Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Insulator Elements”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 325 records · Page 18Linked to original sources
PubMed1998

[Boundaries of structure-functional units (domains) of eukaryotic chromosomes].

Extensive evidence indicates that eukaryotic chromosomes are organized as sets of structural-functional units (domains) that form the basis of independent gene activity. At present, several types of chromosome regions have been suggested for the role of putative domain boundaries between structural-functional units in eukaryotic chromosomes. In this review, the experimental data on several chromosome elements as possible boundaries in eukaryotic chromosomes are presented. They are: scs/scs'-elements of the Drosophila heat-shock locus, DCR, LCR and the insulator of the human and chicken beta-globin locus, as well as MARs (SARs) of plant and animal chromosomes.

Animals↗
PubMed1998

The mouse H19 locus mediates a transition between imprinted and non-imprinted DNA replication patterns.

Genes subject to genomic imprinting generally occur in clusters of hundreds of kilobases. These domains exhibit several gamete of origin-dependent manifestations, including a pattern of asynchronous replication when studied by fluorescence in situ hybridization (FISH). We find a transition from asynchronous replication at the imprinted mouse H19 gene to synchronous replication at the downstream Rpl23 gene, the human homologue of which appears to be non-imprinted. Two-colour FISH demonstrates that this transition is due solely to a difference in replication timing between the upstream and downstream chromatin on the later-replicating (maternal) chromosome. This difference is lost in mice deleted for the H19 gene body and 9.9 kb of upstream DNA when this deletion is maternally inherited, with synchronous replication patterns extending over 110 kb upstream from the deleted area. No effect is seen when the deletion is paternally inherited. The presence of a boundary element in this region has been suggested by observations of position-independent expression of H19 -containing transgenes and the blocking of accessibility of downstream enhancers to the upstream Igf2 and Ins2 genes on the maternal chromosome. The FISH studies presented here demonstrate the insulation of replication patterns within the imprinted domain from downstream, non-imprinted chromatin, mediated by an element at the H19 locus which is subject to genomic imprinting.

Animals↗
PubMed2002

Development of virus vectors for gene therapy of beta chain hemoglobinopathies: flanking with a chromatin insulator reduces gamma-globin gene silencing in vivo.

We have previously described the development of oncoretrovirus vectors for human gamma-globin using a truncated beta-globin promoter, modified gamma-globin cassette, and alpha-globin enhancer. However, one of these vectors is genetically unstable, and both vectors exhibit variable expression patterns in cultured cells, common characteristics of oncoretrovirus vectors for globin genes. To address these problems, we identified and removed the vector sequences responsible for genetic instability and flanked the resultant vector with the chicken beta-globin HS4 chromatin insulator to protect expression from chromosomal position effects. After determining that flanking with the cHS4 element allowed higher, more uniform levels of gamma-globin expression in MEL cell lines, we tested these vectors using a mouse bone marrow transduction and transplantation model. When present, the gamma-globin cassettes from the uninsulated vectors were expressed in only 2% to 5% of red blood cells (RBCs) long term, indicating they are highly sensitive to epigenetic silencing. In contrast, when present the gamma-globin cassette from the insulated vector was expressed in 49% +/- 20% of RBCs long term. RNase protection analysis indicated that the insulated gamma-globin cassette was expressed at 23% +/- 16% per copy of mouse alpha-globin in transduced RBCs. These results demonstrate that flanking a globin vector with the cHS4 insulator increases the likelihood of expression nearly 10-fold, which in turn allows for gamma-globin expression approaching the therapeutic range for sickle cell anemia and beta thalassemia.

3T3 Cells↗
PubMedDate not supplied

Continuous parasacral sciatic nerve block: two case reports.

OBJECTIVE: This study investigated the use of a continuous parasacral sciatic nerve block for anesthesia and postoperative analgesia for lower extremity surgery. METHODS: A continuous parasacral sciatic nerve block was performed in two patients (triple ankle arthrodesis and below-knee amputation). The sacral plexus was identified using an insulated Tuohy needle and a nerve stimulator. A catheter was placed near the elements of the sacral plexus via the Tuohy needle. RESULTS: In both patients, surgical anesthesia was successfully established through the parasacral catheter with lidocaine 1% (1/200,000 epinephrine), and postoperative analgesia was successfully established with a bolus of bupivacaine 0.375% (1/200,000 epinephrine) and maintained with a continuous infusion of bupivacaine 0.1% (8 mL/h) for 48 hours. CONCLUSION: We conclude that continuous parasacral sciatic nerve block can provide anesthesia and long-term analgesia for operative procedures of the foot and leg.

Amputation, Surgical↗
PubMed2006

Adaptive finite element methods in electrochemistry.

In this article, we review some of our previous work that considers the general problem of numerical simulation of the currents at microelectrodes using an adaptive finite element approach. Microelectrodes typically consist of an electrode embedded (or recessed) in an insulating material. For all such electrodes, numerical simulation is made difficult by the presence of a boundary singularity at the electrode edge (where the electrode meets the insulator), manifested by the large increase in the current density at this point, often referred to as the edge effect. Our approach to overcoming this problem has involved the derivation of an a posteriori bound on the error in the numerical approximation for the current that can be used to drive an adaptive mesh-generation algorithm, allowing calculation of the quantity of interest (the current) to within a prescribed tolerance. We illustrate the generic applicability of the approach by considering a broad range of steady-state applications of the technique.

Journal Article↗
PubMed2001

Gene transfer into nonhuman primate hematopoietic stem cells: implications for gene therapy.

Hematopoietic stem cells (HSCs) are desirable targets for gene therapy because of their self-renewal and multilineage differentiation abilities. Retroviral vectors are extensively used for HSC gene therapy. However, the initial human trials of HSC gene marking and therapy showed that the gene transfer efficiency into human HSCs with retroviral vectors was very low in contrast to the much higher efficiency observed in murine experiments. The more quiescent nature of human HSCs and the lower density of retroviral receptors on them hindered the efficient gene transfer with retroviral vectors. Since nonhuman primates have marked similarity to humans in all aspects including the HSC biology, their models are considered to be important to evaluate and improve gene transfer into human HSCs. Using these models, clinically relevant levels (around 10% or even more) of gene-modified cells in peripheral blood have recently been achieved after gene transfer into HSCs and their autologous transplantation. This has been made possible by improving ex vivo transduction conditions such as introduction of Flt-3 ligand and specific fibronectin fragment (CH-296) into ex vivo culture during transduction, and the use of retroviral vectors pseudotyped with the gibbon ape leukemia virus or feline endogenous retrovirus envelope. Other strategies including the use of lentiviral vectors and in vivo selective expansion of gene-modified cells with the drug resistance gene or selective amplifier gene (also designated the molecular growth switch) are now being tested to further increase the fraction of gene-modified cells using nonhuman primate models. In addition to the high gene transfer efficiency, high-level and long-term expression of transgenes in human HSCs and their progeny is also required for effective HSC gene therapy. For this purpose, other backbones of retroviral vectors such as the murine stem cell virus and cis-DNA elements, such as the ss-globin locus control region and the chromatin insulator, also need to be tested in nonhuman primate models. Nonhuman primate studies will continue to provide an important framework for human HSC gene therapy. Well-designed nonhuman primate studies will also offer unique insights into the HSCs, immune system, and transplantation biology characteristic of large animals.

Animals↗
PubMed1998

The A gamma-globin 3' element provides no unique function(s) for human beta-globin locus gene regulation.

The human beta-globin locus is activated transcriptionally by a complex series of events that culminate in appropriate temporal and tissue-specific control over five separate genes during embryonic and early postnatal development. One cis-regulatory element in the locus, originally identified as an enhancer 3' to the Agamma-globin gene, more recently has been suggested to harbor alternative or additional properties, including stage-specific silencer, insulator, nuclear matrix, or chromosome scaffold attachment activities. We have re-evaluated the activity during erythropoiesis that is conferred by this element by deleting it from a yeast artificial chromosome (YAC) containing the entire human beta-globin locus and then assaying for the expression of each gene at each developmental stage after incorporation of the mutant YAC into the mouse germline. The data show that loss of the Agamma-globin 3' element confers no phenotype in six independent lines of intact YAC mutant transgenic mice, thus demonstrating (minimally) that any activities attributable to this element are fully compensated by other DNA sequences within the beta-globin locus.

Animals↗
PubMedDate not supplied

Characterization and mitigation of stray radiofrequency currents during monopolar resectoscopic electrosurgery.

STUDY OBJECTIVE: To determine patterns and range of stray radiofrequency (RF) currents flowing through the working element of monopolar resectoscopes during routine endometrial rollerball ablation or resection; and to determine whether straightforward modifications of the uterine resectoscope and the application of RF monitoring could provide a safe pathway for such currents. DESIGN: Prospective in vivo measurements (Canadian Task Force classification II-1). SETTING: University-affiliated teaching hospital. PATIENTS: Twelve women undergoing resectoscopic surgery. INTERVENTIONS: During routine resectoscopic surgery using 1.5% glycine irrigant solution, three modified 26F Storz resectoscope working elements (model 27070E) were adapted to be continuously monitored with an Encision AEM device for excessive capacitive coupling and other stray currents from insulation failure. Active electrodes used were 3 mm and 5 mm rollberballs and 8 mm-diameter cutting loops powered by ERBE or Valleylab generators at 120 W. Active and working element currents were monitored by Pearson current transformers followed by root-mean-squared detectors based on the Analog Devices AD-637 integrated circuit. Data were recorded using a Fluke 199C oscilloscope, then serially transferred to a notebook computer and analyzed using Flukeview, Excel, and Minitab software. RESULTS: Typical values of working element currents ranged from 0.10 to 0.20 A. Active electrode currents were typically in the range of 0.50 to 1.10 A. Frequently, the working element current exceeded the typical values and ranged up to 0.60 A. These current surges produced a heat factor (I(2)t) of 0.45 A(2).sec in a 10-second period. CONCLUSIONS: During resectoscopic electrosurgery, baseline, most likely capacitive coupled, currents were always present. In addition, high values of working element currents occurred frequently, and they surged up to 0.60 A for significant periods of time. Without the modification of the resectoscopic device, these currents have the capability of flowing through the patient's genital tract and causing burns. Since monopolar electrosurgery remains an integral part of most hysteroscopic procedures, active electrode monitoring may offer a solution in protecting the patient and the surgeon from stray electrosurgical burns.

Adult↗
PubMed2000

A chromatin insulator protects retrovirus vectors from chromosomal position effects.

Recombinant murine retroviruses are widely used as delivery vectors for gene therapy. However, once integrated into a chromosome, these vectors often suffer from profound position effects, with vector silencing observed in vitro and in vivo. To overcome this problem, we investigated whether the HS4 chromatin insulator from the chicken beta-globin locus control region could protect a retrovirus vector from position effects. When used to flank a reporter vector, this element significantly increased the fraction of transduced cells that expressed the provirus in cultures and in mice transplanted with transduced marrow. These results demonstrate that a chromatin insulator can improve the expression performance of a widely used class of gene therapy vectors by protecting these vectors from chromosomal position effects.

3T3 Cells↗
PubMed2000

Comparative genomic sequencing identifies novel tissue-specific enhancers and sequence elements for methylation-sensitive factors implicated in Igf2/H19 imprinting.

A differentially methylated region (DMR) and endoderm-specific enhancers, located upstream and downstream of the mouse H19 gene, respectively, are known to be essential for the reciprocal imprinting of Igf2 and H19. To explain the same imprinting patterns in non-endodermal tissues, additional enhancers have been hypothesized. We determined and compared the sequences of human and mouse H19 over 40 kb and identified 10 evolutionarily conserved downstream segments, 2 of which were coincident with the known enhancers. Reporter assays in transgenic mice showed that 5 of the other 8 segments functioned as enhancers in specific mesodermal and/or ectodermal tissues. We also identified a conserved 39-bp element that appeared repeatedly within the DMR and formed complexes with specific nuclear factors. Binding of one of the factors was inhibited when the target sequence contained methylated CpGs. These complexes may contribute to the presumed boundary function of the unmethylated DMR, which is proposed to insulate maternal Igf2 from the enhancers. Our results demonstrate that comparative genomic sequencing is highly efficient in identifying regulatory elements.

5' Untranslated Regions↗
PubMed2002

A sandwich-designed temperature-gradient incubator for studies of microbial temperature responses.

A temperature-gradient incubator (TGI) is described, which produces a thermal gradient over 34 aluminium modules (15x30x5 cm) intersected by 2-mm layers of partly insulating graphite foil (SigraFlex Universal). The new, sandwich-designed TGI has 30 rows of six replicate sample wells for incubation of 28-ml test tubes. An electric plate heats one end of the TGI, and the other end is cooled by thermoelectric Peltier elements in combination with a liquid cooling system. The TGI is equipped with 24 calibrated Pt-100 temperature sensors and insulated by polyurethane plates. A PC-operated SCADA (Supervisory Control And Data Acquisition) software (Genesis 4.20) is applied for temperature control using three advanced control loops. The precision of the TGI temperature measurements was better than +/-0.12 degrees C, and for a 0-40 degrees C gradient, the temperature at the six replicate sample wells varied less than +/-0.04 degrees C. Temperatures measured in incubated water samples closely matched the TGI temperatures, which showed a linear relationship to the sample row number. During operation for 8 days with a gradient of 0-40 degrees C, the temperature at the cold end was stable within +/-0.02 degrees C, while the temperatures at the middle and the warm end were stable within +/-0.08 degrees C (n=2370). Using the new TGI, it was shown that the fine-scale (1 degrees C) temperature dependence of S(o) oxidation rates in agricultural soil (0-29 degrees C) could be described by the Arrhenius relationship. The apparent activation energy (E(a)) for S(o) oxidation was 79 kJ mol(-1), which corresponded to a temperature coefficient (Q(10)) of 3.1. These data demonstrated that oxidation of S(o) in soil is strongly temperature-dependent. In conclusion, the new TGI allowed a detailed study of microbial temperature responses as it produced a precise, stable, and certifiable temperature gradient by the new and combined use of sandwich-design, thermoelectric cooling, and advanced control loops. The sandwich-design alone reduced the disadvantageous thermal gradient over individual sample wells by 56%.

Bacteria↗
PubMed1996

The Fab-7 element of the bithorax complex attenuates enhancer-promoter interactions in the Drosophila embryo.

Enhancers integrate positive and negative regulatory information to direct localized patterns of gene expression in the Drosophila embryo. Here we present evidence for the occurrence of cis regulatory elements that control the levels of gene expression by modulating enhancer-promoter interactions. For this purpose we have investigated the Drosophila bithorax complex (BX-C) because genetic studies suggest that the BX-C contains as much as 300 kb of cis regulatory information. A specialized DNA element, Fab-7, has been proposed to function as a boundary element that separates the iab-6 and iab-7 cis regulatory regions within the Abd-B domain of the BX-C. A 1.2-kb Fab-7 DNA fragment was placed between divergently transcribed white and lacZ test promoters and challenged with several defined enhancers expressed in the early embryo. These studies suggest that Fab-7 functions as an attenuator, which weakens gene expression by reducing enhancer-promoter interactions. Fab-7 selectively blocks distal enhancers in an orientation-independent fashion, and can function when located far from either the distal enhancer or target promoter. Fab-7 may be related to insulator DNAs, which flank genetic loci and functionally isolate neighboring genes. We propose that specialized DNA elements, such as the Fab-7 attenuator, might play a general role in controlling the levels of gene expression by modulating enhancer-promoter interactions.

Animals↗
PubMedDate not supplied

Positive self-transitions in women child abuse survivors.

Clinicians need information about what factors contribute to women child abuse survivors' mental health and success. Most previous research has focused on negative after-effects of abuse, and correlating types and severity of abuse with specific mental and physical problems in adulthood. Two qualitative studies (total N = 55) were done with low-income women survivors of child abuse and neglect from a midwestern inner city area. The majority of these women were African American. All participants were in recovery from substance abuse problems. A secondary analysis of these accounts was done to examine positive life transitions made by these women after experiences of abuse, and throughout adulthood, despite having serious after-effects of abuse. The investigator and research team categorized themes from the narrative data into two processes--epiphanies and maintaining momentum--and six elements constituting the content areas of self-change: self-centering, ownership, interpersonal insulation, willfulness, seeing options, and spiritual connection. Maintaining momentum and shortening the time between epiphanies was found to be central. Tentative clinical implications are discussed. The study was preliminary in that it was a secondary analysis. Suggestions for further research are outlined.

Adaptation, Psychological↗
PubMed1995

Three-dimensional finite element analysis of current density and temperature distributions during radio-frequency ablation.

This study analyzed the influence of electrode geometry, tissue-electrode angle, and blood flow on current density and temperature distribution, lesion size, and power requirements during radio-frequency ablation. We used validated three-dimensional finite element models to perform these analyses. We found that the use of an electrically insulating layer over the junction between electrode and catheter body reduced the chances of charring and coagulation. The use of a thermistor at the tip of the ablation electrodes did not affect the current density decreased more slowly with distance from the electrode surface. We analyzed the effects of three tissue-electrode angles: 0, 45, and 90 degrees. More power was needed to reach a maximal tissue temperature of 95 degrees C after 120 s when the electrode-tissue angle was 45 degrees. Consequently, the lesions were larger and deeper for a tissue-electrode angle of 45 degrees than for 0 and 90 degrees. The lesion depth, volume, and required power increased with blood flow rate regardless of the tissue-electrode angle. The significant changes in power with the tissue-electrode angle suggest that it is safer and more efficient to ablate using temperature-controlled RF generators. The maximal temperature was reached at locations within the tissue, a fraction of a millimeter away from the electrode surface. These locations did not always coincide with the local current density maxima. The locations of these hottest spots and the difference between their temperature and the temperature read by a sensor placed at the electrode tip changed with blood flow rate and tissue-electrode angle.

Animals↗
PubMed2004

An enhancer element at the Igf2/H19 locus drives gene expression in both imprinted and non-imprinted tissues.

The insulin-like growth factor 2 (Igf2) gene encodes a potent growth factor that is expressed in multiple tissues during embryonic development. Expression at this locus is mediated by genomic imprinting. In the developing endodermal tissues, imprinting of Igf2 is mediated by the interaction of a set of enhancers downstream of the linked H19 gene with a differentially methylated domain (DMD) that lies approximately 2-4 kb upstream of H19 that has a boundary or insulator function in the hypomethylated state. In the remainder of tissues that express Igf2 and H19, the cis elements that drive their correct expression and imprinting are not well understood. In addition, enhancers driving expression of Igf2 in the choroid plexus and leptomeninges, tissues where the gene is thought not to be imprinted, have not been isolated. Here we show that biallelic (non-imprinted) expression within the choroid plexus is restricted to the epithelium, and we provide evidence that a conserved intergenic region functions as an enhancer for Igf2 both in tissues where the gene is imprinted, and where Igf2 is biallelically expressed. The presence of an enhancer for imprinted tissues in the intergenic region argues for the existence of imprinting controls distinct from the DMD, which may be provided by differential methylation at sites proximal to Igf2.

Animals↗
PubMed1998

Polycomb group repression is blocked by the Drosophila suppressor of Hairy-wing [su(Hw)] insulator.

The suppressor of Hairy-wing [SU(HW)] binding region disrupts communication between a large number of enhancers and promoters and protects transgenes from chromosomal position effects. These properties classify the SU(HW) binding region as an insulator. While enhancers are blocked in a general manner, protection from repressors appears to be more variable. In these studies, we address whether repression resulting from the Polycomb group genes can be blocked by the SU(HW) binding region. The effects of this binding region on repression established by an Ultrabithorax Polycomb group Response Element were examined. A transposon carrying two reporter genes, the yellow and white genes, was used so that repression and insulation could be assayed simultaneously. We demonstrate that the SU(HW) binding region is effective at preventing Polycomb group repression. These studies suggest that one role of the su(Hw) protein may be to restrict the range of action of repressors, such as the Polycomb group proteins, throughout the euchromatic regions of the genome.

ATP-Binding Cassette Transporters↗
PubMed2002

Topological constraints governing the use of the chicken HS4 chromatin insulator in oncoretrovirus vectors.

The expression of integrated oncoretrovirus vectors is subject to the inhibitory effects of surrounding chromatin. A previous report from our laboratory indicated that such position effects can be overcome by flanking a reporter vector with the cHS4 chromatin insulator. To characterize this activity more thoroughly, we switched the promoter-gene combinations in the reporter vector and analyzed expression of these vectors flanked with the cHS4 fragment in both orientations following bone marrow transduction and transplantation in mice. The results indicate that the cHS4 fragment can function in both orientations and can insulate both the virus long-terminal-repeat (LTR) promoter and an internal phosphoglycerate kinase (Pgk) promoter. However, insulation of the LTR promoter diminished when the orientation of the cHS4 fragment placed the CTCF-binding core element immediately proximal to the U3 region, suggesting a minimal distance requirement. Moreover, placement of the cHS4 fragment in the U3 region of the 3' LTR dramatically decreased the level of expression from an internal Pgk promoter, presumably by blocking interaction with the 3' LTR enhancer. Finally, sorting studies suggest that the severity of position effects or autonomous promoter silencing increases as transduced progenitors differentiate into mature progeny. These findings have direct implications for the use of chromatin insulators such as cHS4 in oncoretrovirus vectors.

3' Untranslated Regions↗
PubMed2006

Controlled insulator-to-metal transformation in printable polymer composites with nanometal clusters.

Although organic semiconductors have received the most attention, the development of compatible passive elements, such as interconnects and electrodes, is also central to plastic electronics. For this, ligand-protected metal-cluster films have been shown to anneal at low temperatures below 250 degrees C to highly conductive metal films, but they suffer from cracking and inadequate substrate adhesion. Here, we report printable metal-cluster-polymer nanocomposites that anneal to a controlled-percolation nanostructure without complete sintering of the metal clusters. This overcomes the previous challenges while still retaining the desired low transformation temperatures. Highly water- and alcohol-soluble gold clusters (75 mg ml-1) were synthesized and homogeneously dispersed into poly(3,4-ethylenedioxythiophene) to give a material with annealed d.c. conductivity tuneable between 10(-4) and 10(5) S cm-1. These composites can inject holes efficiently into all-printed polymer organic transistors. The insulator-metal transformation can also be electrically induced at 1 MV cm-1, suggesting possible memory applications.

Journal Article↗