Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro.

ABSTRACT: Paracoccidioides brasiliensis is a thermally dimorphic fungus, which causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has been rarely addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25% of the organismM-bM-^@M-^Ys genome, and its utilization in identifying genes and gene expression patterns during growth in vitro. Cloned, amplified inserts from randomly sheared gDNA and known control genes were printed onto glass slides to generate a microarray of over 12,000 elements. To examine gene expression, mRNA was extracted and amplified from mycelial or yeast cultures grown in semi-defined medium for 5, 8 and 14 days. Principal components analysis and hierarchical clustering indicated that yeast gene expression profiles differed greatly from mycelia, especially at earlier time-points, and that mycelial gene expression changed less than it did on yeasts over time. Genes, upregulated in yeasts, were found to be involved in methionine/cysteine metabolism, respiratory processes, metabolic processes (sugars, amino acids, proteins and lipids), transporters (small peptides, sugar, ions and toxin), regulatory proteins and transcription factors. Mycelia genes involved in processes such as cell division, protein catabolism, nucleotide biosynthesis and toxin and sugar transporters showed differential expression. Sequenced clones were compared with Histoplasma capsulatum and Coccidioides posadasii genome sequences to assess potentially common pathways across species, such as sulfur and lipid metabolism, amino acid transporters, transcription factors and genes possibly related to virulence. We also analyzed gene expression with time in culture and found that while transposable elements and components of respiratory pathways tended to increase in expression with time, genes coding for ribosomal structural proteins and protein catabolism tended to sharply decrease in expression over time, particularly in yeast. These findings expand knowledge about the different morphologic forms of P. brasiliensis during growth in culture. Briefly we aimed at generating and comparing gene expression patterns of P. brasiliensis mycelia and yeasts during growth in liquid modified McVeigh/Morton media for 5, 8 and 14 days. We extracted total RNA from two cultures per time point for each morphologic form. Hybridizations were done twice for each total RNA sample. We used sheared genomic DNA as reference sample (Cy3) on every slide. Normalization was done as follows: Raw data underwent global normalization using the R statistical package to perform quantile normalization of the global reference channel by computing the centroid of all global reference signatures, and performing a loess fit of each individual global reference signature to the centroid. The loess curve for a given sample was then used to scale both channels for each gene in that sample. After that data was loaded in GeneSpring GX 7.3.1 and underwent the following normalization steps: -per spot to the gDNA channel -per slide using 102 positive controls (listed below) -per gene to each geneM-bM-^@M-^Ys respective median Positive controls: JM_2d11 JM_5a12 JM_22h5 JM_120a12 JM_61c4 JM_15g2 JM_13g9 JM_27c1 JM_55a6 JM_42a8 JM_14e1 JM_66c4 JM_13a9 JM_109e5 JM_20c1 JM_100a11 JM_62d8 JM_105c11 JM_22e8 JM_6a7 JM_66a5 JM_1e7 JM_42f5 JM_99h9 JM_9c7 JM_123g1 JM_33e5 JM_112a2 JM_25g6 JM_114h6 JM_98b1 JM_38h9 JM_30e11 JM_10e10 JM_22g11 JM_120c9 JM_120g4 JM_87g2 JM_70e12 JM_119e12 JM_79b12 JM_99a8 JM_67b5 JM_41b11 JM_76a2 JM_131e11 JM_65c2 JM_104e12 JM_26h3 JM_18c11 JM_56a7 JM_111c8 JM_21c9 JM_5e1 JM_3g12 JM_64e7 JM_115e5 JM_74c5 JM_117a5 JM_17b12 JM_10a5 JM_118e2 JM_15e5 JM_73e8 JM_89c6 JM_1c5 JM_5c8 JM_2e1 JM_83f10 JM_81e10 JM_83f11 JM_116a3 JM_5b11 JM_4c4 JM_86g6 JM_66e10 JM_44a6 JM_22a1 JM_22b4 JM_16a2 JM_22b5 JM_79g2 JM_26a11 JM_94b5 JM_25c2 JM_17e3 JM_66g1 JM_54a9 JM_84a11 JM_74b11 JM_115c6 JM_14a6 JM_122c9 JM_133e9 JM_11b9 JM_51b4 JM_24c7 JM_52b2 JM_66f3 JM_10c8 JM_57c1 JM_8b2

ORGANISM(S): Paracoccidioides brasiliensis

SUBMITTER: Jomar Monteiro

PROVIDER: E-GEOD-15511 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro.

Monteiro Jomar Patrício JP Clemons Karl V KV Mirels Laurence F LF Coller John A JA Wu Thomas D TD Shankar Jata J Lopes Catalina R CR Stevens David A DA

Microbiology (Reading, England) 20090430 Pt 8

Paracoccidioides brasiliensis is a thermally dimorphic fungus, and causes the most prevalent systemic mycosis in Latin America. Infection is initiated by inhalation of conidia or mycelial fragments by the host, followed by further differentiation into the yeast form. Information regarding gene expression by either form has rarely been addressed with respect to multiple time points of growth in culture. Here, we report on the construction of a genomic DNA microarray, covering approximately 25 % o ...[more]

PMID: 19406900

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Project description:Background: Paracoccidioides is the causative agent of paracoccidioidomycosis, a systemic mycosis endemic to Latin America. Infection is initiated by inhalation of conidia (C) or mycelial (M) fragments, which subsequently differentiate into yeast (Y). Epidemiological studies show a striking predominance of paracoccidioidomycosis in adult men compared to premenopausal women. In vitro and in vivo studies suggest that the female hormone (17M-NM-2-estradiol, E2) regulates or inhibits M-or-C-to-Y transition. In this study we have profiled transcript expression to understand the molecular mechanism of how E2 inhibits M-to-Y transition. Methodology: We assessed temporal gene expression in strain Pb01 in the presence or absence of E2 at various time points through 9 days of the M-to-Y transition using an 11,000 element random-shear genomic DNA microarray and verified the results using quantitative real time-PCR. E2-regulated clones were sequenced to identify genes and biological function. Principal findings: E2-treatment affected gene expression of 550 array elements, with 331 showing up-regulation and 219 showing down-regulation at one or more time points (p M-bM-^IM-$ 0.001). Genes with low expression after 4 or 12 h exposure to E2 belonged to pathways involved in heat shock response (hsp90 and hsp70), energy metabolism, and several retrotransposable elements. Y-related genes, M-NM-1-1,3-glucan synthase, mannosyltransferase and Y20, demonstrated low or delayed expression in E2-treated cultures. Genes potentially involved in signaling, such as palmitoyltransferase (erf2), small GTPase RhoA, phosphatidylinositol-4-kinase, and protein kinase (serine/threonine) showed low expression in the presence of E2, whereas a gene encoding for an arrestin domain-containing protein showed high expression. Genes related to ubiquitin-mediated protein degradation, and oxidative stress response genes were up-regulated by E2. Conclusion: This study characterizes the effect of E2 at the molecular level on the inhibition of the M-to-Y transition and is indicative that the inhibitory actions of E2 may be working through signaling genes that regulate dimorphism. Two-condition experiment, Controls vs. 17M-NM-2-estradiol (E2) treated Paracoccidioides Pb01 cells (at 0, 4, 12 24, 72, 144 or 216 h time points). Biological replicates: 4 controls (2 untreated(C) + 2 ethanol (OH) treated control), 2 (E2)treated at each time points, independently grown and harvested. Two array from each biological samples.

Project description:Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and Analysis of Sequence Counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. K-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes. Axenic T. pseudonana CCMP 1335 was grown at 14Â°C under 24 hour light (120 Âµmol photons m-2 s-1) after Dyhrman et al. (2012) in f/2 plus silica chelated media made from surface Sargasso Sea water. Nitrate, silica, vitamins, and trace metals were at f/2 concentrations (Guillard and Ryther 1962), while iron and phosphate were modified across treatments. In brief, triplicate cultures of replete (36 ÂµM PO4, 400 nM Fe), P-limited (0.4 ÂµM PO4, 400 nM Fe), Fe-limited (36 ÂµM PO4, 40 nM Fe), and Co-limited (0.4 ÂµM PO4, 40 nM Fe) treatments were harvested when growth deviated from the replete control. Growth was monitored by cell counts. Biomass was harvested onto 0.2 Âµm filters and flash frozen in liquid nitrogen and total RNA was extracted as described in Dyhrman et al. (2012). Tag-seq sequencing of the transcriptome was performed by Illumina with a polyA selection and NlaIII digestion, resulting in 21 bp sequence reads or tags (Dyhrman et al., 2012). Libraries were of varied sizes as follows: replete (~12 million), P-limited (~13 million), Fe-limited (~23 million), and Co-limited (~26 million). Tags were mapped to gene models (predicted protein coding regions) with a pipeline designed by Genesifter Inc., requiring 100% identity and covering 9759 genes. Tag counts within a gene were pooled and normalized to the size of the library, with resulting data expressed in tags per million (tpm). Genes with normalized tag counts less than 2.5 tpm for each of the four treatments were excluded (Figure S1) , leaving 7380 genes in the analysis.

Project description:A genomic insight into how an insect pest responds to the infection of a fungal insect pathogen, such as Beauveria bassiana, is critical for alternative strategy of insect pest contol based on fungal insecticides but has not been well probed. Here we constructed three pairs of digital expression libraries (transcriptomes) of Plutella xylostella (global lepidopteran pest) larvae 24, 36 and 48 hours post treatment of infection (hptI) and control (hptC) to reveal the host response to B. bassiana infection at genomic level. The paired libraries comprised 2144, 3200 and 2967 differentially expressed genes (DEGs) of P. xylostella at 24, 36 and 48 hptI/hptC, respectively. These DEGs were enriched in various immune pathways activated by the fungal infection, such as the pathways of complement and coagulation cascades, protein digestion and absorption, and drug metabolism - cytochrome P450. We found that 24 hptI was critical either for the cuticular penetration of B. bassiana or for the initial activation of the host defense system. The host immune response peaked at 36 hptI so that multiple defense mechanisms were activated against the fungal entry into the host hemocoel. At 48 hptI, many host genes involved in immunity and metabolism were downregulated, suggesting a success of fungal localization in the host hemocoel by overcoming the host defense reaction. Finally, we revealed that several fungal pathways could play important roles in the host-pathogen interaction, such as antioxidant activity, peroxidase activity and proteolysis. Up to 1636 fungal genes were co-expressed at the three time points, and 116 of them encode putative secretion proteins. Our results provide a novel insight into the pathogen-insect interaction and help to probe molecular mechanisms involved in the control of P. xylostella by B. bassiana. Here we constructed three pairs of digital expression libraries (transcriptomes) of Plutella xylostella (global lepidopteran pest) larvae 24, 36 and 48 hours post treatment of infection (hptI) and control (hptC) to reveal the host response to B. bassiana infection at genomic level

Dataset Information

DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro.

Publications

Genomic DNA microarray comparison of gene expression patterns in Paracoccidioides brasiliensis mycelia and yeasts in vitro.

Similar Datasets

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