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)

We have examined the structures of unique sequence, A/T-rich DNAs that are predicted to be relatively rigid [oligo(dA).oligo(dT)], flexible [oligo[d(A-T)]], and curved, using the hydroxyl radical as a cleavage reagent. A 50-base-pair segment containing each of these distinct DNA sequences was placed adjacent to the T7 RNA polymerase promoter, a sequence that will strongly position nucleosomes. The final length of the DNA fragments was 142 bp, enough DNA to assemble a single nucleosome. Cleavage of DNA in solution, while bound to a calcium phosphate crystal, and after incorporation into a nucleosome is examined. We find that the distinct A/T-rich DNAs have very different structural features in solution and helical periodicities when bound to a calcium phosphate. In contrast, the organization of the different DNA sequences when associated with a histone octamer is very similar. We conclude that the histone core exerts a dominant constraint on the structure of DNA in a nucleosome and that inclusion of these various unique sequences has only a very small effect on overall nucleosome stability and structure.
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PMID:The histone core exerts a dominant constraint on the structure of DNA in a nucleosome. 165 13

The neural cell adhesion molecule (N-CAM) is an important mediator of calcium independent cell-cell interactions. Variations in the primary structure of the protein are due to alternative splicing of pre-mRNA in the region encoding the extracellular, trans-membrane and cytoplasmic domains. In order to identify the patterns of exon usage during development of skeletal muscle and brain of the mouse, a coupled reverse-transcriptase/polymerase chain reaction was used to identify the murine homologues of the muscle-specific domain (MSD), located between exons 12 and 13 in human N-CAM mRNA. The cDNAs produced have been cloned and sequenced, or analysed directly. The amplification reactions were shown to maintain the concentration ratios of the initial cDNAs. The results indicate that the mouse homologue to exon MSD1a is under tissue and developmental regulation that is independent of exons MSD1b and MSD1c. The inclusion of the triplet exon AAG is also regulated in a cell- and stage-specific manner, which is independent of the other alternatively spliced exons of this domain.
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PMID:The muscle specific domain of mouse N-CAM: structure and alternative splicing patterns. 171 58

We have investigated the organization and expression of the Caulobacter crescentus flbF gene because it occupies a high level in the flagellar gene regulatory hierarchy. The nucleotide sequence comprising the 3' end of the flaO operon and the adjacent flbF promoter and structural gene was determined, and the organization of transcription units within this sequence was investigated. We located the 3' ends of the flaO operon transcript by using a nuclease S1 protection assay, and the 5' end of the flbF transcript was precisely mapped by primer extension analysis. The nucleotide sequence upstream from the 5' end of the flbF transcript contains -10 and -35 elements similar to those found in promoters transcribed by sigma 28 RNA polymerase in other organisms. Mutations that changed nucleotides in the -10 or -35 elements or altered their relative spacing resulted in undetectable levels of flbF transcript, demonstrating that these sequences contain nucleotides essential for promoter function. We identified a 700-codon open reading frame, downstream from the flbF promoter region, that was predicted to be the flbF structural gene. The amino-terminal half of the FlbF amino acid sequence contains eight hydrophobic regions predicted to be membrane-spanning segments, suggesting that the FlbF protein may be an integral membrane protein. The FlbF amino acid sequence is very similar to that of a transcriptional regulatory protein called LcrD that is encoded in the highly conserved low-calcium-response region of virulence plasmid pYVO3 in Yersinia enterocolitica (A.-M. Viitanen, P. Toivanen, and M. Skurnik, J. Bacteriol. 172:3152-3162, 1990).
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PMID:Characterization of the Caulobacter crescentus flbF promoter and identification of the inferred FlbF product as a homolog of the LcrD protein from a Yersinia enterocolitica virulence plasmid. 173 19

A putative ATPase gene was cloned from Trypanosoma brucei genomic DNA. The length of the gene open reading frame is 3,033 bp, predicting a protein of about 110 kDa. The sequence of this protein shares 10 blocks of homology with other eukaryotic ATPases, including the putative phosphorylation site characteristic of P-ATPases. Its hydropathy profile reveals 8-10 potential membrane-spanning regions. While the amino acid sequence of the T. brucei ATPase shows only 25% overall homology with its counterpart from the related kinetoplastid protozoan Leishmania donovani, 49% sequence conservation is found when compared with the calcium-ATPase from rabbit sarcoplasmic reticulum. This gene is present in only one copy, localized in the large chromosome fraction. It is transcribed at a similar level in procyclic and bloodstream forms, as a 4.3-kb mRNA. Run-on assays suggest continuous transcription of the gene and flanking sequences over at least 10 kb, by a RNA polymerase sensitive to alpha-amanitin. Transcription inhibition by UV irradiation suggests that the ATPase gene is more than 4 kb downstream from its promoter.
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PMID:Structure and transcription of a P-ATPase gene from Trypanosoma brucei. 183 43

The expression of genes that code for the large ribosomal RNAs (rRNAs) and tRNAs can be regulated by calcium, serum, insulin and a tumor-promoting phorbol ester, TPA. These effectors can rapidly alter rRNA and tRNA synthesis in dividing and nondividing Drosophila cells. In an in vitro assay system of the nondividing cells of the male accessory glands, calcium, insulin and TPA were shown to increase both rRNA and tRNA synthesis. Exposure of actively dividing Drosophila culture cells to differing serum concentrations or TPA also altered rRNA and tRNA synthesis. Nuclear run-on assays demonstrate that the exposure of these cells to increased serum concentrations coordinately alters RNA polymerase I loading on both 18S and 28S rDNA. These data indicate that calcium, growth factors and a tumor-promoter each can signal changes in ribosomal and tRNA gene expression.
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PMID:Serum, insulin and phorbol esters stimulate rRNA and tRNA gene expression in both dividing and nondividing Drosophila cells. 192 1

A method for the in vitro uncoating of the phi 6 nucleocapsid (NC) was developed. The resulting particle, designated as the NC core, containing the genomic double-stranded (ds) RNA segments and the proteins P1, P2, P4 and P7, was not infectious but had a highly enhanced in vitro transcriptase activity compared to that of the intact NC. The NC shell protein P8 was purified by immunoaffinity chromatography, and it was shown to self-assemble to shell-like structures upon addition of calcium ions. The conditions for the self-assembly of the shell were optimized. Shell reassembly on to the NC cores restored the infectivity but resulted in a decrease of transcriptase activity. No reassembly of the shell on to RNA-less cores (procapsids) produced from a cDNA construction in Escherichia coli was observed. Our results suggest that the intracellular uncoating of the NC is the event activating the phi 6 dsRNA transcriptase and that the NC shell is necessary for infectivity, probably for the passage of the NC through the host cytoplasmic membrane. Packaging of the dsRNA segments into the procapsid appears to be a prerequisite for NC shell assembly.
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PMID:Generation of infectious nucleocapsids by in vitro assembly of the shell protein on to the polymerase complex of the dsRNA bacteriophage phi 6. 201 47

Using a complementation assay, we have evaluated the potential of two eukaryotic expression systems to produce functional virus proteins. The first expression system was based on a bovine papilloma virus (BPV) eukaryotic expression vector which contained a copy of the gene for the membrane glycoprotein G of vesicular stomatitis virus (VSV). This vector was transfected into a mouse cell line, and transformed cell clones constitutively expressing VSV G protein were selected. These cell clones were then screened for their ability to support the replication of a temperature-sensitive G mutant of VSV (tsO45) at the permissive and nonpermissive temperatures. A 100-fold increase in tsO45 titer was observed in some of the G protein-producing cell lines in comparison with nonproducing cells. These results were compared with complementation by VSV G protein expressed from a second expression system utilizing a vaccinia virus (VV) recombinant which produced bacteriophage T7 RNA polymerase. T7 RNA polymerase expressed in cells infected with the vaccinia recombinant produced VSV G transcripts from a plasmid which had been transfected into these cells. This plasmid contained the VSV G gene cloned between T7 RNA polymerase initiation and termination signals. VSV G protein expressed by this system was able to complement tsO45 replication at the nonpermissive temperature, and yielded much greater levels of complemented virus than the BPV system. When calcium phosphate-mediated transfection was used to introduce the VSV G plasmid vector into cells infected with the VV recombinant, a complementation efficiency as high as 1500-fold was obtained. Using lipofectin-mediated transfection, a 15,000-fold increase in virus titer could be obtained in G protein-producing cells in contrast to nonproducing cells. At the nonpermissive temperature, yields of temperature-sensitive virus were within 10-fold of the yields obtained at the permissive temperature. Virus produced in this system was shown to be a pseudotype which contained wild-type G protein in the viral envelope but still maintained the temperature-sensitive genotype. This expression system will be used to study the extent to which the integrity of the G coding sequence of wild-type VSV might be altered in the absence of selection pressure for functional G protein during VSV replication.
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PMID:Complementation of a vesicular stomatitis virus glycoprotein G mutant with wild-type protein expressed from either a bovine papilloma virus or a vaccinia virus vector system. 217 Nov 87

A cDNA clone containing the entire coding region for bovine pre-alpha-lactalbumin (LA) together with 27 base pairs of 5'-noncoding and 268 base pairs of 3'-noncoding sequences was isolated from a bovine mammary cDNA plasmid library in the Okayama-Berg vector system using a synthetic oligonucleotide probe and sequenced. The coding segment for mature LA was subcloned into the T7 expression system of Studier and co-workers (Studier, F.W., and Moffatt, B.A. (1986) J. Mol. Biol. 189, 113-130; Rosenberg, A.H., Lade, B.N., Chui, D.S., Lin, S.W., Dunn, J.J., and Studier, F.W. (1987) Gene (Amst.) 56, 125-135) and expressed as a 21-kDa fusion protein that consisted of the mature bovine LA sequence connected to the NH2-terminal 50 residues of human cathepsin D by a linker sequence containing protease cleavage sites. This fusion protein was expressed in an insoluble form and accumulated to about 50% of the total bacterial protein within 3 h after induction of T7 RNA polymerase synthesis. The protein was solubilized, purified by gel filtration, and converted to an active form by treatment with mixtures of reduced and oxidized glutathione in the presence of Ca2+. The maximum specific activity of the fusion protein was about 25% of that of native LA, suggesting that the attachment of an NH2-terminal extension sterically hinders but does not prevent the interaction with galactosyltransferase. The extension also does not block the binding of the regulatory Ca2+ ion that is required for folding from the reduced denatured state. Trypsin cleaved the folded fusion protein specifically at a Lys-Glu bond at the junction with the mature LA sequence to give a product indistinguishable in structure and activity from native LA.
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PMID:Recombinant bovine alpha-lactalbumin obtained by limited proteolysis of a fusion protein expressed at high levels in Escherichia coli. 268 74

The many different cellular functions so far shown to involve actin and to be regulated by specific actin binding proteins are located primarily, if not exclusively, in the cytoplasm. Actin is also found in the nucleus of various cells, but because of the problems of cell fractionation the significance of nuclear actin has remained unclear. The large amphibian oocyte nucleus (germinal vesicle), however, can be isolated manually with little cytoplasmic contamination. This nucleus contains high concentrations (4-6 mg ml-1) of mostly soluble, although polymerization-competent beta- and gamma-actin, which exists in a nucleocytoplasmic exchange pool. The findings that drastic effects on transcription and chromosome morphology are caused by the injection of actin antibodies or actin binding proteins into germinal vesicles, and that a factor required for accurate transcription by RNA polymerase II is actin, suggest that nuclear actin is involved in specific nuclear functions. We have recently identified two main components in Xenopus laevis oocytes with actin binding activities; one of these activities is Ca2+-dependent, is located predominantly, if not exclusively, in the cytoplasm and is attributable to gelsolin. Here we report that the second component, having a Ca2+-independent activity, is a heterodimeric acting binding protein; this protein is markedly enriched in the nuclei of oocytes and somatic cells of amphibia, but also occurs in nuclei of other vertebrate cells.
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PMID:Identification of a widespread nuclear actin binding protein. 268 84

Protein kinase C (PKC) was purified to near homogeneity from human leukemia ML-1 cells. The purified enzyme showed a single polypeptide band of 80 kDa on SDS-polyacrylamide gel after electrophoresis, and was totally dependent on Ca2+/phospholipid for activity. Diacylglycerol and the tumor-promoting on Ca2/phospholipid for activity. Diacylglycerol and the tumor-promoting phorbol esters stimulated the enzyme activity. Autophosphorylation of PKC purified from phenyl-Sepharose column showed both 80- and 37 kDa polypeptides. Further fractionation of PKC on a hydroxyapatite column revealed two peaks of enzyme activity, indicating that there may be two different forms of protein kinase C present in human leukemia cells. The purified PKC was used to phosphorylate RNA polymerase II of human leukemia cells in vitro and the autoradiogram showed that RNA polymerase II large subunits (240, 220 and 150 kDa) were phosphorylated in a time-dependent manner.
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PMID:Isolation and purification of protein kinase C from human leukemia ML-1 cells phosphorylation of human leukemia RNA polymerase II in vitro. 275 42


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