Project description:S. pombe cells were challenged with 0.5 mM hydrogen peroxide for various periods of time (0, 10, 30, 60, 120, 180 minutes). Total RNA was isolated at each time point, labeled with Cy3 and hybridized on cDNA microarrays competitively with Cy5 labeled RNA pooled from all 6 samples. A biological replicate was performed in analyzed as a dyw swap (pool = Cy3, time series = Cy5).
Project description:In S. pombe, about 5% of genes are meiosis-specific and accumulate little or no mRNA during vegetative growth. Here we use Affymetrix tiling arrays to characterize transcripts in vegetative and meiotic cells. In vegetative cells, many meiotic genes, especially those induced in mid-meiosis, have abundant antisense transcripts. These results suggest that antisense transcription represses sense transcription of meiotic genes in vegetative cells. Although the mechanism(s) of antisense mediated transcription repression need to be further explored, our data indicates that RNAi machinery, such as Rdp1, is not required for repression. Previously, we and others used non-strand specific methods to study splicing regulation of meiotic genes and concluded that 28 mid-meiotic genes are spliced only in meiosis. We now demonstrate that the “unspliced” signal in vegetative cells comes from the antisense RNA, not from unspliced sense RNA, andwe argue against the idea that splicing regulates these mid-meiotic genes. Most of these mid-meiotic genes are induced in mid-meiosis by the forkhead transcription factor Mei4. Interestingly, deletion of a different forkhead transcription factor, Fkh2, allows low levels of sense expression of some mid-meiotic genes in vegetative cells. We propose that expression of mid-meiotic genes is kept tightly off in vegetative cells by two independent ways: antisense transcription and Fkh2 repression.
Project description:BxPc-3, Miapaca-2 and ASPC-1 cell lines were treated with 100 µM NS-398 in DMSO or with DMSO alone (control) for 48 h. Total-RNA from each sample was isolated with the RNeasy kit of Qiagen (Hilden, Germany) according to the manufacturer's protocol. The integrity of the isolated RNA was checked on an Agilent Bioanalyser 2100 using the RNA 6000 Nano Kit (Agilent Technologies, Palo Alto, USA) according to the manufacturer's protocol as recommended by the manufacturer.<br>Microarrays were produced and processed as described before. Human cDNAs representing some 7,000 genes that are highly associated with the occurrence of pancreatic cancer including apoptotic and oncogenic genes, growth factors, angiogenic, cell cycle, metastasis-associated, and housekeeping genes were PCR-amplified using amino-modified M13 universal primers. PCR-products were purified with Multiscreen PCR (Millipore, Schwalbach, Germany), resuspended in spotting solution (TeleChem International, Sunnyvale, USA) and arrayed onto slides with epoxysilane surface (Quantifoil Micro Tools, Jena, Germany). DNA spotting was done with a Micro-Arrayer of Engineering Services Inc. (Virtek's arrayer system, BioRad, Munich, Germany) using SMP3 pins (TeleChem). <br><br>Transcript profiling<br>Fluorescently labelled cDNA samples were prepared from 15 µg total-RNA and Cy3- or Cy5-labelled dCTP was directly incorporated during first-strand synthesis . Hybridisation to the microarrays was done in hybridisation chambers (TeleChem) at 62°C overnight. After washing in 0.1xSSC, fluorescence signals were detected with a confocal ScanArray 5000 scanner (Packard Bioscience, USA) and analysed with GenePix Pro 6 (Axon Instruments, Union City, USA). <br>
Project description:Comparison of wild type and bir6 mutant Arabidopsis thaliana seedlings. The bir6 mutant is resistant to root growth inhibition by buthione sulfoxime, an inhibitor of glutathione biosynthesis.