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)

Alpha-1 tubulin is the principal alpha-tubulin isotype found in the flagella of the unicellular green alga, Chlamydomonas reinhardii. Although the pattern of tubulin mRNA accumulation and utilization has been examined in some detail in Chlamydomonas (Lefebvre and Rosenbaum 1986), the transcriptional mechanisms establishing tubulin mRNA levels are not understood. To begin an analysis of the alpha-1 tubulin gene transcriptional control elements, we studied a number of promoter mutants of this gene from Chlamydomonas. These mutants, assayed by injection into Xenopus oocyte nuclei, delimit the promoter to 36 bp of DNA upstream of the cap site and 73 bp of the untranslated mRNA leader. A major rate-controlling element lies in a short GC-rich sequence positioned between the TATA homology and the mRNA cap site (position + 1). A similar sequence motif has been found in the same position upstream of all four tubulin genes of Chlamydomonas (Brunke et al. 1984). A 10 bp linker insertion within this sequence abolishes transcription. A far upstream sequence, located in a fragment between -400 and -800, is an efficiency element, whose deletion inhibits transcription in vivo by about 30%. The upstream element (ue) also has the unique ability to drive RNA polymerase II (RNAPII) transcription in vivo when isolated from all downstream promoter elements, unlike any control element described to date. These results suggest that a sequence within the upstream element is an entry site for RNAPII into the tubulin transcription unit.
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PMID:Novel control elements in the alpha-1 tubulin gene promoter from Chlamydomonas reinhardii. 323 8

Two antibody single-chain Fv (scFv) fragments carrying five C-terminal histidine residues were expressed in Escherichia coli as periplasmic inclusion bodies. Their variable heavy (VH) and light (VL) domains are derived from the mouse monoclonal antibody 215 (MAb215), specific for the largest subunit of RNA polymerase II of Drosophila melanogaster and rat MAb Yol1/34, specific for pig brain alpha-tubulin. ScFv-215 contains an additional cysteine residue near to its C-terminus. After solubilization of inclusion bodies followed by immobilized metal affinity chromatography (IMAC) in 6M urea and a renaturation procedure, scFv monomers, noncovalent dimers, and aggregated antibody fragments were separated by size exclusion chromatography. In addition, a fraction of disulfide-bonded scFv-215 homodimers (scFv')2 was also isolated. The various antibody forms appear to be in equilibrium after renaturation since first peak composed mainly of aggregates could be resolved into a similar pattern of aggregates, dimers, and monomers after repeating the denaturation/renaturation procedure. All fractions of the recombinant scFv-215 demonstrated high antigen-binding activity and specificity as shown by enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Affinity measurements carried out by competitive immunoassays showed that covalently linked (scFv')2 have binding constants quite close to those of the parental MAbs and fourfold higher than scFv' monomers. ScFv derivatives, specifically biotinylated through the free sulfhydryl group, recognize the corresponding antigen in ELISA and Western blot analysis, thus demonstrating the possibility of using chemically modified scFv antibodies for immunodetection.
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PMID:Bacterial expression and refolding of single-chain Fv fragments with C-terminal cysteines. 852 51

Microtubules play an essential role in cell division. Little is known about possible variations of total tubulin and tubulin isotype expression during the cell cycle. We analyzed the total tubulin content, tubulin polymerization status and tubulin isotype content in resting and dividing human K562 leukemic cells and human MES-SA sarcoma cells. Although the total cellular tubulin content increases as the cells progress toward mitosis, the total tubulin/total protein ratio is stable during the cell cycle. Reverse transcriptase-polymerase chain reaction was applied to analyze the levels of expression of alpha, beta, and gamma-tubulin isotypes. Whereas alpha-tubulin isotype and gamma-tubulin transcripts were found to be expressed at constant levels throughout the cell cycle, some of the beta-tubulin isotype transcripts were found to be more highly expressed in dividing then in resting cells. Both of the class IV beta-tubulin isotype transcripts (human 5 beta and beta 2, Class IVa and IVb, respectively) were expressed in dividing K562 and MES-SA cells at twice the levels found in resting cells. Increased expression of the class IV isotype proteins in dividing cells was confirmed by immunoblotting, both in K562 and in MES-SA cells. A larger fraction of total cell tubulin was found to be polymerized in dividing cells (36-40%) than in resting cells (27-30%). The degree of polymerization of class IV tubulin in dividing and resting cells was similar to that of total tubulin. These results show that total tubulin is expressed as constant levels throughout the cell cycle but that the degree of polymerization is increased as cells are committed to division. The relative overexpression of the two class IV beta-tubulin isotypes in dividing cells suggests functional specificity for these isotypes and a regulatory role of these isotypes on the microtubule network during mitosis.
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PMID:Differential expression of tubulin isotypes during the cell cycle. 887 65

A 1.3Mb chromosome 11-specific yeast artificial chromosome (YAC) that spans a t(1;11) translocation breakpoint associated with major psychosis has been used to enrich cDNAs that are encoded within it and expressed in the human foetal brain. Database analysis of the selected fragments led to the identification of 54 clones matching alpha-tubulin, 4 fragments matching two anonymous human expressed sequence tags (ESTs) and 8 fragments giving no database matches. The clones matching alpha-tubulin led to the identification of a novel alpha-tubulin locus located approximately 250 kb proximal to the translocation breakpoint. Extensive sequence and expression analysis of this locus suggests that this is a processed pseudogene, although a long open reading frame is maintained and the possibility that an abnormally acting protein may be expressed in a highly tissue or developmental specific manner cannot be discounted. The novel cDNA fragments map up to 700 kb proximal to the translocation breakpoint and are associated with potential CpG islands. Reverse transcriptase polymerase chain reaction (RT-PCR) expression analysis and high resolution genomic mapping suggest that they may comprise up to three novel genes. No major disruption of the identified fragments could be detected in the genomic DNA of translocation carriers. The psychosis associated with this translocation may therefore be due to position effects on the transcription of these genes or an involvement of translocated chromosome 1 sequences.
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PMID:Novel transcribed sequences neighbouring a translocation breakpoint associated with schizophrenia. 903 12

It has been long known that neoplastic transformation is accompanied by a lowered requirement for extracellular Ca2+ for growth. The studies presented here demonstrate that human fibroblastic cell lines produce the two commonly found 'housekeeping' isoforms of the plasma membrane Ca(2+)-ATPase (PMCA), PMCA1b and 4b, and at the expression of both is demonstrably lower in cell lines neoplastically transformed by SV40 than in the corresponding parental cell lines. Western blot analyses of lysates from control (GM00037) and SV40-transformed (GM00637) skin fibroblasts revealed a 138 kDa PMCA whose level was significantly lower in the SV40-transformed cells relative to either total cellular protein or alpha-tubulin. Similar analyses of plasma membrane preparations from control WI-38) and SV40-transformed (WI-38VA13) lung fibroblasts revealed 3-4-fold lower levels of PMCA in the SV40-transformed cells. Competitive ELISAs performed on detergent solubilized plasma membrane preparations indicated at least 3-4-fold lower levels of PMCA in the SV40-transformed cell lines compared to controls. Reverse transcriptase coupled-PCR analyses showed that PMCA1b and PMCA4b were the only isoforms expressed in all four cell lines. The PMCA4b mRNA level detected by Northern analysis also was substantially lower in SV40 transformed skin fibroblasts than in non-transformed fibroblasts. Quantitative RT-PCR analyses showed levels of PMCA1b and 4b mRNAs to be 5 and 10-fold lower, respectively, in GM00637 than in GM00037 when the levels of PCR products were normalized to glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA. These results demonstrate that the expression of these distinct PMCA genes is substantially lower in SV40 transformed human skin and lung fibroblasts and may be coordinately regulated in these cells.
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PMID:Analysis of plasma membrane Ca(2+)-ATPase expression in control and SV40-transformed human fibroblasts. 905 77

The experiments described herein were designed to determine whether tumor necrosis factor alpha (TNF-alpha) displays a diurnal variation in various areas of the normal rat brain. TNF-alpha mRNA transcripts were detected by reverse-transcriptase polymerase chain reaction. To monitor diurnal changes in TNF-alpha and alpha-tubulin expression, rats were sacrificed every 4 h for 24 h starting 1 h after light onset; relative mRNA levels were determined for the cerebellum, cortex, hippocampus, hypothalamus and brainstem. TNF-alpha mRNA was higher during the light than in the dark phase in the hypothalamus and hippocampus. alpha-Tubulin mRNA exhibited a similar diurnal variation in the hypothalamus, hippocampus and cortex. In contrast, beta-actin mRNA was lower during the light phase than the dark phase in the hippocampus and cortex. The observed diurnal variations in TNF-alpha mRNA are consistent with the hypothesis that TNF has a physiological role in the brain.
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PMID:Diurnal variations of tumor necrosis factor alpha mRNA and alpha-tubulin mRNA in rat brain. 948 99

The matrix (M) protein of vesicular stomatitis virus (VSV) functions in virus assembly and inhibits host-directed gene expression independently of other viral components. Experiments in this study were carried out to determine the ability of M protein to inhibit transcription directed by each of the three host RNA polymerases (RNA polymerase I [RNAPI], RNAPII, and RNAPIII). The effects of wild-type (wt) VSV, v6 (a VSV mutant isolated from persistently infected cells), and tsO82 viruses on poly(A)+ and poly(A)- RNA synthesis were measured by incorporation of [3H]uridine. v6 and tsO82 viruses, which contain M-gene mutations, had a decreased ability to inhibit synthesis of both poly(A)+ and poly(A)- RNA. Nuclear runoff analysis showed that VSV inhibited transcription of 18S rRNA and alpha-tubulin genes, which was dependent on RNAPI and RNAPII, respectively, but infection with wt virus enhanced transcription of 5S rRNA by RNAPIII. The effect of M protein alone on transcription by RNAPI-, RNAPII-, and RNAPIII-dependent promoters was measured by cotransfection assays. M protein inhibited transcription from RNAPI- and RNAPII-dependent promoters in the absence of other viral gene products. RNAPIII-dependent transcription of the adenovirus VA promoters was also inhibited by M protein. However, as observed during wt VSV infection, M protein enhanced endogenous 5S rRNA transcription, indicating that the inhibition of transcription by RNAPIII was dependent on the nature of the promoter.
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PMID:Effect of vesicular stomatitis virus matrix protein on transcription directed by host RNA polymerases I, II, and III. 973 95

Reverse-transcriptase polymerase chain reaction cloning of the 3'-ends of the alpha-tubulin cDNAs of Tritrichomonas mobilensis in combination with Southern-blot analysis identified seven to eight distinct alpha-tubulin genes. All seven lack a carboxy-terminal tyrosine and the corresponding sequence compatible with posttranslational tyrosination. This indicates that whereas tyrosination of alpha-tubulin has been found in most species, including humans and trypanosomes, it is absent in trichomonads.
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PMID:Alpha-tubulins of Tritrichomonas mobilensis are encoded by multiple genes and are not posttranslationally tyrosinated. 995 70

The use of double-stranded RNA (dsRNA) to disrupt gene expression has become a powerful method of achieving RNA interference (RNAi) in a wide variety of organisms. However, in Trypanosoma brucei this tool is restricted to transient interference, because the dsRNA is not stably maintained and its effects are diminished and eventually lost during cellular division. Here, we show that genetic interference by dsRNA can be achieved in a heritable and inducible fashion. To show this, we established stable cell lines expressing dsRNA in the form of stem-loop structures under the control of a tetracycline-inducible promoter. Targeting a-tubulin and actin mRNA resulted in potent and specific mRNA degradation as previously observed in transient interference. Surprisingly, 10-fold down regulation of actin mRNA was not fatal to trypanosomes. This type of approach could be applied to study RNAi in other organisms that are difficult to microinject or electroporate. Furthermore, to quickly probe the consequences of RNAi for a given gene we established a highly efficient in vivo T7 RNA polymerase system for expression of dsRNA. Using the alpha-tubulin test system we obtained greater than 98% transfection efficiency and the RNAi response lasted at least two to three cell generations. These new developments make it possible to initiate the molecular dissection of RNAi both biochemically and genetically.
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PMID:Genetic interference in Trypanosoma brucei by heritable and inducible double-stranded RNA. 1091 1

The recent advances on the cytoplasmic regulators of the induction of germinal vesicle break down, maturation and degeneration of oocytes, and glycosaminoglycan composition during cumulus expansion of cumulus-oocyte complexes are discussed. A) Inactive mitogen-activated protein kinases (MAPKs) are present in the oocytes at germinal vesicle (GV) stage, and are activated with germinal vesicle breakdown (GVBD), and remain highly active throughout maturation in porcine oocytes. Inactive MAPKs are localized in the cytoplasm of GV-arrested oocytes and active MAPKs were detected in the GV just before GVBD. B) Cumulus expansion of porcine cumulus-oocyte complexes (COCs) was reduced by oocy tectomy. The profile of total glycosaminoglycan synthesis was attributed to hyaluronic acid rather than chondroitin sulfate in intact COCs and oocytectomy reduced hyaluronic acid synthesis. C) The abnormalities of chromosomes and alpha-tubulin morphology were observed in the oocytes of c-mos deficient mice. MAPK activity of c-mos deficient oocytes did not significantly fluctuate throughout maturation and was clearly lower than that of wild-type oocytes. One of the most drastic abnormalities in c-mos knockout mouse oocytes was their entrance into the interphase instead of second meiosis after first polar body emission. D) Reverse transcriptase/polymerase chain reaction-Southern blot hybridization demonstrated positive expression of Fas in intraovarian mouse oocytes. In contrast, expression of Fas ligand was detected in granulosa cells. These findings were histologically confirmed by in situ hybridization with Fas- and FasL-specific probes. Co-culture of intact and zona-free eggs and granulosa cells demonstrated positive TUNEL staining only zona-free eggs.
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PMID:Morphological dynamics of cumulus-oocyte complex during oocyte maturation. 1131 42


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