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Site directed mutations

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https://www.readbyqxmd.com/read/29333573/simultaneous-site-directed-mutagenesis-of-duplicated-loci-in-soybean-using-a-single-guide-rna
#1
Yuhei Kanazashi, Aya Hirose, Ippei Takahashi, Masafumi Mikami, Masaki Endo, Sakiko Hirose, Seiichi Toki, Akito Kaga, Ken Naito, Masao Ishimoto, Jun Abe, Tetsuya Yamada
Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD)...
January 15, 2018: Plant Cell Reports
https://www.readbyqxmd.com/read/29332168/efficient-crispr-cas9-based-genome-editing-in-carrot-cells
#2
Magdalena Klimek-Chodacka, Tomasz Oleszkiewicz, Levi G Lowder, Yiping Qi, Rafal Baranski
The first report presenting successful and efficient carrot genome editing using CRISPR/Cas9 system. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas9) is a powerful genome editing tool that has been widely adopted in model organisms recently, but has not been used in carrot-a model species for in vitro culture studies and an important health-promoting crop grown worldwide. In this study, for the first time, we report application of the CRISPR/Cas9 system for efficient targeted mutagenesis of the carrot genome...
January 13, 2018: Plant Cell Reports
https://www.readbyqxmd.com/read/29330717/posterior-retroperitoneoscopic-resection-of-extra-adrenal-paraganglioma-located-in-the-aorto-caval-space
#3
Sang-Wook Kang, Emad Kandil, Min Jhi Kim, Kwang Soon Kim, Cho Rok Lee, Jong Ju Jeong, Kee-Hyun Nam, Woong Youn Chung, Cheong Soo Park
BACKGROUND: The posterior retroperitoneoscopic adrenalec tomy has several advantages compared with the transperitoneal approach such as a shorter and more direct route to the target organ, no breach of the intraperitoneal space, and no required retraction of the adjacent organs. It also is a safe procedure with a short learning curve.1-5 This report presents a challenging case of an extra-adrenal paraganglioma located in the aorto-caval space and managed using the retroperitoneal approach...
January 12, 2018: Annals of Surgical Oncology
https://www.readbyqxmd.com/read/29329928/cloning-and-characterization-of-%C3%A2-6-%C3%A2-5-fatty-acyl-desaturase-fad-gene-promoter-in-the-marine-teleost-siganus-canaliculatus
#4
Yewei Dong, Jianhong Zhao, Junliang Chen, Shuqi Wang, Yang Liu, Qinghao Zhang, Cuihong You, Óscar Monroig, Douglas R Tocher, Yuanyou Li
The rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the ability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 PUFA precursors, and all genes encoding the key enzymes for LC-PUFA biosynthesis have been cloned and functionally characterized, which provides us a potential model to study the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. As the primary step to clarify such mechanisms, present research focused on promoter analysis of gene encoding ∆6/∆5 fatty acyl desaturase (Fad), a rate-limiting enzyme catalyzing the first step in the conversion of C18 PUFA to LC-PUFA...
January 9, 2018: Gene
https://www.readbyqxmd.com/read/29326159/an-intrinsic-lipid-binding-interface-controls-sphingosine-kinase-1-function
#5
Michael J Pulkoski-Gross, Meredith L Jenkins, Jean-Philip Truman, Mohamed F Salama, Christopher J Clarke, John E Burke, Yusuf A Hannun, Lina M Obeid
Sphingosine kinase 1 (SK1) is a key enzyme which regulates a multitude of cellular processes including cellular growth, immune cell trafficking, and inflammation via the production of sphingosine-1-phosphate (S1P). To produce S1P, SK1 must access sphingosine directly from membranes. Despite the need for membrane access, the molecular mechanisms underlying SK1s direct membrane interaction remain unclear. Here, we identify a novel positively charged site on SK1 which is responsible for electrostatic interactions with membranes...
January 11, 2018: Journal of Lipid Research
https://www.readbyqxmd.com/read/29325769/the-structural-determinants-of-the-bitopic-binding-mode-of-a-negative-allosteric-modulator-of-the-dopamine-d2-receptor
#6
Christopher J Draper-Joyce, Mayako Michino, Ravi Kumar Verma, Carmen Klein Herenbrink, Jeremy Shonberg, Anitha Kopinathan, Peter J Scammells, Ben Capuano, David M Thal, Jonathan A Javitch, Arthur Christopoulos, Lei Shi, J Robert Lane
SB269652 is a negative allosteric modulator of the dopamine D2 receptor (D2R) yet possesses structural similarity to ligands with a competitive mode of interaction. In this study, we aimed to understand the ligand-receptor interactions that confer its allosteric action. We combined site-directed mutagenesis with molecular dynamics simulations using both SB269652 and derivatives from our previous structure activity studies. We identify residues within the conserved orthosteric binding site (OBS) and a secondary binding pocket (SBP) that determine affinity and cooperativity...
January 8, 2018: Biochemical Pharmacology
https://www.readbyqxmd.com/read/29325723/double-function-hydroperoxide-lyases-epoxyalcohol-synthases-cyp74c-of-higher-plants-identification-and-conversion-into-allene-oxide-synthases-by-site-directed-mutagenesis
#7
Yana Y Toporkova, Svetlana S Gorina, Elena K Bessolitsyna, Elena O Smirnova, Valeria S Fatykhova, Fredi Brühlmann, Tatiana M Ilyina, Lucia S Mukhtarova, Alexander N Grechkin
The CYP74C subfamily of fatty acid hydroperoxide transforming enzymes includes hydroperoxide lyases (HPLs) and allene oxide synthases (AOSs). This work reports a new facet of the putative CYP74C HPLs. Initially, we found that the recombinant CYP74C13_MT (Medicago truncatula) behaved predominantly as the epoxyalcohol synthase (EAS) towards the 9(SS)-hydroperoxide of linoleic acid. At the same time, the CYP74C13_MT mostly possessed the HPL activity towards the 13(S)-hydroperoxides of linoleic and α-linolenic acids...
January 8, 2018: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/29323272/dominant-negative-smarca4-mutants-alter-the-accessibility-landscape-of-tissue-unrestricted-enhancers
#8
H Courtney Hodges, Benjamin Z Stanton, Katerina Cermakova, Chiung-Ying Chang, Erik L Miller, Jacob G Kirkland, Wai Lim Ku, Vaclav Veverka, Keji Zhao, Gerald R Crabtree
Mutation of SMARCA4 (BRG1), the ATPase of BAF (mSWI/SNF) and PBAF complexes, contributes to a range of malignancies and neurologic disorders. Unfortunately, the effects of SMARCA4 missense mutations have remained uncertain. Here we show that SMARCA4 cancer missense mutations target conserved ATPase surfaces and disrupt the mechanochemical cycle of remodeling. We find that heterozygous expression of mutants alters the open chromatin landscape at thousands of sites across the genome. Loss of DNA accessibility does not directly overlap with Polycomb accumulation, but is enriched in 'A compartments' at active enhancers, which lose H3K27ac but not H3K4me1...
January 2018: Nature Structural & Molecular Biology
https://www.readbyqxmd.com/read/29322650/molecular-characterization-of-the-%C3%AE-amyloid-4-10-epitope-of-plaque-specific-a%C3%AE-antibodies-by-affinity-mass-spectrometry-using-alanine-site-mutation
#9
Raluca Ștefănescu, Loredana Lupu, Marilena Manea, Roxana E Iacob, Michael Przybylski
Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the β-amyloid(1-42) (Aβ) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aβ-specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aβ antibodies contributes to the elucidation of their mechanism of action in the human organism...
January 2018: Journal of Peptide Science: An Official Publication of the European Peptide Society
https://www.readbyqxmd.com/read/29321962/mutual-influence-of-secondary-and-key-drug-resistance-mutations-on-catalytic-properties-and-thermal-stability-of-tem-type-%C3%AE-lactamases
#10
Vitaly Grigorenko, Igor Uporov, Maya Rubtsova, Irina Andreeva, Dmitrii Shcherbinin, Alexander Veselovsky, Oksana Serova, Maria Ulyashova, Igor Ishtubaev, Alexey Egorov
Highly mutable β-lactamases are responsible for the ability of Gram-negative bacteria to resist β-lactam antibiotics. Using site-directed mutagenesis technique, we have produced in vitro a number of recombinant analogs of naturally occurring TEM-type β-lactamases, bearing the secondary substitution Q39K and key mutations related to the extended-spectrum (E104K, R164S) and inhibitor-resistant (M69V) β-lactamases. The mutation Q39K alone was found to be neutral and hardly affected the catalytic properties of β-lactamases...
January 2018: FEBS Open Bio
https://www.readbyqxmd.com/read/29318463/a-novel-splice-site-mutation-of-the-prkar1a-gene-c-440-5-g-c-in-a-chinese-family-with-carney-complex
#11
J Fu, F Lai, Y Chen, X Wan, G Wei, Y Li, H Xiao, X Cao
BACKGROUND: Carney complex (CNC) is an extremely rare, multiple endocrine neoplasia syndrome that occurs in an autosomal dominant manner. Mutations in PRKAR1A have been reported to be a common genetic cause of CNC. METHODS: In this study, we reported a Chinese pedigree of CNC that manifests mainly as spotty skin pigmentation and primary pigmented nodular adrenocortical disease. Whole blood samples of this pedigree were collected for DNA/RNA analysis. Polymerase chain reaction (PCR) and reverse-transcription polymerase chain reaction analyses were performed to amplify the 11 exons and adjacent introns of PRKAR1A...
January 9, 2018: Journal of Endocrinological Investigation
https://www.readbyqxmd.com/read/29316444/force-generation-via-%C3%AE-cardiac-myosin-titin-and-%C3%AE-actinin-drives-cardiac-sarcomere-assembly-from-cell-matrix-adhesions
#12
Anant Chopra, Matthew L Kutys, Kehan Zhang, William J Polacheck, Calvin C Sheng, Rebeccah J Luu, Jeroen Eyckmans, J Travis Hinson, Jonathan G Seidman, Christine E Seidman, Christopher S Chen
Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation...
January 8, 2018: Developmental Cell
https://www.readbyqxmd.com/read/29313669/design-of-small-molecules-that-compete-with-nucleotide-binding-to-an-engineered-oncogenic-kras-allele
#13
Yan Zhang, Mare-Helene Larraufie, Leila Musavi, Hemanth Akkiraju, Lewis M Brown, Brent R Stockwell
RAS mutations are found in 30% of all human cancers, with KRAS the most frequently mutated among the three RAS isoforms (KRAS, NRAS, HRAS). However, directly targeting oncogenic KRAS with small molecules in the nucleotide-binding site has been difficult due to the high affinity of KRAS for GDP and GTP. We designed an engineered allele of KRAS, and a covalent inhibitor that competes for GTP and GDP. This ligand-receptor combination demonstrates that the high affinity of GTP/GDP for RAS proteins can be overcome with a covalent inhibitor and a suitably engineered binding site...
January 9, 2018: Biochemistry
https://www.readbyqxmd.com/read/29305491/local-sequence-features-that-influence-ap-1-cis-regulatory-activity
#14
Hemangi G Chaudhari, Barak A Cohen
In the genome, most occurrences of transcription factor binding sites (TFBS) have no cis-regulatory activity, which suggests that flanking sequences contain information that distinguishes functional from non-functional TFBS. We interrogated the role of flanking sequences near Activator Protein 1 (AP-1) binding sites that reside in DNase I Hypersensitive Sites (DHS) and regions annotated as Enhancers. In these regions we found that sequence features directly adjacent to the core motif distinguish high from low activity AP-1 sites...
January 5, 2018: Genome Research
https://www.readbyqxmd.com/read/29305214/expression-of-cry1ac-toxin-binding-region-in-plutella-xyllostella-cadherin-like-receptor-and-studying-their-interaction-mode-by-molecular-docking-and-site-directed-mutagenesis
#15
Xiaodan Hu, Xiao Zhang, Jianfeng Zhong, Yuan Liu, Cunzheng Zhang, Yajing Xie, Manman Lin, Chongxin Xu, Lina Lu, Qing Zhu, Xianjin Liu
Cadherin-like protein has been identified as the primary Bacillus thuringiensis (Bt) Cry toxin receptor in Lepidoptera pests and plays a key role in Cry toxin insecticidal. In this study, we successfully expressed the putative Cry1Ac toxin-binding region (CR7-CR11) of Plutella xylostella cadherin-like in Escherichia coli BL21 (DE3). The expressed CR7-CR11 fragment showed binding ability to Cry1Ac toxin under denaturing (Ligand blot) and non-denaturing (ELISA) conditions. The three-dimensional structure of CR7-CR11 was constructed by homology modeling...
January 2, 2018: International Journal of Biological Macromolecules
https://www.readbyqxmd.com/read/29304333/tyrosine-1-of-rna-polymerase-ii-ctd-controls-global-termination-of-gene-transcription-in-mammals
#16
Nilay Shah, Muhammad Ahmad Maqbool, Yousra Yahia, Amal Zine El Aabidine, Cyril Esnault, Ignasi Forné, Tim-Michael Decker, David Martin, Roland Schüller, Stefan Krebs, Helmut Blum, Axel Imhof, Dirk Eick, Jean-Christophe Andrau
The carboxy-terminal domain (CTD) of RNA polymerase (Pol) II is composed of a repetition of YSPTSPS heptads and functions as a loading platform for protein complexes that regulate transcription, splicing, and maturation of RNAs. Here, we studied mammalian CTD mutants to analyze the function of tyrosine1 residues in the transcription cycle. Mutation of 3/4 of the tyrosine residues (YFFF mutant) resulted in a massive read-through transcription phenotype in the antisense direction of promoters as well as in the 3' direction several hundred kilobases downstream of genes...
January 4, 2018: Molecular Cell
https://www.readbyqxmd.com/read/29302050/demographic-analysis-of-cyanobacteria-based-on-the-mutation-rates-estimated-from-an-ancient-ice-core
#17
Takahiro Segawa, Nozomu Takeuchi, Koji Fujita, Vladimir B Aizen, Eske Willerslev, Takahiro Yonezawa
Despite the crucial role of cyanobacteria in various ecosystems, little is known about their evolutionary histories, especially microevolutionary dynamics, because of the lack of knowledge regarding their mutation rates. Here we directly estimated cyanobacterial mutation rates based on ancient DNA analyses of ice core samples collected from Kyrgyz Republic that dates back to ~12,500 cal years before present. We successfully sequenced the 16S rRNA and 16S-23S internal transcribed spacer (ITS) region. Two cyanobacterial operational taxonomic units (OTUs) were detected from the ancient ice core samples, and these OTUs are shared with those from the modern glacier surface...
January 5, 2018: Heredity
https://www.readbyqxmd.com/read/29302027/kshv-episomes-reveal-dynamic-chromatin-loop-formation-with-domain-specific-gene-regulation
#18
Mel Campbell, Tadashi Watanabe, Kazushi Nakano, Ryan R Davis, Yuanzhi Lyu, Clifford G Tepper, Blythe Durbin-Johnson, Masahiro Fujimuro, Yoshihiro Izumiya
The three-dimensional structure of chromatin organized by genomic loops facilitates RNA polymerase II access to distal promoters. The Kaposi's sarcoma-associated herpesvirus (KSHV) lytic transcriptional program is initiated by a single viral transactivator, K-Rta. Here we report the KSHV genomic structure and its relationship with K-Rta recruitment sites using Capture Hi-C analyses. High-resolution 3D viral genomic maps identify a number of direct physical, long-range, and dynamic genomic interactions. Mutant KSHV chromosomes harboring point mutations in the K-Rta responsive elements (RE) significantly attenuate not only the directly proximate downstream gene, but also distal gene expression in a domain-specific manner...
January 4, 2018: Nature Communications
https://www.readbyqxmd.com/read/29301984/stromal-cell-derived-factor-1-cxcl12-activates-integrins-by-direct-binding-to-an-allosteric-ligand-binding-site-site-2-of-integrins-without-cxcr4
#19
Masaaki Fujita, Parastoo Davari, Yoko K Takada, Yoshikazu Takada
Leukocyte arrest on the endothelial cell surface during leukocyte extravasation is induced by rapid integrin activation by chemokines. We recently reported that fractalkine induces integrin activation without its receptor CX3CR1 through binding to the allosteric site (site 2) of integrins. Peptides from site 2 bound to fractalkine and suppressed integrin activation by fractalkine. We hypothesized that this is not limited to membrane-bound fractalkine. We studied ifstromal cell-derived factor 1(SDF1), another chemokine that plays a critical role in leukocyte arrest, activates integrins through binding to site 2...
January 4, 2018: Biochemical Journal
https://www.readbyqxmd.com/read/29291138/shuffling-active-site-substate-populations-affects-catalytic-activity-the-case-of-glucose-oxidase
#20
Dušan Petrović, David Frank, Shina Caroline Lynn Kamerlin, Kurt Hoffmann, Birgit Strodel
Glucose oxidase has wide applications in the pharmaceutical, chemical, and food industries. Many recent studies have enhanced key properties of this enzyme using directed evolution, yet without being able to reveal why these mutations are actually beneficial. This work presents a synergistic combination of experimental and computational methods, indicating how mutations, even when distant from the active site, positively affect glucose oxidase catalysis. We have determined the crystal structures of glucose oxidase mutants containing molecular oxygen in the active site...
September 1, 2017: ACS Catalysis
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