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Results for “regulatory sequence design”

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At least 127 records · Page 7Linked to original sources

PubMed1993

Activation of protooncogene c-jun by the X protein of hepatitis B virus.

A specific viral oncogenic mechanism has not been shown for hepatitis B virus (HBV), although persistent HBV infection has been strongly associated with the development of hepatocellular carcinoma (HCC). Most HCCs in HBV carriers contain integrated viral sequences in host DNA and this raises the question of whether such integrations ever contribute to oncogenesis. HBV does contain a gene (designated the hbx gene) which encodes a transcriptional trans-activator protein capable of activating homologous and heterologous regulatory sequences. Hbx has been detected in some human HCC with HBV integrations and the expressed hbx protein appears to have transcriptional transactivating activity. These findings raise the possibility that hbx expression could contribute to hepatocarcinogenesis by activating cellular genes that could contribute to oncogenicity. The possibility that the hbx protein may activate certain protooncogenes was investigated and we found that hbx can activate the protooncogene c-jun promoter. c-Jun was found to be expressed at a very low level in normal liver tissue but at high levels in HCCs of HBV-infected patients.

Base Sequence↗

PubMed2006

RegRNA: an integrated web server for identifying regulatory RNA motifs and elements.

Numerous regulatory structural motifs have been identified as playing essential roles in transcriptional and post-transcriptional regulation of gene expression. RegRNA is an integrated web server for identifying the homologs of regulatory RNA motifs and elements against an input mRNA sequence. Both sequence homologs and structural homologs of regulatory RNA motifs can be recognized. The regulatory RNA motifs supported in RegRNA are categorized into several classes: (i) motifs in mRNA 5'-untranslated region (5'-UTR) and 3'-UTR; (ii) motifs involved in mRNA splicing; (iii) motifs involved in transcriptional regulation; (iv) riboswitches; (v) splicing donor/acceptor sites; (vi) inverted repeats; and (vii) miRNA target sites. The experimentally validated regulatory RNA motifs are extracted from literature survey and several regulatory RNA motif databases, such as UTRdb, TRANSFAC, alternative splicing database (ASD) and miRBase. A variety of computational programs are integrated for identifying the homologs of the regulatory RNA motifs. An intuitive user interface is designed to facilitate the comprehensive annotation of user-submitted mRNA sequences. The RegRNA web server is now available at http://RegRNA.mbc.NCTU.edu.tw/.

Animals↗

PubMed1997

ABM-1 and ABM-2 homology sequences: consensus docking sites for actin-based motility defined by oligoproline regions in Listeria ActA surface protein and human VASP.

Actin-based motility involves a cascade of binding interactions designed to assemble actin regulatory proteins into functional locomotory units. Listeria ActA surface protein contains a series of nearly identical EFPPPPTDE-type oligoproline sequences for binding vasodilator-stimulated phosphoprotein (VASP). The latter is a tetrameric protein with numerous GPP-PPP docking sites for profilin, a 15 kDa regulatory protein that promotes actin filament assembly. Analysis of known actin regulatory proteins led to the identification of distinct Actin-Based Motility homology sequences ABM-1; (D/E)FPPPPX(D/E); and ABM-2, XPPPPP (where X denotes G, A, L, and S).

Actins↗

PubMed2003

Regulation of FRUA expression during vegetative growth and development of Myxococcus xanthus.

Expression of the fruA gene, encoding a putative transcription factor essential for fruiting body formation of Myxococcus xanthus, is specifically activated during development. In the present study, we have analyzed the mechanism of the transcriptional regulation of fruA expression. From gel retardation and footprinting assays using various fruA regulatory regions as probes and competitors, a protein designated factor X was found to specifically bind to a sequence (xbs) located downstream of the transcription-initiation site (+78 to +94) of the fruA gene. Factor X activity was present during vegetative growth and decreased during early development. Analysis of promoter activities of various segments of the fruA regulatory region using the lacZ reporter gene in vivo indicated that a DNA segment extending 45-bp upstream from the transcription-initiation site was required for developmentally regulated fruA expression at a low level. In addition, cis-acting regulatory regions located upstream and downstream of the fruA promoter region and including C-box and xbs, were found to be involved in regulation of fruA expression during development. When inserted into the vegA gene, the xbs element inhibited vegA expression during vegetative growth. Together with previously reported results, our studies reveal that fruA expression is regulated by both positive and negative mechanisms during the M. xanthus life cycle.

Bacterial Proteins↗

PubMed2006

[PCR-SSCP polymorphism of FSHbeta gene and its relationship with prolificacy of Jining grey goats].

The follicle-stimulating hormone beta-subunit (FSHbeta) gene was studied as a candidate gene for the prolificacy in Jining Grey goats. According to the sequence of ovine FSHb gene, nine pairs of primers were designed to detect single nucleotide polymorphisms of 5' regulatory region, exon 1 and exon 2 of FSHbeta gene in both high fecundity breed (Jining Grey goat) and low fecundity breeds (Liaoning Cashmere goat, Boer goat and Angora goat) by PCR-SSCP. The results indicated that the homology of nucleotide sequence of this fragment between goat and sheep was 98 percent. Only the products amplified by primer P9 displayed polymorphism. Three genotypes (AA, AB and AC) were detected in Jining Grey goats and Liaoning Cashmere goats. Three genotypes (AA, CC and AC) were detected in Boer goats. Six genotypes (AA, BB, CC, AB, AC and BC) were detected in Angora goats. Sequencing revealed a G-->A mutation at 94 bp of exon 2 of FSHbeta gene in the BB genotype in comparison to the AA genotype and a C-->T mutation at 174 bp of exon 2 of FSHbeta gene in the CC genotype in comparison to the AA genotype. The former mutation resulted in an amino acid change: alanine-->threonine, and the latter mutation did not cause any amino acid change. Genotype frequency of AA, AB and AC was 0.686, 0.137 and 0.177 in Jining Grey goats, respectively. The does with genotype AA had 0.78 (P<0.05) or 0.64 (P<0.05) kids more than those with genotype AB or AC in Jining Grey goats, respectively.

Amino Acid Sequence↗

PubMed1996

Characterization of a seventh family of immunoglobulin heavy chain VH gene segments in the channel catfish, Ictalurus punctatus.

A VH gene segment that could not be assigned to any of the six known VH gene families of the channel catfish was identified in a genomic clone containing VH gene segments. This gene segment (designated VH7.1) exhibited the structural features characteristic of vertebrate VH genes, specifically potential upstream regulatory sequences, a leader sequence split by an intron, a reading frame that could be readily divided into framework and complementarity determining regions, and a 3' recombination signal sequence. Two regions of nucleotide deletions coupled with degeneracy in the nonamer sequence indicate that this VH gene segment is a pseudogene. Genomic DNA restricted with different enzymes and hybridized under stringent conditions with probes derived from VH7.1 showed that 8-10 bands were present in Southern blots. Reverse transcriptase PCR approaches were used to determine if any of these related sequences were expressed. Sequence analysis of cloned PCR products indicates that different VH gene segments exhibiting > 80% similarity to germline VH7.1 are expressed. Multiple sequence alignments showed that the expressed cVH7a cDNA sequence shared less than 60% nucleotide similarity with representative cDNA sequences from the other known catfish VH gene families. These combined results thus fulfil the criteria for the definition of a new family of catfish VH gene segments. This newly defined, small VH family is designated VH7.

Amino Acid Sequence↗

PubMed2004

Identification of a negative regulatory element which regulates basal transcription of a multidrug resistance gene CDR1 of Candida albicans.

We have earlier shown that transcriptional activation of the Candida drug resistance gene, CDR1, is linked to various stresses wherein a proximal promoter (-345 bp from the transcription start point (TSP)) was found to be predominantly more responsive. In this study we have examined basal expression of the CDR1 proximal promoter by employing a Renilla luciferase reporter system. We observed that upon sequential deletion of the proximal promoter, there was modulation in basal reporter activity. The reporter activity was highest (2.3-fold) in NGY261 (-261 bp from TSP), and was reduced upon subsequent deletions. DNase I footprinting revealed four protected regions (W1, W2, W3 and W4) in the proximal promoter which could represent possible trans-acting factor binding sites and thus might be involved in CDR1 expression. Site-directed mutational analysis of three of these protected regions did not significantly affect the basal reporter activity, however, the mutation of W1 led to a considerable enhancement in reporter activity (approximately 4-fold) and was designated a negative regulatory element (NRE). Mutation as well as deletion of the W1 sequence in the native promoter (-1147 bp from TSP) and sequential deletion of the 5'-flanking region-harboring W1 (NRE) also resulted in enhanced promoter reporter activity. When the reporter activity of native (NPY1147) and NRE-mutated (NGYM1147) promoter integrants was monitored throughout the growth phase of Candida albicans, there was modulation in reporter activity in both integrants, but interestingly the level of basal reporter activity of the NRE-mutated promoter was always approximately 3-fold higher than that of the native promoter. UV cross-linking and affinity purification confirmed that a purified approximately 55-kDa nuclear protein specifically interacts with the NRE. Taken together, we have identified a NRE and purified its interactive protein, which may be involved in controlling basal expression of CDR1.

Base Sequence↗

PubMed1997

'Locked-on' and 'locked-off' signal transduction mutations in the periplasmic domain of the Escherichia coli NarQ and NarX sensors affect nitrate- and nitrite-dependent regulation by NarL and NarP.

The Escherichia coli NarX, NarQ, NarL and NarP proteins comprise a two-component regulatory system that controls the expression of many anaerobic electron-transport and fermentation-related genes in response to nitrate and nitrite. Either of the two sensor-transmitter proteins, NarX and NarQ, can activate the response-regulator proteins, NarL and NarP, which in turn are able to bind at their respective DNA regulatory sites to modulate gene expression. NarX contains a conserved 17 amino acid sequence, designated the 'P-box' element, that is essential for nitrate sensing. In this study we characterize narQ mutants that also confer altered nitrate control of NarL-dependent nitrate reductase (narGHJI) and fumarate reductase (frdABCD) gene expression. While some narQ mutations cause the constitutive activation or repression of reporter-gene expression even when the cells are grown in the absence of the nitrate signal (i.e. a 'locked-on' phenotype), other mutations abolish nitrate-dependent control (i.e. a 'locked-off' phenotype). Interestingly the narQ (A42-->T) and narQ (R50-->Q) mutations along with the analogous narX18 (A46-->T) and narX902 (R54-->E) mutations also confer a 'locked-on' or a 'locked-off' phenotype in response to nitrite, the second environmental signal detected by NarQ and NarX. Furthermore, these narQ and narX mutations also affect NarP-dependent gene regulation of nitrite reductase (nrfABCDEFG) and aeg-46.5 gene expression in response to nitrite. We therefore propose that the NarQ sensor-transmitter protein also detects nitrate and nitrite in the periplasmic space via its periplasmic domain. A signal transduction model, which we previously proposed for NarX, is now extended to NarQ, in which a nitrate- or nitrite-detection event in the periplasmic region of the cell is followed by a signal transduction event through the inner membrane to the cytoplasmic domain of NarQ and NarX proteins to modulate their protein kinase/phosphatase activities.

Alanine↗

PubMed1994

Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate.

Pseudomonas putida PRS2000 is chemotactic to 4-hydroxybenzoate and other aromatic acids. This behavioral response is induced when cells are grown on 4-hydroxybenzoate or benzoate, compounds that are degraded via the beta-ketoadipate pathway. Isolation of a transposon mutant defective in 4-hydroxybenzoate chemotaxis allowed identification of a new gene cluster designated pcaRKF. DNA sequencing, mutational analysis, and complementation studies revealed that pcaR encodes a regulatory protein required for induction of at least four of the enzymes of the beta-ketoadipate pathway and that pcaF encodes beta-ketoadipyl-coenzyme A thiolase, the last enzyme in the pathway. The third gene, pcaK, encodes a transporter for 4-hydroxybenzoate, and this protein is also required for chemotaxis to aromatic acids. The predicted PcaK protein is 47 kDa in size, with a deduced amino acid sequence indicative of membership in the major facilitator superfamily of transport proteins. The protein, expressed in Escherichia coli, catalyzed 4-hydroxybenzoate transport. In addition, whole cells of P. putida pcaK mutants accumulated 4-hydroxybenzoate at reduced rates compared with that in wild-type cells. The pcaK mutation did not impair growth at the expense of 4-hydroxybenzoate under most conditions; however, mutant cells grew somewhat more slowly than the wild type on 4-hydroxybenzoate at a high pH. The finding that 4-hydroxybenzoate chemotaxis can be disrupted without an accompanying effect on metabolism indicates that this chemotactic response is receptor mediated. It remains to be determined, however, whether PcaK itself is a chemoreceptor for 4-hydroxybenzoate or whether it plays an indirect role in chemotaxis. These findings indicate that aromatic acid detection and transport are integral features of aromatic degradation pathways.

Acetyl-CoA C-Acyltransferase↗

PubMed1997

Optimization of the human factor VIII complementary DNA expression plasmid for gene therapy of hemophilia A.

While the gene delivery vehicle is critical for the efficacy of human factor VIII gene therapy, optimization of the potency and duration of the factor VIII gene that is delivered is equally important in light of the poor transcription and translation characteristics of this gene. We discuss here a systematic approach to optimization of factor VIII complementary DNA expression by analysis of specific elements engineered into the transcription unit and other positions in the expression plasmid. Within the transcription unit we have engineered different 5' and 3' sequence modifications and tested them for factor VIII expression in human liver cells. These changes incorporate liver-specific promoter and enhancer sequences and regulatory elements affecting RNA export. Specifically, the thyroid hormone-binding globulin promoter and alpha 1 microglobulin/bikunin enhancer were tested and a synthetic 5' intron was compared to a 3' post-transcriptional regulatory element on factor VIII expression levels. For translation optimization, a leader sequence was designed to be of optimum length, have no RNA secondary structure and contain the optimal translation initiation sequence. Finally, we discuss areas for plasmid optimization, which include removal of near-consensus splicing sequences, the inclusion of strong transcription termination elements and the use of autonomous replicating plasmid sequences for episomal maintenance and enhanced plasmid retention for duration of gene expression.

Carcinoma, Hepatocellular↗

PubMed2026

Alignment-free integration of single-nucleus ATAC-seq across species with sPYce.

Changes in gene regulation largely contribute to differences in cellular identities and phenotypes between species. Single-nucleus assays for transposase-accessible chromatin with sequencing (snATAC-seq) are an efficient strategy to identify putative gene regulatory elements and provide new insight into evolutionary divergence of regulatory programmes. However, no dedicated framework exists to integrate and compare snATAC-seq data across species, while methods designed for single-cell gene expression data have serious limitations. Here we present sPYce, a cross-species snATAC-seq integration method that relies on sequence composition similarities through k-mer histograms of regulatory regions, removing the need for genome alignments to anchor data from different species. sPYce can embed datasets from multiple species into the same mathematical space and permits further downstream analysis steps. We benchmarked sPYce against existing approaches on two publicly available datasets spanning more than 160 myr of evolution, showing that it successfully uncovers conserved cellular programmes while preserving biologically relevant species-specific differences. By comparing cerebellar development in mice and opossums, sPYce identifies regulatory divergence in granule cell differentiation programmes, particularly driven by nuclear factor 1. As an easy-to-use, alignment-free cross-species snATAC-seq integration approach, sPYce opens new perspectives to compare gene regulatory evolution across species.

Animals↗

PubMed1990

Regulatory DNA sequence conserved in the course of BK virus evolution.

Within the genome of human polyomavirus BK (BKV), there exists a noncoding regulatory region toward the late region side of the origin of DNA replication. In most BKV strains isolated by viral culture, this regulatory region contains tandem repeats varying in size. Recently, however, several laboratories isolated new BKV strains (designated as archetypal strains) lacking such repeat sequences. To examine the genetic relationship between archetypal strains, a phylogenetic tree was constructed for seven BKV strains, including three archetypal strains, from DNA sequence data on the late genes, those for leader protein (agnoprotein), and those for structural proteins (VP1, VP2, and VP3). For three strains data previously reported were used, whereas for the others sequences were determined in this study. From total numbers of nucleotide substitutions in each pair of strains, a phylogenetic tree was constructed by the unweighted pair-group method. The phylogenetic tree obtained reveals that BKV strains containing the archetypal regulatory region do not constitute a cluster of closely related strains and that these strains, together with those carrying the major part of the archetypal regulatory region, are widespread in the BKV population. This finding suggests that the basic structure of the archetypal regulatory region has been conserved in the course of BKV evolution.

BK Virus↗

PubMed2003

The completed human genome: implications for chemical biology.

The recently completed human genome sequence represents an enormous opportunity to understand biology and accelerate the development of new therapeutics. However, it also presents equally large logistical, scientific and paradigmatic challenges to efficiently translate the enormous cache of sequence data into functional information that will be the precursor of new drug development. Small-molecule chemical biology applied on a genomic scale promises to speed this translation to novel therapeutics.

Animals↗

PubMed1993

Identification of a sequence within the mouse metallothionein-I gene promoter mediating its basal transcription and of a protein interacting with this element.

Previous studies in our laboratory have shown that a trans-acting factor, which binds to a 106 bp sequence in the mouse metallothionein-I (MT-I) gene, is responsible for the relatively high level of MT-I gene transcription in the liver. Using electrophoretic mobility shift assay, we have now identified a 26 bp sequence within the 106 bp region, which interacts with a trans-activating factor in the liver nuclear extract. This sequence, designated MRE-c', is located between positions -135 and -110 with respect to the transcription start site and comprises the metal regulatory element MRE-c and part of its 5' and 3' flanking sequences. UV cross-linking and Southwestern analysis showed that a protein of an apparent molecular mass of 33,000 specifically interacts with MRE-c'. Deletion of the MRE-c' region resulted in a six- to sevenfold decrease in the MT-I promoter activity, as measured by reduction in chloramphenicol acetyltransferase activity. A comparison of other regulatory domains of the MT-I gene and the potential factors interacting with these sequences indicates that MRE-c' and probably the 33 kDa polypeptide are involved in the constitutive transcription of the MT-I gene.

Animals↗

PubMed1995

Characterization and mapping of the gene encoding mouse proteasome subunit DELTA (Lmp19).

The proteasome subunit DELTA is unusually closely related to the major histocompatibility complex (MHC)-linked proteasome subunit, LMP2. The sequence of a mouse cDNA for DELTA confirms that this 22,100 M(r) proteasome subunit is highly conserved across species. Sequence analysis of the mouse gene encoding DELTA, designated Lmp19, indicates that it consists of six exons and five introns, similar to the Lmp2 gene. The 5' upstream region lacks a TATA regulatory sequence, which is also absent from proteasome genes isolated from Drosophila. BXD recombinant inbred (RI) mice were used to map the potential chromosomal location of Lmp19, and revealed that the DELTA subunit has related sequences present on two different mouse chromosomes, chromosomes 1 and 11. Typing of 89 progeny from a C57BL/6J X Mus spretus DNA backcross panel (BSS) confirmed the chromosome 1 assignment. Southern hybridization with a polymerase chain reaction-generated Lmp19 intron 2-specific probe indicates that the Lmp19 genomic clone corresponds to the sequence on chromosome 11, and further suggests that the chromosome 1 copy represents a processed pseudogene (Lmp19-ps1).

Amino Acid Sequence↗

PubMed2005

The design of transcription-factor binding sites is affected by combinatorial regulation.

BACKGROUND: Transcription factors regulate gene expression by binding to specific cis-regulatory elements in gene promoters. Although DNA sequences that serve as transcription-factor binding sites have been characterized and associated with the regulation of numerous genes, the principles that govern the design and evolution of such sites are poorly understood. RESULTS: Using the comprehensive mapping of binding-site locations available in Saccharomyces cerevisiae, we examined possible factors that may have an impact on binding-site design. We found that binding sites tend to be shorter and fuzzier when they appear in promoter regions that bind multiple transcription factors. We further found that essential genes bind relatively fewer transcription factors, as do divergent promoters. We provide evidence that novel binding sites tend to appear in specific promoters that are already associated with multiple sites. CONCLUSION: Two principal models may account for the observed correlations. First, it may be that the interaction between multiple factors compensates for the decreased specificity of each specific binding sequence. In such a scenario, binding-site fuzziness is a consequence of the presence of multiple binding sites. Second, binding sites may tend to appear in promoter regions that are subject to low selective pressure, which also allows for fuzzier motifs. The latter possibility may account for the relatively low number of binding sites found in promoters of essential genes and in divergent promoters.

Binding Sites↗

PubMed1992

Absence of mutations in the promoter region of the low density lipoprotein receptor gene in a large number of familial hypercholesterolaemia patients as revealed by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis (DGGE) was used in combination with the polymerase chain reaction (PCR) to detect sequence variations in the promoter region of the low density lipoprotein receptor (LDLR) gene. On the basis of calculated predictive melting properties we designed primers to amplify a 447-bp fragment of the promoter region from position -512 to -66, containing previously identified regulatory sequences. Using a primer with a GC-clamp in combination with restriction enzyme digestion, two melting domains could be analysed simultaneously. By oligonucleotide-directed mutagenesis artificial mutants were generated to optimize the conditions and to test the sensitivity of the method. All mutants were readily detected by electrophoresis in a 9% polyacrylamide gel containing a 10%-60% linear denaturing gradient. Using this method, we analysed DNA samples of 350 heterozygous familial hypercholesterolaemia (FH) patients. No mutations were detected, suggesting that mutations in the regulatory elements of the promoter sequence do not play a significant role in the etiology of FH.

Base Sequence↗

PubMed2003

Integrated analysis of yeast regulatory sequences for biologically linked clusters of genes.

Dramatic progress in deciphering the regulatory controls in Saccharomyces cerevisiae has been enabled by the fusion of high-throughput genomics technologies with advanced sequence analysis algorithms. Sets of genes likely to function together and with similar expression profiles have been identified in diverse studies. By fusing an advanced pattern recognition algorithm for identification of transcription factor binding sites with a new method for the quantitative comparison of binding properties of transcription factors, we provide an integrated means to move from expression data to biological insights. The Yeast Regulatory Sequence Analysis system, YRSA, combines standard functions with a novel pattern characterization procedure in an intuitive interface designed for use by a broad range of scientists. The features of the system include automated retrieval of user-defined promoter sequences, binding site discovery by pattern recognition, graphical displays of the observed pattern and positions of similar sequences in the specified genes, and comparison of the new pattern against a collection of binding patterns for characterized transcription factors. The comprehensive YRSA system was used to study the regulatory mechanisms of yeast regulons. Analysis of the regulatory controls of a battery of genes induced by DNA damaging agents supports a putative mediating role for the cell-cycle checkpoint regulatory element MCB. YRSA is available at http://yrsa.cgb.ki.se. [YRSA: ancient Scandinavian name meaning old she-bear (Latin Ursus arctos = brown bear/grizzly).]

Algorithms↗