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Results for “regulatory element transcripts”

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PubMed1997

The mouse extracellular signal-regulated kinase 2 gene. Gene structure and characterization of the promoter.

ERK2 (extracellular-signal regulated kinase 2, also known as p42 mitogen-activated protein kinase) is an integral member of the mitogen-activated protein kinase cascade that is crucial for many cellular events such as proliferation and differentiation. Here, we determined the genomic organization of the Erk2 gene and characterized its promoter. The Erk2 gene spans over 60 kilobases, and the coding region is split into eight exons. In the coding region, exon-intron organization was exactly conserved between the two mouse genes for ERK2 and ERK1 except one junction shifted by one nucleotide. Primer extension and S1 nuclease analyses identified two major transcription start sites located at -219 and -223 relative to the translation start site. The 5'-flanking sequence lacked TATA box but contained a CCAAT box located approximately 60 base pairs upstream of transcription start sites. Sequencing of the 5'-flanking region also revealed potential cis-acting elements for multiple transcriptional regulatory factors including Sp1, zif268, Ets, CREB, and PuF sites. The promoter activity of the 5'-flanking region was examined using chloramphenicol acetyltransferase as a reporter gene. Transient transfection experiments using Chinese hamster ovary cells defined a maximal promoter activity in a 371-base pair region immediately upstream of the translation start site. Furthermore, we demonstrated, using mouse P19 embryonal carcinoma cells, that this 371-base pair sequence is likely to be sufficient to confer the transcriptional activation of the ERK2 promoter during the retinoic acid-induced differentiation of P19 cells.

Amino Acid Sequence↗

PubMed1998

Isolation and characterization of the mouse ribosomal protein S7 gene.

We have isolated and characterized the single intron-containing gene encoding the mouse ribosomal protein S7. The mouse 129SV S7 gene has seven exons and six introns spanning about 5000 nucleotides. The exon-intron structure of the gene is similar to other vertebrate homologues. Southern blot analysis showed that in addition to the isolated single-copy intron-containing gene, there are 10-12 members in the mouse S7 gene family, which are all most probably processed pseudogenes. The promoter region of rpS7 contains several evolutionarily conserved putative regulatory elements. The main transcription start site was mapped to a T residue within a polypyrimidine tract, 79 nucleotides upstream from AUG codon.

Amino Acid Sequence↗

PubMed2005

A novel class of Pseudoautosomal region 1 deletions downstream of SHOX is associated with Leri-Weill dyschondrosteosis.

Leri-Weill dyschondrosteosis (LWD) is a pseudoautosomal dominant disorder characterized by disproportionate short stature and a characteristic curving of the radius, known as the "Madelung deformity." SHOX mutations resulting in SHOX haploinsufficiency have been found in LWD and in a variable proportion of patients with idiopathic short stature (ISS), whereas homozygous loss of SHOX results in the more severe Langer mesomelic dysplasia (LMD). Defects in SHOX have been identified in approximately 60% of LWD cases, whereas, in the remaining approximately 40%, the molecular basis is unknown. This suggests either genetic heterogeneity or the presence of mutations in unanalyzed regions of SHOX, such as the upstream, intragenic, or downstream regulatory sequences. Therefore, the pseudoautosomal region 1 (PAR1) of 80 patients with LWD, in whom SHOX deletions and mutations had been excluded, was screened for deletions by use of a new panel of microsatellite markers. We identified 12 patients with LWD who presented with a novel class of PAR1 deletions that did not include SHOX. The deletions were of variable size and mapped at least approximately 30-530 kb downstream of SHOX. In our cohort, this type of deletion accounted for 15% of cases. In all cases, the deletions cosegregated with the phenotype. No apparent phenotypic differences were observed between patients with SHOX deletions and those with this new class of PAR1 deletions. Thus, we present here the identification of a second PAR1 region implicated in the etiopathogenesis of LWD. Our findings suggest the presence of distal regulatory elements of SHOX transcription in PAR1 or, alternatively, the existence of an additional locus apparently involved in the control of skeletal development. Deletion analysis of this newly identified region should be included in the mutation screening of patients with LWD, LMD, and ISS.

Base Sequence↗

PubMed2000

Predominant transgene expression in exocrine pancreas directed by the CMV promoter.

The enhancer/promoter of the human cytomegalovirus gene encoding the major immediate-early protein (CMVp) is reputed to be one of the strongest and most promiscuous regulatory elements for directing transcription of heterologous genes in vitro. However, transgene expression under the promoter in adult transgenic mice is often more restricted. We selected a CMVp segment from position -350 to +59 to control expression of transgenes for two secretory fusion proteins. Expression was analyzed by immunohistology staining and quantified by Northern blot, Western blot, and ELISA of secretions from explanted tissues. In all six lines of transgenic mice, the highest expression of transgenes at the mRNA and protein level was observed in the exocrine tissue of the pancreas, although the levels of expression varied among the lines. The results indicate not only that CMVp is not a universal promoter in vivo but indeed that it can be relatively specific for the exocrine pancreas, where expression of the gene it controlled was consistently very high.

Animals↗

PubMed2006

n-3 fatty acids and gene expression.

Accumulating evidence in both humans and animal models clearly indicates that a group of very-long-chain polyunsaturated fatty acids, the n-3 fatty acids (or omega-3), have distinct and important bioactive properties compared with other groups of fatty acids. n-3 Fatty acids are known to reduce many risk factors associated with several diseases, such as cardiovascular diseases, diabetes, and cancer. The mechanisms whereby n-3 fatty acids affect gene expression are complex and involve multiple processes. As examples, n-3 fatty acids regulate 2 groups of transcription factors, such as sterol-regulatory-element binding proteins and peroxisome proliferator-activated receptors, that are critical for modulating the expression of genes controlling both systemic and tissue-specific lipid homeostasis. Modulation of specific genes by n-3 fatty acids and cross-talk between these genes are responsible for many effects of n-3 fatty acids.

Animals↗

PubMed1998

Repression by the 3' UTR of fem-3, a sex-determining gene, relies on a ubiquitous mog-dependent control in Caenorhabditis elegans.

The fem-3 sex-determining gene is repressed post-transcriptionally via a regulatory element in its 3' untranslated region (UTR) to achieve the switch from spermatogenesis to oogenesis in the Caenorhabditis elegans hermaphrodite germ line. In this paper, we investigate the fem-3 3' UTR control in somatic tissues using transgenic reporter assays, and we also identify six genes essential for this control. First, we find that a reporter transgene bearing a wild-type fem-3 3' UTR is repressed in somatic tissues, whereas one bearing a mutant fem-3 3' UTR is derepressed. Moreover, control by mutant 3' UTRs is temperature sensitive as predicted from the temperature sensitivity of the fem-3 gain-of-function (gf) mutations. Secondly, we find a fem-3 3' UTR RNA-binding activity in somatic tissues, in addition to the previously reported germ-line-specific binding by FBF. Thirdly, we find that each of six genes, mog-1-mog-6, is required for repression by the fem-3 3' UTR. Therefore, the mog genes not only affect the sperm/oocyte switch in the germ line, but also function in somatic tissues. We suggest that the mog genes may encode components of a ubiquitous machinery that is used for fem-3 3' UTR-mediated repression and the sperm/oocyte switch.

3' Untranslated Regions↗

PubMed2003

Rationally designed insulator-like elements can block enhancer action in vitro.

Insulators are DNA sequences that are likely to be involved in formation of chromatin domains, functional units of gene expression in eukaryotes. Insulators can form domain boundaries and block inappropriate action of regulatory elements (such as transcriptional enhancers) in eukaryotic nuclei. Using an in vitro system supporting enhancer action over a large distance, the enhancer-blocking insulator activity has been recapitulated in a highly purified system. The insulator-like element was constructed using a sequence-specific DNA-binding protein making stable DNA loops (lac repressor). The insulation was entirely dependent on formation of a DNA loop that topologically isolates the enhancer from the promoter. This rationally designed, inducible insulator-like element recapitulates many key properties of eukaryotic insulators observed in vivo. The data suggest novel mechanisms of enhancer and insulator action.

Base Sequence↗

PubMed2005

Altered pre-lamin A processing is a common mechanism leading to lipodystrophy.

Lipodystrophies are a heterogeneous group of human disorders characterized by the anomalous distribution of body fat associated with insulin resistance and altered lipid metabolism. The pathogenetic mechanism of inherited lipodystrophies is not yet clear; at the molecular level they have been linked to mutations of lamin A/C, peroxisome proliferator-activated receptor (PPARgamma) and other seemingly unrelated proteins. In this study, we examined lamin A/C processing in three laminopathies characterized by lipodystrophic phenotypes: Dunnigan type familial partial lipodystrophy, mandibuloacral dysplasia and atypical Werner's syndrome. We found that the lamin A precursor was specifically accumulated in lipodystrophy cells. Pre-lamin A was located at the nuclear envelope and co-localized with the adipocyte transcription factor sterol regulatory element binding protein 1 (SREBP1). Using co-immunoprecipitation experiments, we obtained the first demonstration of an in vivo interaction between SREBP1 and pre-lamin A. Binding of SREBP1 to the lamin A precursor was detected in patient fibroblasts as well as in control fibroblasts forced to accumulate pre-lamin A by farnesylation inhibitors. In contrast, SREBP1 did not interact in vivo with mature lamin A or C in cultured fibroblasts. To gain insights into the effect of pre-lamin A accumulation in adipose tissue, we inhibited lamin A precursor processing in 3T3-L1 pre-adipocytes. Our results show that pre-lamin A sequesters SREBP1 at the nuclear rim, thus decreasing the pool of active SREBP1 that normally activates PPARgamma and causing impairment of pre-adipocyte differentiation. This defect can be rescued by treatment with troglitazone, a known PPARgamma ligand activating the adipogenic program.

3T3-L1 Cells↗

PubMed1994

Human Fas ligand: gene structure, chromosomal location and species specificity.

Fas ligand (FasL) is a 40 kDa type II membrane protein belonging to the tumor necrosis factor family, which induces apoptosis by binding to its receptor, Fas. In this report, we isolated the chromosomal gene for human FasL. The human FasL gene consists of approximately 8.0 kb and is split into four exons. The human FasL gene was mapped on chromosome 1q23 by in situ hybridization against human metaphase chromosomes. Human FasL cDNA was isolated by the reverse polymerase chain reaction of mRNA prepared from human activated peripheral blood lymphocytes. Human FasL is a type II membrane protein consisting of 281 amino acids with a calculated M(r) of 31,759. It has an identity of 76.9% at the amino acid sequence level with mouse FasL. Both human and mouse recombinant FasL expressed in COS induced apoptosis in the cells expressing either human Fas or mouse Fas, indicating that FasL fully cross-reacts between human and mouse. A comparison of human and mouse FasL chromosomal genes indicated that a approximately 300 bp sequence upstream of the ATG initiation codon is highly conserved between them. Several transcription cis-regulatory elements such as SP-1, NF-kappa B and IRF-1 were recognized in this region.

Adult↗

PubMed2025

Deep learning guided programmable design of Escherichia coli core promoters from sequence architecture to strength control.

Core promoters are essential regulatory elements that control transcription initiation, but accurately predicting and designing their strength remains challenging due to complex sequence-function relationships and the limited generalizability of existing AI-based approaches. To address this, we developed a modular platform integrating rational library design, predictive modelling, and generative optimization into a closed-loop workflow for end-to-end core promoter engineering. Conserved and spacer region of core promoters exert distinct effects on transcriptional strength, with the former driving large-scale variation and the latter enabling finer gradation. Based on this insight, Mutation-Barcoding-Reverse Sequencing approach was used and constructed a synthetic promoter library comprising 112 955 variants with minimal redundancy and a 16 226-fold expression range. A Transformer-based model trained on this dataset achieved a Pearson correlation of 0.87 with experimentally measured promoter strengths. When combined with a conditional diffusion model, the system enabled de novo generation of promoter sequences with defined strengths, achieving a design-to-measurement correlation of 0.95 and maintaining high accuracy (R = 0.93) across varied sequence contexts. The designed promoters consistently preserved their intended strength gradients, demonstrating robust plug-and-play functionality. This work establishes a scalable and extensible platform (www.yudenglab.com) for deep learning-guided programmable design of Escherichia coli core promoters, enabling precise transcriptional control.

Promoter Regions, Genetic↗

PubMed1993

Identification and analysis of the human choline acetyltransferase gene promoter.

Choline acetyltransferase (ChAT) is the key enzyme responsible for the synthesis of the neurotransmitter acetylcholine and is reduced in various central neurodegenerative diseases. From a previously selected 12.6 kb human choline acetyltransferase (hChAT) genomic clone, we have identified and characterized a promoter region of 895 bp. Sequence analysis revealed the presence of a TATA-like box, a CAAT box and several putative regulatory responsive elements. Three transcription initiation sites were determined by primer extension analysis. The Northern blot of poly(A)+ RNA, showed a single band of 2300 Nt in the human nucleus accumbens and facial nucleus. By using transient transfections into NE-1-115 and COS-1 cells of the 5' flanking region of the hChAT gene we identified a sequence of 66 bp upstream of the transcription start site which confers responsiveness to proto-oncogenes c-Fos/c-Jun. These data suggest that the hChAT gene may be a physiological target of c-Fos/c-Jun and therefore may play a role in neuronal responses to various stimuli.

Base Sequence↗

PubMed1994

Characterization of the minimal elements of the hepatitis B virus large surface antigen promoter.

It has been demonstrated that the hepatocyte nuclear factor 1 (HNF1) binding site is critical for the majority of the hepatitis B virus (HBV) large surface antigen promoter activity in differentiated hepatoma cell lines. Examination of a series of clustered point mutations in the minimal large surface antigen promoter demonstrated that the HNF1 and TATA box binding sites are the major regulatory elements required for transcription from this promoter. Synthetic promoter constructs containing the large surface antigen promoter HNF1 binding site and TATA box element upstream of the luciferase open reading frame were tested for their transcriptional activities in HepG2. 1 cells in the absence or presence of an HNF1 expression vector. These synthetic promoter constructs displayed a similar level of transcriptional activity and induction by HNF1 in comparison with the full-length large surface antigen promoter, suggesting that additional HBV sequences are dispensable for full transcriptional activity. The distance between the HNF1 binding site and TATA box element in the synthetic promoter constructs appeared to influence the transcriptional activity modestly and in a periodic manner.

Base Sequence↗

PubMed1994

Characterization of the auxin-inducible SAUR-AC1 gene for use as a molecular genetic tool in Arabidopsis.

The small auxin up RNA (SAUR) genes were originally characterized in soybean, where they encode a set of unstable transcripts that are rapidly induced by auxin. In this report, the isolation of a SAUR gene, designated SAUR-AC1, from Arabidopsis thaliana (L.) Heynh. ecotype Columbia is described. The promoter of the SAUR-AC1 gene contains putative regulatory motifs conserved among soybean SAUR promoters, as well as sequences implicated in the regulation of other genes in response to auxin. The transcribed region is approximately 500 bp in length and contains no introns. Highly conserved sequences located within the SAUR-AC1 transcript include the central portion of the coding region and a putative mRNA instability sequence (DST) located in the 3' untranslated region. Accumulation of SAUR-AC1 mRNA is readily induced by natural and synthetic auxins and by the translational inhibitor cycloheximide. Moreover, several auxin- and gravity-response mutants of Arabidopsis exhibit decreased accumulation of the SAUR-AC1 mRNA in elongating etiolated seedlings. In particular, in the axr2-1 mutant the SAUR-AC1 transcript accumulates to less than 5% of wild-type levels. These studies indicate that SAUR-AC1 will be a useful probe of auxin-induced gene expression in Arabidopsis and will facilitate the functional analysis of both transcriptional and posttranscriptional regulatory elements.

Amino Acid Sequence↗

PubMed1990

Regulation of elastin gene expression: evidence for functional promoter activity in the 5'-flanking region of the human gene.

Analysis of nucleotide sequences in the 5'-flanking region of the human elastin gene has revealed several unusual features, suggesting that regulation of elastin gene expression is complex. To identify any cis-acting regulatory promoter elements, a 35-kb fragment of DNA (CosE) was isolated from a human genomic cosmid library by hybridizations with a human elastin cDNA. Southern blots of EcoRI digests of CosE DNA, utilizing a 5'-end labeled 21-mer oligonucleotide corresponding to the signal sequence of elastin, revealed the presence of a single 7.8-kb genomic fragment. Partial dideoxynucleotide sequencing of this EcoRI genomic subclone revealed that it extended approximately 2.5 kb 3' of the translation initiation site (ATG), encompassing exon 1 and a portion of the first intron, while the remaining DNA encompassed the 5'-flanking region. Exonuclease III digestion (3'----5') was performed to remove sequences of the first intron and first exon, including the ATG site. One clone, approximately 5 kb in size, had the 3' end located 14 bp upstream of the ATG site. A 462-bp 3' portion of this 5-kb fragment was subcloned into a Bluescript/CAT chimeric plasmid (pBS0CAT) to generate an elastin gene promoter/CAT reporter gene construct (pEP6CAT). Transient transfection experiments with pEP6CAT using human skin fibroblasts, human HT-1080, mouse NIH-3T3, or freshly isolated neonatal rat aortic smooth muscle cells revealed significant CAT activity in each cell line. These results suggest that the 5'-flanking region of the elastin gene contains the cis-acting regulatory elements necessary for transcription. The chimeric plasmid pEP6CAT provides a means to study the transcriptional control of elastin gene expression by exogenous affector molecules, as well as in human dermatologic diseases.

Base Sequence↗

PubMed2005

HIV protease inhibitors activate the unfolded protein response in macrophages: implication for atherosclerosis and cardiovascular disease.

Human immunodeficiency virus (HIV) protease inhibitors have been successfully used in highly active antiretroviral therapy for HIV-1 infection. Treatment of patients infected with HIV with HIV protease inhibitors is unfortunately associated with a number of clinically significant metabolic abnormalities and an increased risk of premature atherosclerosis and myocardial infarction. However, the cellular/molecular mechanisms of the HIV protease inhibitor-induced lipid dysregulation and atherosclerosis remain elusive. Macrophages are the most prominent cell type present in atherosclerotic lesions and play essential roles in both early lesion development and late lesion complications. In this study, we demonstrate that three different HIV protease inhibitors (ritonavir, indinavir, and atazanavir) induce endoplasmic reticulum stress and activate the unfolded protein response in mouse macrophages. Furthermore, at therapeutic concentrations (5-15 microM), these HIV protease inhibitors were found to increase the levels of transcriptionally active sterol regulatory element binding proteins, decrease endogenous cholesterol esterification, cause the accumulation of free cholesterol in intracellular membranes, deplete endoplasmic reticulum calcium stores, activate caspase-12, and increase apoptosis in macrophages. These findings provide possible cellular mechanisms by which HIV protease inhibitors promote atherosclerosis and cardiovascular disease in HIV-1 infected patients treated with HIV protease inhibitors.

Animals↗

PubMed1974

Role of the leu-3 cistron in the regulation of the synthesis of isoleucine and valine biosynthetic enzymes of Neurospora.

The production by Neurospora of the enzymes of isoleucine and valine synthesis in response to specific end product-derived signals depends upon the presence of an effective leu-3 regulatory product and its effector alpha-isopropylmalate (alpha-IPM). In leu-3(+) strains, threonine deaminase production is repressed as a function of available isoleucine, acetohydroxy acid synthetase as a function of valine, and the isomeroreductase and dihydroxy acid dehydratase as a function of isoleucine and leucine. In the absence of an effective leu-3 regulatory product, alpha-isopropylmalate, or both, the production of isoleucine and valine biosynthetic enzymes is fixed at or near fully repressed levels even under conditions of severe end product limitation. Thus, in addition to its involvement in the regulation of expression of the three structural genes of leucine synthesis, the leu-3 alpha-IPM regulatory product is necessary for full expression of at least four genes specifying the structure of the enzymes of isoleucine and valine synthesis. It is suggested that the leu-3 alpha-IPM regulatory element may facilitate transcription of the genetically dispersed cistrons either by imposing specificity on ribonucleic acid polymerase for structurally similar promoters adjacent to each of the cistrons or by "opening" promoters after interaction with nearly identical stretches of deoxyribonucleic acid near each of the structural genes.

Alcohol Oxidoreductases↗

PubMed1994

Hox proteins have different affinities for a consensus DNA site that correlate with the positions of their genes on the hox cluster.

The hox genes, members of a family of essential developmental regulators, have the intriguing property that their expression in the developing murine embryo is colinear with their chromosomal organization. Members of the hox gene family share a conserved DNA binding domain, termed the homeodomain, which mediates interactions of Hox proteins with DNA regulatory elements in the transcriptional control regions of target genes. In this study, we characterized the DNA binding properties of five representative members of the Hox family: HoxA5, HoxB4, HoxA7, HoxC8, and HoxB1. To facilitate a comparative analysis of their DNA binding properties, we produced the homeodomain regions of these Hox proteins in Escherichia coli and obtained highly purified polypeptides. We showed that these Hox proteins interact in vitro with a common consensus DNA site that contains the motif (C/G)TAATTG. We further showed that the Hox proteins recognize the consensus DNA site in vivo, as determined by their ability to activate transcription through this site in transient transfection assays. Although they interact optimally with the consensus DNA site, the Hox proteins exhibit subtle, but distinct, preferences for DNA sites that contain variations of the nucleotides within the consensus motif. In addition to their modest differences in DNA binding specificities, the Hox proteins also vary in their relative affinities for DNA. Intriguingly, their relative affinities correlate with the positions of their respective genes on the hox cluster. These findings suggest that subtle differences in DNA binding specificity combined with differences in DNA binding affinity constitute features of the "Hox code" that contribute to the selective functions of Hox proteins during murine embryogenesis.

Amino Acid Sequence↗

PubMed1997

Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression.

Phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK) is a key enzyme in the synthesis of glucose in the liver and kidney and of glyceride-glycerol in white adipose tissue and the small intestine. The gene for the cytosolic form of PEPCK (PEPCK-C) is acutely regulated by a variety of dietary and hormonal signals, which result in alteration of synthesis of the enzyme. Major factors that increase PEPCK-C gene expression include cyclic AMP, glucocorticoids, and thyroid hormone, whereas insulin inhibits this process. PEPCK-C is absent in fetal liver but appears at birth, concomitant with the capacity for gluconeogenesis. Regulatory elements that control transcription of the PEPCK-C gene in liver, kidney, and adipose tissue have been delineated, and many of the transcription factors that bind to these elements have been identified. Transgenic mice have been especially useful in elucidating the physiological roles of specific sequence elements in the PEPCK-C gene promoter and in demonstrating the key role played at these sites by the isoforms of CAAT/enhancer binding protein in patterning of PEPCK-C gene expression during the perinatal period. The PEPCK-C gene provides a model for the metabolic control of gene transcription.

Animals↗