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 purpose of this investigation was to identify and characterize the regulatory elements involved in the transcriptional activation of the beta gamma (leaky-late or gamma 1) genes of herpes simplex virus type 1 (HSV-1) by using the major capsid protein (VP5 or ICP5) gene as model. Gel mobility shift assays with nuclear extracts from uninfected and infected HeLa cells enabled us to identify two major protein-DNA complexes involving the VP5 promoter. The mobilities of these two complexes remained unaltered, and no unique complexes were observed when infected cell nuclear extracts were used. DNase I and orthophenanthroline-Cu+ footprint analyses revealed that the two complexes involve a single binding site, GGCCATCTTGAA, located between -64 and -75 bp relative to the VP5 cap site. To determine the function of this leaky-late binding site (LBS) in VP5 gene activation, we tested the effect of mutations in this region by using transient expression of a cis-linked chloramphenicol acetyltransferase gene. Deletion of the above sequence resulted in a seven- to eightfold reduction in the level of transactivation of the chloramphenicol acetyltransferase gene by superinfection with HSV-1 or by cotransfection of HSV-1 immediate-early genes. From these results, we conclude that the LBS sequence and a cellular factor(s) are involved in the transactivation of the VP5 gene. A search of published gene sequences revealed that sequences related to the LBS exist in a number of other HSV-1, cytomegalovirus, retrovirus, and cellular promoters. Sequence homologies of binding sites and results of unpublished competition binding studies suggest that this leaky-late binding factor may be related to, or the same as, a ubiquitous cellular transcriptional factor called YY1 or common factor-1 (also known as NF-E1, delta, and UCRBP).
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PMID:Transactivation of the major capsid protein gene of herpes simplex virus type 1 requires a cellular transcription factor. 131 6

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

A unique characteristics of thyrotrope-specific gene expression is the coordinated expression and regulation of the alpha- and beta-subunits of TSH. A cell line (alpha TSH) derived from the transplantable mouse thyrotropic tumor MGH101A, which no longer expresses the TSH beta-subunit gene but continues to secrete large amounts of alpha-subunit, was used as a model to study alpha-subunit gene expression independent from the TSH beta-subunit gene and was compared with the expression in TSH-secreting TtT97 tumors. Transient transfection studies showed a striking similarity in the activity of 5' deletions of the mouse alpha-subunit gene promoter in both alpha TSH and TtT97 cells and localized two regions important for expression that spanned 100 base pairs, from -480 to -417 and from -417 to -381. These regions were found to have no activity in nonthyrotrope pituitary GH4 cells and L-cell fibroblasts. Analysis of the alpha-subunit 5' flanking DNA interactions with alpha TSH and TtT97 nuclear extracts showed two DNase I protected sequences, from -474 to -452 and from -447 to -400, both of which colocalized with the functionally important regions. Gel retardation analysis demonstrated the specificity of these interactions, and a similar migration of the DNA-protein complexes suggested that protein factors were similar in the two cell types. We conclude that the nuclear factors necessary for alpha-subunit expression in thyrotropes are retained in alpha TSH cells. Moreover, since alpha TSH cells do not express the TSH beta-subunit gene, the factors that determine the expression of the alpha-subunit may not be sufficient for TSH beta-subunit gene expression.
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PMID:A cell line that produces the glycoprotein hormone alpha-subunit contains specific nuclear factors similar to those present in thyrotropes. 138 77

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 CyI actin gene of the sea urchin, Strongylocentrotus purpuratus, is regulated temporally and spatially within the cells of the early embryo. In an effort to understand the molecular basis for the CyI actin pattern of expression, we have begun analyzing the protein-DNA interactions within regions previously shown to be of potential functional importance (Katula et al., 1987). Using DNase I footprinting, 10 protected regions were identified containing both conserved and apparently novel protein binding sites. Gel mobility shift competition assays confirmed the presence of multiple protein factors which specifically recognize CyI actin upstream sequences. Determination of a relative affinity constant value (Kr) indicated that most of the protein factors preferred their respective oligonucleotide sequences vs. a synthetic competitor DNA in a range of 10(4). The highest affinity binding was observed for proteins binding to the oligonucleotide probe containing the octamer element (Kr approximately 10(6)). Heterologous gel shift competition assays were carried out to investigate the interrelatedness of the protein factors. These studies, combined with other data, indicate there are both unique and redundant protein-DNA interactions in the region being examined. Possible alterations in CyI actin DNA binding proteins were investigated during the period of CyI transcriptional activation by gel mobility shift analysis. An increase in binding activity was observed for most of the factors, indicating that early transcriptional activity of CyI actin may involve a general increase in the amount or activity of specific transcription factors. In addition, qualitative changes, as seen by alterations in the shift patterns, were observed for some of the oligonucleotide probes.
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PMID:Analysis of the DNA binding proteins interacting with specific upstream sequences of the S. purpuratus CyI actin gene. 147 71

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 nitrate reductase operon (narGHJI) of Escherichia coli encodes an anaerobic respiratory enzyme. Previous work has identified two cis-acting sites in the nar operon control region: a proximal site required for anaerobic induction mediated by the activator Fnr and a remote upstream site required for nitrate induction mediated by the activator NarL [Li, S. & DeMoss, J. A. (1988) J. Biol. Chem. 263, 13700-13705]. Our search for nar regulatory mutants yielded one strain with a mutation in himD, the structural gene for one of the subunits of integration host factor (IHF). Strains carrying null alleles of the IHF structural genes, himD and himA, had severe defects in nitrate induction of the nar operon but were normal for nitrate induction of the coordinately regulated fdn operon. Anaerobic expression of both operons was normal in him mutants. Gel-mobility-shift and DNase I protection experiments revealed a single IHF binding site in the nar operon control region, located midway between the upstream activation site and the promoter. We conclude that an IHF-mediated DNA bend is essential for efficient nitrate induction of the sigma 70-dependent nar operon promoter. This requirement of IHF for transcriptional activation had been noted for several sigma 54-dependent promoters.
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PMID:In vivo requirement of integration host factor for nar (nitrate reductase) operon expression in Escherichia coli K-12. 152 82

Initiation of adenovirus DNA synthesis is preceded by the assembly of a nucleoprotein complex at the origin of DNA replication containing three viral proteins, preterminal protein, DNA polymerase and DNA binding protein, and two cellular proteins, nuclear factors I and III. While sequence specific interactions of the cellular proteins with their cognate sites in the origin of DNA replication are well characterized, the question of how the viral replication proteins recognize the origin has remained unanswered. Preterminal protein and DNA polymerase were therefore purified to homogeneity from recombinant baculovirus infected insect cells. Gel filtration demonstrated that while DNA polymerase existed in monomeric and dimeric forms, preterminal protein was predominantly monomeric and when combined the proteins formed a stable heterodimer. In a gel electrophoresis DNA binding assay each of the protein species recognized DNA within the origin of DNA replication with unique specificity. Competition analysis and DNase I protection experiments revealed that although each protein could recognize the origin, the heterodimer did so with enhanced specificity, protecting bases 8-17 from cleavage with the nuclease. Thus the highly conserved 'core' of the origin of DNA replication, present in all human adenoviruses, is recognized by the preterminal protein--DNA polymerase heterodimer.
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PMID:Recognition of the adenovirus type 2 origin of DNA replication by the virally encoded DNA polymerase and preterminal proteins. 153 47


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