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)

A phage contig of 400 kb that extends from the brain-specific promoter at the 5'-end of the human dystrophin gene, through the muscle-specific promoter over 100 kb further downstream, and across most of intron 1 has been assembled. To achieve this, a yeast artificial chromosome (YAC) subcloning approach was used. Total DNA from a yeast strain containing a 400-kb YAC from the dystrophin gene was cloned using a lambda phage vector containing RNA polymerase promoters flanking the cloning sites. Phage containing human DNA inserts were then ordered into an overlapping set by hybridization of end-specific RNA probes from individual clones back to plaque lifts of gridded phage subclones. The clones generated will be useful as reagents for detailed structural and functional analyses of this region of the dystrophin gene.
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PMID:Generation of ordered phage sublibraries of YAC clones: construction of a 400-kb phage contig in the human dystrophin gene. 844 19

The ability to clone and overexpress genes encoding mouse Fab (antigen-binding fragment) proteins in bacteria led to the development of a methodology which has the potential to replace traditional hybridoma technology [Huse et al., Science 246 (1989) 1275-1281]; however, several observations have suggested that clones with desirable chemical properties may be missed in immunoscreens of large combinatorial libraries due to low levels of functional Ab protein. To increase the efficiency of cloning and characterization of Ab gene fragments, we have reconsidered several features of the original cloning vehicles. These studies show that at the present time a unique expression system cannot adequately accommodate the requirements of plaque-lift immunoassays for clonal selection and biochemical assays for further characterization in vitro. A monocistronic arrangement of heavy- and light-chain-encoding genes using two lacP promoters produces sufficient amounts of functional Ab protein for clonal selection from phage lambda libraries and minimizes interference with the lytic cycle of recombinant vectors. In liquid culture, a strong coliphage promoter and a relatively abundant RNA polymerase can be used to produce quantities of Ab protein sufficient for further characterization in vitro. A rapid purification protocol obviates the need for fusing heavy-chain protein to a decapeptide sequence, an affinity-tail sequence which slows the folding and assembly of the Ig heterodimer. These results have been used to formulate a new strategy for cloning and characterization of Ab gene fragments in bacteria.
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PMID:A revised strategy for cloning antibody gene fragments in bacteria. 850 51

The genetic study of RNA viruses is greatly facilitated by the availability of infectious cDNA clones. However, their construction has often been difficult. While exploring ways to simplify the construction of infectious clones, we have successfully modified and applied the newly described technique of "long PCR" to the synthesis of a full-length DNA amplicon from the RNA of a cytopathogenic mutant (HM 175/24a) of the hepatitis A virus (HAV). Primers were synthesized to match the two extremities of the HAV genome. The antisense primer, homologous to the 3' end, was used in both the reverse transcription (RT) and the PCR steps. With these primers we reproducibly obtained a full-length amplicon of approximately 7.5 kb. Further, since we engineered a T7 promoter in the sense primer, RNA could be transcribed directly from the amplicon with T7 RNA polymerase. Following transfection of cultured fetal rhesus kidney cells with the transcription mixture containing both the HAV cDNA and the transcribed RNA, replicating HAV was detected by immunofluorescence microscopy and, following passage to other cell cultures, by focus formation. The recovered virus displayed the cytopathic effect and large plaque phenotype typical of the original virus; this result highlights the fidelity of the modified long reverse transcription-PCR procedure and demonstrates the potential of this method for providing cDNAs of viral genomes and simplifying the construction of infectious clones.
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PMID:Amplification of the full-length hepatitis A virus genome by long reverse transcription-PCR and transcription of infectious RNA directly from the amplicon. 863 73

Bioactivities of 42 didemnin congeners, either isolated from the marine tunicates Trididemnun solidum and Aplidium albicans or prepared synthetically and semisynthetically, have been compared. The growth inhibition of various murine and human tumor cells and plaque reduction of HSV-1 and VSV grown on cultured mammalian cells were used to assess cytotoxicity and antiviral activity. Biochemical assays for macromolecular synthesis (protein, DNA, and RNA) and enzyme inhibition (dihydrofolate reductase, thymidylate synthase, DNA polymerase, RNA polymerase, and topoisomerases I and II) were also performed to specify the mechanisms of action of each analogue. Immunosuppressive activity of the didemnins was determined using a mixed lymphocyte reaction (MLR) assay. These assays revealed that the native cyclic depsipeptide core is an essential structural requirement for most of the bioactivites of the didemnins, especially for cytotoxicities and antiviral activities. The linear side-chain portion of the peptide can be altered with a gain, in some cases, of bioactivities. In particular, dehydrodidemnin B, tested against several types of tumor cells and in in vivo studies in mice, as well as didemnin M, tested for the mixed lymphocyte reaction and graft vs host reaction in murine systems, showed remarkable gains in their in vitro and in vivo activities compared to didemnin B.
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PMID:Structure--activity relationships of the didemnins. 870 12

We have selected and plaque purified a mutant of feline immunodeficiency virus (FIV) that is resistant to 2',3'-dideoxycytidine (ddC). This mutant was selected in cultured cells in the continuous presence of 25 microM ddC. The mutant, designated DCR-5c, was fourfold resistant to ddC, threefold resistant to 2',3'-dideoxyinosine, and more than fourfold resistant to phosphonoformic acid. DCR-5c displayed little or no resistance to (-)-beta-2',3'-dideoxy-3'-thiacytidine, 3'-azido-3'-deoxythymidine, or 9-(2-phosphonylmethoxyethyl) adenine. Reverse transcriptase purified from DCR-5c was less susceptible to inhibition by ddCTP, phosphonoformic acid, ddATP, or azido-dTTP than the wild-type FIV reverse transcriptase. Sequence analysis of DCR-5c revealed a single base change (G to C at nucleotide 2342) in the reverse transcriptase-encoding region of FIV. This mutation results in substitution of His for Asp at codon 3 of FIV reverse transcriptase. The role of this mutation in ddC resistance was confirmed by site-directed mutagenesis.
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PMID:Selection and characterization of a mutant of feline immunodeficiency virus resistant to 2',3'-dideoxycytidine. 884 58

In the present study the effect of the attenuated strain TC-83 of the Venezuelan Equine Encephalitis virus on the nuclear transcription in brain cells of rats was assessed. The transcription activity of the DNA depending RNA polymerases (types I and II) in the isolated nuclei of brain of infected rats and controls was determinated by incorporation of the (3H) UTP. Simultaneously a viral replication curve in the brain and the serum was carried out by plaque forming method in chicken embryo cell cultures. RNA polymerase I activity was only significantly reduced after 25 hours of infection, respect to control values, while polymerase II activity was progressive and significantly diminished from inicial stages of the viral infection at 10, 15, 20 y 25 hours post-infection compared to control values. The virus was not detected in the brain but after 25 hours post-infection with very low titers (< 0.7 log10 P.F.U./ml.), while the viral presence in the blood was demonstrated after a 10 hour period. Our results demonstrated a marked effect of the attenuated strain on the brain nuclear transcription, although the presence of the virus was not detected in the brain of the infected rats. This finding suggest a mechanism of action which deserves further studies to elucidate the cerebral metabolic response and the pathogenesis of the Venezuelan Equine Encephalitis infection.
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PMID:[Effect of the attenuated strain (TC-83) of Venezuelan equine encephalitis virus on nuclear transcription in rat brain cells]. 892 28

Psoriasis is a hyperproliferative inflammatory disease and 70% of patients develop a chronic plaque form. The pathogenesis of psoriasis is not known but evidence exists that T cells play a crucial role. The T cell V-gene receptor repertoire from psoriasis skin (different layers) was compared with peripheral blood T cells by employing RNA polymerase chain reaction (PCR) amplification. T cell receptor (TCR) BV 5.1, 11, 12, 13.1 and 16 were utilized to a significantly higher degree in areas close to the basal layers when compared to CD4+, CD8+ or unfractionated blood T cells from the same patients, whereas only BV11 and 13.1 genes of T cells from deeper layers of the dermis showed such a skewed usage. No biased usage of TCRBV genes was observed in superficial layers or in whole skin. Furthermore, T cell receptor junctional diversity analysed by high resolution gel electrophoresis showed skin psoriatic T cells to be poly- or oligoclonal. In conclusion, we show that TCRBV gene usage from different layers of psoriatic skin has a different pattern compared with the corresponding gene usage in circulating peripheral blood T cells. This pattern may implicate possible skin-associated antigen or superantigens activating a limited number of T cells in areas of skin close to basal layers, which in turn could promote keratinocyte proliferation.
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PMID:RT-PCR topography of chronic psoriasis skin based on analysis of T-cell receptor B variable region gene usage. 916 99

Gene transfer or gene therapy has advantages in the treatment of a variety of disorders due to its selective expression within specific mammalian cells. Interferon-alpha (IFN-alpha) has been used in the management of leukemia but its diverse adverse activities with multiple potential side effects, possibly unrelated to therapeutic targets, may negatively influence the ability of IFN-alpha to treat this disorder. Therefore, we examined the ability of adenovirus (Ad)-IFN-alpha gene construct to transfect normal (CD34+ cells) and chronic myelogenous leukemia (CML) bone marrow mononuclear cells (BMMNC) and the transient overexpression of IFN-alpha in these cells. Ad-cytomegalovirus promoter driven IFN-alpha (AdCMV-IFN-alpha) at multiple doses was assessed to transfect highly purified CD34+ cells in liquid culture, and optimal transduction of CD34+ cells was achieved using 120 plaque forming units. Flow cytometric determinations revealed that there was no significant difference in cell viability for the 4 h or 24 h transfection periods. Immunoassay of IFN-alpha produced by CD34+ cells shows that IFN-alpha levels increased several fold in transfected cells. Transient expression of the IFN-alpha gene did not suppress proliferation of CD34+ progenitors as indicated by BFU-E or colony forming units-granulocyte-macrophage (CFU-GM) growth. Reverse transcriptase/polymerase chain reaction analysis of RNA from CD34+ harvested CFU-GM progenitor cells demonstrated transient IFN-alpha mRNA expression. Similarly, CML BMMNC were transfected with AdCMV-IFN-alpha under similar conditions as described for CD34+ cells. BMMNC cells exposed to adenovirus for 24 h and 48 h were found to express IFN-alpha at a substantial level. This in vitro data suggest that Ad-mediated gene transfer of IFN-alpha into hematopoietic stem cells can be achieved and that the IFN-alpha gene can be translated into its specific mRNA in CD34 progenitor cells.
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PMID:Adenovirus mediated alpha interferon (IFN-alpha) gene transfer into CD34+ cells and CML mononuclear cells. 2739 20

A genetic engineering approach was made to generate a recombinant non-segmented negative-strand RNA virus, Sendai virus (SeV) of the family Paramyxoviridae, that expresses firefly luciferase. The DNA construct containing the entire open reading frame (ORF) of the luciferase gene followed by the SeV transcription stop and restart signals connected with the conserved intergenic three nucleotides was inserted immediately before the ORF of the viral 3'-proximal nucleocapsid (N) protein gene in a full-length SeV cDNA copy. After intracellular expression of full-length antigenomic transcripts from the engineered cDNA and of the viral n ucleocapsid protein and RNA polymerase from the respective plasmids, a recombinant SeV expressing luciferase activity at a high level was recovered, although the tendency of this particular reporter gene product to aggregate in cells made it difficult to estimate the maximum level of expression. The increase in genome length brought about by inserting 1728 nucleotides into the 15,384 nucleotide parental SeV was associated with reduced plaque size, slightly slower replication kinetics and a severalfold decrease in yield of the virus. The inserted luciferase gene was stably maintained after numerous rounds of replication by serial passages in chick embryos. These results indicate the potential utility of SeV as a novel expression vector.
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PMID:Creation of an infectious recombinant Sendai virus expressing the firefly luciferase gene from the 3' proximal first locus. 936 67

The N terminal amino acid of nonstructural protein nsP4, the viral RNA polymerase, is a tyrosine in all sequenced alphaviruses; this is a destabilizing amino acid for the N-end rule pathway and results in rapid degradation of nsP4 produced in infected cells or in reticulocyte lysates. We have constructed 11 mutants of Sindbis virus bearing Phe, Ala, Thr, Cys, Leu, Met, Asn, Gln, Glu, Arg, or Pro at the N terminus of nsP4. Translation of RNAs in reticulocyte lysates showed that cleavage at the nsP3/nsP4 site occurred efficiently for all mutants except for Glu-nsP4, which was cleaved inefficiently, and Pro-nsP4, which was not detectably cleaved, and that Tyr, Cys, Leu, Arg, and Phe destabilized nsP4 but Ala, Met, Thr, Asn, Gln, and Glu stabilized nsP4 to various extents. The viability of the mutants was examined by transfection of chicken cells at 30 or 40 degrees C. The Phe-nsP4 mutant formed large plaques at both temperatures. The Met-nsP4 mutant was also viable but formed small plaques at 30 degrees C and minute plaques at 40 degrees C. The remaining mutants did not form plaques at either temperature. However, after prolonged incubation at 30 degrees C, all the mutants except Glu-nsP4 and Pro-nsP4 produced viable viruses. In the case of Cys-, Leu-, Asn-, Gln-, or Arg-nsP4, revertants that were indistinguishable in plaque phenotype from the wild-type virus arose by same-site reversion to Tyr, Trp, Phe, or His by a single nucleotide substitution in the original mutant codon. Viable viruses also arose from the Ala-, Leu-, Cys-, Thr-, Asn-, Gln-, and Arg-nsP4 mutants that retained the original mutations at the N terminus of nsP4, but these viruses formed smaller plaques than the wild-type virus and many were temperature sensitive. Our results indicate that only nsP4s bearing N-terminal Tyr, Phe, Trp, or His have wild-type or near-wild-type activity for RNA replication and that rapid degradation of nsP4 is not a prerequisite for its function. nsP4s bearing other N-terminal residues, with the exception of Met-nsP4, have only very low or negligible activity, so that no detectable infectious virus can be produced. However, suppressor mutations can arise that enable most such nsP4s to regain significant but still suboptimal activity.
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PMID:Requirement for an aromatic amino acid or histidine at the N terminus of Sindbis virus RNA polymerase. 949 91


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