scholarly journals Cloning and functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.)

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0242949
Author(s):  
Guiying Tang ◽  
Pingli Xu ◽  
Pengxiang Li ◽  
Jieqiong Zhu ◽  
Guangxia Chen ◽  
...  
Keyword(s):  
Transcriptional Start Site ◽  
Functional Characterization ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Histochemical Staining ◽  
Expression Vectors ◽  
Upstream Sequence ◽  
Functional Region ◽  
Chromosomal Walking ◽  
Storage Reserve

LEAFY COTYLEDON1 (LEC1) is a HAP3 subunit of CCAAT-binding transcription factor, which controls several aspects of embryo and postembryo development, including embryo morphogenesis, storage reserve accumulation and skotomorphogenesis. Herein, using the method of chromosomal walking, a 2707bp upstream sequence from the ATG initiation codon site of AhLEC1A which is a homolog of Arabidopsis LEC1 was isolated in peanut. Its transcriptional start site confirmed by 5’ RACE was located at 82 nt from 5’ upstream of ATG. The bioinformatics analysis revealed that there existed many tissue-specific elements and light responsive motifs in its promoter. To identify the functional region of the AhLEC1A promoter, seven plant expression vectors expressing the GUS (β-glucuronidase) gene, driven by 5’ terminal series deleted fragments of AhLEC1A promoter, were constructed and transformed into Arabidopsis. Results of GUS histochemical staining showed that the regulatory region containing 82bp of 5’ UTR and 2228bp promoter could facilitate GUS to express preferentially in the embryos at different development periods of Arabidopsis. Taken together, it was inferred that the expression of AhLEC1A during seed development of peanut might be controlled positively by several seed-specific regulatory elements, as well as negatively by some other regulatory elements inhibiting its expression in other organs. Moreover, the GUS expression pattern of transgenic seedlings in darkness and in light was relevant to the light-responsive elements scattered in AhLEC1A promoter segment, implying that these light-responsive elements harbored in the AhLEC1A promoter regulate skotomorphogenesis of peanut seeds, and AhLEC1A expression was inhibited after the germinated seedlings were transferred from darkness to light.

scholarly journals Cloning and Functional characterization of seed-specific LEC1A promoter from peanut (Arachis hypogaea L.)

2020 ◽  
Author(s):  
Guiying Tang ◽  
Pingli Xu ◽  
Pengxiang Li ◽  
Jieqiong Zhu ◽  
Guangxia Chen ◽  
...  
Keyword(s):  
Transcriptional Start Site ◽  
Functional Characterization ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Histochemical Staining ◽  
Expression Vectors ◽  
Upstream Sequence ◽  
Functional Region ◽  
Chromosomal Walking ◽  
Storage Reserve

AbstractLEAFY COTYLEDON1 (LEC1) is a HAP3 subunit of CCAAT-binding transcription factor, which controls several aspects of embryo and postembryo development, including embryo morphogenesis, storage reserve accumulation and skotomorphogenesis. Herein, using the method of chromosomal walking, a 2707bp upstream sequence from the ATG initiation codon site of AhLEC1A which is a homolog of Arabidopsis LEC1 was isolated in peanut. Its transcriptional start site confirmed by 5’ RACE was located at 82 nt from 5’ upstream of ATG. The bioinformatics analysis revealed that there existed many tissue-specific elements and light responsive motifs in its promoter. To identify the functional region of the AhLEC1A promoter, seven plant expression vectors expressing the GUS (β-glucuronidase) gene, driven by 5’ terminal series deleted fragments of AhLEC1A promoter, were constructed and transformed into Arabidopsis. Results of GUS histochemical staining showed that the regulatory region containing 82bp of 5’ UTR and 2228bp promoter could facilitate GUS to express preferentially in the embryos at different development periods of Arabidopsis. Taken together, it was inferred that the expression of AhLEC1A during seed development of peanut might be controlled positively by several seed-specific regulatory elements, as well as negatively by some other regulatory elements inhibiting its expression in other organs. Moreover, the GUS expression pattern of transgenic seedlings in darkness and in light was relevant to the light-responsive elements scattered in AhLEC1A promoter segment, implying that these light-responsive elements harbored in the AhLEC1A promoter regulate skotomorphogenesis of peanut seeds, and AhLEC1A expression was inhibited after the germinated seedlings were transferred from darkness to light.

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 The Divergently Transcribed Streptococcus parasanguis Virulence-Associated fimA Operon Encoding an Mn2+-Responsive Metal Transporter and pepO Encoding a Zinc Metallopeptidase Are Not Coordinately Regulated

2002 ◽  
Vol 70 (10) ◽  
pp. 5706-5714 ◽  
Author(s):  
Joyce Oetjen ◽  
Paula Fives-Taylor ◽  
Eunice H. Froeliger
Keyword(s):  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Divalent Metal Ions ◽  
Metal Transporters ◽  
Metal Transporter ◽  
Gram Positive Bacteria ◽  
Growth Deficiency ◽  
Domain Mapping ◽  
Transcriptional Start Sites

ABSTRACT The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The Streptococcus parasanguis fimA operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the Streptococcus gordonii ScaR binding domain. Mapping of the promoter and regulatory regions of fimA and the divergently transcribed pepO gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap. fimA had one transcriptional start site, whereas pepO had three. Analysis of truncated versions of the pepO promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the fimA operon promoter and the pepO promoter are not coordinately regulated. The fimA operon is responsive to changes in Mn2+ concentration, but the pepO promoter is not. A S. parasanguis fimA mutant showed a growth deficiency under conditions of limiting Mn2+. This deficiency was not alleviated by compensation with either Mg2+ or Fe3+. Wild-type S. parasanguis could take up Mn2+ and Fe3+, while the fimA mutant showed a marked reduction in this ability. These data suggested that FimA is a component of a metal transporter system capable of transporting both Mn2+ and Fe3+. FimA expression itself was shown to be responsive to Mn2+ concentration, but not to availability of Fe3+ or Mg2+.

scholarly journals Repression and activation of the Drosophila dopa decarboxylase gene in glia.

1992 ◽  
Vol 12 (12) ◽  
pp. 5659-5666 ◽  
Author(s):  
G S Mastick ◽  
S B Scholnick
Keyword(s):  
Germ Line ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Dopa Decarboxylase ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Cell Activator ◽  
Germ Line Transformation

Glial expression of the Drosophila dopa decarboxylase gene (Ddc) is repressed by a regulatory region located approximately 1 kb upstream of the transcriptional start site. We have used in vitro mutagenesis and germ line transformation to determine which elements within the Ddc promoter mediate repression. Our evidence suggests that the hypodermal cell activator elements IIA and IIB play a major role in the transcriptional regulation of Ddc in glial cells. A variety of mutations demonstrate that element IIA is a strong glial activator element and that element IIB is necessary for glial repression. Although these two regulatory elements are nearly identical in sequence, our data suggest that they are not redundant. Altering the wild-type number and spacing of elements IIA and IIB indicates that the wild-type arrangement of this repeat is critical for repression. We conclude that these key elements of the Ddc promoter regulate both activation and repression in glia.

Repression and activation of the Drosophila dopa decarboxylase gene in glia

1992 ◽  
Vol 12 (12) ◽  
pp. 5659-5666
Author(s):  
G S Mastick ◽  
S B Scholnick
Keyword(s):  
Germ Line ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Dopa Decarboxylase ◽  
In Vitro Mutagenesis ◽  
Wild Type ◽  
Cell Activator ◽  
Germ Line Transformation

Glial expression of the Drosophila dopa decarboxylase gene (Ddc) is repressed by a regulatory region located approximately 1 kb upstream of the transcriptional start site. We have used in vitro mutagenesis and germ line transformation to determine which elements within the Ddc promoter mediate repression. Our evidence suggests that the hypodermal cell activator elements IIA and IIB play a major role in the transcriptional regulation of Ddc in glial cells. A variety of mutations demonstrate that element IIA is a strong glial activator element and that element IIB is necessary for glial repression. Although these two regulatory elements are nearly identical in sequence, our data suggest that they are not redundant. Altering the wild-type number and spacing of elements IIA and IIB indicates that the wild-type arrangement of this repeat is critical for repression. We conclude that these key elements of the Ddc promoter regulate both activation and repression in glia.

scholarly journals Molecular and Functional Characterization of theRhodopseudomonas palustris No. 7 Phosphoenolpyruvate Carboxykinase Gene

1999 ◽  
Vol 181 (9) ◽  
pp. 2689-2696 ◽  
Author(s):  
Masayuki Inui ◽  
Kaori Nakata ◽  
Jung Hyeob Roh ◽  
Kenneth Zahn ◽  
Hideaki Yukawa
Keyword(s):  
Carbon Source ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Transcriptional Start Site ◽  
Rhodopseudomonas Palustris ◽  
Functional Characterization ◽  
Carbon Sources ◽  
Pcr Amplification ◽  
Upstream Sequence ◽  
Gene Encoding ◽  
Reading Frame

ABSTRACT The pckA gene, encoding the gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), was cloned by PCR amplification from the purple nonsulfur bacteriumRhodopseudomonas palustris No. 7. Sequencing of a 2.5-kb chromosomal SmaI-PstI fragment containing the structural gene revealed an open reading frame encoding 537 amino acids, homologous to known pckA genes. Primer extension analysis identified a transcriptional start site 72 bp upstream of thepckA initiation codon and an upstream sequence similar to ς70 promoters. Studies of a pckA-lacZ gene fusion indicated that when cells were grown in minimal media with various carbon sources, such as succinate, malate, pyruvate, lactate, or ethanol, under both anaerobic light and aerobic dark conditions, thepckA gene was induced in log phase, irrespective of the carbon source. A R. palustris No. 7 PEPCK-deficient strain showed growth characteristics identical to those of the wild-type strain either anaerobically in the light or aerobically in the dark when a C4-dicarboxylic acid, such as succinate or malate, was used as a carbon source. These results indicate that in R. palustris No. 7, an alternative gluconeogenic pathway may exist in addition to PEPCK.

scholarly journals Identification of the Ω4499 Regulatory Region Controlling Developmental Expression of a Myxococcus xanthus Cytochrome P-450 System

1999 ◽  
Vol 181 (17) ◽  
pp. 5467-5475 ◽  
Author(s):  
Makda Fisseha ◽  
Dvora Biran ◽  
Lee Kroos
Keyword(s):  
Transcriptional Start Site ◽  
Consensus Sequence ◽  
Myxococcus Xanthus ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Open Reading Frame ◽  
Developmental Expression ◽  
Reading Frame ◽  
Dna Elements ◽  
Cytochrome P 450

ABSTRACT Ω4499 is the site of a Tn5 lac insertion in theMyxococcus xanthus chromosome that fuses lacZexpression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn5 lac Ω4499. The DNA upstream of the Ω4499 insertion has been cloned, and the promoter has been localized. Analysis of the DNA sequence downstream of the promoter revealed one complete open reading frame and a second partial open reading frame that is interrupted by Tn5 lac Ω4499. The predicted products of these open reading frames are highly similar to reductase and oxidase components of bacterial cytochrome P-450 systems, which allow catabolism or anabolism of unusual compounds. However, the function of the gene products of the Ω4499 locus remains unclear because M. xanthus containing Tn5 lac Ω4499 exhibits no apparent defect in growth, developmental aggregation, fruiting body formation, or sporulation. Deletion analysis of the Ω4499 regulatory region showed that multiple DNA elements spanning more than 500 bp upstream of the transcriptional start site contribute to developmental promoter activity. At least two DNA elements, one downstream of −49 bp and one between −49 and −218 bp, boosted activity of the promoter in response to intercellular C signaling. Three sequences in the Ω4499 promoter region, centered at −55, −33, and −1 bp, nearly match a 7-bp sequence found in other C signal-dependent promoters. We propose that these sequences, matching the consensus sequence 5′-CAYYCCY-3′, be called C box sequences, and we speculate that these sequences arecis-acting regulatory elements important for the expression of M. xanthus genes that depend upon intercellular C signaling during development.

scholarly journals Transcriptional Regulation of AQP-8, a Caenorhabditis elegans Aquaporin Exclusively Expressed in the Excretory System, by the POU Homeobox Transcription Factor CEH-6

2007 ◽  
Vol 282 (38) ◽  
pp. 28074-28086 ◽  
Author(s):  
Allan K. Mah ◽  
Kristin R. Armstrong ◽  
Derek S. Chew ◽  
Jeffrey S. Chu ◽  
Domena K. Tu ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Regulatory Networks ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Upstream Sequence ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
C Elegans ◽  
Octamer Motif ◽  
Excretory Cell

Due to the ever changing environmental conditions in soil, regulation of osmotic homeostasis in the soil-dwelling nematode Caenorhabditis elegans is critical. AQP-8 is a C. elegans aquaporin that is expressed in the excretory cell, a renal equivalent tissue, where the protein participates in maintaining water balance. To better understand the regulation of AQP-8, we undertook a promoter analysis to identify the aqp-8 cis-regulatory elements. Using progressive 5′ deletions of upstream sequence, we have mapped an essential regulatory region to roughly 300 bp upstream of the translational start site of aqp-8. Analysis of this region revealed a sequence corresponding to a known DNA functional element (octamer motif), which interacts with POU homeobox transcription factors. Phylogenetic footprinting showed that this site is perfectly conserved in four nematode species. The octamer site's function was further confirmed by deletion analyses, mutagenesis, functional studies, and electrophoretic mobility shift assays. Of the three POU homeobox proteins encoded in the C. elegans genome, CEH-6 is the only member that is expressed in the excretory cell. We show that expression of AQP-8 is regulated by CEH-6 by performing RNA interference experiments. CEH-6's mammalian ortholog, Brn1, is expressed both in the kidney and the central nervous system and binds to the same octamer consensus binding site to drive gene expression. These parallels in transcriptional control between Brn1 and CEH-6 suggest that C. elegans may well be an appropriate model for determining gene-regulatory networks in the developing vertebrate kidney.

scholarly journals Deletion of a non-canonical regulatory sequence causes loss of Scn1a expression and epileptic phenotypes in mice

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Jessica L. Haigh ◽  
Anna Adhikari ◽  
Nycole A. Copping ◽  
Tyler Stradleigh ◽  
A. Ayanna Wade ◽  
...  
Keyword(s):  
Regulatory Element ◽  
Transcriptional Start Site ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Dravet Syndrome ◽  
Regulatory Sequence ◽  
Functional Requirements ◽  
Transgenic Mouse Line ◽  
Gene And Protein Expression ◽  
Promoter Mutations

Abstract Background Genes with multiple co-active promoters appear common in brain, yet little is known about functional requirements for these potentially redundant genomic regulatory elements. SCN1A, which encodes the NaV1.1 sodium channel alpha subunit, is one such gene with two co-active promoters. Mutations in SCN1A are associated with epilepsy, including Dravet syndrome (DS). The majority of DS patients harbor coding mutations causing SCN1A haploinsufficiency; however, putative causal non-coding promoter mutations have been identified. Methods To determine the functional role of one of these potentially redundant Scn1a promoters, we focused on the non-coding Scn1a 1b regulatory region, previously described as a non-canonical alternative transcriptional start site. We generated a transgenic mouse line with deletion of the extended evolutionarily conserved 1b non-coding interval and characterized changes in gene and protein expression, and assessed seizure activity and alterations in behavior. Results Mice harboring a deletion of the 1b non-coding interval exhibited surprisingly severe reductions of Scn1a and NaV1.1 expression throughout the brain. This was accompanied by electroencephalographic and thermal-evoked seizures, and behavioral deficits. Conclusions This work contributes to functional dissection of the regulatory wiring of a major epilepsy risk gene, SCN1A. We identified the 1b region as a critical disease-relevant regulatory element and provide evidence that non-canonical and seemingly redundant promoters can have essential function.

scholarly journals Identification and characterization of a biphasic/bidirectional wound-inducible RHA3B gene promoter from Arabidopsis thaliana

2021 ◽  
Author(s):  
Venkateswara Rao ◽  
Vijaybhaskar Virupapuram
Keyword(s):  
Gene Expression ◽  
Regulatory Region ◽  
Inducible Promoter ◽  
Regulatory Elements ◽  
Enhancer Trap ◽  
Upstream Sequence ◽  
List Type ◽  
Post Injury ◽  
Inducible Gene Expression ◽  
Inducible Gene

AbstractConditional promoters such as wound inducible are important tools for plant biotechnology to selectively express agronomically important genes. From a set of Enhancer trap (DsE-uidA) transposant lines we identified wound inducible ET1075 line by injuring leaf blade and screening for Ds linked GUS gene expression. Earliest GUS stain detected at petiolar region at 2hrs post injury with a progressive attainment of maximum visual intensity between 12 and 24hrs wherein, the transcript expression induced in a bidirectional manner. DsE was found to be inserted in the intergenic region between AT4G35480 and AT4G35490. To find the essential transcriptional regulatory region, deletion constructs comprising upstream sequences of pRHA3B fused to GUS reporter gene were functionally tested in Arabidopsis plants by generating stable transgenics. A 481 bp of upstream sequence from ATG is found to be sufficient to promote wound inducible gene expression. Sequence analysis revealed the presence of several regulatory elements implicated in wound inducible gene expression. Comparative analysis with similar wound inducible promoters revealed the presence of common cis-regulatory elements. This promoter can essentially be used in pest control and in molecular pharming to conditionally express agronomically/commercially important genes in plants.HighlightsDsE enhancer trap transposant lines were generated.Identified a novel wound inducible promoter line ET1075.Wound inducible promoter pRHA3B is bidirectional and induces the expression of RHA3B and MRPL11 genes.Wound responsive key cis-elements WRKY, W-box, FORCA are present in the pRHA3B promoter.

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