Screening in E. coli overexpresssion libraries for genes protecting from histone killing
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ABSTRACT: The experiment's aim is to follow the population of E. coli cells before and after treatment with histones. The E. coli libraries consisted of MACH1 pSMART-LCKm carrying random fragments of 1, 2, 4 and 8 kb of E. coli genome. The selection procedure involved three rounds of histones treatment (same dose each time during 3h hours then recovery time of 2h before applying the next histone treatment, at the end a total of 15-16h).
Project description:To investigate the possible range of additional RNase P substrates in vivo a strand-specific, high-density microarray was used to analyze what RNA accumulates with a mutation in the catalytic RNA subunit of nuclear RNase P in Saccharomyces cerevisiae. A wide variety of noncoding RNAs were shown to accumulate, suggesting nuclear RNase P participates in the turnover of normally unstable nuclear RNAs. In some cases, the accumulated noncoding RNAs were shown to be antisense to transcripts that commensurately decreased in abundance. Pre-mRNAs containing introns also accumulated broadly, consistent with either compromised splicing or failure to efficiently turnover pre-mRNAs that do not enter the splicing pathway. Taken together with the high complexity of the nuclear RNase P holoenzyme and its relatively non-specific capacity to bind and cleave mixed sequence RNAs, these data suggest nuclear RNase P facilitates turnover of nuclear RNAs in addition to its role in pre-tRNA biogenesis.
Project description:Clostridium acetobutylicum is a Gram-positive, endospore-forming bacterium that is considered as a strict anaerobe. It ferments sugars to the organic acids acetate and butyrate or shifts to formation of the solvents - ethanol, butanol and acetone. In most bacteria the major regulator of iron homeostasis is Fur (ferric uptake regulator). Analysis of the genome of Clostridium acetobutylicum has revealed three genes encoding Fur-like proteins. The amino acid sequece of one of them showed 70% similarity to the Fur protein of the closely related Bacillus subtilis.<br>Thus, to gain insight into the role of Fur and the mechanisms for maintenance of iron homeostasis in this strict anaerobic organism, we determined its transcriptional profile in response to iron limitation and inactivation of fur.
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:The met31 single delete and the met32 single delete strains were analyzed for genome-wide transcription at different times following Met4 hyeractivation and compared to cells that contain both Met31 and Met32.
Project description:E. coli MG1655 was grown in MOPS minimal glucose medium at 37 degrees C with aeration to an OD600 of 0.4 - 0.5, and HOCl was added to a final concentration of 400 ?M. 0.5 ml samples were collected in liquid nitrogen immediately before, 5 min after, and 10 min after HOCl addition, and total RNA was prepared using the RNeasy? Midi kit (Qiagen). cDNA synthesis, array hybridization to Affymetrix GeneChip E. coli genome 2.0 Arrays, and imaging were performed according to Affymetrix guidelines at the Affymetrix and Microarray Core facility at the University of Michigan, Ann Arbor.
Project description:A dilution series experiment to determine the sensitivity of a microarray method for human papillomavirus genotyping. HPV genotyping is based on multiplex PCR (PGMY-t primers) followed by ligation step where two probes are ligated together if a matching template is present in the mixture. The ligated probes are then detected on microarray. Plasmids containing the full genome of HPV 16 or 18 were used in the experiment. The concentrations were 1000, 100, 10 ,1, 0.1 and 0 femtograms of plasmid as a template in HPV PCR reaction.<br><br>