Transcriptome analysis of Escherichia coli O157:H7 EDL933 during rumen fluid exposure using microarrays
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ABSTRACT: The transcriptome of Escherichia coli K-12 has been widely studied over a variety of conditions for the past decade while such studies involving E. coli O157:H7, its pathogenic cousin, are just now being conducted. To better understand the impact of rumen fluid on E. coli O157:H7, global transcript levels of strain EDL933 cells resuspended in heat clarified rumen fluid for 15 min were compared to cells resuspended in fresh LB using microarrays. Seven independent RNA samples from rumen fluid treated cultures were paired with seven independent RNA samples from control cultures for hybridization to seven two-color microarrays. For three arrays, the control RNA sample was labeled with Cy3 dye and the experimental RNA sample was labeled with Cy5 dye, the dyes were reversed for the other four arrays to overcome dye bias.
Project description:The transcriptome of Escherichia coli K-12 has been widely studied over a variety of conditions for the past decade while such studies involving E. coli O157:H7, its pathogenic cousin, are just now being conducted. To better understand the impact of intracellular life within a ruminant and environmental protozoan on E. coli O157:H7, global transcript levels of strain EDL933 cells inside Acanthamoeba were compared to cell grown in the protozoan media (ATCC PYG712) by microarray. Nine independent RNA samples from cell from within Acanthamoeba were paired with seven independent RNA samples from control cultures for hybridization to nine two-color microarrays. For four arrays, the control RNA sample was labeled with Cy3 dye and the experimental RNA sample was labeled with Cy5 dye, the dyes were reversed for the other five arrays to overcome dye bias.
Project description:The transcriptome of Escherichia coli K-12 has been widely studied over a variety of conditions for the past decade while such studies involving E. coli O157:H7, its pathogenic cousin, are just now being conducted. To better understand the impact of heat shock on E. coli O157:H7, global transcript levels of strain EDL933 cells shifted from 37°C to 50°C for 15 min were compared to cells left at 37°C using microarrays. Keywords: Stress Response Twelve independent RNA samples from heat shocked cultures were paired with twelve independent RNA samples from control cultures for hybridization to twelve two-color microarrays. For six arrays, the control RNA sample was labeled with Cy3 dye and the experimental RNA sample was labeled with Cy5 dye, the dyes were reversed for the other six arrays to overcome dye bias.
Project description:The transcriptome of Escherichia coli K-12 has been widely studied over a variety of conditions for the past decade while such studies involving E. coli O157:H7, its pathogenic cousin, are just now being conducted. To better understand the impact of an anaerobic environment on E. coli O157:H7, global transcript levels of strain EDL933 cells grown aerobically were compared to cells grown anaerobically using microarrays. Eight independent RNA samples from aerobic cultures were paired with eight independent RNA samples from anaerobic cultures for hybridization to eight two-color microarrays. For four arrays, the control RNA sample was labeled with Cy3 dye and the experimental RNA sample was labeled with Cy5 dye, the dyes were reversed for the other six arrays to overcome dye bias.
Project description:Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist. The virulence factors of M. hyopneumoniae are largely unknown and are most probably complex in nature. The transcriptional profile of intergenic regions is investigated using microarray PCR and oligonucleotide probes. It is hypothesized that intergenic regions of the Mhyo genome are being transcribed along with the ORFs. To test for transcripts in IG regions, cDNA was generated from M. hyopneumoniae RNA using random hexamers (IDT; Integrated DNA Technologies, Inc. Coralville, IA) and a set of 129 hexamer oligonucleotide primers designed for M. hyopneumoniae mRNA. A single total RNA sample was split into eight 5 μg samples, and cDNA synthesis was conducted on four of these samples using the random hexamers (IDT); for the other four samples, cDNA was generated using primers designed for M. hyopneumoniae as described by Madsen et al. 2006. In each set of four, two samples were fluorescently labeled with Cy3 and the other two were labeled with Cy5 using an indirect labeling protocol. After labeling and cleanup, all 8 samples were quantified on a Nanodrop ND-1000 spectrophotometer. For each array on the slide, 1 μg of labeled cDNA from each type of primer generation (Cy3 or Cy5, random or specific primer set) was combined and hybridized to a PCR featured array and an oligonucleotide featured array. This resulted in 2 arrays of PCR probes hybridized with dye swaps and 2 arrays of oligonucleotide probes with dye swaps. A second RNA sample was split in four 5 μg samples of total RNA, and the hybridization process was repeated for the oligonucleotide probe array.
Project description:Yersinia pestis is the etiology of plague that is able to sense cell density by quorum sensing. The function of quorum sensing in Y.pestis is not clear. Here, the process of quorum sensing was investigated by comparing transcript profiles when three quorum sensing synthase genes are knocked out. Two strains, â??pgm (pigmentation-negative) mutant R88 as treatment and 3XQS mutant with mutation (â??pgm, â??ypeIR, â??yspIR, and â??luxS) R115 as control are used in this analysis. Six independent RNA samples from R115 cultures were paired with six independent RNA samples from R88 cultures for hybridization to six two-color microarrays. Dye-swap design was used to remove the Cy5 and Cy3 dye bias.
Project description:Yersinia pestis, the etiological agent of plague, is able to sense cell density by quorum sensing. The function of quorum sensing in Y. pestis is not clear. Here, the process of quorum sensing was investigated by comparing transcript profiles when three quorum-sensing synthase genes are knocked out. Two strains, M-bM-^HM-^Fpgm (pigmentation-negative) mutant R88 as treatment and quorum sensing null strain R115 with mutations (M-bM-^HM-^Fpgm, M-bM-^HM-^FypeIR, M-bM-^HM-^FyspIR, and M-bM-^HM-^FluxS) as control, are used in this analysis. Six independent RNA samples from R115 cultures were paired with six independent RNA samples from R88 cultures for hybridization to six two-color microarrays. A dye-swap design was used to remove the Cy5 and Cy3 dye bias.
Project description:Using classical overexpression/knockout studies, we find that miR-126 regulates the duration of the primitive erythroid program, and confirm this in embryonic miR-126-/- yolk sacs. MicroRNA microarray analysis was performed on samples from ES cell differentiation
Project description:Vegetative phase change is the developmental transition from the juvenile phase to the adult phase during which a plant becomes competent for sexual reproduction. Gain of ability to flower is often accompanied by changes in patterns of differentiation in newly forming vegetative organs. In maize, juvenile leaves differ from adult leaves in morphology, anatomy, and cell wall composition. Whereas the normal sequence of juvenile followed by adult is repeated with every sexual generation, this sequence can be altered in maize by the isolation and culture of the shoot apex from an adult phase plant; an âadultâ meristem so treated reverts to forming juvenile vegetative organs. To investigate the molecular differences between the juvenile and adult phases in maize comparisons among two juvenile samples, leaf 4 and culture-derived leaf 3 or 4, and an adult sample (leaf 9) were made using cDNA microarrays. All samples were leaf primordia at plastochron 6. A gene was scored as âphase specificâ if it was up- (or down-) regulated in both juvenile samples compared to the adult sample with at least a twofold-change in gene expression at P-value less than or equal to 0.005. Some 221 ESTs up-regulated in juvenile and 28 ESTs up-regulated in adult were identified. Altered patterns of expression of selected ESTs in the phase change mutants Tp2, d1 and gl15 further confirmed these genes as being phase-specific and allowed us to position these genes in the known genetic hierarchy regulating phase change. Keywords: Transcript profiling among seed-derived juvenile leaf 4 and adult leaf 9 and culture-rejuvenated leaf 3 or 4 in maize To identify juvenile or adult specific ESTs, total RNA from leaf primordia at plastochron 6 (P6) was isolated from leaves 4 (L4) and 9 (L9) from seed-derived plants and leaf 3 or 4 (RL3/4) from culture-derived plants. For each of six biological replications, each of the three pairwise comparisons of P6-staged leaf primordia from L4, L9 and RL3/4 was made on one slide. With six biological replications and three slides per replication (L4 vs. L9, L9 vs. RL3/4, RL3/4 vs. L4), this replicated loop design used a total of 18 slides. To ensure dye balance, each of the 18 target samples was measured once with Cy3 labeling and once with Cy5 labeling.
Project description:During Zea mays (maize) C4 differentiation, mesophyll (M) and bundle sheath (BS) cells accumulate distinct sets of photosynthetic enzymes, with very low photosystem II (PSII) content in BS chloroplasts. Consequently, there is little linear electron transport in the BS and ATP is generated by cyclic electron flow. In contrast, M thylakoids are very similar to those of C3 plants and produce the ATP and NADPH that drive metabolic activities. Regulation of this differentiation process is poorly understood but involves expression and coordination of nuclear and plastid genomes. Here, we identify a recessive allele of the maize Hcf136 homologue that in Arabidopsis thaliana functions as a PSII stability or assembly factor located in the thylakoid lumen. Proteome analysis of the thylakoids and electron microscopy reveal that Zm hcf136 lacks PSII complexes and grana thylakoids in M chloroplasts, consistent with the previously defined Arabidopsis function. Interestingly, hcf136 is also defective in processing the full-length psbB-psbT-psbH-petB-petD polycistron specifically in M chloroplasts. To determine whether the loss of PSII in M cells affects C4 differentiation, we performed cell-type specific transcript analysis of hcf136 and wild-type seedlings. The results indicate that M and BS cells respond uniquely to the loss of PSII, with little overlap in gene expression changes between data sets. These results are discussed in the context of signals that may drive differential gene expression in C4 photosynthesis. To explore the disruption of PSII activity on gene expression, transcript profiles from separated M and BS cells were examined using two-label microarray analysis. Total RNA was isolated from the second leaves of mutant and wild-type silbings. Six biological replicates were used to compare wild-type and mutant transcript profiles in separate M and BS experiments. To maximize biological replication, different seedling pools were used for each of the 12 hybridizations. Microarray experiments and analyses were performed using the Genisphere MPX900 kit and the Maize Array Consortium oligonucleotide platform (GPL5439; GPL5440). Feature intensity values were log-transformed and corrected for local background signal, and a LOWESS procedure (Dudoit et al., 2002) was used to normalize between channels. Features with either low or saturating signal intensity were discarded from further analysis. High expression filtering was less stringent to avoid elimination of previously characterized, high abundance, C4 cell-specific transcripts. After filtering, features that were not assigned an MZ number by the Maize Array Consortium were discarded from further analysis. The moderated t-test (Smyth, 2004) using the R package limma was applied to identify differentially expressed genes. The p-values for each test (gene) were converted to q-values for false discovery rate analysis as described by Storey et al. (2004). To avoid confounding treatment effects associated with direct comparisons of M and BS transcriptomes (Sawers et al., 2007), comparisons were only made using the same cell type across the hcf136 and wild-type sibling genotypes. Bundle sheath (BS) Samples: GSM245063-GSM245164 Mesophyll (M) Samples: GSM245165 - GSM245206