array scanning protocol
Following the instructions from the supplier (Affymetrix).
nucleic acid hybridization to array protocol
Samples were hybridized to GeneChip Yeast Genome 2.0 microarrays following the instructions from the supplier (Affymetrix). Characteristics[experimentator] indicate the different person who carried out the experiments. Comment [Name of reference arrays] indicate the array we used for comparisons.
nucleic acid labeling protocol
Labeling of samples for array analysis was performed using the GeneChip 3IVT labeling assay (Affymetrix) with 100 ng input RNA.
nucleic acid extraction protocol
Total RNA was extracted with the RiboPure-Yeast Kit (Ambion/Applied Biosystems), following the manufacturers protocol. Labeled RNA was biotinylated and purified as described in (Dolken, et al., 2008, RNA 14, 1959-1972). Comment [S. pombe batch] indicate the batch name of the internal standard we used.
In order to study the newly synthesized mRNA, we performed a metabolic labeling protocol useing 4-thioracil. 4-thiouracil (4-tU, Sigma, 2M in DMSO) was added to the media at a final concentration of 5 mM, and cells were harvested after 6 min of labeling by centrifugation at 2465*g for 1 min. The supernatant was discarded and the pellet re-suspended in RNAlater solution (Ambion/Applied Biosystems). The cell concentration was determined by Cellometer N10 (Nexus) before flash-freezing in liquid nitrogen. Prior to Cell lysis, exact 2.25¡Á108 cells were mixed with 0.75¡Á108 cells from labeled Schizosaccharomyces pombe culture that provided an internal standard.
Saccharomyces cerevisiae(Sc) strains RPB1 and rpb1-N488D (GRY 3020 and GRY 3027 respectively) were generously provided by Mikhail Kashlev (Malagon et al. 2006). Genotypes of GRY 3020 and GRY 3027 are mat a, his3delta, leu2delta, lys2delta, met15delta, trp1delta::HISG, URA::CMV-tTA. For cDTA we used Sc wild-type strain BY4741 MATa, his2delta1, leu2delta0, met15delta0, ura3delta0 (Euroscarf) and the isogenic knockout deltapop2 and deltaccr4. deltapop2 was from the YKO library (OpenBiosystem) and deltaccr4 was generated by substituting the target gene for a KanMX cassette using homologous recombination in the same genetic background(Longtine et al. 1998). Schizosaccharomyces pombe(sp) strain FY2317 h+, leu1-32::hENT1-leu1+(pJAH29) his7-366::hsv-tk-his7+(pJAH31) ura4-D18 ade6-M210(Hodson et al. 2003) was kindly provided by Susan Forsburg. YPD medium was inoculated with a single Sc colony. Sp was grown in YES medium.Sc cells were grown in YPD medium overnight, diluted to an OD600 of 0.1, and grown to mid-log phase. OD600 of 0.8 corresponded to 1.75 ¡Á 107 cells per ml.