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Adeline Feri, Raphaël Loll-Krippleber, Pierre-Henri Commere, Corinne Maufrais, Natacha Sertour, Katja Schwartz, Gavin Sherlock, Marie-Elisabeth Bougnoux, Christophe d'Enfert, Mélanie Legrand
: The diploid genome of the yeast Candida albicans is highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratory C. albicans SC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere...
October 11, 2016: MBio
Zhenyu He, Kehkooi Kee
Gene targeting and editing is an essential tool for both basic research and clinical application such as gene therapy. Several endonucleases have been invented to fulfill these purposes, including zinc finger nucleases, TALEN, and CRISPR/Cas9. Although all of these systems can target DNA sequence with high efficiency, they also exert off-target effects and genotoxicity. The off-target effects might not hinder their usage in animal models because the correctly targeted cells can be selected for further studies...
2017: Methods in Molecular Biology
Giulia Bastianello, Hiroshi Arakawa
All three B cell-specific activities of the immunoglobulin (Ig) gene re-modeling system-gene conversion, somatic hypermutation and class switch recombination-require activation-induced deaminase (AID). AID-induced DNA lesions must be further processed and dissected into different DNA recombination pathways. In order to characterize potential intermediates for Ig gene conversion, we inserted an I-SceI recognition site into the complementarity determining region 1 (CDR1) of the Ig light chain locus of the AID knockout DT40 cell line, and conditionally expressed I-SceI endonuclease...
October 3, 2016: Nucleic Acids Research
Veronika Mladenova, Emil Mladenov, George Iliakis
The adverse biological effects of ionizing radiation (IR) are commonly attributed to the generation of DNA double-strand breaks (DSBs). IR-induced DSBs are generated by clusters of ionizations, bear damaged terminal nucleotides, and frequently comprise base damages and single-strand breaks in the vicinity generating a unique DNA damage-clustering effect that increases DSB "complexity." The number of ionizations in clusters of different radiation modalities increases with increasing linear energy transfer (LET), and is thought to determine the long-known LET-dependence of the relative biological effectiveness (RBE)...
2016: Frontiers in Oncology
K Hoshijima, M J Jurynec, D J Grunwald
Simple and efficient methods are presented for creating precise modifications of the zebrafish genome. Edited alleles are generated by homologous recombination between the host genome and double-stranded DNA (dsDNA) donor molecules, stimulated by the induction of double-strand breaks at targeted loci in the host genome. Because several kilobase-long tracts of sequence can be exchanged, multiple genome modifications can be generated simultaneously at a single locus. Methods are described for creating: (1) alleles with simple sequence changes or in-frame additions, (2) knockin/knockout alleles that express a reporter protein from an endogenous locus, and (3) conditional alleles in which exons are flanked by recombinogenic loxP sites...
2016: Methods in Cell Biology
Jesús Prieto, Pilar Redondo, Nekane Merino, Maider Villate, Guillermo Montoya, Francisco J Blanco, Rafael Molina
Homing endonucleases are highly specific DNA-cleaving enzymes that recognize and cleave long stretches of DNA. The engineering of these enzymes provides instruments for genome modification in a wide range of fields, including gene targeting. The homing endonuclease I-SceI from the yeast Saccharomyces cerevisiae has been purified after overexpression in Escherichia coli and its crystal structure has been determined in complex with its target DNA. In order to evaluate the number of ions that are involved in the cleavage process, thus determining the catalytic mechanism, crystallization experiments were performed in the presence of Mn(2+), yielding crystals that were suitable for X-ray diffraction analysis...
June 2016: Acta Crystallographica. Section F, Structural Biology Communications
Agnes Schipler, Veronika Mladenova, Aashish Soni, Vladimir Nikolov, Janapriya Saha, Emil Mladenov, George Iliakis
Chromosome translocations are hallmark of cancer and of radiation-induced cell killing, reflecting joining of incongruent DNA-ends that alter the genome. Translocation-formation requires DNA end-joining mechanisms and incompletely characterized, permissive chromatin conditions. We show that chromatin destabilization by clusters of DNA double-strand-breaks (DSBs) generated by the I-SceI meganuclease at multiple, appropriately engineered genomic sites, compromises c-NHEJ and markedly increases cell killing and translocation-formation compared to single-DSBs...
September 19, 2016: Nucleic Acids Research
Alexandre Alanio, Maud Gits-Muselli, Séverine Mercier-Delarue, Françoise Dromer, Stéphane Bretagne
Pneumocystis jirovecii is an uncultivable fungal pathogen responsible for Pneumocystis pneumonia (PCP) in immunocompromised patients, the physiopathology of which is only partially understood. The diversity of the Pneumocystis strains associated with acute infection has mainly been studied by Sanger sequencing techniques precluding any identification of rare genetic events (< 20% frequency). We used next-generation sequencing to detect minority variants causing infection, and analyzed the complexity of the genomes of infection-causing P...
2016: Frontiers in Microbiology
Nicholas A Willis, Ralph Scully
The Escherichia coli replication fork arrest complex Tus/Ter mediates site-specific replication fork arrest and homologous recombination (HR) on a mammalian chromosome, inducing both conservative "short tract" gene conversion (STGC) and error-prone "long tract" gene conversion (LTGC) products. We showed previously that bidirectional fork arrest is required for the generation of STGC products at Tus/Ter-stalled replication forks and that the HR mediators BRCA1, BRCA2 and Rad51 mediate STGC but suppress LTGC at Tus/Ter-arrested forks...
July 17, 2016: Cell Cycle
Yibin Wang, Shen Li, Krissy Smith, Barbara Criscuolo Waldman, Alan S Waldman
Mutation of Bloom helicase (BLM) causes Bloom syndrome (BS), a rare human genetic disorder associated with genome instability, elevation of sister chromatid exchanges, and predisposition to cancer. Deficiency in BLM homologs in Drosophila and yeast brings about significantly increased rates of recombination between imperfectly matched sequences ("homeologous recombination," or HeR). To assess whether BLM deficiency provokes an increase in HeR in human cells, we transfected an HeR substrate into a BLM-null cell line derived from a BS patient...
May 2016: DNA Repair
So Maezawa, Saori Nakano, Takaaki Kuniya, Osamu Koiwai, Kotaro Koiwai
We have constructed a novel, nonhomologous end-joining (NHEJ) assay vector (NAV), containing mKate2, Venus and ccdB genes. Cotransfection of NAV with a construct expressing the restriction enzyme I-SceI generated a double-strand break (DSB) in NAV that excised mKate2 and ccdB. Repair of this DSB produced an intact vector that expressed Venus, a green fluorescent protein. Because cells bearing the repaired NAV lacked the ccdB gene which slows cell proliferation, the cultures were enriched in cells containing repaired DSBs...
January 2016: FEBS Open Bio
Yukari Ishihara, Motoyuki Esaki, Atsushi Yasuda
The bacterial artificial chromosome (BAC) technology has been a mainstay approach for generating recombinant viruses, and several methods for excision of the mini-F sequences from the viral BAC vectors have been developed. However, these strategies either require complicated procedures or leave scars of inserted sequences. To overcome these problems, a new method to excise the mini-F sequences from viral BAC vectors based on the Removal of Inserted BAC after linearizatiON (RIBON) strategy was developed in this study for herpesvirus of turkeys (HVT)...
August 1, 2016: Journal of Veterinary Medical Science
Jean Paul Ouedraogo, Mark Arentshorst, Igor Nikolaev, Sharief Barends, Arthur F J Ram
We previously showed that creation of a double strand DNA break (DSB) by expressing I-SceI in an engineered Trichoderma reesei (Hypocrea jecorina) strain containing a I-SceI recognition site improved transformation and homologous integration efficiencies. In this study, we further improved homologous integration frequencies by combining I-SceI mediated double strand break with disruption of the tku70 gene. The inability of the tku70 mutant to repair a I-SceI mediated DSB via NHEJ was used to force integration of an expression cassette with homologous flanks surrounding the DSB site...
March 20, 2016: Journal of Biotechnology
Koichi Watanabe, Ulrike Breier, Götz Hensel, Jochen Kumlehn, Ingo Schubert, Bernd Reiss
Gene targeting is becoming an important tool for precision genome engineering in plants. During gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. We have analysed gene targeting in barley (Hordeum vulgare) using a model system based on double-strand break (DSB) induction by the meganuclease I-SceI and a transgenic, artificial target locus. In the plants we obtained, the donor construct was inserted at the target locus by homology-directed DNA integration in at least two transformants obtained in a single experiment and was stably inherited as a single Mendelian trait...
March 2016: Journal of Experimental Botany
Camille Gelot, Josée Guirouilh-Barbat, Tangui Le Guen, Elodie Dardillac, Catherine Chailleux, Yvan Canitrot, Bernard S Lopez
The end joining of distant DNA double-strand ends (DSEs) can produce potentially deleterious rearrangements. We show that depletion of cohesion complex proteins specifically stimulates the end joining (both C-NHEJ and A-EJ) of distant, but not close, I-SceI-induced DSEs in S/G2 phases. At the genome level, whole-exome sequencing showed that ablation of RAD21 or Sororin produces large chromosomal rearrangements (translocation, duplication, deletion). Moreover, cytogenetic analysis showed that RAD21 silencing leads to the formation of chromosome fusions synergistically with replication stress, which generates distant single-ended DSEs...
January 7, 2016: Molecular Cell
Roger D Plaut, Scott Stibitz
A system was previously developed for conducting I-SceI-mediated allelic exchange in Bacillus anthracis. In this system, recombinational loss of a chromosomally-integrated allelic exchange vector is stimulated by creation of a double-stranded break within the vector by the homing endonuclease I-SceI. Although this system is reasonably efficient and represents an improvement in the tools available for allelic exchange in B. anthracis, researchers are nonetheless required to "pick and patch" colonies in order to identify candidate "exchangeants...
2015: PloS One
Fares Osam Yáñez-Cuna, Mildred Castellanos, David Romero
UNLABELLED: Gene conversion, the nonreciprocal transfer of information during homologous recombination, is the main process that maintains identity between members of multigene families. Gene conversion in the nitrogenase (nifH) multigene family of Rhizobium etli was analyzed by using a two-plasmid system, where each plasmid carried a copy of nifH. One of the nifH copies was modified, creating restriction fragment length polymorphisms (RFLPs) spaced along the gene. Once the modified plasmid was introduced into R...
February 2016: Journal of Bacteriology
Soumen Nandy, Shan Zhao, Bhuvan P Pathak, Muthusamy Manoharan, Vibha Srivastava
BACKGROUND: Practical approaches for multigene transformation and gene stacking are extremely important for engineering complex traits and adding new traits in transgenic crops. Trait deployment by gene stacking would greatly simplify downstream plant breeding and trait introgression into cultivars. Gene stacking into pre-determined genomic sites depends on mechanisms of targeted DNA integration and recycling of selectable marker genes. Targeted integrations into chromosomal breaks, created by nucleases, require large transformation efforts...
2015: BMC Biotechnology
Polly Gravells, Sara Ahrabi, Rajani K Vangala, Kazunori Tomita, James T Brash, Lena A Brustle, Christopher Chung, Julia M Hong, Aikaterini Kaloudi, Timothy C Humphrey, Andrew C G Porter
Understanding the mechanisms of chromosomal double-strand break repair (DSBR) provides insight into genome instability, oncogenesis and genome engineering, including disease gene correction. Research into DSBR exploits rare-cutting endonucleases to cleave exogenous reporter constructs integrated into the genome. Multiple reporter constructs have been developed to detect various DSBR pathways. Here, using a single endogenous reporter gene, the X-chromosomal disease gene encoding hypoxanthine phosphoribosyltransferase (HPRT), we monitor the relative utilization of three DSBR pathways following cleavage by I-SceI or CRISPR/Cas9 nucleases...
December 15, 2015: Human Molecular Genetics
Huseyin Tas, Cac T Nguyen, Ravish Patel, Neil H Kim, Thomas E Kuhlman
We describe an optimized system for the easy, effective, and precise modification of the Escherichia coli genome. Genome changes are introduced first through the integration of a 1.3 kbp Landing Pad consisting of a gene conferring resistance to tetracycline (tetA) or the ability to metabolize the sugar galactose (galK). The Landing Pad is then excised as a result of double-strand breaks by the homing endonuclease I-SceI, and replaced with DNA fragments bearing the desired change via λ-Red mediated homologous recombination...
2015: PloS One
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