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Atomic Force Microscope for Topographic Studies
Published in Yuri L. Lyubchenko, An Introduction to Single Molecule Biophysics, 2017
The DNA mismatch repair (MMR) system is biologically one of the most important systems whose function is to correct the DNA synthesis errors that occur during replication (Modrich 2006). As a result, the fidelity of DNA synthesis increases by several orders of magnitude. Progress in the structure-function study of MMR was signified by the 2015 Nobel Prize to P. Modrich for the MMR discovery and its thorough studies. Various techniques were applied to characterize the MMR system with AFM among them.
Toxic and carcinogenic effects of hexavalent chromium in mammalian cells in vivo and in vitro: a recent update
Published in Journal of Environmental Science and Health, Part C, 2022
Shehnaz Islam, Sreejata Kamila, Ansuman Chattopadhyay
Most of the chromate cancer occurs due to microsatellite instability of DNA mismatch repair (MMR) proteins.4, 142–144 DNA hypermethylation responsible for the repression of MLH1 protein (one of MMR protein) was observed in chromate exposed worker.144,145 In the presence of antioxidant ascorbate (Asc), Cr (VI) rapidly reduced to Cr (III) and forms Asc-Cr-DNA crosslink, which activates the MMR system and induces p53 independent cellular apoptosis.142,143 Recruiting gamma H2AX foci by MMR after Cr-DNA adduct formation induces DNA double-strand breaks and p53-mediated apoptosis.4, 143 Loss or deficiency of MMR protein are the major reasons behind chromate cancer (Figure 2).
Characterization of Pseudomonas aeruginosa isolated from various environmental niches: New STs and occurrence of antibiotic susceptible “high-risk clones”
Published in International Journal of Environmental Health Research, 2020
Asma Bel Hadj Ahmed, Mohamed Salah Abbassi, Beatriz Rojo-Bezares, Lidia Ruiz-Roldán, Rabii Dhahri, Ines Mehri, Yolanda Sáenz, Abdennaceur Hassen
Multilocus sequence typing (MLST) was performed for isolates belonging to relevant PFGE pulsotypes and from different sources. Internal fragments of the following seven housekeeping genes were amplified and subsequently sequenced: acsA (acetyl coenzyme A synthetase), aroE (shikimate dehydrogenase), guaA (GMP synthase), mutL (DNA mismatch repair protein), nuoD (NADH dehydrogenase I chain C, D), ppsA (phosphoenolpyruvate synthase) and trpE (anthralite synthetase components I) (Curran et al. 2004). The nucleotide sequences of alleles were compared with those of the MLST database (http://pubmlst.org/paeruginosa/) to obtain the specific Sequence Type (ST). The clonal complex (CC) was determined by using the software PHYLOViZ (Francisco et al. 2012).
Development of capability for genome-scale CRISPR-Cas9 knockout screens in New Zealand
Published in Journal of the Royal Society of New Zealand, 2018
Francis W. Hunter, Peter Tsai, Purvi M. Kakadia, Stefan K. Bohlander, Cristin G. Print, William R. Wilson
We elected to use the improved, version 2 (Sanjana et al. 2014) of the human GeCKO (genome-scale CRISPR knockout) library developed by Feng Zhang at MIT (Shalem et al. 2014). GeCKO v2 is comprised of 123,411 sgRNAs targeting 19,050 genes (six sgRNAs per gene spread across the first three constitutive exons) and 1864 miRNA (four per gene), and includes 1000 negative control sgRNAs that do not align to the human genome (and are thus non-targeting). The important feature of targeting redundancy provides high confidence in discerning on-mechanism effects, as information from multiple, independent sgRNAs targeting the same gene can be synthesised to determine gene-level effect sizes. The two-plasmid variant of GeCKO v2, where Streptococcus pyogenes Cas9 and the sgRNAs are expressed on separate lentiviral vectors, allows for sequential transductions (Figure 4) and confirmation of Cas9 expression prior to committing to scale introduction of the sgRNA library. Importantly, we have identified optimal methods for ultracentrifuge concentration of GeCKO v2 viral particles, which greatly facilitates the performance of screens in hard-to-transduce cell lines, notably including leukaemia cells that grow in suspension. Proof-of-principle screens with the chemotherapeutic agent 6-thioguanine have proven fruitful for functional validation of libraries, as inactivating mutations in thiopurine activation (Doench et al. 2016) and DNA mismatch repair (Swann et al. 1996) are known to confer significant resistance to this agent (Figure 6A). Observing positive selection of sgRNAs targeting 6-thioguanine sensitivity genes (Figure 6B) thus provides confidence in the utility of libraries for screening with agents for which the mechanisms-of-action are less well understood. At the conclusion of selections, PCR amplification of the sgRNA cassette is necessary both to isolate the latter from the genomic DNA of the host cells and to attach the adapters and barcodes that are required for multiplexed sequencing using Illumina NextSeq or HiSeq instruments. Careful optimisation of a three-stage PCR methodology was, therefore, vital for minimising amplification artefacts and reproducibly generating the highly-pure product required for quality (and deep) sequencing.