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At least 739 records · Page 41Linked to original sources
PubMed1998

Distinct rat proteins can recognize CCAAT-homologous sequences of the metallothionein promoter and trans-activate this promoter.

We have previously purified and characterized a rat liver protein C'BP-1 that is either identical or closely related to C/EBPdelta (Aniskovitch and Jacob, 1997). The mouse metallothionein-I (MT-I) promoter contains two C'BP-1 binding sites, one of which includes the MRE-c' region (-135 to -110). The C'BP-1 binding activity was detected by EMSA as a major activity for MRE-c' in nonproliferating adult liver cells but not in rat hepatoma cells. In this study, we purified and characterized a factor, C'BP-2, which had a dominant binding activity for MRE-c' in Morris hepatoma 3924A, a poorly differentiated, fast-growing tissue. C'BP-2 is a 28 kDa protein which exists in solution as a monomer. As observed for C'BP-1, affinity-purified C'BP-2 stimulated transcription from the mMT-I gene promoter. DNase I footprinting revealed two C'BP-2 binding sites in the regions that overlap with the CCAAT homologies of the C'BP-1 binding sites on the mMT-I promoter. C'BP-2 made essential contacts with the CCAAT homology and in the region upstream of this sequence. Competition electrophoretic mobility shift assay and methylation interference analysis revealed that C'BP-2 is a protein closely related, but not identical, to CP2. These data suggest that C'BP-1 and C'BP-2 may play a role in hepatocyte proliferation and/or differentiation.

Animals↗
PubMed2001

Genomic distribution of three promoters of the bovine gene encoding acetyl-CoA carboxylase alpha and evidence that the nutritionally regulated promoter I contains a repressive element different from that in rat.

The enzyme acetyl-CoA carboxylase alpha (ACC-alpha) is rate-limiting for the synthesis of long-chain fatty acids de novo. As a first characterization of the bovine gene encoding this enzyme, we established the entire bovine ACC-alpha cDNA sequence (7041 bp) and used experiments with 5' rapid amplification of cDNA ends to determine the heterogeneous composition of 5' untranslated regions, as expressed from three different promoters (PI, PII and PIII). The individual locations of these promoters have been defined within an area comprising 35 kbp on Bos taurus chromosome 19 ('BTA19'), together with the segmentation of the first 14 exons. Primer extension analyses reveal that the nutritionally regulated PI initiates transcription from at least four sites. PI transcripts are much more abundant in adipose and mammary-gland tissues than in liver or lung. A 2.6 kb promoter fragment drives the expression of reporter genes only weakly in different model cells, irrespective of stimulation with insulin or dexamethasone. Thus bovine PI is basically repressed, like its analogue from rat. Finely graded deletions of PI map two separate elements, which have to be present together in cis to repress bovine PI. The distal component resides within a well-preserved Art2 retroposon element. Thus sequence, structure and evolutionary origin of the main repressor of PI in bovines are entirely different from its functional counterpart in rat, which had been identified as a (CA)(28) microsatellite. We show that, in different mammalian species, unrelated genome segments of different origins have been recruited to express as functionally homologous PI the ancient and otherwise highly conserved ACC-alpha-encoding gene.

Acetyl-CoA Carboxylase↗
PubMed1999

Structural requirements for marbox function in transcriptional activation of mar/sox/rob regulon promoters in Escherichia coli: sequence, orientation and spatial relationship to the core promoter.

The promoters of the mar/sox/rob regulon of Escherichia coli contain a binding site (marbox) for the homologous transcriptional activators MarA, SoxS and Rob. In spite of data from footprinting studies, the marbox has not been precisely defined because of its degeneracy and asymmetry and seemingly variable location with respect to the -10 and -35 hexamers for RNA polymerase (RNP) binding. Here, we use DNA retardation studies and hybrid promoters to identify optimally binding 20 bp minimal marboxes from a number of promoters. This has yielded a more defined marbox consensus sequence (AYnGCACnnWnnRYYAAAYn) and has led to the demonstration that some marboxes are inverted relative to others. Using transcriptional fusions to lacZ, we have found that only one marbox orientation is functional at a given location. Moreover, the functional orientation is determined by marbox location: marboxes that are 15 or more basepairs upstream of the -35 hexamer are oriented opposite those closer to the -35 hexamer. Marbox orientation and the spacing between marbox and signals for RNP binding are critical for transcriptional activation, presumably to align MarA with RNP.

Bacterial Proteins↗
PubMed2004

Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation.

By monitoring the end-to-end extension of a mechanically stretched, supercoiled, single DNA molecule, we have been able directly to observe the change in extension associated with unwinding of approximately one turn of promoter DNA by RNA polymerase (RNAP). By performing parallel experiments with negatively and positively supercoiled DNA, we have been able to deconvolute the change in extension caused by RNAP-dependent DNA unwinding (with approximately 1-bp resolution) and the change in extension caused by RNAP-dependent DNA compaction (with approximately 5-nm resolution). We have used this approach to quantify the extent of unwinding and compaction, the kinetics of unwinding and compaction, and effects of supercoiling, sequence, ppGpp, and nucleotides. We also have used this approach to detect promoter clearance and promoter recycling by successive RNAP molecules. We find that the rate of formation and the stability of the unwound complex depend profoundly on supercoiling and that supercoiling exerts its effects mechanically (through torque), and not structurally (through the number and position of supercoils). The approach should permit analysis of other nucleic-acid-processing factors that cause changes in DNA twist and/or DNA compaction.

DNA↗
PubMed2003

Activation domain-mediator interactions promote transcription preinitiation complex assembly on promoter DNA.

The interaction of activators with mediator has been proposed to stimulate the assembly of RNA polymerase II (Pol II) preinitiation complexes, but there have been few tests of this model. The finding that the major adenovirus E1A and mitogen-activated protein kinase-phosphorylated Elk1 activation domains bind to Sur2 uniquely among the metazoan mediator subunits and the development of transcriptionally active nuclear extracts from WT and sur2-/- embryonic stem cells, reported here, allowed a direct test of the model. We found that whereas VP16, E1A, and phosphorylated Elk1 activation domains each stimulate binding of mediator, Pol II, and general transcription factors to promoter DNA in extracts from WT cells, only VP16 stimulated their binding in extracts from sur2-/- cells. This stimulation of mediator, Pol II, and general transcription factor binding to promoter DNA correlated with transcriptional activation by these activators in WT and mutant extracts. Because the mutant mediator was active in reactions with the VP16 activation domain, the lack of activity in response to the E1A and Elk1 activation domains was not due to loss of a generalized mediator function, but rather the inability of the mutant mediator to be bound by E1A and Elk1. These results directly demonstrate that the interaction of activation domains with mediator stimulates preinitiation complex assembly on promoter DNA.

ATP-Binding Cassette Transporters↗
PubMed1985

Transcription factor Sp1 recognizes promoter sequences from the monkey genome that are simian virus 40 promoter.

A 440-base-pair fragment of African green monkey genomic DNA shares homology with the transcriptional regulatory region of simian virus 40 (SV40) and has been reported to direct transcription in vivo. We find that two regions within this fragment bind the promoter-specific cellular transcription factor Sp1 and are protected in DNase protection ("footprinting") experiments. As in SV40, binding occurs in regions containing multiple copies of the sequence GGGCGG. These regions, when fused to the proximal, or "TATA box," element of the herpes simplex virus thymidine kinase promoter, are able to direct Sp1-dependent transcription in vitro. The finding that Sp1 is capable of productive interaction with sequences taken from a cellular promoter supports the idea that Sp1 may play a role in modulating transcription of cellular genes.

Animals↗
PubMed1987

Two promoter-specific host factors interact with adjacent sequences in an EIA-inducible adenovirus promoter.

We previously reported the identification of a host factor (EIIA-EF) specific for an upstream transcriptional control sequence (-82 to -66) of the EIA-inducible adenovirus EIIA early promoter. The levels of this factor remained unchanged after virus infection of human cells. Another study also identified a factor (EIIF) specific for this same promoter, but the activity of this second factor was shown to increase severalfold after virus infection. We now show that these dramatically different results, both based on gel shift assays on the same promoter, may be explained by variations in protocol details and actually identify two distinct factors. When synthetic DNA copolymers [poly(dI).poly(dC) or poly(dI-dC).poly(dI-dC)] are used as competitors in gel shift assays, a factor specific for DNA sequences between -82 and -66 can be identified, whereas when natural eukaryotic DNAs (salmon sperm or calf thymus) are used as competitors a different factor specific for DNA sequences between -69 and -33 can be identified. We have mapped the DNA-protein contact residues for the EIIF by analyzing a series of linker scan mutants in gel shift assays and methylation interference experiments. The EIIA-EF and EIIF bind to two distinct but adjacent sequences. Competition experiments indicate that these two activities are due to two different factors. Consistent with the earlier reports, the levels of one (EIIA-EF) do not change after virus infection of human cells, whereas the levels of the other (EIIF) are increased severalfold.

Adenoviruses, Human↗
PubMed1991

Exclusive expression of Epstein-Barr virus nuclear antigen 1 in Burkitt lymphoma arises from a third promoter, distinct from the promoters used in latently infected lymphocytes.

Epstein-Barr virus transformation of human B lymphocytes in vitro results in the expression of six viral nuclear antigens (EBNAs) and three viral membrane proteins. However, examination of viral gene expression in fresh Burkitt lymphoma isolates has revealed expression of only one of the nuclear antigens, EBNA-1. Previous transcriptional analyses of the EBNA-encoding genes demonstrated that all these genes are driven from one of two distal promoters located near the left end of the viral genome, raising the question of how exclusive expression of EBNA-1 occurs in Burkitt lymphoma tumors. Although most established Burkitt lymphoma cell lines (group 3) exhibit the full-expression pattern of viral antigens seen in lymphoblastoid cell lines, a few cell lines have been established that retain the restricted pattern of viral gene expression (group 1). In this paper we characterize transcription of the EBNA-1 gene in a group 1 Burkitt lymphoma cell line and show that (i) neither Cp nor Wp, the promoters involved in driving EBNA gene expression in lymphoblastoid cell lines, are active in this cell line; (ii) treatment of this cell line with 5-azacytidine, previously shown to induce expression of all EBNA genes, induced Cp and Wp activity; (iii) sizes of the EBNA-1 transcripts detected in two group 1 Burkitt lymphoma cell lines correlated with each other and were distinct from the size of the EBNA-1 transcript seen in lymphoblastoid cell lines; (iv) the EBNA-1 transcripts in the group 1 Burkitt lymphoma cell lines do not hybridize to a probe containing the common 5' exons present in all the EBNA transcripts from lymphoblastoid cell lines; and (v) anchored-PCR cloning the 5' region of the EBNA-1 transcript from one of the group 1 cell lines identified two exons, FQ and U, upstream of the EBNA-1 coding exon. The FQ exon lies just downstream of a TATAA box, which may represent the promoter for transcription of EBNA-1 in these cells. It is particularly noteworthy that an incomplete EBNA-1 cDNA clone from a nasopharyngeal carcinoma tumor line that expresses EBNA-1, but not the other EBNAs, has been characterized; this EBNA-1 transcript also contains the FQ/U splice junction, suggesting that the organization of exons upstream of the EBNA-1 coding exon is the same and that this organization may reflect a viral program for exclusive EBNA-1 expression.

Animals↗
PubMed1995

Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.

Base Sequence↗
PubMed1996

The ability of the inhibitory domain of the POU family transcription factor Oct-2 to interfere with promoter activation by different classes of activation domains is dependent upon the nature of the basal promoter elements.

The Oct-2 transcription factor contains an inhibitory domain which is able to repress transcription following DNA binding. Here we show that within the neuronally expressed Oct-2.5 form, the inhibitory domain can strongly inhibit activation by transcription factor activation domains which are either composed predominantly of acidic residues or contain the HOB motif, whereas it has a weaker effect or no effect on proline-rich activation domains and on a glutamine-rich domain. In contrast, the isolated inhibitory domain of Oct-2 can efficiently repress all types of activation domains. This effect is observed however, only on TATA box-containing promoters and not on promoters containing an initiator motif. This widespread inhibition of different activation domains and its dependence on the nature of the basal promoter elements indicate that the inhibitory domain is likely to act by contacting a common downstream target of activation domains within the basal transcriptional complex bound at the TATA box rather than quenching specific activation domains by direct interaction. These effects are discussed in terms of the functional role of the inhibitory domain within Oct-2.5 and the mechanism by which it acts.

Animals↗
PubMed2001

Methylation of promoter proximal-transcribed sequences of an embryonic globin gene inhibits transcription in primary erythroid cells and promotes formation of a cell type-specific methyl cytosine binding complex.

The methylation pattern of a 248-base pair proximal transcribed region (rho248) of the avian embryonic rho-globin gene was found to correlate inversely with stage-specific expression in avian erythroid cells. In vitro methylation of the rho248 segment alone (in the absence of promoter methylation) resulted in a 5-fold inhibition of transcription in a transient transfection assay in primary erythroid cells in which the transfected gene is packaged into nucleosomal chromatin. This effect was observed if the rho248 segment was positioned adjacent to the promoter but not when it was located 2.7 kilobases downstream. Fully methylated but not unmethylated rho248 formed a novel cell type-specific methyl cytosine-binding protein complex (MeCPC) that contained methyl binding domain protein-2 (MBD-2) and histone deacetylase 1 proteins but differed from MeCP-1. The histone deacetylase inhibitor trichostatin A failed to relieve methylation-mediated repression of transcription from the rho-gene promoter, supporting the notion of the dominance of methylation over histone deacetylation in silencing through CpG-rich sequences at this locus. These data demonstrate that site-specific methylation of a vertebrate gene 5'-transcribed region alone at the exact CpGs that are methylated in vivo can suppress transcription in homologous primary cells and facilitate binding to a cell type-specific MeCPC.

Animals↗
PubMed2003

Promoting safety--a component in health promotion in primary and secondary schools.

Accidents, especially accidents involving children or young people, are a key health problem that needs to find higher prominence in preventative measures. The school as the central socialisation institution plays a key role in the process of continual improvement. However, to date the prevailing approaches of technical accident prevention and behaviourled safety training have not led to sustained improvements in the safety of children and young people. This article presents the key elements and characteristics of a new approach that not only aims to make significant improvements in safety but is also aimed at schools having an improved perception of their key role--to instruct and educate. The authors assume that greater security can only be achieved in and through schools if safety promotion is at the same time school development. In this understanding, safety promotion focuses on the key players in the school and school life itself, and in particular endeavours to strengthen relationships and behaviour that promote safety and to minimise those that can have a negative effect. It emphasises practical changes to daily school life with regard to construction and fittings, education, training, organisation and politics, and involves the needs of everybody who lives and works at the school.

Accident Prevention↗
PubMed2000

RNA polymerase--promoter recognition. Specific features of electrostatic potential of "early" T4 phage DNA promoters.

Comparative analysis of electrostatic potential distribution for "early" T4 phage promoters was undertaken, along with calculation of topography of electrostatic potential around the native and ADP-ribosylated C-terminal domain of RNA polymerase alpha-subunit. The data obtained indicate that there is specific difference in the patterns of electrostatic potential distribution in far upstream regions of T4 promoters differing by their response to ADP-ribosylation of RNA polymerase. A specific change in profiles of electrostatic potential distribution for the native and ADP-ribosylated forms of RNA polymerase alpha-subunit was observed suggesting that this factor may be responsible for modulating T4 promoter activities in response to the enzyme modification.

Adenosine Diphosphate Ribose↗
PubMed1994

Binding of promoter-associated AP-1 is not altered during induction and subsequent repression of the c-jun promoter by TPA and UV irradiation.

Rapid transient induction of the human c-jun proto-oncogene by 12-O-tetradecanoylphorbol-13-acetate (TPA) and UV irradiation requires the presence of two cis-acting elements, Jun1 and Jun2. Using dimethyl sulfate (DMS) genomic footprinting, in vivo, all protein binding sites in the c-jun promoter, including Jun1 and Jun2, are already fully occupied before induction and the protein--DNA contacts are unchanged during gene activation by TPA and UV and subsequent repression. In vitro binding studies suggest that both sites are recognized with high affinity by protein complexes containing cJun and ATF-2. Jun1 is also recognized by complexes containing Fos and Jun in vitro, but with only a very low affinity. The binding of Jun/ATF-2-containing complexes to Jun1 or Jun2 is not affected during early and late time points after induction. Transcriptional shut-off is caused by neither a loss of binding of an activating protein nor by additional binding of a putative repressor. The lack of detectable changes in DNA binding and factor composition strongly suggests that transcriptional activation and subsequent inactivation of c-jun promoter activity by TPA or UV is mediated by post-translational modifications of prebound cJun and possibly ATF-2. Such pre-formed structures on the promoter could be a general requirement for the rapid and transient transcriptional responses of immediate-early genes to extracellular signals.

Base Sequence↗
PubMed1996

Evaluation of preventive and health promotion activities in 166 primary care practices in Spain. The Coordinating Group For Prevention and Health Promotion in Primary Care in Spain.

BACKGROUND: Since January 1989 the Spanish Society of Family and Community Medicine has supported the Program of Prevention and Health Promotion (PAPPS) with the following objectives: to detect possible difficulties in the implementation of recommendations especially among asymptomatic low-risk adults and children in Spain, to disseminate those recommendations, and to encourage research in prevention and health promotion in primary care. OBJECTIVE: We wished to evaluate the implementation of PAPPS in primary care practices. METHODS: A retrospective audit of medical records in practices enrolled in the programme was carried out. From 166 primary care practices widely distributed in Spain 21,631 patients were selected using systematic sampling. RESULTS: Age was positively associated with correct performance in the adult population, while in the paediatric population it was negatively associated. Females had higher probability than males of having blood pressure measurement (OR 1.34, 95% CI 1.24-1.45) and smoking counselling (OR 1.38, 95% CI 1.33-1.43) correctly performed. Practices not using the preventive chart had a lower probability than user practices of correctly performing blood pressure measurement (OR 0.69, 95% CI 0.62-0.76), alcohol counselling (OR 0.66, 95% CI 0.60-0.73) and smoking counselling (OR 0.69, 95% CI 0.63-0.76). Non-teaching practices had a higher probability than teaching centres of correct performance of blood pressure measurement (OR 1.47, 95% CI 1.35 to 1.59), alcohol counselling (OR 1.67, 95% CI 1.54 to 1.79) and smoking counseling (OR 1.39, 95% CI 1.29 to 1.51). CONCLUSIONS: There is an unequal level of performance depending on the procedure and on the target population. A preventive chart might be useful in improving implementation of periodic health maintenance. Teaching centres with training physicians need to put more emphasis on prevention and health promotion activities.

Adolescent↗
PubMed1998

Promoting breast cancer screening in rural, African American communities: the "science and art" of community health promotion.

Implementing community health promotion programs can be a daunting task for social workers. Aside from the practical skills necessary for engaging diverse groups, it requires an understanding of the complex interplay of several theoretical constructs. Using an eight-year breast cancer prevention project, the North Carolina Breast Cancer Screening Program, as a case study, this article describes both the "science and art" of community health promotion. The article provides examples illustrating the "art" of generating participation by both lay and professional communities and concludes with guidelines for social workers who plan to conduct their own community health promotion programs.

Black or African American↗
PubMed1992

Characterization and purification of Adh distal promoter factor 2, Adf-2, a cell-specific and promoter-specific repressor in Drosophila.

Chromatin footprinting in Drosophila tissue culture cells has detected the binding of a non-histone protein at +8 of the distal Adh RNA start site, on a 10-bp direct repeat motif abutting a nucleosome positioned over the inactive Adh distal promoter. Alternatively the active promoter is bound by a transcription initiation complex. We have characterized and purified a protein Adf-2 that binds specifically to this direct repeat motif 5'TCTCAGTGCA3', present at +8 and -202 of the distal RNA start site. DNase I footprinting, methylation interference, and UV-crosslinking analyses showed that both direct repeats interact in vitro with a nuclear protein of approximately 120 kilodaltons (kDa). We purified Adf-2 through multiple rounds of sequence-specific DNA affinity chromatography. Southwestern analysis showed that the purified 120 KDa polypeptide binds the Adf-2 motif efficiently as a monomer or homomultimer. In vivo titrations of Adf-2 activity with the Adf-2 motif by transient co-transfection competitions in different Drosophila cell lines suggested that Adf-2 is a cell-specific repressor. Adf-2 has been detected ubiquitously in vitro, but is functional in vivo as a sequence-specific DNA binding protein and repressor only in the cells that have the inactive distal promoter. We discuss the possibility that an activation process is required for Adf-2 protein to bind DNA and function in vivo.

Alcohol Dehydrogenase↗
PubMed1994

Transactivation and repression of the alpha-fetoprotein gene promoter by retinoid X receptor and chicken ovalbumin upstream promoter transcription factor.

Retinoic acid (RA) is widely involved in the control of cell proliferation and differentiation, as well as embryo pattern formation. Transcription of the oncodevelopmental protein, alpha-fetoprotein (AFP), is stimulated by retinoic acid (RA) in neoplastic cells. To study RA regulation of AFP gene expression, the 5'-flanking region of AFP gene was cloned and analyzed. In the present study, transfection of deletion mutants and sequence analysis revealed a retinoid X receptor response element (AFP-RXRE) located at position -139 to -127 of the AFP promoter. Synthetic AFP-RXRE was ligated into a reporter construct with the heterologous promoter and chloramphenicol acetyltransferase (CAT). AFP-RXRE conferred a marked RA responsiveness in the cotransfection with retinoid X receptor (RXR), but not with retinoic acid receptors (RARs). Consistent with these data, only RXR bound to AFP-RXRE with high affinity in the mobility shift assays. Chicken ovalbumin upstream promoter transcription factor (COUP-TF), an orphan member of the steroid/thyroid hormone superfamily, also demonstrated specific binding activity to AFP-RXRE in vitro. In cotransfection assays, COUP-TF dramatically repressed the transactivation of RXR on AFP-RXRE. The mechanism of repression by COUP-TF may involve the mutual occupancy of the AFP-RXRE binding site between RXR and COUP-TF.

Animals↗