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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The components required for specific transcription of ribosomal RNA were isolated from logarithmically growing Acanthamoeba castellanii. The transcription initiation factor fraction, TIF, and RNA polymerase I were extracted from whole cells at 0.35 M KCl. The extract was fractionated with polyethylenimine, then chromatographed on phosphocellulose (P11) which resulted in the separation of TIF from RNA polymerase I. The fractions containing TIF were further chromatographed on DEAE cellulose (DE52), Heparin Affigel, and Matrex green agarose, followed by sedimentation through glycerol gradients. TIF was purified approximately 17,000-fold, and shown to have a native molecular weight of 289 kD, and to bind specifically to rRNA promoter sequences by DNase I footprinting. The addition of homogeneous RNA polymerase I to this complex permitted the initiation of specific transcription in vitro. The phosphocellulose fractions containing RNA polymerase I were chromatographed on DEAE cellulose, Heparin-Sepharose, DEAE-Sephadex, and sedimented through sucrose gradients. Polymerase I was purified to apparent homogeneity with a yield of 8.1% and a specific activity of 315. It contained one fewer subunit than previously reported. DNase I protection experiments demonstrated that in both partially purified and homogeneous fractions, RNA polymerase I was capable of stable binding to the TIF-rDNA complex, and correctly initiating transcription on rDNA templates.
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PMID:Purification of components required for accurate transcription of ribosomal RNA from Acanthamoeba castellanii. 162 Jun 19

To identify the DNA sequences that cis-regulate the expression of the rat liver pyruvate kinase (L-PK) genes, a series of constructs in which the chloramphenicol acetyltransferase reporter genes is driven by various deleted fragments of the 3200 base pairs (bp) upstream of the L-PK gene cap site have been assayed for transient expression after introduction into hepatoma HepG2 cells, rat hepatocytes in primary culture, fibroblast LTK- cells, myogenic C2C12 cells, and CHO cells. Four distinct regulatory domains have been characterized. A proximal promoter region containing a binding site for the hepatocyte nuclear factor 1 (HNF1) which is sufficient to confer liver specificity, even in the presence of a ubiquitous enhancer. A distal promoter region (-96 to -283 bp) containing binding sites for the liver-specific factor A1 (LFA1), the ubiquitous nuclear factor 1 (NF1), the major late transcriptional factor (MLTF), and so far unidentified proteins binding to the L5-PK region which is essential to maximally activate expression of the construct in HepG2 cells. An extinguisher region, located between positions -2082 and -1170 bp, which decreases efficiency of the L-PK promoter in HepG2 cells, but not in hepatocytes in primary culture. Finally, a far upstream region (-2900 to -2500 bp) which seems to correspond to a liver-specific DNase I hypersensitive site and which behaves in HepG2 cells as an activating sequence efficient in the absence of the extinguisher.
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PMID:cis-acting DNA elements regulating expression of the liver pyruvate kinase gene in hepatocytes and hepatoma cells. Evidence for tissue-specific activators and extinguisher. 201 72

The fibrosarcoma IC9 is deficient in the expression of the major histocompatibility complex class I genes Kb, Kk, and Dk and expresses only the Db molecule. Because class I deficiency may enable tumor cells to escape the immune response by cytotoxic T lymphocytes, we investigated why the class I genes are not expressed. Expression of the silent class I genes could not be induced, but all known DNA-binding factors specific for class I genes could be detected in nuclear extracts of IC9 cells. After cloning of the silent Kb gene from the IC9 cells and subsequent transfection of this cloned Kb gene into LTK- and IC9 cells, normal Kb antigens were expressed on the cell surface of both cell lines. Digestion of the chromatin of IC9 cells with micrococcal nuclease and DNase I showed a decreased nuclease sensitivity of the silent class I genes in comparison with active genes and the absence of DNase I hypersensitive sites in the promoter region of the silent Dk gene. These findings demonstrate that class I expression is turned off by a cis-acting regulatory mechanism at the level of the chromatin structure.
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PMID:Major histocompatibility complex class I genes in murine fibrosarcoma IC9 are down regulated at the level of the chromatin structure. 250 38

PDGF has been shown to contribute to hypertrophy in vascular smooth muscle cells (VSMC). PDGF-AA differentially promotes protein synthesis in VSMC from spontaneously hypertensive rats (SHR) but not in those from Wistar-Kyoto rats (WKY). This observation has led us to postulate a role for PDGF alpha receptor (PDGFR-alpha) in the hypertensive hypertrophy of blood vessels. Western and Northern blot analyses demonstrated a high and specific expression of the PDGFR-alpha protein and mRNA in SHR cells but not in WKY cells. To clarify the mechanism of the differential expression of the PDGFR-alpha gene, we isolated the promoter region of the gene. Studies on the promoter functions indicated that this promoter is active in SHR cells but not in WKY cells. The regulatory domain responsible for this difference was narrowed to the sequence between -246 and -139, which enhanced the promoter activity of SHR fivefold over the basal activity. DNase I footprinting and gel-shift assay indicated that this sequence specifically interact with nuclear proteins from VSMC through the binding site for CCAAT/enhancer-binding proteins, and members of the C/enhancer-binding protein family play a significant role in the strain-specific transcription of the PDGFR-alpha gene.
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PMID:Molecular structure and transcriptional regulation of the gene for the platelet-derived growth factor alpha receptor in cultured vascular smooth muscle cells. 761 28

In hepatocytes, insulin-like growth factor-binding protein-1 (IGFBP-1) levels are increased by glucocorticoids and by agents that raise intracellular cAMP levels such as glucagon, theophylline, forskolin, and cAMP analogues. In contrast, insulin lowers IGFBP-1 levels, an effect dominant over the glucocorticoid and cAMP effects. Previous studies showed that dibutyryl cAMP (Bt2cAMP) and theophylline increase IGFBP-1 promoter activity in HEP G2 human hepatoma cells and that insulin abolishes this increase. In studies reported here, HEP G2 cells were used to further evaluate the role of cAMP in stimulating IGFBP-1 expression. Initial studies found that either 0.5 or 5.0 mM Bt2cAMP alone, or the combination of 0.5 mM Bt2cAMP and 2 mM theophylline, increased IGFBP-1 protein levels, mRNA levels, and promoter activity, but that the addition of theophylline to Bt2cAMP was required to give a approximately 5-fold increase in promoter activity. Deletion mutations of the IGFBP-1 promoter were used to show that much of the effect of Bt2cAMP and theophylline was conferred by the region between 269 and 246 base pairs (bp) 5' of the IGFBP-1 mRNA cap site. DNase I protection assays showed that HEP G2 nuclear extract footprinted the region from 273 to 249 bp 5' of the cap site; this region, designated P2, has a central CGTCA motif common to cAMP-responsive elements (CREs). Mutating the CGTCA motif in the 1205-bp IGFBP-1 promoter construct to TAGCA led to a 51% decrease in the ability of Bt2cAMP and theophylline to stimulate IGFBP-1 promoter activity above control levels. In addition, cotransfection of the catalytic subunit of cAMP-dependent protein kinase A (PKA) with the native 1205-bp IGFBP-1 promoter construct stimulated IGFBP-1 promoter activity 3.9-fold, but the TAGCA mutation decreased by 73% the ability of PKA to stimulate IGFBP-1 promoter activity above control levels. Mutating the CGTCA motif to TAGCA also blocked the ability of both crude HEP G2 nuclear extract and recombinant CRE-binding protein to bind to the P2 element. These data suggest that the P2 element is a CRE that confers at least part of the stimulatory effect of cAMP on the human IGFBP-1 promoter.
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PMID:Identification of a promoter element which participates in cAMP-stimulated expression of human insulin-like growth factor-binding protein-1. 768 58

Promoter elements accounting for HER2 (c-erbB-2/neu) overexpression were searched for in several human breast cancer cell lines (MDA-453, BT-474, ZR-75-1, MCF-7) known to express constitutively a 30-fold range in HER2 transcripts per gene copy. HER2 overexpressing cells showed a single prominent DNase I hypersensitive site near a conserved and hitherto unrecognized ets response element (GAGGAA), located 38 bases down-stream from the CAAT box and directly 5' of the TATA box in the human HER2 promoter. Transient transfection of HER2 promoter constructs (0.125, 0.5, and 2.0 kilobase pairs (kb)) demonstrated that the most proximal promoter region (0.125 kb) was capable of conferring up to 30-fold enhanced activity in HER2-overexpressing cell lines relative to low HER2-expressing control lines. Site-directed mutagenesis of the ets response element (GAGGAA-->GAGAGA) caused a > or = 60% reduction in promoter activity affecting at least 0.5 kb of upstream HER2 regulatory sequence. Gel-shift assays with nuclear extracts and oligonucleotide sequences spanning the 0.125-kb promoter region detected an ETS-immunoreactive complex, present most abundantly in cells overexpressing HER2, whose high-affinity binding depended on the GAGGAA response element. Methylation interference confirmed the ETS-specific pattern of protein binding by this complex to guanine bases in the ets response element. UV cross-linking and immunoprecipitation implicate a approximately 60-kDa ETS protein, and candidate ETS genes expressed in these breast cancer cells include GABP alpha, elk-1, elf-1, and PEA3.
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PMID:Binding of an ETS-related protein within the DNase I hypersensitive site of the HER2/neu promoter in human breast cancer cells. 791 92

Overexpression of the ERBB2 gene in human breast cancer is associated with a poor prognosis and resistance to hormonal treatment and chemotherapy. Oestrogen receptor (ER) positive tumour-derived cell lines are known to express relatively low levels of ERBB2 protein under oestrogenic conditions, but markedly higher levels following withdrawal of oestrogens or administration of tamoxifen. Expression of the closely related ERBB3 gene, which co-operates with ERBB2 in cellular transformation, is now shown to respond to oestrogenic manipulation in a similar way, both responses being mediated largely by transcriptional changes. Six previously undescribed DNase I hypersensitive sites occur within the first intron of ERBB2 in cells that overexpress the gene. A 409 base pair DNA fragment containing one of these sites conferred ER dependent oestrogen inhibition on the ERBB2 promoter in two types of transient transfection assay. DNase I footprinting revealed four separate transcription factor binding sites within this fragment consistent with a role as a transcriptional enhancer. These findings implicate intron 1 sequences in the control of ERBB2 expression for the first time and demonstrate that one site within this region is involved in mediating the transcriptional response to oestrogens. Additionally, there is likely to be synergism between ERBB2 and ERBB3 signalling when both are overexpressed in response to oestrogen inhibition, thereby driving transformed cell behaviour.
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PMID:An intron 1 enhancer element mediates oestrogen-induced suppression of ERBB2 expression. 924 84

In recent years, significant progress has been made toward understanding skeletal muscle development. However, the mechanisms that regulate smooth muscle development and differentiation are presently unknown. To better understand smooth muscle-specific gene expression, we have focused our studies on the smooth muscle myosin heavy chain (SMHC) gene, a highly specific marker of differentiated smooth muscle cells. The goal of the present study was to isolate and characterize the mouse SMHC gene promoter, since the mouse promoter would be particularly suited for in vivo promoter analyses in transgenic mice and would serve as a tool for targeting genes of interest into smooth muscle cells. We report here the isolation and characterization of the mouse SMHC promoter and its 5' flanking region. DNA sequence analysis of a 2.6-kb portion of the promoter identified several potential binding sites for known transcription factors. Transient transfection analysis of promoter deletion constructs in primary cultures of smooth muscle cells showed that the region between -1208 and -1050 bp is critical for maximal SMHC promoter activity. A comparison of SMHC promoter sequences from mouse, rat, and rabbit revealed the presence of a highly conserved region located between -967 and -1208 bp. This region includes three CArG/CArG*-like elements, two SP-1 binding sites, a NF-1-like element, an Nkx2-5 binding site, and an Elk-1 binding site. Gel mobility shift assay and DNase I footprinting analyses show that all three CArG/CArG*-like elements can form DNA-protein complexes with nuclear extract from vascular smooth muscle cells. Protein binding to the CArG* elements can be competed out by either serum response element or by an authentic CArG element from the cardiac alpha-actin gene. Using a serum response factor (SRF) antibody, we demonstrate that SRF is part of the protein complex. In addition, we show that cotransfection with the SRF dominant-negative mutant expression vector abolishes SMHC promoter activity, suggesting that SRF protein plays a critical role in SMHC gene regulation.
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PMID:Evolutionarily conserved promoter region containing CArG*-like elements is crucial for smooth muscle myosin heavy chain gene expression. 952 61

The Leydig insulin-like gene (Ley I-L), a member of the insulin-related gene family, is specifically expressed in pre- and postnatal Leydig cells of the testis and in postnatal theca cells of the ovary. To determine the functional region of the mouse Ley I-L promoter and factors controlling the Ley I-L gene expression, we used 2.1 kb of the 5'-flanking region of the mouse Ley I-L gene to generate chimeric constructs with the chloramphenicol acetyltransferase gene (CAT). Transient transfections of MA10 Leydig cells, LTK- fibroblasts, and F9 embryonic cells by a series of 5'-deleted mouse Ley I-L promoter-CAT constructs revealed that the sequence between nucleotides -157 to +4 directs the transcription of the reporter gene in MA10 but not in LTK- and F9 cells, indicating that the determinants of Leydig cell-specific expression reside within this region. Deoxyribonuclease I (DNase I) footprint analysis revealed that the sequences designated SF-1/1, SF-1/2, and SF-1/3 within three DNase I-protected regions are homologous to the consensus binding site of the steroidogenic factor-1 (SF-1). Competition and antibody studies showed that the three SF-1-binding sites in the Ley I-L promoter have similar binding affinities for SF-1. Furthermore, transient transfections of MA10 cells with mutant reporter constructs, in which SF-1/1 or both SF-1/2 and SF-1/3 were deleted, demonstrated that all three SF-1-binding sites are required for SF-1-mediated stimulation of Ley I-L transcription. Cotransfection of an SF-1-containing expression vector together with a Ley I-L promoter-CAT construct into HeLa cells, which lack the endogenous SF-1 protein, resulted in CAT gene transcription, which indicated that SF-1 can transactivate the Ley I-L promoter. These data demonstrate an essential role of SF-1 in transcriptional activation of the Ley I-L promoter.
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PMID:Transcription of the Leydig insulin-like gene is mediated by steroidogenic factor-1. 960 33

To characterize the human elk-1 promoter, we mapped the transcriptional start site and isolated elk-1-specific genomic phage clones that contained extensive upstream and downstream sequences. A TATA-like motif was identified immediately upstream of the transcriptional start site. Functional analyses of DNA fragments containing the TATA element and the identification of a DNase I-hypersensitive chromatin site (HS 1) in close proximity to the TATA box suggest that the identified TATA motif is important for elk-1 transcription in vivo. Sequences upstream and downstream from the TATA box were found to contribute to elk-1 promoter activity. A second hypersensitive site (HS 2) was identified within the first intron in pre-monocytic cells, which express Elk-1 only when differentiating to monocytes. In a variety of other cell types, which display a constitutive Elk-1 expression, HS 2 did not exist, suggesting that inducibility of elk-1 expression is associated with the presence of HS 2. Egr-1 and the serum response factor were found to interact specifically with the intronic sequence at +265 and +448, respectively. Because Egr-1 mRNA and protein levels were observed to increase significantly before induction of elk-1 expression, we propose that Egr-1 is important for the regulation of elk-1 transcription in differentiating monocytes.
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PMID:Characterization of the human elk-1 promoter. Potential role of a downstream intronic sequence for elk-1 gene expression in monocytes. 988 May 55


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