Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:APRD00631 (Gel)
 14,881 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The SV40 early (SV40E) transcriptional regulatory element (TRE) is able to direct heterologous gene expression in a variety of eukaryotic cell lines. This ability is conferred, in part, by the presence of several cis-elements. Transfection studies, mutational analyses, and in vitro DNA binding assays have demonstrated that the SV40E TRE is capable of interacting with several cellular transcription (trans) factors. In the present study, we have investigated the inability of the SV40E TRE to direct gene expression in cultured rat anterior pituitary GH-3 cells. Gel shift analysis demonstrated that nuclear factors within these cells can recognize and specifically bind to DNA containing SV40 enhancer sequences. Surprisingly, we have found that both HeLa and GH-3 cells possess relatively equal quantities of Sp1-specific RNA; however, a dramatic decrease in Sp1 protein was seen in GH-3 cells. Transfection studies utilizing CAT reporter plasmids revealed that the intact SV40E TRE is inactive in these cells, and that subsequent deletion of a region(s) where nuclear factor binding occurs does not result in detectable levels of gene expression. Thus, removal of cis-sites potentially involved in repressor binding does not result in activation of the SV40E TRE in these cells. Subcloning an SV40 enhancer fragment upstream of a heterologous TK promoter yielded chimeric TREs that could direct high levels of gene expression in HeLa but not GH-3 cells. Therefore, the prototypic SV40 enhancer, in the context of GH-3 cells, cannot enhance gene expression.
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PMID:The SV40 early transcriptional regulatory element is unable to direct gene expression in pituitary GH-3 cells. 132 82

The 5'-flanking DNA of the mouse RII beta subunit of the cAMP-dependent protein kinase gene was characterized by transient transfection of RII beta-CAT constructs into mouse neuroblastoma cells (NB2a) and Chinese hamster ovary (CHO) cells and by gel mobility shift and footprinting assays. The minimal promoter of the RII beta gene was composed of two adjacent functional elements. A 3'-element which supported enhanced CAT activity was located between base pairs (bp) -267/-168 from the translation initiation start site. CAT plasmids containing these RII beta sequences showed 12- and 16-fold increased CAT activity in the NB2a and CHO cells, respectively, compared to the basic CAT vector. Plasmids containing 20 additional bp 5' to the -267/-168 fragment showed 2-fold more CAT activity than the shorter fragment in NB2a cells, while CAT activity in CHO cells was nearly the same for both constructs. CAT plasmids containing only this 20-bp fragment showed 9- and 13-fold increased CAT activity in NB2a and CHO cells, respectively. The core promoter of the RII beta gene lacked classical TATA and CAT sequences, but contained 3 copies of the Sp1 core consensus sequence. Gel mobility shift assays using 32P-labeled 5'-flanking DNA containing bp -291/-49 and nuclear extracts from NB2a and CHO cells displayed several retarded bands in the gels suggesting complex formation with nuclear DNA-binding factors. Unlabeled DNA containing bp -291/-49 blocked the appearance of all retarded bands. Competition using an oligonucleotide corresponding to the Sp1 DNA-binding site effectively blocked the appearance of the two more slowly migrating bands but did not affect the major rapidly migrating bands. DNase I footprinting analysis using purified Sp1 protein confirmed that Sp1 could bind to the Sp1 sites. Methylation interference and mutational analysis showed that one of the faster migrating bands was the result of factor binding to the DNA sequence adjacent to the Sp1 sites. Additional tissue-specific nuclear-binding factor sequences were detected upstream of the core promoter. Our data suggest that the core promoter of the RII beta gene can initiate transcription from the DNA around the Sp1 sites but that there are tissue-specific nuclear factor-binding sites located distal to the Sp1 sites.
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PMID:Characterization of a minimal promoter element required for transcription of the mouse type II beta regulatory subunit (RII beta) of cAMP-dependent protein kinase. 133 64

To explore the role of transcriptional factors in the genesis of the senescent phenotype, nuclear extracts from 4- and 30-month-old rat brains were analyzed for the presence of DNA-binding proteins able to interact with double-stranded oligonucleotides containing recognition sites for sequence-specific DNA-binding factors. Gel shift assays revealed that the DNA-binding efficiency of Sp1 is significantly reduced in aged animals compared to young ones, whereas CTF/NF1 and AP1 from young and old rat nuclear extracts bind their DNA targets with the same efficiency. The quantitative analysis of Sp1 by immunoblotting indicated that equivalent quantities and degrees of heterogeneity of Sp1 protein are present in both nuclear extracts, suggesting that the observed difference is not due to a different expression of this transcriptional factor. DNase I footprinting of the heavy chain ferritin gene promoter, which contains a Sp1 binding site, demonstrated that the nuclear extract from 30-month-old rat brain does not protect the region involved in the regulation of the H ferritin gene by Sp1. This results in a reduction of about 50% of the expression of the H ferritin mRNA in aged rat brains. Furthermore, the Sp1 binding sites present in the SV40 early promoter are not protected in a DNase I footprinting assay where a nuclear extract from 30-month-old rat brain was used as a source of DNA binding proteins. Liver nuclear extracts prepared from young and aged rats demonstrated that a decrease of Sp1 binding efficiency is similarly present in this tissue.
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PMID:Sp1 DNA binding efficiency is highly reduced in nuclear extracts from aged rat tissues. 138 57

In order to locate the promoter region of the human alpha 2A adrenergic receptor gene we used RNase protection analysis and antisense RNA probes to map the cap site of the alpha 2 transcripts. Prior sequence analysis has shown two potential TATA box motifs in the human alpha 2A adrenergic receptor gene, TATATAT and TATAAAA, located 427 and 1037 base pairs (bp), respectively, upstream of the protein coding region. RNase protection experiments and primer extension show that transcription starts downstream of the distal TATAAAA, indicating that the 5'-untranslated region is approximately 1 kilobase in length. We have used the chloramphenicol acetyltransferase reporter gene and transient transfection into HT29, a human adenocarcinoma cell line that expresses the alpha 2A receptor, to show that as little as 150 bp upstream of the cap site can direct transcription. Sequence analysis shows that although this region contains the TATA box motif it lacks a CCAAT box motif. DNase I footprint analysis of a fragment from -17 to -193 (where +1 is the transcription initiation site), using nuclear extracts from HT29, showed hypersensitive sites (-68/-69) and two protected regions: -70 to -87, which includes a 10-bp palindrome, and -92 to -105, which includes a GC box, a common motif for Sp1 nuclear factor binding. Gel mobility shift assays indicate that Sp1 or a related factor may bind to this GC box. Deletion of the GC box and the palindrome from chloramphenicol acetyltransferase constructs abolishes transcription. We propose that these cis sequences may function in lieu of a CCAAT box to regulate transcription of the human alpha 2A adrenergic receptor gene.
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PMID:Promoter region of the human alpha 2A adrenergic receptor gene. 138 31

Expression of the immediate early 1 and 2 gene (IE-1/2) of human cytomegalovirus, an important pathogen in immunosuppressed patients, is controlled by a strong enhancer/promoter. To define the promoter domain within this large cis-active region of about 550 nucleotides, DNA-protein interactions were studied. DNase I footprinting experiments using procaryotically expressed transcription factor Sp1 revealed an extensive interaction of this transcription factor with both consensus and aberrant recognition elements within the IE-1/2 promoter region. Protection of these Sp1 binding sites could also be observed when nuclear extracts prepared from HeLa cells and permissive human fibroblast cells were used. After in vitro mutagenesis of Sp1 targets and transient expression of mutagenized CAT-expression plasmids, however, no significant reduction in CAT activities was found. By analyzing a series of 5' deletion mutants of the IE-1/2 promoter region, a strong cis-acting element was localized between nucleotides -94 and -78, upstream of sites that interact with Sp1. Gel retardation experiments demonstrated binding of recombinant transcription factor CREB to this motif which reveals it as an aberrant CREB recognition sequence. Thus, this study identifies several previously unknown binding sites for transcription factors Sp1 and CREB within the proximal promoter region of the IE-1/2 gene, which differ markedly in their relevance for constitutive promoter function.
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PMID:Analysis of proteins binding to the proximal promoter region of the human cytomegalovirus IE-1/2 enhancer/promoter reveals both consensus and aberrant recognition sequences for transcription factors Sp1 and CREB. 138 62

The human neurofilament (H) promoter contains multiple binding sites for nuclear proteins including a Proximal (Prox) site centered around the sequence GGTTGGACC and an adjacent pyrimidine (Pyr) tract site centered around the sequence CCCTCCTCCCC. Surprisingly binding to a probe containing the Prox/Pyr region of the NF(H) promoter was competed in gel shifts by an oligonucleotide containing only an Sp1 binding site (GGGGCGGGG). Supershift assays with a polyclonal anti-Sp1 antisera confirmed that Sp1 was part of the complex formed with the Prox/Pyr probe. However neither bacterially expressed Sp1 516C or vaccinia virus expressed full-length Sp1 778C bound to the Prox or Pyr sequences in DNase I footprints or gel shift assays. Gel shift competitions and supershift assays with probes containing either Prox or Pyr tract sites alone demonstrated targeting of Sp1 to the Prox binding site and identified a non-Sp1 containing complex which contains a Prox binding protein. Adding exogenous Sp1 to a HeLa nuclear extract enhanced the Sp1-containing complex but had no effect on the Prox complex. These studies show that Sp1 can be targeted to a non-Sp1 site in the human NF(H) promoter through protein/protein interactions with a distinct sequence specific DNA-binding protein.
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PMID:Targeting of Sp1 to a non-Sp1 site in the human neurofilament (H) promoter via an intermediary DNA-binding protein. 147 89

The expression of type I collagen is regulated developmentally and tissue specifically. Two sets of binding sites for nuclear factor I (NF-I) and Sp1 transcription factors arrayed as an imperfect tandem repeat are critical for high activity of the murine alpha 1(I) collagen gene in NIH-3T3 fibroblasts and are conserved in evolution. Gel retardation analysis combined with methylation interference studies show that NF-I and Sp1 bind to overlapping sites in a mutually exclusive manner. Cotransfection studies using Drosophila Schneider L2 cells, which lack both transcription factors, demonstrate that each factor alone trans-activates the gene, while cotransfection of both factors results in the inhibition of the strong Sp1 trans-activation. In contrast, the herpes simplex virus thymidine kinase promoter, which contains functionally independent NF-I and Sp1 binding sites, is maximally transactivated by the cotransfection of both factors. Because the two NF-I/Sp1 binding sites overlap, the ratio of the activities of the two factors rather than their absolute concentrations determine alpha 1(I) gene expression, characterizing these promoter sequences as transcription factor switch elements.
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PMID:NF-I/Sp1 switch elements regulate collagen alpha 1(I) gene expression. 152 78

Transcription of the delta 1-crystallin gene is developmentally regulated in the embryonic chicken lens. Previous work defined a positive transcription regulatory element between positions -120 and -43 of the delta 1-crystallin promoter. This region contains a putative Sp1 binding site (-78 to -71), adjacent to a CAAT box (-70 to -67). Gel retardation assays using lens nuclear extracts revealed two protein-DNA complexes which involved the Sp1 site. The formation of the complexes increased from day 6 to day 11 of embryogenesis (period of lens organogenesis) peaked between days 11 and 15, then decreased in a non-parallel manner until hatching (day 21). A point mutation in the Sp1 binding site of the delta 1-crystallin promoter abolished formation of one of the complexes (complex 1, slower in mobility), while point mutations in the CAAT box had no effect on the formation of either complex. Studies using purified Sp1 protein and increasing amounts of embryonic chicken lens nuclear extracts showed cooperativity in the formation of both complexes, more remarkable with complex 1.
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PMID:DNA binding factors which interact with the Sp1 site of the chicken delta 1-crystallin promoter are developmentally regulated. 155 May 73

We cloned, sequenced and characterized a promoter region of the mouse homologue of the Alzheimer's disease amyloid precursor protein (APP)-encoding gene. The promoter region is highly homologous to that of the human APP (hAPP) gene. It has a high G+C content, lacks typical 'TATA' and 'CAAT' boxes, and contains possible binding sites for AP-1, heat-shock factor, Sp1 and AP-4. The promoter region was fused with the cat reporter gene, and the fusion genes were transfected to both the NB41A3 (mouse neuroblastoma) and L-cell lines. The promoter activity was monitored by chloramphenicol acetyltransferase (CAT) activity in a transient expression assay. The promoter was equally active in both cell lines. The deletion analysis revealed that there existed a negative regulatory element(s) between -153 and -100 bp and a positive element(s) between -100 and -37 bp. The negative element was shown to suppress the transcriptional activity of heterologous simian virus 40 promoter. DNase I footprinting experiments revealed that three nuclear protein-binding sites existed in the regulatory region, one in the negative and two in the positive regulatory regions. Gel retardation assay showed that Sp1 was one of the factors binding to the positive regulatory region. A nuclear factor binding to the negative regulatory region seemed to be missing in brain.
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PMID:Positive and negative regulatory elements for the expression of the Alzheimer's disease amyloid precursor-encoding gene in mouse. 155 68

Phorbol 12-myristate 13-acetate induces a 3- and 10-fold induction of chloramphenicol acetyltransferase (CAT) activity in HT1080 and HeLa cells, respectively, following transient transfection of a 336-base pair plasminogen activator inhibitor-1 (PAI-1) promoter fragment linked to a CAT reporter gene. Substitution mutations in the regions encompassing nucleotides -78 to -69 (TGGGTGGGGC) or -61 to -54 (TGAGTTCA), but not in the regions -155 to -149 (TGCCTCA) or -84 to -76 (AGTGAGTGG) reduced this induction. Gel electrophoresis of double-stranded -65 to -50 oligonucleotides of the PAI-1 promoter region and nuclear extracts from Hela cells produced a gel shift pattern similar to that obtained with a AP-1 consensus oligomer, and excess unlabeled AP-1 oligomer reverted binding, suggesting that this region of the PAI-1 promoter is an AP-1-like binding site. Gel electrophoresis of double-stranded -82 to -65 oligonucleotides with HeLa nuclear extracts revealed a gel shift pattern of three bands; Sp1 consensus oligomer competed with the binding to two of these bands and AP-2 consensus sequence oligomer with the binding to the third band. The -82 to -65 oligomer also bound to purified AP-2 and Sp1 proteins. Southwestern blotting of HeLa nuclear extracts revealed that the labeled oligomer spanning region -82 to -65 bound to proteins with molecular masses of 52 and 72 kDa. Consensus AP-2 oligonucleotides competed for binding of the labeled -82 to -65 oligonucleotide to the 52-kDa protein, but consensus Sp-1 oligonucleotides did not compete for binding to the 72-kDa compound. The 72-kDa component binding to the -82 to -65 region may represent a new protein involved in transcriptional regulation.
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PMID:Interaction of AP-1-, AP-2-, and Sp1-like proteins with two distinct sites in the upstream regulatory region of the plasminogen activator inhibitor-1 gene mediates the phorbol 12-myristate 13-acetate response. 163 45


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