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PubMed1997

Bidirectional binding of the TATA box binding protein to the TATA box.

By selective attachment of a DNA cleavage agent to specific residues in the yeast TATA box binding protein (yTBP), we demonstrate that, in solution, yTBP binds to the TATA boxes of both the adenovirus major late promoter and the yeast CYC1 promoter with only a modest preference in orientation and binds well to several overlapping binding sites. The general factors TFIIA and TFIIB each increase the rotational and translational selectivity of yTBP but are not sufficient, at least individually, to confer a unique polarity to the preinitiation complex. We conclude that TBP alone cannot define the productive orientation of general factor assembly on a promoter.

Adenoviridae↗

PubMed2001

Fluorescence-based analyses of the effects of full-length recombinant TAF130p on the interaction of TATA box-binding protein with TATA box DNA.

We have used a combination of fluorescence anisotropy spectroscopy and fluorescence-based native gel electrophoresis methods to examine the effects of the transcription factor IID-specific subunit TAF130p (TAF145p) upon the TATA box DNA binding properties of TATA box-binding protein (TBP). Purified full-length recombinant TAF130p decreases TBP-TATA DNA complex formation at equilibrium by competing directly with DNA for binding to TBP. Interestingly, we have found that full-length TAF130p is capable of binding multiple molecules of TBP with nanomolar binding affinity. The biological implications of these findings are discussed.

DNA↗

PubMed2002

Novel mutations affecting SRY DNA-binding activity: the HMG box N65H associated with 46,XY pure gonadal dysgenesis and the familial non-HMG box R30I associated with variable phenotypes.

The SRY gene (sex-determining region of the Y chromosome) initiates the process of male sex differentiation in mammalians. In humans mutations in the SRY gene have been reported to account for 10-15% of the XY sex reversal cases. We describe here two novel missense mutations in the SRY gene after the screening of 17 patients, including 3 siblings, with 46,XY gonadal dysgenesis and 4 true hermaphrodites. One of the mutations, an A to C transversion within the HMG box, causes the N65H substitution and it was found in a patient presenting 46,XY pure gonadal dysgenesis. The Escherichia coli expressed SRY(N65H) protein did not present DNA-binding activity in vitro. The other mutation, a G to T transversion, causes the R30I substitution. This mutation was found in affected and nonaffected members of a family, including the father, two siblings with partial gonadal dysgenesis, a phenotypic female with pure gonadal dysgenesis, and three nonaffected male siblings. The G to T base change was not found in the SRY sequence of 100 normal males screened by ASO-PCR. The R30I mutation is located upstream to the HMG box, within the (29)RRSSS(33) phosphorylation site. The E. coli expressed SRY(R30I) protein was poorly phosphorylated and consequently showed reduced DNA-binding capacity in vitro.

Blotting, Western↗

PubMed1993

The human erythropoietin-encoding gene contains a CAAT box, TATA boxes and other transcriptional regulatory elements in its 5' flanking region.

We have reported the cloning and expression of a human erythropoietin (hEp)-encoding cDNA [Lee-Huang, Proc. Natl. Acad. Sci. USA 81 (1984) 2708-2712]. Using this hEp cDNA as a probe, we isolated a 9.3-kb BamHI genomic Ep clone from a human leukocyte library soon thereafter. The size and restriction map of this clone is in agreement with restriction analysis of human genomic DNA probed with the hEp cDNA, demonstrating that this clone is representative of the single hEp gene. This clone is unique in that it extends beyond any reported hEp genomic clone by 3.9 kb on the 5' side and by 1.8 kb on the 3' side. The promoter function of the newly described 5' flanking region has been demonstrated by the expression of biologically active hEp in transfected cells. We find that, despite reports to the contrary, hEp does contain classic canonical TATA boxes and a CAAT box. The 5'-flanking region also contains cytokine-responsive consensus sequences, tissue-specific and metal-responsive elements, CRE and GRE sites, and binding sites for transcription factors, including AP1, NF-kappa beta and Sp1. These regulatory elements have not been found in the hEp genomic clones thus far reported. The identification of these elements and their precise localization in hEp should be useful in studying the regulation of hEp expression, as well as in gene therapy and physiologic modulation of this hormone.

Activating Transcription Factor 2↗

PubMed1992

The significance of diagnostic imaging in acute and chronic brain damage in boxing. A prospective study in amateur boxing using magnetic resonance imaging (MRI).

The significance and possible extent of structural damage to the central nervous system (CNS) due to boxing are investigated. Bleeding, especially microhematomas, is considered to be one probable cause of the chronic encephalopathy in boxers. In a prospective study, 13 amateur boxers were investigated with the help of MRI several times before and after their fights. The MRI investigations were accompanied by neurologic examinations before and after the fights. Among the 13 boxes, 5 demonstrated focal neurological signs following the fights, without evidence of small hematoma or other structural alterations. The number of head punches did not correlate with the occurrence of neurologic signs. These results indicate that up to now imaging methods cannot clarify the development of chronic encephalopathy.

Acute Disease↗

PubMed2000

Dhr1p, a putative DEAH-box RNA helicase, is associated with the box C+D snoRNP U3.

Putative RNA helicases are involved in most aspects of gene expression. All previously characterized members of the DEAH-box family of putative RNA helicases are involved in pre-mRNA splicing. Here we report the analysis of two novel DEAH-box RNA helicases, Dhr1p and Dhr2p, that were found to be predominantly nucleolar. Both genes are essential for viability, and MET-regulated alleles were therefore created. Depletion of Dhr1p or Dhr2p had no detectable effect on pre-mRNA splicing in vivo or in vitro. Both Dhr1p and Dhr2p were, however, required for 18S rRNA synthesis. Depletion of Dhr2p inhibited pre-rRNA cleavage at sites A(0), A(1), and A(2), while Dhr1p depletion inhibited cleavage at sites A(1) and A(2). No coprecipitation of snoRNAs was detected with ProtA-Dhr2p, but Dhr1p-ProtA was stably associated with the U3 snoRNA. Depletion of Dhr1p inhibited processing steps that require base pairing of U3 to the 5' end of the 18S rRNA. We speculate that Dhr1p is targeted to the preribosomal particles by the U3-18S rRNA interaction and is required for the structural reorganization of the rRNA during formation of the central pseudoknot.

Base Sequence↗

PubMed2005

The up-regulation of Y-box binding proteins (DNA binding protein A and Y-box binding protein-1) as prognostic markers of hepatocellular carcinoma.

PURPOSE: The development of hepatocellular carcinoma is associated with the chronic inflammation of the liver caused by various factors such as hepatitis B or C virus infection. Previously, we reported DNA binding protein A (dbpA) as a candidate molecule that can accelerate inflammation-induced hepatocarcinogenesis. DbpA belongs to the Y-box binding protein family, and Y-box binding protein-1 (YB-1), the prototype member of this family, is reported to be a prognostic marker of malignant diseases other than hepatocellular carcinoma. The purpose of this study is to examine the significance of the expression of dbpA or of the T-to-G transversion in the dbpA promoter region, which enhances the promoter activity in vitro, for the progression of hepatocellular carcinoma. EXPERIMENTAL DESIGN: We studied the expression of dbpA (as well as of YB-1) in 82 formalin-fixed hepatocellular carcinoma tissues by immunohistochemistry and determined the sequence of the dbpA promoter region in 42 frozen hepatocellular carcinoma tissues. We examined the relationship between these findings and the clinicopathologic factors of hepatocellular carcinoma patients. RESULTS: DbpA expression was associated with the advanced stages of hepatocellular carcinoma, and the cases with the nuclear dbpA expression had a poor prognosis. DbpA contributed more significantly to this association than YB-1. Furthermore, the T-to-G transversion in the dbpA promoter region was related to the nuclear localization of dbpA. CONCLUSION: DbpA was a more significant prognostic marker of hepatocellular carcinoma than YB-1. The T-to-G transversion in the dbpA promoter region was suggested to be a predisposing factor for the progression of hepatocellular carcinoma.

Antibodies, Monoclonal↗

PubMed1999

Splicing-independent processing of plant box C/D and box H/ACA small nucleolar RNAs.

Small nucleolar RNAs (snoRNAs) are involved in various aspects of ribosome biogenesis and rRNA maturation. Plants have a unique organisation of snoRNA genes where multiple, different genes are tightly clustered at a number of different loci. The maize gene clusters studied here include genes from both of the two major classes of snoRNAs (box C/D and box H/ACA) and are transcribed as a polycistronic pre-snoRNA transcript from an upstream promoter. In contrast to vertebrate and yeast intron-encoded snoRNAs, which are processed from debranched introns by exonuclease activity, the particular organisation of plant snoRNA genes suggests a different mode of expression and processing. Here we show that single and multiple plant snoRNAs can be processed from both non-intronic and intronic transcripts such that processing is splicing-independent and requires endonucleolytic activity. Processing of these different snoRNAs from the same polycistronic transcript suggests that the processing machineries needed by each class are not spatially separated in the nucleolus/nucleus.

Base Sequence↗

PubMed2003

Isolation and characterization of a cDNA encoding a putative high mobility group (HMG)--box protein from stored mRNA in resting cysts of the ciliate Oxytricha (Sterkiella) nova: ciliate macronuclear gene encoding a putative HMG-box protein.

We have isolated an cDNA after applying a DDRT-PCR analysis on mRNA from mature resting cysts of the ciliate Oxytricha (Sterkiella) nova. From this cDNA fragment the complete macronuclear minichromosome was obtained by using the Mac-End-PCR method. After cloning and sequencing, this cDNA shown certain similarity to HMG-like proteins. The analysis of the inferred amino acid sequence shown that this putative HMG-like protein has one HMG-box interrupted by a intron. The analysis of others characteristics (including a 3D model) confirms that it is a HMGB family protein. It is the first time that a macronuclear gene encoding a putative HMG-box protein is isolated from resting cysts of a stichotrich ciliate. The possible implications of this stored mRNA in the ciliate cryptobiotic stage are discussed.

Amino Acid Sequence↗

PubMed2000

Activation of c-myc promoter P1 by immunoglobulin kappa gene enhancers in Burkitt lymphoma: functional characterization of the intron enhancer motifs kappaB, E box 1 and E box 2, and of the 3' enhancer motif PU.

Deregulated expression of the proto-oncogene c- myc in Burkitt lymphoma (BL) cells carrying a t(2;8) translocation is mediated by a synergistic interaction of the translocated immunoglobulin (Ig) kappa gene intron (kappaEi) and 3' (kappaE3') enhancers and characterized by a strong activation of the promoter P1. We have investigated the functional role of distinct kappa enhancer sequence motifs in P1 activation on both mini-chromosomes and reporter gene constructs. Stable and transient transfections of BL cells revealed critical roles of the kappaEi and kappaE3' elements kappaB and PU, respectively. Joint mutation of kappaB and PU completely abolished P1 activity, implying that an interaction of kappaB- and PU-binding factors is essential for the enhancer synergism. Mutation of the E box 1 and E box 2 motifs markedly decreased P1 activity in transient but not in stable transfection experiments. Co-expression of the NF-kappaB subunit p65(RelA) and Sp1, an essential factor for P1 transcription, in Drosophila melanogaster SL2 cells synergistically enhanced promoter activity. Our results support a model which proposes cross-talk between promoter and enhancer binding factors as the basic mechanism for kappa enhancer-mediated c- myc activation in BL cells.

Animals↗

PubMed1988

The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains.

Transcription of promoters of immunoglobulin genes is controlled by an octanucleotide sequence element. The sequence of a cDNA encoding a B-cell-specific protein, Oct-2, has been determined. This protein specifically recognizes the octanucleotide element and is part of the previously identified NF-A2 family of proteins. The DNA-binding domain of Oct-2 is structurally related to the homeo box consensus and thus contains a potential helix-turn-helix sequence. Oct-2 also possesses a potential 'leucine zipper' domain, where four leucines are each separated by exactly seven residues. Comparisons of Oct-2 with protein Oct-1, which also recognizes the octanucleotide element but is constitutively expressed in all cell types, show high sequence conservation through the 60-residue DNA-binding domain, as well as an adjacent tract of 75 residues. The latter conserved region is also found in regulatory genes expressed in pituitary cells and nematodes and has been termed a POU box. Because two different cDNAs were isolated, it is proposed that the oct-2 gene is expressed as multiple mRNAs that vary in splicing patterns. Most interestingly, the oct-2 cDNA contains a second overlapping open reading frame, 278 residues in length, which might also specify a protein important for B-cell development.

Amino Acid Sequence↗

PubMed1992

TGA1 and G-box binding factors: two distinct classes of Arabidopsis leucine zipper proteins compete for the G-box-like element TGACGTGG.

Regulatory elements containing the sequence ACGT are found in several plant promoters and are recognized by various basic/leucine zipper (bZIP) proteins. The Arabidopsis G-box binding factor 1 (GBF1), initially identified by its ability to bind to the palindromic G-box (CCACGTGG), also interacts with the TGACGT motif if this hexamer sequence is followed by either the dinucleotide GG--as found in the Hex motif of the wheat histone 3 promoter--or GT. Here we describe the isolation of an Arabidopsis bZIP protein, denoted TGA1, that also recognizes ACGT-containing sequences. However, TGA1 differs from members of the GBF family in the spectrum of base pair permutations flanking the ACGT sequence that are required for DNA binding. TGA1 primarily requires a TGACG motif and preferentially binds to those pentamers that are followed by a T residue. We show that although both TGA1 and GBF1 bind to the Hex motif (TGACGTGG), this binding can be distinguished on the basis of their specific DNA-protein contacts. Furthermore, TGA1 also differs from members of the GBF family in that it apparently does not form heterodimers with any member of this family.

Amino Acid Sequence↗

PubMed2006

Identification of novel HrpXo regulons preceded by two cis-acting elements, a plant-inducible promoter box and a -10 box-like sequence, from the genome database of Xanthomonas oryzae pv. oryzae.

A regulatory protein HrpXo of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial leaf blight of rice, is known to control the expression of hrp genes that encode components of a type III secretion system and of some effector protein genes. In this study, we screened novel HrpXo regulons from the genome database of X. oryzae pv. oryzae, searching for ORFs preceded by two predicted sequence motifs, a plant-inducible promoter box-like sequence and a -10 box-like sequence. Using a gus reporter system, nine of 15 ORF candidates were expressed HrpXo dependently. We also showed by base-substituted mutagenesis that both motifs are essential for the expression of the genes.

Bacterial Proteins↗

PubMed2004

Cloning and characterization of granulosa cell high-mobility group (HMG)-box protein-1, a novel HMG-box transcriptional regulator strongly expressed in rat ovarian granulosa cells.

Specific events in the ovary are dependent on gene expression in the tissue. By screening a rat ovarian granulosa cell cDNA library, a cDNA clone encoding a novel transcription factor-like protein containing a high-mobility group-box, referred to as granulosa cell high-mobility group-box protein-1 (GCX-1), was identified. The expression of GCX-1 is restricted to the hypothalamus, pituitary, testis, uterus, and ovary but was not detected in the adrenal gland. An in situ hybridization study revealed that the expression of GCX-1 was restricted to granulosa cell layers in early-stage follicles, and the expression was very low in large antral follicles and the corpus luteum, but localized expression in the testis or pituitary was not clear. Endogenous GCX-1 protein in the granulosa cells was identified by a Western blot analysis, and an analysis using the green fluorescence protein-GCX-1 fusion protein revealed that the GCX-1 protein was localized in the cell nucleus. GAL4 fusion protein-based assays demonstrated that GCX-1 is a potent transcriptional activator, and its putative transactivation domain was mapped to the region between amino acid residues 25 and 63 from the N terminus. These data strongly suggest that GCX-1 is likely a novel transcriptional activator that is exclusively expressed in reproductive tissues involving the hypothalamo-pituitary-gonadal axis, and functions as a specific regulator of follicular development, and may also participate in other specific events related to reproduction, particularly in the female.

Amino Acid Sequence↗

PubMed1996

Choosing a future for epidemiology: II. From black box to Chinese boxes and eco-epidemiology.

Part I of this paper traced the evolution of modern epidemiology in terms of three eras, each with its dominant paradigm, culminating in the present era of chronic disease epidemiology with its paradigm, the black box. This paper sees the close of the present era and foresees a new era of eco-epidemiology in which the deployment of a different paradigm will be crucial. Here a paradigm is advocated for the emergent era. Encompassing many levels of organization--molecular and societal as well as individual--this paradigm, termed Chinese boxes, aims to integrate more than a single level in design, analysis, and interpretation. Such a paradigm could sustain and refine a public health-oriented epidemiology. But preventing a decline of creative epidemiology in this new era will require more than a cogent scientific paradigm. Attention will have to be paid to the social processes that foster a cohesive and humane discipline.

Epidemiologic Methods↗

PubMed1996

HMG box-activating factors 1 and 2, two HMG box transcription factors that bind the human Ig heavy chain enhancer.

We present the isolation of two cDNAs that encode distinct, yet related, proteins that bind the HE2 region of the human Ig heavy chain (IgH) enhancer. Designated HMG box-activating factors (HAF) 1 and 2, the two proteins are new members of the HMG box family of DNA binding proteins. Both are potent transcription activators when expressed 1) as GAL4 fusions targeted to promoters containing GAL4 operators, or 2) as intact proteins targeted to minimal promoters containing binding sites derived from the IgH enhancer. HAF-1 and HAF-2 mRNAs are apparently expressed in both B cells and non-B cells. However, activity generated by the isolated HE2 region in B cells is dependent on both an intact HAF-1/HAF-2 binding site and at least one additional site that has been reported previously to bind a B cell-restricted protein. Our results suggest a collaborative role for either or both HAF-1 and HAF-2 in establishing the B cell activity of the human IgH enhancer.

Amino Acid Sequence↗

PubMed1994

Transcription of the sea urchin U6 gene in vitro requires a TATA-like box, a proximal sequence element, and sea urchin USF, which binds an essential E box.

The tandemly repeated gene set encoding the sea urchin U6 gene has been cloned from the sea urchin Strongylocentrotus purpuratus. The U6 gene is transcribed by RNA polymerase III in a sea urchin nuclear extract. Like that of the vertebrate U6 genes, transcription of the sea urchin U6 gene does not require any internal sequences or 3' sequences but requires only 5' flanking sequences. Only 88 nucleotides of 5' flanking sequence are required for maximal expression in vitro. Mutagenesis experiments demonstrated the requirement for three elements, a CACGTG element at -80, a proximal sequence element at about -55, and the TATA-like box at -25. The major protein in sea urchin extracts that interacts with the CACGTG element is sea urchin USF, and immunodepletion of sea urchin USF greatly reduces transcription. The USF binding site in the U6 gene is highly homologous (11 of 13 nucleotides) with the USF binding sites found in the promoter of the S. purpuratus spec genes.

Animals↗

PubMed2002

Isolation and characterization of cold-shock domain protein genes, Oryzias latipes Y-box protein 2 (OlaYP2) and Fugu rubripes Y-box protein 1 (FruYP1), in medakafish and pufferfish.

The Y-box protein (YP) family shares a nucleic acid binding domain, called cold-shock domain, that has been evolutionarily highly conserved from bacteria to human. The different YPs identified so far in vertebrates are thought to function as transcriptional activators, transcriptional repressors and/or translational repressors. Medakafish and pufferfish are very suitable vertebrate models for the study of developmental genetics and comparative genomics, respectively. Here we report the isolation of two teleost YP genes, medakafish Oryzias latipes (Ola)YP2 and Fugu rubripes (Fru)YP1, which are expressed in multiple tissues. Phylogenetic analysis demonstrated that OlaYP2 and FruYP1 belong to different subclasses of the cold-shock domain protein genes. Future studies in suitable model systems, like the medaka for developmental biology and Fugu for evolutionary genomics, are expected to contribute to our understanding of YPs.

Amino Acid Sequence↗