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Results for “Enhancer mapping”

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PubMed2004

An enhancer directs differential expression of the linked Mrf4 and Myf5 myogenic regulatory genes in the mouse.

The myogenic regulatory factors, Mrf4 and Myf5, play a key role in skeletal muscle formation. An enhancer trap approach, devised to isolate positive-acting elements from a 200-kb YAC covering the mouse Mrf4-Myf5 locus in a C2 myoblast assay, yielded an enhancer, A17, which mapped at -8 kb 5' of Mrf4 and -17 kb 5' of Myf5. An E-box bound by complexes containing the USF transcription factor is critical for enhancer activity. In transgenic mice, A17 gave two distinct and mutually exclusive expression profiles before birth, which correspond to two phases of Mrf4 transcription. Linked to the Tk or Mrf4 minimal promoters, the nlacZ reporter was expressed either in embryonic myotomes, or later in fetal muscle, with the majority of Mrf4 lines showing embryonic expression. When linked to the Myf5 minimal promoter, only fetal muscle expression was detected. These observations identify A17 as a sequence that targets sites of myogenesis in vivo and raise questions about the mutually exclusive modes of expression and possible promoter/enhancer interactions at the Mrf4-Myf5 locus.

Animals↗

PubMed2003

Current status of the ovine genome map.

Genome maps in livestock species have been under development for the last decade. While the sheep map is one of the least advanced for livestock, the amount of available information is noteworthy, in light of the paucity of funding and personnel devoted to this project. These limited resources have been strategically aligned to take advantage of information from the human, mouse and bovine mapping and sequencing efforts. The resulting ovine linkage and physical maps have greatly enhanced the search for genes controlling important traits in sheep. In order to improve the efficiency of these investigations, it is imperative that efforts on the sheep comparative map be continued.

Animals↗

PubMed2000

Substitution of two insulin receptor carboxy-terminal tyrosines with phenylalanine impairs the expression of MAP kinase phosphatase-1 (MKP-1) mRNA.

Cells expressing mutant insulin receptors (Y/F2), in which tyrosines 1316 and 1322 have been replaced with phenylalanine, exhibit enhanced insulin-induced MAP kinase activity and DNA synthesis in comparison with cells expressing wild type insulin receptors (Hirc B). To elucidate the mechanism of enhanced responsiveness, the expression of MAP kinase phosphatase-1 (MKP-1), a negative regulator of MAP kinase activity, was measured in Hirc B and Y/F2 cells incubated in the absence and presence of insulin for various periods of time, and over increasing concentrations of the ligand. Treatment of both cell lines with insulin induced a time and concentration-dependent relative increase in MKP-1 mRNA expression. However, in Y/F2 cells both basal and insulin-stimulated MKP-1 mRNA levels were more than 60% lower than that observed in cells transfected with the wildtype receptors. Cyclic AMP analog (8-Br-cAMP)/inducer (Forskoline) increased MKP-1 mRNA levels in both cell lines, and to a lesser extent in Y/F2 cells. In contrast to insulin the relative increase in MKP-1 mRNA expression induced by 8-Br-cAMP or forskoline was similar in Y/F2 and Hirc B cells. The overexpression of MKP-1 in Y/F2 cells inhibited insulin stimulated DNA synthesis. Transfection of wild type insulin receptors into Y/F2 cells increased basal levels of MKP-1. These results suggest that insulin receptor tyrosine residues 13/16 and 1322 play an important role in the regulation of MKP-1 expression both under basal and insulin stimulated conditions, and are not necessary for the induction of MKP-1 mRNA by cAMP. Furthermore, the enhanced insulin induced mitogenic signaling seen in Y/F2 cells is, at least in part, due to impaired MKP-1 expression.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2003

Intragenic HIV-1 env sequences that enhance gag expression.

Expression of HIV-1 genes is regulated at multiple levels including the complex RNA splicing and transport mechanisms. Multiple cis-acting elements involved in these regulations have been previously identified in various regions of HIV-1 genome. Here we show that another cis-acting element was present in HIV-1 env region. This element enhanced the expression of Gag when inserted together with Rev response element (RRE) into a truncated HIV-1 genome in the presence of Rev. The enhancing activity was mapped to a 263-bp fragment in the gp41 region downstream to RRE. RNA analysis showed that it might function by promoting RNA stability and Rev-dependent RNA export. The enhancement was specific to Rev-dependent expression, since it did not enhance Gag expression driven by Sam68, a cellular protein that has been shown to be able to substitute for Rev in RNA export function.

Base Sequence↗

PubMed1996

Liver-specific enhancer of the glucokinase gene.

Glucokinase gene regions that are important for liver specific expression of the enzyme have been functionally identified using transient transfection of rat hepatocytes. Maximal luciferase activity was elicited by a reporter plasmid with 3.4 kilobase pairs of genomic DNA flanking the liver glucokinase promoter. Deletion of a gene fragment between -1000 and -600 with respect to the start of transcription resulted in a 60% decrease in luciferase activity. Further reduction, close to background level, occurred upon deletion of a 90-base pair sequence between -123 and -34. Reporter plasmids with the liver glucokinase promoter and any length of flanking sequence were minimally active in INS-1 insulinoma cells, and conversely reporters with the beta-cell-specific promoter were ineffective in primary hepatocytes. In FTO-2B hepatoma cells, a differentiated line expressing many liver-specific traits but not the endogenous glucokinase gene, the promoter proximal region between -123 and -34 markedly stimulated the expression of transfected plasmids above background. However, addition of the flanking region up to -1000 inhibited luciferase expression. The gene fragment from -1003 to -707 was shown to be a bona fide, hepatocyte-specific enhancer by the following criteria: 1) it stimulated reporter expression by more than 10- and 5-fold when inserted directly upstream of the glucokinase TATA box or complete promoter, respectively, regardless of orientation; 2) it stimulated gene expression from the heterologous SV 40 promoter 4-fold; 3) it was also effective from a downstream position; and 4) in contrast to the enhancer effect in primary hepatocytes, the sequence acted as a silencer in FTO-2B cells and was neutral in INS-1 cells. Both the promoter proximal and the enhancer regions were marked by DNase I hypersensitive sites in the chromatin of primary hepatocytes but not hepatoma or insulinoma cells. Seven footprinted elements termed A through G were mapped in the enhancer by the in vitro DNase I protection assay. Elements A-C may bind liver enriched factors, because they were not protected by spleen nuclear extract. In hepatocyte transfection, the downstream half of the enhancer containing elements A-C was about half as effective as the complete enhancer in stimulating glucokinase promoter activity. Site-directed mutagenesis of element A virtually abrogated the activity of the half-enhancer, whereas mutation of element C had a more moderate effect. The sequence between -732 and -578 upstream of the liver start of transcription in the human glucokinase gene displays 79% sequence identity with the downstream half of the rat enhancer. The human gene fragment ligated to the minimal rat liver glucokinase promoter was shown to work as an enhancer in the hepatocyte transfection system.

Animals↗

PubMed1990

Identification and functional characterization of the human T-cell receptor beta gene transcriptional enhancer: common nuclear proteins interact with the transcriptional regulatory elements of the T-cell receptor alpha and beta genes.

A transcriptional enhancer has been mapped to a region 5.5 kilobases 3' of the C beta 2 gene in the human T-cell receptor (TCR) beta-chain locus. Transient transfections allowed localization of enhancer activity to a 480-base-pair HincII-XbaI restriction enzyme fragment. The TCR beta enhancer was active on both the minimal simian virus 40 promoter and a TCR beta variable gene promoter in both TCR alpha/beta + and TCR gamma/delta + T cells. It displayed significantly less activity in Epstein-Barr virus-transformed B cells and K562 chronic myelogenous leukemia cells and no activity in HeLa fibroblasts. DNA sequence analysis revealed that the enhancer contains a consensus immunoglobulin kappa E2 motif, as well as an AP-1-binding site and a cyclic AMP response element. DNase I footprint analyses using Jurkat T-cell nuclear extracts allowed the identification of five nuclear protein-binding sites, T beta 1 to T beta 5, within the enhancer element. Deletion and in vitro mutagenesis studies demonstrated that the T beta 2- and T beta 3- and T beta 4-binding sites are each required for full transcriptional enhancer activity. In contrast, deletion of the T beta 1- and T beta 5-binding sites had essentially no effect on enhancer function. Electrophoretic mobility shift assays demonstrated that TCR alpha/beta + and TCR gamma/delta + T cells expressed T beta 2-, T beta 3-, and T beta 4-binding activities. In contrast, non-T-cell lines, in which the enhancer was inactive, each lacked expression of at least one of these binding activities. TCR alpha and beta gene expression may be regulated by a common set of T-cell nuclear proteins in that the T beta 2 element binding a set of cyclic AMP response element-binding proteins that are also bound by the T alpha 1 element of the human TCR alpha enhancer and the decamer element present in a large number of human and murine TCR beta promoters. Similarly, the T beta 5 TCR beta-enhancer element and the T alpha 2 TCR alpha-enhancer element bind at least one common T-cell nuclear protein. Taken together, these results suggest that TCR beta gene expression is regulated by the interaction of multiple T cell nuclear proteins with a transcriptional enhancer element located 3' of the C beta 2 gene and that some of these proteins may be involved in the coordinate regulation of TCR alpha and beta gene expression.

Base Sequence↗

PubMed1997

A transcriptional enhancer required for the differential expression of the human estrogen receptor in breast cancers.

Breast cancers lacking estrogen receptor (ER) expression have an adverse prognosis and fail to respond to endocrine therapy. We have identified a transcriptional enhancer in the human ER gene which is differentially active in ER-positive (ER+) and ER-negative (ER-) human breast cancer cell lines. Enhancer function was mapped to a 35-bp element located from -3778 to -3744 upstream of the major human ER mRNA start site, which we have termed ER-EH0 (for estrogen receptor enhancer). Gel retardation assays with ER+ and ER- cell lines identified multiple DNA-protein complexes which specifically form on this enhancer. One of these complexes could be supershifted by anti-Jun or anti-Fos antibodies, identifying it as an AP-1-containing complex. Methylation interference assays suggest binding of factors to both the AP-1 site and adjacent base pairs. Enhancer activity requires both the AP-1 site and these adjacent sequences. Mutations introduced into ER-EH0 and the recently described proximal promoter element ERF-1 in the context of the full-length promoter confirm ER-EH0 as the dominant cis-acting element involved in differential ER expression.

Base Sequence↗

PubMed2001

Epistasis, complex traits, and mapping genes.

Using a three-locus model wherein two loci regulate a third, candidate locus, I examine physiological epistasis from the 'gene's eye view' of the regulated locus. I show that, depending upon genetic background at the regulatory loci, an allele at the candidate locus can be dominant, additive, recessive, neutral, over-dominant, or under-dominant in its effects on fitness. This kind of variation in allelic effect caused by variation in genetic background from population to population, from time to time in the same population, or sample to sample makes finding and mapping the genes underlying a complex phenotype difficult. The rate of evolution of such genes can also be slowed, especially in genetically subdivided metapopulations with migration. Nevertheless, understanding how variation in genetic background causes variation in allelic effects permits the genetic architecture of such complex traits to be dissected into the interacting component genes. While some backgrounds diminish allelic effects and make finding and mapping genes difficult, other backgrounds enhance allelic effects and facilitate gene mapping.

Alleles↗

PubMed2006

Parametric mapping of the hepatic perfusion index with gadolinium-enhanced volumetric MRI.

The purpose of this study was to adapt the hepatic perfusion index (HPI) methodology previously developed for MRI to derive 3D parametric maps of HPI, and to investigate apparent differences in HPI maps between a group of colorectal cancer patients and controls. To achieve this, a new and simpler approach to HPI calculation which does not require measurements from the aorta or portal vein is introduced, and assessed with large liver regions of interest (ROIs) in patients and controls. Several example HPI maps showing localized variation are then presented. The subject group consisted of 12 patients with known colorectal metastases, and 13 control subjects referred for routine contrast-enhanced spine imaging with no history of neoplastic disease. HPI was evaluated from serial T1 volume acquisitions acquired over the course of a Gd-DTPA bolus injection. Regions of abnormal perfusion were visible on the HPI maps derived for the patient group, manifested as areas of locally increased HPI extending around the visible margins of known metastases evident on the conventional contrast-enhanced images. This method for MR voxel-based parametric mapping of HPI has the potential to demonstrate regional variations in perfusion at the segmental and subsegmental level.

Adult↗

PubMed1996

Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with the link protein gene.

The type II collagen gene (Col2a1) is expressed primarily in chondrocytes. Transcription of Col2a1 is mediated by cell-specific regulatory elements located within the promoter and first intron. Here, we map a minimal enhancer and identify elements that determine cartilage-specific Col2a1 expression by analyzing the activity of a series of chimeric genes consisting of rat Col2a1 first intron deletion mutants ligated to the chloramphenicol acetyltransferase reporter gene. We show that a 100-base pair (bp) segment within the first intron is the minimum size necessary for high level, cell type-specific expression of Col2a1. Sequence analysis of this 100-bp Col2a1 enhancer revealed several sequence motifs similar to motifs present within the regulatory region of the link protein gene, another cartilage gene. These motifs include an AT-rich element, a C1 motif and a C3 motif. Deletion of any of these elements reduced Col2a1 enhancer activity in chick embryo chondrocytes. We also tested enhancer-mediated activity in CFK2 cells which differentiate to a chondrogenic phenotype and begin to express type II collagen mRNA after extended culture. In stably transfected CFK2 cells, constructs containing the 100-bp enhancer were activated during the transition from prechondrogenic to chondrogenic cell populations and deletions within the enhancer strongly down-regulated activity. Chondrocyte-specific DNA-protein complexes were identified using nuclear extracts prepared from chick embryo chondrocytes and 32P-labeled oligonucleotides from these regions of the first intron. These results suggest that interaction of chondrocyte specific nuclear factors with multiple core elements from a small region within the first intron are important for cell-type specific Col2a1 enhancer activity.

Animals↗

PubMed2007

Quantitative analysis of whole-tumor Gd enhancement histograms predicts malignant transformation in low-grade gliomas.

PURPOSE: To quantify subtle gadolinium (Gd) enhancement (signal increase) in whole-tumor histograms and optimize their ability to predict subsequent malignant transformation in low-grade gliomas (LGGs). MATERIALS AND METHODS: We analyzed histograms from 21 adult subjects with LGGs (eight nontransformers and 13 transformers) who had been imaged every six months for periods of two to five years. Before transformation these tumors were reported as radiologically non-enhancing. Imaging included a T(1)-weighted volume sequence before and after a double dose of Gd-DTPA contrast agent. Image data sets were spatially registered and subtracted to obtain maps of percent enhancement (%E). Tumor outlines were defined on fluid-attenuated inversion recovery (FLAIR) images, and the volumes were calculated. Histogram tails were analyzed to obtain the volume (mL) of subtly enhancing tissue (%E > 10%). RESULTS: Baseline enhancing volumes were higher for Ts than for NTs (P < 0.005). Kaplan-Meier survival curves for a threshold of 4 mL showed clear differences at five years (P < 0.04). Pretransformation examinations predicted transformation (corrected threshold = 3.0 mL, P = 0.011). CONCLUSION: Clear histogram differences at presentation suggest that the process of transformation starts very early. It is now possible to identify individuals at high risk for transformation at baseline by quantifying the volume of subtly enhancing tumor tissue, and such findings could have an impact on patient management.

Adult↗

PubMed1995

A 2D crossover-based map of the human X chromosome as a model for map integration.

We have constructed a two-dimensional map of 243 markers on the X chromosome. The average distance between markers ordered by two recombinants is 5.4 centiMorgans (cM), which is reduced to 3.2 cM using a less stringent criterion of one recombinant. Map resolution is enhanced by replacing the usual reference marker format with a 2D format, and the two-recombinant rule is more conservative than the lod 3.0 criterion for order. Taken together, crossover mapping and the 2D format produces maps with greater reliability and higher resolution than maps constructed using currently accepted standards. This first high-density crossover-based map of an entire human chromosome provides a model for integrating physical and genetic maps.

Chromosome Mapping↗

PubMed1997

Selected methodological issues in meiotic mapping of obesity genes in humans: issues of power and efficiency.

This paper focuses on methods for mapping novel obesity genes in humans via meiotic mapping techniques. By novel we mean genes that are as yet unidentified as playing a role in obesity. We begin by presenting a discussion of why we believe it is important to seek out novel obesity genes and, in particular, novel genes of small effect. In light of the arguably Herculean task of finding genes of small effect with conventional gene mapping methods, we discuss alternative methods and procedures that may enhance our ability to map novel obesity genes of small effect. Many of these methods have been discussed previously in the literature and are summarized here. These include reconceptualizing power in the context of genomewide scans, multivariate linkage approaches, the use of phenotypically extreme subjects, and the use of large sibships. These are discussed in the context of linkage studies. Association studies and disequilibrium mapping are also discussed, and again, issues involving the use of extreme phenotypes and multiple testing are included. We also provide a brief discussion of DNA pooling and transmission disequilibrium tests for quantitative traits. Finally, we advocate data pooling techniques (e.g., meta-analysis) to enhance the power and efficiency of the entire field of the genetics of obesity.

Chromosome Mapping↗

PubMed2001

Early development of vasogenic edema in experimental cerebral fat embolism in cats: correlation with MRI and electron microscopic findings.

RATIONALE AND OBJECTIVES: To evaluate the magnetic resonance imaging and electron microscopic findings of the hyperacute stage of cerebral fat embolism in cats and the time needed for the development of vasogenic edema. METHODS: Magnetic resonance imaging was performed at 30 minutes (group 1, n = 9) and at 30 minutes and 1, 2, 4, and 6 hours after embolization with triolein (group 2, n = 10). As a control for group 2, the same acquisition was obtained after embolization with polyvinyl alcohol particles (group 3, n = 5). Magnetic resonance images were analyzed qualitatively and quantitatively. Electron microscopic examination was done in all cats. RESULTS: In group 1, the lesions were iso- or slightly hyperintense on T2-weighted (T2W) and diffusion-weighted (DWIs) images, hypointense on the apparent diffusion coefficient (ADC) map image, and markedly enhanced on the gadolinium-enhanced T1-weighted images (Gd-T1WIs). In group 2 at 30 minutes, the lesions were similar to those in group 1. Thereafter, the lesions became more hyperintense on T2WIs and DWIs and more hypointense on the ADC map image. The lesions were enhanced on Gd-T1WIs at all acquisition times. In group 3, the lesions showed mild hyperintensity on T2WIs at 6 hours but hypointensity on the ADC map image from 30 minutes, with a tendency toward a greater decrease over time. The lesions were not enhanced on Gd-T1WIs at any time point. Electron microscopic findings revealed discontinuity of the capillary endothelial wall, perivascular and interstitial edema, and swelling of glial and neuronal cells in groups 1 and 2. Cellular swelling and interstitial edema were more prominent in group 2. In group 3, interstitial edema was seen; however, discontinuity of the endothelial wall was absent. CONCLUSIONS: The lesions were hyperintense on T2WIs and DWIs, hypointense on the ADC map image, and enhanced on Gd-T1WIs. On electron microscopy, the lesions showed cytotoxic and vasogenic edema with disruption of the blood-brain barrier. Vasogenic edema seems to develop within 30 minutes in cerebral fat embolism in cats.

Animals↗

PubMedDate not supplied

A review on biomedical image processing and future trends.

The last two decades have witnessed a revolutionary development in the field of biomedical and diagnostic imaging. Imaging procedures and modalities which were only in the experimental research phase in the early part of the last two decades, have now become universally accepted clinical procedures. They include computerized tomography (CT), magnetic resonance imaging, ultrasound imaging, nuclear medicine imaging, computerized hematological cell analysis, etc. In the past, the conventional and relatively simple image processing techniques such as image enhancement, gray-level mapping, spectral analysis, region extraction, etc. have been modified for biomedical images and successfully applied for processing and analysis. The role of image enhancement, gray-level mapping, and image reconstruction from projections algorithms in CT and other radiological imaging modalities is well evident. Recently, many advances in biomedical image processing, analysis, and understanding algorithms have shown a great potential for enhancing and interpreting useful diagnostic information from these images more accurately. This paper presents a review on the current state-of-the-art techniques in biomedical image processing and comments on future trends.

Algorithms↗

PubMed2006

Development and genetic mapping of sequence-tagged microsatellites (STMs) in bread wheat (Triticum aestivum L.).

The density of SSRs on the published genetic map of bread wheat (Triticum aestivum L.) has steadily increased over the last few years. This has improved the efficiency of marker-assisted breeding and certain types of genetic research by providing more choice in the quality of SSRs and a greater chance of finding polymorphic markers in any cross for a chromosomal region of interest. Increased SSR density on the published wheat genetic map will further enhance breeding and research efforts. Here, sequence-tagged microsatellite profiling (STMP) is demonstrated as a rapid technique for the economical development of anonymous genomic SSRs to increase marker density on the wheat genetic map. A total of 684 polymorphic sequence-tagged microsatellites (STMs) were developed, and 380 were genetically mapped in three mapping populations, with 296 being mapped in the International Triticeae Mapping Initiative W7984 x Opata85 recombinant inbred cross. Across the three populations, a total of 479 STM loci were mapped. Several technological advantages of STMs over conventional SSRs were also observed. These include reduced marker deployment costs for fluorescent-based SSR analysis, and increased genotyping throughput by more efficient electrophoretic separation of STMs and a high amenability to multiplex PCR.

Chromosome Mapping↗

PubMed2006

Evaluating kriging as a tool to improve moderate resolution maps of forest biomass.

The USDA Forest Service, Forest Inventory and Analysis program (FIA) recently produced a nationwide map of forest biomass by modeling biomass collected on forest inventory plots as nonparametric functions of moderate resolution satellite data and other environmental variables using Cubist software. Efforts are underway to develop methods to enhance this initial map. We explored the possibility of modeling spatial structure to make such improvements. Spatial structure in the field biomass data as well as in residuals from the map was investigated across 18 ecological zones in the Interior Western U.S. Exploratory tools included directional graphs of summary statistics, three dimensional maps, Moran's I correlograms, and variograms. Where spatial pattern was present, field and residual biomass were kriged, and predictions made for an independent test set were evaluated for improvement over predictions in the initial biomass map. While kriging has some potential benefit when analyzing the field data and exploring spatial structure, kriging residuals resulted in little or no improvement in the initial biomass map developed using Cubist software. Stationarity assumptions, variogram behavior, and appropriate model fitting strategies are discussed.

Biomass↗

PubMed1991

Two immunodominant domains of gp41 bind antibodies which enhance human immunodeficiency virus type 1 infection in vitro.

Four of eight human monoclonal antibodies (huMAbs) to gp41 were identified which could enhance human immunodeficiency virus type 1 (HIV-1) infection in vitro by complement-mediated antibody-dependent enhancement (C'-ADE). These enhancing huMAbs were mapped to two distinct domains on the HIV-1 gp41 transmembrane glycoprotein by using synthetic peptides. The first domain, amino acids 579 to 613 (peptide AA579-613), was recognized by three of the four enhancing huMAbs. The AA579-613 peptide blocked C'-ADE of HIV-1 infection in vitro whether it was mediated by these three huMAbs or by human polyclonal anti-HIV serum. The second domain, amino acids 644 to 663, bound the remaining enhancing huMAb. This peptide weakly blocked C'-ADE mediated by the huMAb and by an HIV immune globulin fraction but did not block C'-ADE mediated by a patient's serum. The patient's serum did react with the peptide in an enzyme immunoassay. The huMAbs to the two domains could interact in vitro to enhance HIV-1 infection in a synergistic manner. These two domains, which bind enhancing antibodies, are conserved between HIV-1 isolates as well as between HIV-2 and simian immunodeficiency virus isolates. These data demonstrate the existence of two conserved regions within the HIV-1 gp41 which bind enhancing antibodies; these two domains, amino acids 579 to 613 and 644 to 663, may prove important in HIV-1 vaccine development and in immunopathogenesis of HIV-1 infection.

Amino Acid Sequence↗