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Article Dans Une Revue Nature Année : 2004

Growth-regulated recruitment of the essential yeast ribosomal protein gene activator Ifh1

Stephan Schawalder
  • Fonction : Auteur
Mehdi Kabani
Isabelle Howald
  • Fonction : Auteur
Urmila Choudhury
  • Fonction : Auteur
David Shore
  • Fonction : Auteur
Joseph T Wade
  • Fonction : Auteur
Daniel B Hall
  • Fonction : Auteur
Kevin Struhl
  • Fonction : Auteur

Résumé

an Applied Biosystems 7700 sequence detector. Relative occupancy values were calculated by determining the apparent immunoprecipitation efficiency (amount of PCR product in the immunoprecipitated sample divided by the amount of PCR product in the input sample) and normalized to the level observed at the coding sequence of the POL1 gene, which was defined as 1. This background binding was then subtracted to give a value in 'occupancy units'. Error bars shown reflect the standard deviation of the mean of independent experiments. Western blotting TAP (tandem affinity purification)-tagged Ifh1 was detected with peroxidase-anti-peroxidase antibody (Sigma) and Fhl1 was detected with anti-myc antibody (9e10, Santa Cruz Biotechnology). Microarray analysis Microarrays containing duplicate spots of 6,528 PCR products corresponding to nearly all yeast intergenic regions were hybridized with a mixture of amplified immunoprecipitate (labelled with Cy5 fluorescent dye) and input (labelled with Cy3 dye) samples, as described previously 27. Values shown are an average of two independent experiments. Conserved DNA motifs were identified using AlignACE 28. WebLogo was used to generate motif logos 29. Transcriptional analysis Total RNA was purified using Qiagen RNeasy columns with DNase I treatment. First-strand cDNAwas synthesized using dT 16 , and quantitative PCR in real time was performed on the resulting first-strand complementary DNA using primers specific to the gene of interest 30. RNA levels were determined relative to a control gene, ACT1.
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Dates et versions

hal-02989548 , version 1 (05-11-2020)

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Stephan Schawalder, Mehdi Kabani, Isabelle Howald, Urmila Choudhury, Michel Werner, et al.. Growth-regulated recruitment of the essential yeast ribosomal protein gene activator Ifh1. Nature, 2004, 432 (7020), pp.1058-1061. ⟨10.1038/nature03200⟩. ⟨hal-02989548⟩

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