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Results for “Enhancer mapping”

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PubMed1986

Sequences upstream from the mouse c-mos oncogene may function as a transcription termination signal.

A region upstream from the mouse c-mos proto-oncogene, termed upstream mouse sequence (UMS), prevents expression of mos transforming activity. Previous studies suggested that the UMS prevented transcription readthrough. In this study, we constructed a recombinant DNA clone, pHTS3MS, with the UMS inserted downstream from both the mos gene and a truncated long terminal repeat containing only the U3 enhancer region. In this position UMS did not inhibit mos transforming activity. We examined cells transformed by pHTS3MS for RNA expression. S1 nuclease analysis showed that the UMS provides two polyadenylation signals to mos-containing RNA and nuclear run-on transcription showed that the primary transcripts terminate in UMS. In addition, using portions of the UMS, we found that a 360-bp fragment containing the UMS polyadenylation signals and sites inserted between the herpes simplex virus type 1 (HSV-1) thymidine kinase gene (tk) and its promoter inhibits tk transforming activity by 99% and prevents detectable expression of this construct in transient expression assays. Thus, the UMS must contain signals for polyadenylation and appears to function as a transcription terminator.

Animals↗

PubMed1998

Mouse mammary tumor virus sequences responsible for activating cellular oncogenes.

Integration of mouse mammary tumor virus (MMTV) near the int genes results in the inappropriate expression of these proto-oncogenes and initiates events that lead to the formation of mammary adenocarcinomas. In most cases, the MMTV provirus integrates in a transcriptional orientation opposite that of the int genes. We have used a novel, vector-based system designed to recapitulate the integration of MMTV upstream of the int-2 promoter. Compared to a cellular promoter or another retroviral promoter, the MMTV long terminal repeat (LTR) in this configuration is particularly efficacious at activating the int-2 promoter. The sequences responsible for enhancing the activity of the int-2 promoter map to two domains in the 5' end of the MMTV LTR. One domain is a previously defined element; the second is an element delineated by these studies that acts synergistically with the first. Both of these elements display mammary cell-specific activity. Thus, even though the MMTV promoter itself is weak without hormonal stimulation, viral integration can position the 5' LTR elements to efficiently activate transcription from cellular proto-oncogenes. Other functional elements in the LTR have little effect on the activation of the int-2 promoter. Even stimulation of the MMTV promoter with steroid hormones only modestly activates transcription from the int-2 promoter, suggesting that the 5' elements of the LTR are the predominant determinants of the tissue- and orientation-specific activation of cellular promoters by MMTV.

Animals↗

PubMed1993

Activation of p42 mitogen-activated protein kinase by glutamate receptor stimulation in rat primary cortical cultures.

Recent studies have identified at least two homologous mitogen-activated protein (MAP) kinases that are activated by phosphorylation of both tyrosine and threonine residues by an activator kinase. To help define the role of these MAP kinases in neuronal signalling, we have used primary cultures derived from fetal rat cortex to assess the regulation of their activity by agonist stimulation of glutamate receptors and by synaptic activity. Regulation was assayed by monitoring changes in both tyrosine phosphorylation on western blots and in vitro kinase activity toward a selective MAP kinase substrate peptide. In initial studies, we found that phorbol ester treatment increased tyrosine phosphorylation of p42 MAP kinase and stimulated MAP kinase activity. A similar response was elicited by three agonists of metabotropic glutamate receptors, i.e., trans-(+/-)-1-amino-1,3-cyclopentane dicarboxylic acid, quisqualate, and (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine. MAP kinase activity and p42 MAP kinase tyrosine phosphorylation were also stimulated by the ionotropic glutamate receptor agonist, kainate, but not by N-methyl-D-aspartate. To examine regulation of MAP kinase by synaptic activity, cultures were treated with picrotoxin, an inhibitor of GABAA receptor-mediated inhibition that enhances spontaneous excitatory synaptic activity. Treatment of cultures with picrotoxin elicited activation of MAP kinase. This response was blocked by tetrodotoxin, which suppresses synaptic activity. These results demonstrate that p42 MAP kinase is activated by glutamate receptor agonist stimulation and by endogenous synaptic activity.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1996

Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome.

Pseudomonas fluorescens is a saprophytic bacterium commonly isolated from soil, water, and the surfaces and tissues of plants and animals. The species has important applications in biotechnology because it can enhance plant growth and protect crops against disease. A complete physical map of the 6.63 Mbp P. fluorescens SBW25 chromosome was constructed using data obtained from combinations of one- and two-dimensional electrophoresis of completely or partially digested chromosomal DNA with end labelling. In total, 139 restriction sites (15 PacI, 53 SpeI, 71 XbaI) were placed on the physical map and complete maps of the circular chromosome were obtained for both PacI and SpeI; only XbaI fragments linking SpeI fragments were positioned. The average resolution of restriction sites was 48 kbp. A genetic map was derived from the physical map by southern hybridization and 31 genes were positioned including oriC, rDNA operons (rnnA-E), recA, gacA, and pyvD.

Blotting, Southern↗

PubMed1993

Duck lactate dehydrogenase B/epsilon-crystallin gene. Lens recruitment of a GC-promoter.

In duck the single copy lactate dehydrogenase B (LDH-B) gene also encodes an abundant lens protein, epsilon-crystallin. The LDH-B/epsilon-crystallin gene consists of eight exons, of which the first is non-coding. The promoter region lacks a TATA box, is very GC-rich and contains multiple consensus Sp1 binding sites. The gene has two discrete transcription start sites located 28 base-pairs apart. Both sites are used about equally in heart tissue, while transcription from the downstream start site predominates in the lens. For maximal promoter activity in lens or heart, sequences from the first intron are required. The enhancer(s) in this intron is promoter specific as it could not activate the tk promoter. Studies at the RNA level show that the overexpression of the LDH-B/epsilon-crystallin gene in the lens is regulated at the transcriptional level, yet no tissue-specific regulatory elements could be detected in a region spanning from -1.9 kb (1 kb = 10(3) bases or base-pairs) up to the translation initiation site in the second exon. The basis for the differential expression of the LDH-B/epsilon-crystallin gene in duck heart and lens is the usage of the downstream transcription initiation site. However, our results do not allow a distinction between activation of the downstream transcription start site in the lens or repression of the use of this site in heart.

Animals↗

PubMed2003

MEK1,2 response element mediates angiotensin II-stimulated plasminogen activator inhibitor-1 promoter activation.

The MEK1,2 (MAPK/ERK kinase 1 and 2) pathway mediates the up-regulation of plasminogen activator inhibitor-1 (PAI-1) expression in vascular smooth muscle cells by a variety of hormones, including angiotensin II. Transfection of constitutively active MEKK-1, an upstream activator of the mitogen-activated protein (MAP) kinase pathways, was used to isolate an enhancer element located between -89 and -50 bp in PAI-1 promoter that was activated by MEKK-1 and selectively blocked by the MEK1,2 inhibitor PD98059. Mutational analysis revealed that the MEKK-1 response element (MRE) contained 2 cis-acting Sp1- and AP-1-like sequences, located between -75 to -70 and -63 to -52 bp, respectively. Overexpression of Sp1 enhanced MEKK-1-induced MRE promoter activity and a dominant-negative c-Fos blocked this Sp1 response. The combination of Sp1 and c-Jun or c-Fos was required to activate this MRE. Angiotensin II (Ang II) stimulation increased c-Fos, c-Jun, and Sp1 binding to the MRE by 100-, 4.9-, and 1.9-fold, respectively, and these responses were inhibited by PD98059 and AT1 receptor antagonist candesartan. Intravenous Ang II infusion in rats increased aortic c-Fos binding to the MRE. This MRE sequence mediated a 4-fold increase of MEK1,2-dependent PAI-1/luciferase mRNA expression by angiotensin II stimulation. This report identifies the MEK1,2 response element that mediates angiotensin II-stimulated PAI-1 promoter activation and shows that activation of this element requires Sp1 and AP-1 co-activation.

Adenoviridae↗

PubMedDate not supplied

Evaluation of therapeutic effectiveness of transarterial chemoembolization for hepatocellular carcinoma: correlation of dynamic susceptibility contrast-enhanced echoplanar imaging and hepatic angiography.

The objective of this study was to evaluate the therapeutic effectiveness of transarterial chemoembolization (TACE) for hepatocellular carcinoma (HCC) with dynamic susceptibility contrast-enhanced magnetic resonance imaging (DSC-MRI). Seventeen patients with histopathologically proven HCC were included in this study. All patients underwent MR examinations with conventional T1- and T2-weighted images, gadolinium-enhanced images, and DSC-MRI before TACE treatment. Hepatic blood volume (HBV) maps were reconstructed from the time-intensity curves. The same MRI sequences and techniques were repeated 24 h and 6 weeks after TACE. Serial changes in tumor perfusion on HBV maps were correlated with vascularity in hepatic angiography. All tumors were hypointense on T1-weighted images and hyperintense on T2-weighted images. Heterogeneous enhancement was observed in all tumors before and immediately after TACE. Hyperperfusion was noted in most of the tumors on HBV map before TACE and moderate to marked hypoperfusion following TACE. The degree of tumor perfusion on HBV map correlated well with the vascularity in angiography. In conclusion, the noninvasive nature of DSC-MRI is useful to evaluate the effectiveness of TACE. Invasive procedures, such as angiography, are seldom necessary.

Aged↗

PubMed2001

Molecular cloning, genetic mapping, and expression analysis of four zebrafish c/ebp genes.

The CCAAT/enhancer binding protein family (C/EBP) are transcription factors that play integral roles in the development and function of many organ systems, including hematopoietic cells, adipose tissues, and liver. We have identified and characterized putative zebrafish orthologs of mammalian C/EBP alpha, beta, gamma, and delta using low-stringency hybridization screening and computer searches of the GenBank EST database. c/ebpa and g were mapped within 1 cM of each other on linkage group (LG) 7, syntenic with human CEBPA and G genes on chromosome 19. c/ebpb was mapped to LG8, and c/ebpd was mapped to LG24, on the same LG as a recently identified unique c/ebp in zebrafish, c/ebp1. The mapping of these genes established new syntenic relationships between LG8 and human chromosome 20, extended existing synteny between LG7 and human chromosome 19, and confirmed the synteny between LG24 and human chromosome 8. In addition, these syntenies between zebrafish and human chromosomes are also conserved in the mouse genome. To characterize the expression of these genes, RNA in situ hybridization in embryos of wild type and a hematopoietic mutant, cloche, was performed. The results showed that zebrafish c/ebpa, b, g, and d were expressed in many embryonic tissues. c/ebpa and b were expressed in a subset of hematopoietic cells in a region consistent with myeloid expression. In addition, there was expression of c/ebpa and b in the liver and c/ebpa, b, and d in regions of the gastrointestinal tract. The expression of the c/ebps may serve as important markers for analysis of myelopoiesis, hepatic development, and other developmental processes in the future.

Amino Acid Sequence↗

PubMed2004

Applications of manganese-enhanced magnetic resonance imaging (MEMRI) to image brain plasticity in song birds.

The song control system of song birds is an excellent model for studying brain plasticity and has thus far been extensively analyzed by histological and electrophysiological methods. However, these approaches do not provide a global view of the brain and/or do not allow repeated measures, which are necessary to establish correlations between alterations in neural substrate and behavior. Application of in vivo manganese-enhanced MRI enabled us for the first time to visualize the song control system repeatedly in the same bird, making it possible to quantify dynamically the volume changes in this circuit as a function of seasonal and hormonal influences. In this review, we introduce and explore the song control system of song birds as a natural model for brain plasticity to validate a new cutting edge technique, which we called 'repeated dynamic manganese enhanced MRI' or D-MEMRI. This technique is based on the use of implanted permanent cannulae--for accurate repeated manganese injections in a defined target area--and the subsequent MRI acquisition of the dynamics of the accumulation of manganese in projection brain targets. A compilation of the D-MEMRI data obtained thus far in this system demonstrates the usefulness of this new method for studying brain plasticity. In particular it is shown to be a perfect tool for long-term studies of morphological and functional responses of specific brain circuits to changes in endocrine conditions. The method was also successfully applied to obtain quantitative measures of changes in activity as a function of auditory stimuli in different neuronal populations of a same nucleus that project to different targets. D-MEMRI, combined with other MRI techniques, clearly harbors potential for unraveling seasonal, hormonal, pharmacological or even genetically driven changes in a neuronal circuit, by simultaneously measuring changes in morphology, activity and connectivity.

Adaptation, Physiological↗

PubMed1989

Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein.

This paper presents the results of experiments that determine the chromosomal location of the mouse gene encoding CCAAT/enhancer binding protein (C/EBP) and measure its expression as a function of tissue type and temporal period of development in mice and rats. Three alleles of the C/EBP gene were identified according to restriction fragment length polymorphisms. The strain distribution pattern of the three alleles was determined in recombinant inbred mouse strains and compared to that of other mouse genes. These results mapped the gene to a position within 2.5 centimorgans (cM) of the structural gene encoding glucose phosphate isomerase on chromosome 7 of the mouse. The expression pattern of the C/EBP gene was studied by a combination of nucleic acid hybridization and antibody staining assays. High levels of C/EBP mRNA were observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates. These included liver, fat, intestine, lung, adrenal gland, and placenta. More detailed analysis of two of these tissues, liver and fat, showed that C/EBP expression was limited to fully differentiated cells. Moreover, analysis of the temporal pattern of expression of C/EBP mRNA in two tissues, liver and intestine, revealed a coordinated induction just prior to birth. These observations raise the possibility that the synthesis of C/EBP may be responsive to humoral factors and that modulation in C/EBP expression might mediate coordinated changes in gene expression that facilitate adaptive challenges met during development or during the fluctuating physiological states of adult life.

Adipose Tissue↗

PubMed1986

Cis-acting sequences responsible for the transcriptional activation of human T-cell leukemia virus type I constitute a conditional enhancer.

Transcription from the long terminal repeat promoter of human T-cell leukemia virus type I is activated in the presence of a trans-activator protein, TA-I, encoded in the 3' part of the genome. A series of long terminal repeat mutants and hybrid promoter constructs have been studied in a transient expression assay for their ability to be activated in the presence of the trans-activator protein. The sequences responsible for trans-activation have properties similar to those of transcription enhancer elements. They act relatively independent of position and orientation and activate both the homologous as well as heterologous promoters only in the presence of the trans-activator protein. Therefore, the trans-activator protein of human T-cell leukemia virus type I acts via an inducible enhancement mechanism.

Base Sequence↗

PubMed1999

Genomic structure and chromosomal mapping of the human sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) gene.

Sterol regulatory element binding protein (SREBP) cleavage-activating protein (SCAP) is a central regulator of lipid synthesis and uptake in mammalian cells. The entire genomic structure of the human SCAP gene was cloned in a 110-kb region covered by overlapping genomic clones. The SCAP gene was localized to chromosome 3p21.3 by fluorescence in situ hybridization. The human SCAP gene is over 30 kb in length and contains 23 exons and 22 introns. The transcription initiation site within exon 1 is separate from the initiation codon coded in exon 2. Analysis of exon/intron structure revealed that the gene consists of a mosaic of exons encoding functional protein domains. Exon 1 encodes the 5' non-coding region. Exons 2, 3, 7, 8, 9, 10, 11, 13, and 15, respectively, encode each of the eight transmembrane regions. Of these, exons 7-11 encode the sterol-sensing domain. Exons 15-23 encode the hydrophilic carboxyl-terminal domains containing four copies of a motif called the Trp-Asp (WD) repeats that interact with and regulate SREBP and the site-1 protease. Sequence analysis of the 5'-flanking region showed that it comprised a high G/C-rich region and contained adipocyte determination and differentiation-dependent factor 1 (ADD1)/SREBP-1 binding sites in addition to Sp1 and AP2 sites. This suggests that SCAP gene expression is under the control of SREBP-1, a key regulator of the expression of genes essential for intracellular lipid metabolism. Our data establish the basis of investigation for molecular variants in this gene that may result in alterations in plasma lipoprotein levels and/or derangement of intracellular lipid metabolism.

Base Sequence↗

PubMed1984

Xenon/CT blood flow mapping of the kidney and liver.

A noninvasive technique for measuring blood flow by xenon-enhanced X-ray transmission CT has been developed and reported quite extensively in recent years. In this method nonradioactive xenon gas is inhaled, and the temporal changes in radiographic enhancement produced by the inhalation are measured by sequential CT. Time-dependent xenon concentration within various tissue segments is used to derive local blood flow maps. The method has been amply discussed in relation to assessment of local cerebral blood flow. Its application to other body organs is explored in this paper, in which results from six preliminary blood flow studies in the liver and kidneys of nonhuman primates are reported. Blood flow in renal cortex ranged from 150 to 280 ml/100 cc/min and hepatic tissue perfusion from 80 to 120 ml/100 cc/min. The advantages and limitations of the method in such applications are discussed.

Animals↗

PubMed2005

Genetic analysis of the human papillomavirus type 31 differentiation-dependent late promoter.

Human papillomaviruses infect stratifying squamous epithelia, causing benign and malignant lesions. Upon differentiation of the host keratinocyte, the virus undergoes a dramatic increase in both DNA replication and transcription from the late promoter, leading to expression of late genes and virion morphogenesis. In human papillomavirus type 31 (HPV31), the late promoter is designated p742 and includes multiple start sites embedded within the E7 gene. In this report, we mapped viral DNA elements that control transcriptional activity from p742. Enhancer elements in the viral upstream regulatory region positively regulate this promoter. The region containing the transcriptional start sites is dispensable for activity, and at least two separate elements in the E6/E7 region are capable of supporting transcription. Of these, we mapped one to a 150-bp region of the E7 open reading frame and designate it the core p742 promoter. Using GF109203X, an inhibitor of protein kinase C signaling, we show that p742 activation is independent of viral genome amplification. Finally, we mapped elements in the region of p742 that confer responsiveness to differentiation and show that the upstream regulatory region does not contribute to the differentiation response of p742. These studies are an important step toward understanding the functioning and regulation of this multiple-start promoter.

Cells, Cultured↗

PubMed1987

Insertion of dominant selectable markers into the human genome.

In the above discussion, I have outlined the current status of our efforts to use retroviral vectors to introduce selectable markers into the human genome and to use these markers for mapping specific chromosomal regions. I have not reviewed in detail the extensive characterization of the mouse H-2 region carried out by David Nelson and John Weis based on the insertion of a single retroviral element. This analysis has provided a model for the application of retroviral elements into various regions of the human genome. The prospects for increasing the resolution of the human genetic map and identifying many genes relevant to human health and development are likely to be enhanced by increasing the precision of this methodology.

Chromosome Mapping↗

PubMed1997

Activator effect of coinjected enhancers on the muscle-specific expression of promoters in zebrafish embryos.

The transient expression of reporter gene constructs in embryos provides a powerful tool to characterise cis-acting transcriptional elements of the genes involved in development. In the present study, we have analysed the expression pattern of several muscle-specific and ubiquitous regulatory sequences in microinjected zebrafish embryos. By using a fast and reproducible coinjection strategy, the mosaic expression of lacZ reporter gene was monitored in wholemount embryos injected with sequences containing putative enhancer elements and a carp myosin heavy chain promoter/lacZ reporter construct. We have found that a 0.9-kb myosin heavy chain (MyHC) proximal promoter containing several putative myogenic regulatory factors (MRF) binding sites is sufficient to restrict lacZ expression to the skeletal muscle fibres of prim-6 stage zebrafish embryos. Expression of a rat-derived foetal myosin light chain enhancer (MyLC) and different fragments of a carp beta-actin regulatory region together with the MyHC promoter were compared by accumulating the type, number and spatial distribution of beta-galactosidase-expressing cells on an expression map. beta-galactosidase activity increased similarly whether the MyLC enhancer was ligated to the promoter/ reporter construct directly or when coinjected as a separate fragment whilst skeletal muscle specificity was retained. The coinjection of two different forms of the beta-actin regulatory elements also showed a marked effect on the MyHC promoter activity. The coinjection of putative enhancers with minimal promoter constructs and subsequent analysis of the transient expression pattern in the developing embryos provides a rapid and simple technique to identify cis acting activator elements of genes expressed in the vertebrate embryo.

Actins↗

PubMed2005

Heavy metal interference with growth hormone signalling in trout hepatoma cells RTH-149.

We have studied the effects of heavy metals (Hg2+, Cu2+, Cd2+) on growth hormone (GH) activation of tyrosine kinase and Ca2+ signaling in the trout (Oncorhynchus mykiss) hepatoma cell line RTH-149. Molecular cloning techniques using primer designed on Oncorhynchus spp. growth hormone receptor (GHR) genes allowed to isolate a highly homologous cDNA fragment from RTH-149 mRNA. Thereafter, cells were analysed by Western blotting or, alternatively, with Ca2+ imaging using fura-2/AM. Exposure of cells to ovine GH alone produced a stimulation of the JAK2/STAT5 pathway and intracellular free Ca2+ variations similar to what has been observed in mammalian models. Cell pre-exposure to Cu2+, Hg2+ or Cd2+ affected cell response to GH by enhancing (Cu2+) or inhibiting (Cd2+) the phosphorylation of JAK2 and STAT5. Heavy metals induced the activation of the MAP kinase p38, and pre-exposure to Hg2+ or Cu2+ followed by GH enhanced the effect of metal alone. Image analysis of fura2-loaded cells indicated that pre-treatment with Hg2+ prior to GH produced a considerable increase of the [Ca2+]i variation produced by either element, while using Cu2+ or Cd2+ the result was similar but much weaker. Data suggest that heavy metals interfere with GH as follows: Hg2+ is nearly ineffective on JAK/STAT and strongly synergistic on Ca2+ signaling; Cu2+ is activatory on JAK/STAT and slightly activatory on Ca2+; Cd2+ is strongly inhibitory on JAK/STAT and slightly activatory on Ca2+; heavy metals could partially activate STAT via p38 independently from GH interaction.

Amino Acid Sequence↗

PubMed1990

Structure and function of the enhancer 3' to the human A gamma globin gene.

An enhancer is located immediately 3' to the A gamma globin gene. We have used DNase I footprinting to map the sites of interaction of nuclear proteins with the DNA sequences of this enhancer. Eight footprints were discovered, distributed over 600 base pairs of DNA. Three of these contain a consensus binding site for the erythroid specific factor GATA-I. Each of these GATA-1 sites had an enhancer activity when inserted into a reporter plasmid and tested in human erythroleukemia cells. Other footprints within the enhancer contained consensus binding sequences for the ubiquitous, positive regulatory proteins AP2 and CBP-1. An Sp1-like recognition sequence was also identified. Synthetic oligonucleotides encompassing two of the footprints generated a slowly migrating complex in gel mobility shift assays. The same complex forms on a fragment of the human gamma globin gene promoter extending from -260 to -200. The DNaseI footprint of this protein complex with the enhancer overlapped a sequence, AGGAGGA, found within the binding site for a protein that interacts with the chicken beta globin promoter and enhancer, termed the stage selector element. We propose that this complex of proteins may be involved in the human gamma globin promoter-enhancer interaction.

Animals↗