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At least 163 records · Page 9Linked to original sources
PubMed1991

A cell cycle-responsive transcriptional control element and a negative control element in the gene encoding DNA polymerase alpha in Saccharomyces cerevisiae.

Transcription of the POL1 gene of Saccharomyces cerevisiae, which encodes DNA polymerase alpha, the DNA polymerase required for the initiation of DNA replication, has previously been shown to be cell cycle regulated. To understand how the POL1 gene senses cell cycle position, we have investigated the cis-acting elements that respond to the factors that govern cell cycle progression. In this report we demonstrate that a region of 54 nucleotides containing the repeated element ACGCGT, which conforms to an Mlu I restriction endonuclease recognition site, contains all information necessary for transcriptional activation and cell cycle responsiveness. Although oligonucleotides lacking either one or both of the repeated Mlu I sites can function as an upstream activating sequence, the presence of at least one Mlu I site stimulates expression and, moreover, is absolutely essential for cell cycle regulation. A synthetic oligonucleotide corresponding to a 19-base-pair sequence in the POL1 promoter containing one Mlu I site can function as an autonomous cell cycle-responsive upstream element (upstream activation sequence) with temporal regulation indistinguishable from that previously described for the POL1 gene. Thus, the Mlu I site is an essential part of a cis-acting element responsible for the observed periodic activation. This sequence differs from previously defined cell cycle-responsive transcriptional control elements in the yeast HO endonuclease and histone genes. We also present evidence for a negative regulatory element in the 5' flanking region of the Mlu I upstream activation sequence.

Base Sequence↗
PubMed1996

An Alu element in the myeloperoxidase promoter contains a composite SP1-thyroid hormone-retinoic acid response element.

An Alu element preceding the myeloperoxidase gene (MPO) contains four hexamer motifs related to the consensus recognition sequence for nuclear hormone receptors (AGGTCA), arranged as direct repeats with spacing of 2, 4, and 2 nucleotides (DR-2-4-2). Gel shift experiments and transient transfection assays demonstrate that these sequences include binding sites for retinoic acid and thyroid hormone receptors and function in vivo to activate transcription of a chloramphenicol acetyltransferase reporter gene. The first DR-2 elements of the series do not bind known receptors but do bind the SP1 transcription factor. Two alleles of the MPO gene exist that differ at one position within this element, resulting in one allele with and one without a strong SP1 binding site. The element with the SP1 site activates transcription by 25-fold in transient transfection assays, while the alternative allele confers severalfold less transcriptional activity. Most cases of acute myelocytic leukemia are homozygous for the allele with the SP1 binding site, suggesting this element plays an important role in regulating the MPO gene in myeloid leukemias. This MPO-Alu is a representative of an Alu subclass numbering approximately 400,000 copies, suggesting many genes may be regulated by such elements.

Alleles↗
PubMed1997

A transforming growth factor beta (TGFbeta) control element drives TGFbeta-induced stimulation of smooth muscle alpha-actin gene expression in concert with two CArG elements.

The goal of the present study was to determine the molecular mechanism whereby transforming growth factor beta (TGFbeta) increases smooth muscle (SM) alpha-actin expression. Confluent, growth-arrested rat aortic smooth muscle cells (SMC) were transiently transfected with various SM alpha-actin promoter/chloramphenicol acetyltransferase deletion mutants and stimulated with TGFbeta (2.5 ng/ml). Results demonstrated that the first 125 base pairs of the SM alpha-actin promoter were sufficient to confer TGFbeta responsiveness. Three cis elements were shown to be required for TGFbeta inducibility: two highly conserved CArG boxes, designated A (-62) and B (-112) and a novel TGFbeta control element (TCE) (-42). Mutation of any one of these elements completely abolished TGFbeta-induced reporter activity. Results of electrophoretic mobility shift assays demonstrated that nuclear extracts from TGFbeta-treated SMC enhanced binding activity of serum response factor to the CArG elements and binding of an as yet unidentified factor to the TCE. Northern analysis showed that TGFbeta also stimulated transcription of two other SM (SM myosin heavy chain) differentiation marker genes, SM myosin heavy chain and h1 calponin, whose promoters also contained a TCE-like element. In summary, we identified a TGFbeta response element in the SM alpha-actin promoter that may contribute to coordinate regulation of expression of multiple cell-type specific proteins during SMC differentiation.

Actins↗
PubMed1994

Localization of an RNA binding element of the iron responsive element binding protein within a proteolytic fragment containing iron coordination ligands.

The iron responsive element binding protein (IRE-BP) regulates iron storage and uptake in response to iron. This control results from the interaction of the IRE-BP with the iron responsive element (IRE), a conserved sequence/structure element located near the 5' end of all ferritin mRNAs and in the 3' UTR of transferrin receptor mRNAs. Proteolysis was used to probe for functional elements of the IRE-BP. Partial chymotrypsin digestion generates a simple digestion pattern yielding fragments of 68, 56, 41, and 30 kDa. The 68 and 30 kDa fragments are derived from a single cleavage at Trp623. Further cleavages of the 68 kDa polypeptide yield the 56 and 41 kDa peptides. A combination of UV-crosslinking and chymotrypsin digestion was used to localize an RNA binding element within the C-terminus of the 68 kDa fragment, between amino acid residues 480 and 623. This region includes cysteine residues 503 and 506 which have been shown to be required for iron-sulfur cluster assembly and for iron regulation of the IRE-BP. Proteolytic fragments of the IRE-BP that contain this RNA binding region can be crosslinked to the IRE but do not bind with high affinity, suggesting that elements within the IRE-BP, in addition to those located between residues 480 and 623, are required for high affinity binding to the IRE.

Amino Acid Sequence↗
PubMed1997

HIV-1 Tat protein can transactivate a heterologous TATAA element independent of viral promoter sequences and the trans-activation response element.

OBJECTIVE: To determine whether the HIV-1 transactivator protein Tat acts as a DNA sequence-specific transcription factor and activates transcription from a heterologous TATAA element in the absence of the trans-activation response (TAR) element and other sequences in the HIV-1 long terminal repeat (LTR). DESIGN: Activating protein-1 (AP-1) and Tat-induced transcription were assessed using Jun and hybrid Tat/Jun-expression plasmids and reporter gene constructs which contained AP-1 binding sites upstream of the rat prolactin TATAA element or an HIV-1 LTR construct in which AP-1 binding sites replaced the TAR element. METHODS: Tat-induced transcription was determined following transient transfection of colon epithelial cell lines with reporter gene constructs and Tat/Jun-expression plasmids in which Tat was fused to the DNA binding domain of Jun. Activation of prolactin (PL) and LTR reporter genes was assessed by luciferase (LUC) or chloramphenicol acetyltransferase (CAT) activity in cellular extracts. RESULTS: Cotransfection of cells with Tat/Jun and the AP-1 PL LUC or LTR AP-1 CAT reporter plasmid resulted in a marked increase in reporter gene activity which was comparable with that induced by transfection of cells with several different AP-1 expression plasmids (e.g., JunD, JunB, c-Fos), or that elicited by stimulation of the cells transfected with LTR AP-1 CAT plasmids with phorbol ester or tumor necrosis factor-alpha. Tat-induced transcription was DNA-mediated since both a Jun DNA binding domain fused to Tat as well as AP-1 binding sites within the promoter were required for the induction of CAT expression. CONCLUSIONS: Tat-activated transcriptor can occur strictly through a heterologous TATAA element independent of TAR and Sp1 binding sites or other HIV-1 LTR sequences. Tat appears to increase transcription initiated through the TATAA element by mechanisms similar to that of DNA sequence-specific transcription factors.

Animals↗
PubMed2004

Plane-wave basis finite elements and boundary elements for three-dimensional wave scattering.

Classical finite-element and boundary-element formulations for the Helmholtz equation are presented, and their limitations with respect to the number of variables needed to model a wavelength are explained. A new type of approximation for the potential is described in which the usual finite-element and boundary-element shape functions are modified by the inclusion of a set of plane waves, propagating in a range of directions evenly distributed on the unit sphere. Compared with standard piecewise polynomial approximation, the plane-wave basis is shown to give considerable reduction in computational complexity. In practical terms, it is concluded that the frequency for which accurate results can be obtained, using these new techniques, can be up to 60 times higher than that of the conventional finite-element method, and 10 to 15 times higher than that of the conventional boundary-element method.

Journal Article↗
PubMed1998

A comparative evaluation of mandibular finite element models with different lengths and elements for implant biomechanics.

Further validity of finite element analysis (FEA) in implant biomechanics requires an increase of modelled range and mesh refinement, and a consequent increase in element number and calculation time. To develop a new method that allows a decrease of the modelled range and element number (along with less calculation time and less computer memory), 10 FEA models of the mandible with different mesio-distal lengths and elements were constructed based on three-dimensional graphic data of the bone structure around an osseointegrated implant. Analysis of stress distribution followed by 100 N loading with the fixation of the most external planes of the models indicated that a minimal bone length of 4.2 mm of the mesial and distal sides was acceptable for FEA representation. Moreover, unification of elements located far away from the implant surface did not affect stress distribution. These results suggest that it may be possible to develop a replica FEA implant model of the mandible with less range and fewer elements without altering stress distribution.

Biomechanical Phenomena↗
PubMed2005

Lamb mode conversion at edges. A hybrid boundary element-finite-element solution.

Two general and flexible numerical techniques based on the finite-element and boundary element methods developed by the authors in a previous paper are applied to study Lamb wave propagation in multilayered plates and Lamb mode conversion at free edges for frequencies beyond the first cutoff frequency. Both techniques are supported by a meshing criterion which guarantees the accuracy of the results when a condition is fulfilled. A finite-element formulation is directly applicable to study Lamb wave propagation and reflection by simple obstacles such as a flat edge. In order to tackle Lamb wave diffraction problems by defects with more complex geometries, a hybrid boundary element-finite-element formulation is used. This technique provides a major improvement with respect to the only previous boundary element application on Lamb waves: the connecting boundary might be placed as close to the reflector as desired, reducing greatly the requirement on mesh size. Two main application problems on practical metallic plates are studied and compared with reported numerical, theoretical, and experimental results: (1) Lamb wave propagation in degraded titanium diffusion bonds, and (2) Lamb mode conversion at inclined or perpendicular free edges of steel plates for frequencies beyond the first cutoff frequency.

Journal Article↗
PubMed1999

Utilization of splicing elements and polyadenylation signal elements in the coupling of polyadenylation and last-intron removal.

Polyadenylation (PA) is the process by which the 3' ends of most mammalian mRNAs are formed. In nature, PA is highly coordinated, or coupled, with splicing. In mammalian systems, the most compelling mechanistic model for coupling arises from data supporting exon definition (2, 34, 37). We have examined the roles of individual functional components of splicing and PA signals in the coupling process by using an in vitro splicing and PA reaction with a synthetic pre-mRNA substrate containing an adenovirus splicing cassette and the simian virus 40 late PA signal. The effects of individually mutating splicing elements and PA elements in this substrate were determined. We found that mutation of the polypyrimidine tract and the 3' splice site significantly reduced PA efficiency and that mutation of the AAUAAA and the downstream elements of the PA signal decreased splicing efficiency, suggesting that these elements are the most significant for the coupling of splicing and PA. Although mutation of the upstream elements (USEs) of the PA signal dramatically decreased PA, splicing was only modestly affected, suggesting that USEs modestly affect coupling. Mutation of the 5' splice site in the presence of a viable polypyrimidine tract and the 3' splice site had no effect on PA, suggesting no effect of this element on coupling. However, our data also suggest that a site for U1 snRNP binding (e.g., a 5' splice site) within the last exon can negatively effect both PA and splicing; hence, a 5' splice site-like sequence in this position appears to be a modulator of coupling. In addition, we show that the RNA-protein complex formed to define an exon may inhibit processing if the definition of an adjacent exon fails. This finding indicates a mechanism for monitoring the appropriate definition of exons and for allowing only pre-mRNAs with successfully defined exons to be processed.

HeLa Cells↗
PubMed1995

One of three CCArGG box/serum response elements of the beta-actin gene is an insulin-responsive element.

The cytoskeletal actins are abundant proteins in mammalian nonmuscle cells. We have previously reported that physiological concentrations of insulin induced beta-actin transcription in rat H4 hepatoma cells. To define whether one or more of the three CCArGG box elements or other elements within the beta-actin gene promoter is an insulin response element, we transfected H4 cells with regions of the human beta-actin gene promoter fused to the chloramphenicol acetyltransferase gene. A 350-basepair DNA fragment was isolated that mediates both insulin and serum effects. This fragment contains at least two up-stream elements, a CCAAT box and a CCArGG box, and accounts for more than 70% of the basal activity of the beta-actin promoter in H4 cells. There was a small, but significant, stimulatory effect of insulin over maximal serum induction, suggesting a difference in their mechanisms of action. Mutation of the CCAAT box drastically reduced basal expression, with no effect on insulin induction. In contrast, a mutation of the CCArGG element reduced basal expression and completely abolished insulin inducibility. Electrophoretic mobility shift assays suggested that insulin regulated the activity, but not the binding, of a factor(s) that associates with the CCArGG box. These data demonstrate that in H4 cells, insulin induction of beta-actin gene expression was mediated at least in part through one of the three beta-actin CCArGG elements.

Actins↗
PubMed1991

Identification of a thyroid hormone response element in the phosphoenolpyruvate carboxykinase (GTP) gene. Evidence for synergistic interaction between thyroid hormone and cAMP cis-regulatory elements.

Transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) in the liver is regulated by many hormones including thyroid hormone (T3). In order to identify the elements in the promoter which are required for transcriptional induction by T3, we cotransfected a T3 receptor expression vector with a PEPCK-CAT reporter gene into HepG2 cells. Using vectors with deletions in the PEPCK promoter, we identified a single T3 response element (TRE) between positions -332 and -308. This element binds [125I]T3-labeled T3 receptor contained in nuclear extracts prepared from rat liver. Furthermore, the P3(I) element (-250 to -234), a previously described cis-sequence involved in mediating the induction of PEPCK gene transcription by cAMP, is also required for the T3 responsiveness of the promoter. In the absence of either the TRE or the P3(I) binding sites, no stimulation of transcription from the PEPCK promoter by T3 was observed, indicating that both elements are required for the T3 transcriptional regulation. Finally, a synergistic induction of PEPCK gene transcription by T3 and cAMP is described. This interaction requires both T3- and cAMP-responsive cis-acting elements.

Base Sequence↗
PubMed1990

Interaction of cytokine- and glucocorticoid-response elements of acute-phase plasma protein genes. Importance of glucocorticoid receptor level and cell type for regulation of the elements from rat alpha 1-acid glycoprotein and beta-fibrinogen genes.

Dexamethasone increased the transscriptional activity of several acute-phase plasma protein genes in cytokine-treated HepG2 cells, suggesting the presence of functional glucocorticoid receptors (GR). The level of GR was, however, insufficient for stimulation of transiently transfected gene constructs containing glucocorticoid-response elements (GRE). By complementation of HepG2 cells with a GR expression vector, a cell system was generated that allowed analysis of the interaction between GRE and cytokine-response elements of the rat alpha 1-acid glycoprotein gene and identification of the principal regulatory elements of the rat beta-fibrinogen gene. Although the expression of plasmid-derived GR mRNA was reduced by dexamethasone treatment, the concentration of GR was sufficient for full, short-term stimulation of GRE-containing vectors. By comparing the pattern of regulation of the cloned GRES in HepG2 and mouse L-cells, an equivalent, cell-type independent dexamethasone response was monitored for the alpha 1-acid glycoprotein element but a response limited to HepG2 cells was found for the beta-fibrinogen element. The data indicate that, although substantial differences exist in the organization and composition of the regulatory elements of the two genes, the overall function is, nevertheless, remarkably similar.

Acute-Phase Proteins↗
PubMed1991

[Elemental, semi-elemental, polymeric diets. Choice, indications, rational use].

In this review paper the main properties of elemental, semi-elemental and polymeric diets utilized in enteral nutrition are compared. The theoretical value of elemental diets (they do not require digestion) is upset by their effects on secretion. Semi-elemental diets are not hyperosmolar and are easily absorbed. Polymeric formulas acan be given in almost all indications of enteral nutrition, and in view of the high cost of elemental and semi-elemental diets, the latter should be reserved to elective indications, notably antigenic arrest of the digestive tract and major nitrogen malabsorption.

Digestive System Diseases↗
PubMed1993

Sequence-selective interactions of transcription factor elements with tandem glucocorticoid-responsive elements at physiological steroid concentrations.

Synergism in transcription is said to occur when the combined response from two DNA elements for the binding of trans-acting factors is greater than the sum of the responses from each element in isolation. The synergism of steroid receptors with themselves or with other trans-acting factors at saturating concentrations of steroid has proved to be an important component of steroid-regulated gene transcription. We have recently described a glucocorticoid modulatory element (GME) of the rat tyrosine aminotransferase gene that, in conjunction with a trans-acting factor, modulates the transcriptional activity of receptor-glucocorticoid and -antiglucocorticoid complexes with homologous and heterologous genes and promoters (Oshima, H., and Simons, S. S., Jr. (1992) Mol. Endocrinol. 6, 416-428). We now report that, under certain circumstances, the GME displays synergistic activity with a glucocorticoid-responsive element (GRE). However, several properties of GME action are different from those previously observed for synergism. The effects of the GME were marked at subsaturating or physiological concentrations of glucocorticoids but insignificant at saturating concentrations, which are the established conditions for synergism. The GME was found to increase the agonist activity of partial antiglucocorticoids, while synergism involving antisteroids has yet to be reported. Furthermore, the GRE was active in conjunction with two tandem repeats of a GRE, which was a combination that did not support conventional synergism. Most importantly, the effects of the GME were greater than with any other trans-acting factor binding element tested, indicative of a sequence-selective activity. The efficacy of the GME was also insensitive to the spacing between elements. Thus, the GME provides a mechanism for selective transcriptional modulation by physiological concentrations of steroid, and by antisteroids, of a common class of genes that are under the control of two or more GREs.

Animals↗
PubMed1994

cis-Acting elements controlling transcription from rat serine protease inhibitor 2.1 gene promoter. Characterization of two growth hormone response sites and a dominant purine-rich element.

The cis-acting elements that are functionally important for the basal, the growth hormone (GH), and the glucocorticoid hormone (GC) regulation of expression of the rat serine protease inhibitor 2.1 gene (spi 2.1) were mapped. Normal rat hepatocytes were transiently transfected with constructs harboring deleted or mutated versions of the spi 2.1 proximal promoter region fused to the chloramphenicol acetyltransferase gene. A purine-rich sequence (GAGA box, nucleotides -57 to -45), whose mutation or deletion almost completely knocks out both basal and hormone-stimulated promoter activities, plays the role of a key control element. A positive GC response element, spanning nucleotides -88 to -74, confers GC responsiveness to a heterologous promoter. Two structurally unrelated GH-response elements (GHRE) were identified. GHRE-II (nucleotides -136 to -104) contains a CCAAT enhancer binding protein binding site whose mutation completely abolishes its GH-dependent enhancer function. GHRE-I, which spans nucleotides -61 to +8, is not an enhancer element. Its GH-dependent activity depends on the preservation of the distance separating the GAGA box and elements of the basic transcriptional machinery. Taken together, these results have revealed the existence of an apparently new type of promoter functioning that strictly depends on the integrity of a key regulatory (G + A) motif.

Animals↗
PubMed1994

3,5,3'-L-triiodothyronine (thyroid hormone)-induced protein-DNA interactions in the thyroid hormone response elements and cell type-specific elements of the rat growth hormone gene revealed by in vivo dimethyl sulfate footprinting.

The cell type-specific element and 3,5,3'-L-triiodothyronine (thyroid hormone) (T3) response element of the rat growth hormone gene act synergistically to produce cell type-specific, T3-regulated expression. Pit-1 is a pituitary cell type-specific transcription factor that binds the cell type-specific element and is essential to its activity. T3 receptors bind as homodimers and heterodimers to the T3 response element and are essential for its activity. Here, we report the use of ligation-mediated polymerase chain reaction in vivo dimethyl sulfate footprinting to study the effects of T3 on protein-DNA interactions in the rat growth hormone gene promoter in vivo. T3-responsive guanine methylation was detected only in and near the T3 response element and the Pit-1 binding sites. The results indicate that within 2 h, T3 induces occupancy of the T3 response element and Pit-1 sites by their respective trans-acting factors in vivo.

Animals↗
PubMed1994

The avian cardiac alpha-actin promoter is regulated through a pair of complex elements composed of E boxes and serum response elements that bind both positive- and negative-acting factors.

The chicken alpha-cardiac actin is one of the earliest contractile protein genes selectively expressed during embryonic skeletal and cardiac muscle differentiation. Cardiac actin promoter elements were examined in these two sarcomeric cell types. A portion of the alpha-cardiac actin promoter responsible for striated muscle specificity has been delineated (1, 2) and shown to contain four serum response elements (SRE). Previously, SRE3 was shown to be part of a complex element in conjunction with a functional E box (2), and we now show that SRE4 is also part of an upstream SRE.E box cis-element complex. The SREs function similarly, but the E boxes have dissimilar properties within and between striated muscle types. The SRE3.E1 box binds myogenic basic helix-loop-helix factors and is required for cardiac actin trans-activation in primary muscle cell cultures but functions as a negative regulatory element in cardiac muscle cells. The SRE4.E2 box, on the other hand, fails to bind basic helix-loop-helix (bHLH) factors, is negative acting in skeletal muscle cells, and is positive acting in cardiac myocytes. A DNA binding factor similar to HF1a (3) was identified that interacts specifically with the SRE4.E2 box. This study shows that the avian cardiac actin promoter elements are differentially used between skeletal and cardiac striated muscle cell lineages.

Actins↗
PubMed1993

Multiple parameters control the selectivity of nuclear receptors for their response elements. Selectivity and promiscuity in response element recognition by retinoic acid receptors and retinoid X receptors.

A number of nuclear receptors are capable of binding to specific DNA sequences known as response elements that correspond to two 5'-PuG(G/T)TCA motifs. We have systematically compared the selectivity of DNA sequence recognition by the estrogen receptor and the retinoic acid receptor (RAR) and retinoid X receptor (RXR), using a variety of synthetic oligonucleotides related to natural response elements. The importance of the spacing and relative orientation of the PuG(G/T)TCA motifs as well as the possible role of the flanking sequences were investigated for each receptor. We find that the three receptors have different response element preferences and that their intrinsic DNA binding properties cannot be simply ascribed to spacing or orientation rules. For example, RARs bind with similar efficiencies to response elements containing directly repeated PuGTTCA motifs separated by 2, 3, 4, or 5 base pairs, and RXRs bind to response elements in which the directly repeated motifs are separated by 1, 2, and 5 base pairs. The actual sequence of the repeated motifs and the nature of their flanking bases appear to be important parameters in determining RAR and RXR binding efficiencies. The possibility that the binding specificity of nuclear receptors can be modified by interactions with other proteins, in particular other nuclear receptors, is discussed in relation to the patterns of transcriptional activation observed with either endogenous or transiently expressed receptors on chimeric promoters containing various response elements.

Animals↗