplasma gondii by a gene trap with the positive and negative selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase. 944 77

An RNA virus designated hepatitis G virus (HGV) has been recently identified in patients with acute and chronic liver disease. HGV is transfusion transmissible, it has global distribution, and it is present in the volunteer blood donor population in the United States. One hundred sixty patients undergoing maintenance hemodialysis at the University of Miami-affiliated unit were evaluated. There were 99 men and 61 women ranging in age from 22 to 80 years. Sixty percent had a history of blood transfusion, 6% had a history of drug abuse, and 9% were infected with the human immunodeficiency virus. HGV-RNA was detected by reverse-transcriptase polymerase chain reaction with amplification of two independent regions (5'-nontranslated region and NS5a coding region). Detection of digoxigenin-labeled amplification products with specific capture probes to the coding and noncoding regions was performed with the Enzymun-test DNA on an ES-300 Immunoassay System (Boehringer-Mannheim, Mannheim, Germany). Hepatitis C antibodies were measured with anti-hepatitis C virus enzyme-linked immunosorbent third-generation assays and hepatitis C virus RNA by reverse-transcriptase polymerase chain reaction. There were 32 (20%) patients with detectable HGV RNA with both primer pairs. Because of possible mutations, the HGV virus may be detectable only with one primer pair. We considered the latter as indeterminate: 12 had detectable levels to the NS5a region only, seven to the 5'-nontranslated region, and six had borderline results. Detectable and indeterminate samples were confirmed by repeat measurements in a new blood sample. Seven of 24 (29%) patients with detectable hepatitis C virus RNA had coexisting HGV with one or both HGV primer pairs (four with both and three with one). Five patients were hepatitis B surface antigen positive and HGV negative. We conclude that HGV infection is prevalent in our dialysis patients. The clinical significance of HGV infection remains to be established.
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PMID:Prevalence of hepatitis C and G virus infection in chronic hemodialysis patients. 946 14


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