Analysis of the asparagus (Asparagus officinalis) asparagine synthetase gene promoter identifies evolutionarily conserved cis-regulatory elements that mediate Suc-repression

2004 ◽  
Vol 31 (1) ◽  
pp. 63 ◽  
Author(s):  
Somrutai Winichayakul ◽  
Richard L. Moyle ◽  
Simon A. Coupe ◽  
Kevin M. Davies ◽  
Kevin J. F. Farnden
Keyword(s):  
Reporter Gene ◽  
Specific Interaction ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Asparagus Officinalis ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Gus Reporter ◽  
Foliar Senescence ◽  
Responsive Element

In asparagus (Asparagus officinalis L.), increased levels of asparagine (Asn) and Asn synthetase (AS) transcript are detected during foliar senescence and in harvested spears, possibly triggered by signals from a reduced supply of carbohydrate. To identify cis-elements mediating this regulation, the asparagus AS gene promoter was isolated and analysed by DNA sequencing, followed by expression of AS::GUS (β-glucuronidase) reporter-gene constructs in transgenic tissue, and electrophoretic mobility shift assays (EMSA). The 1958-base pair (bp) region of the AS promoter upstream of the translation initiation ATG (–1958 bp region) was sufficient to confer sucrose (Suc)-regulated expression on the GUS reporter gene in asparagus callus and protoplasts, which were transformed by particle bombardment and electroporation, respectively. Removal of Suc from callus or protoplast media resulted in the induction of GUS activity. Deletion analysis of this 1958-bp fragment identified elements in the –640 to –266�bp region as important for both high GUS levels and mediating the Suc response. This was supported by EMSA results, which showed the formation of three nuclear protein–DNA complexes with the –558 to –284 bp fragment of the promoter. A 20-bp oligonucleotide, designed to match the sequence from –423 to –404 bp, was able to out-compete formation of one of these protein-DNA complexes, suggesting a specific interaction with this sequence. This region of the promoter, overlapping with the 20-bp oligonucleotide sequence, contains a 10-bp stretch identical to a sequence previously shown to mediate low Suc induction of an Oryza sativa (rice) α-amylase gene, and may thus represent a conserved Suc-responsive element.

Functional overload increases β-MHC promoter activity in rodent fast muscle via the proximal MCAT (βe3) site

2002 ◽  
Vol 282 (3) ◽  
pp. C518-C527 ◽  
Author(s):  
Julia M. Giger ◽  
Fadia Haddad ◽  
Anqi X. Qin ◽  
Kenneth M. Baldwin
Keyword(s):  
Gene Promoter ◽  
Firefly Luciferase ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Type I ◽  
Luciferase Reporter Gene ◽  
Mobility Shift ◽  
Slow Type ◽  
Responsive Element ◽  
Gel Mobility Shift

Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, β-MHC isoform. Different length rat β-MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of −3,500, −914, −408, and −215 bp promoters increased in response to 1 wk of OL. The smallest −171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the −171 and −408 bp region were performed. The −408 bp promoters containing mutations of the βe1, distal muscle CAT (MCAT; βe2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (βe3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the βe3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the βe3 site functions as a putative OL-responsive element in the rat β-MHC gene promoter.

scholarly journals RETRACTED ARTICLE: The specific MYB binding sites bound by TaMYB in the GAPCp2/3 promoters are involved in the drought stress response in wheat

2019 ◽  
Vol 19 (1) ◽  
Author(s):  
Lin Zhang ◽  
Zhiqiang Song ◽  
Fangfang Li ◽  
Xixi Li ◽  
Haikun Ji ◽  
...  
Keyword(s):  
Gene Expression ◽  
Abiotic Stress ◽  
Drought Stress ◽  
Reporter Gene ◽  
Binding Sites ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Cis Elements ◽  
Gus Reporter ◽  
Almost All

Abstract Background Drought stress is one of the major abiotic stresses that affects plant growth and productivity. The GAPCp genes play important roles in drought stress tolerance in multiple species. The aim of this experiment was to identify the core cis-regulatory elements that may respond to drought stress in the GAPCp2 and GAPCp3 promoter sequences. Results In this study, the promoters of GAPCp2 and GAPCp3 were cloned. The promoter activities were significantly improved under abiotic stress via regulation of Rluc reporter gene expression, while promoter sequence analysis indicated that these fragments were not almost identical. In transgenic Arabidopsis with the expression of the GUS reporter gene under the control of one of these promoters, the activities of GUS were strong in almost all tissues except the seeds, and the activities were induced after abiotic stress. The yeast one-hybrid system and EMSA demonstrated that TaMYB bound TaGAPCp2P/3P. By analyzing different 5′ deletion mutants of these promoters, it was determined that TaGAPCp2P (− 1312~ − 528) and TaGAPCp3P (− 2049~ − 610), including the MYB binding site, contained enhancer elements that increased gene expression levels under drought stress. We used an effector and a reporter to co-transform tobacco and found that TaMYB interacted with the specific MYB binding sites of TaGAPCp2P (− 1197~ − 635) and TaGAPCp3P (− 1456~ − 1144 and − 718~ − 610) in plant cells. Then, the Y1H system and EMSA assay demonstrated that these MYB binding sites in TaGAPCp2P (− 1135 and − 985) and TaGAPCp3P (− 1414 and − 665) were the target cis-elements of TaMYB. The deletion of the specific MYB binding sites in the promoter fragments significantly restrained the drought response, and these results confirmed that these MYB binding sites (AACTAAA/C) play vital roles in improving the transcription levels under drought stress. The results of qRT-PCR in wheat protoplasts transiently overexpressing TaMYB indicated that the expression of TaGAPCp2/3 induced by abiotic stress was upregulated by TaMYB. Conclusion The MYB binding sites (AACTAAA/C) in TaGAPCp2P/3P were identified as the key cis-elements for responding to drought stress and were bound by the transcription factor TaMYB.

scholarly journals A natural sequence consisting of overlapping glucocorticoid-responsive elements mediates glucocorticoid, but not androgen, regulation of gene expression

2000 ◽  
Vol 350 (1) ◽  
pp. 123-129 ◽  
Author(s):  
Charbel MASSAAD ◽  
Michèle GARLATTI ◽  
Elizabeth M. WILSON ◽  
Françoise CADEPOND ◽  
Robert BAROUKI
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Transcriptional Activation ◽  
Regulation Of Gene Expression ◽  
Gene Promoter ◽  
Mobility Shift ◽  
Liver And Kidney ◽  
Androgen Regulation ◽  
Responsive Element ◽  
Half Site

Cytosolic aspartate aminotransferase (cAspAT) is regulated by glucocorticoids in rat liver and kidney. Part of this regulation is mediated by an unusual glucocorticoid-responsive element (GRE)-like sequence called GRE A. GRE A is composed of two overlapping imperfect GREs, each comprising a conserved half-site (half-sites 1 and 4 respectively) and a poorly conserved half-site (half-sites 2 and 3 respectively). The sequence binds co-operatively two dimers of the glucocorticoid receptor (GR) and mediates efficient glucocorticoid regulation of gene expression. Analysis of deletions of the cAspAT gene promoter and subcloning of GRE A upstream of the thymidine kinase promoter indicate that this sequence is responsive to glucocorticoids, but not to androgens. Electrophoretic mobility shift assays indicate that the GRE A unit does not bind the androgen receptor (AR). The modification of three nucleotides in the poorly conserved half-sites 2 and 3, converting GRE A into two overlapping high-affinity GREs (ov-cGRE), resulted in co-operative binding of the AR. Furthermore, ov-cGRE efficiently mediated androgen regulation of the thymidine kinase promoter. A single base modification in half-site 2 or 3 in GRE A allowed the binding of the AR as one or two dimers respectively, and restored transcriptional activation by androgens only in the latter case. Thus the poor affinity of the AR for half-sites 2 and 3 prevented its binding to GRE A, indicating that the overlapping GRE A sequence of the cAspAT gene promoter discriminates a glucocorticoid-mediated from an androgen-mediated response.

Cloning of the Promoter Region of Human Endoglin, the Target Gene for Hereditary Hemorrhagic Telangiectasia Type 1

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4677-4690 ◽  
Author(s):  
Carlos Rı́us ◽  
Joshua D. Smith ◽  
Nuria Almendro ◽  
Carmen Langa ◽  
Luisa M. Botella ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Hereditary Hemorrhagic Telangiectasia ◽  
Promoter Region ◽  
Transforming Growth Factor ◽  
Specific Activity ◽  
Specific Interaction ◽  
Regulatory Elements ◽  
Mobility Shift ◽  
Isolation And Characterization

Endoglin (CD105) is a cell surface component of the transforming growth factor-β (TGF-β) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5′-flanking sequence of the human endoglin gene has been isolated. The 5′-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFκB, and Mad, as well as TGF-β–, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5′ RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream −400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the −81/+350 fragment as well as major transcriptional regulatory elements within the −400/−141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position −68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-β1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.

Cloning of the Promoter Region of Human Endoglin, the Target Gene for Hereditary Hemorrhagic Telangiectasia Type 1

Blood ◽  
1998 ◽  
Vol 92 (12) ◽  
pp. 4677-4690 ◽  
Author(s):  
Carlos Rı́us ◽  
Joshua D. Smith ◽  
Nuria Almendro ◽  
Carmen Langa ◽  
Luisa M. Botella ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Hereditary Hemorrhagic Telangiectasia ◽  
Promoter Region ◽  
Transforming Growth Factor ◽  
Specific Activity ◽  
Specific Interaction ◽  
Regulatory Elements ◽  
Mobility Shift ◽  
Isolation And Characterization

Abstract Endoglin (CD105) is a cell surface component of the transforming growth factor-β (TGF-β) receptor complex highly expressed by endothelial cells. Mutations in the endoglin gene are responsible for the hereditary hemorrhagic telangiectasia type 1 (HHT1), also known as Osler-Weber-Rendu syndrome (OMIM 187300). This is an autosomal dominant vascular disorder probably caused by a haploinsufficiency mechanism displaying low levels of the normal protein. To understand the mechanisms underlying the regulated expression of endoglin, a genomic DNA clone containing 3.3 kb of the 5′-flanking sequence of the human endoglin gene has been isolated. The 5′-flanking region of the endoglin gene lacks consensus TATA and CAAT boxes, but contains two GC-rich regions and consensus motifs for Sp1, ets, GATA, AP-2, NFκB, and Mad, as well as TGF-β–, glucocorticoid-, vitamin D-, and estrogen-responsive elements. As determined by primer extension and 5′ RACE experiments, a cluster of transcriptional start sites was found to be located 350 bp upstream from the translation initiation codon. To analyze the endoglin promoter activity, the upstream −400/+341 fragment was fused to the luciferase gene and transient transfections were conducted in several cell types. This construct displayed a tissue-specific activity in human and bovine endothelial cells. Analysis of various deletion constructs showed the existence of a basal promoter region within the −81/+350 fragment as well as major transcriptional regulatory elements within the −400/−141 fragment. Electrophoretic mobility shift assays demonstrated the specific interaction of a member of the ets family with a consensus motif located at position −68. A promoter construct mutated at this ets sequence showed a much reduced activity as compared with the wild-type construct, supporting the involvement of this ets motif in the basal activity of the promoter. The endoglin promoter exhibited inducibility in the presence of TGF-β1, suggesting possible therapeutic treatments in HHT1 patients, in which the expression level of the normal endoglin allele might not reach the threshold required for its function. Isolation and characterization of the human endoglin promoter represents an initial step in elucidating the controlled expression of the endoglin gene.

The harvest-responsive region of the Asparagus officinalis sparagine synthetase promoter reveals complexity in the regulation of the harvest response

2008 ◽  
Vol 35 (12) ◽  
pp. 1212 ◽  
Author(s):  
Donald A. Hunter ◽  
Lyn M. Watson
Keyword(s):  
Promoter Region ◽  
Constitutive Expression ◽  
Wound Response ◽  
Asparagus Officinalis ◽  
Minimal Promoter ◽  
Nucleotide Substitutions ◽  
Cis Acting ◽  
Gus Reporter ◽  
Highly Active ◽  
Responsive Element

The activity of a 1915-bp asparagine synthetase (AS) promoter of Asparagus officinalis L. was induced in mature leaves of transgenic Arabidopsis thaliana (L.) Heynh. plants when the leaves were detached and held in water for 24 h. To understand this induction by harvest, variants of the AS promoter were linked to the β-glucuronidase GUS reporter gene. Harvest induction in the leaves required detachment and was not simply a wound response. Two regions in the AS promoter (Region A, –640 to –523; Region B, –524 to –383) were independently able to confer harvest response to the otherwise unresponsive –383AS (minimal) promoter. Region A was studied in further detail. Various truncations, deletions, or nucleotide substitutions of Region A affected activity and fold induction of the minimal promoter. However, no harvest-inducible cis-acting element within Region A was identified. Although the minimal promoter contained a dehydration-responsive element and ACGT elements similar to ABA-responsive regulatory motifs these were not needed by the upstream regulatory regions for directing harvest response. When four copies of Region A were linked to the minimal promoter it became highly active in leaves before harvest. Deletions within Region A showed that it required its complete 117 bp for driving harvest response, yet the region cannot simply be thought of as a harvest-responsive module, since its concatemerisation led to constitutive expression.

scholarly journals Divergent regulation of the human atrial natriuretic peptide gene by c-jun and c-fos.

1992 ◽  
Vol 12 (1) ◽  
pp. 292-301 ◽  
Author(s):  
B Kovacic-Milivojević ◽  
D G Gardner
Keyword(s):  
Atrial Natriuretic Peptide ◽  
Natriuretic Peptide ◽  
Transcriptional Activation ◽  
Gene Promoter ◽  
Regulatory Elements ◽  
Neonatal Rat ◽  
Regulatory Sequence ◽  
Mobility Shift ◽  
Human Atrial Natriuretic Peptide ◽  
Atrial Natriuretic

Employing transient transfection analysis in neonatal rat cardiocytes, we have demonstrated that overexpression of c-jun results in a dose-dependent induction of the human atrial natriuretic peptide (hANP) gene promoter. Studies using a series of mutations in the hANP gene promoter identified a TRE-like, cis-acting regulatory sequence which conferred c-jun sensitivity. This same region was shown to interact with the c-jun/c-fos complex in an in vitro gel mobility shift assay. Selective mutation of this site suppressed basal activity of the hANP promoter and significantly reduced c-jun-dependent activation. Overexpression of c-fos had a biphasic effect on hANP gene promoter activity. At low levels, in concert with c-jun, it activated, while at higher levels it suppressed, transcription from the hANP gene promoter. This inhibition was both cell and promoter specific. hANP gene promoter sequences which mediate c-fos-dependent inhibition appear to be separable from those responsible for the induction. In addition, the protein domains on c-fos responsible for transcriptional activation and repression can be segregated topographically, with the inhibitory activity being localized to the carboxy-terminal domain. Thus, c-fos can activate or repress hANP gene expression through two separate functional domains that act on distinct regulatory elements in the hANP gene promoter. These data imply that the ANP gene may be a physiological target for c-fos- and c-jun-dependent activity in the heart and suggest a potential mechanism linking environmental stimuli to its expression.

scholarly journals Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter

2021 ◽  
Author(s):  
Malgorzata Gorniak-Walas ◽  
Karolina Nizinska ◽  
Katarzyna Lukasiuk
Keyword(s):  
Transcriptional Regulation ◽  
Reporter Gene ◽  
Hippocampal Neurons ◽  
Gene Promoter ◽  
Promoter Sequence ◽  
Regulatory Elements ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay

AbstractTweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

scholarly journals Interaction of CCAAT/enhancer-binding protein α and β with the rat caeruloplasmin gene promoter

1993 ◽  
Vol 294 (2) ◽  
pp. 473-479 ◽  
Author(s):  
C D Bingle ◽  
R E Fleming ◽  
J D Gitlin
Keyword(s):  
Rat Liver ◽  
Specific Binding ◽  
Binding Protein ◽  
Specific Interaction ◽  
Gene Promoter ◽  
Nuclear Extract ◽  
Mobility Shift ◽  
Specific Expression ◽  
Enhancer Binding Protein ◽  
Ccaat Enhancer Binding Protein

To determine the mechanisms of expression of the rat caeruloplasmin gene, the promoter region was analysed by DNAase I footprinting. Using nuclear extract from rat liver, a prominent site of protein-DNA interaction was detected from -93 to -48 upstream of the caeruloplasmin gene transcription start and sequence analysis of this region revealed three potential CCAAT/enhancer-binding protein (C/EBP) consensus elements. Mobility-shift analysis using an oligonucleotide encoding this region identified specific binding of proteins from rat liver nuclear extract, and some of these complexes were supershifted using antisera to the C/EBP alpha and beta family members. Mobility-shift studies using a polypeptide encoding the DNA-binding domain of C/EBP alpha also revealed a specific interaction with this region of the caeruloplasmin promoter, and DNAase I footprinting using this polypeptide protected the identical region from -93 to -48. Co-transfection of expression plasmids encoding C/EBP alpha or a related leucine-zipper factor D-binding protein (DBP) revealed a C/EBP-specific increase in reporter gene activity in HepG2 cells transfected with caeruloplasmin-chloramphenicol acetyltransferase containing the -93 to -48 region. A similar result was obtained when these constructs were co-transfected into mouse L cells which were shown not to express the endogenous caeruloplasmin gene. Taken together, these data indicate a role for C/EBP alpha and beta in mediating transcription from the caeruloplasmin gene promoter and suggest that this region of the promoter is not responsible for tissue-specific expression.

scholarly journals Genomic organization, chromosomal mapping and promoter analysis of the mouse selenocysteine tRNA gene transcription-activating factor (mStaf) gene

2000 ◽  
Vol 346 (1) ◽  
pp. 45-51 ◽  
Author(s):  
Kazushige ADACHI ◽  
Masato KATSUYAMA ◽  
Shigeaki SONG ◽  
Takami OKA
Keyword(s):  
Transcriptional Activation ◽  
Trna Gene ◽  
Zinc Finger Protein ◽  
Regulatory Elements ◽  
Chromosomal Mapping ◽  
Proximal Promoter ◽  
Mobility Shift ◽  
Responsive Element ◽  
Specificity Protein ◽  
Electrophoretic Mobility Shift Assays

mStaf is a zinc-finger protein that activates the transcription of the mouse selenocysteine tRNA gene. The mStaf gene is approx. 35 kb long and split into 16 exons. All exon-intron junction sequences conform to the GT/AG rule. The transcription start site is located 83 bp upstream of the initiation codon. Chromosomal mapping localized the gene to mouse chromosome 7, region E3-F1. Sequence analysis of the proximal promoter region revealed several potential regulatory elements; these include the recognition elements of Sp1, Nkx, CP2, E2A, SIF (SIS-inducible factor), TFII-I and cAMP-responsive element (CRE), but no TATA sequences. Transfection experiments demonstrated that the 5ʹ-flanking region (-1894 to +37) of the mStaf gene drives transcription in mouse NMuMG cells and that a construct containing a fragment from -387 to +37 showed the highest transcriptional activity. Deletion and mutation experiments suggested that four Sp1 sites played an important role for the basal promoter activity. Furthermore, electrophoretic mobility-shift assays demonstrated that Sp3 but not other Sp (specificity protein) family members binds to three of the Sp1 sites. Our present study suggests that Sp3 is involved in the basal transcriptional activation of the mStaf gene.

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