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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have analyzed P-element-transformed lines carrying hsp26/lacZ transgenes with various deletions and substitutions within the Drosophila melanogaster hsp26 promoter region in order to identify the sequences required for the formation of the DNase I hypersensitive sites (DH sites). DH sites are generally found associated with promoters and enhancer elements of active and inducible eukaryotic genes, and are thought to be nucleosome-free regions of DNA that interact with regulatory proteins and the transcriptional machinery. There are two major DH sites located within the promoter region of the hsp26 gene, centered at -50 and at -350 (relative to the hsp26 transcription start site). The sequences from -135 to -85, which contain (CT)n.(GA)n repeats, contribute significantly to the formation of the DH sites in the hsp26 promoter region. Deletion or substitution of this (CT)n region drastically reduces the accessibility of the DNA at these sites to DNase I. This reduction in accessibility was quantified by measuring the susceptibility of the DNA within nuclei to cleavage at a restriction site within the DH site. In addition to the (CT)n region and the promoter at -85 to +11 (region P), one of two other regions must be present for effective creation of the DH sites: sequences between -351 and -135 (region A), or sequences between +11 and +632 (region D). Disruption of the wild-type chromatin structure, as assayed by the loss of accessibility to the DH sites, is correlated with a decrease in inducible transcriptional activity, even when the TATA box and heat shock regulatory elements are present in their normal positions.
J Mol Biol 1992 Jun 20
PMID:Promoter sequence containing (CT)n.(GA)n repeats is critical for the formation of the DNase I hypersensitive sites in the Drosophila hsp26 gene. 137 79

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.
Mol Cell Biol 1992 Aug
PMID:Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene. 137 30

Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor alpha (TGF-alpha). TGF-alpha mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha promoter complex was detected in response to 5-azaC treatment. This 5-azaC-induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-alpha promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5-azaC-induced complex produced a footprint on the TGF-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha expression by augmenting the Sp1 activity. However, neither the Sp1 mRNA nor its protein was induced by 5-azaC. These results suggest that in HA-A cells, TGF-alpha expression is down-modulated by DNA methylation. In addition, this process may involve the specific regulation of Sp1 activity without altering the amount of the transcription factor.
Mol Cell Biol 1992 Sep
PMID:5-Azacytidine treatment of HA-A melanoma cells induces Sp1 activity and concomitant transforming growth factor alpha expression. 138 Jun 48

Nucleotide excision repair in Escherichia coli is initiated by the UvrA, UvrB and UvrC proteins. UvrA is the damage recognition subunit, makes an A2B1 complex with the targeting subunit UvrB, and the complex binds to the lesion site; UvrA dissociates leaving behind a very stable UvrB-DNA complex that is recognized by the trigger subunit, UvrC, and the ensuing UvrB-UvrC heterodimer makes two incisions, one on either side of the lesion. Using electron microscopy, we investigated the structures of these early A, A-B intermediates on DNA containing ultraviolet light photoproducts. UvrA, which is known to bind to DNA as a dimer and produce a DNase I footprint of 33 base-pairs does not change the trajectory of DNA appreciably. The A2B1 complex clearly shows a bipartite structure and its effect on the trajectory of the DNA was not consistently straight or kinked. In contrast, the DNA in the preincision UvrB-DNA complex appears to be severely kinked; 43% of the molecules are bent by 80 degrees or more, with an average bending angle of 127 degrees. It appears that protein-induced bending is an important step on the pathway leading to excision of the damaged nucleotide by (A)BC excinuclease.
J Mol Biol 1992 Jul 20
PMID:Electron microscopic study of (A)BC excinuclease. DNA is sharply bent in the UvrB-DNA complex. 138 87

Using gel retardation and DNase I protection techniques, we have demonstrated that the Escherichia coli integration host factor (IHF) stabilizes the interaction between Mu repressor and its cognate operator-binding sites in vitro. These results are discussed in terms of a model in which IHF may commit the phage to the lytic or lysogenic pathway depending on the occupancy of the operator sites by the repressor.
Mol Microbiol 1992 Jun
PMID:Escherichia coli integration host factor stabilizes bacteriophage Mu repressor interactions with operator DNA in vitro. 138 45

When preadipocytes differentiate into adipocytes, several differentiation-linked genes are activated. Lipoprotein lipase (LPL) is one of the first genes induced during this process. To investigate early events in adipocyte development, we have focused on the transcriptional activation of the LPL gene. For this purpose, we have cloned and fused different parts of intragenic and flanking sequences with a chloramphenicol acetyltransferase reporter gene. Transient transfection experiments and DNase I hypersensitivity assays indicate that several positive as well as negative elements contribute to transcriptional regulation of the LPL gene. When reporter gene constructs were stably introduced into preadipocytes, we were able to monitor and compare the activation patterns of different promoter deletion mutants at selected time points representing the process of adipocyte development. We could delimit two cis-regulatory elements important for gradual activation of the LPL gene during adipocyte development in vitro. These elements, LP-alpha (-702 to -666) and LP-beta (-468 to -430), contain a striking similarity to a consensus sequence known to bind the transcription factors HNF-3 and fork head. Results of gel mobility shift assays and DNase I and exonuclease III in vitro protection assays indicate that factors with DNA-binding properties similar to those of the HNF-3/fork head family of transcription factors are present in adipocytes and interact with LP-alpha and LP-beta. We also demonstrate that LP-alpha and LP-beta were both capable of conferring a differentiation-linked expression pattern to a heterolog promoter, thus mimicking the expression of the endogenous LPL gene during adipocyte differentiation. These findings indicate that interactions with LP-alpha and LP-beta could be a part of a differentiation switch governing induction of the LPL gene during adipocyte differentiation.
Mol Cell Biol 1992 Oct
PMID:Characterization of the human lipoprotein lipase (LPL) promoter: evidence of two cis-regulatory regions, LP-alpha and LP-beta, of importance for the differentiation-linked induction of the LPL gene during adipogenesis. 140 52

Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.
Mol Cell Biol 1992 Nov
PMID:Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers. 140 73

Transcription of the human erythropoietin (Epo) gene is stimulated by exposure to hypoxia and/or cobalt in whole animals and in Hep3B cells. We have systematically investigated the promoter and 3' enhancer elements necessary for this induction by transient transfection of Hep3B cells. We define a promoter region of 53 bp and an enhancer region of 43 bp that confer hypoxia and cobalt inducibility. Each element gives rise to a 6- to 10-fold induction alone. In combination they produce a 50-fold induction after stimulation, similar to the 50- to 100-fold induction of the endogenous Epo gene. Two areas of DNA sequence homology are present in these regions. We demonstrate specific DNA-protein interactions in the enhancer and the ability of the promoter element to compete with these interactions in electrophoretic mobility shift assays. DNase I footprinting and methylation interference data further refine the cis-acting element in the 43-bp enhancer to a short region containing a direct repeat of a steroid/thyroid hormone receptor response element half-site separated by a 2-bp gap. Two half-site consensus sequences are also present in the 53-bp promoter. Site-specific mutation of the half-site sequences in the enhancer destroys the functional activity of the enhancer.
Mol Cell Biol 1992 Dec
PMID:Hypoxic induction of the human erythropoietin gene: cooperation between the promoter and enhancer, each of which contains steroid receptor response elements. 144 72

We have identified a 50-nucleotide enhancer from the human erythropoietin gene 3'-flanking sequence which can mediate a sevenfold transcriptional induction in response to hypoxia when cloned 3' to a simian virus 40 promoter-chloramphenicol acetyltransferase reporter gene and transiently expressed in Hep3B cells. Nucleotides (nt) 1 to 33 of this sequence mediate sevenfold induction of reporter gene expression when present in two tandem copies compared with threefold induction when present in a single copy, suggesting that nt 34 to 50 bind a factor which amplifies the induction signal. DNase I footprinting demonstrated binding of a constitutive nuclear factor to nt 26 to 48. Mutagenesis studies revealed that nt 4 to 12 and 19 to 23 are essential for induction, as substitutions at either site eliminated hypoxia-induced expression. Electrophoretic mobility shift assays identified a nuclear factor which bound to a probe spanning nt 1 to 18 but not to a probe containing a mutation which eliminated enhancer function. Factor binding was induced by hypoxia, and its induction was sensitive to cycloheximide treatment. We have thus defined a functionally tripartite, 50-nt hypoxia-inducible enhancer which binds several nuclear factors, one of which is induced by hypoxia via de novo protein synthesis.
Mol Cell Biol 1992 Dec
PMID:A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. 144 77

The fission yeast pap1+ gene encodes an AP-1-like transcription factor that contains a leucine zipper motif. We identified a target gene of pap1, the p25 gene. The 5' upstream region of the p25 gene contains an AP-1 site, and by DNase I footprint analysis, we showed that the pap1 protein binds to the AP-1 site as well as to a 14-bp palindrome sequence. p25 is overproduced when the pap1+ gene is overexpressed, whereas p25 is not produced at all in the pap1 deletion mutant. p25 was previously found to be overproduced in strains carrying cold-sensitive crm1 mutations whose gene product is essential for viability and is thought to play an important role in maintenance of a proper chromosomal architecture. Deletion and site-directed mutagenesis of sequences upstream of the p25 gene demonstrated that the AP-1 site as well as the palindrome sequence are crucial for transcriptional activation either by pap1 overproduction or by the cold-sensitive crm1 mutation; pap1+ is apparently negatively regulated by crm1+. Moreover, we found that cold-sensitive crm1 mutations are suppressed by the deletion of pap1+, further indicating a close relationship between crm1+ and pap1+. The crm1 protein is highly conserved; the budding yeast homolog, CRM1, which complements the fission yeast cold-sensitive crm1 mutation, was isolated and found to also be essential for viability. These results suggest the functional importance of chromosome structure on the regulation of gene expression through the pap1 transcription factor.
Mol Cell Biol 1992 Dec
PMID:Fission yeast pap1-dependent transcription is negatively regulated by an essential nuclear protein, crm1. 144 80


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