Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional Profile of the Shigella flexneri Response to Berberine

ABSTRACT: Berberine is a natural isoquinoline alkaloid found in Chinese medicinal herbs which is active against a variety of microbial infections. To examine the potential effects of berberine on Shigella flexneri, a whole-genome DNA microarray was constructed and transcriptome analysis of the cellular responses of S.flexneri when exposed to Berberine Chloride (BC) was performed. When cultures were grown to early exponential growth phase, drug was added. Meanwhile, DMSO solution without BC was added to control cultures to the same final concentration. At 30 min after treatment, samples were collected and washed twice with phosphate buffered saline at 25°C for subsequent RNA isolation. To prepare biological replicates for RNA isolation, each experiment was independently performed three times. After drug treatment, total RNA was extracted using SV RNA Isolation System (Promega). Reverse transcription of 20 ug RNA from each sample was performed with Superscript II system (Invitrogen) to prepare cDNA. According to the manufacturer's instructions, the cDNA was labeled with incorporation of fluorescent dyes (Amersham). Cy3-dCTP monofunctional dye was selected for control cDNA samples and Cy5-dCTP was used for drug-treated samples. Each pair of labeled cDNA was mixed and dried by vacuum centrifugation. The pairs of the labeled cDNA samples were hybridized to the DNA microarrays following the protocol available at http://www.ifr.ac.uk/safety/microarrays/. The slides were scanned by using a GenePix 4100A scanner. Fluorescent spots and local background intensities were quantified using GenePix Pro 6.0 software (Axon). Signal intensities were corrected by subtracting the local background value. The data set was filtered so that spots flagged as "not found," "bad," and "empty" were excluded from further analysis. Moreover, spots either with signal-to-noise ratio below the detection limit of 3 or containing less than 55% of pixels two standard deviations above background in both channels were discarded from the data set. Lowess normalization was performed with the fluorescent signal ratios (Cy5/Cy3) using MIDAS software (http://www.tigr.org/software/tm4) with a 0.33 smooth parameter.

ORGANISM(S): Shigella flexneri

SUBMITTER: Hua Fu

PROVIDER: E-GEOD-16714 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Ants provide remarkable examples of equivalent genotypes developing into divergent and discrete phenotypes. Diploid eggs can develop either into queens, which specialize in reproduction, or workers, which participate in cooperative tasks such as building the nest, collecting food, and rearing the young. In contrast, the differentiation between males and females generally depends upon whether eggs are fertilized, with fertilized (diploid) eggs giving rise to females and unfertilized (haploid) eggs giving rise to males. To obtain a comprehensive picture of the relative contributions of gender (sex), caste, developmental stage, and species divergence to gene expression evolution, we investigated gene expression patterns in pupal and adult queens, workers, and males of two species of fire ants, Solenopsis invicta and Solenopsis richteri. Microarray hybridizations revealed that variation in gene expression profiles is influenced more by developmental stage than by caste membership, sex, or species identity. The second major contributor to variation in gene expression was the combination of sex and caste. Although workers and queens share equivalent diploid nuclear genomes, they have highly distinctive patterns of gene expression in both the pupal and the adult stages, as might be expected given their extraordinary level of phenotypic differentiation. Overall, the difference in the proportion of differentially expressed genes was greater between workers and males than between workers and queens or queens and males, consistent with the fact that workers and males share neither gender nor reproductive capability. Moreover, between-species comparisons revealed that the greatest difference in gene expression patterns occurred in adult workers, a finding consistent with the fact that adult workers most directly experience the distinct external environments characterizing the different habitats occupied by the two species. Thus, much of the evolution of gene expression in ants may occur in the worker caste, despite the fact that these individuals are largely or completely sterile. Analyses of gene expression evolution revealed a combination of positive selection and relaxation of stabilizing selection as important factors driving the evolution of such genes. Transcriptome analysis is a powerful tool for unveiling the distribution and magnitude of genetic incompatibilities between hybridizing taxa. The nature of such incompatibilities is closely associated with the evolutionary histories of the parental species and may differ across tissues and between the sexes. In eusocial insects, the presence of distinct castes that experience divergent selection regimes may result in additional distinct patterns of caste-specific hybrid incompatibilities. We analyzed levels of expression of >14,000 genes in two life stages of each caste and sex in the fire ants Solenopsis invicta and Solenopsis richteri and in their hybrids. We found strong contributions of both developmental stage and caste to gene expression patterns. In contrast, variability in expression was only weakly associated with species, with hybrid scores falling between those of the two parental species in both life stages and all castes. Hybrid incompatibilities were surprisingly modest, with only 16 genes in pupae and 18 genes in adults being mis-expressed, and indicating low levels of disruption in gene regulation in hybrids. The castes differed in numbers of mis-expressed genes, with males and workers each mis-expressing at least seven times as many genes as queens in both life stages. Interestingly, homologues of many of the mis-expressed genes have been implicated in behavioral variation in Drosophila melanogaster. General expression profiles of hybrids consistently were more similar to those of Solenopsis richteri than Solenopsis invicta, presumably because Solenopsis richteri trans-regulatory elements tend to be dominant to those of Solenopsis invicta and/or because there is an overall bias in the genetic composition of the hybrids towards Solenopsis richteri. Altogether, our results suggest that selection acting on each caste may contribute differently to interspecific divergence and speciation in this group of ants. We analyzed samples from two species, their hybrids: a total of 140 samples, consisting of either workers, queens or males (1-5 pooled individuals) and collected either as larvae, pupae or adults. Each sample was tested once (i.e. single biological replicate) against a common reference (dye-swap across experiments)

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Transcriptional Profile of the Shigella flexneri Response to Berberine

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