Promoter deletion analysis reveals root-specific expression of the alkenal reductase gene (OsAER1) in Oryza sativa

2019 ◽  
Vol 46 (4) ◽  
pp. 376
Author(s):  
Aniversari Apriana ◽  
Atmitri Sisharmini ◽  
Hajrial Aswidinnoor ◽  
Kurniawan R. Trijatmiko ◽  
Sudarsono Sudarsono
Keyword(s):  
Oryza Sativa ◽  
Transgenic Plants ◽  
Promoter Activity ◽  
Deletion Analysis ◽  
Specific Expression ◽  
Cis Acting ◽  
Promoter Fragment ◽  
Gus Reporter ◽  
Promoter Deletion Analysis

Root-specific promoters are useful in plant genetic engineering, primarily to improve water and nutrient absorption. The aim of this study was to clone and characterise the promoter of the Oryza sativa L. alkenal reductase (OsAER1) gene encoding 2-alkenal reductase, an NADPH-dependent oxidoreductase. Expression analysis using quantitative real-time PCR confirmed the root-specific expression of the OsAER1 gene. Subsequently, a 3082-bp fragment of the OsAER1 promoter was isolated from a local Indonesian rice cultivar, Awan Kuning. Sequencing and further nucleotide sequence analysis of the 3082-bp promoter fragment (PA-5) revealed the presence of at least 10 root-specific cis-regulatory elements putatively responsible for OsAER1 root-specific expression. Using the 3082-bp promoter fragment to drive the expression of the GUS reporter transgene confirmed that the OsAER1 promoter is root-specific. Further, the analysis indicated that OsAER1 promoter activity was absent in leaves, petioles and shoots during sprouting, vegetative, booting and generative stages of rice development. In contrast, the promoter activity was present in anthers and aleurone layers of immature seeds 7–20 days after anthesis. Moreover, there was no promoter activity observed in the aleurone layers of mature seeds. The OsAER1 promoter activity is induced by Al-toxicity, NaCl and submergence stresses, indicating the OsAER1 promoter activity is induced by those stresses. Exogenous treatments of transgenic plants carrying the PA-5 promoter construct with abscisic acid and indoleacetic acid also induced expression of the GUS reporter transgene, indicating the role of plant growth regulators in controlling OsAER1 promoter activity. Promoter deletion analysis was conducted to identify the cis-acting elements of the promoter responsible for controlling root-specific expression. The GUS reporter gene was fused with various deletion fragments of the OsAER1 promoter and the resulting constructs were transformed in rice plants to generate transgenic plants. The results of this analysis indicated that cis-acting elements controlling root-specific expression are located between −1562 to −1026bp of the OsAER1 CDS. Here we discusses the results of the conducted analyses, the possible role of OsAER1 in rice growth and development, possible contributions and the potential usage of these findings in future plant research.

scholarly journals A complex array of positive and negative elements regulates the chicken alpha A-crystallin gene: involvement of Pax-6, USF, CREB and/or CREM, and AP-1 proteins.

1994 ◽  
Vol 14 (11) ◽  
pp. 7363-7376 ◽  
Author(s):  
A Cvekl ◽  
C M Sax ◽  
E H Bresnick ◽  
J Piatigorsky
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Cellular Differentiation ◽  
Ocular Lens ◽  
Mobility Shift ◽  
Specific Expression ◽  
Negative Element ◽  
Cis Acting ◽  
Direct Data ◽  
Gel Mobility Shift

The abundance of crystallins (> 80% of the soluble protein) in the ocular lens provides advantageous markers for selective gene expression during cellular differentiation. Here we show by functional and protein-DNA binding experiments that the chicken alpha A-crystallin gene is regulated by at least five control elements located at sites A (-148 to -139), B (-138 to -132), C (-128 to -101), D (-102 to -93), and E (-56 to -41). Factors interacting with these sites were characterized immunologically and by gel mobility shift experiments. The results are interpreted with the following model. Site A binds USF and is part of a composite element with site B. Site B binds CREB and/or CREM to enhance expression in the lens and binds an AP-1 complex including CREB, Fra2 and/or JunD which interacts with USF on site A to repress expression in fibroblasts. Sites C and E (which is conserved across species) bind Pax-6 in the lens to stimulate alpha A-crystallin promoter activity. These experiments provide the first direct data that Pax-6 contributes to the lens-specific expression of a crystallin gene. Site D (-104 to -93) binds USF and is a negative element. Thus, the data indicate that USF, CREB and/or CREM (or AP-1 factors), and Pax-6 bind a complex array of positive and negative cis-acting elements of the chicken alpha A-crystallin gene to control high expression in the lens and repression in fibroblasts.

Analysis of a promoter for the NADH - glutamate synthase gene in rice (Oryza sativa): cell type-specific expression in developing organs of transgenic rice plants

2000 ◽  
Vol 27 (9) ◽  
pp. 787 ◽  
Author(s):  
Soichi Kojima ◽  
Michiko Kimura ◽  
Yukine Nozaki ◽  
Tomoyuki Yamaya
Keyword(s):  
Oryza Sativa ◽  
Oryza Sativa L ◽  
Glutamate Synthase ◽  
Gus Activity ◽  
Start Codon ◽  
Assimilate Transport ◽  
Upstream Region ◽  
Vascular Bundles ◽  
Specific Expression ◽  
Gus Reporter

This paper originates from a presentation at the International Conference on Assimilate Transport and Partitioning, Newcastle, NSW, August 1999 The entire 3.7 kbp 5´-upstream region (–2840 to +886) from the translational start codon of NADH–glutamate synthase (NADH–GOGAT, EC 1.4.1.14) gene from rice (Oryza sativa L.) or the region sequentially deleted from the 5´-end was fused with the β−glucuronidase (GUS) reporter gene. The chimeric gene was introduced into calli derived from rice scutellum via Agrobacterium tumefaciens-mediated transformation and tissue-specific GUS activity determined in T0 generations. When the entire region was fused, GUS activity was detected in vascular bundles of the developing leaf blade and in dorsal and lateral vascular bundles of developing grains. This corresponds with our previous immunodetection of NADH–GOGAT protein (Hayakawa et al., Planta 193, 455–460, 1994). A series of deletion experiments showed that a 149-nucleotide region between –142 and +7 was essential for promoter activity in the NADH–GOGAT gene.

A complex array of positive and negative elements regulates the chicken alpha A-crystallin gene: involvement of Pax-6, USF, CREB and/or CREM, and AP-1 proteins

1994 ◽  
Vol 14 (11) ◽  
pp. 7363-7376
Author(s):  
A Cvekl ◽  
C M Sax ◽  
E H Bresnick ◽  
J Piatigorsky
Keyword(s):  
Gene Expression ◽  
Promoter Activity ◽  
Cellular Differentiation ◽  
Ocular Lens ◽  
Mobility Shift ◽  
Specific Expression ◽  
Negative Element ◽  
Cis Acting ◽  
Direct Data ◽  
Gel Mobility Shift

The abundance of crystallins (> 80% of the soluble protein) in the ocular lens provides advantageous markers for selective gene expression during cellular differentiation. Here we show by functional and protein-DNA binding experiments that the chicken alpha A-crystallin gene is regulated by at least five control elements located at sites A (-148 to -139), B (-138 to -132), C (-128 to -101), D (-102 to -93), and E (-56 to -41). Factors interacting with these sites were characterized immunologically and by gel mobility shift experiments. The results are interpreted with the following model. Site A binds USF and is part of a composite element with site B. Site B binds CREB and/or CREM to enhance expression in the lens and binds an AP-1 complex including CREB, Fra2 and/or JunD which interacts with USF on site A to repress expression in fibroblasts. Sites C and E (which is conserved across species) bind Pax-6 in the lens to stimulate alpha A-crystallin promoter activity. These experiments provide the first direct data that Pax-6 contributes to the lens-specific expression of a crystallin gene. Site D (-104 to -93) binds USF and is a negative element. Thus, the data indicate that USF, CREB and/or CREM (or AP-1 factors), and Pax-6 bind a complex array of positive and negative cis-acting elements of the chicken alpha A-crystallin gene to control high expression in the lens and repression in fibroblasts.

scholarly journals The 26-Amino Acid ß-Motif of the Pit-1ß Transcription Factor Is a Dominant and Independent Repressor Domain

2009 ◽  
Vol 23 (9) ◽  
pp. 1371-1384 ◽  
Author(s):  
Matthew D. Jonsen ◽  
Dawn L. Duval ◽  
Arthur Gutierrez-Hartmann
Keyword(s):  
Transcription Factor ◽  
Amino Acid ◽  
Promoter Activity ◽  
Pituitary Cells ◽  
Heterologous Promoter ◽  
Specific Expression ◽  
Dna Binding Sites ◽  
Repressor Activity ◽  
Sequence Position

Abstract The POU-homeodomain transcription factor Pit-1 governs the pituitary cell-specific expression of Pit-1, GH, prolactin (PRL), and TSHß genes. Alternative splicing generates Pit-1ß, which contains a 26-amino acid ß-domain inserted at amino acid 48, in the middle of the Pit-1 transcription activation domain (TAD). Pit-1ß represses GH, PRL, and TSHß promoters in a pituitary-specific manner, because Pit-1ß activates these same promoters in HeLa nonpituitary cells. Here we comprehensively analyze the role of ß-domain sequence, position, and context, to elucidate the mechanism of ß-dependent repression. Repositioning the ß-motif to the Pit-1 amino terminus, hinge, linker, and carboxyl terminus did not affect its ability to repress basal rat (r) PRL promoter activity in GH4 pituitary cells, but all lost the ability to repress Ras-induced rPRL promoter activity. To determine whether ß-domain repression is independent of Pit-1 protein and DNA binding sites, we generated Gal4-Pit-1TAD, Gal4-Pit-1ßTAD, and Gal4-ß-domain fusions and demonstrated that the ß-motif is sufficient to actively repress VP16-mediated transcription of a heterologous promoter. Moreover, ß-domain point mutants had the same effect whether fused to Gal4 or within the context of intact Pit-1ß. Surprisingly, Gal4-ß repression lost histone deacetylase sensitivity and pituitary specificity. Taken together, these results reveal that the ß-motif is a context-independent, modular, transferable, and dominant repressor domain, yet the ß-domain repressor activity within Pit-1ß contains cell type, promoter, and Pit-1 protein context dependence.

scholarly journals A38 TRPV1 VISCERAL AFFERENTS CONTROL CENTRAL SENSITIZATION IN IBD

2021 ◽  
Vol 4 (Supplement_1) ◽  
pp. 278-279
Author(s):  
M Defaye ◽  
N Abdullah ◽  
M Iftinca ◽  
C Altier
Keyword(s):  
Chronic Pain ◽  
Central Sensitization ◽  
Microglial Activation ◽  
Visceral Pain ◽  
Purinergic Signaling ◽  
Designer Drugs ◽  
Visceral Afferents ◽  
Peripheral Sensitization ◽  
Specific Expression

Abstract Background Long-lasting changes in neural pain circuits precipitate the transition from acute to chronic pain in patients living with inflammatory bowel diseases (IBDs). While significant improvement in IBD therapy has been made to reduce inflammation, a large subset of patients continues to suffer throughout quiescent phases of the disease, suggesting a high level of plasticity in nociceptive circuits during acute phases. The establishment of chronic visceral pain results from neuroplasticity in nociceptors first, then along the entire neural axis, wherein microglia, the resident immune cells of the central nervous system, are critically involved. Our lab has shown that spinal microglia were key in controlling chronic pain state in IBD. Using the Dextran Sodium Sulfate (DSS) model of colitis, we found that microglial G-CSF was able to sensitize colonic nociceptors that express the pain receptor TRPV1. While TRPV1+ nociceptors have been implicated in peripheral sensitization, their contribution to central sensitization via microglia remains unknown. Aims To investigate the role of TRPV1+ visceral afferents in microglial activation and chronic visceral pain. Methods We generated DREADD (Designer Receptors Exclusively Activated by Designer Drugs) mice in which TRPV1 sensory neurons can be inhibited (TRPV1-hM4Di) or activated (TRPV1-hM3Dq) in a time and tissue specific manner using the inert ligand Clozapine-N-Oxide (CNO). To test the inhibition of TRPV1 neurons in DSS-induced colitis, TRPV1-hM4Di mice were treated with DSS 2.5% or water for 7 days and received vehicle or CNO i.p. injection twice daily. To activate TRPV1 visceral afferents, TRPV1-hM3Dq mice received vehicle or CNO daily for 7 days, by oral gavage. After 7 days of treatment, visceral pain was evaluated by colorectal distension and spinal cords tissues were harvested to measure microglial activation. Results Our data validated the nociceptor specific expression and function of the DREADD in TRPV1-Cre mice. Inhibition of TRPV1 visceral afferents in DSS TRPV1-hM4Di mice was able to prevent the colitis-induced microglial activation and thus reduce visceral hypersensitivity. In contrast, activation of TRPV1 visceral afferents in TRPV1-hM3Dq mice was sufficient to drive microglial activation in the absence of colitis. Analysis of the proalgesic mediators derived from activated TRPV1-hM3Dq neurons identified ATP as a key factor of microglial activation. Conclusions Overall, these data provide novel insights into the mechanistic understanding of the gut/brain axis in chronic visceral pain and suggest a role of purinergic signaling that could be harnessed for testing effective therapeutic approaches to relieve pain in IBD patients. Funding Agencies CCCACHRI (Alberta Children’s Hospital Research Institute) and CSM (Cumming School of Medicine) postdoctoral fellowship

scholarly journals MiRNA Expression in Neuroendocrine Neoplasms of Frequent Localizations

2021 ◽  
Vol 7 (3) ◽  
pp. 38
Author(s):  
Alexandra Korotaeva ◽  
Danzan Mansorunov ◽  
Natalya Apanovich ◽  
Anna Kuzevanova ◽  
Alexander Karpukhin
Keyword(s):  
Mirna Expression ◽  
Malignant Tumors ◽  
Neuroendocrine Neoplasms ◽  
Specific Expression ◽  
Clinical Biomarkers ◽  
Different Types ◽  
Functional Features ◽  
First Time ◽  
Potential Biomarkers

Neuroendocrine neoplasms (NEN) are infrequent malignant tumors of a neuroendocrine nature that arise in various organs. They occur most frequently in the lungs, intestines, stomach and pancreas. Molecular diagnostics and prognosis of NEN development are highly relevant. The role of clinical biomarkers can be played by microRNAs (miRNAs). This work is devoted to the analysis of data on miRNA expression in NENs. For the first time, a search for specificity or a community of their functional characteristics in different types of NEN was carried out. Their properties as biomarkers were also analyzed. To date, more than 100 miRNAs have been characterized as differentially expressed and significant for the development of NEN tumors. Only about 10% of the studied miRNAs are expressed in several types of NEN; differential expression of the remaining 90% was found only in tumors of specific localizations. A significant number of miRNAs have been identified as potential biomarkers. However, only a few miRNAs have values that characterized their quality as markers. The analysis demonstrates the predominant specific expression of miRNA in each studied type of NEN. This indicates that miRNA’s functional features are predominantly influenced by the tissue in which they are formed.

scholarly journals POPULATION GENETICS OF DROSOPHILA AMYLASE. IV. SELECTION IN LABORATORY POPULATIONS MAINTAINED ON DIFFERENT CARBOHYDRATES

Genetics ◽  
1983 ◽  
Vol 103 (4) ◽  
pp. 675-689
Author(s):  
Jeffrey R Powell ◽  
Marko Andjelković
Keyword(s):  
Structural Gene ◽  
Amylase Activity ◽  
Enzyme System ◽  
Specific Expression ◽  
Carbohydrate Source ◽  
Laboratory Populations ◽  
Consistent Change ◽  
Regulation Changes ◽  
Starch Medium

ABSTRACT Two polymorphic systems impinging on α-amylase in Drosophila pseudoobscura have been studied in laboratory populations maintained on medium in which the only carbohydrate source was starch (the substrate of amylase) and replicas maintained on medium in which the only carbohydrate source was maltose (the product of amylase). The two polymorphic systems were alleles at the structural gene (Amy) coding for the enzyme (allozymes) and variation in the tissue-specific expression along the adult midgut controlled by several genes. In the seven populations on maltose medium little consistent change was noted in either system. In the seven populations on starch medium, both polymorphisms exhibited selective changes. A midgut pattern of very limited expression of amylase rose in frequency in all starch populations, as did the frequency of the "fast" (1.00) Amy allele. The overall specific amylase activity did not differ between starch-adapted and maltose-adapted flies.—The results, along with previous studies, indicate that when a gene-enzyme system is specifically stressed in laboratory populations, allozymes often exhibit selective differences. Such results make the selectionist hypothesis at least tenable. Furthermore, the fact that both types of polymorphisms responded to selection indicates the role of structural gene vs. gene regulation changes in adaptive evolution is not an either/or question but one of relative roles and interactions.

scholarly journals Characterization of two cis-acting elements, P1BS and W-box, in the regulation of OsPT6 responsive to phosphors deficiency

2021 ◽  
Author(s):  
Qingchun Zhao ◽  
Zhenzhen Luo ◽  
Jiadong Chen ◽  
Hongfang Jia ◽  
Penghui Ai ◽  
...  
Keyword(s):  
Gus Expression ◽  
Element Analysis ◽  
P Deficiency ◽  
Targeted Deletion ◽  
Cis Acting ◽  
Gus Reporter ◽  
Pi Starvation ◽  
Expression Activity ◽  
Underlying Mechanisms ◽  
Diverse Plant Species

AbstractPhosphorus (P) deficiency is one of the major nutrient stresses restricting plant growth. The uptake of P by plants from soil is mainly mediated by the phosphate (Pi) transporters belonging to the PHT1 family. Multiple PHT1 genes from diverse plant species have been shown to be strongly up-regulated upon Pi starvation, however, the underlying mechanisms for the Pi-starvation-induced (PSI) up-regulation have not been well deciphered for most Pi transporter genes. Here, we reported a detailed dissection of the promoter activity of a PSI rice Pi transporter gene OsPT6, using the β-glucuronidase (GUS) reporter gene. OsPT6 promoter could drive GUS expression strongly in both roots and blades of rice plants grown under low P, but not high P. Cis-acting element analysis identified one copy of the P1BS motif and two copies of the W-box motif in OsPT6 promoter. Targeted deletion of the P1BS motif caused almost complete abolition of GUS induction in response to Pi starvation, irrespective of the presence or absence of the W-box motif, Four repeats of the P1BS motif fused to the CaMV35S minimal promoter was sufficient to induce GUS expression responsive to Pi starvation. Targeted deletion of the upstream W-box motif (W1) did not affect the GUS expression activity compared with the full-length OsPT6 promoter, while targeted deletion of the downstream W-box motif (W2) or both of the W-box motifs remarkably reduced the GUS induction rate upon Pi starvation. Our results proposed that the PSI response of OsPT6 was positively regulated by at least two elements, the sole P1BS and the downstream W-box, in its promoter, and the W-box-mediated up-regulation of OsPT6 might be highly dependent on the P1BS motif.

Sign in / Sign up

Export Citation Format

Share Document