Unknown,Transcriptomics,Genomics,Proteomics

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Chlamydomonas ecoevo v2

ABSTRACT: Chemostat experiments: Predator-prey dynamics were followed in four replicate chemostats (continuous-flow aquatic culture vessels) supplied with nitrogen-limited sterile medium that was otherwise sufficient in all nutrient constituents (Yoshida et al. 2003; Becks et al. 2010). Three chemostats contained 380 mL of medium, while the fourth, used for our algal gene expression study, contained 3000 mL so as to provide sufficient material for genetic analysis. Brachionus calyciflorus was isolated originally from Milwaukee harbor, Wisconsin, and provided by M. Boraas. Chlamydomonas reinhardtii was obtained from the University of Texas algal culture collection (UTEX 89). Both species reproduced asexually in our chemostat system. Though B. calyciflorus is naturally cyclically parthenogenetic, the rotifers in our stock culture have evolved not to produce males because the bubbling in our chemostats prevents mating; we used only one mating type of Chlamydomonas. Absence of sexual reproduction does not preclude the processes of microevolution (Bell 2009) that we study, and it is commonly accepted that asexual species can evolve (Lenski et al. 1991; Barrett et al. 2005). We used a Chlamydomonas lineage, founded from our original Chlamydomonas stock and then exposed continuously to Brachionus predation for six months in chemostat culture prior to this study during which Chlamydomonas evolved to form clumps (the 'grazed Chlamydomonas' lineage in Becks et al. 2010). We tracked changes in Chlamydomonas gene expression throughout two complete cycles of algal and rotifer abundance and concurrent cycles in algal defense. Algal samples were taken from one chemostat at intervals of approximately every ten days (8 dates total), chosen to capture critical transitions in cell clumping. A sample taken at the start of the experiment from the culture that was used to inoculate the chemostat was the standard against which algae from later samples were compared (i.e., the M-^Sgrazed ChlamydomonasM-^T). To obtain sufficient material for microarray analysis, ca. 1 - 1.5 L algal samples were collected and filtered through a 10-um mesh to exclude rotifers and rotifer eggs. The algal samples of ca. 100 ml of chemostat medium were concentrated by centrifugation and frozen. Samples were shock frozen in liquid nitrogen and transferred to -80C.

ORGANISM(S): Chlamydomonas reinhardtii

SUBMITTER: Lutz Becks

PROVIDER: E-MEXP-3562 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Despite the scientific and applied interest in anaerobic metabolism of Saccharomyces cerevisiae, not all genes whose transcription is up-regulated under anaerobic conditions have yet been linked to known transcription factors. Experiments with a reporter construct in which the promoter of the anaerobically up-regulated TIR1 gene was fused to LacZ revealed a complete loss of anaerobic up-regulation in a snf7Δ mutant. Anaerobic up-regulation was restored by expression of a truncated allele of RIM101 that encodes for a constitutively active Rim101p transcription factor. Analysis of LacZ expression in several deletion mutants confirmed that the effect of Snf7p on anaerobic up-regulation of TIR1 involved Rim101p and did not require a functional multi-vesicular body sorting pathway (in which Snf7p also participates). Transcriptome analysis in anaerobic chemostat cultures revealed that 26 additional genes exhibited a Snf7p/Rim101p dependent anaerobic up-regulation. Since, in its activated form, Rim101p is generally known as a transcriptional repressor, its role in anaerobic up regulation of TIR1 and other ‘anaerobic’ yeast genes must involve additional factors. Further studies with deletion mutants in NRG1, NRG2 and SMP1, which were previously shown to be regulated by Rim101p, showed that these genes were not involved in the regulation of TIR1. However, the aerobic repression mechanism of TIR1 involved the general repressor Ssn6p-Tup1p complex. The physiological relevance of Snf7p/Rim101p-mediated transcriptional up-regulation of several genes in anaerobic yeast cultures was evident from reduced growth of a snf7Δ under anaerobic conditions. Experiment Overall Design: The aim of the present study is to investigate the role of SNF7 in the regulation of TIR1, to assess whether this role involves the Rim101p pathway, and to investigate whether Snf7p and/or Rim101p are also involved in regulation of other ‘anaerobic’ S. cerevisiae genes. After analyzing transcriptional regulation of TIR1 in several genetic backgrounds, a chemostat-based transcriptome analysis was performed on snf7Δ and rim101Δ strains as well as on the isogenic reference strain. Sets of genes that were differentially expressed in the snf7 and rim101 deletion strains were then compared to sets of genes that were previously shown to be transcriptionally up-regulated in anaerobic chemostat cultures of S. cerevisiae (Piper et al., 2002; Tai et al., 2005) Experiment Overall Design: Piper MD, Daran-Lapujade P, Bro C, Regenberg B, Knudsen S, Nielsen J & Pronk JT (2002) Reproducibility of oligonucleotide microarray transcriptome analyses. An interlaboratory comparison using chemostat cultures of Saccharomyces cerevisiae. J Biol Chem 277: 37001-37008. Experiment Overall Design: Tai SL, Boer VM, Daran-Lapujade P, Walsh MC, de Winde JH, Daran JM & Pronk JT (2005) Two-dimensional transcriptome analysis in chemostat cultures. Combinatorial effects of oxygen availability and macronutrient limitation in Saccharomyces cerevisiae. J Biol Chem 280: 437-447.

Project description:Cells must adjust their gene expression in order to compete in a constantly changing environment. Two alternative strategies could in principle ensure optimal coordination of gene expression with physiological requirement. First, the internal physiological state itself could feedback to regulated gene expression. Second, the expected physiological state could be inferred from the external environment, using evolutionary-tuned signaling pathways. Coordination of ribosomal biogenesis with the requirement for protein synthesis appears to be particularly important, since cells devote a large fraction of their biosynthetic capacity for ribosomal biogenesis. To define the relative importance of internal vs. external sensing to the regulation of ribosomal biogenesis gene expression, we subjected S. cerevisiae cells to conditions which decoupled the actual vs. environmentally-expected growth rate. Gene expression followed the environmental signal according to the expected, but not the actual, growth rate. Simultaneous monitoring of gene expression and growth rate in chemostat-grown cultures further confirmed that ribosome biogenesis genes responded rapidly to changes in the environments but were oblivious to longer-term changes in growth rate. Our results suggest that the capacity to anticipate and prepare for environmental changes presented a major selection force during yeast evolution. Keywords: Saccharomyces_Cerevisiae, Stress response, ADH1 deletion, time courses, chemostat Experiment 1: ADH1 deletion cells, which grow faster on glycerol rather then glucose medium, were grown in both glucose and glycerol medium. The cells expression on glycerol is compared to their expression glucose (5 microarrays). Experiment 2: Time course of ADH1 deletion cells upon growth on glycerol and on glucose (7 microarrays). Experiment 3: Response of steady state grown cells to environmental perturbations. Cells were grown in glucose or histidine limited chemostats and reached steady state. The cells were then subjected to perturbations such as DTT, heat shock, NaCl, Clotrimazole, H2O2, and addition of limiting factors (histidine and glucose, respectively). Overall we have examined 10 timecourses with 83 microarrays.

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Chlamydomonas ecoevo v2

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