Project description:The first 4 samples belong to the RNA-IP using in situ TAP tagged ZC3H30 in procyclic (insect) form of the parasite T. brucei Lister 427, 2 samples are Elu or eluate, and 2 are FL or flowthrough (unbound) sample. The other 8 samples are also from procyclic cells. 4 samples belong to DKO(ZC3H30 gene double knockout), 2 are non-stressed and 2 are heat shocked samples; the rest 4 samples are DKO-ectopic (ZC3H30 double knockouts, expressing, ectopic copy of ZC3H30) 2 are non-stressed and 2 are heat shocked samples. Heat Shock experiment was done at 39 degree Celsius.
Project description:Trypanosomes are a globally important group of parasites which together kill and debilitate millions of people world-wide. In trypanosomes, genes do not have individual promoters, rather ~10000 genes share ~200 promoters and all gene expression is thus regulated post-transcriptionally. While effector proteins which modulate the expression of many genes have been described, the mechanisms by which trypanosomes sense changes in their environment and manifest changes in gene expression remain elusive. This study demonstrates that trypanosomes sense changes in their environment through temperature sensitive RNA secondary structure. We show that the majority of observed mRNA abundance changes which distinguish insect adapted and bloodstream adapted life cycle stages can be explained through change in temperature alone.
Project description:RNA-coimmunopurifications with TAP-tagged Puf proteins from Saccharomyces cereviseae. Untagged strain (BY4741) served as a control. Cells were grown to midlog phase and harvested by centrifugation. TAP-tagged Puf proteins were affinity purified from cell-free extracts with IgG sepharose and eluted with TEV protease. RNA was isolated from extract (=input)and from purified protein samples by phenol-chloroform extraction. RNA samples were reverse transcribed using a mixture of oligo-dT and random nonamer oligos in the presence of amino-allyl dUTP/ dNTP mixture. cDNAs were fluorescently labeled and hybridized on yeast DNA microarrays over night at 65 degrees. For a detailed procedure see http://microarray-pubs.stanford.edu/yeast_puf and also Gerber AP et al. PLoS Biology, 2004.
Project description:MCF-7 TET Off cells (MCF-7 wt) were used to produce stable clones expressing ER-beta tagged with TAP-tag respectively at the C-term and at the N-term (C-TAP-ER-beta and N-TAP-ER-beta) or expressing ER-alpha tagged (C-TAP-ER-alpha). All were grown in Dulbecco's modified Eagle's medium (DMEM). Then cells were lysed and RNA extracted were pooled. For mRNA expression profiling, 500 ng total RNA were reverse transcribed and used for synthesis of cDNA and biotinylated cRNA. Finally cRNA were hybridized for 18 hours on Illumina HumanHT-12 v3.0 BeadChips and after scanning, data analysis was performed.