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Site directed mutations

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https://www.readbyqxmd.com/read/28817611/the-directed-migration-of-gonadal-distal-tip-cells-in-caenorhabditis-elegans-requires-ngat-1-a-%C3%A3-1-4-n-acetylgalactosaminyltransferase-enzyme
#1
Joseph Veyhl, Robert J Dunn, Wendy L Johnston, Alexa Bennett, Lijia W Zhang, James W Dennis, Harry Schachter, Joseph G Culotti
Glycoproteins such as growth factor receptors and extracellular matrix have well-known functions in development and cancer progression, however, the glycans at sites of modification are often heterogeneous molecular populations which makes their functional characterization challenging. Here we provide evidence for a specific, discrete, well-defined glycan modification and regulation of a stage-specific cell migration in Caenorhabditis elegans. We show that a chain-terminating, putative null mutation in the gene encoding a predicted β1,4-N-acetylgalactosaminyltransferase, named ngat-1, causes a maternally rescued temperature sensitive (ts) defect in the second phase of the three phase migration pattern of the posterior, but not the anterior, hermaphrodite Distal Tip Cell (DTC)...
2017: PloS One
https://www.readbyqxmd.com/read/28815502/flping-genes-on-and-off-in-drosophila
#2
Bonnie M Weasner, Jinjin Zhu, Justin P Kumar
The fruit fly, Drosophila melanogaster, has been a favorite experimental system of developmental biologists for more than a century. One of the most attractive features of this model system is the clarity by which one can analyze mutant phenotypes. Most genes are found in single copies, and loss-of-function mutants often have obvious phenotypes that can be analyzed during development and in adulthood. As with all metazoans, a significant fraction of Drosophila genes are used during both embryonic and postembryonic development, and null mutants often die during embryogenesis thereby precluding the analysis of postembryonic tissues...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28814578/genetic-human-prion-disease-modelled-in-prp-transgenic-drosophila
#3
Alana M Thackray, Alzbeta Cardova, Hanna Wolf, Lydia Pradl, Ina Vorberg, Walker S Jackson, Raymond Bujdoso
Inherited human prion diseases, such as FFI and familial CJD (fCJD), are associated with autosomal dominant mutations in the human prion protein gene PRNP and accumulation of PrPSc, an abnormal isomer of the normal host protein PrPC, in the brain of affected individuals. PrPSc is the principal component of the transmissible neurotoxic prion agent. It is important to identify molecular pathways and cellular processes that regulate prion formation and prion-induced neurotoxicity. This will allow identification of possible therapeutic interventions for individuals with, or at risk from, genetic human prion disease...
August 16, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28813625/detection-of-first-line-drug-resistance-mutations-and-drug-protein-interaction-dynamics-from-tuberculosis-patients-in-south-india
#4
Somanna Ajjamada Nachappa, Sumana M Neelambike, Chokkanna Amruthavalli, Nallur B Ramachandra
Diagnosis of drug-resistant tuberculosis predominantly relies on culture-based drug susceptibility testing, which take weeks to produce a result and a more time-efficient alternative method is multiplex allele-specific PCR (MAS-PCR). Also, understanding the role of mutations in causing resistance helps better drug designing. AIMS: To evaluate the ability of MAS-PCR in the detection of drug resistance and to understand the mechanism of interaction of drugs with mutant proteins in Mycobacterium tuberculosis...
August 16, 2017: Microbial Drug Resistance: MDR: Mechanisms, Epidemiology, and Disease
https://www.readbyqxmd.com/read/28811710/single-amino-acid-mutation-of-sr-bi-decreases-infectivity-of-hepatitis-c-virus-derived-from-cell-culture-in-a-cell-culture-model
#5
Rong Gao, Wei Gao, Gang Xu, Jie Xu, Hao Ren
AIM: To investigate the effect of a single amino acid mutation in human class B scavenger receptor I (SR-BI) on the infectivity of cell culture-derived hepatitis C virus (HCVcc) in SR-BI knock-down Huh7-siSR-BI cells. METHODS: Site-directed mutagenesis was used to construct the SR-BI S112F mutation, and the mutation was confirmed by nucleotide sequencing. SR-BI knock-down Huh7-siSR-BI cells were transfected with SR-BI S112F, SR-BI wild type (WT) and control plasmids, and then infected with HCVpp (HCV pseudoparticles) and hepatitis C virus derived from cell culture (HCVcc)...
July 28, 2017: World Journal of Gastroenterology: WJG
https://www.readbyqxmd.com/read/28806064/conditional-displacement-hybridization-assay-for-multiple-snp-phasing
#6
Tsz Wing Fan, Henson L Lee Yu, I-Ming Hsing
The two chromosomal copies of the human genome are highly polymorphic, and the allelic content on each strand can dictate a person's biological outcomes. While many of the current diagnostic tools are able to detect the presence of multiple mutations at the same time, most cannot determine the phase of these mutations unless long-range PCR or sequencing techniques are used or if templates are compartmentalized into single copies prior to amplification. Here, an enzyme-coupled hybridization assay, named Conditional Displacement Hybridization Assay (CDHA), is described for the concurrent and rapid determination of the presence and phase of SNP variants...
August 14, 2017: Analytical Chemistry
https://www.readbyqxmd.com/read/28803966/a-chimeric-prokaryotic-eukaryotic-pentameric-ligand-gated-ion-channel-reveals-interactions-between-the-extracellular-and-transmembrane-domains-shape-neurosteroid-modulation
#7
Borna Ghosh, Tzu-Wei Tsao, Cynthia Czajkowski
Pentameric ligand-gated ion channels (pLGICs) are the targets of several clinical and endogenous allosteric modulators including anesthetics and neurosteroids. Molecular mechanisms underlying allosteric drug modulation are poorly understood. Here, we constructed a chimeric pLGIC by fusing the extracellular domain (ECD) of the proton-activated, cation-selective bacterial channel GLIC to the transmembrane domain (TMD) of the human ρ1 chloride-selective GABAAR, and tested the hypothesis that drug actions are regulated locally in the domain that houses its binding site...
August 10, 2017: Neuropharmacology
https://www.readbyqxmd.com/read/28801462/protein-glutaminylation-is-a-yeast-specific-posttranslational-modification-of-elongation-factor-1a
#8
Thomas Jank, Yury Belyi, Christophe Wirth, Sabine Rospert, Zehan Hu, Jörn Dengjel, Tina Tzivelekidis, Gregers Rom Andersen, Carola Hunte, Andreas Schlosser, Klaus Aktories
Ribosomal translation factors are fundamental for protein synthesis and highly conserved in all kingdoms of life. The essential eukaryotic elongation factor 1A (eEF1A), delivers aminoacyl tRNAs to the A-site of the translating 80S ribosome. Several studies have revealed that eEF1A is posttranslationally modified. Using MS analysis, site-directed mutagenesis, and X-ray structural data analysis of Saccharomyces cerevisiae eEF1A, we identified a posttranslational modification in which the alpha amino group of mono-L-glutamine is covalently linked to the side chain of glutamate 45 in eEF1A...
August 11, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28800953/col7a1-editing-via-crispr-cas9-in-recessive-dystrophic-epidermolysis-bullosa
#9
Stefan Hainzl, Patricia Peking, Thomas Kocher, Eva M Murauer, Fernando Larcher, Marcela Del Rio, Blanca Duarte, Markus Steiner, Alfred Klausegger, Johann W Bauer, Julia Reichelt, Ulrich Koller
Designer nucleases allow specific and precise genomic modifications and represent versatile molecular tools for the correction of disease-associated mutations. In this study, we have exploited an ex vivo CRISPR/Cas9-mediated homology-directed repair approach for the correction of a frequent inherited mutation in exon 80 of COL7A1, which impairs type VII collagen expression, causing the severe blistering skin disease recessive dystrophic epidermolysis bullosa. Upon CRISPR/Cas9 treatment of patient-derived keratinocytes, using either the wild-type Cas9 or D10A nickase, corrected single-cell clones expressed and secreted similar levels of type VII collagen as control keratinocytes...
July 13, 2017: Molecular Therapy: the Journal of the American Society of Gene Therapy
https://www.readbyqxmd.com/read/28798948/hoxb13-mutations-and-binding-partners-in-prostate-development-and-cancer-function-clinical-significance-and-future-directions
#10
Hannah Brechka, Raj R Bhanvadia, Calvin VanOpstall, Donald J Vander Griend
The recent and exciting discovery of germline HOXB13 mutations in familial prostate cancer has brought HOX signaling to the forefront of prostate cancer research. An enhanced understanding of HOX signaling, and the co-factors regulating HOX protein specificity and transcriptional regulation, has the high potential to elucidate novel approaches to prevent, diagnose, stage, and treat prostate cancer. Toward our understanding of HOX biology in prostate development and prostate cancer, basic research in developmental model systems as well as other tumor sites provides a mechanistic framework to inform future studies in prostate biology...
June 2017: Genes & Diseases
https://www.readbyqxmd.com/read/28798394/orthosteric-versus-allosteric-dependent-activation-of-the-gabaa-receptor-requires-numerically-distinct-subunit-level-rearrangements
#11
Jahanshah Amin, Meena S Subbarayan
Anaesthetic molecules act on synaptic transmission via the allosteric modulation of ligand-gated chloride channels, such as hetero-oligomeric α1β2γ2 GABAA receptors. To elucidate the overall activation paradigm via allosteric versus orthosteric sites, we used highly homologous, but homo-oligomeric, ρ1 receptors that are contrastingly insensitive to anaesthetics and respond partially to several full GABA α1β2γ2 receptor agonists. Here, we coexpressed varying ratios of RNAs encoding the wild-type and the mutated ρ1 subunits, which are anaesthetic-sensitive and respond with full efficacy to partial GABA agonists, to generate distinct ensembles of receptors containing five, four, three, two, one, or zero mutated subunits...
August 10, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28797116/calcium-imaging-with-genetically-encoded-sensor-case12-facile-analysis-of-%C3%AE-7-%C3%AE-9-nachr-mutants
#12
Irina Shelukhina, Ekaterina Spirova, Denis Kudryavtsev, Lucy Ojomoko, Markus Werner, Christoph Methfessel, Michael Hollmann, Victor Tsetlin
Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method...
2017: PloS One
https://www.readbyqxmd.com/read/28796488/direct-interaction-of-chivosazole-f-with-actin-elicits-cell-responses-similar-to-latrunculin-a-but-distinct-from-chondramide
#13
Ireos Filipuzzi, Jason Ray Thomas, Verena Pries, David Estoppey, Michael Salcius, Christian Studer, Markus Schirle, Dominic Hoepfner
The microbial metabolite Chivosazole F has been described to affect the cytoskeleton and to inhibit actin polymerization in vitro. Applying orthogonal genomic and proteomics approaches, we now show for the first time that Chivosazole F exerts its effect by directly interacting with actin and demonstrate the cellular impact of Chivosazole F in an unbiased, genome-wide context in yeast and in mammalian cells. Furthermore, mutation-based resistance mapping identifies two SNPs located in the putative Chivosazole F binding site of actin...
August 15, 2017: ACS Chemical Biology
https://www.readbyqxmd.com/read/28794039/the-ul21-tegument-protein-of-herpes-simplex-virus-type-1-is-differentially-required-for-the-syncytial-phenotype
#14
Akua Sarfo, Jason Starkey, Erica Mellinger, Dan Zhang, Pooja Chadha, Jillian Carmichael, John W Wills
The initial goal of this study was to re-examine the requirement of UL21 for herpes simplex virus 1 (HSV-1) replication. Previous studies suggested that UL21 is dispensable for replication in cell cultures, but a recent report on HSV-2 challenges those findings. As was done for the HSV-2 study, a UL21-null virus was made and propagated on complementing cells to discourage selection of compensating mutations. This HSV-1 mutant was able to replicate in non-complementing cells, even at a low MOI, though a reduction in titer was observed...
August 9, 2017: Journal of Virology
https://www.readbyqxmd.com/read/28782937/regulation-of-focal-adhesion-kinase-through-a-direct-interaction-with-an-endogenous-inhibitor
#15
Taylor J Zak, Yevgenia E Koshman, Allen M Samarel, Seth L Robia
Focal adhesion kinase (FAK) plays a key role in integrin and growth factor signaling pathways. FAK related non-kinase (FRNK) is an endogenous inhibitor of FAK that shares its primary structure with the C-terminal third of FAK. FAK S910 phosphorylation is known to regulate FAK protein-protein interactions, but the role of the equivalent site on FRNK (S217) is unknown. Here we determined that S217 is highly phosphorylated by ERK in cultured rat aortic smooth muscle cells. Blocking phosphorylation by mutation (S217A) greatly increased FRNK inhibitory potency, resulting in strong inhibition of FAK autophosphorylation at Y397 and induction of smooth muscle cell apoptosis...
August 7, 2017: Biochemistry
https://www.readbyqxmd.com/read/28781081/specific-interaction-of-the-human-mitochondrial-uncoupling-protein-1-with-free-long-chain-fatty-acid
#16
Linlin Zhao, Shuqing Wang, Qianli Zhu, Bin Wu, Zhijun Liu, Bo OuYang, James J Chou
The mitochondrial uncoupling protein 1 (UCP1) generates heat by causing proton leak across the mitochondrial inner membrane that requires fatty acid (FA). The mechanism by which UCP1 uses FA to conduct proton remains unsolved, and it is also unclear whether a direct physical interaction between UCP1 and FA exists. Here, we have shown using nuclear magnetic resonance that FA can directly bind UCP1 at a helix-helix interface site composed of residues from the transmembrane helices H1 and H6. According to the paramagnetic relaxation enhancement data and molecular dynamics simulation, the FA acyl chain appears to fit into the groove between H1 and H6 while the FA carboxylate group interacts with the basic residues near the matrix side of UCP1...
July 19, 2017: Structure
https://www.readbyqxmd.com/read/28780871/identification-and-functional-characterization-of-the-glycogen-synthesis-related-gene-glycogenin-in-pacific-oysters-crassostrea-gigas
#17
Busu Li, Jie Meng, Li Li, Sheng Liu, Ting Wang, Guofan Zhang
High glycogen levels in the Pacific oyster (Crassostrea gigas) contribute to its flavor, quality, and hardiness. Glycogenin (CgGN) is the priming glucosyltransferase that initiates glycogen biosynthesis. We characterized the full sequence and function of C. gigas CgGN. Three CgGN isoforms (CgGN-α, β, and γ) containing alternative exon regions were isolated. CgGN expression varied seasonally in the adductor muscle and gonadal area and was the highest in the adductor muscle. Autoglycosylation of CgGN can interact with glycogen synthase (CgGS) to complete glycogen synthesis...
August 7, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28776993/improving-the-catalytic-behavior-of-dfa-i-forming-inulin-fructotransferase-from-streptomyces-davawensis-with-site-directed-mutagenesis
#18
Shuhuai Yu, Yanmin Zhang, Yingying Zhu, Tao Zhang, Bo Jiang, Wanmeng Mu
Previously, a α-D-fructofuranose-β-D-fructofuranose 1,2':2,1'-dianhydride (DFA I)-forming inulin fructotransferase (IFTase), namely SdIFTase, was identified. The enzyme doesn't show high performances. In this work, to improve catalytic behavior including activity and thermostability, the enzyme was modified using site-directed mutagenesis on the basis of structure. The mutated residues were divided into three groups. Those in group I are located at central tunnel including G236, A257, G281, T313, and A314S...
August 4, 2017: Journal of Agricultural and Food Chemistry
https://www.readbyqxmd.com/read/28768733/central-catalytic-domain-of-brap-rnf52-recognizes-the-types-of-ubiquitin-chains-and-utilizes-oligo-ubiquitin-for-ubiquitylation
#19
Shisako Shoji, Kazuharu Hanada, Noboru Ohsawa, Mikako Shirouzu
RING-finger protein 52 (RNF52), an E3 ubiquitin ligase, is found in eukaryotes from yeast to humans. Human RNF52 is known as breast cancer type 1 susceptibility protein-associated protein 2 (BRAP or BRAP2). The central catalytic domain of BRAP comprises four subdomains: nucleotide-binding α/β plait (NBP), really interesting new gene zinc finger (RING), deubiquitinase-like zinc finger (ZfUBP), and coiled-coil (CC). This domain architecture is conserved in RNF52 orthologs; however, the domain's function in the ubiquitin system has not been delineated...
August 2, 2017: Biochemical Journal
https://www.readbyqxmd.com/read/28768481/piil-visualization-of-dna-methylation-and-gene-expression-data-in-gene-pathways
#20
Behrooz Torabi Moghadam, Neda Zamani, Jan Komorowski, Manfred Grabherr
BACKGROUND: DNA methylation is a major mechanism involved in the epigenetic state of a cell. It has been observed that the methylation status of certain CpG sites close to or within a gene can directly affect its expression, either by silencing or, in some cases, up-regulating transcription. However, a vertebrate genome contains millions of CpG sites, all of which are potential targets for methylation, and the specific effects of most sites have not been characterized to date. To study the complex interplay between methylation status, cellular programs, and the resulting phenotypes, we present PiiL, an interactive gene expression pathway browser, facilitating analyses through an integrated view of methylation and expression on multiple levels...
August 2, 2017: BMC Genomics
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