ABSTRACT: Two sets of microarray experiments examining the transcriptional response to Ni deprivation. One set was for growth on ammonium and the other for growth on urea Pairwise replicated comparisons (Ni deprivation in urea and ammonia)
Project description:au11-02_cho-thf - shoots and roots treated with methotrexate and 5-formyl-thf - Folates and 1C metabolism - The objective of this study is to investigate the changes on the plant expression profiles due to 5-CHO-THF assimilation and transformation. Additionally, plants supplemented with methotrexate (MTX) (an inhibitor of folate biosynthesis) will complement the study. Results from this work will serve to better understand the effects of folate accumulation in plants. 12 dye-swap - treated vs untreated comparison
Project description:fapesp-bra-inra-10-01_bioen_hypocotyl - dark hypocotyls tor rnai - Transcriptional comparison between 2 TOR RNAi mutants versus GUS control. - Sowing after 24h imbibition at 4M-BM-0C in the dark, on MS1/5, no sucrose, 10 mM ethanol, 8 g/l agar, vertical growth with 3h light, 6 days growth in the dark (20M-BM-0C), hypocotyls were harvested under green light ; cotyledons and root were removed. 4 dye-swap - normal vs rnai mutant comparaison
Project description:Curli are adhesive fimbriae of Enterobactericaeae and are involved in surface attachment, cell aggregation and biofilm formation. We previously reported that natural curli variants of E. coli O157:H7 (EcO157) displayed distinct acid resistance; however, this difference was not linked to the curli fimbriae per se. Here, we investigated the underlying molecular basis of this phenotypic divergence between the curli variants. Among curli-producing (C+) variants isolated from the 1993 U.S. hamburger-associated outbreaks strains, we identified large deletions in the rcsB gene that encodes the response regulator of RcsCDB two-component signal transduction system of rcsB ,. Further comparison of stress fitness revealed that C+ variants were also significantly more sensitive to heat shock, but remained similar resistance to osmotic stress and oxidative damage as curli-deficient (C-) variants. Transcriptomics analysis uncovered a large number of differentially expressed genes between the curli variants, characterized by the enhanced expression of genes related to biofilm formation, virulence, catabolic activity and nutrients uptake, but marked decrease in transcription of genes related to various stress resistance in C+ variants. Supplying C+ variants with a functional rcsB restored cells resistance to heat shock and acid challenge, but blocked the curli production, confirming that inactivation of RcsB in C+ variants was the basis of fitness segregation within the EcO157population. This study provides an example of how genome instability of EcO157promotes the intra-population diversification, generating sub-populations carrying an array of distinct phenotypes that may confer the pathogen survival advantages in host and nonhost environments. Three replicates for isolates RM6607R, RM6607W, RM6608R, RM6608W independently grown from single colonies in LB broth and incubated at 28M-BM-0C overnight on a shaker (150 rpm). The cells were collected by centrifugation, washed once in LBNS broth, and inoculated in 25ml of LBNS broth with a concentration equivalent to 0.001OD600 ml-1. The cultures were incubated at 28M-BM-0C for 24 h with aeration (150 rpm). At the end of incubation, ice-cold phenol-ethanol (5%:95%) solution was immediately added to culture at a final concentration of 20% (volume), and the mixture was incubated on ice for 30 min. The cells were collected by centrifugation at 4M-BM-0C and the pellets were stored at -80M-BM-0C until RNA extraction. The cells were collected by centrifugation, washed once in LBNS broth, and inoculated in 25ml of LBNS broth with a concentration equivalent to 0.001OD600 ml-1. The cultures were incubated at 28M-BM-0C for 24 h with aeration (150 rpm). At the end of incubation, ice-cold phenol-ethanol (5%:95%) solution was immediately added to culture at a final concentration of 20% (volume), and the mixture was incubated on ice for 30 min. The cells were collected by centrifugation at 4M-BM-0C and the pellets were stored at -80M-BM-0C until RNA extraction. RNA was isolated using Promega SV Total RNA kit. A type 2 gene expression experimental design was used, with fluorescently labeled genomic DNA as a reference channel in each experiment as described by Lucchini, S., et al. 2005. Infect Immun 73:88-102.
Project description:Investigation of differentially expressed genes in human HCT116 cells after knockdown of FBXO28 for 16h and 36h. FBXO28 knockdown and control HCT116 cells. 4 replicates per time point (16h, 36h), including dye swaps.
Project description:Murine bronchioalveolar stem cells play a key role in pulmonary epithelial maintenance and repair but their molecular profile is poorly described so far. In this study, we used antibodies directed against Sca-1 and CD34, two markers originally ascribed to pulmonary cells harboring regenerative potential, to isolate single putative stem cells from murine lung tissue. The mean detection rate of positive cells was 8 per 106 lung cells. We then isolated and globally amplified the mRNA of positive cells to analyze gene expression in single cells. The resulting amplicons were then used for molecular profiling by transcript specific polymerase chain reaction (PCR) and global gene expression analysis using microarrays. Single marker-positive cells displayed a striking heterogeneity for the expression of epithelial and mesenchymal transcripts on the single cell level. Nevertheless, they could be subdivided into two cell populations: Sca-1+/CD34- and Sca-1+/CD34+ cells. In these subpopulations, transcripts of the epithelial marker Epcam (CD326) were exclusively detected in Sca-1+/CD34- cells (p = 0.03), whereas mRNA of the mesenchymal marker PdgfrM-NM-1 (CD140a) was detected in both subpopulations and more frequently in Sca-1+/CD34+ cells (p = 0.04). FACS analysis confirmed the existence of a PdgfrM-NM-1 positive subpopulation within Epcam+/Sca-1+/CD34- epithelial cells. Gene expression analysis by microarray hybridization identified transcripts differentially expressed between the two cell types as well as between epithelial reference cells and Sca-1+/CD34+ single cells, and selected transcripts were validated by quantitative PCR. Our results suggest a more mesenchymal commitment of Sca-1+/CD34+ cells and a more epithelial commitment of Sca-1+/CD34- cells. In summary, the study shows that single cell analysis enables the identification of novel molecular markers in yet poorly characterized populations of rare cells. Our results could further improve our understanding of Sca-1+/CD34+,- cells in the biology of the murine lung. Single cells of 10 Sca-1+/CD34+/CD31-/CD45-, 7 Sca-1+/CD34-/CD31-/CD45-, and 12 Sca-1-/CD34-/CD31-/CD45- were analyzed. Although the raw data are two channel only Cy5 signal of each file as analyzed. The Cy5 channel for each gene is normalized to the average Cy5 intensity of the gene across all samples.
Project description:Gene expression analysis of murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. MC3T3-E1 cells were cultured in vitro for up to 28 days in the presence of 8-glycerol phosphate and ascorbic acid to induce osteoblastic differentiation. Total RNA was isolated after 2, 5, 10 and 28 days in culture to sample proliferating preosteoblasts (Day 2), growth arrested preosteoblasts (Day 5), differentiating osteoblasts (Day 10), and mature osteoblasts (Day 28). Timing of sample collection was based on direct measurements of cell number, alkaline phosphatase expression, type 1A collagen synthesis, and matrix mineralization in parallel cultures. Triplicate samples from each time point were hybridized to slide arrays printed with the Operon Mouse Oligo set, version 2.0.
Project description:Effect of the inactivation of locus PP4959 upon gene expression of Pseudomonas putida KT2440 in the stationary phase of growth in rich medium LB. This locus encodes the unique dual GGDEF/EAL domains response regulator in KT2440. To identify those genes with altered expression, cells were cultivated in LB at 24 M-:C to reach a turbidity at 660 nm of 3.3.
Project description:WD40 motif-containing Msi1-like (MSIL) proteins play pleiotropic cellular functions as a negative regulator of the Ras/cAMP-pathways and a component of chromatin assembly factor-I (CAF-I), and yet have not been studied in fungal pathogens. Here we identified and characterized an MSIL protein, Msl1, in Cryptococcus neoformans, which can cause fatal meningoencephalitis in humans. Notably, Msl1 was not a functional ortholog for the yeast Msi1 but played pleiotropic roles in C. neoformans in both cAMP-dependent and -independent manners but mainly Ras-independently. Msl1 negatively controlled antioxidant melanin production and sexual differentiation, which can be repressed by inhibiting the cAMP-signaling pathways. In contrast, Msl1 controlled thermotolerance, diverse stress responses, and antifungal drugs resistance in Ras/cAMP-independent manners. Cac2, which is the second CAF-I component, appeared to play both redundant and distinct function with Msl1. Msl1 is required for full virulence of C. neoformans. Transcriptome and proteomic analysis identified a group of Msl1-regulated genes or -interacting proteins, respectively, which mostly include stress-related genes, including HSP12, HSP78, SSA1, SSA4, and STM1. Furthermore, we identified the third putative component of CAF-1, Rlf2, in C. neoformans. In conclusion, this study demonstrated the pleiotropic roles of Msl1 in human fungal pathogen C. neoformans, providing a novel antifungal therapeutic target. There is more than 95% genome homology between JEC21 and H99. Therefore, 6 slides of JEC21 (Cryptococcus neoformans var. neoformans serotype D) 70-mer oligos are used in this analysis. Total RNAs are extracted from 2 strains from H99 (H99 wild-type strain (Cryptococcus neoformans var. grubii serotype A), msl1M-NM-^T). 3 biological replicate experiments are performed for each strain. We use the mix of all total RNAs from this experiment as the control RNA. We use Cy3 as the test sample dye and Cy5 as the control dye.
Project description:Salvia is an important genus from the Lamiaceae with approximately 1000 species distributed globally. Several Salvia species are commercially important because of their medicinal and culinary properties. We report the construction of the first fingerprinting array for Salvia species enriched with polymorphic and divergent DNA sequences and demonstrate the potential of this array for fingerprinting several economically important members of this genus. In order to generate the Salvia Subtracted Diversity Array (SDA), a Suppression Subtractive Hybridization (SSH) was performed between a pool of ten Salvia species and a pool of non-angiosperm and angiosperms (excluding the Lamiaceae) to selectively isolate Salvia-specific sequences. A total of 285 subtracted genomic DNA (gDNA) fragments were amplified and arrayed. DNA fingerprints were obtained for fifteen Salvia genotypes including three that were not part of the original subtraction pool. Hierarchical cluster analysis indicated that the Salvia-specific SDA was capable of differentiating closely related species of S. officinalis and S. miltiorrhiza and was also able to reveal genetic relationships consistent with geographical origins. Species-specific features were also found for S. elegans, S. officinalis, S. sclarea, S. przewalskii and S. runcinata.
Project description:Although Candida species, more specifically C. albicans, are the most common pathogenic yeasts, little is known about the mechanism involved in eicosanoid production, a virulence factor in these organisms. This is an important area of research which may aid in the understanding of the complex interactions between host and pathogen and may possibly lead to the identification of novel antifungals or drug targets. In this study, genomic hybridization studies using C. albicans DNA microarrays were used to evaluate the regulation of C. albicans biofilm genes during incubation in the presence of arachidonic acid, the major precursor for prostaglandin E2 production. The results obtained indicated that the genes differentially expressed had diverse functions not normally required for cell growth. Two-condition experiment, Control biofilms vs. Arachidonic acid treated biofilms at different time intervals. Biological replicates: 3 Control, 3 Arachidonic acid treated, independently grown and harvested. Technical replicate: dye swap included. Triplicate per array.