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Affy data from WT, FHL1 deleted and FHL1,IFH1 double deleted strains.

ABSTRACT: Affymetrix experiment performed on RNA isolated from Wild type, FHL1 deleted and FHL1,IFH1 double deleted strains. Data aquired in duplicate. In S. cerevisiae the mRNAs from the 138 ribosomal protein (RP) genes are amongst the most abundant in the cell, and their transcription is regulated tightly so that they are the most prominent cluster in most transcriptome experiments. It has recently been observed that the proteins Fhl1p and Ifh1p are found almost exclusively at RP genes (Lee et al., Science, 298, 799-804, 2002;Jorgensen et al., Genes Dev, 18, 2491-2505 2004; Schawalder et al Nature in press; Rudra et al , EMBO J. submitted), and data suggests they are the true transcription factors. This experiment utilizes Affymetrix arrays to measure the level of all mRNAs in cells with a deletion of FHL1 or of both FHL1 and IFH1, compared to the wild type strain, W303. Such mutant cells are viable but grow very slowly (Hermann-Le Denmat S. et al., Mol Cell Biol, 14, 2905-2913, 1994; Cherel and Thuriaux, Yeast, 11, 261-270, 1995). Total RNA was made from log phase cells, amplified according to conventional methods, and hybridized to the array. (Since the total RNA/cell of the mutants is only a fifth of the wt, each mutant required five times the number of cells to provide the same amount of RNA.) The experiment was carried out in duplicate and the six tables provide the raw data from the wt, W303, delFHL1, and delFHL1, delIFH1 strains. Although we expected that the RP genes would be substantially reduced in these mutant cells, they are only slightly less abundant than normal in comparison to all other mRNAs! The interesting result is that the cells respond to a severe deficiency of ribosomes by reducing the level of nearly all mRNAs to match the capacity of the translational apparatus. Keywords = Ribosome Keywords = FHL1 Keywords = IFH1. Keywords: parallel sample

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE2096 | GEO | 2004/12/22

SECONDARY ACCESSION(S): PRJNA91789

REPOSITORIES: GEO

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Project description:Array design -Platform: amino-silane coated glass slides (GAPS II, Corning) -S. cerevisiae intergenic regions amplified from S288C genomic DNA (ResGen) using the intergenic region primer oligonucleotides (ResGen) (Harismendy et al. EMBO J. 22(18): 4738-4747, 2003). The primers allow the amplification of the sequence located on either side of elements such as open reading frames, tRNAs, small nuclear RNAs, Ty elements, solo δ, etc. A complete description of the primers can be obtained on the web site (ftp://ftp.resgen.com/pub/genepairs/yeast_intergenic). -The PCR products were purified and spotted using an automated arrayer (MicroGrid II, BioRobotics) Experiment design -Type of experiment: genome wide binding analysis -Experimental factor: genotype (Wild-Type, Fhl1-myc, Ifh1-myc); drug (rapamycin) -Number of hybridization performed in the experiment: 15 (four conditions with three to five biological replicates each) -Reference used for each condition: ChIP from the wild-type strain -Quality control: one dye-swap per condition Samples used, extract preparation and labeling -The origin of the biological sample: o Species: S. cerevisiae o Cell type: (1) W303; (2) W303+IFH1-13myc-TRP1MX6;(3) W303+FHL1-13myc-HIS3MX6. -Growth conditions: log phase (2x107/mL) at 30ºC in YPD medium; when indicated rapamycin was added at the final concentration of 200 ng/mL for 90 min. -Cross-linking for 30 min at 30ºC -Chromatin immunoprecipitation: Dynabeads M280 coupled to sheep anti-mouse IgG or sheep anti-rabbit IgG and mouse monoclonal antibodies against the myc epitope (culture supernatant of 9E10) were used for immunoprecipitation according to standard techniques. -Labeling protocol(s): each IP was blunt-ended, ligated to linker DNA, then subjected to Ligation Mediated (LM)-PCR in the presence of amino-allyl dUTP. Labeling was performed for 1 hour at room temperature in the presence of NHS-ester Cy3 or Cy5 in 0.1 M Sodium Carbonate. The labeling reaction was stopped with 2 M Hydroxylamine and the labeled DNA purified. Hybridization procedures and parameters -Standard protocols were used Measurement data and specifications -Software for scanning: GenePix Pro 4.0 (Axon Instruments, Inc.) -Scanner: GenePix 4000A (Axon Instruments, Inc.) Keywords: dose response

Project description:Array design -Platform: amino-silane coated glass slides (GAPS II, Corning) -S. cerevisiae intergenic regions amplified from S288C genomic DNA (ResGen) using the intergenic region primer oligonucleotides (ResGen) (Harismendy et al. EMBO J. 22(18): 4738-4747, 2003). The primers allow the amplification of the sequence located on either side of elements such as open reading frames, tRNAs, small nuclear RNAs, Ty elements, solo Î´, etc. A complete description of the primers can be obtained on the web site (ftp://ftp.resgen.com/pub/genepairs/yeast_intergenic). -The PCR products were purified and spotted using an automated arrayer (MicroGrid II, BioRobotics) Experiment design -Type of experiment: genome wide binding analysis -Experimental factor: genotype (Wild-Type, Fhl1-myc, Ifh1-myc); drug (rapamycin) -Number of hybridization performed in the experiment: 15 (four conditions with three to five biological replicates each) -Reference used for each condition: ChIP from the wild-type strain -Quality control: one dye-swap per condition Samples used, extract preparation and labeling -The origin of the biological sample: o Species: S. cerevisiae o Cell type: (1) W303; (2) W303+IFH1-13myc-TRP1MX6;(3) W303+FHL1-13myc-HIS3MX6. -Growth conditions: log phase (2x107/mL) at 30ÂºC in YPD medium; when indicated rapamycin was added at the final concentration of 200 ng/mL for 90 min. -Cross-linking for 30 min at 30ÂºC -Chromatin immunoprecipitation: Dynabeads M280 coupled to sheep anti-mouse IgG or sheep anti-rabbit IgG and mouse monoclonal antibodies against the myc epitope (culture supernatant of 9E10) were used for immunoprecipitation according to standard techniques. -Labeling protocol(s): each IP was blunt-ended, ligated to linker DNA, then subjected to Ligation Mediated (LM)-PCR in the presence of amino-allyl dUTP. Labeling was performed for 1 hour at room temperature in the presence of NHS-ester Cy3 or Cy5 in 0.1 M Sodium Carbonate. The labeling reaction was stopped with 2 M Hydroxylamine and the labeled DNA purified. Hybridization procedures and parameters -Standard protocols were used Measurement data and specifications -Software for scanning: GenePix Pro 4.0 (Axon Instruments, Inc.) -Scanner: GenePix 4000A (Axon Instruments, Inc.) Keywords: dose response

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Affy data from WT, FHL1 deleted and FHL1,IFH1 double deleted strains.

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