The Experts below are selected from a list of 171 Experts worldwide ranked by ideXlab platform
O A Dontsova - One of the best experts on this subject based on the ideXlab platform.
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the last rrna methyltransferase of e coli revealed the yhir Gene encodes adenine n6 methyltransferase specific for modification of a2030 of 23s ribosomal rna
RNA, 2012Co-Authors: Anna Y Golovina, Margarita M Dzama, Ilya A Osterman, Petr V Sergiev, Marina V Serebryakova, A. A. Bogdanov, O A DontsovaAbstract:The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR Gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR Gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR Knockout Gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.
Anna Y Golovina - One of the best experts on this subject based on the ideXlab platform.
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the last rrna methyltransferase of e coli revealed the yhir Gene encodes adenine n6 methyltransferase specific for modification of a2030 of 23s ribosomal rna
RNA, 2012Co-Authors: Anna Y Golovina, Margarita M Dzama, Ilya A Osterman, Petr V Sergiev, Marina V Serebryakova, A. A. Bogdanov, O A DontsovaAbstract:The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR Gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR Gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR Knockout Gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.
Roberto Docampo - One of the best experts on this subject based on the ideXlab platform.
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A CRISPR/Cas9-riboswitch-Based Method for Downregulation of Gene Expression in Trypanosoma cruzi.
Frontiers in Cellular and Infection Microbiology, 2020Co-Authors: Noelia Lander, Teresa Cruz-bustos, Roberto DocampoAbstract:ABSTRACT. Few Genetic tools were available to work with Trypanosoma cruzi until the recent introduction of the CRISPR/Cas9 technique for Gene Knockout, Gene knock-in, Gene complementation, and endogenous Gene tagging. Riboswitches are naturally occurring selfcleaving RNAs (ribozymes) that can be ligand-activated. Results from our laboratory recently demonstrated the usefulness of the glmS ribozyme from Bacillus subtilis, which has been shown to control reporter Gene expression in response to exogenous glucosamine, for Gene silencing in Trypanosoma brucei. In this work we used the CRISPR/Cas9 system for endogenously tagging T. cruzi glycoprotein 72 (TcGP72) and vacuolar proton pyrophosphatase (TcVP1) with the active (glmS) or inactive (M9) ribozyme. Gene tagging was confirmed by PCR and protein downregulation was verified by western blot analyses. Further phenotypic characterization was performed by immunofluorescence analysis and quantification of growth in vitro. Our results indicate that the method was successful in silencing the expression of both Genes without the need of glucosamine in the medium, suggesting that T. cruzi produces enough levels of endogenous glucosamine 6-phosphate to stimulate the glmS ribozyme activity under normal growth conditions. This method could be useful to obtain knockdowns of essential Genes in T. cruzi and to validate potential drug targets in this parasite.
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Genome Editing by CRISPR/Cas9 in Trypanosoma cruzi.
Methods of Molecular Biology, 2019Co-Authors: Noelia Lander, Miguel Angel Chiurillo, Roberto DocampoAbstract:The Genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 system for genome editing in this organism. The system was initially used for Gene Knockout in T. cruzi, later on for endogenous Gene tagging and more recently for Gene complementation. Mutant cell lines obtained by CRISPR/Cas9 have been used for the functional characterization of proteins in different stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter we describe the methodology to achieve genome editing by CRISPR/Cas9 in T. cruzi. Our method involves the utilization of a template cassette (donor DNA) to promote double-strand break repair by homologous directed repair (HDR). In this way, we have Generated homoGeneous populations of Genetically modified parasites in 4-5 weeks without the need of cell sorting, selection of clonal populations, or insertion of more than one resistance marker to modify both alleles of the Gene. The methodology has been organized according to three main Genetic purposes: Gene Knockout, Gene complementation of Knockout cell lines Generated by CRISPR/Cas9, and C-terminal tagging of endogenous Genes in T. cruzi. In addition, we refer to the specific results that have been published using each one of these strategies.
Ilya A Osterman - One of the best experts on this subject based on the ideXlab platform.
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the last rrna methyltransferase of e coli revealed the yhir Gene encodes adenine n6 methyltransferase specific for modification of a2030 of 23s ribosomal rna
RNA, 2012Co-Authors: Anna Y Golovina, Margarita M Dzama, Ilya A Osterman, Petr V Sergiev, Marina V Serebryakova, A. A. Bogdanov, O A DontsovaAbstract:The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR Gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR Gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR Knockout Gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.
Petr V Sergiev - One of the best experts on this subject based on the ideXlab platform.
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the last rrna methyltransferase of e coli revealed the yhir Gene encodes adenine n6 methyltransferase specific for modification of a2030 of 23s ribosomal rna
RNA, 2012Co-Authors: Anna Y Golovina, Margarita M Dzama, Ilya A Osterman, Petr V Sergiev, Marina V Serebryakova, A. A. Bogdanov, O A DontsovaAbstract:The ribosomal RNA (rRNA) of Escherichia coli contains 24 methylated residues. A set of 22 methyltransferases responsible for modification of 23 residues has been described previously. Herein we report the identification of the yhiR Gene as encoding the enzyme that modifies the 23S rRNA nucleotide A2030, the last methylated rRNA nucleotide whose modification enzyme was not known. YhiR prefers protein-free 23S rRNA to ribonucleoprotein particles containing only part of the 50S subunit proteins and does not methylate the assembled 50S subunit. We suggest renaming the yhiR Gene to rlmJ according to the rRNA methyltransferase nomenclature. The phenotype of yhiR Knockout Gene is very mild under various growth conditions and at the stationary phase, except for a small growth advantage at anaerobic conditions. Only minor changes in the total E. coli proteome could be observed in a cell devoid of the 23S rRNA nucleotide A2030 methylation.