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At least 667 records · Page 37Linked to original sources
PubMed1995

The genome organization of banana bunchy top virus: analysis of six ssDNA components.

We have cloned, sequenced and analysed an additional five circular ssDNA components of banana bunchy top virus (BBTV) which we have called components 2, 3, 4, 5 and 6. These components were present in all BBTV infections tested. Four of these components (components 3, 4, 5 and 6) had one large open reading frame (ORF) in the virion sense located 3' of a stem-loop structure. Each ORF had a potential TATA box and one or two potential polyadenylation signals associated with it and each polyadenylation signal had an associated GC-rich region containing the trinucleotide sequence TTG. A number of ORFs were identified in component 2 but none of these had appropriately located potential TATA boxes and polyadenylation signals associated with them. None of the ORF amino acid sequences nor the full DNA sequences of any of the components had significant sequence identity with any known protein or nucleic acid sequences. However, the ORF of component 4 encoded a 30 residue hydrophobic domain which may indicate that this ORF encoded a transmembrane protein. Further, the ORFs of components 3 and 5 potentially encoded proteins of about 20 kDa, the size of the BBTV coat protein. There were two regions of sequence identity between the five components described here and the previously described component 1. Each component contained a conserved stem-loop structure and a nonanucleotide potential TATA box which was 5' of the large virion-sense ORF in five of the components. The stem-loop structures were incorporated in a common region (CR-SL) of 69 nucleotides which was 62% identical between components. All six BBTV components also contained a major common region (CR-M) which was located 5' of the CR-SL in each component, in the non-coding region and was 76% identical over 92 nucleotides. Each CR-M contained a near-complete 16 nucleotide direct repeat and a GC-box which was similar to the rightward promoter element found in wheat dwarf geminivirus. From these results, BBTV appears to belong to an undescribed plant virus group which could also include subterranean clover stunt virus, coconut foliar decay virus, faba bean necrotic yellows virus and milk vetch dwarf virus.

Amino Acid Sequence↗
PubMed1987

Analyses of alpha/beta-type gliadin genes from diploid and hexaploid wheats.

The alpha/beta-gliadin genes isolated from both hexaploid wheat (cv. Yamhill) and the diploid A genome progenitor Triticum urartu had remarkably similar sequences and differ by only a few point mutations. Primer extension analysis indicated that the transcriptional start points for individual genes in the family cluster within a few nucleotides. Comparison of the promoter region of several alpha/beta-gliadin and B-hordein genes reveals two conserved regions at about -130 and -250 bp. DNA from the hexaploid cultivars, Cheyenne and Chinese Spring, and the diploid progenitors T. urartu and Aegilops squarrosa was analysed by Southern blotting. Restriction fragment lengths of the alpha/beta-gliadin genes varied only slightly between the various wheats, although the overall copy number varied significantly. A region between approx. -1700 and -700 bp upstream from the TATA box was highly repeated in all three wheat genomes. For the hexaploid-derived gene, over 1700 bp of sequence upstream from the TATA box was determined, revealing an additional open reading frame between approx. -1550 and -1250 bp relative to the gliadin TATA box. Northern blot analysis indicated that RNA homologous to this repeated sequence family was present only in developing seed and accumulated to a maximum at late stages of maturation.

Base Sequence↗
PubMed2002

Human calcium-sensing receptor gene. Vitamin D response elements in promoters P1 and P2 confer transcriptional responsiveness to 1,25-dihydroxyvitamin D.

The calcium-sensing receptor (CASR), expressed in parathyroid chief cells, thyroid C-cells, and cells of the kidney tubule, is essential for maintenance of calcium homeostasis. Here we show parathyroid, thyroid, and kidney CASR mRNA levels increased 2-fold at 15 h after intraperitoneal injection of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in rats. Human thyroid C-cell (TT) and kidney proximal tubule cell (HKC) CASR gene transcription increased approximately 2-fold at 8 and 12 h after 1,25(OH)2D3 treatment. The human CASR gene has two promoters yielding alternative transcripts containing either exon 1A or exon 1B 5'-untranslated region sequences that splice to exon 2 some 242 bp before the ATG translation start site. Transcriptional start sites were identified in parathyroid gland and TT cells; that for promoter P1 lies 27 bp downstream of a TATA box, whereas that for promoter P2, which lacks a TATA box, lies in a GC-rich region. In HKC cells, transcriptional activity of a P1 reporter gene construct was 11-fold and of P2 was 33-fold above basal levels. 10(-8) m 1,25(OH)2D3 stimulated P1 activity 2-fold and P2 activity 2.5-fold. Vitamin D response elements (VDREs), in which half-sites (6 bp) are separated by three nucleotides, were identified in both promoters and shown to confer 1,25(OH)2D3 responsiveness to a heterologous promoter. This responsiveness was lost when the VDREs were mutated. In electrophoretic mobility shift assays with either in vitro transcribed/translated vitamin D receptor and retinoid X receptor-alpha, or HKC nuclear extract, specific protein-DNA complexes were formed in the presence of 1,25(OH)2D3 on oligonucleotides representing the P1 and P2 VDREs. In summary, functional VDREs have been identified in the CASR gene and provide the mechanism whereby 1,25(OH)2D up-regulates parathyroid, thyroid C-cell, and kidney CASR expression.

Alternative Splicing↗
PubMed1991

Promoter activity and distance constraints of one versus two Sp1 binding sites.

In the simple adenovirus 2 early 1B (E1B) promoter, moving the single Sp1 transcription factor binding site (GC box) from its wild-type position eight base pairs (bp) from the TATA box to 30 bp upstream is equivalent to its deletion (Wu, L., and Berk, A. (1988) Genes & Dev. 2, 403-411). In more complex promoters, multiple upstream elements regulate transcription over greater distances. To understand these spacing constraints, we placed two GC boxes in phase at various distances from the E1B TATA box. Whereas one GC box at eight bp from TATA increases transcription in vivo 5-fold compared with TATA alone (Wu, L., Rosser, D. S. E., Schmidt, M. C., and Berk, A. (1987) Nature 326, 512-515), two GC boxes increased in vivo transcription 25-30-fold. Transcriptional stimulation by two GC boxes fell off rapidly in vivo at 30 bp from TATA, remained approximately constant through 50 bp, then decreased again at 70 bp. Consequently, two GC boxes have a multiplicative effect at eight bp from TATA and continue to stimulate transcription at a greater distance from TATA than a single Sp1 site. Quantitatively different results were observed for in vitro transcription using a nuclear extract; a more linear fall off with increasing distance from TATA was observed. Separating the two GC boxes progressively decreased transcription. E1A stimulation of these promoters in vivo indicates that Sp1 transcription control is independent of E1A transactivation.

Adenoviridae↗
PubMed1996

DNA bending between upstream activator sequences increases transcriptional synergy.

To determine if DNA bending enhances transcriptional synergy between tandem upstream activators, we constructed a series of synthetic promoters containing either an AP1 or an NF1 site linked to four copies of the putative transcription factor binding site ACGTGA incorporated into an intrinsic DNA bending sequence. In transient transfection, the intrinsic bending sequence between AP1 and the TATA box or between NF1 and the TATA box elicited a strong synergistic activation of transcription. The synergistic activation of transcription was greatly reduced when the intrinsic bending sequence was replaced with a mutant sequence which retains the binding site, but does not bend the DNA, and when the intrinsic bending sequence was upstream of the AP1 or NF1 sites. These data indicate that bent DNA located between upstream activator sequences can facilitate transcriptional synergism between bound activators.

Animals↗
PubMed1987

The inducible lac operator-repressor system is functional in mammalian cells.

We have investigated the use of the Escherichia coli lac operator-repressor system to regulate expression of transfected genes in mammalian cells. We show that lac repressor produced in mouse L cells by transfection of a lacl expression vector blocks transcription of an MSV-CAT fusion gene when the lac operator is inserted at any one of the following sites within the promoter region: between the initiation codon (ATG) and the transcription start site; between the transcription start and TATA box regions; or upstream of the TATA box region. This last result suggests that the repressor may prevent protein-protein interactions involved in transcription activation. The inducer IPTG causes a marked derepression of CAT expression. The lac repressor-operator complex may be useful as an on/off "switch" in the regulation of gene expression for gene transfer experiments.

Animals↗
PubMed1997

Cloning and characterization of the type I inositol 1,4,5-trisphosphate receptor gene promoter. Regulation by 17beta-estradiol in osteoblasts.

The inositol 1,4,5-trisphosphate (InsP3) receptor is essential for signal Ca2+ release from intracellular stores and for capacitative Ca2+ entry. We have isolated the promoter and proximal DNA segments of the human type I InsP3 receptor gene. Transcription initiation in human G-292 osteosarcoma and HL-60 promyelocytic leukemia cells was shown to occur predominantly from an adenine residue located 39 base pairs downstream of a consensus TATA box element. Upstream DNA including the TATA box promoted directional transcription of a chloramphenicol acetyltransferase reporter gene when transfected into G-292 cells. A negative regulatory element in the distal promoter and a positive element in the proximal region were identified by deletion mapping and transcription assays. The proximal region enhanced transcription in response to 12-O-tetradecanoylphorbol-13-acetate or serum, but conferred transcriptional repression in response to 1,25-dihydroxyvitamin D3 or 17beta-estradiol. The repressive effect of 17beta-estradiol was mediated by the nuclear estrogen receptor, as estrogen-dependent transcriptional repression was inhibited by the antiestrogen tamoxifen and the estrogen receptor antagonist ICI 182,780. This is the first study of the type I InsP3 receptor gene promoter, and the results suggest a mechanism by which chronic estrogen treatment of osteoblasts affects type I InsP3 receptor gene expression, signal transduction, and secretion.

Base Sequence↗
PubMed1996

Genomic structure and regulation of Kcn1, a cGMP-gated potassium channel.

We recently cloned a novel rabbit gene that encodes a 725-amino acid protein (Kcn1) (Y. Yao, A. S. Segal, P. Welling, X. Zhang, C. M. McNicholas, D. Engel, E. L. Boulpaep, and G. Desir. Proc. Natl. Acad. Sci. USA 92: 11711-11715, 1995). Kcn1 RNA injected in Xenopus oocytes leads to the expression of potassium channels that are specifically activated by guanosine 3',5'-cyclic monophosphate (cGMP). Northern blot and ribonuclease (RNase) protection analysis show that Kcn1 is differentially expressed in kidney, aorta, brain, and heart. The purpose of present study is to determine the structure of Kcn1 gene, analyze the promoter region, and identify cis-regulatory elements responsible for transcription. We find that the coding region of Kcn is intronless. The major transcription initiation site was identified by primer extension. Sequence analysis of the 5'-flanking region indicates that, although the gene lacks a typical TATA box, it does have a TATA-box-like region (-TAT-). Using luciferase reporter constructs transfected in the porcine kidney cell line (LLC-PK1), the promoter region and a 5' enhancer element were identified by deletion analysis. Phorbol esters (12-O-tetradecanoylphorbol-13-acetate) and forskolin stimulated Kcn1 gene expression 2.5- and 3.5-fold, respectively. In conclusion, we have identified the region of the novel potassium channel gene, Kcn1, that contains the promoter, a 5' enhancer, and several cis-regulatory elements and shown that gene transcription is stimulated by cAMP and phorbol esters.

Animals↗
PubMed1996

Characterization of the promoter region for flt-1 tyrosine kinase gene, a receptor for vascular endothelial growth factor.

The flt-1 gene encodes a Vascular Endothelial Growth Factor receptor, (Flt-1), whose expression is restricted to vascular endothelial cells. To characterize the cell type specificity of flt-1 gene expression, we isolated the upstream genomic DNA of the human flt-1 gene and identified a single transcription initiation site 29 bp downstream of a TATA box. DNA sequencing revealed that one TATA box, four GC boxes, nine ETS motifs and one CRE motif were present in the upstream 489 bp region of exon 1. Functional analyses using CAT plasmids in 293E1 cells, which express significant levels of the flt-1 gene, showed that the -229 to +8 region is essential for the cell type-specific expression of this gene. Deletion mutant analysis also pointed to the possible existence of negative and positive regulatory elements in the region -911 to -435, and +8 to +276, respectively. These results suggest that multiple regulatory factors are involved in the transcriptional regulation of the flt-1 gene expression in a cell type-specific, or a more ubiquitous manner.

Base Sequence↗
PubMed1990

Weight matrix descriptions of four eukaryotic RNA polymerase II promoter elements derived from 502 unrelated promoter sequences.

Optimized weight matrices defining four major eukaryotic promoter elements, the TATA-box, cap signal, CCAAT-, and GC-box, are presented; they were derived by comparative sequence analysis of 502 unrelated RNA polymerase II promoter regions. The new TATA-box and cap signal descriptions differ in several respects from the only hitherto available base frequency Tables. The CCAAT-box matrix, obtained with no prior assumption but CCAAT being the core of the motif, reflects precisely the sequence specificity of the recently discovered nuclear factor NY-I/CP1 but does not include typical recognition sequences of two other purported CCAAT-binding proteins, CTF and CBP. The GC-box description is longer than the previously proposed consensus sequences but is consistent with Sp1 protein-DNA binding data. The notion of a CACCC element distinct from the GC-box seems not to be justified any longer in view of the new weight matrix. Unlike the two fixed-distance elements, neither the CCAAT- nor the GC-box occurs at significantly high frequency in the upstream regions of non-vertebrate genes. Preliminary attempts to predict promoters with the aid of the new signal descriptions were unexpectedly successful. The new TATA-box matrix locates eukaryotic transcription initiation sites as reliably as do the best currently available methods to map Escherichia coli promoters. This analysis was made possible by the recently established Eukaryotic Promoter Database (EPD) of the EMBL Nucleotide Sequence Data Library. In order to derive the weight matrices, a novel algorithm has been devised that is generally applicable to sequence motifs positionally correlated with a biologically defined position in the sequences. The signal must be sufficiently over-represented in a particular region relative to the given site, but need not be present in all members of the input sequence collection. The algorithm iteratively redefines the set of putative motif representatives from which a weight matrix is derived, so as to maximize a quantitative measure of local over-representation, an optimization criterion that naturally combines structural and positional constancy. A comprehensive description of the technique is presented in Methods and Data.

Algorithms↗
PubMed1994

Basal components of the transcription apparatus (RNA polymerase II, TATA-binding protein) contain activation domains: is the repetitive C-terminal domain (CTD) of RNA polymerase II a "portable enhancer domain"?

Regions rich in serine, threonine, and proline residues can be found in transcriptional activation domains, as well as in the N-terminal parts of mammalian TATA-binding proteins, where they are interrupted by polyglutamine stretches. Likewise, the C-terminal domain of the largest subunit of RNA polymerase II contains multiple repeats of the consensus heptapeptide sequence YSPTSPS. To test directly for possible activation functions, we fused the GAL4 DNA-binding domain to the N-terminal domain of human TBP or subdomains of it, and to the C-terminal domain (CTD) of mouse RNA polymerase II or synthetic polymers of a CTD consensus repeat. We found that these chimeric proteins were able to activate transcription when bound to a GAL4 site in front of the TATA box, a function characteristic of transcription factors. However, while subdomains of TBP functioned only from a position close to the TATA box ("promoter" position), multiple repeats of the CTD consensus sequence were also able to mediate transcriptional activation from a remote ("enhancer") position. Our findings suggest that a region of TBP that is unique to mammals functionally cooperates with "proximal" activation domains of promoter-bound transcription factors. They also imply that the C-terminal domain of RNA polymerase II includes a function that is otherwise confined to remote activation domains of enhancer-bound transcription factors. We suggest that the CTD of RNA polymerase II contains a "portable" remote activation domain that may also facilitate chromatin opening within the transcription unit.

Amino Acid Sequence↗
PubMed1993

Regulated activity of the distal promoter-like element of the human corticotropin-releasing hormone gene and secondary structural features of its corresponding transcripts.

Corticotropin-releasing hormone (CRH) plays a major role in the coordination of the stress response. Its gene is expressed in multiple brain regions, the peripheral sympathetic system and the placenta, as well as in peripheral inflammatory sites where CRH acts as a pro-inflammatory cytokine. The human (h) CRH gene, in addition to its primary promoter (TATA box I), has a second distal promoter-like structure (TATA box II) and a functional cyclic adenosine monophosphate-responsive element, all of which are preserved in the rat and ovine genes. To examine the functionality of TATA II, we positioned a 881-bp-long segment of the 5' flanking region of the hCRH gene containing TATA II, but lacking TATA I, upstream from a chloramphenicol acetyltransferase (CAT) reporter gene cloned in a pUC vector. We transfected COS-7 cells with this construct and examined responsiveness of CAT activity to potential stimulants and inhibitors. Phorbol ester (TPA) and forskolin had mild but clear stimulatory effects on CAT expression (approximately 1.5- and approximately 1.3-fold, respectively), with a combined effect of approximately 1.9-fold. Dexamethasone (DEX) inhibited TPA-stimulated CAT activity by approximately 2.6-fold. In contrast, in the presence of a co-transfected glucocorticoid receptor cDNA expression plasmid, DEX augmented TPA-stimulated CAT expression by approximately 3.1-fold. The predicted secondary structures of the primary transcripts employing the distal and proximal promoters had significant differences, which could affect their stability and translatability.2

Base Sequence↗
PubMed1995

Host factors LR1 and Sp1 regulate the Fp promoter of Epstein-Barr virus.

The Epstein-Barr virus EBNA-1 gene product is essential for latent replication of the virus. In transformed cells characterized by the most restricted patterns of viral latent gene expression, EBNA-1 transcription is driven from the Fp promoter. We have used genetic and biochemical techniques to study the promoter-proximal elements that regulate Fp expression in B cells. We show that a 114-bp fragment of DNA spanning the Fp "TATA" box functions as a remarkably active transcriptional regulatory element in B cells. Two host factors, Sp1 and LR1, regulate Fp transcription from the promoter-proximal region. Sp1 binds a single site just downstream of the TATA box, and LR1 binds two sites just upstream of the TATA box. Transcripts from both the viral genome and the minimal promoter initiate at the same unique site, and one function of LR1 at Fp is to direct initiation to this unique start site. In contrast to Sp1, which is ubiquitous, LR1 is present only in activated B cells and may contribute to cell-type-specific transformation by Epstein-Barr virus.

Antigens, Viral↗
PubMed1994

Molecular heterogeneity of naturally occurring sn-glycerol-3-phosphate dehydrogenase low-activity variants in Drosophila melanogaster.

Northern analyses of two low-activity sn-glycerol-3-phosphate dehydrogenase (Gpdh) alleles extracted from natural populations of Drosophila melanogaster showed that one of them, GpdhACyg22, produced wild-type levels of a normal sized (1.7-kb) mRNA but the other, GpdhAMB5, had very low levels of a 1.7-kb mRNA together with low levels of a transcript 200 bp larger. The two variant genes were cloned and sequenced. Compared with normal activity alleles, there were two nucleotide differences in the DNA sequence of GpdhACyg22 which were in first-codon positions and would be expected to give rise to Asn-13-->Tyr and Arg-272-->Cys substitutions. The second of these changes is most likely to account for the altered properties of the enzyme. In contrast, none of the nucleotide differences in GpdhAMB5 would give rise to amino acid substitutions, but a 76-bp deletion in the 5' region removed the normal TATA box and there was a 20-bp insertion in the same region. One of the two transcripts was derived from the use of a substitute TATA box sequence in the insertion, but the 1.9-kb transcript had heterogeneous 5' ends that were not associated with substitute TATA box sequences. The two transcripts either are produced at a lower rate or are less stable than the normal mRNA.

Alleles↗
PubMed1998

Implication of 5'-flanking sequence elements in expression of a plant tRNA(Leu) gene.

A comparison of 5'-flanking sequences from 68 different nuclear plant tRNA genes was analyzed to find consensus sequences. Three conserved features stood out, all of which are present in the tRNA(Leu) gene used in this study: (1) a high proportion of A and T residues upstream of all tRNA genes; (2) a region of low duplex stability about 30-35 bp before the coding sequence, often containing a TATA-box like motif; (3) a CAA triplet in the region of the presumed transcription start. The effect of replacement of the AT-rich upstream sequences with GC-rich sequences or unrelated AT-rich sequences was tested by progressive deletions and by inserting randomly cloned sequences upstream of the tRNA gene. GC-rich 5'-flanking sequences were found to be generally incompatible with high levels of expression. The TATA-box like motifs and the CAA triplet were removed or altered by deletion or directed mutagenesis. Mutation of the CAA triplet significantly decreased expression of the tRNA(Leu) gene, suggesting that this CAA triplet is important for transcription efficiency, but mutation or elimination of the TATA-box like motifs generally had little effect. The presence or absence of each of these features in tRNA genes from other organisms is discussed; there are clear and interesting differences between plant tRNA genes and those of yeast and mammals.

Base Sequence↗
PubMed1982

The yeast his3 promoter contains at least two distinct elements.

Phenotypic analysis of 65 mutations indicates that the yeast his3 promoter is composed of at least two separate regions of DNA. Each is necessary, but neither is sufficient for wild-type levels of his3 expression. Deletion mutations that destroy either promoter element express his3 poorly or not at all. The upstream element is located between 112 and 155 base pairs before the site of transcriptional initiation (nucleotides -112 to -155). A comparison of derivatives strongly suggests that the downstream element maps somewhere between nucleotides -32 and -52 and includes a sequence between nucleotides -45 and -52. This location coincides with sequences conserved before most eukaryotic genes(the TATA box region). By using derivatives in which his3 sequences are replaced by a small fragment of coliphage M13 DNA, three properties of the his3 promoter were established. First, his3 TATA box deletions fail to express his3 because they lack specific sequences and not because they disrupt spacing relationships between other sequences. Second, the TATA box region can be replaced functionally by the one orientation of the M13 DNA fragment that contains a TATA-like sequence. Third, the distance between the two elements (normally 90 base pairs) can be varied between 40 and 160 base pairs without markedly affecting promoter function. These results strongly suggest that yeast RNA polymerase II, unlike its Escherichia coli counterpart, does not bind simultaneously to both promoter elements, and they add further support to the view that the upstream element is not part of a transcriptionally competent binding site. This ability of the his3 upstream promotor element to act at a long and variable distance is similar to properties of viral enhancer sequences and is reminiscent of position effects in yeast.

Base Sequence↗
PubMed2003

A shared surface of TBP directs RNA polymerase II and III transcription via association with different TFIIB family members.

The TATA box binding protein TBP is highly conserved and the only known basal factor that is involved in transcription by all three eukaryotic nuclear RNA polymerases from promoters with or without a TATA box. By mutagenesis and analysis on a selected set of four model pol II and pol III TATA box-containing and TATA-less promoters, we demonstrate that human TBP utilizes two modes to achieve its versatile functions. First, it uses a different set of surfaces on the conserved and structured TBP core domain to direct transcription from each of the four model promoters. Second, unlike yeast TBP, human TBP can use a shared surface to interact with two different TFIIB family members--TFIIB and Brf2--to initiate transcription by different RNA polymerases.

DNA↗
PubMed2004

Expression of the p16INK4a gene and methylation pattern of CpG sites in the promoter region in rat tumor cell lines.

Loss of p16(INK4a) protein expression has frequently been related to DNA methylation in association with gene silencing. Although the methylation status of exon1alpha for p16(INK4a) involvement in various cancers has been extensively analyzed, it has been pointed out that some inconsistencies existed in its relationship to gene silencing of p16(INK4a). In this study, we focused on the expression and methylation status in the regions of nt -478 to -201, containing a putative TATA box (nt -401 to -396), and nt -233 to 26, both in a recently cloned 5' upstream region of rat p16(INK4a). We showed that rat lung adenocarcinoma RLCNR did not express the p16(INK4a) gene, whereas rat osteosarcoma COS1NR and malignant fibrous histiocytoma MFH1NR both expressed it at levels similar to normal fibroblasts, even though the region of nt -233 to 26 was hypermethylated in COS1NR rather than RLCNR. In contrast, the CpG islands near the putative TATA box region were consistently methylated in RLCNR, but not in COS1NR and MFH1NR, as well as in normal fibroblasts. Treatment with 5-aza 2'-deoxycytidine induced expression of p16(INK4a) gene in RLCNR after 48 h, but no changes were observed in COS1NR and MFH1NR. The results indicated that methylation of CpG islands near a TATA box region played a critical role for gene silencing of the rat p16(INK4a) gene, rather than that of other regions.

Adenocarcinoma↗