scholarly journals Specific connectivity between photoreceptors and horizontal cells in the zebrafish retina

2016 ◽  
Vol 116 (6) ◽  
pp. 2799-2814 ◽  
Author(s):  
Lauw J. Klaassen ◽  
Wim de Graaff ◽  
Jorrit B. van Asselt ◽  
Jan Klooster ◽  
Maarten Kamermans
Keyword(s):  
Promoter Activity ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Horizontal Cell ◽  
Feedback Signal ◽  
Kinetic Properties ◽  
Strongly Coupled ◽  
H1 Cells ◽  
Green Fluorescent ◽  
Biphasic Responses

The functional and morphological connectivity between various horizontal cell (HC) types (H1, H2, H3, and H4) and photoreceptors was studied in zebrafish retina. Since HCs are strongly coupled by gap junctions and feedback from HCs to photoreceptors depends strongly on connexin (Cx) hemichannels, we characterized the various HC Cxs (Cx52.6, Cx52.7, Cx52.9, and Cx55.5) in Xenopus oocytes. All Cxs formed hemichannels that were conducting at physiological membrane potentials. The Cx hemichannels differed in kinetic properties and voltage dependence, allowing for specific tuning of the coupling of HCs and the feedback signal from HCs to cones. The morphological connectivity between HC layers and cones was determined next. We used zebrafish expressing green fluorescent protein under the control of Cx promoters. We found that all HCs showed Cx55.5 promoter activity. Cx52.7 promoter activity was exclusively present in H4 cells, while Cx52.9 promoter activity occurred only in H1 cells. Cx52.6 promoter activity was present in H4 cells and in the ventral quadrant of the retina also in H1 cells. Finally, we determined the spectral sensitivities of the HC layers. Three response types were found. Monophasic responses were generated by HCs that contacted all cones (H1 cells), biphasic responses were generated by HCs that contacted M, S, and UV cones (H2 cells), and triphasic responses were generated by HCs that contacted either S and UV cones (H3 cells) or rods and UV cones (H4 cells). Electron microscopy confirms that H4 cells innervate cones. This indicates that rod-driven HCs process spectral information during photopic and luminance information during scotopic conditions.

OKP cells express the Na-dicarboxylate cotransporter NaDC-1

2004 ◽  
Vol 287 (1) ◽  
pp. C64-C72 ◽  
Author(s):  
Seiji Aruga ◽  
Ana M. Pajor ◽  
Kiyoshi Nakamura ◽  
Liping Liu ◽  
Orson W. Moe ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Xenopus Oocytes ◽  
Fluorescent Protein ◽  
Stone Formation ◽  
Chronic Metabolic Acidosis ◽  
Opossum Kidney ◽  
Citrate Transport ◽  
Increased Risk ◽  
Green Fluorescent ◽  
Urinary Citrate

Urinary citrate concentration, a major factor in the formation of kidney stones, is primarily determined by its rate of reabsorption in the proximal tubule. Citrate reabsorption is mediated by the Na-dicarboxylate cotransporter-1 (NaDC-1). The opossum kidney (OKP) cell line possesses many characteristics of the renal proximal tubule. The OKP NaDC-1 (oNaDC-1) cDNA was cloned and encodes a 2.4-kb mRNA. When injected into Xenopus oocytes, the cotransporter is expressed and demonstrates Na-coupled citrate transport with a stoichiometry of ≥3 Na:1 citrate, specificity for di- and tricarboxylates, pH-dependent citrate transport, and pH-independent succinate transport, all characteristics of the other NaDC-1 orthologs. Chronic metabolic acidosis increases proximal tubule citrate reabsorption, leading to profound hypocitraturia and an increased risk for stone formation. Under the conditions studied, endogenous OKP NaDC-1 mRNA abundance is not regulated by changes in media pH. In OKP cells transfected with a green fluorescent protein-oNaDC-1 construct, however, media acidification increases Na-dependent citrate uptake, demonstrating posttranscriptional acid regulation of NaDC-1 activity.

scholarly journals Construction and use of aCupriavidus necatorH16 soluble hydrogenase promoter (PSH) fusion togfp(green fluorescent protein)

PeerJ ◽  
2016 ◽  
Vol 4 ◽  
pp. e2269 ◽  
Author(s):  
Bat-Erdene Jugder ◽  
Jeffrey Welch ◽  
Nady Braidy ◽  
Christopher P. Marquis
Keyword(s):  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Small Scale ◽  
Growth Media ◽  
Reporter System ◽  
Screening Assay ◽  
Fluorescent Reporter ◽  
Fusion Construct ◽  
Green Fluorescent

Hydrogenases are metalloenzymes that reversibly catalyse the oxidation or production of molecular hydrogen (H2). Amongst a number of promising candidates for application in the oxidation of H2is a soluble [Ni–Fe] uptake hydrogenase (SH) produced byCupriavidus necatorH16. In the present study, molecular characterisation of the SH operon, responsible for functional SH synthesis, was investigated by developing a green fluorescent protein (GFP) reporter system to characterise PSHpromoter activity using several gene cloning approaches. A PSHpromoter-gfp fusion was successfully constructed and inducible GFP expression driven by the PSHpromoter under de-repressing conditions in heterotrophic growth media was demonstrated in the recombinantC. necatorH16 cells. Here we report the first successful fluorescent reporter system to study PSHpromoter activity inC. necatorH16. The fusion construct allowed for the design of a simple screening assay to evaluate PSHactivity. Furthermore, the constructed reporter system can serve as a model to develop a rapid fluorescent based reporter for subsequent small-scale process optimisation experiments for SH expression.

scholarly journals Nanoluciferase-Based Method for Detecting Gene Expression in Caenorhabditis elegans

Genetics ◽  
2019 ◽  
Vol 213 (4) ◽  
pp. 1197-1207 ◽  
Author(s):  
Ivana Sfarcic ◽  
Theresa Bui ◽  
Erin C. Daniels ◽  
Emily R. Troemel
Keyword(s):  
Gene Expression ◽  
Caenorhabditis Elegans ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Fluorescent Reporters ◽  
C Elegans ◽  
Link Type ◽  
Highly Sensitive ◽  
Green Fluorescent

Genetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, animal autofluorescence limits the sensitivity of GFP and other fluorescent reporters in whole-animal settings like in the nematode Caenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in a multiwell format to detect constitutive and inducible gene expression in C. elegans. We optimize detection of bioluminescent signals from NanoLuc in C. elegans and show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by the vha-6 promoter. We can reliably detect signal in single vha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from a 1/100 dilution of lysate from a single vha-6p::Nanoluc-expressing adult and from a single vha-6p::Nanoluc-expressing adult “hidden” in a pool of 5000 N2 wild-type animals. We also optimize various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof-of-concept, we used NanoLuc to monitor the promoter activity of the pals-5 stress/immune reporter and were able to measure 300- and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring whole-animal gene expression in C. elegans.

Determinants of the enzymatic activity and the subcellular localization of aspartate N-acetyltransferase

2011 ◽  
Vol 441 (1) ◽  
pp. 105-112 ◽  
Author(s):  
Gaëlle Tahay ◽  
Elsa Wiame ◽  
Donatienne Tyteca ◽  
Pierre J. Courtoy ◽  
Emile Van Schaftingen
Keyword(s):  
Catalytic Activity ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Catalytic Domain ◽  
Kinetic Properties ◽  
Conserved Residues ◽  
Membrane Bound ◽  
Green Fluorescent ◽  
Necessary And Sufficient ◽  
Membrane Region

Aspartate N-acetyltransferase (NAT8L, N-acetyltransferase 8-like), the enzyme that synthesizes N-acetylaspartate, is membrane-bound and is at least partially associated with the ER (endoplasmic reticulum). The aim of the present study was to determine which regions of the protein are important for its catalytic activity and its subcellular localization. Transfection of truncated forms of NAT8L into HEK (human embryonic kidney)-293T cells indicated that the 68 N-terminal residues (regions 1 and 2) have no importance for the catalytic activity and the subcellular localization of this enzyme, which was exclusively associated with the ER. Mutation of conserved residues that precede (Arg81 and Glu101, in region 3) or follow (Asp168 and Arg220, in region 5) the putative membrane region (region 4) markedly affected the kinetic properties, suggesting that regions 3 and 5 form the catalytic domain and that the membrane region has a loop structure. Evidence is provided for the membrane region comprising α-helices and the catalytic site being cytosolic. Transfection of chimaeric proteins in which GFP (green fluorescent protein) was fused to different regions of NAT8L indicated that the membrane region (region 4) is necessary and sufficient to target NAT8L to the ER. Thus NAT8L is targeted to the ER membrane by a hydrophobic loop that connects two regions of the catalytic domain.

Differential CTX-M Expression from a Conserved Promoter: Role of Promoter-Associated Spacer Sequences Downstream of the blaCTX-M Regulon

2016 ◽  
Vol 26 (4) ◽  
pp. 284-290 ◽  
Author(s):  
Lin Liu ◽  
Xiangyan Zhang ◽  
Siyuan Yang ◽  
Yao Zhai ◽  
Weijia Liu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Expression Vector ◽  
Fluorescent Protein ◽  
Gfp Expression ◽  
Qrt Pcr ◽  
Key Factor ◽  
Green Fluorescent

<b><i>Aims:</i></b> The aim of this project was to explore the different CTX-M expression levels occurring from a single conserved promoter with different spacer sequences, the variation of which is hypothesized to be a key factor in fluctuating levels of CTX-M. <b><i>Methods:</i></b> The <i>bla</i><sub>CTX-M</sub> promoter fragments with five different spacer sequences were amplified, sequenced and cloned into the pUA66 expression vector carrying the green fluorescent protein (GFP) gene. The expression of <i>bla</i><sub>CTX-M</sub> in the transconjugants was analyzed using fluorescence microscopy, flow cytometry and qRT-PCR. <b><i>Results:</i></b> The promoters of all the <i>bla</i><sub>CTX-M</sub> genes were provided by IS<i>Ecp1 </i>and were extremely conserved. The promoter-associated spacer sequences varied from 42 to 127 bp and variations in GFP expression in the five transconjugants were observed. A nucleic acid deletion and point mutation were detected in the spacer sequences by variations in which the expression of <i>bla</i><sub>CTX-M</sub> was influenced. <b><i>Conclusion:</i></b> The different spacer sequences have a significant impact on the activity of the conserved promoter. The shorter spacer sequence between the conserved promoter and the <i>bla</i><sub>CTX-M</sub> gene does not specifically enhance the expression of<i> bla</i><sub>CTX-M</sub>, contrary to previous reports. The expression of <i>bla</i><sub>CTX-M</sub> may be regulated by changes in promoter activity caused by diverse spacer sequences.

scholarly journals Human UMP-CMP Kinase 2, a Novel Nucleoside Monophosphate Kinase Localized in Mitochondria

2007 ◽  
Vol 283 (3) ◽  
pp. 1563-1571 ◽  
Author(s):  
Yunjian Xu ◽  
Magnus Johansson ◽  
Anna Karlsson
Keyword(s):  
Fluorescent Protein ◽  
Lymphoblastic Leukemia ◽  
Nucleoside Analogs ◽  
Term Treatment ◽  
Myelogenous Leukemia ◽  
Kinetic Properties ◽  
Protein Fusion ◽  
Mtdna Depletion ◽  
Long Term Treatment ◽  
Green Fluorescent

Enzyme deficiency in the salvage pathway of deoxyribonucleotide synthesis in mitochondria can cause mtDNA depletion syndromes. We have identified a human mitochondrial UMP-CMP kinase (UMP-CMPK, cytidylate kinase; EC 2.7.4.14), designated as UMP-CMP kinase 2 (UMP-CMPK2). The C-terminal domain of this 449-amino acid protein contains all consensus motifs of a nucleoside monophosphate kinase. Phylogenetic analysis showed that UMP-CMPK2 belonged to a novel nucleoside monophosphate kinase family, which was closer to thymidylate kinase than to cytosolic UMP-CMP kinase. Subcellular localization with green fluorescent protein fusion proteins illustrated that UMP-CMPK2 was localized in the mitochondria of HeLa cells and that the mitochondrial targeting signal was included in the N-terminal 22 amino acids. The enzyme was able to phosphorylate dUMP, dCMP, CMP, and UMP with ATP as phosphate donor, but the kinetic properties were different compared with the cytosolic UMP-CMPK. Its efficacy to convert dUMP was highest, followed by dCMP, whereas CMP and UMP were the poorest substrates. It also phosphorylated the monophosphate forms of the nucleoside analogs ddC, dFdC, araC, BVDU, and FdUrd, which suggests that UMP-CMPK2 may be involved in mtDNA depletion caused by long term treatment with ddC or other pyrimidine analogs. UMP-CMPK2 mRNA expression was exclusively detected in chronic myelogenous leukemia K-562 and lymphoblastic leukemia MOLT-4 among eight studied cancer cell lines. Particular high expression in leukemia cells, dominant expression in bone marrow, and tight correlation with macrophage activation and inflammatory response suggest that UMP-CMPK2 may have other functions in addition to the supply of substrates for mtDNA synthesis.

scholarly journals Green Fluorescent Protein as a Quantitative Reporter of Relative Promoter Activity in E. coli

BioTechniques ◽  
2000 ◽  
Vol 28 (1) ◽  
pp. 82-89 ◽  
Author(s):  
J.L. Lissemore ◽  
J.T. Jankowski ◽  
C.B. Thomas ◽  
D.P. Mascotti ◽  
P.L. deHaseth
Keyword(s):  
Green Fluorescent Protein ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
E Coli ◽  
Green Fluorescent
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