Project description:Population adaptation to strong selection can occur through the sequential or parallel accumulation of competing beneficial mutations. The dynamics, diversity and rate of fixation of beneficial mutations within and between populations are still poorly understood. To study the changes in the mutational landscape across populations during adaptation, we performed experimental evolutions on seven parallel populations of Saccharomyces cerevisiae continuously cultured in limiting sulfate medium. By combining qPCR, array CGH, restriction, digestion and CHEF gels, and whole genome sequencing, we followed the trajectory of evolution to determine the identity and fate of beneficial mutations. Over a period of 200 generations, the yeast populations displayed parallel evolutionary dynamics that are driven by the coexistence of independent beneficial mutations. Segmental amplifications are rapidly gained under this selective pressure, including, common inverted amplifications containing the sulfate transporter gene SUL1. Detailed analysis of the populations uncovers a deep complexity where by multiple subpopulations arise and compete with each another. The most common trajectories to adaptation in these populations are incomplete soft sweeps, with adaptive variants replacing one another. These are CGH arrays. Each experiment compares the DNA content of an experimentally evolved strain with its ancestor.
Project description:The availability of human genome sequence has transformed biomedical research over the past decade. However, an equivalent map for the human proteome with direct measurements of proteins and peptides was lacking. To this end, Akhilesh Pandey's lab reported a draft map of the human proteome based on high resolution Fourier transform mass spectrometry-based proteomics technology, which included an in-depth proteomic profiling of 30 histologically normal human samples including 17 adult tissues, 7 fetal tissues and 6 purified primary hematopoietic cells ( http://dx.doi.org/10.1038/nature13302 ). The profiling resulted in identification of proteins encoded by greater than 17,000 genes accounting for ~84% of the total annotated protein-coding genes in humans. This large human proteome catalog (available as an interactive web-based resource at http://www.humanproteomemap.org) complements available human genome and transcriptome data to accelerate biomedical research in health and disease. Pandey's lab and collaborators request that those considering use of this primary dataset for commercial purposes contact pandey@jhmi.edu. The full details of this study can be found in the PRIDE database: www.ebi.ac.uk/pride/archive/projects/PXD000561/. This ArrayExpress entry represents a top level summary of the metadata only which formed the basis of the reanalysis performed by Joyti Choudhary's team ( jc4@sanger.ac.uk ), results of which are presented in the Expression Atlas at EMBL-EBI : http://www.ebi.ac.uk/gxa/experiments/E-PROT-1.
Project description:We Investigate of the mice hearts from embryonic day 10.5 to postnatal week 8 and reveal developmental changes in phosphoproteome, proteome encompassing cardiogenesis and cardiac maturation.
Project description:Primary hyperoxaluria type I (PH1) is a genetic disease caused by a deficiency in the peroxisomal alanine:glyoxylate aminotransferase (AGT) activity. Mutations in AGT mostly cause protein mistargeting and enhanced aggregation, although the molecular and structural basis of these mechanisms are unknown. In this work, we use hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) to provide novel insight into these pathogenic mechanisms. We characterize the wild-type (WT) protein, the LM variant (containing the mutations P11L and I340M, a haplotype more frequent in PH1 patients) and the LM G170R (the most common genotype in PH1, introducing the G170R mutation on the LM background). Our study provides the first experimental analysis of the local stability and dynamics of AGT, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants destabilize locally the structure. Enzymatic transamination of the PLP bound to AGT hardly affects stability. Our study thus supports that AGT misfolding is not caused by dramatic effects on the stability and dynamics of the holo-protein.
Project description:We employed CapitalBio Corporation to investigate the global transcriptional profiling of Saccharomyces cerevisiae treated with thymol. Keywords: gene expression array-based, count S. cerevisiae strain L1190 was incubated in a rotary shaker. Subsequently, thymol was added to the culture and DMSO to the control. Then, the yeast cells were further incubated for a certain time and total RNA was extracted by a hot acidic phenol method. The experimental sample and control sample were incorporated and dissolved in hybridization solution after labelled. Arrays hybridization was preformed in a CapitalBio BioMixerTM II Hybridization Station overnight and washed with two consecutive solutions. Arrays were scanned with a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software. A space- and intensity-dependent normalization based on a LOWESS program was employed.
Project description:We employed CapitalBio Corporation to investigate the global transcriptional profiling of Saccharomyces cerevisiae treated with dictamnine. S. cerevisiae strain L1190 was incubated in a rotary shaker. Subsequently, dictamnine was added to the culture and DMSO to the control. Then, the yeast cells were further incubated for a certain time and total RNA was extracted by a hot acidic phenol method. The experimental sample and control sample were incorporated and dissolved in hybridization solution after labelled. Arrays hybridization was preformed in a CapitalBio BioMixerTM II Hybridization Station overnight and washed with two consecutive solutions. Arrays were scanned with a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software. A space- and intensity-dependent normalization based on a LOWESS program was employed.
Project description:Response of Saccharomyces cerevisiae to Ammonium, L-alanine, or L-glutamine Limitation. The protrotophic laboratory strain CEN.PK113-7D (MAT a) was grown in laboratory fermentors with a working volume of 1 litre at dilution rate (D) of 0.20 per hour (in triplicate for each nitrogen limited condition). At steady state, samples from each of the 12 continuous cultures were taken and cooled below 2 degree C within ten seconds by mixing 40% sample and 60% crushed ice.
Project description:To gain insights into molecular mechanisms of tolerance to heat stress, we conducted a transcript profiling experiment to identify heat-responsive genes in contrasting peanut mini core accessions, either un-acclimated or acclimated to heat stress. Plants at reproductive stage were exposed to 28 °C (control), 45 °C for 15 d (un-acclimated) or 45 °C for 1 d followed by 7 d recovery and 15 d stress (acclimated). Two contrasting genotypes showing diverse response to stress were selected based on a bioassay involving chlorophyll fluorescence yield under elevated respiratory demand and membrane thermostability. Transcript profiling was performed using 4 x 44k custom oligo microarrays containing 22k peanut EST sequences. The microarray analysis identified 710 stress-induced and 770 stress-repressed putative heat-responsive transcripts in the tolerant genotype. Gene enrichment analysis was performed using Blast2GO program and genes with homology to known proteins were categorized into detailed molecular functional groups. Majority of stress-responsive genes assigned to KEGG pathways belonged to starch, sucrose and galactose metabolism followed by amino acid metabolism, and secondary metabolite biosynthesis. Differentially expressed transcripts from samples obtained from first year’s experiment were validated in the samples from second year by quantitative real-time PCR. Transcripts of eight genes involved in terpenoid and flavanoid biosynthesis were induced after second and seventh day, respectively, in leaves under heat stress. Metabolite analysis confirmed increases in metabolites of selected pathways under heat stress. The heat up-regulated genes in tolerant COC041 mini-core accession are potential candidate genes for engineering stress-tolerant peanuts and unraveling molecular mechanisms of peanut adaptation to heat stress. We used Agilent peanut microarrays to identify putative heat stress-responsive genes. Directly heat-stressed leaf tissues of the peanut genotypes COC041 (tolerant) and COC166 (susceptible) were used in the study. Three replications of microarray experiments were carried out by hybridizing the cRNA from different time points and stress conditions in a loop design on 4 x 44k microarray.
Project description:We employed CapitalBio Corporation to investigate the global transcriptional profiling of Saccharomyces cerevisiae treated with allicin. S. cerevisiae strain L1190 was incubated in a rotary shaker. Subsequently, allicin was added to the culture and DMSO to the control. Then, the yeast cells were further incubated for a certain time and total RNA was extracted by a hot acidic phenol method. The experimental sample and control sample were incorporated and dissolved in hybridization solution after labelled. Arrays hybridization was preformed in a CapitalBio BioMixerTM II Hybridization Station overnight and washed with two consecutive solutions. Arrays were scanned with a confocal LuxScanTM scanner and the images obtained were then analyzed using LuxScanTM 3.0 software. A space- and intensity-dependent normalization based on a LOWESS program was employed.