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At least 991 records · Page 55Linked to original sources

PubMed1998

Three-dimensional matrix primes mesangial cells to down-regulation of alpha-smooth muscle actin via deactivation of CArG box elements.

Prolonged culture of mesangial cells forms multifocal nodular structures, termed "hillocks," composed of cells and extracellular matrix (ECM), which may mimic the situation in the glomerular mesangium. Mesangial cells incorporated in hillocks show repressed expression of alpha-smooth muscle actin, a marker of mesangial cell activation/dedifferentiation. The aim of this study is to elucidate molecular mechanisms involved in this phenomenon, focusing on the activity of CArG box elements located in 5'-flanking region of the alpha-smooth muscle actin gene. Reporter mesangial cells were created to monitor the activity of CArG elements. These clones expressed beta-galactosidase gene (lacZ) under the control of CArG boxes. Within the hillocks, reporter cells showed repressed expression of lacZ as well as alpha-smooth muscle actin compared to the cells in two-dimensional cultures. Consistent with this result, the reporter cells embedded in collagen gel exhibited down-regulation of lacZ and alpha-smooth muscle actin transcripts. Deactivation of CArG box elements by transfection with either a dominant negative mutant of serum response factor or a dominant negative form of ternary complex factor Elk-1 led to depressed expression of alpha-smooth muscle actin gene. These data suggested that three-dimensional ECM primes mesangial cells to down-regulation of alpha-smooth muscle actin via deactivation of CArG box elements.

Actins↗

PubMed1985

Effect of boxing on some metabolic indices of muscular contraction.

Muscular contraction, especially if it is protracted or particularly intense, causes notable changes in the substrate and enzymes involved. Marked changes have been found in the serum levels of myoglobin and CK in individuals subjected to very stressful physical work. This phenomenon has usually been attributed to the repeated and intense muscular traumas inherent in certain sporting activities. In the light of these facts, the authors decided to perform a study on amateur boxers aimed at measuring the difference between simple muscular work (shadow boxing) and traumatic activity (real boxing) in terms of their effect on a number of humoral parameters, especially myoglobin and CK. The serum levels of these two latter substances were found to increase more after real boxing than after shadow boxing.

Adult↗

PubMed2004

Arabidopsis EIN3-binding F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene action and promote growth by directing EIN3 degradation.

Ubiquitination of various intracellular proteins by ubiquitin-protein ligases (or E3s) plays an essential role in eukaryotic cell regulation primarily through its ability to selectively target proteins for degradation by the 26S proteasome. Skp1, Cullin, F-box (SCF) complexes are one influential E3 class that use F-box proteins to deliver targets to a core ligase activity provided by the Skp1, Cullin, and Rbx1 subunits. Almost 700 F-box proteins can be found in Arabidopsis, indicating that SCF E3s likely play a pervasive role in plant physiology and development. Here, we describe the reverse genetic analysis of two F-box proteins, EBF1 and -2, that work coordinately in SCF complexes to repress ethylene action. Mutations in either gene cause hypersensitivity to exogenous ethylene and its precursor 1-aminocyclopropane-1-carboxylic acid. EBF1 and -2 interact directly with ethylene insensitive 3 (EIN3), a transcriptional regulator important for ethylene signaling. Levels of EIN3 are increased in mutants affecting either EBF1 or -2, suggesting that the corresponding SCF complexes work together in EIN3 breakdown. Surprisingly, double ebf1 ebf2 mutants display a substantial arrest of seedling growth and have elevated EIN3 levels, even in the absence of exogenous ethylene. Collectively, our results show that the SCF(EBF1/EBF2)-dependent ubiquitination and subsequent removal of EIN3 is critical not only for proper ethylene signaling but also for growth in plants.

Amino Acid Sequence↗

PubMed2002

Systematic assembly of the double molecular boxes: [Cs subset [CpCo(CN)(3)](4)[Cp*Ru](3)] as a tridentate ligand.

Cubic cage compounds composed of Co-CN-Ru linkages have been prepared which illustrate the following features: (i) new motifs for alkali metal ion complexation (i.e., cationic receptors for cations), (ii) a new family of triaza-metalloligands, and (iii) a double box-like cage. The cages are synthesized by the condensation of [CpCo(CN)(3)](-) and [Cp*Ru(NCMe)(3)](+) (cyclopentadienyl, Cp; pentamethylcyclopentadienyl, Cp*) the presence of Cs(+). The species [Cs subset[[CpCo(CN)(3)](4)[Cp*Ru](4)](+) and [Cs subset[[CpCo(CN)(3)](4)[Cp*Ru](3)[Cp*Rh]](2+) illustrate the box-completion reaction Cs subset Co(4)Ru(3) + M (M = Cp*Rh(2+), Cp*Ru(+)). With the naked ion precursors [Na(NCMe)(6)](+) and [Fe(NCMe)(6)](2+), the box-completion reactions afforded [Na[Cs subset[CpCo(CN)(3)](4)[Cp*Ru](3)]](2)](+) and [Fe[Cs subset [CpCo(CN)(3)](4)[Cp*Ru](3)]](2)](2+). These cages provide the first examples, to our knowledge, of the double-box motif.

Bridged-Ring Compounds↗

PubMed2001

A conserved RNA structure (thi box) is involved in regulation of thiamin biosynthetic gene expression in bacteria.

The thiCOGE genes of Rhizobium etli code for enzymes involved in thiamin biosynthesis. These genes are transcribed with a 211-base untranslated leader that contains the thi box, a 38-base sequence highly conserved in the 5' regions of thiamin biosynthetic and transport genes of Gram-positive and Gram-negative organisms. A deletion analysis of thiC-lacZ fusions revealed an unexpected relationship between the degree of repression shown by the deleted derivatives and the length of the thiC sequences present in the transcript. Three regions were found to be important for regulation: (i) the thi box sequence, which is absolutely necessary for high-level expression of thiC; (ii) the region immediately upstream to the translation start codon of thiC, which can be folded into a stem-loop structure that would mask the Shine-Dalgarno sequence; and (iii) the proximal part of the coding region of thiC, which was shown to contain a putative Rho-independent terminator. A comparative phylogenetic analysis revealed a possible folding of the thi box sequence into a hairpin structure composed of a hairpin loop, two helices, and an interior loop. Our results show that thiamin regulation of gene expression involves a complex posttranscriptional mechanism and that the thi box RNA structure is indispensable for thiCOGE expression.

5' Untranslated Regions↗

PubMed2003

The L box regulon: lysine sensing by leader RNAs of bacterial lysine biosynthesis genes.

Expression of amino acid biosynthesis genes in bacteria is often repressed when abundant supplies of the cognate amino acid are available. Repression of the Bacillus subtilis lysC gene by lysine was previously shown to occur at the level of premature termination of transcription. In this study we show that lysine directly promotes transcription termination during in vitro transcription with B. subtilis RNA polymerase and causes a structural shift in the lysC leader RNA. We find that B. subtilis lysC is a member of a large family of bacterial lysine biosynthesis genes that contain similar leader RNA elements. By analogy with related regulatory systems, we designate this leader RNA pattern the "L box." Genes in the L box family from Gram-negative bacteria appear to be regulated at the level of translation initiation rather than transcription termination. Mutations of B. subtilis lysC that disrupt conserved leader features result in loss of lysine repression in vivo and loss of lysine-dependent transcription termination in vitro. The identification of the L box pattern also provides an explanation for previously described mutations in both B. subtilis and Escherichia coli lysC that result in lysC overexpression and resistance to the lysine analog aminoethylcysteine. The L box regulatory system represents an example of gene regulation using an RNA element that directly senses the intracellular concentration of a small molecule.

5' Untranslated Regions↗

PubMed1986

Homeo box gene expression in anterior and posterior regions of the Drosophila embryo.

The homeo box is a 180-base-pair coding sequence that has been implicated in the control of Drosophila development. A common feature of the nine previously reported homeo box genes is their involvement in the establishment of the segmentation pattern of the embryo. In this report we describe the isolation and properties of two additional homeo box genes, F90-2 and S67. Transcripts encoded by the two genes are detected in embryonic tissues that derive from regions near the anterior and posterior poles of the embryo, which are outside the limits of expression of known homeotic genes. These results suggest that at least some homeo box genes specify positional identity along the anterior-posterior body axis that is independent of the process of segmentation.

Amino Acid Sequence↗

PubMed1986

The homeo domain of a murine protein binds 5' to its own homeo box.

Nuclear protein extracts from day 12.5 mouse embryos were used to study protein binding to DNA sequences 5' of the Hox 1.5 homeo box. Embryos of this developmental stage are known to express this gene. DNA binding protein blotting and retardation gel techniques show that murine embryonic nuclear proteins specifically bind a 753-base pair (bp) DNA fragment from the region upstream of the Hox 1.5 homeo box. A fusion protein containing the Hox 1.5 homeo domain constructed in lambda gt11 also binds the same 753-bp DNA fragment. Specific binding of the fusion protein to the upstream DNA fragment shows that the homeo box contains the sequences required for specific protein-DNA interactions, and the 753-bp fragment contains a homeo domain binding site. These results support the hypothesis that murine homeo boxes are DNA binding domains of proteins involved in the regulation of embryonic development.

Amino Acid Sequence↗

PubMed1988

No strict alignment is required between a transcriptional activator binding site and the "TATA box" of a yeast gene.

GAL4 is a transcriptional activator of the galactose metabolism genes in the yeast Saccharomyces cerevisiae. We show that GAL4 expressed in yeast activated transcription equally well when a single GAL4 binding site was placed at any of nine positions upstream of the GAL1 (galactokinase gene) "TATA box." We chose a sufficient number of positions for the binding site to ensure that, in several of these positions, GAL4 was on the opposite side of the DNA helix with respect to the TATA box. Smaller GAL4 derivatives were similar to wild-type GAL4 in that they also activated transcription in a manner independent of the side of the DNA helix they bound with respect to the TATA box. Unlike wild-type GAL4, however, these smaller GAL4 derivatives activated transcription better when we placed a binding site progressively closer to the TATA box over a distance of 34 base pairs.

Bacterial Proteins↗

PubMed1990

Coordinate occupancy of AP-1 sites in the vitamin D-responsive and CCAAT box elements by Fos-Jun in the osteocalcin gene: model for phenotype suppression of transcription.

Osteocalcin, a bone-specific protein and marker of the mature osteoblast, is expressed only in nonproliferating osteoblasts in a mineralizing extracellular matrix, while type I collagen is expressed in proliferating cells. The nuclear proteins encoded by the c-fos and c-jun protooncogenes are expressed during the proliferation period of osteoblast phenotype development. We present evidence that AP-1 (HeLa cell-activating protein 1) sites residing within two promoter elements of the osteocalcin gene bind the Fos-Jun protein complex: the osteocalcin box (OC box; nucleotides -99 to -76), which contains a CCAAT motif as a central element and influences tissue-specific basal levels of osteocalcin gene transcription, and the vitamin D-responsive element (VDRE; nucleotides -462 to -440), which mediates enhancement of osteocalcin gene transcription. Gel electrophoretic mobility-shift analysis demonstrated high AP-1 binding activity in proliferating osteoblasts and dramatic changes in this activity after the down-regulation of proliferation and the initiation of extracellular-matrix mineralization in primary cultures of normal diploid osteoblasts. Methylation interference analysis established at single nucleotide resolution that purified recombinant Fos and Jun proteins bind in a sequence-specific manner to the AP-1 sites within the VDRE and OC box. Similarly, an AP-1 motif within a putative VDRE of the alkaline phosphatase gene, which is also expressed after the completion of proliferation, binds the Fos-Jun complex. These results support a model in which coordinate occupancy of the AP-1 sites in the VDRE and OC box in proliferating osteoblasts may suppress both basal level and vitamin D-enhanced osteocalcin gene transcription as well as transcription of other genes associated with osteoblast differentiation--a phenomenon we describe as phenotype suppression. This model is further supported by binding of the Fos-Jun complex at an AP-1 site in the type alpha I collagen promoter that is contiguous with, but not overlapping, the VDRE. Such a sequence organization in the collagen VDRE motif is compatible with vitamin D modulation of collagen but not with osteocalcin and alkaline phosphatase expression in proliferating osteoblasts.

Animals↗

PubMed1990

Engrailed, a homeodomain protein, can repress in vitro transcription by competition with the TATA box-binding protein transcription factor IID.

Engrailed (En) is a homeodomain protein that binds to a consensus sequence (NP) and plays an important role during Drosophila development. Purified En, which is produced in Escherichia coli, binds not only to this consensus sequence but also to the TATA box of the Drosophila Hsp70 promoter and of other eukaryotic promoters. Interestingly, En represses transcription of these promoters in an in vitro-reconstituted mammalian transcription system and footprint analyses show that En competes with the TATA box-binding protein transcription factor IID for binding to the TATA box. In contrast, a stable template-committed complex formed by preincubation of transcription factor IID with the promoter is not disrupted by addition of En, and in this case transcription is not repressed. These in vitro studies suggest a transcriptional repression mechanism, involving competition between En and transcription factor IID for TATA box binding, that may be involved in En-mediated repression in vivo.

Animals↗

PubMed1993

Amplification of a DEAD box protein gene in retinoblastoma cell lines.

DEAD box proteins, characterized by the conserved motif Asp-Glu-Ala-Asp, are putative RNA helicases implicated in a number of cellular processes involving alteration of RNA secondary structure such as translation initiation, nuclear and mitochondrial splicing, and ribosome and spliceosome assembly. Based on their distribution patterns, some members of this family are believed to be involved in embryogenesis, spermatogenesis, and cellular growth and division. Here, we report that the mRNA encoding a DEAD box protein, designated HuDBP-RB, is present at elevated levels in two of six retinoblastoma (RB) cell lines tested and is preferentially expressed in fetal tissues of neuroectodermal origin. It is not possible to classify HuDBP-RB as a member of any of the DEAD box protein subgroups identified to date since the regions of amino acid similarity between HuDBP-RB and other DEAD box proteins are restricted to the conserved motifs found in all members of this family. The HuDBP-RB gene, which has been mapped to chromosome band 2p24, is amplified in the RB cell lines that overexpress HuDBP-RB RNA. Furthermore, the MYCN gene is also present in multiple copies in these two cell lines, suggesting coamplification of the two genes.

Amino Acid Sequence↗

PubMed1994

Transcriptional regulation of the human insulin gene is dependent on the homeodomain protein STF1/IPF1 acting through the CT boxes.

Insulin gene transcription is a unique feature of the pancreatic beta cells and is increased in response to glucose. The recent cloning of insulin promoter factor 1 (IPF1) and somatostatin transcription factor 1 (STF1) unexpectedly revealed that these are mouse and rat homologues of the same protein mediating transactivation through binding of CT box-like elements in rat insulin 1 and somatostatin promoter/enhancer regions, respectively. By using oligonucleotides representing each of the three CT boxes of the human insulin (HI) gene enhancer and nuclear extracts from the mouse islet tumor cell lines beta TC3 and alpha TC1, we have identified a beta-cell-specific binding activity as reported for IPF1, which has maximal affinity toward the CT2 box. However, in pluripotent, HI-transfected rat islet tumor cells, NHI-6F, this binding activity is present prior to induction of (human) insulin gene transcription. Its migration is identical to that of in vitro translated STF1 in electrophoretic mobility-shift assays; it is specifically recognized by anti-STF1 antibodies and has an apparent molecular mass of 46 kDa. Mutation of the CT2 box decreases transcriptional activity of a HI reporter plasmid by approximately 65% in beta TC3 cells and blocks the glucose response in isolated newborn rat islet cells. Furthermore, cotransfection with STF1 cDNA into the glucagon-producing alpha TC1 cells increases the activity of the HI enhancer 4- to 5-fold, suggesting that STF1/IPF1 can confer on alpha TC1 cells the ability to transcribe the HI gene. We conclude that STF1/IPF1 is a necessary but not sufficient key regulator of insulin gene activity, possibly also involved in glucose-regulated transcription.

Animals↗

PubMed1994

Human transcription factor IIIC box B binding subunit.

Transcription factor IIIC (TFIIIC) is a multisubunit basic TF for RNA polymerase III. It initiates transcription complex assembly on tRNA and related genes by binding to the internal box B promoter element and is also required for transcription of 5S rRNA and other stable nuclear and cytoplasmic RNAs transcribed by polymerase III. In mammalian cells, regulation of TFIIIC activity controls overall polymerase III transcription in response to growth factors and viral infection. Here, we report the cloning and sequencing of a full-length cDNA (and genomic DNA from the transcription initiation region) encoding the box B binding subunit of human TFIIIC, the 243-kDa alpha subunit. Specific antisera raised against the encoded protein super shifts a TFIIIC-box B DNA complex during an electrophoretic mobility shift assay and immunodepletes TFIIIC transcriptional activity from a partially purified TFIIIC fraction, proving that the cDNA encodes a component of TFIIIC. The human protein shows surprisingly little similarity to the box B binding subunit of yeast TFIIIC.

Amino Acid Sequence↗

PubMed1997

Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP), an ethylene-inducible, GCC box DNA-binding protein interacts with an ocs element binding protein.

Ocs elements are a group of promoter sequences required for the expression of both pathogen genes in infected plants and plant defense genes. Genes for ocs element binding factors (OBFs), belonging to a specific class of basic-region leucine zipper (bZIP) transcription factors, have been isolated in a number of plants. Using protein-protein interaction screening with OBF4 we have isolated AtEBP, an Arabidopsis protein that contains a novel DNA-binding domain, the AP2/EREBP domain. One class of proteins that contain this domain are the tobacco ethylene-responsive element binding proteins (EREBPs). The EREBPs bind the GCC box that confers ethylene responsiveness to a number of pathogenesis related (PR) gene promoters. AtEBP expression is inducible by exogenous ethylene in wild-type plants and AtEBP transcripts are increased in the ctr1-1 mutant, where ethylene-regulated pathways are constitutively active. Electrophoretic mobility-shift assay and DNase I footprint analysis revealed that AtEBP can specifically bind to the GCC box. Interestingly, the highest level of AtEBP expression was detected in callus tissue, where ocs elements are very active. Synergistic effects of the GCC box with ocs elements or the related G-box sequence have been previously observed, for example, in the ethylene-induced expression of a PR gene promoter. Our results suggest that cross-coupling between EREBP and bZIP transcription factors occurs and may therefore be important in regulating gene expression during the plant defense response.

Amino Acid Sequence↗

PubMed1998

Mutation of the polo-box disrupts localization and mitotic functions of the mammalian polo kinase Plk.

Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation. In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic domain, termed the polo-box. The function of the polo-box is currently undefined. The mammalian polo-like kinase Plk is a functional homologue of Saccharomyces cerevisiae Cdc5. Here, we show that Plk localizes at the spindle poles and cytokinetic neck filaments. Without impairing kinase activity, a conservative mutation in the polo-box disrupts the capacity of Plk to complement the defect associated with a cdc5-1 temperature-sensitive mutation and to localize to these subcellular structures. Our data provide evidence that the polo-box plays a critical role in Plk function, likely by directing its subcellular localization.

Amino Acid Sequence↗

PubMed1995

An E box element is required for the expression of the ad4bp gene, a mammalian homologue of ftz-f1 gene, which is essential for adrenal and gonadal development.

Ad4BP, also known as SF-1, is a cell type-specific transcription factor regulating all the steroidogenic P-450 genes. Recently, the targeted disruption of the mouse ftz-f1 gene encoding Ad4BP/SF-1 has established its essential function in both adrenal and gonadal development and sexual differentiation. As an initial step toward understanding its role in the cascade of gene activations necessary for the differentiation of the steroidogenic tissues and the sex differentiation of the gonads, we isolated and characterized the rat ad4bp gene. A sequence analysis of the ad4bp gene revealed that another nuclear factor ELP was also transcribed from the same gene by alternative promoter usage and splicing. The promoter of the ad4bp gene showed activities in the steroidogenic cells such as Y-1 adrenocortical cells and I-10 testicular Leydig cells when examined by transient transfection assays. Using deletion analysis and site-directed mutagenesis, we identified a cis-element at the position from -82 bp to -77 bp in the 5'-upstream region. The cis-element was identical to the consensus E box element, which is the binding site for the basic-helix-loop-helix proteins. Gel mobility shift analyses revealed the amount of a binding factor to this E box in the nuclear extract prepared from the rat testes attained a maximal level 1 week after birth and then decreased dramatically thereafter, and only trace amounts were detected in adult rats. In contrast, the binding factor in the ovaries attained a maximal level just after birth and kept its level thereafter. These dimorphic expressions of the binding factor to the E box correlated well with those of Ad4BP, and thus suggested that the expression of Ad4BP, and thus suggested that the expression of Ad4BP is transcriptionally regulated through this E box element.

Adrenal Cortex↗

PubMed1995

Transcriptional regulation of the genes encoding cytochromes P450BM-1 and P450BM-3 in Bacillus megaterium by the binding of Bm3R1 repressor to Barbie box elements and operator sites.

We previously reported (Liang, Q., He, J.-S., and Fulco, A.J. (1995) J. Biol. Chem. 270, 4438-4450) that Bm3R1, a repressor regulating the expression of P450BM-3 in Bacillus megaterium, could bind to Barbie box sequences in the 5'-flanking regions of barbiturate-inducible genes. We've now shown that pentobarbital does not inhibit in vitro binding of Bm3R1 to the P450BM-3 and P450BM-1 Barbie boxes (BB3 and BB1), although the palindromic operator sequence (OIII) of P450BM-3 did have a strong competitive effect on such binding. G39E-Bm3R1, a mutant of Bm3R1, did not bind to either Barbie box. In the presence of Bm3R1, portions of the regulatory regions of P450BM-3 and P450BM-1 were protected from DNase I digestion. These included 11 of the 15 base pairs of BB3 plus 7 base pairs 3' to BB3, BB1 plus 16 base pairs 3' to BB1, and, in the 5'-flanking region of P450BM-1, segments covering most of two palindromic sequences (OII and OIII) of 24 and 52 base pairs. These DNase I-protected regions (including OIII) showed considerable sequence identity, especially in a conserved poly(A) motif. Barbiturates did not inhibit binding of Bm3R1 to OI. OII in vitro while G39E-Bm3R1 did not bind. The regulatory effects of Bm3R1 on P450BM-1 and P450BM-3 were also evaluated in vivo using heterologous chloramphenicol acetyltransferase constructs and Western blotting. In the G39E mutant strain, both P450BM-1 and P450BM-3 were constitutively expressed, and the regulatory proteins Bm1P1 and Bm3P1, although still pentobarbital-inducible, had significantly higher basal levels of synthesis. In toto, our results show that Bm3R1 represses both P450BM-1 and P450BM-3 expression and that it may effect this by coordinate binding to operator and Barbie box sequences to produce looping of the P450BM-1 and P450BM-3 regulatory regions through protein-protein interaction.

Bacillus megaterium↗