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)

Pre-mRNA synthesis in eukaryotic cells is preceded by the formation of a transcription initiation complex and binding of unphosphorylated RNA polymerase II (Pol II) at the promoter region of a gene. Transcription initiation and elongation are accompanied by the hyperphosphorylation of the carboxy-terminal domain (CTD) of Pol II large subunit. Recent biochemical studies provided evidence that RNA processing factors, including those required for splicing, associate with hyperphosphorylated CTDs forming "transcription factories." To directly visualize the existence of such factories, we simultaneously detected human cytomegalovirus immediate-early (IE) DNA and RNA with splicing factors and Pol II in rat 9G cells inducible for IE gene expression. Combined in situ hybridization and immunocytochemistry revealed that, after induction, both splicing factors and Pol II are present at the sites of IE mRNA synthesis and of IE mRNA processing that extend from the transcribing gene. Noninduced cells revealed no such associations. When IE mRNA-synthesizing cells were treated with a transcription inhibitor, these associations disappeared within 30 min. Our results show that the association of Pol II and splicing factors with IE DNA is dependent on its transcriptional activity and furthermore suggest that splicing factors are still associated with Pol II during active splicing.
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PMID:RNA polymerase II localizes at sites of human cytomegalovirus immediate-early RNA synthesis and processing. 988 60

The gene for a 45 kDa merozoite surface protein (MSA-2) of the human malaria parasite Plasmodium falciparum was PCR amplified and cloned into eukaryotic expression vectors VR1012 and pcDNA3 to yield plasmids P1 and P2, respectively. The coding sequences for two N-terminal fragments of the 185 kDa merozoite surface protein (MSA-1) gene were similarly PCR amplified and cloned into vectors VR1020 and VR1012 to yield plasmids P3 and P4, respectively. The MSA-1 signal peptide sequence, present in P4, was replaced with the human tissue plasminogen activator signal sequence in P3. The four plasmids expressed the cloned genes under the control of the cytomegalovirus promoter and carried 3' bovine growth hormone termination/poly A signals. P1, P3 and P4 also contained the cytomegalovirus intron A enhancer sequence. MSA-1 expression was more readily detected than MSA-2 in Cos cells transfected with P3/P4 and P1/P2 respectively. The MSA-2 gene was also cloned into the phagemid pBluescript IISK+ with and without a 3' poly A tail composed of 35 A residues. MSA-2 was synthesised in HeLa cells infected with a recombinant vaccinia virus carrying T7 RNA polymerase when MSA-2 recombinant pBluescript was transfected into the cells. Inoculation with P1 intramuscularly or intradermally and with P2 intradermally into rabbits led to the production of antibodies to MSA-2 detectable by immunofluorescence and Western blotting. Antibodies were also produced against MSA-1 after intramuscular/intradermal inoculation with P3 and P4. Inoculation of rabbits with MSA-2 mRNA yielded better antibody titres when a poly A tail was present. Antibody levels were maintained for > 9 weeks after the final immunisation. However the immune sera failed to inhibit in vitro parasite growth.
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PMID:Mammalian cell expression of malaria merozoite surface proteins and experimental DNA and RNA immunisation. 998 40

Reverse transcriptase of human immunodeficiency virus type I is a vitalenzyme in the HIV-1 replication cycle and an attractive target of attempts to arrest a primary viral infection. We designed a vector for eukaryotic expression of the 66 kDa subunit of reverse transcriptase under the control of the immediate early cytomegalovirus promoter. Efficient transient expression of the 66 kDa subunit of reverse transcriptase was achieved in a variety of cells. Immunostaining of the transfected cells revealed the cytoplasmatic localization of reverse transcriptase. Reverse transcriptase activity was detected in all transfected cell lines. Injection of this plasmid encoding the 66 kDa subunit of reverse transcriptase into mice resulted in strong reverse transcriptase-specific immune responses indicating that the 66 kDa subunit of reverse transcriptase is expressed in vivo. Sera from DNA-immunized mice inhibited reverse transcription in vitro.
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PMID:Eukaryotic expression of enzymatically active human immunodeficiency virus type 1 reverse transcriptase. 1021 52

Actinomycin D and alpha-amanitin are commonly used to inhibit transcription. Unexpectedly, however, the transcription of the human immunodeficiency virus (HIV-1) long terminal repeats (LTR) is shown to be activated at the level of elongation, in human and murine cells exposed to these drugs, whereas the Rous sarcoma virus LTR, the human cytomegalovirus immediate early gene (CMV), and the HSP70 promoters are repressed. Activation of the HIV LTR is independent of the NFkappaB and TAR sequences and coincides with an enhanced average phosphorylation of the C-terminal domain (CTD) from the largest subunit of RNA polymerase II. Both the HIV-1 LTR activation and the bulk CTD phosphorylation enhancement are prevented by several CTD kinase inhibitors, including 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole. The efficacies of the various compounds to block CTD phosphorylation and transcription in vivo correlate with their capacities to inhibit the CDK9/PITALRE kinase in vitro. Hence, the positive transcription elongation factor, P-TEFb, is likely to contribute to the average CTD phosphorylation in vivo and to the activation of the HIV-1 LTR induced by actinomycin D.
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PMID:The transcriptional inhibitors, actinomycin D and alpha-amanitin, activate the HIV-1 promoter and favor phosphorylation of the RNA polymerase II C-terminal domain. 1034 61

Pyrrole-imidazole polyamides are ligands that bind in the minor groove of DNA with high affinity and sequence selectivity. Molecules of this class have been shown to disrupt specific transcription factor-DNA interactions and to inhibit basal and activated transcription from various RNA polymerase II and III promoters. A set of eight-ring hairpin-motif pyrrole-imidazole polyamides has been designed to bind within the binding site for the human cytomegalovirus (CMV) UL122 immediate early protein 2 (IE86). IE86 represses transcription of the CMV major immediate early promoter (MIEP) through its cognate cis recognition sequence (crs) located between the TATA box and the transcription initiation site. The designed polyamides bind to their target DNA sequence with nanomolar affinities and with a high degree of sequence selectivity. The polyamides effectively block binding of IE86 protein to the crs in DNase I footprinting experiments. A mismatch polyamide, containing a single imidazole to pyrrole substitution, and also a polyamide binding to a site located 14 base pairs upstream of the repressor binding site, do not prevent IE86 binding to the crs. IE86-mediated transcriptional repression in vitro is relieved by a match polyamide but not by a mismatch polyamide. Transcription from a DNA template harboring a mutation in the crs is not affected either by IE86 protein or by the match polyamides. These results demonstrate that this new class of small molecules, the pyrrole-imidazole polyamides, are not only effective inhibitors of basal and activated transcription, but also can be used to activate transcription by blocking the DNA-binding activity of a repressor protein.
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PMID:Anti-repression of RNA polymerase II transcription by pyrrole-imidazole polyamides. 1045 76

The nonreplicating vaccinia virus MVA/T7 RNA polymerase hybrid system was tested with Petri dish electroporation for ectopic gene expression in human umbilical vein endothelial cells (HUVECs). A range of voltages (150-450 V), pulse times (10-40 ms), DNA concentrations (0-20 microg/ml) and infection levels (0-15 multiplicities of infection) were tested for effects on T7 promoter-directed chloramphenicol acetyltransferase (CAT) activity after MVA/T7RP infection. MVA/T7RP-directed expression was transient and at least 10 000-fold in excess of nonviral, cytomegalovirus enhancer-directed expression. Use of a Petri dish electrode with the MVA/T7RP system showed increased viability compared with a cuvette electrode. Overexpression of interleukin-2 alpha subunit (IL2Ralpha) pro- tein followed by anti-IL2Ralpha-directed magnetic immunoaffinity cell sorting allowed isolation of the transfected population. The high fidelity of cellular sorting was shown by segregation of CAT activity in the IL2Ralpha-sorted population after transfection of T7 promoter-directed bicistronic IL2Ralpha/CAT DNA. Expression of a panel of proteins including the fluorophore green fluorescent protein as detected by fluorescence microscopy and p21cip1, p27kip1, pp60c-src, FGF-1, pRb, p107 and pRb2/p130 proteins was also achieved. Thus, use of the nonreplicating vaccinia virus/T7 RNA polymerase expression system with Petri dish electroporation is feasible for certain applications for the manipulation of HUVECs by gene transfer.
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PMID:Endothelial cell DNA transfer and expression using petri dish electroporation and the nonreplicating vaccinia virus/T7 RNA polymerase hybrid system. 1049 Jul 72

Ca2+ plays a key role in many pathological processes, including viral infections. Rotavirus, the major etiological agent of viral gastroenteritis in children and young animals, provides a useful model to study a number of Ca2+ dependent virus-cell interactions. Rotavirus entry, activation of transcription, morphogenesis, cell lysis, particle release, and the distant action of viral proteins are Ca2+ dependent processes. In the extracellular medium, Ca2+ stabilizes the structure of the viral capsid. During entry into the cell the low cytoplasmic Ca2+ concentration induced the solubilization of the outer protein layer of the capsid and transcriptase activation. Viral protein synthesis modifies Ca2+ homeostasis which, in turn, favours viral morphogenesis and induces cell death. The generation of diarrhea is a multifactorial process involving Ca2+ dependent secretory processes of mediators and water and electrolytes, as well as the induction of cell death in the different cell types that compose the intestinal epithelium. The discovery of the non-structural viral protein NSP4 as a viral enterotoxin and the possible participation of the enteric nervous system in the pathogenesis of diarrhea represent significant advances in its understanding. Ca2+ also plays a role in the replication cycles and pathogenesis of other viral diseases such as poliovirus, Coxsackie virus, cytomegalovirus, vaccinia and measles virus and HIV.
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PMID:Role of Ca2+in the replication and pathogenesis of rotavirus and other viral infections. 1102 Mar 76

The bone marrow (BM) in myelodysplastic syndromes (MDS) undergoes pathobiological changes that mimic an inflammatory process, and hence, an infectious etiology was suspected in these disorders. In the present report, we examined the bone marrow mononuclear cells (BMMNC) of 19 MDS patients and seven normal donors for the expression of one latency-related (Latency membrane protein 1 (LMP-1) and immediate early protein (IEP)) and one activation-related (BZLF and DNA-Pol) m-RNA each for two herpes viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), respectively. Reverse transcriptase polymerase chain reaction was used for this purpose. The latency-related transcripts (EBV-LMP-1 and CMV-IEP) were present in all the MDS and normal specimens. Intriguingly, 10/19 MDS specimens ( approximately 53%) and 2/7 normal donors ( approximately 28%) were positive for active EBV-BZLF (P=0.0067), while 2/19 MDS specimens ( approximately 11%) with 1/7 normal ( approximately 14%) showed active CMV-DNA-Pol (P=0.1588). Later, from another set of MDS patients (n=7) and normal donors (n=4), BM stromal cultures were established, which, at a 75% confluency, were overlaid with cord blood mononuclear cells (CBMNC). IEP was detectable in the CBMNC before and after co-incubation with MDS, as well as normal stroma. So, it was also present both in MDS and normal stromal cells. The other three were absent both in MDS and normal stromal layers. In CBMNC though, active EBV-BZLF and CMV-DNA-Pol m-RNA were detectable in one of seven MDS co-cultures each, albeit from different patients. None of the normal co-cultures showed active virus, either in stroma or CBMNC. Thus, the present report demonstrates, for the first time, the presence of active herpes viruses in the BMMNC of MDS patients and reveals the ability of the MDS stroma to support the viral activation.
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PMID:Presence of activation-related m-RNA for EBV and CMV in the bone marrow of patients with myelodysplastic syndromes. 1117 35

Vectors based on recombinant SV40 viruses (rSV40) are highly effective in delivering transgene expression driven by constitutive promoters. We tested here whether these vectors could be used with conditional promoters and promoters using RNA polymerase III transcription, with inhibition of HIV-1 by Tat activation response (TAR) decoys as a functional measure of effective transgene delivery and activity. TAR decoys inhibit HIV-1 Tat, a trans-activator of HIV-1 transcription. Tat acts early in the viral replicative cycle and is essential for efficient viral replication. We evaluated rSV40 gene delivery using two different inhibitors of Tat. One was a dual function polyTAR gene encoding 25 sequential TAR elements (TAR(25)), plus an antisense tat, driven either by HIV-1 long terminal repeat (HIV-LTR) as a conditional promoter, or by cytomegalovirus immediate-early promoter (CMV-IEP) as a constitutive promoter. The other inhibitor was a single TAR decoy, driven by the U6 small nuclear RNA promoter (U6-P). These decoys were delivered to unselected cells in two different human T lymphocyte lines and to unstimulated primary human peripheral blood mononuclear cells (pbmc). Gene delivery was confirmed by PCR, and expression by RT-PCR. By in situ hybridization analysis, >95% of cells were transduced. These transgene constructs protected all cell types tested from HIV-1, as measured by syncytia formation and p24 antigen release. Somewhat better inhibition of HIV-1 replication was achieved with HIV-1 long terminal repeat (HIV-1 LTR) as a conditional promoter than with the constitutive CMV-IEP. The U6-P was also very effective, driving a TAR(1) transcript. Cell viability was not detectably affected by TAR decoy expression. Thus, rSV40 vectors effectively deliver HIV-1-inhibitory RNAs using either constitutive or conditional pol II promoters, or using a pol III promoter. The versatility of this gene delivery system may prove to be useful in anti-HIV-1 therapeutics.
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PMID:SV40-derived vectors provide effective transgene expression and inhibition of HIV-1 using constitutive, conditional,and pol III promoters. 1143 38

RNA ligands were generated against various gene products present in HeLa nuclear extract. Functional profiling was performed to identify RNA ligands that modulate RNA polymerase II (pol II)-mediated transcription. Unexpectedly, four of the eight inhibitor ligands identified by this screen contained an 11-nucleotide sequence identical to the 5'-splice site of eukaryotic pre-mRNAs. Such ligands were shown to impede pre-initiation complex assembly on a cytomegalovirus promoter. In addition, U1 small nuclear ribonucleoprotein particles (snRNP) and pol II had been co-immunoprecipitated in the absence of transcription. These results suggest a role for U1 snRNP in co-ordinating transcription and RNA splicing.
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PMID:RNA ligands generated against complex nuclear targets indicate a role for U1 snRNP in co-ordinating transcription and RNA splicing. 1174 4


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