scholarly journals Functional characterization of threeCYP21A2sequence variants (p.A265V, p.W302S, p.D322G) employing a yeast co-expression system

2008 ◽  
Vol 30 (2) ◽  
pp. E443-E450 ◽  
Author(s):  
Caroline Bleicken ◽  
Lourdes Loidi ◽  
Vivek Dhir ◽  
Silvia Parajes ◽  
Celsa Quinteiro ◽  
...  
Keyword(s):  
Expression System ◽  
Functional Characterization

scholarly journals Harnessing a Transient Gene Expression System in Nicotiana benthamiana to Explore Plant Agrochemical Transporters

Plants ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 524
Author(s):  
Bingqi Wu ◽  
Zhiting Chen ◽  
Xiaohui Xu ◽  
Ronghua Chen ◽  
Siwei Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transient Expression ◽  
Nicotiana Benthamiana ◽  
Heterologous Gene Expression ◽  
Expression System ◽  
Functional Characterization ◽  
Transient Gene Expression ◽  
High Mobility ◽  
Gene Expression System

Functional characterization of plant agrichemical transporters provided an opportunity to discover molecules that have a high mobility in plants and have the potential to increase the amount of pesticides reaching damage sites. Agrobacterium-mediated transient expression in tobacco is simple and fast, and its protein expression efficiency is high; this system is generally used to mediate heterologous gene expression. In this article, transient expression of tobacco nicotine uptake permease (NtNUP1) and rice polyamine uptake transporter 1 (OsPUT1) in Nicotiana benthamiana was performed to investigate whether this system is useful as a platform for studying the interactions between plant transporters and pesticides. The results showed that NtNUP1 increases nicotine uptake in N. benthamiana foliar discs and protoplasts, indicating that this transient gene expression system is feasible for studying gene function. Moreover, yeast expression of OsPUT1 apparently increases methomyl uptake. Overall, this method of constructing a transient gene expression system is useful for improving the efficiency of analyzing the functions of plant heterologous transporter-encoding genes and revealed that this system can be further used to study the functions of transporters and pesticides, especially their interactions.

Cloning and functional characterization of an uncoupling protein homolog in hummingbirds

2001 ◽  
Vol 5 (3) ◽  
pp. 137-145 ◽  
Author(s):  
CLAUDIA R. VIANNA ◽  
THILO HAGEN ◽  
CHEN-YU ZHANG ◽  
ERIC BACHMAN ◽  
OLIVIER BOSS ◽  
...  
Keyword(s):  
Uncoupling Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Pectoral Muscle ◽  
Mitochondrial Fraction ◽  
Mrna Levels ◽  
Coding Region ◽  
Reading Frame ◽  
Rapid Amplification

The cDNA of an uncoupling protein (UCP) homolog has been cloned from the swallow-tailed hummingbird, Eupetomena macroura. The hummingbird uncoupling protein (HmUCP) cDNA was amplified from pectoral muscle (flight muscle) using RT-PCR and primers for conserved domains of various known UCP homologs. The rapid amplification of cDNA ends (RACE) method was used to complete the cloning of the 5′ and 3′ ends of the open reading frame. The HmUCP coding region contains 915 nucleotides, and the deduced protein sequence consists of 304 amino acids, being ∼72, 70, and 55% identical to human UCP3, UCP2, and UCP1, respectively. The uncoupling activity of this novel protein was characterized in yeast. In this expression system, the 12CA5-tagged HmUCP fusion protein was detected by Western blot in the enriched mitochondrial fraction. Similarly to rat UCP1, HmUCP decreased the mitochondrial membrane potential as measured in whole yeast by uptake of the fluorescent potential-sensitive dye 3′,3-dihexyloxacarbocyanine iodide. The HmUCP mRNA is primarily expressed in skeletal muscle, but high levels can also be detected in heart and liver, as assessed by Northern blot analysis. Lowering the room’s temperature to 12–14°C triggered the cycle torpor/rewarming, typical of hummingbirds. Both in the pectoral muscle and heart, HmUCP mRNA levels were 1.5- to 3.4-fold higher during torpor. In conclusion, this is the first report of an UCP homolog in birds. The data indicate that HmUCP has the potential to function as an UCP and could play a thermogenic role during rewarming.

scholarly journals Rhinella marina oocytes: a suitable alternative expression system for functional characterization of aquaglyceroporins

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Vania Rojas ◽  
Yulexi Y. Ortiz ◽  
Sheridan Rodríguez ◽  
Vladimir Araque ◽  
Alexis Rodríguez-Acosta ◽  
...  
Keyword(s):  
Expression System ◽  
Functional Characterization ◽  
Suitable Alternative ◽  
Rhinella Marina ◽  
Alternative Expression

Functional Characterization of Glycosylation-Deficient Human P-Glycoprotein Using A Vaccinia Virus Expression System

2000 ◽  
Vol 173 (3) ◽  
pp. 203-214 ◽  
Author(s):  
J.J. Gribar ◽  
M. Ramachandra ◽  
C.A. Hrycyna ◽  
S. Dey ◽  
S.V. Ambudkar
Keyword(s):  
Vaccinia Virus ◽  
Expression System ◽  
Functional Characterization ◽  
P Glycoprotein ◽  
Virus Expression

Functional characterization of the human dopamine D4.2 receptor using vaccinia virus as an expression system

1995 ◽  
Vol 290 (1) ◽  
pp. 11-17 ◽  
Author(s):  
Claudia Bouvier ◽  
James R. Bunzow ◽  
Hong-Chang Guan ◽  
Anja Unteutsch ◽  
Olivier Civelli ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Expression System ◽  
Functional Characterization

scholarly journals Clinical and Functional Characterization of a Novel URAT1 Dysfunctional Variant in a Pediatric Patient with Renal Hypouricemia

2019 ◽  
Vol 9 (17) ◽  
pp. 3479
Author(s):  
Blanka Stiburkova ◽  
Jana Bohata ◽  
Iveta Minarikova ◽  
Andrea Mancikova ◽  
Jiri Vavra ◽  
...  
Keyword(s):  
Pediatric Patient ◽  
Primary Care Physicians ◽  
Expression System ◽  
Functional Characterization ◽  
Functional Study ◽  
Sequencing Analysis ◽  
Coding Region ◽  
Cytoplasmic Staining ◽  
Renal Hypouricemia

Renal hypouricemia (RHUC) is caused by an inherited defect in the main (reabsorptive) renal urate transporters, URAT1 and GLUT9. RHUC is characterized by decreased concentrations of serum uric acid and an increase in its excretion fraction. Patients suffer from hypouricemia, hyperuricosuria, urolithiasis, and even acute kidney injury. We report the clinical, biochemical, and genetic findings of a pediatric patient with hypouricemia. Sequencing analysis of the coding region of SLC22A12 and SLC2A9 and a functional study of a novel RHUC1 variant in the Xenopus expression system were performed. The proband showed persistent hypouricemia (67–70 µmol/L; ref. range 120–360 µmol/L) and hyperuricosuria (24–34%; ref. range 7.3 ± 1.3%). The sequencing analysis identified common non-synonymous allelic variants c.73G > A, c.844G > A, c.1049C > T in the SLC2A9 gene and rare variants c.973C > T, c.1300C > T in the SLC22A12 gene. Functional characterization of the novel RHUC associated c.973C > T (p. R325W) variant showed significantly decreased urate uptake, an irregular URAT1 signal on the plasma membrane, and reduced cytoplasmic staining. RHUC is an underdiagnosed disorder and unexplained hypouricemia warrants detailed metabolic and genetic investigations. A greater awareness of URAT1 and GLUT9 deficiency by primary care physicians, nephrologists, and urologists is crucial for identifying the disorder.

Functional characterization of protein variants of the human multidrug transporter ABCC2 by a novel targeted expression system in fibrosarcoma cells

2012 ◽  
Vol 33 (4) ◽  
pp. 750-762 ◽  
Author(s):  
Rudolf Arlanov ◽  
Andrew Porter ◽  
Dennis Strand ◽  
Rachel Brough ◽  
Darja Karpova ◽  
...  
Keyword(s):  
Expression System ◽  
Functional Characterization ◽  
Multidrug Transporter ◽  
Fibrosarcoma Cells ◽  
Targeted Expression ◽  
Protein Variants

scholarly journals The Barley Stripe Mosaic Virus expression system reveals the wheat C2H2 zinc finger protein TaZFP1B as a key regulator of drought tolerance

2019 ◽  
Author(s):  
Mario Houde ◽  
Arnaud Cheuk ◽  
François Ouellet
Keyword(s):  
Drought Stress ◽  
Drought Tolerance ◽  
Mosaic Virus ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Expression System ◽  
Functional Characterization ◽  
Barley Stripe Mosaic Virus ◽  
Finger Protein

Abstract Drought stress is one of the major factors limiting wheat production globally. Improving drought tolerance is important for agriculture sustainability. Although various morphological, physiological and biochemical responses associated with drought tolerance have been documented, the molecular mechanisms and regulatory genes that are needed to improve drought tolerance in crops require further investigation. We have used a novel 4-component version (for overexpression) and a 3-component version (for underexpression) of a barley stripe mosaic virus-based (BSMV) system for functional characterization of the C2H2-type zinc finger protein TaZFP1B in wheat. These expression systems avoid the need to produce transgenic plant lines and greatly speeds up functional gene characterization.Results We show that overexpression of TaZFP1B stimulates plant growth and up-regulates different oxidative stress-responsive genes under well-watered conditions. Plants that overexpress TaZFP1B are more drought tolerant at critical periods of the plant’s life cycle. Furthermore, RNA-Seq analysis revealed that plants overexpressing TaZFP1B reprogram their transcriptome, resulting in physiological and physical modifications that help wheat to grow and survive under drought stress. In contrast, plants transformed to underexpress TaZFP1B are significantly less tolerant to drought and growth is negatively affected.Conclusions This study clearly shows that the two versions of the BSMV system can be used for fast and efficient functional characterization of genes in crops. The extent of transcriptome reprogramming in plants that overexpress TaZFP1B indicates that the encoded transcription factor is a key regulator of drought tolerance in wheat.

scholarly journals Expression, purification and characterization of the Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) DNA polymerase and interaction with the SpliNPV non-hr origin of DNA replication

2001 ◽  
Vol 82 (7) ◽  
pp. 1767-1776 ◽  
Author(s):  
Jianhe Huang ◽  
David B. Levin
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Spodoptera Littoralis ◽  
Expression System ◽  
Functional Characterization ◽  
Eukaryotic Expression ◽  
Replication Assay ◽  
E Coli ◽  
Origin Of Dna Replication

The DNA polymerase from Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) was expressed in, and purified from, prokaryotic and eukaryotic expression systems. While less protein was obtained from the E. coli expression system, SpliNPV DNAPOL purified from E. coli displayed similar biochemical characteristics to DNAPOL expressed in, and subsequently purified from, insect cells (Sf9) using a baculovirus expression system. Biochemical analyses suggested that the DNA polymerase and the 3′–5′ exonuclease activities are intrinsic to the protein. Deletion of the first 80 amino acid residues from the N terminus of the DNAPOL affected neither the DNA polymerase nor the exonuclease activities of the enzyme. Replication products from single-stranded M13 DNA demonstrated that the DNA synthesis activity of SpliNPV DNAPOL is highly processive. Transient expression assays with a set of deletion clones containing the putative SpliNPV non-hr origin of DNA replication permitted functional characterization of sequence elements within the origin fragment. Purified SpliNPV DNAPOL stimulated origin-dependent DNA replication in a cell-free replication assay.

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