scholarly journals RNAi-Mediated Knockdown of Imaginal Disc Growth Factors (IDGFs) Genes Causes Developmental Malformation and Mortality in Melon Fly, Zeugodacus cucurbitae

2021 ◽  
Vol 12 ◽  
Author(s):  
Shakil Ahmad ◽  
Momana Jamil ◽  
Muhammad Fahim ◽  
Shujing Zhang ◽  
Farman Ullah ◽  
...  
Keyword(s):  
Growth Factors ◽  
Imaginal Disc ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Small Body ◽  
Functional Characterization ◽  
Oral Feeding ◽  
Developmental Defects ◽  
Melon Fly ◽  
Green Fluorescent

This study reports the first successful use of oral feeding dsRNA technique for functional characterization of imaginal disc growth factors (IDGFs) genes (IDGF1, IDGF3_1, IDGF4_0, IDGF4_1, and IDGF6) in melon fly Zeugodacus cucurbitae. Phylogenetic and domain analysis indicates that these genes had high similarity with other Tephritidae fruit flies homolog and contain only one conserved domain among these five genes, which is glyco-18 domain (glyco-hydro-18 domain). Gene expression analysis at different developmental stages revealed that these genes were expressed at larval, pupal, and adult stages. To understand their role in different developmental stages, larvae were fed dsRNA-corresponding to each of the five IDGFs, in an artificial diet. RNAi-mediated knockdown of IDGF1 shows no phenotypic effects but caused mortality (10.4%), while IDGF4_0 caused malformed pharate at the adult stage where insects failed to shed their old cuticle and remained attached with their body, highest mortality (49.2%) was recorded compared to dsRNA-green fluorescent protein (GFP) or DEPC. Silencing of IDGF3_1 and IDGF4_1 cause lethal phenotype in larvae, (17.2%) and (40%) mortality was indexed in Z. cucurbitae. IDGF6 was mainly expressed in pupae and adult stages, and its silencing caused a malformation in adult wings. The developmental defects such as malformation in wings, larval–larval lethality, pupal–adult malformation, and small body size show that IDGFs are key developmental genes in the melon fly. Our results provide a baseline for the melon fly management and understanding of IDGFs specific functions in Z. cucurbitae.

scholarly journals Biochemical and functional characterization of a mitochondrial citrate carrier in Arabidopsis thaliana

2020 ◽  
Vol 477 (9) ◽  
pp. 1759-1777 ◽  
Author(s):  
Danielle S. Brito ◽  
Gennaro Agrimi ◽  
Lennart Charton ◽  
Dominik Brilhaus ◽  
Maria Gabriella Bitetto ◽  
...  
Keyword(s):  
Gene Expression Analysis ◽  
Nitrogen Assimilation ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Functional Characterization ◽  
Radicle Growth ◽  
Citrate Carrier ◽  
Green Fluorescent ◽  
Lower Expression

A homolog of the mitochondrial succinate/fumarate carrier from yeast (Sfc1p) has been found in the Arabidopsis genome, named AtSFC1. The AtSFC1 gene was expressed in Escherichia coli, and the gene product was purified and reconstituted in liposomes. Its transport properties and kinetic parameters demonstrated that AtSFC1 transports citrate, isocitrate and aconitate and, to a lesser extent, succinate and fumarate. This carrier catalyzes a fast counter-exchange transport as well as a low uniport of substrates, exhibits a higher transport affinity for tricarboxylates than dicarboxylates, and is inhibited by pyridoxal 5′-phosphate and other inhibitors of mitochondrial carriers to various degrees. Gene expression analysis indicated that the AtSFC1 transcript is mainly present in heterotrophic tissues, and fusion with a green-fluorescent protein localized AtSFC1 to the mitochondria. Furthermore, 35S-AtSFC1 antisense lines were generated and characterized at metabolic and physiological levels in different organs and at various developmental stages. Lower expression of AtSFC1 reduced seed germination and impaired radicle growth, a phenotype that was related to reduced respiration rate. These findings demonstrate that AtSFC1 might be involved in storage oil mobilization at the early stages of seedling growth and in nitrogen assimilation in root tissue by catalyzing citrate/isocitrate or citrate/succinate exchanges.

scholarly journals Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.)

2019 ◽  
Vol 20 (17) ◽  
pp. 4081 ◽  
Author(s):  
Lin Chen ◽  
Xiaohong Liu ◽  
Xiaojia Huang ◽  
Wei Luo ◽  
Yuming Long ◽  
...  
Keyword(s):  
Cell Wall ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Protein Fusion ◽  
Drought Treatment ◽  
Vacuolar Invertase ◽  
Maize Leaves ◽  
Green Fluorescent ◽  
Proteinaceous Inhibitor

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

scholarly journals A Mutation in a Novel Yeast Proteasomal Gene,RPN11/MPR1, Produces a Cell Cycle Arrest, Overreplication of Nuclear and Mitochondrial DNA, and an Altered Mitochondrial Morphology

1998 ◽  
Vol 9 (10) ◽  
pp. 2917-2931 ◽  
Author(s):  
Teresa Rinaldi ◽  
Carlo Ricci ◽  
Danilo Porro ◽  
Monique Bolotin-Fukuhara ◽  
Laura Frontali
Keyword(s):  
Mitochondrial Dna ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Mitochondrial Morphology ◽  
Nonpermissive Temperature ◽  
Protein Fusion ◽  
In The Wild ◽  
A Cell ◽  
Green Fluorescent ◽  
Cytoplasmic Structures

We report here the functional characterization of an essentialSaccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed. For this study we made use of thempr1-1 mutation that causes the following pleiotropic defects. At 24°C growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36°C on either carbon source. Microscopic observation of cells growing on glucose at 24°C shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered. A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide. Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA. Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1proteasomal gene. Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins. Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures. For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype. We actually showed, by the use of [rho°] cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA. Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain. These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Four novel splice variants of sulfonylurea receptor 1

2002 ◽  
Vol 283 (2) ◽  
pp. C587-C598 ◽  
Author(s):  
Annette Hambrock ◽  
Regina Preisig-Müller ◽  
Ulrich Russ ◽  
Anke Piehl ◽  
Peter J. Hanley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Splice Variants ◽  
Functional Characterization ◽  
Pcr Analysis ◽  
Sulfonylurea Receptor ◽  
Transmembrane Segments ◽  
Sulfonylurea Receptor 1 ◽  
Green Fluorescent ◽  
Splice Forms

ATP-sensitive K+ (KATP) channels are composed of pore-forming Kir6.x subunits and regulatory sulfonylurea receptor (SUR) subunits. SURs are ATP-binding cassette proteins with two nucleotide-binding folds (NBFs) and binding sites for sulfonylureas, like glibenclamide, and for channel openers. Here we report the identification and functional characterization of four novel splice forms of guinea pig SUR1. Three splice forms originate from alternative splicing of the region coding for NBF1 and lack exons 17 (SUR1Δ17), 19 (SUR1Δ19), or both (SUR1Δ17Δ19). The fourth (SUR1C) is a COOH-terminal SUR1-fragment formed by exons 31–39 containing the last two transmembrane segments and the COOH terminus of SUR1. RT-PCR analysis showed that these splice forms are expressed in several tissues with strong expression of SUR1C in cardiomyocytes. Confocal microscopy using enhanced green fluorescent protein-tagged SUR or Kir6.x did not provide any evidence for involvement of these splice forms in the mitochondrial KATP channel. Only SUR1 and SUR1Δ17 showed high-affinity binding of glibenclamide ( K d≈ 2 nM in the presence of 1 mM ATP) and formed functional KATPchannels upon coexpression with Kir6.2.

scholarly journals Functional Characterization of Candida albicans ABC Transporter Cdr1p

2003 ◽  
Vol 2 (6) ◽  
pp. 1361-1375 ◽  
Author(s):  
Suneet Shukla ◽  
Preeti Saini ◽  
Smriti ◽  
Sudhakar Jha ◽  
Suresh V. Ambudkar ◽  
...  
Keyword(s):  
Binding Sites ◽  
Fluorescent Protein ◽  
Mutational Analysis ◽  
Substrate Binding ◽  
Functional Characterization ◽  
Drug Binding ◽  
Transmembrane Segment ◽  
Green Fluorescent ◽  
Substrate Binding Sites ◽  
First Time

ABSTRACT In view of the importance of Candida drug resistance protein (Cdr1p) in azole resistance, we have characterized it by overexpressing it as a green fluorescent protein (GFP)-tagged fusion protein (Cdr1p-GFP). The overexpressed Cdr1p-GFP in Saccharomyces cerevisiae is shown to be specifically labeled with the photoaffinity analogs iodoarylazidoprazosin (IAAP) and azidopine, which have been used to characterize the drug-binding sites on mammalian drug-transporting P-glycoproteins. While nystatin could compete for the binding of IAAP, miconazole specifically competed for azidopine binding, suggesting that IAAP and azidopine bind to separate sites on Cdr1p. Cdr1p was subjected to site-directed mutational analysis. Among many mutant variants of Cdr1p, the phenotypes of F774A and ΔF774 were particularly interesting. The analysis of GFP-tagged mutant variants of Cdr1p revealed that a conserved F774, in predicted transmembrane segment 6, when changed to alanine showed increased binding of both photoaffinity analogues, while its deletion (ΔF774), as revealed by confocal microscopic analyses, led to mislocalization of the protein. The mislocalized ΔF774 mutant Cdr1p could be rescued to the plasma membrane as a functional transporter by growth in the presence of a Cdr1p substrate, cycloheximide. Our data for the first time show that the drug substrate-binding sites of Cdr1p exhibit striking similarities with those of mammalian drug-transporting P-glycoproteins and despite differences in topological organization, the transmembrane segment 6 in Cdr1p is also a major contributor to drug substrate-binding site(s).

scholarly journals The rice annexin gene OsAnn5 is a positive regulator of cold stress tolerance at the seedling stage

2020 ◽  
Author(s):  
chunxiu shen ◽  
Zhiqun Que ◽  
Qineng Lu ◽  
Tao Liu ◽  
Shengqiang Li ◽  
...  
Keyword(s):  
Cold Stress ◽  
Stress Tolerance ◽  
Stress Responses ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Survival Rates ◽  
Seedling Stage ◽  
Positive Regulator ◽  
Annexin Gene ◽  
Green Fluorescent

Abstract Annexins exist widely in plants as multigene families and play critical roles in stress responses and a range of cellular processes. In this study, we report on the cloning and functional characterization of the rice annexin gene OsAnn5. We found that the expression of OsAnn5 was induced by cold stress treatment at the seedling stage of rice. GUS staining assay indicated that the expression of OsAnn5 was non tissue-specific and was detected in almost all rice tissues. Subcellular localization indicated that OsAnn5-GFP (green fluorescent protein) signals were found in the endoplasmic reticulum apparatus. Compared with wild type rice, overexpression of OsAnn5 significantly increased survival rates at the seedling stage under cold stress, while knocking out OsAnn5 using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated proteins) mediated genome editing resulted in sensitivity to cold treatments. These results indicate that OsAnn5 is a positive regulator of cold stress tolerance at the seedling stage.

scholarly journals Green fluorescent protein-based assays for high-throughput functional characterization and ligand-binding studies of biotin protein ligase

2016 ◽  
Vol 8 (2) ◽  
pp. 418-424 ◽  
Author(s):  
Samuel P. Askin ◽  
Thomas E. H. Bond ◽  
Patrick M. Schaeffer
Keyword(s):  
Green Fluorescent Protein ◽  
Ligand Binding ◽  
High Throughput ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Binding Studies ◽  
Biotin Protein Ligase ◽  
Green Fluorescent

Rapid functional characterization of GFP-tagged biotin protein ligase (BirA-GFP) with a high-throughput DSF-GTP assay.

scholarly journals Identification and functional characterization of a novel human and rat riboflavin transporter, RFT1

2008 ◽  
Vol 295 (3) ◽  
pp. C632-C641 ◽  
Author(s):  
Atsushi Yonezawa ◽  
Satohiro Masuda ◽  
Toshiya Katsura ◽  
Ken-ichi Inui
Keyword(s):  
Amino Acid ◽  
Fluorescent Protein ◽  
Rat Kidney ◽  
Functional Characterization ◽  
Hek 293 ◽  
Reading Frame ◽  
Transporter Family ◽  
Acid Protein ◽  
Green Fluorescent ◽  
Interfering Rna

Absorption of riboflavin is mediated by transporter(s). However, a mammalian riboflavin transporter has yet to be identified. In the present study, the novel human and rat riboflavin transporters hRFT1 and rRFT1 were identified on the basis of our rat kidney mRNA expression database (Horiba N, Masuda S, Takeuchi A, Saito H, Okuda M, Inui K. Kidney Int 66: 29–45, 2004). hRFT1 and rRFT1 cDNAs have an open reading frame encoding 448- and 450-amino acid proteins, respectively, that exhibit 81.1% identity and 96.4% similarity to one another. In addition, an inactive splice variant of hRFT1, hRFT1sv, was also cloned. The hRFT1sv cDNA, which encodes a 167-amino acid protein, retains an intron between exons 2 and 3 of hRFT1. Real-time PCR revealed that the sum of hRFT1 and hRFT1sv mRNAs was expressed strongly in the placenta and small intestine and was detected in all tissues examined. In addition, hRFT1 and hRFT1sv were expressed in human embryonic kidney (HEK)-293 and Caco-2 cells. HEK-293 cells transfected with green fluorescent protein-tagged hRFT1 and rRFT1 exhibited a fluorescent signal in the plasma membrane. Overexpression of hRFT1 and rRFT1, but not hRFT1sv, increased the cellular accumulation of [3H]riboflavin. The transfection of small interfering RNA targeting both hRFT1 and hRFT1sv significantly decreased the uptake of [3H]riboflavin by HEK-293 and Caco-2 cells. Riboflavin transport is Na+, potential, and pH independent. Kinetic analyses demonstrated that the Michaelis-Menten constants for the uptake by HEK-293 and Caco-2 cells were 28.1 and 63.7 nM, respectively. We propose that hRFT1 and rRFT1 are novel mammalian riboflavin transporters, which belong to a new mammalian riboflavin transporter family.

scholarly journals Differentiation- and stress-dependent nuclear cytoplasmic redistribution of myopodin, a novel actin-bundling protein

2001 ◽  
Vol 155 (3) ◽  
pp. 393-404 ◽  
Author(s):  
Astrid Weins ◽  
Karin Schwarz ◽  
Christian Faul ◽  
Laura Barisoni ◽  
Wolfgang A. Linke ◽  
...  
Keyword(s):  
Heart Muscle ◽  
Nuclear Export ◽  
Fluorescent Protein ◽  
Functional Characterization ◽  
Actin Binding ◽  
Immunogold Electron Microscopy ◽  
Structural Protein ◽  
Western Blots ◽  
Green Fluorescent ◽  
Stress Dependent

We report the cloning and functional characterization of myopodin, the second member of the synaptopodin gene family. Myopodin shows no significant homology to any known protein except synaptopodin. Northern blot analysis resulted in a 3.6-kb transcript for mouse skeletal and heart muscle. Western blots showed an 80-kD signal for skeletal and a 95-kD signal for heart muscle. Myopodin contains one PPXY motif and multiple PXXP motifs. Myopodin colocalizes with α-actinin and is found at the Z-disc as shown by immunogold electron microscopy. In myoblasts, myopodin shows preferential nuclear localization. During myotube differentiation, myopodin binds to stress fibers in a punctuated pattern before incorporation into the Z-disc. Myopodin can directly bind to actin and contains a novel actin binding site in the center of the protein. Myopodin has actin-bundling activity as shown by formation of latrunculin-A–sensitive cytosolic actin bundles and nuclear actin loops in transfected cells expressing green fluorescent protein–myopodin. Under stress conditions, myopodin accumulates in the nucleus and is depleted from the cytoplasm. Nuclear export of myopodin is sensitive to leptomycin B, despite the absence of a classical nuclear export sequence. We propose a dual role for myopodin as a structural protein also participating in signaling pathways between the Z-disc and the nucleus.

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