It is a paper using deep sequencing and obtains very interesting results! When I first look at it, I recall a paper I read long time ago "Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells." Cell. 1997 Nov 14;91(4):501-10. PMID: 9390559 .
Reconstruction, the most convincing biological experimental approach. Exceptional!
It is a good idea to read "Rejoice — RNAi for Yeast" by Danesh Moazed to understand the current status of RNAi research. It won Nobel and progress very very fast ...
Here is the paper I am talking about.
Science. 2009 Sep 10.
RNAi in Budding Yeast.
Drinnenberg IA, Weinberg DE, Xie KT, Mower JP, Wolfe KH, Fink GR, Bartel DP.Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge, MA 02142, USA.; Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
RNA interference (RNAi), a gene-silencing pathway triggered by double-stranded RNA, is conserved in diverse eukaryotic species but has been lost in the model budding yeast, Saccharomyces cerevisiae. Here, we show that RNAi is present in other budding-yeast species, including Saccharomyces castellii and Candida albicans. These species use noncanonical Dicer proteins to generate small interfering RNAs, which mostly correspond to transposable elements and Y' subtelomeric repeats. In S. castellii, RNAi mutants are viable but have excess Y' mRNA levels. In S. cerevisiae, introducing Dicer and Argonaute of S. castellii restores RNAi, and the reconstituted pathway silences endogenous retrotransposons. These results identify a novel class of Dicer proteins, bring the tool of RNAi to the study of budding yeasts, and bring the tools of budding yeast to the study of RNAi.
Why we need RNAi in budding yeast? Although yeast genetics is already very powerful, being able to perform RNAi in them will - " be particularly useful in C. albicans, an obligate diploid for which both gene deletions and genetic screens are not trivial (8). Even in S. cerevisiae, RNAi might have advantages for repressing repetitive gene families. RNAi also enables an inducible repression system that might provide an alternative to existing technologies ... Perhaps more important, the tools of budding yeast can now be applied to the study of RNAi, either by developing reagents to investigate the endogenous pathway in S. castellii or by applying existing technologies to examine the reconstituted pathway in S. cerevisiae."
There will be a flood of paper using RNAi in budding yeasts very soon.
By the way, I am also interested the way to manipulate S. castellii genome.
Growth conditions and genetic manipulations
S. castellii was grown at 25°C on standard S. cerevisiae plate and liquid media (e.g., YPD and SC). Transformations were performed as described (S1) with some modifications. Either 0.5–2 μg plasmid DNA or 1–7 μg linear DNA was added to 5 μl single-stranded DNA (10 mg/ml salmon sperm DNA, Sigma D7656), mixed with 50 μl yeast (~3 x 108 cells in 100 mM lithium acetate), and added to transformation buffer (a mixture of 240 μl 40% PEG 3350 and 36 μl 1 M lithium acetate). After incubation at 25°C for 30–90 min, 35 μl of DMSO was added, and the entire mixture was incubated at 42°C for 10 min, resuspended, and then plated on selective media.
Other species. Growth temperatures were as follows, unless otherwise noted: K. polysporus, 25°C; S. cerevisiae, S. bayanus, and C. albicans, 30°C; E. coli, 37°C.
I feel the same way as the authors "if, in the past, S. castellii rather than S. cerevisiae had been chosen as the model budding yeast, the history of RNAi research would have been dramatically different." The chosen rules. Harry Potter believes he is the chosen and he rocks. What about you? Believe yourself!
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