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At least 199 records · Page 11Linked to original sources
PubMed1985

A 46-nucleotide promoter segment from an IFN-alpha gene renders an unrelated promoter inducible by virus.

A hybrid gene consisting of the human IFN-alpha 1 promoter and a beta-globin transcription unit is expressed correctly only after viral induction. To determine the region required for inducibility, 25 hybrid promoters consisting of varying upstream IFN-alpha 1 and downstream beta-globin promoter moieties were analyzed, and 5'-deletion analysis was performed on an inducible hybrid promoter. An IFN promoter region from position -109 to -64 conferred maximal inducibility on downstream beta-globin promoter segments and even on the intact beta-globin promoter. This region is strikingly conserved among human IFN-alpha and -beta genes. As constitutive expression of the beta-globin gene was not diminished by placing IFN promoter fragments in various positions, induction is attributed largely to positive, rather than to negative control.

Animals↗
PubMed1998

Analysis of a variant surface glycoprotein gene expression site promoter of Trypanosoma brucei by remodelling the promoter region.

Trypanosoma brucei survives in the mammalian bloodstream by antigenic variation of its variant surface glycoprotein (VSG) coat. VSG genes are found in telomeric expression sites (ESs), and only one ES is fully transcribed at a time. The parasite changes its coat by either bringing another VSG gene into the active ES, or by switching on another ES and silencing the first. It has previously been shown that the promoter of an active ES can be replaced by a ribosomal promoter without affecting the function of the ES. This study has now analysed the conserved sequences flanking the ES promoter by deletion or replacement of these sequences in intact trypanosomes. The results show that the sequences 3' of the promoter and extending down to the first protein-coding gene, ESAG 7, are not required in the bloodstream-form parasite either for high-level transcription or for switching of the ES. Transformants in which the sequences 5' of the promoter extending up to simple-sequence 50-bp repeats had been removed were not obtained unless the 5' ES sequences were replaced with exogenous DNA, or unless the ES promoter was replaced by a ribosomal promoter, and even these transformants were rare. Transformants lacking the 5' ES sequences displayed a less complete transcriptional repression of silent ESs. These results indicate that the area 5' of an ES promoter is required for optimal functioning of an ES.

Animals↗
PubMed1985

In vivo and in vitro effect of mutations in tetA promoter from pSC101: insertion of poly(dA.dT) stretch in the spacer region does not inactivate the promoter.

Two mutants, mapping at the HindIII site (between the consensus sequences) of the pSC101 tetA promoter, were studied: MA2 corresponds to a 4 bp deletion between positions -12 and -15; B30 bears a 44 bp insertion C(TA)21 G at the HindIII site. Both mutants were assayed in vivo (ability of the plasmid to confer resistance to tetracycline, plasmid-directed protein synthesis, S1-mapping of mRNA) and in vitro (abortive initiation assay). Compared to w.t., MA2 is a poor promoter in vivo; RNA polymerase binding, complex activation and rate of initial oligonucleotide synthesis are strongly reduced in vitro; this is in keeping with the known effects of altering the consensus elements in E. coli promoters. In contrast, B30 shows in vivo a promoter activity only slightly reduced in comparison to that of the w.t. tetA promoter; both in vivo and in vitro, the transcription start site is outside and downstream the (TA)21 stretch, 5-7 bp upstream that found in the w.t. To adjust the behaviour of B30 and the claimed consensus distance between the E. coli promoter consensus sequences, some structural modification in the (TA)21 stretch -either spontaneous or induced by RNA polymerase- can be hypothesized. Unless the (TA)21 stretch itself plays the role of a relatively good promoter, the results suggest that promoter-specific elements may be distributed along the DNA sequences over distances longer, but seldom less, than the 17 +/- 2 bp consensus distance.

Base Sequence↗
PubMed1997

Torsional constraints on the formation of open promoter complexes on DNA minicircles carrying sigma 54-dependent promoters.

A topoisomer gel retardation assay has been used to examine the topological requirements for the formation of open promoter complexes on DNA minicircles carrying sigma 54-dependent promoters. In the absence of intercalators, individual topoisomers carrying both the nifL and nifF promoters could be resolved as discrete species by electrophoresis, but exhibited anomalous electrophoretic behavior at relatively high negative superhelical density, indicative of a structural transition. In the presence of phosphorylated activator protein NTRC, ATP, and sigma 54 RNA polymerase holoenzyme, discrete topoisomer shifts were detected associated with the formation of open promoter complexes. At the nifL promoter open complexes could be formed on all negatively supercoiled topoisomers examined as well as on nicked circular DNA, but not on the DeltaLk = 0 topoisomer or positively supercoiled DNA. Minicircles carrying the sigma 54-dependent glnAp2 promoter could not be resolved in the electrophoresis system, but using a combination of potassium permanganate footprinting and topoisomerase I relaxation assays, we found in contrast to the nifL promoter, that open complexes were formed not only on negatively supercoiled topoisomers but also on relaxed minicircles and the Delta Lk = +1 topoisomer. These results indicate there is a thermodynamic barrier to the formation of open complexes on DNA minicircles carrying the nifL promoter which is not evident at glnAp2.

Bacterial Proteins↗
PubMed1998

Promoter recognition by Escherichia coli RNA polymerase: effects of the UP element on open complex formation and promoter clearance.

Escherichia coli promoters for transcription of ribosomal and tRNAs are greatly activated by an A+T-rich "UP" element upstream of the -35 region. These same promoters have also been found to otherwise deviate in several respects from the consensus promoter sequence. Here we present the results of a kinetic characterization of the interaction of Escherichia coli RNA polymerase with UP element-containing promoters which by virtue of consensus or near-consensus sequence features should be among the most optimal that can be encountered by Escherichia coli RNA polymerase. We show that for such promoters, (1) the second-order rate constant describing formation of the initial (closed) complex is close to that expected for a diffusion-limited process, (2) the extent of activation by the UP element is temperature-sensitive, (3) the UP element accelerates a process after DNA binding by RNA polymerase, and (4) the presence of the UP element delays promoter clearance upon addition of nucleoside triphosphates to preformed RNA polymerase-promoter complexes. Finally, we provide evidence in support of models which describe the DNA melting process accompanying open complex formation as initiating in the -10 promoter region and progressing in the downstream direction.

Base Sequence↗
PubMed2003

Cestrum yellow leaf curling virus (CmYLCV) promoter: a new strong constitutive promoter for heterologous gene expression in a wide variety of crops.

Appropriately regulated gene expression requires a suitable promoter. A number of promoters have been isolated and shown to be functional in plants, but only a few of them activate transcription of transgenes at high levels constitutively. We report here the cloning and characterization of a novel, constitutively expressed promoter isolated from Cestrum yellow leaf curling virus (CmYLCV), a double-stranded DNA plant pararetrovirus belonging to the Caulimoviridae family. The CmYLCV promoter is highly active in callus, meristems and vegetative and reproductive tissues in Arabidopsis thaliana, Nicotiana tabacum, Lycopersicon esculentum, Zea mays and Oryza sativa. Furthermore, the level of expression is comparable to, or higher than, that from the CaMV 35S, the 'super-promoter' or the maize ubiquitin 1 promoters, three frequently used promoters in agricultural biotechnology. The heritable, strong and constitutive activity in both monocotyledonous and dicotyledonous plants, combined with the extremely narrow CmYLCV host range, makes the CmYLCV promoter an attractive tool for regulating transgene expression in a wide variety of plant species.

Arabidopsis↗
PubMed2004

Analysis of the maize polyubiquitin-1 promoter heat shock elements and generation of promoter variants with modified expression characteristics.

The maize polyubiquitin-1 (Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock. The promoter contains two overlapping sequences with similarity to defined heat shock elements and we show that these sequences are also present upstream of the Ubi-1 homologue isolated from teosinte. Both the maize and teosinte promoters can mediate a heat shock response in transgenic maize. We have dissected the overlapping maize Ubi-1 promoter heat shock elements and demonstrate that the 3' element is required to mediate a heat shock response. The Ubi-1 promoter is particularly active in tissues consisting of rapidly dividing cells, and within the seed it is strongly biased towards driving expression in the embryo. However, replacement of the heat shock elements with a trimer of a basic domain/leucine zipper factor binding site of a pea lectin promoter shifts the balance in seed expression towards the endosperm. The Ubi-1 variants described here differ in their overall activity in the seed, but they all show potential for driving high levels of heterologous gene expression in maize.

Base Sequence↗
PubMed1993

The Escherichia coli cysG promoter belongs to the 'extended -10' class of bacterial promoters.

The Escherichia coli cysG promoter has been subcloned and shown to function constitutively in a range of different growth conditions. Point mutations identify the -10 hexamer and an important 5'-TGN-3' motif immediately upstream. The effects of different deletions suggest that specific sequences in the -35 region are not essential for the activity of this promoter in vivo. This conclusion was confirmed by in vitro run-off transcription assays. The DNAase I footprint of RNA polymerase at the cysG promoter reveals extended protection upstream of the transcript start, and studies with potassium permanganate as a probe suggest that the upstream region is distorted in open complexes. Taken together, the results show that the cysG promoter belongs to the 'extended -10' class of promoters, and the base sequence is similar to that of the P1 promoter of the E. coli galactose operon, another promoter in this class. In vivo, messenger initiated at the cysG promoter appears to be processed by cleavage at a site 41 bases downstream from the transcript start point.

Base Sequence↗
PubMed1994

Abscisic acid promotes novel DNA-binding activity to a desiccation-related promoter of Craterostigma plantagineum.

Abscisic acid-treated callus of the resurrection plant Craterostigma plantagineum tolerates extreme desiccation. Nuclear proteins from tolerant callus bind specific sequence elements in the promoter region of the ABA and desiccation-inducible CDeT27-45 gene. One specific region of the promoter, which is protected from DNAase I treatment by DNA-binding activities, is different from previously reported ABA response elements. Four complexes of nuclear proteins and this DNA region are detected by electrophoretic mobility shift assay: two of these complexes (I and II) are readily detectable in untreated samples and are increased by ABA treatment while two other complexes (III and IV) accumulate only following ABA treatment and are prevented from accumulating by protein synthesis inhibitors. When a fragment containing the novel binding site is deleted from the wild-type promoter the ABA responsiveness of the promoter is removed; however, gain of function experiments using synthetic promoters in a protoplast transient assay suggest that besides the binding site other promoter elements are required. A second region of the promoter, containing the sequence element ACGT which is found in abscisic acid response elements, is also bound by nuclear proteins. The level of this second binding activity is similar in both untreated and ABA- treated cells and promoter/reporter gene constructs which contain only the four ACGT elements of the CDeT27-45 promoter are not ABA responsive in a C. plantagineum transient assay system.

Abscisic Acid↗
PubMed2002

Promoter-proximal tethering elements regulate enhancer-promoter specificity in the Drosophila Antennapedia complex.

Insulator DNAs and promoter competition regulate enhancer-promoter interactions within complex genetic loci. Here we provide evidence for a third mechanism: promoter-proximal tethering elements. The Scr-ftz region of the Antennapedia gene complex includes two known enhancers, AE1 and T1. AE1 selectively interacts with the ftz promoter to maintain pair-rule stripes of ftz expression during gastrulation and germ-band elongation. The T1 enhancer, located 3' of the ftz gene and approximately 25 kb 5' of the Scr promoter, selectively activates Scr expression in the prothorax and posterior head segments. A variety of P element minigenes were examined in transgenic embryos to determine the basis for specific AE1-ftz and T1-Scr interactions. A 450-bp DNA fragment located approximately 100 bp 5' of the Scr transcription start site is essential for T1-Scr interactions and can mediate long-range activation of a ftz/lacZ reporter gene when placed 5' of the ftz promoter. We suggest that the Scr450 fragment contains tethering elements that selectively recruit T1 to the Scr promoter. Tethering elements might regulate enhancer-promoter interactions at other complex genetic loci.

Animals↗
PubMed2001

The promoter of a lysosomal membrane transporter gene, CTNS, binds Sp-1, shares sequences with the promoter of an adjacent gene, CARKL, and causes cystinosis if mutated in a critical region.

Although >55 CTNS mutations occur in patients with the lysosomal storage disorder cystinosis, no regulatory mutations have been reported, because the promoter has not been defined. Using CAT reporter constructs of sequences 5' to the CTNS coding sequence, we identified the CTNS promoter as the region encompassing nucleotides -316 to +1 with respect to the transcription start site. This region contains an Sp-1 regulatory element (GGCGGCG) at positions -299 to -293, which binds authentic Sp-1, as shown by electrophoretic-mobility-shift assays. Three patients exhibited mutations in the CTNS promoter. One patient with nephropathic cystinosis carried a -295 G-->C substitution disrupting the Sp-1 motif, whereas two patients with ocular cystinosis displayed a -303 G-->T substitution in one case and a -303 T insertion in the other case. Each mutation drastically reduced CAT activity when inserted into a reporter construct. Moreover, each failed either to cause a mobility shift when exposed to nuclear extract or to compete with the normal oligonucleotide's mobility shift. The CTNS promoter region shares 41 nucleotides with the promoter region of an adjacent gene of unknown function, CARKL, whose start site is 501 bp from the CTNS start site. However, the patients' CTNS promoter mutations have no effect on CARKL promoter activity. These findings suggest that the CTNS promoter region should be examined in patients with cystinosis who have fewer than two coding-sequence mutations.

Adult↗
PubMed1995

Interferon-gamma inhibits transgene expression driven by SV40 or CMV promoters but augments expression driven by the mammalian MHC I promoter.

The use of mammalian gene expression vectors has become increasingly important for transgenics and gene therapy as well as basic research. Essential for the success of these vectors in medical research applications is the proper choice of promoter linked to the gene of interest. Many mammalian expression vectors use promoter elements from pathogenic viruses, including simian virus 40 (SV40) and cytomegalovirus (CMV). Lymphokines produced by the immune response to proteins expressed by these vectors could inhibit further transcription initiation by viral promoters. Our objective was to determine the effect of interferon-gamma (IFN-gamma) on transgene expression driven by a viral SV40 or CMV promoter/enhancer and the mammalian promoter/enhancer for the major histocompatibility complex class I (MHC I) gene. We transfected the luciferase gene driven by these three promoters into 14 cell lines of many tissues and several species. Luciferase assays of transfected cells untreated or treated with IFN-gamma indicated that, although the viral promoters could drive luciferase production in all cell lines tested to greater or lesser levels than the MHC I promoter, treatment with IFN-gamma caused inhibition of transgene expression in most of the cell lines and amplification of the MHC I promoter-driven transgene expression in all cell lines. These data indicate that the SV40 and CMV promoter/enhancers may not be a suitable choice for gene delivery especially for immune response applications or in patients where IFN levels may be elevated. The MHC I promoter/enhancer, on the other hand, may be an ideal transgene promoter for applications involving the immune system.

Animals↗
PubMed1998

Transcriptional activation and redox regulation of the tumor necrosis factor-alpha promoter in human T cells: role of the CRE/kappa3 promoter region.

Aberrancies in T cell expression of tumor necrosis factor-alpha (TNF-alpha) are frequently observed in inflammatory states characterized by oxidative stress due to excessive generation of hydrogen peroxide (H2O2) and other reactive oxygen species (ROS). In this study, we examined the possible effects of oxidative stress on the expression of TNF-alpha protein and transcriptional activation of the TNF-alpha promoter in human T cells. Results show that exposure of resting T cells to micromolar concentrations of H2O2 did not induce TNF-alpha protein production or transcriptional activation of the TNF-alpha promoter. However, oxidative signals resulted in a dose-dependent suppression of TNF-alpha protein production and transcriptional activation in T cells stimulated with the lectin phytohemagglutinin (PHA) and phorbol myristate acetate (PMA). Optimal suppression of TNF-alpha promoter activity was observed when cells were exposed to oxidative stress during early T cell activation, and other experiments demonstrated that the transactivation responses of the TNF-alpha promoter were quite susceptible to inhibition by both oxidative and reducing changes in cellular redox. Furthermore, reporter gene assays with 5' deletion mutants of the TNF-alpha promoter showed that the CRE/kappa3 composite site played a major role in activation of the TNF-alpha promoter by dual stimulatory signals and suppression of the TNF-alpha promoter by oxidative signals. Thus, T cell expression of TNF-alpha at the protein and transcriptional levels is highly regulated by changes in cellular redox, and the CRE/kappa3 composite site is important for both activation and redox regulation of the TNF-alpha promoter.

Humans↗
PubMed2001

Health promoting hospitals: a typology of different organizational approaches to health promotion.

This paper draws on a review of the literature about the types of health promotion activities conducted by health promoting hospitals and an observation of how some Australian hospitals have structured the organizational arrangements to be more health promoting. This paper also draws on the experiences of one of the authors (A.J.) in managing and evaluating an organizational change process at a major specialist hospital in Adelaide, South Australia, that sought to re-orientate the hospital towards placing more emphasis on health promotion. From these three sources, a typology of four approaches of organizational arrangement to health promotion is presented. These approaches are: 'doing a health promotion project'; 'delegating it to the role of a specific division, department or staff'; 'being a health promotion setting'; and 'being a health promotion setting and improving the health of the community'. For the re-orientation of the specialist hospital to occur and be sustainable, the research indicated that over the case study period of 1994-1998 there had to be strong organizational commitment to change, supported at multiple levels of the organization, and reflected in policy and practice change. The paper concludes that more evaluative research of this type will be important if the rhetoric of healthy settings is to become a reality.

Attitude of Health Personnel↗
PubMed1993

Increase in the basal transcriptional activity of the human foamy virus internal promoter by the homologous long terminal repeat promoter in cis.

The human foamy or spumaretrovirus HFV is a complex and exogenous retrovirus that encodes several bel genes besides the three classical retroviral genes gag, pol, and env. HFV was recently reported to contain two functionally active promoters that are both strongly trans-activated by the HFV trans-activator protein Bel 1. The occurrence of a second internal cap site underscores the complexity of the HFV genome. We have analysed whether there is interference between the HFV long terminal repeat promoter and the internal promoter located in the 3' end of env upstream of the bel genes. Recombinant clones were constructed that carry two different indicator genes, one under the control of the U3 promoter, the other under the control of the internal promoter. The portion of the basal transcriptional activity of the internal promoter that is not trans-activated by Bel 1 was increased two- to threefold in the presence of the long terminal repeat promoter. The rate of trans-activation by Bel 1 of both HFV promoters was not altered in these constructs.

Animals↗
PubMed2003

Solution structure of the influenza A virus cRNA promoter: implications for differential recognition of viral promoter structures by RNA-dependent RNA polymerase.

Influenza A virus replication requires the interaction of viral RNA-dependent RNA polymerase (RdRp) with promoters in both the RNA genome (vRNA) and the full-length complementary RNA (cRNA) which serve as templates for the generation of new vRNAs. Although RdRp binds both promoters effectively, it must also discriminate between them because they serve different functional roles in the viral life cycle. Even though the inherent asymmetry between two RNA promoters is considered as a cause of the differential recognition by the RdRp, the structural basis for the ability of the RdRp to recognize the RNA promoters and discriminate effectively between them remains unsolved. Here we report the structure of the cRNA promoter of influenza A virus as determined by heteronuclear magnetic resonance spectroscopy. The terminal region is extremely unstable and does not have a rigid structure. The major groove of the internal loop is widened by the displacement of a novel A*(UU) motif toward the minor groove. These internal loop residues show distinguishable dynamic characters, with differing motional timescales for each residue. Comparison of the cRNA promoter structure with that of the vRNA promoter reveals common structural and dynamic elements in the internal loop, but also differences that provide insight into how the viral RdRp differentially recognizes the cRNA and vRNA promoters.

Base Sequence↗
PubMed2005

Information theory based T7-like promoter models: classification of bacteriophages and differential evolution of promoters and their polymerases.

Molecular information theory was used to create sequence logos and promoter models for eight phages of the T7 group: T7, phiA1122, T3, phiYeO3-12, SP6, K1-5, gh-1 and K11. When these models were used to scan the corresponding genomes, a significant gap in the individual information distribution was observed between functional promoter sites and other sequences, suggesting that the models can be used to identify new T7-like promoters. When a combined 76-site model was used to scan the eight phages, 108 of the total 109 promoters were found, while none were found for other T7-like phages, phiKMV, P60, VpV262, SIO1, PaP3, Xp10, P-SSP7 and Ppu40, indicating that these phages do not belong to the T7 group. We propose that the T7-like transcription system, which consists of a phage-specific RNA polymerase and a set of conserved T7-like promoters, is a hallmark feature of the T7 group and can be used to classify T7-like phages. Phylogenetic trees of the T7-like promoter models and their corresponding RNA polymerases are similar, suggesting that the eight phages of the T7 group can be classified into five subgroups. However the SP6-like polymerases have apparently diverged from other polymerases more than their promoters have diverged from other promoters.

DNA-Directed RNA Polymerases↗
PubMed1992

The Commelina yellow mottle virus promoter is a strong promoter in vascular and reproductive tissues.

Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects the monocot Commelina diffusa. Although CoYMV and cauliflower mosaic virus (CaMV; another double-stranded DNA virus) probably replicate by a similar mechanism, the particle morphology and host range of CoYMV place it in a distinct group. We present evidence that a prompter fragment isolated from CoYMV confers a tissue-specific pattern of expression that is different from that conferred by the CaMV 35S promoter. When the CoYMV promoter is used to drive expression of the beta-glucuronidase reporter gene in stably transformed tobacco plants, beta-glucuronidase activity occurs primarily in the phloem, the phloem-associated cells, and the axial parenchyma of roots, stems, leaves, and flowers. Activity is also detected throughout the anther, with highest activity in the tapetum. In contrast, the CaMV 35S promoter is active in most cell types. The CoYMV promoter is a strong promoter, and when the activity of the CoYMV promoter is compared with that of a duplicated CaMV 35S promoter, it is 30% as active in tobacco suspension cells and up to 25% as active in maize suspension cells. These properties of the CoYMV promoter make it potentially useful for high-level expression of engineered genes in vascular cells.

Base Sequence↗