Cryptococcus neoformans gene expression in minimal media of wild type, ALL2 mutant, and ALL1ALL2 mutant cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Cryptococcus neoformans
SUBMITTER: Jinsheng Yu
PROVIDER: E-GEOD-74219 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:To examine the comparative transcription profile of WT and ALL1 mutant cells in in vitro conditions to determine genes resposible for hypervirulent phenotype of ALL1 mutant cells. Using transcriptional profiling, we determined that many genes involved in oxidation reduction processes and iron homeostasis were differentially transcribed between the wild type and the mutant strain For arrays, total RNA was isolated from WT and ALL1 mutant using Qiagen RNeasy Kit. For RNA extraction, Wt and ALL1 mutant were grown in two different culture medium conditions including minimal media and YPD broth. Cells were grown for overnight, diluted 1:100 and then again grown overnight or up to an OD of 0.6-0.7 with agitation (150rpm) at 37°C.
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Project description:Little is known about transcriptional control of neurite branching or presynaptic differentiation, events that occur relatively late in neuronal development. Using the C. elegans mechanosensory circuit as an in vivo model, we show that SAM-10, an ortholog of mammalian Single-Stranded DNA binding Protein (SSDP), functions cell-autonomously in the nucleus to regulate synaptic differentiation as well as positioning of a single neurite branch. PLM mechanosensory neurons in sam-10 mutants exhibit abnormal placement of the neurite branch point, and defective synaptogenesis, characterized by an overextended synaptic varicosity, underdeveloped synaptic morphology and disrupted co-localization of active zone and synaptic vesicles. SAM-10 functions coordinately with LDB-1 (Lim Domain Binding protein-1), demonstrated by our observations that 1) mutations in either gene show similar defects in PLM neurons; and 2) LDB-1 is required for SAM-10 nuclear localization. SAM-10 regulates PLM synaptic differentiation by suppressing transcription of prk-2, which encodes an ortholog of the mammalian Pim kinase family. PRK-2-mediated activities of SAM-10 are specifically involved in PLM synaptic differentiation, but not other sam-10 phenotypes such as neurite branching. Thus, these data reveal a novel transcriptional signaling pathway that regulates neuronal specification of neurite branching and presynaptic differentiation. Animals were grown on fresh 8P plates (Wormbook.org) until gravid. Eggs were collected by bleaching and hatched in M9 buffer overnight. L1 animals were fed on fresh food for 2 hours at room temperature, harvested and separated from worm debris using a 25μm mesh. RNA was isolated using a standard Trizol protocol. RNA preps were stored at -80C. Quality of RNA preps was first estimated using Agilent 2100 Bioanalyzer. RNA was then reverse-transcribed using the Genisphere Array 350 kit, which generate tagged cDNA. Resulted products are hybridized to the microarrays. Four independent high quality RNA preps (RIN>=7) were chosen for microarray assay using long oligomer-based spotted microarray (Washington University, St. Louis, MO). For dye-swap controls, we ran a technical replicate hybridization for each pair of samples in which the dyes are reversed. Gene spots with “found/good” signals (defined by ScanArray, channel flag 3) in all scans were chosen for gene expression analysis.
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Project description:To examine the comparative transcription profile of WT and all1all2Πmutant cells in in vitro conditions to determine genes resposible for hyporvirulent phenotype of all1all2Πmutant cells. Using transcriptional profiling, we determined that many genes involved in amino acid transport were differentially transcribed between the wild type and the all1all2Πmutant strain. The genes differentially expressed in double mutant of ALL1 ALL2 did not overlap with ALL1 and ALL2 comparative transcriptomes with RC2 (WT). For arrays, total RNA was isolated from WT and all1all2Πmutant using Qiagen RNeasy Kit. For RNA extraction, WT and all1all2Πmutant were grown in minimal media. Cells were grown for overnight, diluted 1:100 and then again grown overnight or up to an OD of 0.6-0.7 with agitation (150rpm) at 37°C.
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Project description:Virulence of Cryptococcus neoformans for mammals was proposed to emerge from evolutionary pressures on its natural environment by protozoan predators, which selected for strategies that allow survival within macrophages. In fact, Acanthamoeba castellanii ingests yeast cells, which then replicate intracellularly. In addition, most fungal factors needed to establish infection in the mammalian host are also important for survival within the amoeba. To better understand the origin of C. neoformans virulence, we compared the transcriptional profile of yeast cells internalized by amoebae and murine macrophages after 6 h of infection. Our results showed 656 and 293 genes whose expression changed at least two-fold in response to the intracellular environments of amoebae and macrophages, respectively. Among the genes common to both groups, we focused on the ORF CNAG_05662, which was potentially related to sugar transport. We constructed a mutant strain and evaluated its ability to grow on various carbon sources. The results showed that this gene, named PTP1 (Polyol Transporter Protein 1), is involved in the transport of 5- and 6-carbon polyols but its absence had no effect on virulence. Overall, our results are consistent with the hypothesis that mammalian virulence originated from fungal-protozoal interactions and provide a better understanding of how C. neoformans adapts to the mammalian host. Four conditions, pairwise-compared: cells in vegetative growth at 28C versus cells within amoebae at 28C; and cells in vegetative growth at 37C/5% CO2 versus cells within macrophages at 37C/5% CO2. Three biological replicates for each condition. One replicate per array.
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Project description:To examine the comparative transcription profile of WT and all2Πmutant cells in in vitro conditions to determine genes responsible for hypervirulent phenotype of all2Πcells. Using transcriptional profiling, we determined that many genes involved in transport were differentially transcribed between the wild type and the mutant strain. With the help of further experiments we could rule out that ALL2 has a unique function in maintaining the intracellular pH under acidic conditions. For arrays, total RNA was isolated from WT and all2Πmutant using Qiagen RNeasy Kit. For RNA extraction, WT and all2Πmutant were grown in minimal media. Cells were grown for overnight, diluted 1:100 and then again grown overnight or up to an OD of 0.6-0.7 with agitation (150rpm) at 37°C.
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
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