Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data

Lyne, Rachel; Burns, Gavin; Mata, Juan; Penkett, Chris; Rustici, Gabriella; Chen, Dongrong; Langford, Cordelia; Vetrie, David; Bähler, Jürg

doi:10.1186/1471-2164-4-27

Cited by 193 publications

(169 citation statements)

References 36 publications

(42 reference statements)

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“…RNA preparation, labeling, microarray production, and analysis are described in ref. 43. Microarrays were scanned with a Genepix 4000B scanner and analyzed with Genepix software (Axon Instruments, Union City, CA).…”

Section: Methodsmentioning

confidence: 99%

Global roles of Ste11p, cell type, and pheromone in the control of gene expression during early sexual differentiation in fission yeast

Mata

Bähler

2006

Proc. Natl. Acad. Sci. U.S.A.

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125

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Fission yeast cells belong to one of two specialized cell types, M or P. Specific environmental conditions trigger sexual differentiation, which leads to an internal program starting with pheromone signaling between M and P cells, followed by mating, meiosis, and sporulation. The initial steps of this process are controlled by Ste11p, a master transcriptional regulator that activates the expression of cell type-specific genes (only expressed in either M or P cells) as well as genes expressed in both M and P cells. Pheromone signaling is activated by Ste11p-dependent transcription and, in turn, enhances some of this transcription in a positive feedback. To obtain a genomewide view of Ste11p target genes, their cell-type specificity, and their dependence on pheromone, we used DNA microarrays along with different genetic and environmental manipulations of fission yeast cells. We identified 78 Ste11p-dependent genes, 12 and 4 of which are only expressed in M and P cells, respectively. These genes show differing grades of pheromone dependencies for Ste11p-activated transcription, ranging from complete independence to complete dependence on pheromone. We systematically deleted all novel cell type-specific genes and characterized their phenotype during sexual differentiation. A comparison with a similar data set from the distantly related budding yeast reveals striking conservation in both number and types of the proteins that define cell types. Given the divergent mechanisms regulating cell type-specific gene expression, our results highlight the plasticity of regulatory circuits, which evolve to allow adaptation to changing environments and lifestyles.expression profiling ͉ master transcriptional regulator ͉ mating type ͉ microarray ͉ Schizosaccharomyces pombe C ellular differentiation is driven to a large extent by specific programs of gene expression. Yeast has distinct cell types that are required for mating, meiosis, and sporulation; this sexual differentiation provides a useful model system to understand the differentiation into specialized cell types in multicellular organisms. How the regulation of gene expression leads to specialized cell types is relatively well understood at a genomewide level in the budding yeast Saccharomyces cerevisiae (1). In this yeast, two haploid cell types, a and ␣, mate with each other at the first opportunity to form diploid a͞␣ cells, which can undergo meiosis and sporulation under appropriate environmental conditions (reviewed in ref.2).Cells of the fission yeast Schizosaccharomyces pombe also come in two specialized cell types, called P (or hϩ) and M (or h-) mating types. Heterothallic strains have a fixed mating type (P or M), whereas homothallic strains (h90) can switch between both types. Unlike budding yeast, however, fission yeast cells will mate only under specific environmental conditions, most notably nitrogen starvation; the resulting diploid cells then immediately undergo meiosis and sporulation (reviewed in refs. 3 and 4). The two distantly related yeasts thus provid...

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Section: Methodsmentioning

confidence: 99%

Global roles of Ste11p, cell type, and pheromone in the control of gene expression during early sexual differentiation in fission yeast

Mata

Bähler

2006

Proc. Natl. Acad. Sci. U.S.A.

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125

151

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“…To avoid perturbation from stress response induced by higher cell density, 4 we maintained the culture density at A 600 Յ 0.5 by diluting the culture when harvesting cells at 1-h post 3AT treatment if necessary. 50 ml of cells were harvested before or 1 and 3 h after the 3AT treatment and then total RNA was isolated using a hot-phenol protocol (27). We used DNA microarrays carrying all known and predicted S. pombe genes.…”

Section: Methodsmentioning

confidence: 99%

“…20 g of total RNA was labeled by either Cy3-dCTP (reference RNA: wild-type time 0 RNA) or Cy5-dCTP (sample RNAs). Hybridization and initial data analysis were performed as previously described (27). The normalized data were analyzed with GeneSpring software (Agilent).…”

Section: Methodsmentioning

confidence: 99%

“…This suggests that, in S. pombe, longer periods of exposure to 3AT can trigger stress arrangements that induce eIF2 phosphorylation, extending beyond histidine starvation. Importantly, the finding of similar (or slightly stronger) eIF2␣ The following primers were designed by the Bähler group in their microarray studies (27).…”

Section: Genome-wide Expression Profiles Of Fission Yeast In Responsementioning

confidence: 99%

“…A, strains used in Fig. 1B were cultured in EMM-C and treated with 30 mM 3AT for the indicated times, followed by RNA extraction and cDNA microarray analysis (27). Shown is the clustering of genes whose transcripts were changed Ն2-fold by 3AT-induced starvation in wild-type cells.…”

Section: Transcriptional Profiling Of Int6⌬ and Gcn2⌬ Mutants Identifmentioning

confidence: 99%

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Int6/eIF3e Promotes General Translation and Atf1 Abundance to Modulate Sty1 MAPK-dependent Stress Response in Fission Yeast

Udagawa

Nemoto

Wilkinson

et al. 2008

Journal of Biological Chemistry

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int-6 is one of the frequent integration sites for mouse mammary tumor viruses. Although its product is the e-subunit of translation initiation factor eIF3, other evidence indicates that it interacts with proteasomes or other proteins to regulate protein stability. Here we report that the fission yeast int6؉ is required for overcoming stress imposed by histidine starvation, using the drug 3-aminotriazole (3AT). Microarray and complementary Northern studies using wild-type, int6⌬ or gcn2⌬ mutants indicate that 3AT-treated wild-type yeast induces core environmental stress response (CESR) genes in addition to typical general amino acid control (GAAC) genes whose transcription depends on the eIF2 kinase, Gcn2. In agreement with this, Sty1 MAPK and its target transcription factor Atf1, which signal the CESR, are required for overcoming 3AT-induced starvation. We find that Int6 is required for maintaining the basal level of Atf1 and for rapid transcriptional activation of the CESR on 3AT-insult. Pulse labeling experiments indicate that int6⌬ significantly slows down de novo protein synthesis. Moreover, Atf1 protein half-life was reduced in int6⌬ cells. These effects would account for the compromised Atf1 activity on 3AT-induced stress. Thus, the robust protein synthesis promoted by intact eIF3 appears to be a part of the requisites for sound Sty1 MAPK-dependent signaling governed by the activity of the Atf1 transcription factor.Mouse mammary tumor virus integrates into the mouse genome, altering proteins or their expression and thereby eliciting mammary tumors. The sites of mouse mammary tumor virus integration were identified to delineate the molecular basis of the mammary tumorigenesis (see Refs. 1 and 2 for reviews). These so-called int sites include members of the Wnt (Wnt-1/int-1, Wnt-3, and Wnt-10b) and Fgf families (Fgf-3/ int-2, Fgf-4/hst, and Fgf-8/AIGF), Notch-4/int-3 and eIF3e/ int-6. Thus, all of the Int proteins except Int-6 are a well characterized growth factor or transmembrane receptor. Int-6 is an abundant protein and found to be identical to the e (p48)-subunit of eukaryotic translation initiation factor-3 (eIF3), 3 a multisubunit protein (of 11-13 subunits in mammals and plants) required for initiating protein synthesis (3). The eIF3 directly binds to the 40 S ribosomal subunit and mediates Met-tRNA i Met and mRNA binding to the ribosome (4). Int-6/eIF3e was recently determined to be a part of the functional core of mammalian eIF3, comprising only 6 subunits (5). Curiously, eIF3 isolated from the budding yeast Saccharomyces cerevisiae does not contain the homologue of Int-6/eIF3e (6, 7). Yet, eIF3 isolated from the fission yeast Schizosaccharomyces pombe includes its homologue Int6 (also known as Yin6) (8, 9). In addition to its role in translation, human Int-6 appears to regulate protein stability by directly binding to HIF2␣ (10) and MCM7 (11). In fission yeast, Int6 interacts with the 26 S proteasome to promote ubiquitin-dependent degradation of cyclin/Cdc13 and securin/Cut2 (12). However, the me...

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Toward an atomistic model for predicting transcription‐factor binding sites

Endres

Schulthess

Wingreen³

2004

Proteins

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Identifying the specific DNA-binding sites of transcription-factor proteins is essential to understanding the regulation of gene expression in the cell. Bioinformatics approaches are fast compared to experiments, but require prior knowledge of multiple binding sites for each protein. Here, we present an atomistic force-field method to predict binding sites based only on the X-ray structure of a related bound complex. Specific flexible contacts between the protein and DNA are modeled by a library of amino acid side-chain rotamers. Using the example of the mouse transcription factor, Zif268, a well-studied zinc-finger protein, we show that the protein sequence alone, without the detailed experimental structure, gives a strong bias toward the consensus binding site.

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Whole-genome microarrays of fission yeast: characteristics, accuracy, reproducibility, and processing of array data

Cited by 193 publications

References 36 publications

Global roles of Ste11p, cell type, and pheromone in the control of gene expression during early sexual differentiation in fission yeast

Global roles of Ste11p, cell type, and pheromone in the control of gene expression during early sexual differentiation in fission yeast

Int6/eIF3e Promotes General Translation and Atf1 Abundance to Modulate Sty1 MAPK-dependent Stress Response in Fission Yeast

Toward an atomistic model for predicting transcription‐factor binding sites

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