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)

Infection of human cells with adenovirus serotype 12 (Ad12) induces metaphase fragility of four, and apparently only four, chromosomal loci. Surprisingly, each of these four loci corresponds to a cluster of genes encoding a small abundant structural RNA: the RNU1 and RNU2 loci contain tandemly repeated genes encoding U1 and U2 small nuclear RNAs (snRNAs), respectively; the PSU1 locus is a cluster of degenerate U1 genes; and the RN5S locus contains the tandemly repeated genes encoding 5S rRNA. These observations suggested that high local levels of transcription, in combination with Ad12 early functions, can interfere with metaphase chromatin packing. In support of this hypothesis, we and others found that an artificial tandem array of transcriptionally active, but not inactive, U2 snRNA genes would generate a novel Ad12-inducible fragile site. Although U1 and U2 snRNA are both transcribed by RNA polymerase II and share similar enhancer, promoter, and terminator signals, the human U1 promoter is clearly more complex than that of U2. In addition, the natural U1 tandem repeat unit exceeds 45 kb, whereas the U2 tandem repeat unit is only 6.1 kb. We therefore asked whether an artificial array of minimal U1 genes would also generate a novel Ad12-inducible fragile site. The exogenous U1 genes were marked by an innocuous U72C point mutation within the U1 coding region so that steady-state levels of U1 snRNA derived from the artificial array could be quantified by a simple primer extension assay. We found that the minimal U1 genes were efficiently expressed and were as effective as minimal U2 genes in generating a novel Ad12-inducible fragile site. Thus, despite significant differences in promoter architecture and overall gene organization, the active U1 transcription units suffice to generate a new virally inducible fragile site.
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PMID:A tandem array of minimal U1 small nuclear RNA genes is sufficient to generate a new adenovirus type 12-inducible chromosome fragile site. 955 9

Enteric infection of mice with reovirus serotype 1 elicits antibody and cytotoxic T-lymphocytes in gut-associated lymphoid tissue (GALT). This led to the hypothesis that T-helper 1 (Th1) and T-helper 2 (Th2) responses develop in GALT. Reverse transcriptase-polymerase chain reactions on RNA from Peyer's patches (PP), intraepithelial lymphocytes (IEL), and lamina propria (LP) lymphocytes demonstrated that interferon (IFN)-gamma message was increased in PP and IEL, but not in LP following infection. No increase in mRNA for interleukin (IL)-4, IL-5, or IL-6 was detected. IFN-gamma, IL-5, and IL-6 were produced in in vitro cultures of PP 4-10 days postinfection. PP and spleen lymphocytes from infected mice produced IFN-gamma, but no IL-5 following in vitro restimulation. Infection also induced production of mRNA for the beta2 chain of the IL-12 receptor in PP. We conclude that reovirus induces robust Th1 and weak Th2 cell responses in GALT.
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PMID:T-Helper 1 and T-helper 2 cytokine responses in gut-associated lymphoid tissue following enteric reovirus infection. 974 58

Mouse-adapted influenza A virus, FM-MA, interferes with the replication of wild-type strains on co-infection. The interference phenotype was previously mapped to FM-MA segment 2 encoding a mutant PB1 protein, the catalytic component of the RNA polymerase complex. To identify the point at which FM-MA interferes with wild-type A/HK/1/68 (HK), the relative levels of transcription and genome replication of the PB1, NP and M1 genes were determined for FM-MA and HK viruses in co-infected cells using RT-PCR. All stages of HK macromolecular synthesis (primary and secondary transcription, genomic RNA, complementary RNA and protein synthesis) were suppressed relative to FM-MA. Infection with HK virus alone resulted in the accumulation of similar or greater amounts of RNA at late times post-infection relative to FM-MA thus indicating that the presence of FM-MA specifically compromised HK transcription and replication in co-infected cells. However early in infection FM-MA was ten times more active in mRNA transcription than HK or its parental strain FM. FM-MA's ability to interfere was primarily due to an increased capacity for primary transcription. FM-MA genomes were also selectively assembled into progeny virus from cells co-infected with HK and FM-MA, a step which was distinct from the capacity for enhanced RNA synthesis. This suggests that interference of HK growth by FM-MA in mixed infections results from two distinct events: a preferential synthesis of FM-MA-specific macromolecules which is then augmented by a preferential assembly of FM-MA genomes.
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PMID:Interference by a non-defective variant of influenza A virus is due to enhanced RNA synthesis and assembly. 983 88

The recent discovery of chemokine receptors as coreceptors for human immunodeficiency virus-type 1 (HIV-1) entry offers new avenues for investigating the pathogenesis of acquired immunodeficiency syndrome (AIDS)-related cytopenias. To this end, we sought to (1) phenotype human hematopoietic cells for CD4 and the HIV-1 coreceptors CXCR4, CCR5, CCR3, and CCR2b; (2) correlate CD4 and chemokine receptor expression with their susceptibility to HIV-1 infection; and (3) examine any potential interplay between inflammatory cytokines released during HIV-1 infection and regulation of chemokine receptor expression. Fluorescence-activated cell sorting (FACS) analysis of bone marrow mononuclear cells (BMMNC), cells derived from serum-free expanded hematopoietic lineages (colony-forming unit-granulocyte-macrophage [CFU-GM], colony-forming unit-megakaryocyte [CFU-Meg], and burst-forming unit-erythroid [BFU-E]), and CD34(+) cells showed differential expression of chemokine receptors and CD4 with some lineage specificity. Significantly, FACS-sorted CXCR4(+)/CD34(+) cells had the same clonogeneic potential as CXCR4(-)/CD34(+) cells. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of FACS-sorted human candidate stem cells (HSC; CD34(+), c-kit+, Rho123(low)) showed the presence of CXCR4 mRNA but not CD4 mRNA. Infection studies with HIV-1 Env-pseudotyped luciferase reporter viruses indicated that X4 Env (CXCR4-using) pseudotypes infected megakaryocytic cells, whereas R5 Env (CCR5-using) pseudotypes did not. Similarly, R5 but not X4 Env-pseudotyped viruses infected granulocyte-macrophage cells in a CD4/CCR5-dependent manner. Erythroid cells were resistant to R5 or X4 viral infection. Finally, we found that gamma-interferon treatment upregulated CXCR4 expression on primary hematopoietic cells. In summary, the delineation of chemokine receptor expression on primary hematopoietic cells is a first step towards dissecting the chemokine-chemokine receptor axes that may play a role in hematopoietic cell proliferation and homing. Furthermore, susceptibility of hematopoietic cells to HIV-1 infection is likely to be more complicated than the mere physical presence of CD4 and the cognate chemokine receptor. Lastly, our results suggest a potential interplay between gamma-interferon secretion and CXCR4 expression.
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PMID:Coreceptor/chemokine receptor expression on human hematopoietic cells: biological implications for human immunodeficiency virus-type 1 infection. 994 56

Reverse transcriptase (RT) isolated from Rous sarcoma virus (RSV) consists of heterodimeric RTalphabeta, RTalpha, and RTbeta. The alpha subunit (63 kDa) contains an N-terminal polymerase and a C-terminal RNase H domain. The N terminus of beta (95 kDa) corresponds to alpha with the integrase domain attached to the C terminus (32 kDa). We have constructed baculoviruses expressing the genes for alpha or beta or the entire pol (99 kDa). Infection of insect cells with recombinant virus yielded highly active and soluble RSV RT enzymes that could be purified to >90% homogeneity. HPLC gel filtration showed that alpha is a dimeric enzyme that can be partially monomerized upon the addition of 45% Me(2)SO. DNA synthesis on DNA-DNA and DNA-RNA primer-templates in the presence of competitor substrates revealed that alphabeta and beta as well as alpha are processive polymerases. However, the affinity of beta and alphabeta for primer-template substrates appears to be higher than that of alpha. All RSV enzymes investigated have the potential to displace RNA-RNA duplexes more efficiently than human immunodeficiency virus type 1 RT. Unlike human immunodeficiency virus type 1 RT, RSV RTs can catalyze an initial RNase H endonucleolytic cleavage of the RNA template but not a 3' --> 5' directed processing activity.
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PMID:Soluble Rous sarcoma virus reverse transcriptases alpha, alphabeta, and beta purified from insect cells are processive DNA polymerases that lack an RNase H 3' --> 5' directed processing activity. 1047 89

There are two types of infection caused by pathogenic microorganisms, intracellular infection and intercellular infection. Infection of pathogenic leptospira is an intercellular infection. The immunological reaction of host to intercellular infection is unique. The potential immunogen of an expressed protein should meet three criteria: it can be degraded (by antigen-present cells in the host); it should have antigenic epitope which can be recognized by specific antibodies and have at least one epitope that can be recognized by an MHC II protein and T cell receptor. In this study we report the cloning of an L. interrogans protein in plasmid rpDJt and the immunogencity of the expressed protein derivative. A genomic library of L. interrogans serovar lai strain 017 was constructed with the plasmid vector pUC18. Recombinant plasmids, designated pDJH2 and pDJ8 were screened from the bank. EcoRI-inserted fragment of 1. 9 kb recombinant DNA of pDJH2 was ligated into T7 RNA polymerase/promoter vectors (pT7-7). Then they were transformed into E. coli JM109 (De3), one of subclones, designated rpDJt was achieved. SDS-PAGE showed that the molecular weights of expression proteins were 68 kd and 23 kd respectively, designated p68 and p23. Purifying and isolating p68 and p23, we separated them from SDS-Polyacrylamide gels by using Side-Strip method. After fragmenting and electroeluting, p68 and p23 were injected into guinea pigs and rabbits. An extremely strong immune response to p68 was obtained since an anti-p68 antibody response could be detected to a dilution 1:524,288 (guinea pigs) and 1:262,144 (rabbits) by ELISA while anti-P23 antibody being 1:1024 (the same to guinea pigs and rabbits). The results of improved MTT and conA 3HTdR transformation methods showed the activities and proliferation of Th-cells were increased in guinea pigs after p68 immunization (IL-6, 83.25 IU/ml, IL-2, 28.75 IU/ml; RPI, 2.04, SI, 65.62%) Thlymphocyte existed in two subclasses, the Th1- and Th2-cells. A major role of Th2-cells is to "help" B-cells differentiate, replicate, and secrete antibody. The properties of these interactions explain why p68 makes good antigen and p23 does not. The antigens responsible for eliciting the production of protective antibodies are not known; however, several outer membrane proteins on L. interrogans are candidates for vaccine. Our results suggest that expresion protein p68 from recombinants (rpDJt) may be a candidate for gene engineered subunit vaccine for Leptospirosis.
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PMID:[Immunogenecity of expressed protein p68 from recombinant plasmid rpDJt in L. interrogans serovar lai]. 1068 17

Infection with high-risk human papillomaviruses (HPV), is the most significant risk factor for cervical cancer and it may be possible to prevent this malignancy by immunisation. Before immunisation programmes can be designed, however, it is necessary to know the age of acquisition and all routes of infection for these viruses. Sexual transmission is well documented and vertical transmission has also been demonstrated, although the frequency of transmission remains controversial. We previously showed that vertical transmission frequently results in persistent infection, and now present data on the prevalence of HPV-16 DNA (the most prevalent high-risk HPV type) in healthy children. Buccal samples from 267 healthy children aged 3-11 years were tested for HPV DNA by generic PCR (MY09/MY11), and a HPV-16 specific nested PCR. Reverse transcriptase (RT)-PCR was used to determine the prevalence of transcriptionally active HPV-16 infection in a subset of children. HPV-16 DNA was detected by nested PCR in 138 of 267 (51.7%) samples, whereas HPV DNA was detected in only 45 (16.8%) specimens by generic PCR, that has a lower analytical sensitivity. There were no significant differences in prevalence according to age or sex. Early region mRNA was detected by RT-PCR in six (11.3%) of 53 HPV-16 E5 DNA positive samples. HPV-16 E5 DNA sequences from 10 children confirmed the identity of the sequences detected and identified 13 HPV-16 variants.
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PMID:High prevalence of human papillomavirus type 16 infection among children. 1074 35

The aim of this study was to generate urgently needed data on respiratory pathogens in German children using an economical and efficient tool. Nasopharyngeal aspirates of hospitalized children 0-16 years of age with an acute respiratory tract infection were tested by a nine-valent multiplex reverse-transcriptase polymerase chain reaction. Of 1281 children, 449 (35%) had an acute respiratory tract infection caused by at least one of the organisms studied; there were 29 cases of dual infection. At least 34-42% of severe acute respiratory tract infections in children under 5 years of age were caused by viruses. In children over 5 years of age, this proportion was 23% (P<0.001). Infection during the first 2 years of life was most frequently due to respiratory syncytial virus (n = 162 cases). Parainfluenza virus type 3 (n = 22) and type 1 (n = 14) were detected almost exclusively in children under 5 years of age. Influenza A (n = 90) and adenoviruses (n = 98) were prevalent in all age groups. The frequency of influenza B virus isolation (n = 17) rose significantly after the age of 5 years. Mycoplasma pneumoniae infection (n = 24 cases, 5.2%) was most frequent in 5- to 16-year-old patients. Only one case of Chlamydia pneumoniae infection was found. Since the distribution of pathogens within the different types of lower respiratory tract infections is very similar, it seems that host factors determine which form of lower respiratory tract infection develops in an individual patient. The multiplex reverse-transcriptase polymerase chain reaction may, in the future, become an important tool for epidemiological studies as well as for individual diagnosis.
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PMID:Epidemiological investigation of nine respiratory pathogens in hospitalized children in Germany using multiplex reverse-transcriptase polymerase chain reaction. 1089 33

Canine distemper virus (CDV) has been rescued from a full-length cDNA clone. Besides Measles virus (MV) and Rinderpest virus, a third morbillivirus is now available for genetic analysis using reverse genetics. A plasmid p(+)CDV was constructed by sequential cloning using the Onderstepoort vaccine strain large-plaque-forming variant. The presence of a T7 promoter allowed transcription of full-length antigenomic RNA by a T7 RNA polymerase, which was provided by a host range mutant of vaccinia virus (MVA-T7). Plasmids expressing the nucleocapsid protein, the phosphoprotein, and the viral RNA-dependent RNA polymerase, also under control of a T7 promoter, have been generated. Infection of HeLa cells with MVA-T7 and subsequent transfection of p(+)CDV plus the helper plasmids led to syncytium formation and release of infectious recombinant (r) CDV. Comparison of the rescued virus with the parental virus revealed no major differences in the progression of infection or in the shape and size of syncytia. A genetic tag, consisting of two nucleotide changes within the coding region of the L protein, has been identified in the rCDV genome. Expression by rCDV of all the major viral structural proteins has been demonstrated by immunofluorescence.
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PMID:Establishment of a rescue system for canine distemper virus. 1104 18

Chagas' disease, caused by the parasite Trypanosoma cruzi, is an important cause of heart disease. Previous studies from this laboratory revealed that microvascular spasm and myocardial ischemia were observed in infected mice. Infection of endothelial cells with this parasite increased the synthesis of biologically active endothelin-1 (ET-1). Therefore. in the myocardium of T. cruzi-infected mice, we examined ET-1 expression and the p42/44-mitogen activated protein kinase (MAPK)-AP-1 pathway that regulates the expression of ET-1. There was parasitism and myonecrosis in the myocardium of infected C57BL/6 mice. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed elevated mRNA expression of transcription factor AP-1 (c-jun and c-fos) and increased AP-1 DNA binding activity as determined by electrophoretic mobility shift assay (EMSA). Western blot analysis demonstrated an increase in the phosphorylated forms of extracellular signal-regulated kinase (ERK1/2). ET-1 mRNA was upregulated in the myocardium of infected mice. Immunohistochemical and immunoelectron microscopy using anti-ET-1 antibody detected increased expression in cardiac myocytes and endothelium of these mice. These data suggest that ET-1 contributes to chagasic cardiomyopathy and that the mechanism of the increased expression of ET-1 is a result of the activation of the MAPK pathway by T. cruzi infection.
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PMID:Trypanosoma cruzi infection (Chagas' disease) of mice causes activation of the mitogen-activated protein kinase cascade and expression of endothelin-1 in the myocardium. 1107 62


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