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Results for “regulatory element transcripts”

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PubMed2003

Differential DNA binding of Ku antigen determines its involvement in DNA replication.

Ku antigen (Ku70/Ku80) is a regulatory subunit of DNA-dependent protein kinase, which participates in the regulation of DNA replication and gene transcription through specific DNA sequences. In this study, we have compared the mechanism of action of Ku from A3/4, a DNA sequence that appears in mammalian origins of DNA replication, and NRE1, a transcriptional regulatory element in the long terminal repeat of mouse mammary tumor virus through which Ku antigen and its associated kinase, DNA-dependent protein kinase (DNA-PK(cs)), act to repress steroid-induced transcription. Our results indicate that replication from a minimal replication origin of ors8 is independent of DNA-PK(cs) and that Ku interacts with A3/4-like sequences and NRE1 in fundamentally different ways. UV crosslinking experiments revealed differential interactions of the Ku subunits with A3/4, NRE1, and two other proposed Ku transcriptional regulatory elements. In vitro footprinting experiments showed direct contact of Ku on A3/4 and over the region of ors8 homologous to A3/4. In vitro replication assays using ors8 templates bearing mutations in the A3/4-like sequence suggested that Ku binding to this element was necessary for replication. By contrast, in vitro replication experiments revealed that NRE1 was not involved in DNA replication. Our results establish A3/4 as a new class of Ku DNA binding site. Classification of Ku DNA binding into eight categories of interaction based on recognition and DNA crosslinking experiments is discussed.

Antigens, Nuclear↗

PubMed2003

REDUCE: An online tool for inferring cis-regulatory elements and transcriptional module activities from microarray data.

REDUCE is a motif-based regression method for microarray analysis. The only required inputs are (i) a single genome-wide set of absolute or relative mRNA abundances and (ii) the DNA sequence of the regulatory region associated with each gene that is probed. Currently supported organisms are yeast, worm and fly; it is an open question whether in its current incarnation our approach can be used for mouse or human. REDUCE uses unbiased statistics to identify oligonucleotide motifs whose occurrence in the regulatory region of a gene correlates with the level of mRNA expression. Regression analysis is used to infer the activity of the transcriptional module associated with each motif. REDUCE is available online at http://bussemaker.bio.columbia.edu/reduce/. This web site provides functionality for the upload and management of microarray data. REDUCE analysis results can be viewed and downloaded, and optionally be shared with other users or made publicly accessible.

Algorithms↗

PubMed2001

Robust prostate-specific expression for targeted gene therapy based on the human kallikrein 2 promoter.

Tissue-specific transcriptional regulatory elements can increase the safety of gene therapy vectors. Unlike prostate-specific antigen (PSA/hK3), whose expression displays an inverse correlation with prostate cancer grade and stage, human glandular kallikrein 2 (hK2) is upregulated in higher grade and stage disease. Therefore, our goal was to develop a strong and prostate-specific hK2-based promoter for targeted gene therapy. We identified the minimum "full-strength" hK2 enhancer and built transcriptional regulatory elements composed of multiple tandem copies of this 1.2-kb enhancer, fused to the hK2 minimal promoter. Relative to the weak induction of the minimal hK2 promoter by androgen analog (R1881) in androgen receptor (AR)-positive LNCaP cells, transcriptional activity was increased by 25-, 44-, 81-, and 114-fold when one to four enhancers were spliced to the hK2 promoter, respectively. In contrast, the enhancer/promoter elements were inactive in the AR(-) prostate cancer line PC-3 and in a panel of nonprostate lines, including 293, U87, MCF-7, HuH-7, and HeLa cells. Furthermore, we generated a recombinant adenovirus, ADV.hK2-E3/P-EGFP, expressing enhanced green fluorescent protein (EGFP) under the control of the hK2 triplicate enhancer/promoter, and compared its properties with ADV.CMV-EGFP expressing EGFP under the control of the cytomegalovirus (CMV) enhancer/promoter. Unlike the CMV promoter, the hK2-E3/P promoter was at least 100-fold inducible by R1881 in the adenoviral backbone. Compared with in situ injection of subcutaneous LNCaP tumors with ADV.CMV-EGFP, which led to detectable EGFP expression in tumor, liver, and brain tissue, ADV.hK2-E3/P-EGFP injection led to robust but tumor-restricted EGFP expression. These results suggest that the hk2 multienhancer/promoter should be a powerful novel reagent for safer targeted gene therapy of prostate cancer.

Adenoviridae↗

PubMed1992

Regulatory elements and transcriptional regulation by testosterone and retinoic acid of the rat nerve growth factor receptor promoter.

The low-affinity nerve growth factor receptor (LNGFR) is a membrane-associated glycoprotein which is thought to participate in some of the biological activities of nerve growth factor (NGF). Expression of the LNGFR gene is known to be regulated both during development and in response to various agents in cell culture. However, molecular mechanisms responsible for the regulation have not been described. We report here an analysis of a 4.8-kb sequence from the 5'-flanking region of the rat LNGFR gene. Several regulatory elements were identified in this region by transfection of plasmid constructs containing sequences from LNGFR fused to a bacterial cat reporter gene. The proximal part of the promoter region (0.4-kb) was shown to be sufficient to support cat expression in all cell types used. A silencer element located between -1.5 kb and -1.8 kb from the start of translation, as well as an enhancer element in more upstream regions of the promoter, were identified in the phaeochromocytoma cell line, PC12, and in the Sertoli cell line, TM4, that express the LNGFR gene. Treatment of TM4 cells with retinoic acid (RA) increases the level of LNGFR mRNA twofold, while testosterone treatment results in a tenfold decrease. Regions of the promoter responsive to testosterone and RA in TM4 cells were found at -610 to -860 bp and -1840 to -4800 bp upstream from the translation start codon, respectively. A RA-responsive element active in PC12 cells is located between bp -610 to -860 from the start codon.

Animals↗

PubMed1987

Expression of antibody cDNA in murine myeloma cells: possible involvement of additional regulatory elements in transcription of immunoglobulin genes.

Expression vectors for cDNA of the kappa and gamma 1 chains of a monoclonal antibody directed against creatine kinase were introduced into murine myeloma cells. Kappa and gamma 1 cDNA were either under the control of the SV40 early promoter or of the cognate promoters and enhancers of the light- and heavy-chain genes. Secretion of immuno-reactive kappa and gamma 1 chains into the culture medium was demonstrated with the SV40 promoter as well as with the cognate promoters. Expression of gamma 1 cDNA with the SV40 early promoter was about twice as high as with the heavy-chain promoter and enhancer. Expression of kappa cDNA under the control of the SV40 early promoter was about 17 times higher than with the light-chain promoter and enhancer. These expression levels were compared to those of a genomic immunoglobulin (Ig) kappa determinant, including introns. Such an entire kappa gene led to expression of the light chain at levels double those with the kappa cDNA construction using the SV40 promoter and about 35 times as high when using kappa cDNA and the cognate promoter and enhancer. This result might indicate that, besides the cognate promoter and enhancer elements, other intragenic elements are involved in the regulation of Ig expression. However, the SV40 early promoter seems to be able to compensate for the absence of these postulated regulatory elements probably located in the introns.

Animals↗

PubMed1990

Role of alpha-fetoprotein regulatory elements in transcriptional activation in transient heterokaryons.

The requirements for activation of the mouse alpha-fetoprotein (AFP) gene in transient heterokaryons were investigated. For this purpose, the 7-kilobases of DNA flanking the 5' end of the AFP gene were linked to a mouse major histocompatibility complex (MHC) class I structural gene. The fusion gene was stably integrated at different sites into mouse L-cells, which do not transcribe the AFP gene. Transient heterokaryon fusions demonstrated that the silent AFP-MHC gene and the endogenous AFP gene were activated by factors present in HepG2 cells, a liver-derived cell line, but not by those in HeLa cells. Activation was detected at the protein level in single heterokaryons by using monoclonal antibodies against the cell surface protein and at the mRNA level in populations of cells. The AFP promoter alone was sufficient for activation could be used for DNA transfer strategies to identify genes which can activate AFP promoter elements in trans.

Animals↗

PubMed2001

Regulatory elements governing transcription in specialized myofiber subtypes.

Skeletal myofibers of vertebrates acquire specialized metabolic and physiological properties as a consequence of developmental cues in the embryo and different patterns of contractile activity in the adult. The myoglobin gene is regulated stringently in muscle fibers, such that high myoglobin expression is observed in mitochondria-rich, oxidative myofibers (Types I and IIa) compared with glycolytic fibers (Type IIb). Using germ-line transgenesis and somatic cell gene transfer methods, we defined discrete regions of the murine and human genes encoding myoglobin that are sufficient to confer muscle- and fiber type-specific expression to reporter genes. Mutational analysis confirms the importance of A/T-rich, MEF2-binding motifs in myoglobin gene regulation, as suggested by previous studies using different experimental approaches. In addition, we demonstrated a previously unsuspected role for an intragenic E-box motif as a negative regulatory element contributing to the tightly regulated variation in myoglobin gene expression among particular myofiber subtypes.

Animals↗

PubMed1992

cis-acting elements that confer lung epithelial cell expression of the CC10 gene.

To define cis-acting genetic elements responsible for cell-specific transcriptional regulation of the CC10 gene, DNA sequences spanning nucleotides -2338 to +49 of the rat CC10 gene were linked to a reporter gene coding for chloramphenicol acetyltransferase (CAT). In transient expression assays, CC10 sequences were capable of restricting CAT expression to a human lung adenocarcinoma cell line similar to pulmonary Clara cells. Transgenic mice harboring the hybrid RtCC10-CAT construct expressed high levels of CAT activity specifically within protein extracts of lung and trachea. Transcripts for the CAT reporter gene colocalized with those for the endogenous murine CC10 gene within the airways of transgenic mice. Functional analysis of deletion mutants identified stimulatory, inhibitory, and cell type-specific transcriptional regulatory elements. The results of gel retention and DNaseI protection assays suggest that a transcriptional stimulatory region located between -320 and -175, and a cell type-specific regulatory element located between -175 and +49, result from a series of protein-DNA interactions occurring at -220 to -205 and -128 to -86, respectively. Lung epithelial specific transcriptional regulatory elements described herein will be useful for expression of chimeric genes within epithelial cells lining the trachea, bronchi, and bronchioles of mice.

Adenocarcinoma↗

PubMed1991

Molecular cloning, structure, promoters and regulatory elements for transcription of the Bacillus megaterium encoded regulon for xylose utilization.

The xylA and xylB genes of Bacillus subtilis BR151 encoding xylose isomerase and xylulokinase, respectively, were disrupted by gene replacement rendering the constructed mutant strain unable to grow on xylose as the sole carbon source. The Bacillus megaterium encoded xyl genes were cloned by complementation of this strain to xylose utilization. The nucleotide sequence of about 4 kbp of the insertion indicates the presence of the xylA and xylB genes on the complementing plasmid. Furthermore, a regulatory gene, xylR, is located upstream of xylA and has opposite polarity to it. The intergenic region between the divergently oriented reading frames of xylR and xylA contains palindromic sequences of 24 bp spaced by five central bp and 29 bp spaced by 11 bp, respectively, and two promoters with opposite orientation as determined by primer extension analysis. They overlap with one nucleotide of their--35 consensus boxes. Transcriptional fusions of lacZ to xylA, xylB and xylR were constructed and revealed that xylA and xylB are repressed in the absence and can be 200-fold induced in the presence of xylose. The increased level of xylAB mRNA in induced and its absence in repressed cells confirms that this regulation occurs on the level of transcription. Deletion of the xylR gene encoding the Xyl repressor results in constitutive expression of xylAB. The transcription of xylR is autoregulated and can be induced 9-fold by xylose. The mechanism of this regulation is not clear. While the apparent xyl operator palindrome is upstream of the xylR promoter, the potential recognition of another palindrome downstream of this promoter by Xyl repressor is discussed.

Aldose-Ketose Isomerases↗

PubMed1990

Cloning and expression of the human S100 beta gene.

S100 protein is a low molecular weight, EF-hand, Ca2(+)-binding protein widely distributed and conserved in the central nervous system of vertebrates. The gene coding for the beta subunit of human S100 protein (S100 beta) has been recently mapped to chromosome 21. In order to study the expression of this gene in normal and abnormal brain development, we have isolated and characterized overlapping genomic clones spanning the region coding for human S100 beta and its flanking sequences. The intron-exon organization of the human S100 beta gene is similar to that of the genes coding for several other members of the S100 protein subfamily of EF-hand proteins. The human S100 beta gene is composed of 3 exons, the first of which specifies the 5'-untranslated region, while the second and third each encode a single EF-hand, Ca2(+)-binding domain. The promoter region contains several potential regulatory transcription elements including the cAMP-responsive elements CRE and AP-2. A novel sequence motif, the S100 protein element, situated in close proximity to the TATA box of the genes of several members of the S100 protein subfamily, has been identified. In addition, multiple repeats with similar nucleotide sequence and location to the recently reported beta globin direct repeat elements have been also found in the human S100 beta promoter. A full length (17.3 kilobases) copy of the human S100 beta gene was constructed and transfected into rat glioma C6 cells. Stable transfectants were shown to express correctly initiated transcripts of the human S100 beta gene, indicating that the cloned sequences contain functional regulatory transcription elements.

Amino Acid Sequence↗

PubMed2005

Woodchuck post-transcriptional element induces nuclear export of myotonic dystrophy 3' untranslated region transcripts.

The woodchuck post-transcriptional regulatory element (WPRE) can naturally accumulate hepatitis transcripts in the cytoplasm, and has been recently exploited as an enhancer of transgene expression. The retention of mutant myotonic dystrophy protein kinase (DMPK) transcripts in the nucleus of myotonic dystrophy (DM) cells has an important pathogenic role in the disease, resulting in pleiotropic effects including delayed myoblast differentiation. In this study, we report the first use of WPRE as a tool to enhance nuclear export of an aberrantly retained messenger RNA. Stable cell lines expressing the normal and mutant DMPK 3' UTR (3' untranslated region) complementary DNA, with or without WPRE, were produced. It is noteworthy that WPRE stimulated extensive transport of mutant transcripts to the cytoplasm. This was associated with repair of the defective cellular MyoD levels and a subsequent increase in myoblast differentiation. These results provide the basis for a cellular model that can be exploited in DM and in the study of RNA transport mechanisms.

3' Untranslated Regions↗

PubMed2002

Protecting against promiscuity: the regulatory role of insulators.

Eukaryotic genomes contain transcriptional regulatory elements that alter promoter activity through long-range interactions. Many control elements show a broad range of promoter interactions, suggesting that these elements are capable of inappropriate transcription. The identification of a novel class of directing regulatory elements, called insulators, has provided clues into mechanisms used in eukaryotic genomes to maintain transcription fidelity. Insulators contribute to the organization of independent domains of gene function by restricting enhancer and silencer function. This review describes the properties of insulators and related elements that have been isolated from several eukaryotic genomes. Two classes of models of insulator function are considered. These models provide insights into possible mechanisms used by these diverse elements to provide regulatory autonomy.

Animals↗

PubMed1989

Characterization of a single copy gene encoding ferredoxin I from pea.

We have isolated, mapped, and sequenced a genomic clone containing the ferredoxin I (Fed-1) gene from Pisum sativum. The gene is present as a single copy per haploid genome. It has no introns, and it specifies a 753-nucleotide transcript encoding a 149-amino acid protein including a 52-residue transit peptide. Upstream sequences from Fed-1 contain several elements with similarity to transcriptional regulatory elements from RbcS and Cab genes, and gel mobility shift assays show that nuclear extracts from light-grown pea leaves contain one or more DNA binding activities specific for Fed-1 5'-flanking sequences. RbcS and Cab regulatory sequences are only weak competitors for this binding, however, and the RbcS and Cab similarities mostly lie outside of the region essential for binding. These data are discussed in terms of previously observed physiological differences between the light responses of Fed-1 and other genes.

Amino Acid Sequence↗

PubMed2005

In vitro selection of DNA binding sites for ABF1 protein from Saccharomyces cerevisiae.

The autonomously replicating sequence-binding factor 1 (ABF1) from Sacchramoyces cerevisiae is known as a multifunctional DNA binding protein that is involved in transcriptional regulation, DNA-replication, and in restructuring of chromatin via nucleosome remodelling. ABF1 binds to DNA sequences found in ARS elements and in various transcriptional regulatory elements. This led to the early definition of the consensus motive 5'-CGTnnnnnnnGA(G/C)-3'. We have used a SELEX approach to expand and better characterize the DNA sequence requirements of ABF1. Starting from a pool of oligonucleotides randomized at a sequence of 30 nucleotides, we used EMSA to select for sequences with high affinity for ABF1. We obtained the sequences of 106 aptamers after the 15th SELEX round. A 16 nucleotide consensus was derived from this pool by analysis with the motif search programme MEME. Quantitative EMSA experiments verified our experimental approach since binding sequences which were bound with high affinity occurred more often in the pool and resembled the derived consensus to a higher degree. We found DNA sequences that are bound by ABF1 with nearly two-magnitude higher affinity as compared to the hitherto accepted ABF1 consensus sequence. This led us to postulate a strong recognition motive: 5'-TnnCGTnnnnnnTGAT-3'.

Base Sequence↗

PubMed1999

A novel downstream positive regulatory element mediating transcription of the human high mobility group (HMG) I-C gene.

The high mobility group (HMG) I proteins are small, non-histone chromosomal proteins that promote gene activation during development and within rapidly dividing cells. They do so by facilitating enhanceosome formation on inducible genes, via both protein/DNA and protein/protein interactions. The HMG I-C gene is tightly regulated, normally being expressed exclusively during embryonic development. However, HMG I-C expression is also observed frequently in a number of tumor types, and this expression has been shown to contribute to the malignant transformation process. With the aim of dissecting pathways that lead to aberrant expression of HMG I-C in tumor cells, we have analyzed HMG I-C gene regulation in the human hepatoma cell line PLC/PRF/5. One of the two HMG I-C transcripts detected in this cell line originates from a novel downstream initiation site at nucleotide -161 relative to the first methionine. Transcription from the downstream initiation site is mediated by a PRE located between nt -222 and -217. We show here that the Sp1 and Sp3 transcription factors interact with the PRE and transactivate the HMG I-C promoter in a cooperative fashion. This study provides the first characterization of this downstream HMG I-C promoter.

Animals↗

PubMed1991

Regulatory sequences of duck hepatitis B virus C gene transcription.

The regulatory elements involved in transcription of the C gene of duck hepatitis B virus (DHBV) were investigated. Several DHBV DNA fragments were assayed for C gene promoter, enhancer, and silencer activity by using a chloramphenicol acetyltransferase (CAT) reporter gene and transfection of established liver and nonliver cell lines. A major transcript initiating at nucleotide positions 2532 and 2533 and three minor transcripts initiating at positions 2453/2454 and 2461 were identified in cells containing these constructs. These positions correspond to the 5' end of the C mRNA and were close to that of the pre-C mRNAs, respectively, found in infected livers. The pre-C mRNAs were only detected when sequences located between the initiation sites of the pre-C and C mRNAs were deleted. These sequences downregulated, in an orientation-independent fashion, a heterologous promoter and were found to contain a consensus motif common to negative transcriptional regulatory elements previously characterized in other cellular and viral genes. C gene promoter activity was only observed in highly differentiated liver cells and was dependent on a short DHBV DNA fragment containing an enhancer core consensus motif. These data indicate that transcription of the DHBV C gene is regulated by positive, negative, and differentiation factor-responsive elements.

Animals↗

PubMed1992

Transcriptional regulation during T-cell development: the alpha TCR gene as a molecular model.

The regulation of gene expression during lymphocyte differentiation is a complex process involving interactions between multiple positive and negative transcriptional regulatory elements. In this article, transcriptional regulation of the archetypal T-cell-specific gene, alpha TCR, is discussed. Major recent developments, including the identification of novel families of transcription factors that regulate multiple T-cell genes during thymocyte ontogeny and T-cell activation, are described.

Base Sequence↗

PubMed1987

Properties of a distal regulatory element controlling transcription of the U2 small nuclear RNA.

The upstream region of human U2 genes contains a distal transcriptional control element, previously mapped between nucleotide (nt) positions -198 and -258 (Westin et al., 1984b). In the present study we show that it resembles transcriptional enhancers in being active even from a distance of 1.4 kb. However, in contrast to most other enhancers it functions unidirectionally in Xenopus laevis oocytes. The distal control element was further mapped by construction of truncated templates for U2 RNA transcription. The results showed that templates, which extended to either of nt positions -214 and -218, were inactive. Templates comprising sequences to nt positions -225 or -226 displayed an intermediate level of activity whereas templates which extend to nt -258 were fully active. It has previously been shown that the human U2 enhancer contains binding sites for the so-called octamer binding protein and for transcription factor Sp1 [Janson et al., Nucl. Acids Res. 15 (1987) 4997-5016]. The partially active templates included one binding site for the octamer binding protein, whereas the fully active template included, in addition, two Sp1 binding sites, thus indicating that these transcription factors are of importance for U2 RNA transcription. The structure of the enhancer was also probed by inserting a pair of complementary synthetic oligodeoxynucleotides which represented the region between nt positions -235 and -215 into a truncated template which lacked the enhancer. The oligodeoxynucleotide enhanced transcription to approximately 50% of the level obtained with templates extending to position -258.

Animals↗