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Results for “regulatory sequence design”

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At least 109 records · Page 6Linked to original sources

PubMed1999

BTEB2, a Krüppel-like transcription factor, regulates expression of the SMemb/Nonmuscle myosin heavy chain B (SMemb/NMHC-B) gene.

We have recently characterized the promoter region of the rabbit embryonic smooth muscle myosin heavy chain (SMemb/NMHC-B) gene and identified the 15-bp sequence, designated SE1, located at -105 from the transcriptional start site as an important regulatory element for its transcriptional activity in a smooth muscle cell (SMC) line. In this study, we attempted to isolate cDNA clones encoding for the transcription factors that control the expression of the SMemb gene through binding to this cis-regulatory element. We screened a lambdagt11 cDNA library prepared from C2/2 cells, a rabbit-derived SMC line, by using a radiolabeled concatenated oligonucleotide containing SE1 as a probe. Sequence analysis revealed that one of the cDNA clones corresponds to the rabbit homologue of basic transcriptional element binding protein-2 (BTEB2), which has previously been identified as one of the Krüppel-like transcription factor. Gel mobility shift assays and antibody supershift analyses with nuclear extracts from C2/2 cells indicate that BTEB2 is a major component of nuclear factor:SE1 complexes. Furthermore, a glutathione S-transferase-BTEB2 fusion protein binds to the SE1 in a sequence-specific manner. In support of the functionality of BTEB2 binding, basal promoter activity and BTEB2-induced transcriptional activation were markedly attenuated by the disruption of the SE1. In adult rabbit tissues, BTEB2 mRNA was most highly expressed in intestine, urinary bladder, and uterus. BTEB2 mRNA levels were downregulated in rabbit aorta during normal development. Moreover, immunohistochemical analysis indicated a marked induction of BTEB2 protein in the neointimal SMC after balloon injury in rat aorta. These results suggest that BTEB2 mediates the transcriptional regulation of the SMemb/NMHC-B gene and possibly plays a role in regulating gene expression during phenotypic modulation of vascular SMC.

Age Factors↗

PubMed1989

Isolation, characterization, and nucleotide sequence of appY, a regulatory gene for growth-phase-dependent gene expression in Escherichia coli.

A plasmid carrying a regulator gene, designated appY, was found in the screening of an Escherichia coli gene library for clones overproducing AppA, an acid phosphatase which is induced as a culture approaches the stationary phase. In cells containing multicopy plasmids carrying the appY gene, the expression of the chromosomal appY gene was stimulated 10- to 40-fold in the stationary phase and more than 100-fold during exponential growth. The appA plasmid also changed the rate of synthesis of more than 30 other proteins in a growth-phase-dependent way. The appY gene was mapped to 13 min on the E. coli genetic map. The position of the appY gene on the 4.9-kilobase HindIII fragment of the original clone was located by Tn5 mutagenesis and deletion analysis, and the nucleotide sequence of a 1.9-kilobase region containing the gene was determined. The appY gene product was identified as a weakly expressed 243-amino-acid polypeptide which contains a stretch of 20 amino acids showing very good similarity to the conserved DNA-binding domain of repressors and transcriptional activators.

Amino Acid Sequence↗

PubMed2003

DNA vaccines: improving expression of antigens.

DNA vaccination is a relatively recent development in vaccine methodology. It is now possible to undertake a rational step-by-step approach to DNA vaccine design. Strategies may include the incorporation of immunostimulatory sequences in the backbone of the plasmid, co-expression of stimulatory molecules, utilisation of localisation/secretory signals, and utilisation of the appropriate delivery system, for example. However, another important consideration is the utilisation of methods designed to optimise transgene expression. In this review we discuss the importance of regulatory elements, kozak sequences and codon optimisation in transgene expression.

Journal Article↗

PubMed1994

High-copy suppression of glucose transport defects by HXT4 and regulatory elements in the promoters of the HXT genes in Saccharomyces cerevisiae.

HXT4, a new member of the hexose transporter (HXT) family in Saccharomyces cerevisiae was identified by its ability to suppress the snf3 mutation in multicopy. Multicopy HXT4 increases both high and low affinity glucose transport in snf3 strains and increases low and high transport in wild-type strains. Characterization of HXT4 led to the discovery of a new class of multicopy suppressors of glucose transport defects: regulatory elements in the promoters of the HXT genes. We have designated these sequences DDSEs (DNA sequence dependent suppressing element). Multicopy HXT4 and DDSEs in the HXT1, HXT2, HXT3 and HXT4 promoters were found to restore growth to snf3 and grr1 strains on low glucose media. The DDSE in the HXT4 promoter was refined to a 340-bp sequence 450 bp upstream of the HXT4 translational start. This region was found to contain an 183-amino acid open reading frame. Extensive analysis indicates that the DNA sequence itself and not the encoded protein is responsible for suppression. The promoters of SNF3 and of other glycolytic genes examined did not suppress snf3 in multicopy. Suppression of snf3 by DDSE is dependent on the presence of either HXT2 or HXT3.

Amino Acid Sequence↗

PubMed1998

Fertile homozygous transgenic mice expressing a functional truncated herpes simplex thymidine kinase delta TK gene.

Dividing cells expressing the Herpes simplex type 1 thymidine kinase (TK) can be killed upon ganciclovir treatment. Likewise, conditional cell knock-out can be obtained in transgenic mice expressing a TK gene placed under the control of tissue-specific regulatory sequences. Such animals provide powerful experimental systems for assessing the functional role of specific cell populations through their time-controlled ablation. However, whatever the regulatory sequences used, a leaky toxic overexpression of TK in testis renders male TK-transgenic mice sterile and prevents the generation of homozygous TK-expressing animals. To solve this problem, we designed a truncated TK variant (delta TK) not expressed in the testis. We generated transgenic mice expressing delta TK under the control of lymphocyte-specific regulatory sequences derived from the CD4 gene. The delta TK protein expressed in T-lymphocytes allowed the conditional ablation of activated T-cells in vitro and in vivo. Importantly, for one transgenic line we could generate fertile homozygous mice harboring a functional delta TK transgene. delta TK should thus dramatically facilitate the development of transgenic mice expressing a conditional suicide gene.

Animals↗

PubMed1994

Circadian clock- and phytochrome-regulated transcription is conferred by a 78 bp cis-acting domain of the Arabidopsis CAB2 promoter.

The Arabidopsis CAB2 promoter was used to define the terminal genomic targets that are subject to regulation by the circadian clock. An in vivo cab::luciferase bioluminescent marker was used to enable the assaying of the expression of chimeric constructs with unprecedented sensitivity and time resolution in living seedlings. Dissection of -322 to +1 of the CAB2 promoter has revealed several interesting features: it was demonstrated that the 323 bp fragment contains at least one strong general positive element. The positive element contains an ACGT core sequence specifically bound by a protein activity, termed CUF-1, and contributes to high level expression but is not required for phytochrome- or circadian-regulation. Moreover, a 78 bp domain was defined that confers both circadian- and phytochrome-regulation upon heterologous promoters. Conserved GATA sequences within the 78 bp regulatory domain are specifically bound by a protein factor designated CGF-1. The binding specificity of CGF-1 appears to be related to the GT-family of trihelix DNA-binding proteins. The role of these DNA-protein interactions is discussed in terms of clock- and phytochrome regulation, and their relevance as targets for pathways defined by photomorphogenic mutants.

Arabidopsis↗

PubMed1996

Multiple nuclear regulatory proteins bind a single cis-acting promoter element to control basal transcription of the human alpha 4 integrin gene in corneal epithelial cells.

Expression of the fibronectin-binding integrin alpha 4 beta 1 has been postulated to be an important event in the process of corneal epithelial wound healing. In a previous study, we identified upstream positive and negative cis-acting regulatory elements that are needed to modulate the transcriptional activity of the human alpha 4 integrin subunit gene promoter in primary cultures of rabbit corneal epithelial cells. We have shown that most of the basal activity directed by this promoter was dependent on the presence of a cis-acting DNA sequence designated the alpha 4.1 element, centered at position -45 relative to the human alpha 4 mRNA start site. Here, we demonstrate that five distinct nuclear regulatory proteins (designated Bp1 to Bp5) from rabbit corneal epithelial cells possess the ability to bind the alpha 4.1 element in a specific manner in vitro. However, when they are combined together, only two of them (Bp2 and Bp5) retained their ability to interact with their specific target sequence in in vitro assays. The apparent molecular masses of the Bp1 to Bp5 proteins were determined and found to be of 91, 74, 59, 45, and 39 kD, respectively. Electrophoretic mobility-shift assays (EMSAs) indicated that only Bp2 also possesses the ability to bind the alpha 4.2 element, a site homologous to alpha 4.1 which plays a minor role in alpha 4 gene expression. Despite the presence of three Ets binding sites in the immediate vicinity of alpha 4.1, competition experiments in EMSA clearly indicate that Bp1, Bp2, Bp4, and Bp5 do not belong to the Ets family of transcription factors. Insertion of both alpha 4.1 and alpha 4.2 upstream from the basal promoter of the mouse p12 gene provided evidence that both elements have the ability to modulate basal expression driven from a heterologous promoter. alpha 4.1 was shown to function as an activator, whereas alpha 4.2 acted as a repressor in a manner that is dependent on its orientation, further stressing the critical regulatory function played by these two elements on alpha 4 gene basal expression.

Animals↗

PubMed1998

Amplification of JC virus regulatory DNA sequences from cerebrospinal fluid: diagnostic value for progressive multifocal leukoencephalopathy.

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease in the central nervous system caused by a ubiquitous human polyomavirus designated as JC virus (JCV). PML affects individuals with decreased immune competence and is now one of the common opportunistic infections in patients with AIDS. JCV DNAs in the brain of PML patients contain various PML-type regulatory regions that were generated from the archetypal regulatory region during persistence. Recently, many studies have suggested that detection of JCV DNA from the cerebrospinal fluid (CSF) may offer a tool for diagnosing PML. However, in all of these studies, coding sequences within the T antigen or capsid protein gene have been targeted for amplification. To amplify the JCV regulatory region, we established a nested PCR that could efficiently amplify the regulatory region from most JCV subtypes prevalent in the world. Using this PCR, we amplified JCV regulatory regions from the CSF samples from 4 patients strongly suspected of PML, whereas amplification was negative from 80 CSF samples from patients without PML. Sequencing of the amplified fragments revealed that they had unique deletions and/or duplications. Furthermore, in 3 PML patients, we analyzed the structures of regulatory regions derived from the brain as well as CSF. In each of these cases, the major regulatory sequence of both origins were identical. This finding indicates that JCV DNA in brain lesions is excreted in the CSF. Since the structures of PML-type JCV regulatory regions are unique to individual patients, the current PCR, if the amplified fragments are sequenced, can eliminate false positives that may arise from contaminations.

Brain↗

PubMed1992

Regulation of CYP1A1 expression.

CYP1A1 is considered to be involved mainly in oxidative metabolism of exogenous chemicals and drugs. Synthesis of this hemoprotein is induced in livers, lungs, and other tissues of experimental animals by the administration of these chemicals. Regulatory mechanisms of the induction process of the protein have been investigated by the DNA transfer method using the isolated genomic DNA. At least two kinds of cis-acting regulatory DNA sequences are localized 5' upstream of the gene. One is distributed five times in a relatively wide range from -0.5 to -3.5 kb and functions as an inducible enhancer-designated xenobiotic responsive element or XRE. The other is localized just upstream of the TATA sequence and acts as a regulatory element for the constitutive expression. The two DNA elements are required for a high level of the inducible expression. Their cognate DNA binding factors are recognized in the nuclear extracts of Hepa-1 cells and rat liver cells which show the inducible expression of CYP1A1 in response to the inducer. This paper discusses the regulatory mechanisms of CYP1A1 gene expression by summarizing the present state of knowledge about properties of the DNA regulatory elements and their cognate DNA-binding factors.

Amino Acid Sequence↗

PubMed1998

Pharmacodynamic aspects of peptide administration biological response modifiers.

Cytokines, growth factors and other recombinant proteins have been one of the most rapidly growing areas of pharmaceuticals. Further, the development of these bio-engineered drugs is occurring at an astonishing pace with rapid preclinical and clinical development and licensing by regulatory agencies. In addition, the availability of the gene sequences and rational drug design technologies have resulted in a rapid development of engineered genes, proteins and peptidomimetics. In contrast to traditional pharmacophores, which are developed based on the identification of the maximum tolerated dose (MTD), most recombinant proteins have abnormal biodistributions, and pharmacodynamic and pharmacokinetic attributes. Within this chapter, representative cytokines including interferon-alpha (IFN-alpha), IFN-gamma and interleukin-2 are used to discuss the pharmacodynamic aspects of protein/peptide administration that are important in the development of these drugs. This includes the conceptual need for chronic immunoaugmentation for optimal therapeutic activity; the need to consider the pharmacokinetics of administration to optimize drug delivery and the nonlinear dose response relationship, which can result in a bell shaped dose response. Furthermore, these therapeutics have maximal potential in an adjuvant protocol and their development in combination with high-dose chemotherapy and stem cell rescue is discussed. The strategies for combination chemotherapy and immunotherapy, while holding great promise, require close attention to the pharmacodynamics of protein administration in order to impact on failure free and overall survival.

Journal Article↗

PubMed1998

Identification of csrR/csrS, a genetic locus that regulates hyaluronic acid capsule synthesis in group A Streptococcus.

The hyaluronic acid capsule of group A Streptococcus (GAS) is an important virulence factor, but little is known about mechanisms that regulate capsule expression. Transposon Tn916 mutagenesis of the poorly encapsulated M-type 3 GAS strain DLS003 produced a transconjugant that exhibited a mucoid colony morphology, reflecting increased hyaluronic acid capsule production. Analysis of chromosomal DNA sequence immediately downstream of the transposon insertion identified two open reading frames, designated csrR and csrS, which exhibited sequence similarity to bacterial two-component regulatory systems. We constructed an in-frame deletion mutation within csrR, which encodes the putative response component. Replacement of the native csrR gene in the DLS003 chromosome with the mutant allele resulted in a sixfold increase in capsule production and a corresponding increase in transcription of the has operon, which contains the essential genes for hyaluronic acid synthesis. Increased capsule production by the csrR mutant strain was associated with enhanced resistance to complement-mediated opsonophagocytic killing in vitro and with a 500-fold increase in virulence in mice. These results establish CsrR as a negative regulator of hyaluronic acid capsule synthesis and suggest that it is part of a two-component regulatory system that influences capsule expression and virulence.

Amino Acid Sequence↗

PubMed2006

Using local gene expression similarities to discover regulatory binding site modules.

BACKGROUND: We present an approach designed to identify gene regulation patterns using sequence and expression data collected for Saccharomyces cerevisae. Our main goal is to relate the combinations of transcription factor binding sites (also referred to as binding site modules) identified in gene promoters to the expression of these genes. The novel aspects include local expression similarity clustering and an exact IF-THEN rule inference algorithm. We also provide a method of rule generalization to include genes with unknown expression profiles. RESULTS: We have implemented the proposed framework and tested it on publicly available datasets from yeast S. cerevisae. The testing procedure consists of thorough statistical analyses of the groups of genes matching the rules we infer from expression data against known sets of co-regulated genes. For this purpose we have used published ChIP-Chip data and Gene Ontology annotations. In order to make these tests more objective we compare our results with recently published similar studies. CONCLUSION: Results we obtain show that local expression similarity clustering greatly enhances overall quality of the derived rules, both in terms of enrichment of Gene Ontology functional annotation and coherence with ChIP-Chip binding data. Our approach thus provides reliable hypotheses on co-regulation that can be experimentally verified. An important feature of the method is its reliance only on widely accessible sequence and expression data. The same procedure can be easily applied to other microbial organisms.

Binding Sites↗

PubMed1996

The osteocalcin gene promoter provides a molecular blueprint for regulatory mechanisms controlling bone tissue formation: role of transcription factors involved in development.

Characterization of regulatory sequences and their cognate binding factors in the bone-specific osteocalcin (OC) gene promoter has provided insight into mechanisms that control expression of the gene under diverse biological conditions. We present evidence for AP-1 motifs and two multipartite conserved regulatory sequences, the OC Box I (nt-99 to-76) and a site designated OC Box II (nt-136 to-130) in contributing to developmental and tissue-specific expression of osteocalcin. OC Box I is characterized by a homeodomain binding site and OC Box II is a recognition sequence for AML-1 (also called PEBP2 alpha), a runt homology-related DNA binding protein. Functional activity of the elements was established in osseous and non-osseous cell lines and is in part related to the binding of osteoblast-specific complexes which enhance OC transcription. The contribution of several elements and binding of multiple classes of transcription factors to independent elements in both these domains serve to illustrate the complexity of control required for tissue-specific OC expression.

Animals↗

PubMed1996

Structure and function of the zeta-globin upstream regulatory element.

The human zeta-globin promoter contains a strong positive regulatory element in the 5' flanking region, designated the zeta-globin upstream regulatory element (URE). In this study, we define the minimal sequences required for URE function and characterize the associated protein-DNA interactions. Deletion experiments show that the URE spans a 60 bp region located between 220 and 279 bp 5' to the transcription start site. Further subdivision of this region shows that multiple cis acting sequences are present. Electrophoretic mobility shift assays demonstrate that the erythroid transcription factor GATA-1 binds a site at -230, and Sp1 and an unidentified factor bind a CCACC site at -240. The unidentified CCACC factor is distinct from two other CCACC factors, EKLF and BKLF/TEF-2. A third complex contains a novel DNA-binding activity that interacts with a site in the -269 to -255 region, designated URE binding factor (URE-BF). This factor is present in K562 cells that express zeta-globin, but is absent in the OCIM1 cell line, a human erythroid cell line that does not express zeta-globin. URE-BF appears to interact with a GATA factor, since formation of the URE-BF complex can be prevented by the presence of unlabeled oligonucleotides containing GATA sites. Finally, increasing the distance from the -230 GATA site to the two upstream sites causes a progressive decrease in zeta-globin promoter activity. There is no indication of a requirement for GATA-1 to be on the same side of the DNA helix as the other upstream factors. These results show that zeta-globin promoter function is highly dependent on a 60 bp region to which at least three different factors bind. Two of these factors may represent DNA-binding proteins not previously identified as important for regulation of globin gene expression. It is likely that these factors interact physically to create a functional regulatory unit.

Cell Line↗

PubMed2005

Randomized algorithms for motif detection.

MOTIVATION: Motif detection for DNA sequences has many important applications in biological studies, e.g. locating binding sites regulatory signals, designing genetic probes etc. In this paper, we propose a randomized algorithm, design an improved EM algorithm and combine them to form a software tool. RESULTS: (1) We design a randomized algorithm for consensus pattern problem. We can show that with high probability, our randomized algorithm finds a pattern in polynomial time with cost error at most x l for each string, where l is the length of the motif and can be any positive number given by the user. (2) We design an improved EM algorithm that outperforms the original EM algorithm. (3) We develop a software tool, MotifDetector, that uses our randomized algorithm to find good seeds and uses the improved EM algorithm to do local search. We compare MotifDetector with Buhler and Tompa's PROJECTION which is considered to be the best known software for motif detection. Simulations show that MotifDetector is slower than PROJECTION when the pattern length is relatively small, and outperforms PROJECTION when the pattern length becomes large. AVAILABILITY: It is available for free at http://www.cs.cityu.edu.hk/~lwang/software/motif/index.html, subject to copyright restrictions.

Algorithms↗

PubMed1994

Molecular analysis of cis-regulatory sequences of the alpha-amylase gene in D. melanogaster: a short 5'-flanking region of Amy distal gene is required for full expression of Amy proximal gene.

The structural locus for alpha-amylase (AMY) in Drosophila melanogaster is duplicated and divergently transcribed. These two genes are designated as Amy-p and Amy-d, respectively. We searched for the cis-acting regulatory elements for full expression of the duplicated Amy-p and Amy-d loci, by injecting plasmid constructs containing sequences from the Amy locus into preblastoderm embryos of an AMY-null strain and measuring exogenous AMY activity produced in transformed host larvae (i.e., the transient expression assay). Relative activities of endogenous amylase isozymes, AMY-1 and AMY-3, in extracts of AMY1,3 larvae of a Canton-S are almost the same. However, three independently isolated Amy-p1 constructs with only the 5' upstream regions of Amy-p1 expressed a very low AMY-1 activity. Two other Amy-p1 constructs with the 5' upstream region of Amy-d3 in addition to that of Amy-p1 produced a high activity. Thus, the 5' upstream region of Amy-d3 is necessary for full expression of Amy-p1. In order to locate cis-regulatory elements within the 5' region of Amy-d3, a series of hybrid constructs including this region were tested to locate them. Our results clearly show that the cis-acting regulatory sequences required for full expression of Amy-p1 are located between the base pairs at -304 and -372 upstream of Amy-d3 gene. In other words, only a short region located upstream of Amy-d3 was found to be necessary and sufficient for the full expression of Amy-p1 in addition to its promoter. This region seems also necessary for the expression of Amy-d3.

Animals↗

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↗