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)

An enzyme immunoassay was developed to detect human immunodeficiency virus type 1 (HIV-1) DNA amplified by polymerase chain reaction (PCR-EIA). A set of primers (outer set) was used in PCR to amplify a segment of the HIV-1 gag gene from peripheral blood mononuclear cells. Hybrids between the amplified DNA and a RNA probe were measured in a microtiter plate immunoassay using a beta-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids and a fluorescent substrate. A second set of primers (nested set) located within the outer set was used in PCR with a known template to prepare the probe. One primer of the nested set included the T7 RNA polymerase promoter at its 5' end allowing transcription of a single-stranded RNA probe. Ten copies of HIV-1 DNA could be detected by PCR-EIA (42 fluorescent units with a background of 18 fluorescent units) compared with a detection limit of 1000 copies by ethidium bromide-stained agarose gel. HIV-1 DNA was detected by PCR-EIA in peripheral blood mononuclear cells from 32 of 33 seropositive patients (range 54-810 fluorescent units), and 0 of 25 seronegative patients (range 20-40 fluorescent units) (sensitivity 97%; specificity 100%). PCR-EIA offers a practical and nonisotopic method to objectively measure PCR-amplified HIV-1 DNA and has the potential for the measurement of other microbial pathogens in human body fluids.
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PMID:Enzyme immunoassay for detection of hybrids between PCR-amplified HIV-1 DNA and a RNA probe: PCR-EIA. 174 86

The adenovirus EIA and pseudorabies virus immediate early (IE) proteins induce transcription from transfected viral and nonviral genes transcribed by RNA polymerase II (class II genes). These proteins have now been shown also to activate transcription of transfected genes transcribed by RNA polymerase III (class III genes). As previously observed for class II genes, this stimulation of class III gene transcription was much greater for transfected genes than for the major endogenous cellular class III genes. Extracts made from cell lines stably expressing a transfected pseudorabies virus IE gene were 10 to 20 times more active in the in vitro transcription of exogenously added class III genes than extracts of the parental cell line. These results indicate that the E1A and IE proteins stimulate the expression of class III genes by a mechanism similar to the mechanism for stimulation of class II gene transcription by these proteins.
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PMID:Transcription of class III genes activated by viral immediate early proteins. 299 35

The promoter for eukaryotic genes transcribed by RNA polymerase B can be divided into the TATA box (located at -30) and startsite (+1), the upstream element (situated between -40 and about -110), and the enhancer (no fixed position relative to the startsite). Trans-acting factors, which bind to these elements, have been identified and at least partially purified. The role of the TATA box is to bind factors which focus the transcription machinery to initiate at the startsite. The upstream element and the enhancer somehow modulate this interaction, possibly through direct protein-protein interactions. Another class of transcription factors, typified by viral proteins such as the adenovirus EIA products, do not appear to require binding to a particular DNA sequence to regulate transcription. The latest findings in these various subjects are discussed.
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PMID:Transcription elements and factors of RNA polymerase B promoters of higher eukaryotes. 304 89

Astroviruses cause outbreaks of diarrhea in children attending day care centers (DCCs). Reverse transcriptase-polymerase chain reaction (RT-PCR) was compared with EIA detection of astrovirus in stool specimens to characterize further the molecular epidemiology of an outbreak of astrovirus-associated gastroenteritis. Three hundred sixty-eight stool specimens collected prospectively from 36 children enrolled in a DCC during an 11-week outbreak of diarrhea were evaluated by EIA and RT-PCR. Astrovirus was detected in 32% of specimens by RT-PCR versus 10% by EIA (P < .001) and in 89% of children by RT-PCR versus 50% by EIA. The median duration of astrovirus excretion episodes detected by EIA was 1.5 days versus 4 days by RT-PCR (P = .06). Astrovirus was excreted for prolonged periods by immunocompetent children during this outbreak. RT-PCR was more sensitive than EIA for detection of astrovirus in stool specimens and redefined the epidemiology of astrovirus infection in this setting.
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PMID:Virologic features of an astrovirus diarrhea outbreak in a day care center revealed by reverse transcriptase-polymerase chain reaction. 759

The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 31 percent of morphologically typical human caliciviruses (HuCV) and 57% of small round structured viruses (SRSVs) produced a product of 470 bp similar to the NV control, NV 8FIIa/68/US. Alignment of the amino acid sequences of morphologically typical HuCVs with previously published sequences for SRSVs, NV, and Snow Mountain agent (SMA) showed a high degree of homology (90-92%) with SMA and a lesser extent of homology with NV (60-61%). The amino acid sequence of two strains of HuCV, HuCV/3C/92/UK, and HuCV/5C/92/UK differed by only one or two amino acids respectively in the RNA dependent RNA polymerase region from that of two strains of SRSV obtained from children in the United Kingdom, SRSV/4S/90/UK and Japan, SRSV/OTH-25/89/J which were found to have identical amino acid sequences. The use of an EIA for detection of NV antigen employing antisera raised to recombinant NV protein indicated that HuCVs and SRSVs obtained from children and adults in the United Kingdom were antigenically distinct from the prototype Norwalk virus, NV/8fIIa/68/US.
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PMID:Sequence similarity of human caliciviruses and small round structured viruses. 793 Nov 87

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.
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PMID:Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters. 927 77

We identified a Norwalk-like calicivirus (CV) whose genome likely was derived from naturally occurring recombination. This strain (Arg320) was detected by the EIA developed against recombinant Mexico virus (rMxV) capsids, but the viral RNA polymerase sequence was closer to Lordsdale virus, in a separate genetic cluster of Norwalk-like viruses. A 3.3 kb cDNA from the RNA polymerase region to the 3' end of the genome of Arg320 was cloned and sequenced. The sequence demonstrated that the capsid region of Arg320 shared 95% amino acid identity with MxV, but 68% identity with Lordsdale virus, while the RNA polymerase region shared 95% identity with Lordsdale virus, but 87% identity with MxV. Pair-wise sequence comparisons identified a potential recombination site at the polymerase/capsid junction. This is the first example of a naturally occurring recombinant in the CV family. Further studies to search for and characterize other strains may be necessary for understanding the genetic diversity of the family.
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PMID:Characterization of a novel human calicivirus that may be a naturally occurring recombinant. 1066 91

Human caliciviruses were detected by EIA and/or RT-PCR in stool specimens from children with diarrhea treated at out- or in-patient facilities between 1995 and 1998 in Mendoza, Argentina. Mexico virus-like strains detected by primers NV36/51 were transiently prevalent in 1995/1996. Significantly more human caliciviruses were detected when primers were designed from contemporaneously circulating strains. Nucleotide sequences of a highly conserved region in the RNA polymerase gene of 10 selected human caliciviruses were determined. Eight strains were Norwalk-like viruses and two strains were Sapporo-like viruses. Seven of the eight Norwalk-like viruses also were positive by the recombinant Mexico virus antigen EIA. The seven Mexico virus EIA-positive strains revealed two patterns in the RNA polymerase sequences: two strains were closest to Mexico virus and the other five strains were closest to Lordsdale virus. One of the five "Lordsdale" viruses was found to be a naturally occurring recombinant between the Mexico virus and Lordsdale human calicivirus genetic clusters [Jiang et al., (1999b) Archives of Virology 144:2377-2387]. The Mexico virus EIA-negative strain had 73-77% nucleotide identity with the closest related Norwalk-like viruses, indicating it might belong to a new genetic cluster of the Norwalk-like virus genus. The two Sapporo-like viruses were distinct genetically; one belonged to the Houston/90 or Parkville cluster and the other to a new cluster. Some strains appeared to have short periods of prevalence and locally adapted primer pairs significantly increased detection rates. The finding of high diversity of circulating strains, including recombinant strains and strains with previously unrecognized genetic identities, highlights a need for studies of human caliciviruses in these children and other populations.
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PMID:Sequence diversity of human caliciviruses recovered from children with diarrhea in Mendoza, Argentina, 1995-1998. 1199 92

Human myxoid liposarcoma contains a characteristic t(12;16) chromosomal translocation that results in fusion of the N-terminal domain of the translocated in liposarcoma (TLS) protein to the C/EBP homologous protein (CHOP). TLS possesses structural motifs that suggest it may participate in RNA processing. We demonstrate that in human myxoid liposarcoma cells, wild-type TLS binds to RNA polymerase II (Pol II) via its N-terminal domain and to the transcription and translation factor Y-box binding protein-1 (YB-1) through its C-terminal domain. The liposarcoma fusion protein TLS/CHOP retains the ability to bind RNA Pol II but lacks the ability to recruit YB-1 due to replacement of the C-terminal domain of TLS by CHOP. In an in vivo splicing assay, YB-1 promotes splicing of adenovirus EIA pre-mRNA predominantly to the 13S isoform. The oncogenic TLS/CHOP fusion protein inhibits this splicing function of YB-1 in a dominant negative manner. When considered in conjunction with studies on other sarcoma fusion proteins, these data suggest that aberrant RNA splicing may be a common feature of human sarcomas.
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PMID:RNA splicing mediated by YB-1 is inhibited by TLS/CHOP in human myxoid liposarcoma cells. 1216 60

The human ov-serpin monocyte neutrophil elastase inhibitor (MNEI) is encoded by a single gene SERPINB1. It is a highly efficient inhibitor of neutrophil granule proteases. Four murine genes with high sequence identity with MNEI were identified and fully sequenced, and these were named EIA, EIB, EIC, and EID. EIA, EIB and EIC showed the same seven-exon gene structure as SERPINB1. However, EIC included an additional, alternatively spliced, exon due to the insertion of an endogenous retrovirus-like sequence. EID lacked several exons and is a pseudogene. Reverse transcriptase-PCR showed that EIA, like MNEI, is expressed at high levels in many tissues. EIB is mainly expressed in brain, and EIC was only expressed as splicing variants unlikely to encode a functional serpin. Upon incubation with serine proteases, EIA formed inhibitory covalent complexes with pancreatic and neutrophil elastases, cathepsin G, proteinase-3, and chymotrypsin, as previously shown for MNEI, whereas EIB was only able to do so with cathepsin G. According to the new serpin nomenclature, the genes encoding EIA, EIB, EIC, and EID will be called Serpinb1, Serpinb1b, Serpinb1c, and Serpinb1-ps1. These data demonstrate that the four murine homologs of MNEI have met different evolutionary fates, and that EIA is the mouse ortholog of MNEI.
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PMID:Characterization of four murine homologs of the human ov-serpin monocyte neutrophil elastase inhibitor MNEI (SERPINB1). 1218 54


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