By Dipesh Chakrabarty
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14. , Ringpis, G. , Gershon, P. , Lathrop, R. , and Aphasizhev, R. (2009) Novel TUTase associates with an editosome-like complex in mitochondria of Trypanosoma brucei. RNA 15, 1322–1337. 15. , and Bastin, P. (2005) Functional complementation of RNA interference mutants in trypanosomes. BMC Biotechnol. 5, 6. 16. , Stuart, K. , and Hol, W. G. (2004) High resolution crystal structure of a key editosome enzyme from Trypanosoma brucei: RNA editing ligase 1. J. Mol. Biol. 343, 601–613. 17. , Ernst, N.
Transfer at least 3–5 clones, but continue incubating the 96-well plate for a few more days to ensure lack of cell growth in wells with higher dilutions. If this occurs, discard previously collected clones and transfer new ones from the highest dilution wells. For RNAi knockdown of essential genes, induced and mockinduced cell should be harvested at the same cell density. Typically, RNAi is induced at 106 cells/mL and the culture is maintained for 24–48 h; cell density should not exceed 5–6 × 106 cells/mL.
RNA and DNA Editing: Methods and Protocols, Methods in Molecular Biology, vol. 1007/978-1-61779-018-8_3, © Springer Science+Business Media, LLC 2011 41 42 Staber et al. A-to-I editing. Because inosine is recognized by the cellular machinery as guanosine (7), editing can lead to amino-acid coding changes, and does so at a majority of known editing sites in flies. Thus, a spectrum of proteins that are not directly encoded by the genome are created through a simple chemical modification. As mentioned earlier, the binding of dADAR to coding mRNAs is facilitated by the formation of a dsRNA structure comprising the region surrounding the edited exon and a cis-acting complementary sequence, the ECS (exon complementary sequence), which is generally located in an adjacent intron (8, 9).