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https://www.readbyqxmd.com/read/28813668/subnuclear-relocalization-of-structure-specific-endonucleases-in-response-to-dna-damage
#1
Irene Saugar, Alberto Jiménez-Martín, José Antonio Tercero
Structure-specific endonucleases contribute to the maintenance of genome integrity by cleaving DNA intermediates that need to be resolved for faithful DNA repair, replication, or recombination. Despite advances in the understanding of their function and regulation, it is less clear how these proteins respond to genotoxic stress. Here, we show that the structure-specific endonuclease Mus81-Mms4/EME1 relocalizes to subnuclear foci following DNA damage and colocalizes with the endonucleases Rad1-Rad10 (XPF-ERCC1) and Slx1-Slx4...
August 15, 2017: Cell Reports
https://www.readbyqxmd.com/read/28813413/polylox-barcoding-reveals-haematopoietic-stem-cell-fates-realized-in-vivo
#2
Weike Pei, Thorsten B Feyerabend, Jens Rössler, Xi Wang, Daniel Postrach, Katrin Busch, Immanuel Rode, Kay Klapproth, Nikolaus Dietlein, Claudia Quedenau, Wei Chen, Sascha Sauer, Stephan Wolf, Thomas Höfer, Hans-Reimer Rodewald
Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites, viral barcodes, and strategies based on transposons and CRISPR-Cas9 genome editing; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system...
August 16, 2017: Nature
https://www.readbyqxmd.com/read/28811350/defining-the-dna-binding-site-recognized-by-the-fission-yeast-zn2cys6-transcription-factor-pho7-and-its-role-in-phosphate-homeostasis
#3
Beate Schwer, Ana M Sanchez, Angad Garg, Debashree Chatterjee, Stewart Shuman
Fission yeast phosphate homeostasis entails transcriptional induction of genes encoding phosphate-mobilizing proteins under conditions of phosphate starvation. Transcription factor Pho7, a member of the Zn2Cys6 family of fungal transcription regulators, is the central player in the starvation response. The DNA binding sites in the promoters of phosphate-responsive genes have not been defined, nor have any structure-function relationships been established for the Pho7 protein. Here we narrow this knowledge gap by (i) delineating an autonomous DNA-binding domain (DBD) within Pho7 that includes the Zn2Cys6 module, (ii) deploying recombinant Pho7 DBD in DNase I footprinting and electrophoretic mobility shift assays (EMSAs) to map the Pho7 recognition sites in the promoters of the phosphate-regulated pho1 and tgp1 genes to a 12-nucleotide sequence motif [5'-TCG(G/C)(A/T)xxTTxAA], (iii) independently identifying the same motif as a Pho7 recognition element via in silico analysis of available genome-wide ChIP-seq data, (iv) affirming that mutations in the two Pho7 recognition sites in the pho1 promoter efface pho1 expression in vivo, and (v) establishing that the zinc-binding cysteines and a pair of conserved arginines in the DBD are essential for Pho7 activity in vivoIMPORTANCE Fungi respond to phosphate starvation by inducing the transcription of a set of phosphate acquisition genes that comprise a phosphate regulon...
August 15, 2017: MBio
https://www.readbyqxmd.com/read/28809452/neurotoxin-synthesis-is-positively-regulated-by-the-sporulation-transcription-factor-spo0a-in-clostridium-botulinum-type-e
#4
Gerald Mascher, Anna Mertaoja, Hannu Korkeala, Miia Lindström
Clostridium botulinum produces the most potent natural toxin, the botulinum neurotoxin (BoNT), probably to create anaerobiosis and nutrients by killing the host, and forms endospores that facilitate survival in harsh conditions and transmission. Peak BoNT production coincides with initiation of sporulation in C. botulinum cultures, which suggests common regulation. Here we show that Spo0A, the master regulator of sporulation, positively regulates BoNT production. Insertional inactivation of spo0A in C. botulinum type E strain Beluga resulted in significantly reduced BoNT production and in abolished or highly reduced sporulation in relation to wild-type controls...
August 15, 2017: Environmental Microbiology
https://www.readbyqxmd.com/read/28807928/role-of-translesion-synthesis-dna-polymerases-in-dna-replication-in-the-presence-of-a-weak-dna-polymerase-%C3%AE-in-saccharomyces-cerevisiae
#5
Likui Zhang, Yanchao Huang, Xinyuan Zhu, Yuxiao Wang, Haoqiang Shi, Min Chen, Kunming Dong, Xiaojian Zhou
Translesion synthesis (TLS) DNA polymerases (pols) are often mutagenic for lesion bypass due to their low fidelity. Here, we isolated a weak yeast DNA pol δ mutant that possessed amino acid substitution V592G by genetic selection. The pol3-V592G cells were sensitive to hydroxyurea (HU), which increases the requirement for dNTPs, and their HU sensitivity was suppressed by L612M substitution. We also demonstrated that V592G substitution suppressed the phosphonoacetic acid (PAA) sensitivity of pol3-L612M cells, suggesting a cycle of mutual suppression in the HU- and PAA-sensitive yeast DNA pol δ mutants as similarly observed in the optA1- and PAA-sensitive T4 DNA pol mutants...
August 14, 2017: G3: Genes—Genomes—Genetics
https://www.readbyqxmd.com/read/28807293/sarcocystis-neurona-and-neospora-caninum-in-brazilian-opossums-didelphis-spp-molecular-investigation-and-in-vitro-isolation-of-sarcocystis-spp
#6
Leane S Q Gondim, Rogério F Jesus, Müller Ribeiro-Andrade, Jean C R Silva, Daniel B Siqueira, Maria F V Marvulo, Felipe M Aléssio, Jean-François Mauffrey, Fred S Julião, Elisa San Martin Mouriz Savani, Rodrigo M Soares, Luís F P Gondim
Sarcocystis neurona and Neospora spp. are protozoan parasites that induce neurological diseases in horses and other animal species. Opossums (Didelphis albiventris and Didelphis virginiana) are definitive hosts of S. neurona, which is the major cause of equine protozoal myeloencephalitis (EPM). Neospora caninum causes abortion in cattle and infects a wide range of animal species, while N. hughesi is known to induce neurologic disease in equids. The aims of this study were to investigate S. neurona and N. caninum in tissues from opossums in the northeastern Brazil, and to isolate Brazilian strains of Sarcocystis spp...
August 30, 2017: Veterinary Parasitology
https://www.readbyqxmd.com/read/28806522/development-of-a-recombinant-multifunctional-biomacromolecule-for-targeted-gene-transfer-to-prostate-cancer-cells
#7
Arash Hatefi, Zahra Karjoo, Alireza Nomani
The objective of this study was to genetically engineer a fully functional single chain fusion peptide composed of motifs from diverse biological and synthetic origins that can perform multiple tasks including DNA condensation, cell targeting, cell transfection, particle shielding from immune system and effective gene transfer to prostate tumors. To achieve the objective, a single chain biomacromolecule (vector) consisted of four repetative units of histone H2A peptide, fusogenic peptide GALA, short elastin-like peptide and PC-3 cell targeting peptide was designed...
August 14, 2017: Biomacromolecules
https://www.readbyqxmd.com/read/28804739/selection-of-genetically-modified-bacteriophages-using-the-crispr-cas-system
#8
Miriam Manor, Udi Qimron
We present a CRISPR-Cas based technique for deleting genes from the T7 bacteriophage genome. A DNA fragment encoding homologous arms to the target gene to be deleted is first cloned into a plasmid. The T7 phage is then propagated in Escherichia coli harboring this plasmid. During this propagation, some phage genomes undergo homologous recombination with the plasmid, thus deleting the targeted gene. To select for these genomes, the CRISPR-Cas system is used to cleave non-edited genomes, enabling isolation of the desired recombinant phages...
August 5, 2017: Bio-protocol
https://www.readbyqxmd.com/read/28803959/thymosin-%C3%AE-4-is-involved-in-the-antimicrobial-immune-response-of-golden-pompano-trachinotus-ovatus
#9
Yun Sun, Xiaojuan Chen, Yue Xu, Qiaohong Liu, Xue Jiang, Shifeng Wang, Weiliang Guo, Yongcan Zhou
Thymosin beta belongs to the thymosin family, which consists of a series of highly conserved peptides involved in various biological processes. In teleosts, understanding of the immunological functions of thymosin beta is limited, particularly in vivo, which is essentially unknown. In the current study, we cloned and identified thymosin beta 4 from the teleost fish Golden pompano (Trachinotus ovatus), which we have named TroTβ4. We investigated the expression patterns and functions of TroTβ4 in both in vivo and in vitro assays...
August 10, 2017: Fish & Shellfish Immunology
https://www.readbyqxmd.com/read/28803676/ranavirus-phylogenomics-signatures-of-recombination-and-inversions-among-bullfrog-ranaculture-isolates
#10
Sieara C Claytor, Kuttichantran Subramaniam, Nelmarie Landrau-Giovannetti, V Gregory Chinchar, Matthew J Gray, Debra L Miller, Carla Mavian, Marco Salemi, Samantha Wisely, Thomas B Waltzek
Ranaviruses are emerging pathogens of fish, amphibians, and reptiles that threaten aquatic animal industries and wildlife worldwide. Our objective was to genetically characterize ranaviruses isolated during separate bullfrog Lithobates catesbeianus die-offs that occurred eight years apart on the same North American farm. The earlier outbreak was due to a highly pathogenic strain of common midwife toad virus (CMTV) previously known only from Europe and China. The later outbreak was due to a chimeric ranavirus that displayed a novel genome arrangement and a DNA backbone typical for Frog virus 3 (FV3) strains except for interspersed fragments acquired through recombination with the CMTV isolated earlier...
August 10, 2017: Virology
https://www.readbyqxmd.com/read/28803330/bime2-a-novel-gene-required-for-interhomolog-meiotic-recombination-in-the-protist-model-organism-tetrahymena
#11
Anura Shodhan, Maria Novatchkova, Josef Loidl
Meiotic recombination is initiated by DNA double-strand breaks (DSBs). Most DSBs are converted into nonreciprocal exchanges (gene conversions) or crossovers (COs) between sister chromatids. Only a minority of DSBs are processed toward interhomolog COs, the precursors of the chiasmata that connect homologous chromosomes. Dmc1, the meiosis-specific paralog of the universal recombination protein Rad51, is required for interhomolog COs; in its absence, univalents are primarily formed. Here, we report a ciliate-specific novel meiotic gene, BIME2, which also promotes interhomolog crossing over...
August 12, 2017: Chromosome Research
https://www.readbyqxmd.com/read/28802127/investigations-on-the-molecular-mode-of-action-of-the-novel-immunostimulator-zelnate-activation-of-the-cgas-sting-pathway-in-mammalian-cells
#12
Thomas Ilg
Bovine respiratory disease (BRD) is usually prevented or treated with vaccines and/or antibiotics. The use of antibiotics is, however, of concern due to the potential promotion of microbial resistance and the occurrence of residues. Recently an alternative aid in the treatment of BRD, the cationic lipid/bacterial plasmid DNA liposome-based immunomodulator ZelNate, has entered the veterinary market. In the present study, we provide data on the molecular mode of action of ZelNate. Despite the presence of numerous non-methylated CpG motifs in its plasmid DNA, ZelNate proved to be inactive on human and mouse toll-like receptor 9 (TLR9) in cell culture, in both recombinant and natural cellular receptor settings...
August 9, 2017: Molecular Immunology
https://www.readbyqxmd.com/read/28801912/computational-sequence-design-with-r2odna-designer
#13
James T MacDonald, Velia Siciliano
Recently developed DNA assembly methods have enabled the rapid and simultaneous assembly of multiple parts to create complex synthetic gene circuits. A number of groups have proposed the use of computationally designed orthogonal spacer sequences to guide the ordered assembly of parts using overlap-directed or homologous recombination-based methods. This approach is particularly useful for assembling multiple parts with repetitive elements. Orthogonal spacer sequences (sometimes called UNSs-unique nucleotide sequences) also have a number of other potential uses including in the design of synthetic promoters regulated by novel regulatory elements...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28801911/a-modular-approach-to-building-complex-synthetic-circuits
#14
Yinqing Li, Ron Weiss
Efficient assembly of genetic circuits is a critical element in synthetic biology for implementing complex gene expression systems. Here we present an assembly framework that integrates recent developments in DNA recombination technologies and unique nucleotide sequences for modular and reliable construction of complex genetic circuits. All relevant protocols are described in detail. Implementing these protocols relies on readily available reagents and basic molecular biology techniques without the need for specialized equipment...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28801604/recombineering-using-recet-in-corynebacterium-glutamicum-atcc14067-via-a-self-excisable-cassette
#15
Yuanyuan Huang, Lu Li, Shan Xie, Nannan Zhao, Shuangyan Han, Ying Lin, Suiping Zheng
Gene manipulation is essential for metabolic engineering and synthetic biology, but the current general gene manipulation methods are not applicable to the non-model strain Corynebacterium glutamicum (C. glutamicum) ATCC14067, which is used for amino acid production. Here, we report an effective and sequential deletion method for C. glutamicum ATCC14067 using the exonuclease-recombinase pair RecE + RecT (RecET) for recombineering via a designed self-excisable linear double-strand DNA (dsDNA) cassette, which contains the Cre/loxP system, to accomplish markerless deletion...
August 11, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28801534/cgas-is-activated-by-dna-in-a-length-dependent-manner
#16
Stefanie Luecke, Andreas Holleufer, Maria H Christensen, Kasper L Jønsson, Gerardo A Boni, Lambert K Sørensen, Mogens Johannsen, Martin R Jakobsen, Rune Hartmann, Søren R Paludan
Cytosolic DNA stimulates innate immune responses, including type I interferons (IFN), which have antiviral and immunomodulatory activities. Cyclic GMP-AMP synthase (cGAS) recognizes cytoplasmic DNA and signals via STING to induce IFN production. Despite the importance of DNA in innate immunity, the nature of the DNA that stimulates IFN production is not well described. Using low DNA concentrations, we show that dsDNA induces IFN in a length-dependent manner. This is observed over a wide length-span of DNA, ranging from the minimal stimulatory length to several kilobases, and is fully dependent on cGAS irrespective of DNA length...
August 10, 2017: EMBO Reports
https://www.readbyqxmd.com/read/28801461/structural-basis-of-human-prdm9-allele-c-specific-recognition-of-its-cognate-dna-sequence
#17
Anamika Patel, Xing Zhang, Robert M Blumenthal, Xiaodong Cheng
PRDM9 is the only mammalian gene that has been associated with speciation. The PR/SET domain 9 (PRDM9) protein is a major determinant of meiotic recombination hotspots, and acts through sequence-specific DNA binding via its C2H2 zinc finger (ZF) tandem array, which is highly polymorphic within and between species. The most common human variant, PRDM9 allele A (PRDM9a), contains 13 fingers (ZF1-13). Allele C (PRDM9c) is the second-most common among African populations, and differs from PRDM9a by an arginine-to-serine change (R764S) in ZF9, and by replacement of ZF11 with two other fingers, yielding 14 fingers in PRDM9c...
August 11, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28801308/egfr-mutations-compromise-hypoxia-associated-radiation-resistance-through-impaired-replication-fork-associated-dna-damage-repair
#18
Mohammad Saki, Haruhiko Makino, Prashanthi Javvadi, Nozomi Tomimatsu, Lianghao Ding, Jennifer E Clark, Elaine Gavin, Kenichi Takeda, Joel Andrews, Debabrata Saha, Michael D Story, Sandeep Burma, Chaitanya Nirodi
Epidermal growth factor receptor (EGFR) signaling has been implicated in hypoxia-associated resistance to radiation or chemotherapy. Non-small cell lung carcinomas (NSCLC) with activating L858R or ΔE746-E750 EGFR mutations exhibit elevated EGFR activity and downstream signaling. Here, relative to wild type (WT) EGFR, mutant (MT) EGFR expression significantly increases radiosensitivity in hypoxic cells. Gene expression profiling in human bronchial epithelial cells (HBEC) revealed that MT-EGFR expression elevated transcripts related to cell cycle and replication in aerobic and hypoxic conditions and down-regulated RAD50, a critical component of non-homologous end-joining (NHEJ) and homologous recombination (HR) DNA repair pathways...
August 11, 2017: Molecular Cancer Research: MCR
https://www.readbyqxmd.com/read/28801067/dna-methylation-in-cho-cells
#19
Anna Wippermann, Thomas Noll
Chinese hamster ovary (CHO) cells account for the production of the majority of biopharmaceutical molecules - however, the molecular basis for their versatile properties is not entirely understood yet and the underlying cellular processes need to be characterized in detail. One such process that is supposed to contribute significantly to CHO cell phenotype is methylation of DNA at cytosine residues. DNA methylation was shown to be involved in several central biological processes in humans and to contribute to diseases like cancer...
August 8, 2017: Journal of Biotechnology
https://www.readbyqxmd.com/read/28800637/biologically-active-recombinant-human-erythropoietin-expressed-in-hairy-root-cultures-and-regenerated-plantlets-of-nicotiana-tabacum-l
#20
Poornima Devi Gurusamy, Holger Schäfer, Siva Ramamoorthy, Michael Wink
Hairy root culture is a potential alternative to conventional mammalian cell culture to produce recombinant proteins due to its ease in protein recovery, low costs and absence of potentially human pathogenic contaminants. The current study focussed to develop a new platform of a hairy root culture system from Nicotiana tabacum for the production of recombinant human EPO (rhEPO), which is regularly produced in mammalian cells. The human EPO construct was amplified with C-terminal hexahistidine tag from a cDNA of Caco-2 cells...
2017: PloS One
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