Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027819 (neuroblastoma)
 27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

It has been shown that the untranscribed regulatory region of polyoma virus (Py) is hypersensitive (Hs) to DNase I treatment, and that this hypersensitivity is located in two areas which correspond to the A and B domains of the enhancer. We mapped the DNase I hypersensitive sites in the Py regulatory region of wild-type (PyA2) and of mutants, selected in neuroblastoma cells (PyNB), which are characterized by an extensive duplication involving the A domain, with or without deletion of the B domain. The experiments were performed in both a permissive host (3T6 mouse fibroblasts) and in a restrictive host (41A3 mouse neuroblasts). No significant differences were observed between the two hosts. Our results show that four sites, in addition to the ones already described, can be identified in the wild-type A2 strain. These newly identified sites coincide with the domains of the enhancer region as they have recently been established. In PyNB mutants duplications and deletions are generally correlated to the gain or loss of the corresponding hypersensitive sites. However, a new site is formed in one of the duplicated sequences, even if no corresponding hypersensitive site is present in the other identical sequence. A region protected from DNase I digestion occurs in the PyNB mutants which corresponds to the junction of the duplication which is absent in the wild-type strain. In this region, as a consequence of the rearrangement, a GGCGGG motif which is very similar to the one (GGGCGG) present at the binding sites of the cellular regulatory protein SP1, is found.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Sites hypersensitive to, and protected from, nuclease digestion in the regulatory region of wild-type and mutant polyoma chromatin. 303 Jul 31

Expression of the human galanin gene was analysed using a 3.5-kb DNA fragment comprising the 5'-flanking sequence of the gene. This sequence contains a TATA box (ATATATA) preceded by numerous potential binding sites for transcription factors such as SP1, AP2, and NF kappa B. Three half-palindromic estrogen response elements (EREs, GGTCA) are also found at positions -1,162, -361, and -122 bp relative to the transcription start site. To localize functionally important portions of the promoter region, several shorter fragments of the galanin 5'-flanking region were placed upstream from the chloramphenicol acetyltransferase (CAT) reporter gene. In transient transfection assays, all constructs demonstrated substantial transcriptional activity in both rat glioma/mouse neuroblastoma hybrid cells (NG108-15) and Chinese hamster ovary (CHO-K1) cells. Comparison of the basal expression levels of the different constructs suggests the presence of a negative modulator between positions -1,891 and -207. When cotransfected into NG108-15 cells with the human estrogen receptor cDNA, estrogen did not induce transcription of the human galanin gene at physiological levels of estrogen receptor, although transcription was induced up to 30-fold in the presence of high levels of receptor.
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PMID:Characterization of the 5'-flanking region of the human preprogalanin gene. 753 7

The combined factors that regulate the expression of cell adhesion molecules (CAMs) during development of the nervous system are largely unknown. To identify such factors for Ng-CAM, the neuron-glia CAM, constructs containing portions of the 5' end of the Ng-CAM gene were examined for activity after transfection into N2A neuroblastoma and NIH3T3 cells. Positive regulatory elements active in both cell types included an Ng-CAM proximal promoter with SP1 and cAMP response element motifs extending 447 base pairs upstream of a single RNA start site and a region within the first exon corresponding to 5'-untranslated sequences. Negative regulatory elements included five neuron-restrictive silencer elements (NRSEs) and a binding site for Pax gene products in a 305-base pair segment of the first intron. Constructs containing the promoter together with the entire first intron were active in N2A cells but were silenced in NIH3T3 cells. This silencer activity was mapped to the NRSEs. In contrast, the Pax motif inhibited activity of Ng-CAM constructs in both cell types. The DNA elements defined in these transfection experiments were examined for their ability to bind nuclear factors. The region within the first exon formed a DNA-protein complex after exposure to nuclear extracts prepared from both NIH3T3 and N2A cells. The NRSE region formed a more prominent complex with proteins prepared from NIH3T3 cells than it did with extracts from N2A cells. A member of the Pax protein family, Pax-3 bound to the Pax motif. Mutations introduced within the Pax motif in its ATTA sequence eliminated this binding whereas mutations in its GTTCC sequence did not, suggesting that paired homeodomain interactions are important for the recognition of Pax-3 by this DNA target sequence. The combined data suggest that negative regulation by NRSEs and Pax proteins may play a key role in the place-dependent expression patterns of Ng-CAM during development.
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PMID:Silencer elements modulate the expression of the gene for the neuron-glia cell adhesion molecule, Ng-CAM. 754 67

In this work, we have studied the activity of a tetracycline modulatable trans-activator (tTA) generated by fusing the DNA binding domain of the tetracycline repressor to the trans-activation domain of the Herpes simplex virus protein 16 (HSV VP16) (plasmid pUHD15-1Neo). In the three different cell lines studied (HTC, rat hepatoma; T47D, human breast cancer; SK-N-BE, human neuroblastoma), the expression of the luciferase gene under the control of a tetracycline operator sequence (plasmid pUHC13-3) was used as a control of the incorporation and the functionality of the trans-activator. Clones selected from these cells responded in a time and dose-dependent manner to the withdrawal of tetracycline. In all these clones, the tTA trans-activator not only modulates the activity of the luciferase gene, but also modulates the activity of a number of endogenous proteins, including C/EBP beta, the glucocorticoid receptor (GR), and SP1. In the transfected cells, the level of these transcription factors was strongly inhibited in the presence of tetracycline and was highly increased after tetracycline removal. Electrophoresis mobility shift assay (EMSA) and footprint experiments proved that the induced proteins are perfectly efficient in binding the DNA. Their transcriptional activity was also determined. In HTC/A9 cells, the level of the chloramphenicol acetyltransferase (CAT) expression driven by the promoter of the alpha 1-glycoprotein (AGP) gene was strongly enhanced at 72-84 hr following removal of tetracycline from the growth media. The accumulation of the endogenous AGP mRNA also increased at 84 hr. In the T47D/TA11 and SK-N-BE/C2.6 cells, a general activation of protein synthesis was also evidenced.
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PMID:Specificity of action of a herpes virus VP16/tetracycline-dependent trans-activator in mammalian cell cultures. 764 13

We have isolated and characterized the 5'-flanking region and the proximal polyadenylation site of the human 5-HT transporter gene. The major gene transcript is 2,793 bp in length and it contains 208 bp of 5'-untranslated region (5'-UTR) and 694 bases of 3'-UTR. While only a single mRNA species occurs in rats and mice, the most proximal signal for polyadenylation in the human gene appears to be highly degenerate in comparison to the rat and murine motif. This polyadenylation signal-like motif may lead to alternate usage of additional polyadenylation sites resulting in multiple mRNA species in humans. A TATA-like motif and several potential binding sites for transcription factors including AP1, AP2, SP1, and a cAMP response element (CRE)-like motif are present in the 5'-flanking region. A approximately 1.7 kb fragment beginning 217 bp downstream from the transcription start site, which had been ligated into a luciferase reporter vector and transiently expressed in JAR human placental choriocarcinoma cells, displayed both constitutive and forskolin/cholera toxin-induced promoter activity. Functional promoter mapping revealed that there are negative attenuating elements between bp -1,428 and -1,185 and positive elements between bp -1,184 and -78 from the transcription initiation site. Studies with deletional mutants also indicated that core promoter sequences are contained within 78 bp of the transcription start site and that regulation of cAMP-inducible promoter activity depends on multiple cis-acting elements including two AP1 binding sites and a single CRE-like element located at bp -99. Our findings suggest that (1) the 5-HT transporter gene promoter is active in human JAR cells, but inactive in 5-HT transporter-deficient human SK-N-SH neuroblastoma and HeLa cells, (2) the information contained within 1.4 kb of 5'-flanking sequence is sufficient to confer its cell-specific expression, (3) the promoter responds to cAMP induction, and (4) the expression of the 5-HT transporter gene is regulated by a combination of positive and negative cis-acting elements operating through a basal promoter unit defined by a TATA-like motif.
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PMID:Functional promoter and polyadenylation site mapping of the human serotonin (5-HT) transporter gene. 878 73

A 5.4 kilobase-pair segment of DNA flanking the 5' end of Hel-N1 was isolated and characterized. Primer extension studies with normal human brain and neuroblastoma cells revealed a major and minor transcription-initiation site. Sequence analysis of the initial 536 bp upstream to the major start site revealed a core promoter (-1 to -181) which contained two CCAAT boxes, a weakly-conserved TATA box, and an SP1 site. This region was also moderately GC-rich (62%). Using a transient luciferase-reporter-gene assay, the core promoter was found to be essential for basal transcription both in neural (PC12) and non-neural (HeLa and glial) cell types. Two positive regulatory elements, however, were identified in the initial 536 bp (-1 to -181 and -182 to -350) which produced a five- to six-fold increase in transcriptional activity in PC12 cells vs. HeLa or glial cells. These elements, therefore, were sufficient to confer cell-specific enhanced transcription and likely contribute to the neuronal specificity of Hel-N1 mRNA expression.
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PMID:Cloning the 5' flanking region of neuron-specific Hel-N1: evidence for positive regulatory elements governing cell-specific transcription. 881 91

The human cytoplasmic serine hydroxymethyltransferase (CSHMT) gene was isolated, sequenced and its expression characterized in human MCF-7 mammary carcinoma and SH_5Y5Y neuroblastoma cells. The 23-kb gene contains 12 introns and 13 exons; all splice junctions conform to the gt/ag rule. The open reading frame is interrupted by 10 introns, two of which are positionally conserved within the human mitochondrial SHMT gene. The gene is expressed with 330 nucleotides of 5' untranslated message within three exons. The 5' promoter region does not contain a consensus TATA, and primer extension and 5'-RACE studies suggest that transcription initiation occurs at multiple sites. Consensus motifs for several regulatory proteins, including SP1, mammary and neuronal-specific elements, NF1, a Y-box, and two steroid hormone response elements, are present within the first 408 nucleotides of the 5' promoter region. The human gene is expressed as multiple splice variants in both the 5' untranslated region and within the open reading frame, all due to exon excision. The splicing pattern is cell-specific. At least six CSHMT mRNA splice forms are present in MCF-7 cells; the gene is expressed as a full-length message as well as splice forms that lack exon(s) 2, 9 and 10. In 5Y cells, the predominant form of the message lacks exon 2, which encodes part of the 5' untranslated region, but does not contain deletions within the open reading frame. Western analysis suggests that the CSHMT gene is expressed as a single full-length protein in 5Y cells, but as multiple forms in MCF-7 cells. Multiple tissue Northern blots suggest that the CSHMT message levels and alternative splicing patterns display tissue-specific variations.
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PMID:Molecular cloning, characterization and alternative splicing of the human cytoplasmic serine hydroxymethyltransferase gene. 957 90

Eukaryotic transcriptional regulation in different cells involves large numbers and arrangements of cis and trans elements. To survey the number of cis regulatory elements that are active in different contexts, we have devised a high-throughput selection procedure permitting synthesis of active cis motifs that enhance the activity of a minimal promoter. This synthetic promoter construction method (SPCM) was used to identify >100 DNA sequences that showed increased promoter activity in the neuroblastoma cell line Neuro2A. After determining DNA sequences of selected synthetic promoters, database searches for known elements revealed a predominance of eight motifs: AP2, CEBP, GRE, Ebox, ETS, CREB, AP1, and SP1/MAZ. The most active of the selected synthetic promoters contain composites of a number of these motifs. Assays of DNA binding and promoter activity of three exemplary motifs (ETS, CREB, and SP1/MAZ) were used to prove the effectiveness of SPCM in uncovering active sequences. Up to 10% of 133 selected active sequences had no match in currently available databases, raising the possibility that new motifs and transcriptional regulatory proteins to which they bind may be revealed by SPCM. The method may find uses in constructing databases of active cis motifs, in diagnostics, and in gene therapy.
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PMID:Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. 1072 47

UDP-galactose:ceramide galactosyltransferase (CGT, EC 2.4.1.45) is a key enzyme in the biosynthesis of galactocerebroside, the most abundant glycosphingolipid in the myelin sheath. An 8 kb fragment upstream from the transcription initiation site of CGT gene was isolated from a human genomic DNA library. Primer extension analysis revealed a single transcription initiation site 329 bp upstream from the ATG start codon. Neither a consensus TATA nor a CCAAT box was identified in the proximity to the transcription start site; however, this region contains a high GC content and multiple putative regulatory elements. To investigate the transcriptional regulation of CGT, a series of 5' deletion constructs of the 5'-flanking region were generated and cloned upstream from the luciferase reporter gene. By comparing promoter activity in the human oligodendroglioma (HOG) and human neuroblastoma (LAN-5) cell lines, we found that the CGT promoter functions in a cell type-specific manner. Three positive cis-acting regulatory regions were identified, including a proximal region at -292/-256 which contains the potential binding sites for known transcription factors (TFs) such as Ets and SP1 (GC box), a distal region at -747/-688 comprising a number of binding sites such as the ERE half-site, NF1-like, TGGCA-BP, and CRE, and a third positive cis-acting region distally localized at -1325/-1083 consisting of binding sites for TFs such as nitrogen regulatory, TCF-1, TGGCA-BP, NF-IL6, CF1, bHLH, NF1-like, GATA, and gamma-IRE. A negative cis-acting domain localized in a far distal region at -1594/-1326 was also identified. Our results suggest the presence of both positive and negative cis-regulatory regions essential for the cell-specific expression in the TATA-less promoter of the human CGT gene.
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PMID:Characterization of the human UDP-galactose:ceramide galactosyltransferase gene promoter. 1134 20

A comparison between retinoic acid (RA) differentiation-resistant and differentiation-sensitive SK-N-BE neuroblastoma (NB) cell lines revealed an association between resistance to differentiation, exhibited by N-myc stable transfected SK-N-BE 9N cells, with sensitivity to RA induction of p50/p65 nuclear factor kappaB (NF-kappaB) transcription factor activity and induction of matrix metalloproteinase (MMP)-9 expression leading to enhanced invasive behavior in vitro. These effects were not observed in differentiation-sensitive parental SK-N-BE or control-transfected SK-N-BE 2N counterparts. RA activated a MMP-9 promoter reporter gene construct in SK-N-BE 9N but not parental SK-N-BE or SK-N-BE 2N cells through a NF-kappaB element (-600) in association with enhanced p50 mRNA expression, reduced cytoplasmic inhibitor of nuclear factor kappaBalpha protein levels, and the induction of nuclear p50/p65 containing MMP-9 NF-kappaB site binding activity. RA activation of the MMP-9 promoter was inhibited by transient overexpression of a dominant-negative inhibitor of nuclear factor kappaBalpha protein and stimulated by transient p50 but not p65 overexpression in the absence of RA. A limited, nonessential function for activator protein 1 (-74), Ets (-540), and SP1 (-560) elements within the MMP-9 promoter was revealed by point mutation but was not associated with changes in the binding or position of complexes constitutive to differentiation-sensitive or -resistant cells. Our data indicates that in this model of NB resistance to differentiation that results from uncoupled RA regulation of N-myc expression, RA stimulates malignant NB cell behavior by inducing nuclear NF-kappaB transcription factor activity, which in turn induces MMP-9 expression and stimulation of basement membrane invasive capacity involving MMP-9 activity.
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PMID:All-trans-retinoic acid induces nuclear factor kappaB activation and matrix metalloproteinase-9 expression and enhances basement membrane invasivity of differentiation-resistant human SK-N-BE 9N neuroblastoma Cells. 1219 73


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