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At least 631 records · Page 35Linked to original sources
PubMed2002

Functional characterization of human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2 promoters, and comparison with the C1s promoter.

The 5'-flanking regions of the genes encoding human mannose-binding lectin-associated serine protease (MASP)-1/3 and MASP-2, key enzymes in the lectin complement pathway, were isolated and characterized. The features of their promoters were compared with those of the human gene for C1s, the effector component of the classical pathway. The sequences upstream from the transcription start sites of the three genes contained the elements essential for transcription and liver-specific expression. Transient expression of constructs of these genes fused to the luciferase reporter gene confirmed their liver-specific expression and showed that the MASP promoters were slightly up-regulated by the presence of IL-1beta. The stimulatory effects of IL-1beta on MASP1/3 and MASP2 gene expression were abolished by the simultaneous presence of IL-6. MASP-1/3 promoter activity was also down-regulated by IFN-gamma. In contrast, C1s promoter activity was strongly up-regulated by IL-6, IL-1beta and IFN-gamma. These results indicate that IL-6 and IFN-gamma affect the expression of the MASP genes in a different fashion from that of the C1s gene, implying differential regulatory effects of these cytokines on the biosynthesis of lectin pathway-specific serine proteases and classical pathway-specific serine proteases.

5' Flanking Region↗
PubMed1987

Specific cellular proteins bind to critical promoter sequences of the adenovirus early EIIa promoter.

As an approach to the identification of essential factors required for specific expression of the adenovirus type 2 EIIaE early (EIIaE) promoter, an in vitro system was established. Under appropriate conditions, using crude extracts of non-infected HeLa cells, efficient and accurate EIIaE expression has been reproduced. As in vivo, this transcription was strongly dependent upon the integrity of two non-consensus TATA-like elements, T1 and T2, corresponding to the major (EIIaE1) and minor (EIIaE2) start sites, respectively, as well as upon intact upstream elements (A, between -40 and -50 and B, between -70 and -90) common to both overlapping promoters. The implication of specific DNA-binding proteins in the transcriptional effects mediated by these elements was demonstrated by DNAse I footprinting analyses. Both crude nuclear extracts and partially purified fractions confer specific protection against DNAse I digestion to the T1 and B promoter elements defined above, and to a far upstream region (element C, between -110 and -150), which has previously been identified as a weaker promoter element by in vivo transcriptional studies. Separation of the T1 recognition factor from those which bind to the upstream elements B and C by chromatographic fractionation of the extracts has also been achieved.

Adenovirus Early Proteins↗
PubMed1987

Inducible gene expression from multiple promoters by the tumor-promoting agent, PMA.

Phorbol ester tumor promoters affect a broad scope of changes in mammalian cells. This report describes the activation of expression of an introduced chloramphenicol acetyltransferase (CAT) reporter gene by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), in a variety of fibroblast and hematopoietic cell lines. PMA-mediated activation appears to be promoter region specific, yet widespread. Enhanced gene expression is observed for four out of five promoter systems tested, and, in some cases, is dependent on the cellular environment. Further experiments indicate that PMA mediates elevated gene expression by rapidly increasing steady state levels of CAT mRNA. The broad range of promoters affected by PMA may help explain the high potency of this agent in tumor production.

Cell Line↗
PubMed1988

Functional activity of the two promoters of the myosin alkali light chain gene in primary muscle cell cultures: comparison with other muscle gene promoters and other culture systems.

Proximal upstream flanking sequences of the mouse myosin alkali light chain gene encoding MLC1F and MLC3F, the mouse alpha-cardiac actin gene and the chicken gene for the alpha-subunit of the acetylcholine receptor were linked to the bacterial chloramphenicol acetyl transferase (CAT) gene and transfected into primary cultures derived from mouse skeletal muscle or into myogenic cell lines. We demonstrate that the mouse MLC1F/MLC3F gene has two functional promoters. In primary muscle cultures, a 1200 bp sequence flanking exon 1 (MLC1F) and a 438 bp sequence flanking exon 2 (MLC3F) direct CAT activity in myotubes, but not in myoblasts or in non myogenic 3T6 and CV1 cells. Developmentally regulated expression is also seen with the alpha-cardiac actin (320 bp) and acetylcholine receptor alpha-subunit (850 bp) upstream sequences in the primary culture system. Transfection experiments with myogenic cell lines show different results with a given promoter construct, reflecting possible differences in the levels of regulatory factors between lines. Different muscle gene promoters behave differently in a given cell line, suggesting different regulatory factor requirements between these promoters.

Actins↗
PubMed1994

Rat argininosuccinate lyase promoter: the dyad-symmetric CCAAT box sequence CCAATTGG in the promoter is recognized by NF-Y.

The gene for rat argininosuccinate lyase (AL), which catalyzes the last step of arginine biosynthesis, is expressed in many tissues, though its expression is much higher in the liver where the enzyme is involved in the ornithine cycle (urea cycle), and moderately higher in the kidney. In transient transfection analysis using various cell lines, the AL promoter exhibited considerable levels of activity in all tested cell lines, with no apparent liver cell specificity, presumably reflecting the ubiquitous expression of this gene. Analysis of 5'-deletion mutants of the promoter region revealed several cis-regulatory elements, whose contribution to the promoter activity seemed to vary according to cell type. The most prominent positive regulatory region was detected around the position -80 base pairs, and it overlapped with a dyad-symmetric CCAAT box sequence CCAATTGG. A factor(s) interacting with this sequence element was found to be present ubiquitously. Gel shift competition analysis and antibody double shift analysis showed that this factor was identical or highly similar to a transcription factor, nuclear factor-Y (NF-Y), which consists of two different subunits and recognizes CCAAT motifs in various promoters. Point mutagenesis at each half site of the palindrome CCAATTGG and the introduction of a space between the half-site motifs abolished the binding with NF-Y. Thus, it seems that NF-Y recognizes the correctly spaced dyad-symmetric CCAAT box.

Animals↗
PubMedDate not supplied

Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering.

Chimeric phage-plasmid expression vectors were constructed from pUC18/19 plasmids by cloning a single-stranded DNA (ssDNA) origin of replication from bacteriophage f1 and inserting a bacteriophage T7 promoter within the beta-galactosidase gene. A T7 promoter permits in vivo or in vitro expression of single proteins by the translation of T7 RNA polymerase transcripts. Insertional inactivation of the T7 promoter-containing beta-galactosidase gene permits a simple blue-to-white color cloning assay. Compared with several helper phages that were examined, superinfection with M13K07 resulted in the highest yields of the pTZ plasmids as ssDNA viral particles. These ssDNA promoter plasmids are uniquely suited for protein engineering because they simplify cloning, oligonucleotide directed mutagenesis, verification by enzymatic sequence analysis, and expression of mutant proteins from a single vector. These vectors were utilized to eliminate an efficient transcriptional terminator of T7 RNA polymerase in the cDNA of bovine preproparathyroid hormone by oligonucleotide directed mutagenesis. The mutation changed the codon for phenylalanine-19 in the signal peptide to alanine. In a cell-free system the mutant cDNA transcripts were translated into preproparathyroid hormone, which was converted to proparathyroid hormone in the presence of microsomal membranes.

Animals↗
PubMed1999

The politics of health promotion: influences on public health promoting nursing practice in Ontario, Canada from Nightingale to the nineties.

The marked and significant differences in the various meanings ascribed to health promotion in professional literature provide evidence of the concept's evolution over the last half of the 20th century and testify both to the powerful influences of dominant ideologies and the invisibility of others. The "new public health" marks a return to a conceptualization of health that is consistent with a nursing paradigm and thus potentially useful in supporting nursing health promotion practice. To take full advantage of this knowledge, however, it is critical that nurses reclaim their legacy in health promotion, critically appraise outside influences that threaten to undermine their work, and educate the public and other disciplines about nursing's unique focus on health promotion.

Health Promotion↗
PubMed2003

Unique Epstein-Barr virus (EBV) latent gene expression, EBNA promoter usage and EBNA promoter methylation status in chronic active EBV infection.

Chronic active Epstein-Barr virus infection (CAEBV) has been considered to be a non-neoplastic T-cell lymphoproliferative disease associated with Epstein-Barr virus (EBV) infection. In EBV-associated diseases, the cell phenotype-dependent differences in EBV latent gene expression may reflect the strategy of the virus in relation to latent infection. We previously reported that EBV latent gene expression was restricted; EBV nuclear antigen 1 (EBNA1) transcripts were consistently detected in all spleen samples from five CAEBV patients, but EBNA2 transcripts were detected in only one sample. EBV latent gene expression is controlled by distinct usage of three EBNA promoters (Cp, Wp and Qp). In this study, we examined the EBNA promoter usage by RT-PCR and the methylation status in the Cp and Wp regions using bisulfite PCR analysis in spleen samples from CAEBV patients. EBNA1 transcripts were unexpectedly initiated not from Qp but from Cp in all samples in spite of the restricted form of latency. Furthermore, while Cp was active, Cp was heavily methylated, indicating that CAEBV has unique EBV latent gene expression, EBNA promoter usage and EBNA promoter methylation status, in part due to unique splicing of Cp-initiated transcripts and an activation mechanism in hypermethylated Cp.

Adult↗
PubMed1989

ASF-2: a factor that binds to the cauliflower mosaic virus 35S promoter and a conserved GATA motif in Cab promoters.

We have used nuclear extracts prepared from tobacco leaf tissue to characterize a factor binding site, designated as-2 (activating sequence-2), at the -100 region of the cauliflower mosaic virus 35S promoter. The activity of this factor, called ASF-2 (activating sequence factor-2), is not detected in tobacco root extracts. as-2 includes two GT motifs with sequence homology to the SV40 enhancer core A element and the Box II element of pea rbcS. Nevertheless, oligomers of these sequence elements do not compete for ASF-2 binding in gel retardation assays, indicating that the GT motifs may not be involved. Methylation interference studies identify two guanines (G93 and G98) that are required for interaction with ASF-2. Sequences surrounding these two critical guanines display homologies to a GATA repeat conserved among several light-responsive promoters. One such sequence from a petunia Cab promoter is able to compete with as-2 for factor binding. In transgenic plants, a tetramer of as-2 is able to confer leaf expression when fused 5' to the -90 derivative of the 35S promoter. The expression is not dependent on light and, thus, the as-2 tetramer does not function as a light-responsive element in this context. Histochemical localization of the reporter gene product suggests that the as-2 tetramer directs expression in trichomes, vascular elements, and epidermal and mesophyll cells.

Base Sequence↗
PubMed1991

Maize rbcS promoter activity depends on sequence elements not found in dicot rbcS promoters.

Although the molecular mechanisms of dicot photosynthetic gene regulation have been pursued actively, comparable studies of monocot regulation have been slow to come forth. We show here that monocot (maize and wheat) but not dicot (pea, tobacco, and Arabidopsis) ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) gene promoters are active in maize mesophyll protoplasts. The evolutionarily conserved GT and G boxes of dicot rbcS promoters are not essential for light-responsive expression in monocot leaf cells. Instead, at least six constitutive and light-sensitive regulatory elements are likely important for maize rbcS expression. Synergism between upstream and downstream promoter elements is required. Whereas in dicots, light triggers coupled leaf development and photosynthetic gene expression, in monocots, light regulation of rbcS is uncoupled from leaf development. Light regulation of maize rbcS may be divided into direct and indirect contributions mediated by different regulatory elements. Because wheat and maize rbcS promoters show sequence homologies and similar expression patterns in monocot and dicot leaf cells, it appears likely that monocots share conserved regulatory elements irrespective of whether they utilize the C3 or C4 pathway for carbon fixation.

Base Sequence↗
PubMed2004

The - 564 A/G polymorphism in the promoter region of the proteinase 3 gene associated with Wegener's granulomatosis does not increase the promoter activity.

Proteinase 3 is the major autoantigen in patients with Wegener's granulomatosis. Earlier studies have shown that circulating leucocytes from patients with Wegener's granulomatosis show elevated proteinase 3 surface expression and mRNA levels. Wegener's granulomatosis patients also have increased levels of proteinase 3 in plasma. A single nucleotide polymorphism (SNP) (-564 A/G SNP) in the promoter region has been associated with disease. This SNP introduces a new potential Sp1 transcription factor binding site that may be responsible for the observed up-regulated expression of proteinase 3. To investigate this a 740 base pair long region of the promoter was cloned from genomic DNA. The disease-associated -564 A/G, as well as a control -621 A/G exchange, were introduced by polymerase chain reaction mutagenesis and cloned into a luciferase reporter vector. Endogenous expression levels of proteinase 3 mRNA and promoter activity of the cloned constructs were measured in three myeloid cell lines, HL-60, U937 and NB-4, and in epithelial HeLa cells. The results demonstrate a good correlation between the endogenous proteinase 3 mRNA expression and the promoter activity, as judged by luciferase activity. However, no significant differences in activity between the wild-type, polymorphic and the mutated control variant were found. In conclusion, the -564 A/G polymorphism is not responsible for the increased expression levels seen in myeloid cells from patients with Wegener's granulomatosis.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1989

CRP/cAMP- and CytR-regulated promoters in Escherichia coli K12: the cdd promoter.

Transcriptional regulation of the deoP2 promoter by the cyclic AMP/cyclic AMP receptor protein complex (cAMP/CRP) and the CytR repressor requires two high-affinity CRP targets located around -41 and -93 bp preceding the start site for transcription. Here we report the structure of cddP, another CRP/CytR-regulated promoter. In common with what was found in deo, the cdd promoter also contains multiple CRP targets. Thus, using the DNasel footprinting procedure, tandem CRP binding sites were identified around -41 and -93. These findings support a general model for CytR binding and CytR regulation, in which (i) CytR and the CRP/cAMP complex bind to similar or identical targets, (ii) two or more targets are necessary for proper binding of CytR to a promoter region, and (iii) CytR represses transcription by antagonizing cAMP/CRP activation.

Base Sequence↗
PubMed2004

Mechanism of promoter activity of the beta-amyloid precursor protein gene in different cell lines: identification of a specific 30 bp fragment in the proximal promoter region.

The amyloid beta-protein (Abeta) deposited in brains of Alzheimer's disease (AD) patients is proteolytically derived from a large Abeta precursor protein (APP). APP gene expression patterns in the AD brain region indicate that abnormalities of gene regulation may be important in AD pathology. To understand the contribution of different cell types to APP gene expression, we studied it at four levels: promoter activity (by reporter gene assay of transfected cells), DNA-nuclear protein interaction (by electrophoretic mobility shift assay), RNA message and protein (by northern and western blotting, respectively). APP mRNA and protein expression levels were greater in neuroblastoma and PC12 cells than in glial or cervix epithelial cells. Relative activity among 12 different promoter regions and within single regions varied according to cell type/cell line. An upstream regulatory region containing a GATA-1 site is necessary for activity in PC12 and glial cells but not in neuroblastoma cells. DNA-protein interactions were examined in three distal and one proximal promoter elements in nuclear extracts belonging to neuronal and non-neuronal cells. The proximal promoter region is important for cell line-specific APP gene expression. Characterization of the APP regulatory region's interaction with cell type-specific nuclear factor(s) is important to understand tissue-specific expression of APP seen in AD subjects.

Amyloid beta-Protein Precursor↗
PubMed1995

Cloning and partial characterization of regulated promoters from Lactococcus lactis Tn917-lacZ integrants with the new promoter probe vector, pAK80.

Transposon Tn917-LTV1 was used to produce a collection of Lactococcus lactis strains with fusion of a promoterless lacZ gene to chromosomal loci. Screening 2,500 Tn917-LTV1 integrants revealed 222 that express beta-galactosidase on plates at 30 degrees C. Pulsed-field gel electrophoresis revealed Tn917-LTV1 insertions in at least 13 loci in 15 strains analyzed. Integrants in which beta-galactosidase expression was regulated by temperature or pH and/or arginine concentration were isolated. In most cases, the regulation observed on plates was reproducible in liquid medium. One integrant, PA170, produces beta-galactosidase at pH 5.2 but not at pH 7.0, produces more beta-galactosidase at 15 degrees C than at 30 degrees C, and has increased beta-galactosidase activity in the stationary phase. DNA fragments potentially carrying promoters from selected Lactococcus lactis integrants were cloned in Escherichia coli. A new promoter probe vector, pAK80, containing promoterless beta-galactosidase genes from Leuconostoc mesenteroides subsp. cremoris and the Lactococcus lactis subsp. lactis biovar diacetylactis citrate plasmid replication region was constructed, and the lactococcal fragments were inserted. Plasmid pAK80 was capable of detecting and discriminating even weak promoters in Lactococcus lactis. When inserted in pAK80, the promoter cloned from PA170 displayed a regulated expression of beta-galactosidase analogous to the regulation observed in PA170.

Amino Acid Sequence↗
PubMed1993

Early and late responses of TOL promoters to pathway inducers: identification of postexponential promoters in Pseudomonas putida with lacZ-tet bicistronic reporters.

Transcriptional lacZ fusions to the Pu and Pm promoters of the TOL (toluene degradation) plasmid inserted in monocopy in the chromosome of Pseudomonas putida showed a very different responsiveness to their respective aromatic effectors regarding growth phase. While a substantial XylS-dependent activation of Pm-lacZ was detected nearly instantly after m-toluate addition, XylR- and xylene-mediated induction of the sigma 54 promoter Pu became significant only after cells slowed down exponential growth and entered stationary phase. When Pu and Pm were fused to lacZ-tet reporters (i.e., promoterless lacZ genes coupled to a tet gene which confers resistance to tetracycline when cotranscribed with the leading gene) instead of lacZ alone, the resulting colonies displayed a distinct phenotype consisting of hyperfluorescence on agar plates after being sprayed with 4-methylumbelliferyl-beta-D-galactoside, simultaneously with being either sensitive (Pu) or resistant (Pm) to tetracycline. To examine whether the same phenotype could be scored in strains carrying transcriptional fusions of the lacZ-tet cassettes to other genes or promoters whose expression is silenced during growth and activated in stationary phase, we constructed mini-Tn5 lacZ-tet transposons for random genetic probing of promoters preferentially active at advanced stages of growth. Chromosomal insertions of this mobile element were selected by means of the constitutive resistance to kanamycin which is also specified by the transposon. A number of kanamycin-resistant colonies which are hyperfluorescent with 4-methylumbelliferyl-beta-D-galactosidase but sensitive to tetracycline and which reached full induction only at postexponential growth stages were obtained.

Biotransformation↗
PubMed2000

Characterization of the Mycobacterium tuberculosis iniBAC promoter, a promoter that responds to cell wall biosynthesis inhibition.

The cell wall provides an attractive target for antibiotics against Mycobacterium tuberculosis. Agents such as isoniazid and ethambutol that work by inhibiting cell wall biosynthesis are among the most highly effective antibiotics against this pathogen. Although considerable progress has been made identifying the targets for cell wall active antibiotics, little is known about the intracellular mechanisms that are activated as a consequence of cell wall injury. These mechanisms are likely to have an important role in growth regulation and in the induction of cell death by antibiotics. We previously discovered three isoniazid-induced genes (iniB, iniA, and iniC) organized in tandem on the M. tuberculosis genome. Here, we investigate the unique features of the putative iniBAC promoter. This promoter was specifically induced by a broad range of inhibitors of cell wall biosynthesis but was not inducible by other conditions that are toxic to mycobacteria via other mechanisms. Induction required inhibitory concentrations of antibiotics and could be detected only in actively growing cells. Analysis of the iniBAC promoter sequence revealed both a regulatory element upstream and a potential repressor binding region downstream of the transcriptional start site. The induction phenotype and structure of the iniBAC promoter suggest that a complex intracellular response occurs when cell wall biosynthesis is inhibited in M. tuberculosis and other mycobacteria.

Amino Acid Sequence↗
PubMed2003

Sigma 32-dependent promoter activity in vivo: sequence determinants of the groE promoter.

The Escherichia coli transcription factor sigma 32 binds to core RNA polymerase to form the holoenzyme responsible for transcription initiation at heat shock promoters, utilized upon exposure of the cell to higher temperatures. We have developed two ways to assay sigma 32-dependent RNA synthesis in E. coli. The plasmid-borne reporter gene for both is lacZ (beta-galactosidase), driven by the groE promoter. In one application, the cells are exposed to a temperature of 42 degrees C in order to induce accumulation of endogenous sigma 32. The other involves isopropylthiogalactopyranoside (IPTG)-induced synthesis of sigma 32 at 30 degrees C from a gene contained on a second plasmid. The latter employs DnaK(-) cells, which additionally contained a second mutation, inactivating the endogenous sigma 32 gene (Bukau and Walker, EMBO J. 9:4027-4036, 1990). These assays were used to delineate the sequences CTTGA (-37 to -33) and GNCCCCATNT (-18 to -9) as important for sigma 32 promoter activity. At each of the specified base pairs, substitutions were found which reduced promoter activity by greater than 75%. Activity was also dependent upon the number of base pairs separating the two regions.

Bacterial Proteins↗
PubMed1990

The mammalian upstream element factor recognizes two sites in the adenovirus type 2 IVa2-major late promoter intergenic region and stimulates both promoters.

The adenovirus type 2 major late upstream element factor (UEF) recognizes two similar elements that lie between the major late promoter (MLP) and IVa2 promoter cap sites (the previously characterized MLP-UE from nucleotides -49 to -67 and the IVa2-UE from nucleotides -98 to -122). DNase I footprinting and gel retention assays showed that the UEF has a lower affinity for the IVa2-UE than for the MLP-UE. In vitro transcription experiments demonstrated first that the IVa2 promoter, which lacks a consensus TATA box, may work, as does the MLP, in the absence of its proximal upstream element and second that the IVa2-UE stimulated IVa2 transcription two- to threefold, as MLP-UE did for the MLP. In addition, we demonstrated that the more distal upstream element has a weak stimulatory effect on transcription of both promoters.

Adenoviruses, Human↗