scholarly journals Involvement of AP-2 in regulation of the R-FABP gene in the developing chick retina.

1997 ◽  
Vol 17 (10) ◽  
pp. 5935-5945 ◽  
Author(s):  
D A Bisgrove ◽  
E A Monckton ◽  
R Godbout
Keyword(s):  
Binding Site ◽  
Retinal Development ◽  
Regulatory Elements ◽  
Dnase I ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Dna Transfection ◽  
Fatty Acid Binding ◽  
Chick Retina ◽  
Fabp Gene

Little is known regarding the molecular pathways that underlie the retinal maturation process. We are studying the regulation of the retinal fatty-acid-binding protein (R-FABP) gene, highly expressed in retinal precursor cells, to identify DNA regulatory elements and transcriptional factors involved in retinal development. Although the upstream sequence of the R-FABP gene is extremely GC rich, CpG methylation in this region is not implicated in the regulation of this gene because the 5' flanking DNA remains unmethylated with tissue differentiation when there is a dramatic decrease in R-FABP transcript levels. Using a combination of DNase I hypersensitivity experiments, gel shift assays, and DNase I footprinting, we have found three sites of DNA-protein interaction within 205 bp of 5' flanking DNA in the undifferentiated retina and four sites in the differentiated retina. DNA transfection analysis indicates that the first two footprints located within 150 bp of 5' flanking DNA are required for high levels of transcription in primary undifferentiated retinal cultures. The first footprint includes a putative TATA box and Spl binding sites while the second footprint contains a consensus AP-2 DNA binding site. Supershift experiments using antibodies to AP-2 and methylation interference experiments indicate that an AP-2-like transcription factor present in both late-proliferative-stage retina and differentiated retina binds to the upstream region of the R-FABP gene. A combination of data including the expression profile of AP-2 during retinal development and DNA transfection analysis using constructs mutated at critical residues within the AP-2 binding site suggests that AP-2 is a repressor of R-FABP transcription.

scholarly journals Regulatory elements in the upstream region of metallothionein gene in tilapia species

2021 ◽  
Vol 30 (1) ◽  
pp. 95-103
Author(s):  
Mohammad Shamimul Alam ◽  
Israt Jahan ◽  
Sadniman Rahman ◽  
Hawa Jahan ◽  
Kaniz Fatema
Keyword(s):  
Tissue Specificity ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Metal Resistance ◽  
Genomic Region ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Oreochromis Aureus ◽  
Metallothionein Gene ◽  
Genomic Regions

Tilapia is a hardy fish which can survive in water bodies polluted with heavy metals. Metal resistance is conferred by higher expression of metallothionein gene (mt) in many organisms. Level, time and tissue-specificity of gene expression is regulated through transcription factor binding sites (TFBS) which may be present in the upstream, downstream, or even in the introns of a gene. So, as a candidate regulatory region, the 5’upstream sequence of mt gene in three tilapia species, Oreochromis aureus, O. niloticus and O. mossambicus was studied. The targeted region was PCR-amplified and then sequenced using a pair of custom-designed primer. A total of only 2.7% variation was found in the sequenced genomic region among the three species. Metal-related TFBS were predicted from these sequences. A total of twenty eight TFBS were found in O. aureus and twenty nine in O. mossambicus and O. niloticus. The number of metalrelated TFBS predicted in the targeted sequence was significantly higher compared to that found in randomly selected other genomic regions of same size from O. niloticus genome. Thus, the results suggest the presence of putative regulatory elements in the targeted upstream region which might have important role in the regulation of mt gene function. Dhaka Univ. J. Biol. Sci. 30(1): 95-103, 2021 (January)

scholarly journals A conserved GATA motif in a tissue-specific DNase I hypersensitive site of the cardiac α-myosin heavy chain gene

1997 ◽  
Vol 325 (1) ◽  
pp. 47-51 ◽  
Author(s):  
Weei-Yuarn HUANG ◽  
Choong-Chin LIEW
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Developmental Stages ◽  
Regulatory Elements ◽  
Dnase I ◽  
Upstream Region ◽  
Chain Gene ◽  
Nuclear Extracts ◽  
Hypersensitive Sites ◽  
Gata Motif

Transgenic analysis has indicated that far upstream regulatory elements of the cardiac α-myosin heavy chain (MyHC) gene are required for appropriate transgene expression [Subramaniam, Gulick, Neumann, Knotts and Robbins (1993) J. Biol. Chem. 268, 4331–4336]. In an attempt to identify these as-yet-undefined regulatory elements, we mapped the DNase I hypersensitive sites (DHSs) in the 4 kb upstream region of the hamster cardiac α-MyHC gene. When using nuclei isolated from late-gestational and adult heart ventricles, a strong DHS was identified in the -1.9 kb region (α-1.9 kb site). It cannot be detected in kidney, liver or cardiofibroblast nuclei. Within this site, we found a conserved GATA-motif that interacts specifically with GATA-binding factors in nuclear extracts of cardiomyocytes at various developmental stages. These data provide further evidence to support the role of GATA factors in the regulation of cardiac α-MyHC gene expression.

scholarly journals Multiple muscle-specific regulatory elements are associated with a DNase I hypersensitive site of the cardiac β-myosin heavy-chain gene

1997 ◽  
Vol 327 (2) ◽  
pp. 507-512 ◽  
Author(s):  
Weei-Yuarn HUANG ◽  
Jin-Jer CHEN ◽  
N.-L. SHIH ◽  
Choong-Chin LIEW
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Specific Interaction ◽  
Regulatory Elements ◽  
Dnase I ◽  
Proximal Promoter ◽  
Upstream Region ◽  
Chain Gene ◽  
Kinase Gene ◽  
Mouse Muscle

Using nuclei isolated from neonatal cardiomyocytes, we have mapped the DNase I hypersensitive sites (DHSs) residing within the 5ʹ-upstream regions of the hamster cardiac myosin heavy-chain (MyHC) gene. Two cardiac-specific DHSs within the 5 kb upstream region of the cardiac MyHC gene were identified. One of the DHSs was mapped to the -2.3 kb (β-2.3 kb) region and the other to the proximal promoter region. We further localized the β-2.3 kb site to a range of 250 bp. Multiple, conserved, muscle regulatory motifs were found within the β-2.3 kb site, consisting of three E-boxes, one AP-2 site, one CArG motif, one CT/ACCC box and one myocyte-specific enhancer factor-2 site. This cluster of regulatory elements is strikingly similar to a cluster found in the enhancer of the mouse muscle creatine kinase gene (-1256 to -1050). The specific interaction of the motifs within the β-2.3 kb site and the cardiac nuclear proteins was demonstrated using gel mobility-shift assays and footprinting analysis. In addition, transfection analysis revealed a significant increase in chloramphenicol acetyltransferase activity when the β-2.3 kb site was linked to a heterologous promoter. These results suggest that previously undefined regulatory elements of the β-MyHC gene may be associated with the β-2.3 kb site.

Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene

1988 ◽  
Vol 8 (8) ◽  
pp. 3467-3475
Author(s):  
P G Quinn ◽  
T W Wong ◽  
M A Magnuson ◽  
J B Shabb ◽  
D K Granner
Keyword(s):  
Cyclic Amp ◽  
Binding Site ◽  
Promoter Activity ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Dnase I ◽  
Wild Type ◽  
Basal Promoter Activity ◽  
Regulated Expression

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.

scholarly journals Epithelial cell differentiation in normal and transgenic mouse intestinal isografts.

1991 ◽  
Vol 113 (5) ◽  
pp. 1183-1192 ◽  
Author(s):  
D C Rubin ◽  
K A Roth ◽  
E H Birkenmeier ◽  
J I Gordon
Keyword(s):  
Spatial Differentiation ◽  
Human Growth ◽  
Regulatory Elements ◽  
Enteroendocrine Cells ◽  
Lower Percentage ◽  
Inhibitory Peptide ◽  
Fatty Acid Binding ◽  
Cell Subpopulations ◽  
Inappropriate Expression ◽  
Fabp Gene

Transgenes consisting of segments of the rat liver fatty acid-binding protein (L-FABP) gene's 5' non-transcribed domain linked to the human growth hormone (hGH) gene (minus its regulatory elements) have provided useful tools for analyzing the mechanisms that regulate cellular and spatial differentiation of the continuously renewing gut epithelium. We have removed the jejunum from normal and transgenic fetal mice before or coincident with, cytodifferentiation of its epithelium. These segments were implanted into the subcutaneous tissues of young adult CBY/B6 nude mouse hosts to determine whether the bipolar, migration-dependent differentiation pathways of gut epithelial cells can be established and maintained in the absence of its normal luminal environment. Immunocytochemical analysis of isografts harvested 4-6 wk after implantation revealed that activation of the intact endogenous mouse L-FABP gene (fabpl) in differentiating enterocytes is perfectly recapitulated as these cells are translocated along the crypt-to-villus axis. Similarly, Paneth and goblet cells appear to appropriately differentiate as they migrate to the crypt base and villus tip, respectively. The enteroendocrine cell subpopulations present in intact 4-6-wk-old jejunum are represented in these isografts. Their precise spatial distribution along the crypt-to-villus axis mimics that seen in the intact gut. A number of complex interrelationships between enteroendocrine subpopulations are also recapitulated. In both "intact" and isografted jejunum, nucleotides -596 to +21 of the rat L-FABP gene were sufficient to direct efficient expression of the hGH reporter to enterocytes although precocious expression of the transgene occurred in cells located in the upper crypt, before their translocation to the villus base. Inappropriate expression of hGH occurred in a high percentage (greater than 80%) of secretin, gastrin, cholecystokinin, and gastric inhibitory peptide producing enteroendocrine cells present in the intact jejunum of 4-6-wk-old L-FABP-596 to +21/hGH transgenics. Addition of nucleotides -597 to -4,000 reduced the percentage of cells co-expressing this reporter four- to eightfold in several of the subpopulations. Jejunal isografts from each transgenic pedigree studied contained a lower percentage of hGH positive enteroendocrine cells than in the comparably aged intact jejunum.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals An Upstream Sequence Element Required for NucC-Dependent Expression of the Serratia marcescensExtracellular Nuclease

1998 ◽  
Vol 180 (22) ◽  
pp. 6064-6067 ◽  
Author(s):  
Robert H. Winslow ◽  
Bryan Julien ◽  
Richard Calendar ◽  
Gail E. Christie
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Upstream Region ◽  
Upstream Sequence ◽  
Gene Encoding ◽  
Sequence Element ◽  
Activator Proteins ◽  
Footprint Analysis ◽  
Extracellular Nuclease ◽  
Dyad Symmetry

ABSTRACT The Serratia marcescens extracellular nuclease gene,nucA, is positively regulated by the product of thenucC gene. In this study, the upstream region required for NucC-dependent nuclease expression was defined by using fusions to the gene encoding chloramphenicol acetyltransferase (cat). This sequence includes an element of hyphenated dyad symmetry identified previously as the binding site for the P2 Ogr family of activators. Footprint analysis confirmed that members of this family of activator proteins bind to this site, protecting a region between −76 and −59 relative to the start of transcription. The activator binding site in the nucA promoter lies one turn of the helix upstream from the corresponding sites in the P2 and P4 late promoters. The effects of deletions between the downstream end of the activator binding site and the putative −35 region are consistent with a strict helical phasing requirement for activation.

scholarly journals Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.

1988 ◽  
Vol 8 (8) ◽  
pp. 3467-3475 ◽  
Author(s):  
P G Quinn ◽  
T W Wong ◽  
M A Magnuson ◽  
J B Shabb ◽  
D K Granner
Keyword(s):  
Cyclic Amp ◽  
Binding Site ◽  
Promoter Activity ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Regulatory Elements ◽  
Initiation Site ◽  
Dnase I ◽  
Wild Type ◽  
Basal Promoter Activity ◽  
Regulated Expression

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.

scholarly journals CadC Activates pH-Dependent Expression of the Vibrio vulnificus cadBA Operon at a Distance through Direct Binding to an Upstream Region

2005 ◽  
Vol 187 (22) ◽  
pp. 7870-7875 ◽  
Author(s):  
Jee Eun Rhee ◽  
Kun-Soo Kim ◽  
Sang Ho Choi
Keyword(s):  
Binding Site ◽  
Vibrio Vulnificus ◽  
Direct Interaction ◽  
Dnase I ◽  
Upstream Region ◽  
Dependent Manner ◽  
Protection Assay ◽  
Direct Binding ◽  
Ph Dependent ◽  
Single Promoter

ABSTRACT The Vibrio vulnificus cadBA genes were transcribed as a transcriptional operon by a single promoter, P cadBA , which was activated by CadC in a pH-dependent manner. A direct interaction between CadC and the P cadBA DNA was demonstrated, and a CadC binding site centered at −233.5 was mapped by deletion analyses of P cadBA  and confirmed by a DNase I protection assay.

Characterization of Nucleotide Binding ­Site-Encoding Genes in Sweetpotato, Ipomoea batatas(L.) Lam., and Their Response to Biotic and Abiotic Stresses

2021 ◽  
pp. 1-15
Author(s):  
Zengzhi Si ◽  
Yake Qiao ◽  
Kai Zhang ◽  
Zhixin Ji ◽  
Jinling Han
Keyword(s):  
Binding Site ◽  
Abiotic Stresses ◽  
Ipomoea Batatas ◽  
Expression Profiles ◽  
Nucleotide Binding ◽  
Regulatory Elements ◽  
Nucleotide Binding Site ◽  
Biotic And Abiotic Stresses ◽  
Encoding Genes

Sweetpotato, <i>Ipomoea batatas</i> (L.) Lam., is an important and widely grown crop, yet its production is affected severely by biotic and abiotic stresses. The nucleotide binding site (NBS)-encoding genes have been shown to improve stress tolerance in several plant species. However, the characterization of NBS-encoding genes in sweetpotato is not well-documented to date. In this study, a comprehensive analysis of NBS-encoding genes has been conducted on this species by using bioinformatics and molecular biology methods. A total of 315 NBS-encoding genes were identified, and 260 of them contained all essential conserved domains while 55 genes were truncated. Based on domain architectures, the 260 NBS-encoding genes were grouped into 6 distinct categories. Phylogenetic analysis grouped these genes into 3 classes: TIR, CC (I), and CC (II). Chromosome location analysis revealed that the distribution of NBS-encoding genes in chromosomes was uneven, with a number ranging from 1 to 34. Multiple stress-related regulatory elements were detected in the promoters, and the NBS-encoding genes’ expression profiles under biotic and abiotic stresses were obtained. According to the bioinformatics analysis, 9 genes were selected for RT-qPCR analysis. The results revealed that <i>IbNBS75</i>, <i>IbNBS219</i>, and <i>IbNBS256</i> respond to stem nematode infection; <i>Ib­NBS240</i>, <i>IbNBS90</i>, and <i>IbNBS80</i> respond to cold stress, while <i>IbNBS208</i>, <i>IbNBS71</i>, and <i>IbNBS159</i> respond to 30% PEG treatment. We hope these results will provide new insights into the evolution of NBS-encoding genes in the sweetpotato genome and contribute to the molecular breeding of sweetpotato in the future.

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