scholarly journals Distinct cis-acting elements mediate clock, light, and developmental regulation of the Neurospora crassa eas (ccg-2) gene.

1996 ◽  
Vol 16 (2) ◽  
pp. 513-521 ◽  
Author(s):  
D Bell-Pedersen ◽  
J C Dunlap ◽  
J J Loros
Keyword(s):  
Neurospora Crassa ◽  
Developmental Regulation ◽  
Reporter System ◽  
Mobility Shift ◽  
Developmental Control ◽  
Control Of Gene Expression ◽  
Cis Acting ◽  
Regulated Expression ◽  
Complex Regulation ◽  
Electrophoretic Mobility Shift Assays

The Neurospora crassa eas (ccg-2) gene, which encodes a fungal hydrophobin, is transcriptionally regulated by the circadian clock. In addition, eas (ccg-2) is positively regulated by light and transcripts accumulate during asexual development. To sort out the basis of this complex regulation, deletion analyses of the eas (ccg-2) promoter were carried out to localize the cis-acting elements mediating clock, light, and developmental control. The primary sequence determinants of a positive activating clock element (ACE) were found to reside in a 45-bp region, just upstream from the TATA box. Using a novel unregulated promoter/reporter system developed for this study, we show that a 68-bp sequence encompassing the ACE is sufficient to confer clock regulation on the eas (ccg-2) gene. Electrophoretic mobility shift assays using the ACE reveal factors present in N. crassa protein extracts that recognize and bind specifically to DNA containing this element. Separate regions of the eas (ccg-2) promoter involved in light induction and developmental control are identified and shown not to be required for clock-regulated expression of eas (ccg-2). The distinct nature of the ACE validates its use as a tool for the identification of upstream regulatory factors involved in clock control of gene expression.

The extrachromosomal replication of Dictyostelium plasmid Ddp2 requires a cis-acting element and a plasmid-encoded trans-acting factor

1990 ◽  
Vol 10 (7) ◽  
pp. 3727-3736
Author(s):  
B Leiting ◽  
I J Lindner ◽  
A A Noegel
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Wild Strain ◽  
Open Reading Frame ◽  
Mobility Shift ◽  
Reading Frame ◽  
Cis Acting ◽  
Transformation Vector ◽  
Cis And Trans ◽  
Electrophoretic Mobility Shift Assays

Dictyostelium discoideum plasmid Ddp2 from the wild strain WS380B is a 5.8-kilobase (kb) supercoiled circle with a copy number of 300 per haploid genome. We previously described the construction of an extrachromosomally replicating transformation vector pnDeI carrying 4.7 kb of Ddp2 sequences (B. Leiting, and A. Noegel, Plasmid 20:241-248, 1988). In order to reduce the sequences required for extrachromosomal maintenance in D. discoideum, we characterized Ddp2 by sequence analysis, by deletion experiments, by transcription mapping, by electrophoretic mobility shift assays, and by expression of its single open reading frame in Escherichia coli. Two elements were involved in replication of Ddp2: a cis-acting sequence located on a 592-base-pair (bp) fragment that consisted of 220 bp of essential and 372 bp of auxiliary sequences, and a 2.7-kb open reading frame which most likely encodes a trans-acting factor. The cis- and trans-acting elements did not overlap and were shown to act independently from the location of the sequences encoding the trans-acting factor.

scholarly journals Ohr and OhrR Are Critical for Organic Peroxide Resistance and Symbiosis in Azorhizobium caulinodans ORS571

Genes ◽  
2020 ◽  
Vol 11 (3) ◽  
pp. 335
Author(s):  
Yang Si ◽  
Dongsen Guo ◽  
Shuoxue Deng ◽  
Xiuming Lu ◽  
Juanjuan Zhu ◽  
...  
Keyword(s):  
Nitrogenase Activity ◽  
Organic Peroxide ◽  
Nodule Formation ◽  
Azorhizobium Caulinodans ◽  
Reporter System ◽  
Mobility Shift ◽  
Organic Hydroperoxide ◽  
Direct Binding ◽  
Plant Assays ◽  
Electrophoretic Mobility Shift Assays

Azorhizobium caulinodans is a symbiotic nitrogen-fixing bacterium that forms both root and stem nodules on Sesbania rostrata. During nodule formation, bacteria have to withstand organic peroxides that are produced by plant. Previous studies have elaborated on resistance to these oxygen radicals in several bacteria; however, to the best of our knowledge, none have investigated this process in A. caulinodans. In this study, we identified and characterised the organic hydroperoxide resistance gene ohr (AZC_2977) and its regulator ohrR (AZC_3555) in A. caulinodans ORS571. Hypersensitivity to organic hydroperoxide was observed in an ohr mutant. While using a lacZ-based reporter system, we revealed that OhrR repressed the expression of ohr. Moreover, electrophoretic mobility shift assays demonstrated that OhrR regulated ohr by direct binding to its promoter region. We showed that this binding was prevented by OhrR oxidation under aerobic conditions, which promoted OhrR dimerization and the activation of ohr. Furthermore, we showed that one of the two conserved cysteine residues in OhrR, Cys11, was critical for the sensitivity to organic hydroperoxides. Plant assays revealed that the inactivation of Ohr decreased the number of stem nodules and nitrogenase activity. Our data demonstrated that Ohr and OhrR are required for protecting A. caulinodans from organic hydroperoxide stress and play an important role in the interaction of the bacterium with plants. The results that were obtained in our study suggested that a thiol-based switch in A. caulinodans might sense host organic peroxide signals and enhance symbiosis.

DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion

1984 ◽  
Vol 4 (12) ◽  
pp. 2890-2898
Author(s):  
W Nellen ◽  
C Silan ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Gene Fusion ◽  
Regulatory Sequences ◽  
Control Of Gene Expression ◽  
Cis Acting ◽  
Regulated Expression ◽  
Promoter Gene ◽  
Flanking Region ◽  
Average Copy ◽  
Flanking Regions

We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.

Regulation of sucrase and lactase in Caco-2 cells: relationship to nuclear factors SIF-1 and NF-LPH-1

1996 ◽  
Vol 271 (4) ◽  
pp. G707-G713 ◽  
Author(s):  
W. A. Olsen ◽  
M. Lloyd ◽  
H. Korsmo ◽  
Y. Z. He
Keyword(s):  
Gene Expression ◽  
Cellular Differentiation ◽  
Tissue Specificity ◽  
Developmental Regulation ◽  
Mobility Shift ◽  
Nuclear Factors ◽  
Relationship Of ◽  
Electrophoretic Mobility Shift Assays ◽  
The Relationship

Recent studies suggest the importance of two transcription factors, Cdx-2 and NF-LPH-1, in the regulation of sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH) gene expression, respectively. Cdx-2 accounts for the tissue specificity of sucrase expression (16), and NF-LPH-1 varies with postnatal changes in lactase activity, suggesting a role in its developmental regulation (22). We used electrophoretic mobility shift assays to study the relationship of Cdx-2 and NF-LPH-1 to SI and LPH gene expression in Caco-2 cells to provide evidence regarding the role of these factors in the development of sucrase and lactase with cellular differentiation. We found that Cdx-2 levels correlated with SI expression and that NF-LPH-1 did not correlate with LPH expression. These studies suggest a role for Cdx-2 but not for NF-LPH-1 in the development of carbohydrase expression in these cells.

scholarly journals The extrachromosomal replication of Dictyostelium plasmid Ddp2 requires a cis-acting element and a plasmid-encoded trans-acting factor.

1990 ◽  
Vol 10 (7) ◽  
pp. 3727-3736 ◽  
Author(s):  
B Leiting ◽  
I J Lindner ◽  
A A Noegel
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Wild Strain ◽  
Open Reading Frame ◽  
Mobility Shift ◽  
Reading Frame ◽  
Cis Acting ◽  
Transformation Vector ◽  
Cis And Trans ◽  
Electrophoretic Mobility Shift Assays

Dictyostelium discoideum plasmid Ddp2 from the wild strain WS380B is a 5.8-kilobase (kb) supercoiled circle with a copy number of 300 per haploid genome. We previously described the construction of an extrachromosomally replicating transformation vector pnDeI carrying 4.7 kb of Ddp2 sequences (B. Leiting, and A. Noegel, Plasmid 20:241-248, 1988). In order to reduce the sequences required for extrachromosomal maintenance in D. discoideum, we characterized Ddp2 by sequence analysis, by deletion experiments, by transcription mapping, by electrophoretic mobility shift assays, and by expression of its single open reading frame in Escherichia coli. Two elements were involved in replication of Ddp2: a cis-acting sequence located on a 592-base-pair (bp) fragment that consisted of 220 bp of essential and 372 bp of auxiliary sequences, and a 2.7-kb open reading frame which most likely encodes a trans-acting factor. The cis- and trans-acting elements did not overlap and were shown to act independently from the location of the sequences encoding the trans-acting factor.

scholarly journals DNA-mediated transformation in Dictyostelium discoideum: regulated expression of an actin gene fusion.

1984 ◽  
Vol 4 (12) ◽  
pp. 2890-2898 ◽  
Author(s):  
W Nellen ◽  
C Silan ◽  
R A Firtel
Keyword(s):  
Dictyostelium Discoideum ◽  
Gene Fusion ◽  
Regulatory Sequences ◽  
Control Of Gene Expression ◽  
Cis Acting ◽  
Regulated Expression ◽  
Promoter Gene ◽  
Flanking Region ◽  
Average Copy ◽  
Flanking Regions

We have constructed a new vector for transformation that carries a fusion of the Dictyostelium discoideum actin 6 promoter gene and 5' flanking region with the bacterial Tn5 NeoR (KanR) gene which can confer resistance to the aminoglycoside G418. This vector can be used to transform D. discoideum cells. Approximately 200 to 2,000 transformants were obtained per 10(7) cells. Transformed cell populations carried vector DNA at an average copy number of ca. 5 per cell, and the DNA was stable for more than 40 generations in the absence of selection. We have shown that transformed cells synthesize functional kanamycin phosphotransferase and that initiation of transcription of the actin 6-NeoR gene fusion occurs at the actin 6 cap site. Moreover, analysis of RNA isolated from transformed and untransformed cells during vegetative growth and during development indicated that the actin 6-NeoR gene fusion was regulated in parallel with the endogenous actin 6 gene, suggesting that the upstream flanking regions of actin 6 contain the cis-acting regulatory sequences sufficient for differential regulation of this gene during D. discoideum development. These results indicate that this system can be used to examine control of gene expression during D. discoideum development.

scholarly journals DIMR, a Yeast-Based Synthetic Reporter System for Probing Oligomeric Transcription Factor DNA Binding

2019 ◽  
Author(s):  
Zachary A. Myers ◽  
Swadhin Swain ◽  
Shannan Bialek ◽  
Samuel Keltner ◽  
Ben F. Holt
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Reporter System ◽  
Mobility Shift ◽  
Genomic Locus ◽  
Biological Regulation ◽  
Transcriptional Regulatory ◽  
Regulatory Capacity ◽  
Electrophoretic Mobility Shift Assays ◽  
Y Family

AbstractTranscription factors (TFs) are fundamental components of biological regulation, facilitating the basal and differential gene expression necessary for life. TFs exert transcriptional regulation through interactions with both DNA and other TFs, ultimately influencing the action of RNA polymerase at a genomic locus. Current approaches are proficient at identification of binding site requirements for individual TFs, but few methods have been adapted to study oligomeric TF complexes. Further, many approaches that have been turned toward understanding DNA binding of TF complexes, such as electrophoretic mobility shift assays, require protein purification steps that can be burdensome or scope-limiting when considering more exhaustive experimental design. In order to address these shortfalls and to facilitate a more streamlined approach to understanding DNA binding by TF complexes, we developed the DIMR (Dynamic, Interdependent TF binding Molecular Reporter) system, a modular, yeast-based synthetic transcriptional activity reporter. As a proof of concept, we focused on the NUCLEAR FACTOR-Y (NF-Y) family of obligate heterotrimeric TFs in Arabidopsis thaliana. The DIMR system was able to reproduce the strict DNA-binding requirements of an experimentally validated NF-YA2/B2/C3 complex with high fidelity, including recapitulation of previously characterized mutations in subunits that either break NF-Y complex interactions or are directly involved in DNA binding. The DIMR system is a novel, powerful, and easy-to-use approach to address questions regarding the binding of oligomeric TFs to DNA.One sentence summaryThe DIMR system provides an accessible and easy-to-use platform to elucidate DNA binding and transcriptional regulatory capacity of oligomeric transcription factor complexes

Identification of cis elements necessary for glucocorticoid induction of growth hormone gene expression in chicken embryonic pituitary cells

2012 ◽  
Vol 302 (5) ◽  
pp. R606-R619 ◽  
Author(s):  
Kristina Heuck-Knubel ◽  
Monika Proszkowiec-Weglarz ◽  
Jyoti Narayana ◽  
Laura E. Ellestad ◽  
Nattiya Prakobsaeng ◽  
...  
Keyword(s):  
Growth Hormone ◽  
Protein Synthesis ◽  
Luciferase Reporter ◽  
Growth Hormone Gene ◽  
Pituitary Cells ◽  
Mobility Shift ◽  
Cis Acting ◽  
Mrna Induction ◽  
Gh Gene ◽  
Electrophoretic Mobility Shift Assays

Glucocorticoid (GC) treatment of rat or chicken embryonic pituitary (CEP) cells induces premature production of growth hormone (GH). GC induction of the GH gene requires ongoing protein synthesis, and the GH genes lack a canonical GC response element (GRE). To characterize cis-acting elements and identify trans-acting proteins involved in this process, we characterized the regulation of a luciferase reporter containing a fragment of the chicken GH gene (−1727/+48) in embryonic day 11 CEP cells. Corticosterone (Cort) increased luciferase activity and mRNA expression, and mRNA induction was blocked by protein synthesis inhibition. Through deletion analysis, we identified a GC-responsive region (GCRR) at −1045 to −954. The GCRR includes an ETS-1 binding site and a degenerate GRE (dGRE) half site. Nuclear proteins, including ETS-1, bound to a GCRR probe in electrophoretic mobility shift assays, and Cort regulated protein binding. Using chromatin immunoprecipitation, we found that ETS-1 and GC receptor (GR) were associated with the GCRR in CEP cells, and Cort increased GR recruitment to the GCRR. Mutation of the ETS-1 site or dGRE site in the −1045/+48 GH reporter abolished Cort responsiveness. We conclude that GC regulation of the GH gene during development requires cis-acting elements in the GCRR and involves ETS-1 and GR binding to these elements. Similar ETS-1 elements/dGREs are located in the 5′-flanking regions of GH genes in mammals, including rodents and humans. This is the first study to demonstrate involvement of ETS-1 in GC regulation of the GH gene during embryonic development in any species, enhancing our understanding of GH regulation in vertebrates.

Identification of a cis-acting DNA antisilencer element which modulates vimentin gene expression

1992 ◽  
Vol 12 (5) ◽  
pp. 2230-2240
Author(s):  
D M Stover ◽  
Z E Zehner
Keyword(s):  
Gene Expression ◽  
Developmental Regulation ◽  
Gene Activity ◽  
Intermediate Filament Protein ◽  
Mobility Shift ◽  
Cis Acting ◽  
Gel Mobility Shift ◽  
Silencer Element ◽  
Vimentin Gene

Vimentin is a tissue-specific, developmentally regulated member of the intermediate filament protein family normally expressed in cells of mesenchymal origin. Transcription factors which recognize specific cis-acting elements of the chicken gene include Sp-1 and the 95-kDa silencer protein which binds to a 40-bp silencer element at -608 (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we have identified a region upstream of the silencer element which restores gene activity. This region has been further delineated into two functional subelements of 75 and 260 bp. In transient transfection assays, the 75-bp element overrides the silencer effect of pStkCAT by 100%, while the 260-bp element is about half as active. Neither element affects gene activity when the silencer element is absent. Therefore, these elements do not function as enhancers, but they may serve only to override the silencer element and therefore can be viewed as antisilencers. In addition, the 75-bp element binds a specific 140-kDa protein, as determined by gel mobility shift assays and Southwestern (DNA-protein) blots, the binding site of which has been delineated to a 10- to 17-bp element by DNase I protection experiments. During myogenesis, a direct correlation can be made between the binding efficiency of the 140-kDa protein, the silencer protein, and gene activity in vivo. Genes known to contain a functional silencer element also contain at least one antisilencer element, as determined by sequence identity. Therefore, we have identified an antisilencer element and protein important in the developmental regulation of vimentin gene expression which may be involved in the regulation of other genes.

scholarly journals Identification of a cis-acting DNA antisilencer element which modulates vimentin gene expression.

1992 ◽  
Vol 12 (5) ◽  
pp. 2230-2240 ◽  
Author(s):  
D M Stover ◽  
Z E Zehner
Keyword(s):  
Gene Expression ◽  
Developmental Regulation ◽  
Gene Activity ◽  
Intermediate Filament Protein ◽  
Mobility Shift ◽  
Cis Acting ◽  
Gel Mobility Shift ◽  
Silencer Element ◽  
Vimentin Gene

Vimentin is a tissue-specific, developmentally regulated member of the intermediate filament protein family normally expressed in cells of mesenchymal origin. Transcription factors which recognize specific cis-acting elements of the chicken gene include Sp-1 and the 95-kDa silencer protein which binds to a 40-bp silencer element at -608 (F. X. Farrell, C. M. Sax, and Z. E. Zehner, Mol. Cell. Biol. 10:2349-2358, 1990). In this study, we have identified a region upstream of the silencer element which restores gene activity. This region has been further delineated into two functional subelements of 75 and 260 bp. In transient transfection assays, the 75-bp element overrides the silencer effect of pStkCAT by 100%, while the 260-bp element is about half as active. Neither element affects gene activity when the silencer element is absent. Therefore, these elements do not function as enhancers, but they may serve only to override the silencer element and therefore can be viewed as antisilencers. In addition, the 75-bp element binds a specific 140-kDa protein, as determined by gel mobility shift assays and Southwestern (DNA-protein) blots, the binding site of which has been delineated to a 10- to 17-bp element by DNase I protection experiments. During myogenesis, a direct correlation can be made between the binding efficiency of the 140-kDa protein, the silencer protein, and gene activity in vivo. Genes known to contain a functional silencer element also contain at least one antisilencer element, as determined by sequence identity. Therefore, we have identified an antisilencer element and protein important in the developmental regulation of vimentin gene expression which may be involved in the regulation of other genes.

Sign in / Sign up

Export Citation Format

Share Document