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PubMed1999

Long-distance RNA-RNA interactions and conserved sequence elements affect potato virus X plus-strand RNA accumulation.

Conserved octanucleotide sequences located upstream of two major potato virus X (PVX) subgenomic RNAs (sgRNAs), as well as elements in the 5' end of the genome, affect accumulation of sgRNA. To determine if complementarity between these sequences is important for PVX RNA accumulation, we analyzed the effects of mutations within these elements and compensatory mutations in a tobacco protoplast system and in plants. Mutations in the 5' nontranslated region (NTR mutants) that reduced complementarity resulted in lower genomic RNA (gRNA) and sgRNA levels, whereas mutations to the octanucleotide elements affected only the corresponding sgRNA levels. However, for both the NTR and octanucleotide mutants, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Mutants containing changes in the NTR and compensatory changes in one of the octanucleotide elements restored levels of gRNA and the other sgRNA species with an unaltered octanucleotide element to those of wild-type. Although compensatory changes significantly increased levels of the sgRNA species with the modified octanucleotide element, levels were not restored to those of wild-type. Our data indicate that long distance RNA-RNA interactions and the sequences of the interacting elements are required for PVX plus-strand RNA accumulation.

5' Untranslated Regions↗

PubMed1999

A selection system for functional internal ribosome entry site (IRES) elements: analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES.

Picornavirus internal ribosome entry site (IRES) elements direct cap-independent internal initiation of protein synthesis within mammalian cells. These RNA elements (about 450 nt) contain extensive secondary structure including a hairpin loop with a conserved GNRA motif. Such loops are important in RNA-RNA and RNA-protein interactions. Plasmids that express dicistronic mRNAs of the structure GUS/IRES/HOOK have been constructed. The HOOK sequence encodes a cell-surface-targeted protein (sFv); the translation of this open reading frame within mammalian cells from these dicistronic mRNAs requires a functional IRES element. Cells that express the sFv can be selected from nonexpressing cells. A pool of up to 256 mutant encephalomyocarditis virus IRES elements was generated by converting the wild-type hairpin loop sequence (GCGA) to NNNN. Following transfection of this pool of mutants into COS-7 cells, plasmids were recovered from selected sFv-expressing cells. These DNAs were amplified in Escherichia coli and transfected again into COS-7 cells for further cycles to enrich for plasmids encoding functional IRES elements. The sequence of individual selected IRES elements was determined. All functional IRES elements had a tetraloop with a 3' terminal A residue. Optimal IRES activity, assayed in vitro and within cells, was obtained from plasmids encoding an IRES with the hairpin loop sequence fitting a RNRA consensus. In contrast, IRES elements containing YCYA tetraloops were severely defective.

Animals↗

PubMed1992

Contributions of phylogenetically variable structural elements to the function of the ribozyme ribonuclease P.

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme which participates in processing precursor tRNAs. The RNA subunit contains the catalytic site and is capable of catalysis in the absence of the protein subunit. RNase P RNAs from various eubacteria consist of a core of conserved sequence and secondary structure which is evolutionarily modified in different organisms by the presence of discrete helical elements at various sites in the RNAs. The variable occurrence of these helical elements suggests that they have no important functional role in the enzyme. The Escherichia coli RNase P RNA contains four such elements. It has been shown that simultaneous deletion of all four of them produces an RNA that is functional but has several significant defects which could arise from general disruption of the RNA or from the loss of element-specific functions. This paper describes a more detailed analysis of the role of the variable elements in E. coli RNase P RNA. Removal of one of the elements had no apparent effect on RNase P activity in vitro. Two other elements are required for correct folding of the RNA: their absence confers a requirement for extremely high monovalent salt concentrations, apparently to reduce intramolecular electrostatic repulsion. The fourth element that was tested participates in a long-range structural interaction (pseudoknot) which contributes to the structural stability of the enzyme and affects substrate binding affinity. In the absence of this helix, the RNA becomes temperature-sensitive, and the KM increases 100-fold.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗

PubMed1996

Identification of functional positive and negative thyroid hormone-responsive elements in the rat apolipoprotein AI promoter.

Transcription of the antiatherogenic protein apolipoprotein AI is regulated by the thyroid hormone, L-triiodothyronine. Transient transfection and electrophoretic mobility shift assays were used to identify the cis-acting elements involved. In transient transfection assays, hormone bound to either thyroid hormone receptor alpha or beta exerts a positive effect through a thyroid hormone response element, site A (-208 to -193). In the absence of site A, liganded receptor alpha or beta have a negative effect on promoter activity. This negative effect is mediated by a 40 bp fragment spanning nucleotides -46 to -7. Closer examination of this region of the gene shows there to be a negative thyroid hormone response element at position -25 to -20 which is fused to the 3' end of the TATA element. Electrophoretic mobility shift assays show that bacterially expressed chicken or rat thyroid hormone receptor alpha 1 binds to site A, either as a homodimer or as a heterodimer with the human 9-cis-retinoic acid receptor alpha. In contrast, the negative thyroid hormone responsive element binds chicken thyroid hormone receptor alpha exclusively as a monomer. Site-directed mutagenesis of the negative thyroid hormone response element abolished the inhibitory effects of the hormone and increased basal promoter activity by up to 40-fold. These data suggest that functional positive and negative thyroid hormone response elements coexist within the rat apolipoprotein AI promoter and both elements contribute to the control of apolipoprotein AI gene expression.

Animals↗

PubMed2005

Microbial passage in low pressure membrane elements with compromised integrity.

The main objective of this study was to evaluate microbial passage through membrane elements with various levels of compromised integrity while installed within full-scale UF/MF systems. Challenge tests were performed for this purpose with the microbial contaminant surrogates Bacillus subtilis spores at two drinking water treatment plants. The experimental units used were one of several parallel racks part of each plant in which one of the elements was installed with various levels of compromised integrity achieved by precutting several hollow fibers and inserting removable pin-plugs atthe corresponding ends of the broken fiber segments. The UF rack was operated with 38 elements online (the rack has 50 elements) and was designed for an inside-out operation, and the MF rack included 50 elements and was operated in an outside-in mode (with a permeate outlet at one fiber end only). Spore removals observed for both the UF and MF racks with all precut fibers plugged were equal to or greater than 99.9992%, and as expected the removal efficiency deteriorated with an increasing number of unplugged fibers. Predictions made with a model based on the use of the Hagen-Poisseuille equation for laminar flow and the Darcy-Weisbach expression for turbulent flow inside broken fibers were found to provide an adequate conservative representation of experimental results. Additional simulations performed with the verified model revealed the occurrence of a greater microbial passage for an inside-out configuration compared to an outside-in mode. A lower microbial passage was predicted for the outside-in element configuration with one permeate outlet as compared to an element with permeate outlets at both fiber ends. The model offers a useful tool that together with other considerations such as membrane fouling, cleaning, and durability would assist in the selection of low-pressure membrane element configuration.

Bacillus subtilis↗

PubMed2005

Cell-membrane damage and element leaching in transplanted Parmelia sulcata lichen related to ambient SO2, temperature, and precipitation.

Measurements were performed in lichen (Parmelia sulcata) transplants, to gain insight into the lichen vitality as possibly affected by both element deposition or lichen element content and further ambient atmospheric conditions (temperature, precipitation, SO2 levels). The electrical conductivity of rinsing solutions was used to assess cell-membrane damage in Parmelia sulcata in an experiment, which ran from August 2001 until August 2002. Element contents of the solutions and lichens were determined by inductively coupled plasma-optical emission spectrometry and by k0-instrumental neutron activation analysis, respectively. Factor analysis (MCATTFA) was used to determine grouping of elements of similar origin and/ or behavior. All in all, the data indicate that, apartfrom lichen Na and Cl levels, and for temperature and precipitation, no clear relationships with conductivity could be observed. Conductivity was mostly related to released Na, Cl, K, Mg, and Cs. On the basis of concentrations, Na, Cl, and K could be considered as largely determining the conductivity. The data suggest a different origin for K than that for Na and Cl: the latter two are most probably due to effects from sea salt sprays. Parmelia sulcata was sensitive enough to reflect appreciable ambient rises in air SO2 and resistant enough to recover afterward. MCATTFA on selected elements (K, Sc, Cu, V, As, and Sb) indicated the absence of any comparability between K and V, As and Sb, suggesting differences in origin and/or chemico-physical occurrence. Generally speaking, the present data suggest that the comparability of lichen vitality in large geographical areas may be limited and governed by the area's variability in temperature and precipitation rather than by variability in metal deposition rates. The leaching data on all elements and element groups, however, strongly suggest that wet deposition may also severely affect lichen elemental levels. This latter observation means that comparing outcomes for time or spatial series of lichen samples should be accompanied by a comparably careful monitoring of (preceding) ambient conditions.

Air Pollutants↗

PubMed2001

The effect of MAR elements on variation in spatial and temporal regulation of transgene expression.

The level of transgene expression often differs among independent transformants. This is generally ascribed to different integration sites of the transgene into the plant genome in each independently obtained transformant (position effect). It has been shown that in tobacco transformants expressing, for example, a cauliflower mosaic virus (CaMV) 35S promoter-driven beta-glucuronidase (GUS) reporter gene, these position-induced quantitative differences among individual transformants were reduced by the introduction of matrix-associated regions (MAR elements) on the T-DNA. We have previously shown by imaging of in planta firefly luciferase (luc) reporter gene activity that quantitative differences in transgene activity can be the result of either a variation in (1) level, (2) spatial distribution and/or (3) temporal regulation of transgene expression between independent transformants. It is not known which of these three different aspects of transgene expression is affected when the transgene is flanked by MAR elements. Here we have used the firefly luciferase reporter system to analyse the influence of MAR elements on the activity of a CaMV 35S-luc transgene in a population of independently transformed tobacco plants. Imaging of in planta LUC activity in these tobacco plant populations showed that the presence of MAR elements does not result in less variation in the average level of transgene expression between individual transformants. This result is different from that obtained previously with a 35S-GUS reporter gene flanked by MAR elements and reflects the differences in the stability of the LUC and GUS reporter proteins. Also the variation in spatial patterns of in vivo LUC activity is not reduced between independent transformants when the transgene is flanked by MAR elements. However, MAR elements do seem to affect the variation in temporal regulation of transgene expression between individual transformants. The potential effects of MAR elements on the variability of transgene expression and the relation to the stability of the (trans)gene product are discussed.

Gene Expression Regulation, Developmental↗

PubMed1999

The GATA-1 and Spi-1 transcriptional factors bind to a GATA/EBS dual element in the Fli-1 exon 1.

Fli-1 is a proto-oncogene which is rearranged in tumors induced by three different retroviruses, Cas-Br-E, F-MuLV, and 10A1. This gene is a member of the Ets gene family, a class of transcription factors that recognize and bind to a DNA motif known as the Ets binding site (EBS). Our laboratory has previously cloned and characterized the promoter region of both human and mouse Fli-1 genes. We had then identified several regulatory elements conserved between the two species. Two of them, an exon 1 GATA/EBS dual element and an EBS element located in the 5' end of intron 1, were analysed in the present study. EMSA analysis performed with nuclear extracts from different cell lines showed that the EBS element in intron 1 (EBSi) was bound by one potential Ets-related ubiquitous factor. The GATA/EBS element was bound by several factors that seemed Ets-related, one of which was found to be specifically expressed in hematopoietic cells. the GATA/EBS dual element was thus chosen for further analysis. A human Fli-1-derived genomic fragment containing the GATA/EBS led to enhanced transcription when positioned upstream of the SV40 promoter in the erythroleukemic HEL cell line. In addition, an increasing number of GATA/EBS oligonucleotides upstream of this same promoter resulted in a copy number-dependent increase in luciferase activity which was greatly reduced when the EBS consensus sequence was mutated. One of the factors binding to the GATA/EBS region was identified to be Spi-1 by supershift analysis and was also shown to bind to the EBS element of the human Ets-2 gene. Supershift analysis also demonstrated the binding of the GATA-1 factor to the GATA/EBS dual element. Our results suggest that Spi-1 and GATA-1 might play a key role in the regulation of Fli-1.

Animals↗

PubMed2002

SEC-ICP-MS studies for elements binding to different molecular weight fractions of humic substances in compost extract obtained from urban solid waste.

In this work, the speciation of elements in compost was studied with emphasis on their binding to humic substances. In order to assess the distribution of As, Cd, Co, Cr, Cu, Mn, Mo, Ni, Pb, U, Th and Zn among molecular weight fractions of humic substances, the compost extract (extracted by 0.1 mol l(-1) sodium pyrophosphate) was analyzed by size exclusion chromatography coupled on-line with UV-Vis spectrophotometric and ICP-MS detection. Similar chromatograms were obtained for standard humic acid (Fluka) and for compost extract (254 nm, 400 nm) and three size fractions were operationally defined that corresponded to the apparent molecular weight ranges > 15 kDa, 1-15 kDa and < 1 kDa. The percentage of total element content in compost that was leached to the extract ranged from 30% up to 100% for different elements. The elution profiles of Co, Cr, Cu, Ni and Pb (ICP-MS) followed that of humic substances, while for other elements the bulk elution peak matched the retention time observed for the element in the absence of compost extract. Spiking experiments were carried out to confirm elements' binding and to estimate the affinity of individual elements for humic substances derived from compost. The results obtained indicated the following order of decreasing affinity: Cu > Ni > Co > Pb > Cd > (Cr, U, Th) >> (As, Mn, Mo, Zn). After standard addition, further binding of Cu, Ni and Co with the two molecular weight fractions of humic substances was observed, indicating that humic substances derived from compost were not saturated with these elements.

Chromatography, Gel↗

PubMed2002

Priorities for disclosure of the elements of informed consent for research: a comparison between parents and investigators.

BACKGROUND: Disclosure is a key element of the informed consent process. This study examines and compares the priorities for disclosure of the elements of informed consent between parents of paediatric research subjects and investigators. METHODS: The study sample comprised 184 parents who had been approached for permission to allow their child to participate in a clinical anaesthesia or surgery study. Parents were asked to rank 11 elements of informed consent that they believed were most important for them to know before allowing their child to participate in a research study. In addition, 38 investigators were asked to rank, in order of importance, the same elements that they felt were most important for parents to know. RESULTS: The results showed that risk was the most important element considered by both parents and investigators. However, parents placed significantly greater importance on knowledge of the potential benefits to their child (direct) and to other children (indirect) compared with investigators, and less importance on the details of the protocol and the element of voluntariness. CONCLUSIONS: These results demonstrated differences in the priorities for disclosure of the elements of consent between parents and investigators. As such, they may be important in directing the investigator to focus on the elements that are most important to parents and thus maximize their ability to provide truly informed consent.

Adult↗

PubMed1990

Context superiority in a detection task with line-element stimuli: a low-level effect.

Detection and identification performances for vertical and horizontal target elements embedded within an array of oriented noise elements were measured as a function of the orientation difference between the target and noise elements. Detection performances obtained with one vertical and three horizontal target elements clustered together and displayed such that they formed a schematic face-like pattern were significantly better than those obtained with the same clustered target elements displayed in an arbitrary, symmetrical or asymmetrical, configuration. This was so even though the identification of the face and nonface stimuli was well below the detection threshold of their parts. Detection thresholds for the clustered nonface patterns were slightly but significantly lower than those for the same target elements dispersed among the noise elements. Probability summation calculations based on the detection results obtained with one single target element predict detection thresholds which are intermediate between those of the clustered and dispersed targets, suggesting that inhibitory and facilitatory spatial interactions respectively are at work for the two types of stimuli. The existence of a context(face)-superiority effect at the detection level indicates top-down/bottom-up interactions between remote visual processing stages.

Attention↗

PubMed1987

Identification of an essential upstream element in the nopaline synthase promoter by stable and transient assays.

We studied the fine structure of the nopaline synthase (nos) promoter, which is active constitutively in a wide range of plant tissues, by both transient and stable transformation expression analyses. 3' and 5' deletion fragments were linked to form a set of internal deletion and duplication mutants that scanned the nos promoter. These mutated promoters were linked to the gene for the marker chloramphenicol acetyltransferase (CATase) as a means to readily assay promoter strength. The stable transformation analysis revealed the functional importance of an extended CCAAT box region (-97 to -63). Deletion of an upstream region (-112 to -101) containing an octameric repeated element resulted in a reduction in promoter strength by a factor of 30. A further deletion (-119 to -101) disrupted a potential Z-DNA-forming element as well, totally eliminating promoter function. Thus, a 19-base deletion across a repeated octamer and a potential Z-DNA-forming element identifies an essential upstream activator in the nos promoter. Duplication of the upstream element tripled promoter activity. Electroporation-mediated transient analysis was unable to distinguish downstream promoter elements. However, the upstream element behaved similarly in both assays in that deletion of the entire upstream element resulted in no promoter activity and that duplication of the element significantly enhanced the promoter strength.

Journal Article↗

PubMed1988

Mobility of P elements in drosophilids and nondrosophilids.

The mobility properties of the Drosophila melanogaster P element in drosophilid and nondrosophilid species has been determined using a P-element mobility assay that is conducted transiently in insect embryos. P elements are mobilizable in all drosophilids tested, including species outside the genus Drosophila but not in the related Tephritidae (order: Diptera), although the P-element gene necessary for mobility, transposase, is transcribed. These results show that without modifications P elements will not serve as general insect gene vectors and suggest that nonconserved host-encoded factors participate in the transposition of P elements. Our methods will be generally useful for analyzing the cis- and trans-acting factors required for P-element mobility in vivo and could be used to analyze the mobility properties of other transposable elements in insects.

Journal Article↗

PubMed1988

Two adjacent promoter elements mediate nerve growth factor activation of the c-fos gene and bind distinct nuclear complexes.

Protooncogene fos is rapidly and transiently induced by nerve growth factor (NGF) in rat pheochromocytoma PC12 cells. Two adjacent promoter elements have been identified to mediate the NGF response. One element colocalizes with the serum response element (SRE) centered at position -308, previously shown to confer inducibility by serum, phorbol 12-myristate 13-acetate, and epidermal growth factor, whereas the other element, termed SRE-2, maps approximately 20 base pairs downstream of the SRE and contains several sequence repeats. This element also confers serum responsiveness. Gel mobility shift assays have demonstrated that there are specific nucleoprotein complexes associated with each element and that these exist in the cell prior to NGF induction. The NGF response is independent of the cAMP-regulatory element(s) and does not require cAMP-dependent protein kinase II, as induction by NGF is retained in the mutant PC12 cell line A126-1B2. Finally, the human heat shock HSP70 promoter is also transcriptionally activated by NGF and appears to bind the same nuclear complex as the SRE-2 element of the c-fos promoter.

Animals↗

PubMed1992

Vitamin D-responsive protein-DNA interactions at multiple promoter regulatory elements that contribute to the level of rat osteocalcin gene expression.

The observation that vitamin D-mediated enhancement of osteocalcin (OC) gene expression is dependent on and reciprocally related to the level of basal gene expression suggests that an interaction of the vitamin D responsive element (VDRE) with basal regulatory elements of the OC gene promoter contributes to both basal and vitamin D-enhanced transcription. Protein-DNA interactions at the VDRE of the rat OC gene (nucleotides -466 to -437) are reflected by direct sequence-specific and antibody-sensitive binding of the endogenous vitamin D receptor present in ROS 17/2.8 osteosarcoma nuclear protein extracts. In addition, a vitamin D-responsive increase in OC gene transcription is accompanied by enhanced non-vitamin D receptor-mediated protein-DNA interactions in the "TATA" box region (nucleotides -44 to +23), which also contains a potential glucocorticoid responsive element. Evidence for proximity of the VDRE with the basal regulatory elements is provided by two features of nuclear architecture. (i) Nuclear matrix attachment elements in the rat OC gene promoter that bind nuclear matrix proteins with sequence specificity may impose structural constraints on promoter conformation. (ii) Limited micrococcal nuclease digestion and Southern blot analysis indicate that three nucleosomes can be accommodated in the sequence spanning the OC gene VDRE, the OC/CCAAT box (nucleotides -99 to -76), and the TATA/glucocorticoid responsive element, and thereby the potential distance between the VDRE and the basal regulatory elements can be reduced. A model is presented for the contribution of both the VDRE and proximal promoter elements to the enhancement of OC gene transcription in response to vitamin D. The vitamin D receptor plus accessory proteins may function cooperatively with basal regulatory factors to modulate the extent to which the OC gene is transcribed.

Animals↗

PubMed1994

ocs element promoter sequences are activated by auxin and salicylic acid in Arabidopsis.

ocs elements are a group of promoter elements that have been exploited by two distinct groups of plant pathogens, Agrobacterium and certain viruses, to express genes in plants. We examined the activity of single and multiple ocs elements linked to a minimal plant promoter and the uidA reporter gene in transgenic Arabidopsis. beta-Glucuronidase activity was detected only in root tips and in callus tissue after auxin treatment. A more sensitive assay revealed that auxin treatment also increased ocs element activity in aerial parts of the plant, although the absolute levels of ocs element activity were greater in roots. The response of ocs elements to exogenous auxin began within 1 h. Salicylic acid, a disease-resistance signal in plants, also increased ocs element activity in both roots and aerial parts of the plant. The question of whether the induction in ocs element activity is mediated through auxin and/or salicylic acid signal transduction pathways or is part of a more general stress response is discussed.

Arabidopsis↗

PubMed1995

Identification of a DNA element determining synaptic expression of the mouse acetylcholine receptor delta-subunit gene.

mRNAs for acetylcholine receptor genes are highly concentrated in the endplate region of adult skeletal muscle largely as a result of a transcription restricted to the subneural nuclei. To identify the regulatory elements involved, we employed a DNA injection of a plasmid containing a fragment of the acetylcholine receptor delta-subunit gene promoter (positions -839 to +45) linked to the reporter gene lacZ with a nuclear localization signal. Injection of the wild-type construct into mouse leg muscles yielded preferential expression of the reporter gene in the synaptic region. Analysis of various mutant promoters resulted in the identification of a DNA element (positions -60 to -49), referred to as the N box, that plays a critical role in subneural expression. Disruption of this 12-bp element in the context of a mouse delta-subunit promoter from positions -839 to +45 gives widespread expression of the reporter gene throughout the entire muscle fiber, indicating that this element is a silencer that represses delta-subunit gene transcription in extrajunctional areas. On the other hand, this element inserted upstream of a heterologous basal promoter preferentially enhances expression in the endplate region. This element therefore regulates the restricted expression of the delta-subunit gene both as an enhancer at the endplate level and as a silencer in extrajunctional areas. Furthermore, gel-shift experiments with mouse muscle extracts reveal an activity that specifically binds the 6-bp sequence TTCCGG of this element, suggesting that a transcription factor(s) controls the expression of the delta-subunit gene via this element.

Animals↗

PubMed1995

Cooperation between core promoter elements influences transcriptional activity in vivo.

Core promoters for RNA polymerase II frequently contain either (or both) of two consensus sequence elements, a TATA box and/or an initiator (Inr). Using test promoters consisting of prototypical TATA and/or Inr elements, together with binding sites for sequence-specific activators, we have analyzed the function of TATA and Inr elements in vivo. In the absence of activators, the TATA element was significantly more active than the Inr, and the combination of elements was only slightly more effective than the TATA-only promoter. In the presence of any of several coexpressed activator proteins, the TATA elements was again most active, but here addition of the Inr allowed significant increases in activity, indicating a cooperative interaction between the two elements. An interesting exception was observed with the activator Sp1, which was more effective with the Inr-only promoter, and addition of a TATA box did not enhance activity. Finally, in all cases the TATA plus Inr promoters were found to be partially or completely resistant to the dominant negative effects of a transcription factor TFIIB mutant previously shown to interfere with expression from TATA-only promoters. This result strengthens the conclusion that TATA and Inr elements can cooperate in vivo.

Animals↗