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recombinant DNA technology

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https://www.readbyqxmd.com/read/29159422/fine-mapping-of-qtls-for-individual-and-total-isoflavone-content-in-soybean-glycine-max-l-using-a-high-density-genetic-map
#1
Zhandong Cai, Yanbo Cheng, Zhuwen Ma, Xinguo Liu, Qibin Ma, Qiuju Xia, Gengyun Zhang, Yinghui Mu, Hai Nian
Fifteen stable QTLs were identified using a high-density soybean genetic map across multiple environments. One major QTL, qIF5-1, contributing to total isoflavone content explained phenotypic variance 49.38, 43.27, 46.59, 45.15 and 52.50%, respectively. Soybeans (Glycine max L.) are a major source of dietary isoflavones. To identify novel quantitative trait loci (QTL) underlying isoflavone content, and to improve the accuracy of marker-assisted breeding in soybean, a valuable mapping population comprised of 196 F7:8-10 recombinant inbred lines (RILs, Huachun 2 × Wayao) was utilized to evaluate individual and total isoflavone content in plants grown in four different environments in Guangdong...
November 20, 2017: TAG. Theoretical and Applied Genetics. Theoretische und Angewandte Genetik
https://www.readbyqxmd.com/read/29153834/precise-editing-at-dna-replication-forks-enables-multiplex-genome-engineering-in-eukaryotes
#2
Edward M Barbieri, Paul Muir, Benjamin O Akhuetie-Oni, Christopher M Yellman, Farren J Isaacs
We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with an efficiency of >40%, without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation...
November 15, 2017: Cell
https://www.readbyqxmd.com/read/29145601/identification-and-functional-analysis-of-potential-prophage-derived-recombinases-for-genome-editing-in-lactobacillus-casei
#3
Yongping Xin, Tingting Guo, Yingli Mu, Jian Kong
Numerous LAB bacteriophage genomes have been sequenced, while the functional genes are yet to be exploited. In this study, a λ Red-like recombinase operon LCABL_13040-50-60 was identified from a prophage PLE3 in Lactobacillus casei BL23 genome, and its recombination function was confirmed by replacement of a 167 bp galK fragment with chloramphenicol-resistant gene (cat) in the L. casei BL23 genome. Further functional analysis showed that LCABL_13040 and LCABL_13060 were analogs to the host nuclease inhibitor (Redγ) and 5'-3' exonuclease (Redα/RecE), respectively...
November 14, 2017: FEMS Microbiology Letters
https://www.readbyqxmd.com/read/29136861/quality-by-design-approach-in-the-development-of-an-ultra-high-performance-liquid-chromatography-method-for-bexsero-meningococcal-group-b-vaccine
#4
Luca Nompari, Serena Orlandini, Benedetta Pasquini, Cristiana Campa, Michele Rovini, Massimo Del Bubba, Sandra Furlanetto
Bexsero is the first approved vaccine for active immunization of individuals from 2 months of age and older to prevent invasive disease caused by Neisseria meningitidis serogroup B. The active components of the vaccine are Neisseria Heparin Binding Antigen, factor H binding protein, Neisseria adhesin A, produced in Escherichia coli cells by recombinant DNA technology, and Outer Membrane Vesicles (expressing Porin A and Porin B), produced by fermentation of Neisseria meningitidis strain NZ98/254. All the Bexsero active components are adsorbed on aluminum hydroxide and the unadsorbed antigens content is a product critical quality attribute...
February 1, 2018: Talanta
https://www.readbyqxmd.com/read/29116512/magnetic-nanoparticle-based-semi-automated-panning-for-high-throughput-antibody-selection
#5
Angela Chiew Wen Ch'ng, Nurul Hamizah Binti Hamidon, Zoltán Konthur, Theam Soon Lim
The application of recombinant human antibodies is growing rapidly mainly in the field of diagnostics and therapeutics. To identify antibodies against a specific antigen, panning selection is carried out using different display technologies. Phage display technology remains the preferred platform due to its robustness and efficiency in biopanning experiments. There are both manual and semi-automated panning selections using polystyrene plastic, magnetic beads, and nitrocellulose as the immobilizing solid surface...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29114024/mechanisms-of-surface-antigenic-variation-in-the-human-pathogenic-fungus-pneumocystis-jirovecii
#6
Emanuel Schmid-Siegert, Sophie Richard, Amanda Luraschi, Konrad Mühlethaler, Marco Pagni, Philippe M Hauser
Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes...
November 7, 2017: MBio
https://www.readbyqxmd.com/read/29070887/new-reverse-genetics-and-transfection-methods-to-rescue-arboviruses-in-mosquito-cells
#7
Thérèse Atieh, Antoine Nougairède, Raphaëlle Klitting, Fabien Aubry, Anna-Bella Failloux, Xavier de Lamballerie, Stéphane Priet
Reverse genetics is a critical tool to decrypt the biological properties of arboviruses. However, whilst reverse genetics methods have been usually applied to vertebrate cells, their use in insect cells remains uncommon due to the conjunction of laborious molecular biology techniques and of specific difficulties surrounding the transfection of such cells. To leverage reverse genetics studies in both vertebrate and mosquito cells, we designed an improved DNA transfection protocol for insect cells and then demonstrated that the simple and flexible ISA (Infectious Subgenomic Amplicons) reverse-genetics method can be efficiently applied to both mammalian and mosquito cells to generate in days recombinant infectious positive-stranded RNA viruses belonging to genera Flavivirus (Japanese encephalitis, Yellow fever, West Nile and Zika viruses) and Alphavirus (Chikungunya virus)...
October 25, 2017: Scientific Reports
https://www.readbyqxmd.com/read/29060747/motifmark-finding-regulatory-motifs-in-dna-sequences
#8
Hamid Reza Hassanzadeh, Pushkar Kolhe, Charles L Isbell, May D Wang
The interaction between proteins and DNA is a key driving force in a significant number of biological processes such as transcriptional regulation, repair, recombination, splicing, and DNA modification. The identification of DNA-binding sites and the specificity of target proteins in binding to these regions are two important steps in understanding the mechanisms of these biological activities. A number of high-throughput technologies have recently emerged that try to quantify the affinity between proteins and DNA motifs...
July 2017: Conference Proceedings: Annual International Conference of the IEEE Engineering in Medicine and Biology Society
https://www.readbyqxmd.com/read/29058367/crispr-cas9-based-counterselection-boosts-recombineering-efficiency-in-pseudomonas-putida
#9
Tomás Aparicio, Víctor de Lorenzo, Esteban Martínez-García
While adoption of single-stranded (ss) DNA recombineering techniques has greatly eased genetic design of the platform strain Pseudomonas putida KT2440, available methods still produce the desired modifications/deletions at low frequencies. This makes isolation of mutants that do not display selectable or conspicuous phenotypes considerably difficult. To overcome this limitation, we have merged ssDNA recombineering with CRISPR/Cas9 technology in this bacterium for efficient killing of unmodified cells and thus non-phenotypic selection of bacteria bearing the mutations of interest...
October 23, 2017: Biotechnology Journal
https://www.readbyqxmd.com/read/29050667/toward%C3%A2-genome-based-metabolic-engineering-in-bacteria
#10
Sabine Oesterle, Irene Wuethrich, Sven Panke
Prokaryotes modified stably on the genome are of great importance for production of fine and commodity chemicals. Traditional methods for genome engineering have long suffered from imprecision and low efficiencies, making construction of suitable high-producer strains laborious. Here, we review the recent advances in discovery and refinement of molecular precision engineering tools for genome-based metabolic engineering in bacteria for chemical production, with focus on the λ-Red recombineering and the clustered regularly interspaced short palindromic repeats/Cas9 nuclease systems...
2017: Advances in Applied Microbiology
https://www.readbyqxmd.com/read/29046683/the-diversity-of-sequence-and-chromosomal-distribution-of-new-transposable-element-related-segments-in-the-rye-genome-revealed-by-fish-and-lineage-annotation
#11
Yingxin Zhang, Chengming Fan, Shuangshuang Li, Yuhong Chen, Richard R-C Wang, Xiangqi Zhang, Fangpu Han, Zanmin Hu
Transposable elements (TEs) in plant genomes exhibit a great variety of structure, sequence content and copy number, making them important drivers for species diversity and genome evolution. Even though a genome-wide statistic summary of TEs in rye has been obtained using high-throughput DNA sequencing technology, the accurate diversity of TEs in rye, as well as their chromosomal distribution and evolution, remains elusive due to the repetitive sequence assembling problems and the high dynamic and nested nature of TEs...
2017: Frontiers in Plant Science
https://www.readbyqxmd.com/read/29034209/bridge-induced-translocation-between-nup145-and-top2-yeast-genes-models-the-genetic-fusion-between-the-human-orthologs-associated-with-acute-myeloid-leukemia
#12
Valentina Tosato, Nicole West, Jan Zrimec, Dmitri V Nikitin, Giannino Del Sal, Roberto Marano, Michael Breitenbach, Carlo V Bruschi
In mammalian organisms liquid tumors such as acute myeloid leukemia (AML) are related to spontaneous chromosomal translocations ensuing in gene fusions. We previously developed a system named bridge-induced translocation (BIT) that allows linking together two different chromosomes exploiting the strong endogenous homologous recombination system of the yeast Saccharomyces cerevisiae. The BIT system generates a heterogeneous population of cells with different aneuploidies and severe aberrant phenotypes reminiscent of a cancerogenic transformation...
2017: Frontiers in Oncology
https://www.readbyqxmd.com/read/29030838/cloning-and-identification-of-recombinant-argonaute-bound-small-rnas-using-next-generation-sequencing
#13
Pooja Gangras, Daniel M Dayeh, Justin W Mabin, Kotaro Nakanishi, Guramrit Singh
Argonaute proteins (AGOs) are loaded with small RNAs as guides to recognize target mRNAs. Since the target specificity heavily depends on the base complementarity between two strands, it is important to identify small guide and long target RNAs bound to AGOs. For this purpose, next-generation sequencing (NGS) technologies have extended our appreciation truly to the nucleotide level. However, the identification of RNAs via NGS from scarce RNA samples remains a challenge. Further, most commercial and published methods are compatible with either small RNAs or long RNAs, but are not equally applicable to both...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29020515/manufacturing-history-of-etanercept-enbrel-%C3%A0-consistency-of-product-quality-through-major-process-revisions
#14
Brian Hassett, Ena Singh, Ehab Mahgoub, Julie O'Brien, Steven M Vicik, Brian Fitzpatrick
Etanercept (ETN) (Enbrel®) is a soluble protein that binds to, and specifically inhibits, tumor necrosis factor (TNF), a proinflammatory cytokine. ETN is synthesized in Chinese hamster ovary cells by recombinant DNA technology as a fusion protein, with a fully human TNFRII ectodomain linked to the Fc portion of human IgG1. Successful manufacture of biologics, such as ETN, requires sophisticated process and product understanding, as well as meticulous control of operations to maintain product consistency. The objective of this evaluation was to show that the product profile of ETN drug substance (DS) has been consistent over the course of production...
October 11, 2017: MAbs
https://www.readbyqxmd.com/read/28994753/genome-wide-analysis-of-hdac-inhibitor-mediated-modulation-of-micrornas-and-mrnas-in-b-cells-induced-to-undergo-class-switch-dna-recombination-and-plasma-cell-differentiation
#15
Helia N Sanchez, Tian Shen, Dawn Garcia, Zhao Lai, Paolo Casali, Hong Zan
Antibody responses are accomplished through several critical B cell-intrinsic processes, including somatic hypermutation (SHM), class-switch DNA recombination (CSR), and plasma cell differentiation. In recent years, epigenetic modifications or factors, such as histone deacetylation and microRNAs (miRNAs), have been shown to interact with B-cell genetic programs to shape antibody responses, while the dysfunction of epigenetic factors has been found to lead to autoantibody responses. Analyzing genome-wide miRNA and mRNA expression in B cells in response to epigenetic modulators is important for understanding the epigenetic regulation of B-cell function and antibody response...
September 20, 2017: Journal of Visualized Experiments: JoVE
https://www.readbyqxmd.com/read/28990505/biological-therapy-of-hematologic-malignancies-toward-a-chemotherapy-free-era
#16
Pavel Klener, Tomas Etrych, Pavel Klener
Less than 70 years ago, the vast majority of hematologic malignancies were untreatable diseases with fatal prognoses. The development of modern chemotherapy agents, which had begun after the Second World War, was markedly accelerated by the discovery of the structure of DNA and its role in cancer biology and tumor cell division. The path travelled from the first temporary remissions observed in children with acute lymphoblastic leukemia treated with single-agent antimetabolites until the first cures achieved by multi-agent chemotherapy regimens was incredibly short...
October 6, 2017: Current Medicinal Chemistry
https://www.readbyqxmd.com/read/28989973/cloning-assembly-and-modification-of-the-primary-human-cytomegalovirus-isolate-toledo-by-yeast-based-transformation-associated-recombination
#17
Sanjay Vashee, Timothy B Stockwell, Nina Alperovich, Evgeniya A Denisova, Daniel G Gibson, Kyle C Cady, Kristofer Miller, Krishna Kannan, Daniel Malouli, Lindsey B Crawford, Alexander A Voorhies, Eric Bruening, Patrizia Caposio, Klaus Früh
Genetic engineering of cytomegalovirus (CMV) currently relies on generating a bacterial artificial chromosome (BAC) by introducing a bacterial origin of replication into the viral genome using in vivo recombination in virally infected tissue culture cells. However, this process is inefficient, results in adaptive mutations, and involves deletion of viral genes to avoid oversized genomes when inserting the BAC cassette. Moreover, BAC technology does not permit the simultaneous manipulation of multiple genome loci and cannot be used to construct synthetic genomes...
September 2017: MSphere
https://www.readbyqxmd.com/read/28979241/crispr-unlock-multipurpose-cas9-based-strategies-for-conversion-of-yeast-libraries-and-strains
#18
Emily Roggenkamp, Rachael M Giersch, Emily Wedeman, Muriel Eaton, Emily Turnquist, Madison N Schrock, Linah Alkotami, Thitikan Jirakittisonthon, Samantha E Schluter-Pascua, Gareth H Bayne, Cory Wasko, Megan Halloran, Gregory C Finnigan
Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome...
2017: Frontiers in Microbiology
https://www.readbyqxmd.com/read/28973384/characterization-of-breakpoint-regions-of-large-structural-autosomal-mosaic-events
#19
Mitchell J Machiela, Lea Jessop, Weiyin Zhou, Meredith Yeager, Stephen J Chanock
Recent studies have reported a higher than anticipated frequency of large clonal autosomal mosaic events >2 Mb in size in the aging population. Mosaic events are detected from analyses of intensity parameters of linear stretches with deviations in heterozygous probes of single nucleotide polymorphism microarrays. The non-random distribution of detected mosaic events throughout the genome suggests common mechanisms could influence the formation of mosaic events. Here we use publicly available data tracks from the University of California Santa Cruz Genome Browser to investigate the genomic characteristics of the regions at the terminal ends of two frequent types of large structural mosaic events: telomeric neutral events and interstitial losses...
November 15, 2017: Human Molecular Genetics
https://www.readbyqxmd.com/read/28964330/effective-purification-of-recombinant-peptide-ligands-for-gpcr-research
#20
Anisha Ashokan, Gopala K Aradhyam
Peptide purification from natural sources and chemical synthesis is cumbersome with various shortcomings such as low yield, high cost of production, error prone, and restricted by nature of amino acids. Though recombinant DNA technology had overcome all these setbacks for larger proteins, it is still a challenge to produce peptides that are salt free and without impurities. Our approach discussed in this chapter deals with easy and effective purification of peptides of varying sizes (up to 10kDa), expressed as fusion proteins in bacterial system...
2017: Methods in Cell Biology
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