Functional characterization of wild-type and mutant human sialin

AbstractMonotremes are the most basal egg-laying mammals comprised of two extant genera, which are largely nocturnal. Visual pigments, the first step in the sensory transduction cascade in photoreceptors of the eye, have been examined in a variety of vertebrates, but little work has been done to study the rhodopsin of monotremes. We isolated the rhodopsin gene of the nocturnal short-beaked echidna (Tachyglossus aculeatus) and expressed and functionally characterized the protein in vitro. Three mutants were also expressed and characterized: N83D, an important site for spectral tuning and metarhodopsin kinetics, and two sites with amino acids unique to the echidna (T158A and F169A). The λmax of echidna rhodopsin (497.9 ± 1.1 nm) did not vary significantly in either T158A (498.0 ± 1.3 nm) or F169A (499.4 ± 0.1 nm) but was redshifted in N83D (503.8 ± 1.5 nm). Unlike other mammalian rhodopsins, echidna rhodopsin did react when exposed to hydroxylamine, although not as fast as cone opsins. The retinal release rate of light-activated echidna rhodopsin, as measured by fluorescence spectroscopy, had a half-life of 9.5 ± 2.6 min−1, which is significantly shorter than that of bovine rhodopsin. The half-life of the N83D mutant was 5.1 ± 0.1 min−1, even shorter than wild type. Our results show that with respect to hydroxylamine sensitivity and retinal release, the wild-type echidna rhodopsin displays major differences to all previously characterized mammalian rhodopsins and appears more similar to other nonmammalian vertebrate rhodopsins such as chicken and anole. However, our N83D mutagenesis results suggest that this site may mediate adaptation in the echidna to dim light environments, possibly via increased stability of light-activated intermediates. This study is the first characterization of a rhodopsin from a most basal mammal and indicates that there might be more functional variation in mammalian rhodopsins than previously assumed.

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2066 Functional characterization of mutant BRCA1

Journal of Clinical and Translational Science ◽

10.1017/cts.2018.77 ◽

2018 ◽

Vol 2 (S1) ◽

pp. 13-13

Author(s):

John Barrows ◽

David Long

Keyword(s):

Functional Characterization ◽

Coiled Coil ◽

Wild Type ◽

Coiled Coil Region ◽

Egg Extracts ◽

Study Population ◽

Xenopus Egg ◽

Xenopus Egg Extracts

OBJECTIVES/SPECIFIC AIMS: The objective of this work is to determine the mechanistic consequences of BRCA1 mutants in inter-strand crosslink (ICL) repair. METHODS/STUDY POPULATION: Our lab uses Xenopus egg extracts to study ICL repair. These extracts can be depleted of endogenous BRCA1 by immunoprecipitation. The goal of this work is to rescue endogenous depletion with in vitro translated, wild type BRCA1. Once achieved, we can supplement the depleted extract with BRCA1 mutants to access their function in ICL repair. RESULTS/ANTICIPATED RESULTS: We hypothesize that the BRCT and RING domain mutations will abrogate ICL repair, while mutations in the coiled coil region will not affect repair. DISCUSSION/SIGNIFICANCE OF IMPACT: These findings will have an immense impact on the understanding of BRCA1 domains. Importantly these results will spur personalized therapy of BRCA1 mutants by showing which domains are sensitive to cross-linking agents.

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Crystal Structures and Functional Characterization of Wild-Type CYP101D1 and Its Active Site Mutants

Biochemistry ◽

10.1021/bi401330c ◽

2013 ◽

Vol 52 (49) ◽

pp. 8898-8906 ◽

Cited By ~ 13

Author(s):

Dipanwita Batabyal ◽

Thomas L. Poulos

Keyword(s):

Crystal Structures ◽

Active Site ◽

Functional Characterization ◽

Wild Type ◽

Active Site Mutants

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Functional Characterization of Wild-type and 49 CYP2D6 Allelic Variants for N-Desmethyltamoxifen 4-Hydroxylation Activity

Drug Metabolism and Pharmacokinetics ◽

10.2133/dmpk.dmpk-14-rg-014 ◽

2014 ◽

Vol 29 (5) ◽

pp. 360-366 ◽

Cited By ~ 25

Author(s):

Yuka Muroi ◽

Takahiro Saito ◽

Masamitsu Takahashi ◽

Kanako Sakuyama ◽

Yui Niinuma ◽

...

Keyword(s):

Functional Characterization ◽

Wild Type ◽

Allelic Variants ◽

Hydroxylation Activity

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Functional characterization of canine wild type glucocorticoid receptor and an insertional mutation in a dog

BMC Veterinary Research ◽

10.1186/s12917-019-2129-9 ◽

2019 ◽

Vol 15 (1) ◽

Author(s):

Kosei Yamanaka ◽

Masaru Okuda ◽

Takuya Mizuno

Keyword(s):

Glucocorticoid Receptor ◽

Cushing Syndrome ◽

Functional Characterization ◽

Wild Type ◽

Key Factors ◽

Truncated Form ◽

Glucocorticoid Sensitivity ◽

Almost All

Abstract Background Glucocorticoids, among the most widely utilized drugs in veterinary medicine, are employed to treat a wide variety of diseases; however, their use often induces adverse events in dogs. The efficacy of glucocorticoids usually depends on dosage, although differences in sensitivity to glucocorticoids in individual animals have been reported. Glucocorticoids bind to the cytoplasmic glucocorticoid receptor (GR), which is expressed in almost all cells. These receptors are key factors in determining individual sensitivity to glucocorticoids. This study examined individual differences in glucocorticoid sensitivity in dogs, focusing on reactivity of the GR to prednisolone. Results We first molecularly cloned the GR gene from a healthy dog. We discovered a mutant GR in a dog suspected to have iatrogenic Cushing syndrome. The mutant GR had extra nucleotides between exons 6 and 7, resulting in a truncated form of GR that was 98 amino acids shorter than the wild-type dog GR. The truncated GR exhibited very low reactivity to prednisolone, irrespective of concentration. Conclusions We have identified the truncated form of canine GR in a dog with iatrogenic Cushing syndrome. This truncated form showed the very less sensitivity to glucocorticoid in vitro, unfortunately, we could not elucidate its clinical significance. However, our data is a first report about the function of canine GR, and will facilitate the analysis of canine glucocorticoid sensitivity.

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Key wheat GRF genes constraining wheat tillering of mutant dmc

PeerJ ◽

10.7717/peerj.11235 ◽

2021 ◽

Vol 9 ◽

pp. e11235

Author(s):

Jing Zhang ◽

Junchang Li ◽

Yongjing Ni ◽

Yumei Jiang ◽

Zhixin Jiao ◽

...

Keyword(s):

Triticum Aestivum ◽

Acetic Acid ◽

Indole Acetic Acid ◽

Expression Profiles ◽

Functional Characterization ◽

Tissue Expression ◽

Wild Type ◽

Gene Structures ◽

Wheat Mutant

Tillering is a key agronomy trait for wheat (Triticum aestivum L.) production. Previously, we have reported a dwarf-monoculm wheat mutant (dmc) obtained from cultivar Guomai 301 (wild type, WT), and found growth regulating factors (GRFs) playing important roles in regulating wheat tillering. This study is to systematically investigate the roles of all the wheat GRFs (T. aestivum GRFs, TaGRFs) in regulating tillering, and screen out the key regulators. A total of 30 TaGRFs were identified and their physicochemical properties, gene structures, conserved domains, phylogenetic relationships and tissue expression profiles were analyzed. The expression levels of all the TaGRFs were significantly lower in dmc than those in WT at early tillering stage, and the abnormal expressions of TaGRF2-7(A, B, D), TaGRF5-7D, TaGRF10-6(A, B, D) and TaGRF11-2A were major causes constraining the tillering of dmc. The transcriptions of TaGRFs were significantly affected by exogenous indole acetic acid (IAA) and gibberellin acid (GA3) applications, which suggested that TaGRFs as well as IAA, GA signaling were involved in controlling wheat tillering. This study provided valuable clues for functional characterization of GRF genes in wheat.

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Functional Characterization of WaaL, a Ligase Associated with Linking O-Antigen Polysaccharide to the Core of Pseudomonas aeruginosa Lipopolysaccharide

Journal of Bacteriology ◽

10.1128/jb.187.9.3002-3012.2005 ◽

2005 ◽

Vol 187 (9) ◽

pp. 3002-3012 ◽

Cited By ~ 84

Author(s):

Priyanka D. Abeyrathne ◽

Craig Daniels ◽

Karen K. H. Poon ◽

Mauricia J. Matewish ◽

Joseph S. Lam

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Functional Characterization ◽

Wild Type ◽

The Core ◽

O Antigen ◽

Repeat Units ◽

Cytoplasmic Face ◽

O Antigens

ABSTRACT The O antigen of Pseudomonas aeruginosa B-band lipopolysaccharide is synthesized by assembling O-antigen-repeat units at the cytoplasmic face of the inner membrane by nonprocessive glycosyltransferases, followed by polymerization on the periplasmic face. The completed chains are covalently attached to lipid A core by the O-antigen ligase, WaaL. In P. aeruginosa the process of ligating these O-antigen molecules to lipid A core is not clearly defined, and an O-antigen ligase has not been identified until this study. Using the sequence of waaL from Salmonella enterica as a template in a BLAST search, a putative waaL gene was identified in the P. aeruginosa genome. The candidate gene was amplified and cloned, and a chromosomal knockout of PAO1 waaL was generated. Lipopolysaccharide (LPS) from this mutant is devoid of B-band O-polysaccharides and semirough (SR-LPS, or core-plus-one O-antigen). The mutant PAO1waaL is also deficient in the production of A-band polysaccharide, a homopolymer of d-rhamnose. Complementation of the mutant with pPAJL4 containing waaL restored the production of both A-band and B-band O antigens as well as SR-LPS, indicating that the knockout was nonpolar and waaL is required for the attachment of O-antigen repeat units to the core. Mutation of waaL in PAO1 and PA14, respectively, could be complemented with waaL from either strain to restore wild-type LPS production. The waaL mutation also drastically affected the swimming and twitching motilities of the bacteria. These results demonstrate that waaL in P. aeruginosa encodes a functional O-antigen ligase that is important for cell wall integrity and motility of the bacteria.

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Purification and Functional Characterization of Wild-Type and Mutant HIV-1 and HIV-2 Tat Proteins Expressed inEscherichia coli

Protein Expression and Purification ◽

10.1006/prep.1996.0096 ◽

1996 ◽

Vol 8 (2) ◽

pp. 238-246 ◽

Cited By ~ 1

Author(s):

Michael J. Orsini ◽

León F. Garcı́a-Martı́nez ◽

Gopinath Mavankal ◽

Richard B. Gaynor ◽

Christine M. Debouck

Keyword(s):

Functional Characterization ◽

Inescherichia Coli ◽

Wild Type ◽

Hiv 1

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Functional characterization of FlgM in the regulation of flagellar synthesis and motility in Yersinia pseudotuberculosis

Microbiology ◽

10.1099/mic.0.026294-0 ◽

2009 ◽

Vol 155 (6) ◽

pp. 1890-1900 ◽

Cited By ~ 18

Author(s):

Lisha Ding ◽

Yao Wang ◽

Yangbo Hu ◽

Steve Atkinson ◽

Paul Williams ◽

...

Keyword(s):

Yersinia Pseudotuberculosis ◽

Functional Characterization ◽

Direct Interaction ◽

Site Directed Mutagenesis ◽

Wild Type ◽

C Terminus ◽

In Trans ◽

Differential Gene ◽

Flagellar Biogenesis

We describe here the functional characterization of the flgM gene in Yersinia pseudotuberculosis. Direct interaction of FlgM with the alternative sigma factor σ 28 (FliA) was first confirmed. A conserved region in the C-terminus of FlgM was found which included the σ 28 binding domain. By site-directed mutagenesis, bacterial two-hybrid analysis and Western blotting, the primary FlgM binding sites with σ 28 were shown to be Ile85, Ala86 and Leu89. A role for FlgM in swimming motility was demonstrated by inactivation of flgM and subsequent complementation in trans. Transcriptional fusion analyses showed differential gene expression of flhDC, fliA, flgM and fliC in the fliA and flgM mutants compared with the wild-type. flhDC expression was not influenced by σ 28 or FlgM while fliA expression was abolished in the fliA mutant and considerably reduced in the flgM mutant when compared to the wild-type, indicating that both FliA and FlgM can activate fliA transcription. Conversely, flgM transcription was higher in the fliA mutant when compared to the wild-type, suggesting that flgM transcription was repressed by σ 28. Interestingly, fliC expression was markedly increased in the flgM mutant, suggesting a negative regulatory role for FlgM in fliC expression. The transcription of other σ-dependent genes (cheW, flgD, flaA, csrA and fliZ) was also examined in fliA and flgM mutant backgrounds and this revealed that other σ-factors apart from σ 28 may be involved in flagellar biogenesis in Y. pseudotuberculosis. Taking together the motility phenotypes and effects of flgM mutation on the regulation of these key motility genes, we propose that the mechanisms regulating flagellar biogenesis in Y. pseudotuberculosis may differ from those described for other bacteria.

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