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Results for “Enhancer”

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PubMed1989

A yeast ribosomal DNA-binding protein that binds to the rDNA enhancer and also close to the site of Pol I transcription initiation is not important for enhancer functioning.

Using the gel retardation assay we have identified a protein that can specifically bind to a site within the enhancer of the 37S pre-ribosomal RNA operon in yeast, as well as to a site 210 bp upstream of the site of transcription initiation of this operon. This protein (RBP1) has been partially purified by means of heparin-agarose chromatography and protects 20 bp in the rDNA enhancer, and 25 bp in the initiation region, against DNase I in an in vitro footprinting assay. In vivo footprinting studies using methylation of intact yeast cells with dimethylsulphate, indicate that the same binding sites are occupied in vivo as well. Deletions that abolish binding of RBP1 to the enhancer in vitro, as well as linker insertions into the RBP1 binding site in the initiation region that strongly diminish in vitro binding of RBP1, have no effect whatsoever on the enhancement of rDNA transcription in vivo. This was studied by deletion/mutation of the RBP1 binding site in vitro in an artificial ribosomal minigene and measuring the effect on the minigene transcription in vivo in yeast cells, transformed with the deleted/mutated minigenes. It can therefore be concluded that binding of RBP1 is not an important parameter in the functioning of the rDNA enhancer in yeast. Using the same minigene system we also show that RBP1 is not involved in termination of RNA polymerase I (Pol I) transcription at the main terminator T2.

Base Sequence↗

PubMed1996

Identification of ovarian enhancer-binding factors which bind to ovarian enhancer 1 of the Drosophila genes yp1 and yp2.

It has been reported that three different DNA regions-the fat body enhancer and ovarian enhancers 1 and 2-direct the tissue-specific expression of yp1 and yp2 in Drosophila melanogaster. In the present study, we identified ovarian enhancer 1-specific binding proteins. Electrophoretic mobility shift assay revealed that these proteins are present in the adult ovary, but not in adult testis or fat body. Southwestern blot analysis showed that about 130 kDa and 40 kDa proteins, designated OEF1 and OEF2, respectively, from ovarian nuclear or crude extracts bind specifically to the ovarian enhancer 1. The two proteins were partially purified by streptavidin/agarose-DNA affinity chromatography, and their binding activity was confirmed by electrophoretic mobility shift assay. These ovarian enhancer factors may play an important role in the regulation of transcription of yp1 and yp2 in the ovary.

Animals↗

PubMed1992

Transformation by human papillomavirus type 16 (HPV16) DNA but not HPV6b DNA is enhanced by addition of the human cytomegalovirus enhancer.

Primary human cervical epithelial cells immortalized by human papillomavirus type 16 (HPV16) DNA exhibit altered morphology and differentiation characteristic of transformation, but show a lack of transformed phenotype relative to HPV18 DNA immortalized cells in terms of anchorage-independent growth (Pecoraro, Lee, Morgan, and Defendi, 1991, Am. J. Pathol. 138, 1-8). This is completely corrected by inserting a strong heterologous enhancer derived from human cytomegalovirus DNA upstream from the HPV16 long control region. The cells immortalized by this DNA form colonies in agar comparable to those formed by HPV18 DNA immortalized cells. The enhanced transformation capability correlates with increased levels of HPV16 E6-E7 and E5 transcripts. The HPV16 DNA containing this strong enhancer also transforms C127 mouse cells with increased efficiency and strength relative to the natural HPV16 DNA, as measured by the numbers and size of the colonies in agar. The positive effects of this strong enhancer appear specific for HPVs associated with genital malignancies such as HPV16, since HPV6b DNA (primarily in benign tumors) with or without the strong cytomegalovirus enhancer is incapable of immortalizing primary human cervical epithelial cells or allowing efficient growth of C127 mouse cells in agar. These results suggest that the diminished oncogenic properties of HPV16 versus HPV18 DNA in cultured cells and in human malignancies may reside in the long control regions of these viruses and, additionally, may define another difference in the oncogenic properties of HPVs associated with benign or malignant genital neoplasia.

Animals↗

PubMed2006

Multiphasic contrast-enhanced multidetector-row CT of liver: contrast-enhancement theory and practical scan protocol with a combination of fixed injection duration and patients' body-weight-tailored dose of contrast material.

The introduction of multidetector-row CT (MDCT) has provided the abdominal radiologists with dramatic changes for the imaging of the liver. At just present, establishment of new and optimal injection protocol in multiphasic contrast-enhanced MDCT of the liver has been yearned because the difficulty and the importance to optimize the scan timing for each phase imaging have obviously increased because of the extremely rapid scan speed with MDCT scanners. Among technical factors employed in an injection protocol for multiphasic contrast-enhanced MDCT of the liver, to make an injection duration of contrast material fixed is the most important for the precise estimation of the scan timing for each phase imaging. With the fixed injection duration, the peak enhancement time of the aorta, portal vein, and liver constantly appear approximately 10, 20, and 30s after the any fixed injection durations (completion of injection of contrast material), respectively. Moreover, using a combination of the fixed injection duration and patients' body-weight-tailored dose of contrast material, similarity of time-density curves of each organ in shape and pattern can be achieved for individual patient with different body weight. In this issue, we will attempt to determine an ultimately optimal, but practical injection/scan protocol to successfully realize multiphasic contrast-enhanced MDCT of the liver based on the contrast-enhancement theory.

Body Weight↗

PubMed2002

[Evaluation of myocardial viability with contrast-enhanced magnetic resonance imaging--comparison of the late enhancement technique with positronemission tomography].

PURPOSE: To compare contrast-enhanced magnetic resonance imaging (MRI) and positron emission tomography (PET) in the evaluation of myocardial viability. METHODS: [ (18)F]-FDG-PET, [ (201)Tl]-TlCl-SPECT and contrast-enhanced MRI were performed in 29 patients with proven coronary artery disease and impaired left ventricular function to assess myocardial viability. MRI scans were done on a 1.5 T scanner (Magnetom Sonata, Siemens, Germany). After the steady-state free precession cine study, 0.2 mmol/kg BW of Gd-DPTA (Magnevist(R), Schering, Germany) were administered i. v. For the detection of "late enhancement" (LE) indicating scar, left ventricular long axes and contiguous short axis slices of 8 mm thickness were scanned using an inversion recovery turbo gradient echo sequence (TR 8.0 ms; TE 4.0 ms; TI 180 - 240 ms; FA 20 degrees ). The evaluation of LE and FDG uptake in PET with perfusion defect in SPECT was done using an 8 (basal, mid) and 4 (apical) segment model in all short axes to cover the entire ventricle. The transmural extent of LE was assessed using a 4-point score system. Comparison between the two modalities was performed on a segmental basis. RESULTS: A total of 1753 segments were assessed. In MRI, 40 % of the segments showed myocardial scar, whereas PET revealed impaired uptake in 25 %. MRI obtained a very low interobserver variability in detecting myocardial scar (kappa 0.92). Using PET as the standard of reference in the segmental comparison, contrast-enhanced MRI yielded a sensitivity of 84 % and a specificity of 76 % for the detection of scar. 18 % of all segments showed LE but normal FDG uptake, 83 % of them referred to subendocardial scars. CONCLUSIONS: There is close agreement between contrast-enhanced MRI and PET in detecting transmural myocardial scars. Superior spatial resolution enables MRI to detect and quantify even subendocardial scar. Therefore, larger studies using functional recovery after revascularisation as an endpoint have to prove whether MRI might replace PET as the standard of reference in the assessment of myocardial viability.

Aged↗

PubMed2003

[Detection of liver lesions with gadolinium-enhanced VIBE sequence in comparison with SPIO-enhanced MRI].

PURPOSE: Detection of malignant liver lesions with gadolinium-enhanced volumetric interpolated breath-hold examination (VIBE) in comparison with SPIO-enhanced MRT (four different SPIO-enhanced T2w sequences) and histological and imaging follow-up in non-cirrhotic livers at 1.5 T. PATIENTS AND METHODS: Twenty-two patients with suspected focal liver lesions prospectively underwent a gadolinium-enhanced 3D VIBE. Four T2w sequences (HASTE sequence, fast spin-echo-sequence with and without fat-saturation, FLASH 2D gradient echo-sequence) after administration of superparamagnetic iron oxide (SPIO) served as gold standard combined with histological work-up in 17 patients and histological and imaging follow-up in five patients. The image quality was evaluated and the detectability of intrahepatic lesions was rated by the alternative free-response receiver operating characteristic (AFROC) analysis. In addition, the contrast-to-noise ratio was compared. RESULTS: Altogether 49 malignant and 35 benign liver lesions were found. Concerning the image quality, VIBE turned out to be of slightly poorer image quality than the SPIO-enhanced examination with HASTE sequence (4.95 vs. 5.0). The fast spin-echo-sequence without fat-saturation demonstrated the highest contrast-to-noise ratio. All sequences showed a comparable certainty in detecting lesion (area under the curve 0.68-0.73) and identifying malignant liver lesions. CONCLUSION: Despite the small number of patients, VIBE seems to be a comparable, inexpensive and fast method in diagnosing malignant liver lesions.

Adult↗

PubMed1996

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

Alleles↗

PubMed1998

p53 binds and represses the HBV enhancer: an adjacent enhancer element can reverse the transcription effect of p53.

The transcription program of the hepatitis B virus (HBV) genome is regulated by an enhancer element that binds multiple ubiquitous and liver-enriched transcription activators. HBV transcription and replication are repressed in the presence of p53. Here we describe a novel molecular mechanism that is responsible for this repression. The p53 protein binds to a defined region within the HBV enhancer in a sequence-specific manner, and this, surprisingly, results in p53-dependent transcriptional repression in the context of the whole HBV enhancer. This unusual behavior of the HBV enhancer can be reconstituted by replacing its p53-binding region with the p53-binding domain of the mdm2 promoter. Remarkably, mutation of the EP element of the enhancer reversed the effect of p53 from repression to transcriptional stimulation. Furthermore, EP-dependent modulation of p53 activity can be demonstrated in the context of the mdm2 promoter, suggesting that EP is not only required but is also sufficient to convert p53 activity from positive to negative. Our results imply that the transcriptional effect of DNA-bound p53 can be dramatically modulated by the DNA context and by adjacent DNA-protein interactions.

Base Sequence↗

PubMed1999

Enhancer blocking by the Drosophila gypsy insulator depends upon insulator anatomy and enhancer strength.

Insulators are specialized DNA sequences that prevent enhancer-activated transcription only when interposed between an enhancer and its target promoter. The Drosophila gypsy retrotransposon contains an insulator composed of 12 degenerate binding sites for the Suppressor of Hairy-wing [Su(Hw)] protein that are separated by AT-rich DNA possessing sequence motifs common to matrix/scaffold attachment regions (MARs/SARs). To further understand mechanisms of insulator function, the parameters required for the gypsy insulator to prevent enhancer-activated transcription were examined. Synthetic binding regions were created by reiteration of a single Su(Hw) binding site that lacked the MAR/SAR motifs. These synthetic binding regions reconstituted insulator activity, suggesting that the property of enhancer blocking may be distinct from matrix association. We found that the number and spacing of Su(Hw) binding sites within the gypsy insulator, as well as the strength of the enhancer to be blocked, were important determinants of insulator function. These results provide a link between transcription and insulation, suggesting that these processes may be mechanistically interconnected.

Base Sequence↗

PubMed1988

Foreign transcriptional enhancers in yeast. I. Interactions of papovavirus transcriptional enhancers and a quiescent pseudopromoter on supercoiled plasmids.

We have constructed simple test-plasmids to study transcriptional enhancers in yeast. In this paper the reporter-gene is a plasmid borne deletion-substitution derivative (his-del4) of the Saccharomyces cerevisiae HIS3 gene in which the native promoter has been replaced by a dormant, susceptible pseudopromoter. We investigate the function in yeast of foreign control elements, the polyomavirus enhancer and some of its derivatives, inserted in either orientation at the 3' or 5' ends of the reporter gene. The polyoma enhancer (and, although less thoroughly studied, the SV40 enhancer) will strongly activate transcription from latent start sites within the pseudo-promoter sequence. The rules we draw for the polyoma enhancer effect in yeast are, with a few interesting exceptions, remarkably similar to those discovered by experimentation in mammalian cells.

Base Sequence↗

PubMed2003

The ERV-9 LTR enhancer is not blocked by the HS5 insulator and synthesizes through the HS5 site non-coding, long RNAs that regulate LTR enhancer function.

A solitary long terminal repeat (LTR) of ERV-9 human endogenous retrovirus is located upstream of the HS5 site in the human beta-globin locus control region and possesses unique enhancer activity in erythroid K562 cells. In cells transfected with plasmid LTR-HS5-epsilonp-GFP, the LTR enhancer activates the GFP reporter gene and is not blocked by the interposed HS5 site, which has been reported to have insulator function. The LTR enhancer initiates synthesis of long RNAs from the LTR promoter through the intervening HS5 site into the epsilon-globin promoter and the GFP gene. Synthesis of the sense, long LTR RNAs is correlated with high level synthesis of GFP mRNA from the epsilon-globin promoter. Mutations of the LTR promoter and/or the epsilon-globin promoter show that (i) the LTR enhancer can autonomously initiate synthesis of LTR RNAs independent of the promoters and (ii) the LTR RNAs are not processed into GFP mRNA or translated into GFP. However, reversing the orientation of the LTR in plasmid (LTR)rev-HS5-epsilonp-GFP, thus reversing the direction of synthesis of LTR RNAs in the antisense direction away from the epsilon-globin promoter and GFP gene drastically reduces the level of GFP mRNA and thus LTR enhancer function. The results suggest that the LTR-assembled transcription machinery in synthesizing non-coding, LTR RNAs can reach the downstream epsilon-globin promoter to activate transcription of the GFP gene.

Base Sequence↗

PubMed1992

Thin-rim enhancement in Gd-DTPA-enhanced magnetic resonance images of tuberculoma: a new finding of potential differential diagnostic importance.

The article describes rim enhancement seen on Gd-DTPA-enhanced magnetic resonance (MR) images of two patients with tuberculoma. Pathologic examination of the surgical specimens disclosed that the peripheral portion of the tuberculoma, which showed contrast enhancement, was composed mainly of a fibrous capsule and epithelioid granulomas and that the central zone, which showed no contrast enhancement, was composed of caseous necrotic material. In a retrospective study of MR images from 20 consecutive cases of surgically proven bronchogenic carcinoma, none showed a thin enhancing rim on postcontrast MR images. Although confirmation of these findings must await further experience, the postcontrast MR findings described here may prove to be of value in distinguishing tuberculoma and possibly other granulomatous lesions from bronchogenic carcinoma.

Adult↗

PubMed1990

Ovarian follicle cell enhancers from the Drosophila yolk protein genes: different segments of one enhancer have different cell-type specificities that interact to give normal expression.

This paper examines ovarian transcription of the divergently oriented yolk protein genes 1 and 2 (yp1 and yp2) of Drosophila melanogaster. We report germ line transformation results demonstrating that yp1 and yp2 are transcribed in the same subpopulations of ovarian follicle cells. Our results show that this expression pattern is directed by two enhancers: ovarian enhancer 1, located between the genes, and ovarian enhancer 2, located within the first exon of yp2. Analysis of the expression pattern resulting from alterations in ovarian enhancer 1 demonstrates that different segments of this enhancer have different positive or negative effects on the cell-type specificity of transcription.

Animals↗

PubMed1993

Pi, a pre-B-cell-specific enhancer element in the immunoglobulin heavy-chain enhancer.

We have identified a new immunoglobulin heavy-chain enhancer element, designated pi, between the microE2 and microE3 elements. The pi enhancer element is transcriptionally active primarily during early stages of B-cell development but becomes virtually inactive during B-cell maturation at about the stage of immunoglobulin kappa light-chain gene rearrangement. Mutational analysis suggests that the pi element is crucial for immunoglobulin heavy-chain enhancer activity at the pre-B-cell stage but is almost irrelevant for enhancer activity at the mature B-cell or plasma-cell stage. The activity of the pi enhancer element correlates with the presence of an apparently pre-B-cell-specific protein-DNA complex. The similarity of the pi site to recognition sequences for members of the ets gene family suggests that the protein(s) interacting with the pi site most likely are ets-related transcription factors.

3T3 Cells↗

PubMed2000

Phosphorylation of SOX9 by cyclic AMP-dependent protein kinase A enhances SOX9's ability to transactivate a Col2a1 chondrocyte-specific enhancer.

Sox9 is a high-mobility-group domain-containing transcription factor required for chondrocyte differentiation and cartilage formation. We used a yeast two-hybrid method based on Son of Sevenless (SOS) recruitment to screen a chondrocyte cDNA library and found that the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase A (PKA-Calpha) interacted specifically with SOX9. Next we found that two consensus PKA phosphorylation sites within SOX9 could be phosphorylated by PKA in vitro and that SOX9 could be phosphorylated by PKA-Calpha in vivo. In COS-7 cells cotransfected with PKA-Calpha and SOX9 expression plasmids, PKA enhanced the phosphorylation of wild-type SOX9 but did not affect phosphorylation of a SOX9 protein in which the two PKA phosphorylation sites (S(64) and S(211)) were mutated. Using a phosphospecific antibody that specifically recognized SOX9 phosphorylated at serine 211, one of the two PKA phosphorylation sites, we demonstrated that addition of cAMP to chondrocytes strongly increased the phosphorylation of endogenous Sox9. In addition, immunohistochemistry of mouse embryo hind legs showed that Sox9 phosphorylated at serine 211 was principally localized in the prehypertrophic zone of the growth plate, corresponding to the major site of expression of the parathyroid hormone-related peptide (PTHrP) receptor. Since cAMP has previously been shown to effectively increase the mRNA levels of Col2a1 and other specific markers of chondrocyte differentiation in culture, we then asked whether PKA phosphorylation could modulate the activity of SOX9. Addition of 8-bromo-cAMP to chondrocytes in culture increased the activity of a transiently transfected SOX9-dependent 48-bp Col2a1 chondrocyte-specific enhancer; similarly, cotransfection of PKA-Calpha increased the activity of this enhancer. Mutations of the two PKA phosphorylation consensus sites of SOX9 markedly decreased the PKA-Calpha activation of this enhancer by SOX9. PKA phosphorylation and the mutations in the consensus PKA phosphorylation sites of SOX9 did not alter its nuclear localization. In vitro phosphorylation of SOX9 by PKA resulted in more efficient DNA binding. We conclude that SOX9 is a target of cAMP signaling and that phosphorylation of SOX9 by PKA enhances its transcriptional and DNA-binding activity. Because PTHrP signaling is mediated by cAMP, our results support the hypothesis that Sox9 is a target of PTHrP signaling in the growth plate and that the increased activity of Sox9 might mediate the effect of PTHrP in maintaining the cells as nonhypertrophic chondrocytes.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed2005

Binding of hnRNP L to the pre-mRNA processing enhancer of the herpes simplex virus thymidine kinase gene enhances both polyadenylation and nucleocytoplasmic export of intronless mRNAs.

Liu and Mertz (Genes Dev. 9:1766-1780, 1995) previously identified a 119-nt pre-mRNA processing enhancer (PPE) element within the herpes simplex virus type 1 thymidine kinase gene that enables intron-independent gene expression in higher eukaryotes by binding heterogeneous nuclear ribonucleoprotein L (hnRNP L). Here, we identify a 49-nt subelement within this PPE that enhanced stability, polyadenylation, and cytoplasmic accumulation of transcripts synthesized in CV-1 cells from an intronless variant of the human beta-globin gene when present in two or more tandem copies. This 2xTK49 PPE also enhanced (i) the efficiency of polyadenylation of intronless beta-globin RNA in a cell-free polyadenylation system and (ii) the kinetics of nucleocytoplasmic export of an intronless variant of adenovirus major late leader region RNA in Xenopus oocytes. This 2xTK49 PPE bound only hnRNP L. Analysis of 2xTK49 PPE mutants showed a strong positive correlation existed between binding hnRNP L and enhancement of intronless beta-globin gene expression. hnRNP L was found to associate with both the mRNA export factor TAP and the exon-exon junction complex protein Aly/REF. Thus, we conclude that hnRNP L plays roles in enhancing stability, polyadenylation, and nucleocytoplasmic export; it does so, at least in part, by directly recruiting to intronless PPE-containing RNAs cofactors normally recruited to intron-containing RNAs.

Animals↗

PubMed1989

The Xenopus ribosomal DNA 60- and 81-base-pair repeats are position-dependent enhancers that function at the establishment of the preinitiation complex: analysis in vivo and in an enhancer-responsive in vitro system.

Although it is generally believed that the 60- and 81-base-pair (60/81-bp) repeats of the Xenopus laevis ribosomal DNA (rDNA) spacer are position-independent transcriptional enhancers, this has not been shown directly. We have now developed a critical assay which proves that the 60/81-bp repeats do, in fact, stimulate transcription from promoters in cis and that they function in both orientations and when up to 1 kilobase pair from the initiation site. However, contrary to the widely accepted view, these elements are found to be highly position dependent, for they have no net effect when downstream of the initiation site within the transcribed region and they behave as transcriptional silencers of promoters in cis when moved greater than 2 kilobase pairs upstream of the initiation site. The 60/81-bp elements therefore are position-dependent 5' enhancers. We also found that this rDNA enhancer was polymerase I specific and that it was composed of duplicated, individually functional elements. Finally, we report an in vitro system that reproduces both cis enhancement and trans competition by the 60/81-bp repeats. Sequential-addition studies in this system demonstrated that the rDNA enhancer functions in trans at or before establishment of the stable transcription complex, not subsequently at each round of transcription.

Animals↗

PubMed1993

Islet cell tumors: comparison of dynamic contrast-enhanced CT and MR imaging with dynamic gadolinium enhancement and fat suppression.

Ten patients with 11 islet cell tumors underwent dynamic contrast material-enhanced computed tomography (CT) and magnetic resonance (MR) imaging within a 1-month period. MR imaging depicted all 11 tumors, and CT depicted seven of the 11 tumors. CT did not depict four of seven tumors that measured 2.5 cm in diameter or less. Islet cell tumors had low signal intensity on T1-weighted fat-suppressed MR images, and gastrinomas were best shown with this technique. Two of three insulinomas less than 1.5 cm in diameter were best shown on dynamic contrast-enhanced fast low-angle shot (FLASH) images as uniform areas of high signal intensity. Hepatic metastases were seen in five patients and showed peripheral ringlike enhancement best demonstrated on dynamic gadolinium-enhanced FLASH images. Hepatic lesions were most conspicuous on T2-weighted fat-suppressed spin-echo images. MR imaging with dynamic gadolinium enhancement and fat suppression is a promising tool in the investigation of islet cell tumors.

Adenoma, Islet Cell↗