Genotyping of S. aureus SH1000 transposon mutants to identify mutant positions
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ABSTRACT: A library of transposon mutants were generated in S.aureus strain SH1000 using a custom Mariner transposon with an outward-facing T7 promoter. Genomic DNA was extracted from the library and digested with an appropriate restriction enzyme (AluI or RsaI). Labelled RNA run-offs were produced from the T7 promoter and hybridised to a tiling microarray to determine the position of the mutant. This was done using a library of a million S. aureus mutants, and genes not disrupted by a transposon were inferred to be essential for replication and growth in vitro.
Project description:A library of transposon mutants was generated in Salmonella enterica serovar Typhimurium strain SL1344 using custom Tn5 and Mu transposons with outward-facing T7 and SP6 promoters. The library was grown up in vitro, and used as the input pool for mouse infection experiments. An in vivo output pool was obtained from the mouse livers 2 days post-infection.<br><br>Genomic DNA was extracted from the input and output pools and digested with the restriction enzyme RsaI. Cy5-labelled RNA run-offs were produced seperately from the T7 and SP6 promoters and hybridised to a tiling microarray along with the Cy3-labelled in vitro transcription products from an untransposed SL1344 wild type control. Analysis of the microarray data allows the location of the transposon inserts to be determined. Mutants that are present in the input pool but that are absent or less prevalent in the output pools are inferred to be attenuated in vivo.
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:Analysis of PerR regulon. Global transcription profile of wild type GAS M1 (SF370) as compared to perR deletion mutant in mid- and late- log growth phases
Project description:Serum-free Fibrocytes, Serum-containing Fibrocytes, CD14++CD16- Monocytes, CD14++CD16+ Monocytes, CD14+CD16++ Monocytes, Macrophages were all generated from up to 3 biological replicates from each of 3 separate donors. RNA was extracted (Ambion RNAqueous), labelled with cy3, mixed with cy5 labelled human reference (Stratagene), and hybridised to slides printed with Human AROS v4.0 oligonucleotides (Operon). Slides were scanned using a Perkin Elmer GX plus, and the data then normalised with GEPAS v4.0 and collated. Final data analysis was carried out using TMEV 4.0. SAM was performed using a 0.1% FDR. PCA were plotted from this list, and interrogation carried out using DAVID to determine pathway enrichment.
Project description:Study of gene expression differences between wild type and genetically modified Leishmania donovani protozoan parasites. A centrin-gene-deleted cell line after 40 hours in culture conditions that induce failure of cell division is compared to wild type cells under the same culture conditions.
Project description:Fibroblasts, Serum-free Fibrocytes, Serum-containing Fibrocytes, Monocytes, Macrophages, Osteoclasts, immature Dendritic Cells, and mature Dendritic Cells were all generated from 3 biological replicates from each of 3 separate donors. RNA was extracted (Ambion RNAqueous), labelled with cy3, mixed with cy5 labelled human reference (Stratagene), and hybridised to slides printed with Human AROS v4.0 oligonucleotides (Operon). Slides were scanned using a Perkin Elmer GX plus, and the data then normalised with GEPAS v4.0 and collated. Final data analysis was carried out using TMEV 4.0. SAM was performed using a 0.1% FDR. HCL and PCA were plotted from this list, and interrogation carried out using DAVID to determine pathway enrichment. After initial analysis, the Fibroblast cell type was found to significantly bias the data. Due to this, these samples were removed from the analysis, and the data re-normalised. This second data file will be submitted separately.
Project description:CD4 T cells taken from 6 donors were sorted into CD25hi CD45RA+ (na�ve Treg), CD25hi CD45RO+ (memory Treg), CD25- CD45RA+ (na�ve responder) and CD25- CD45RO+ (memory responder) populations. <br><br>RNA was extracted (Ambion RNAqueous), amplified (SMART), labelled with cy3, mixed with cy5 labelled human reference (Stratagene), and hybridised to slides printed with Human AROS v4.0 oligonucleotides (Operon).<br><br>Slides were scanned using a Perkin Elmer GX plus, and the data then normalised with GEPAS v4.0 and collated. One sample was deemed to be of too low hybridisation quality, and was hence removed at this point.<br><br>Final data analysis was carried out using TMEV 4.0. SAM was performed using a 10% FDR. HCL and PCA were plotted from this list, and interrogation carried out using DAVID to determine pathway enrichment<br>