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Gene Expression Profiles of Fission Yeast

Gene Expression Profile upon Nitrogen Starvation with or without Mating Pheromone

Construction of DNA microarray for ORFs

Probe DNA used for microarray was generated by PCR from the coding region of each gene that has been described in annotated fission yeast genome sequences. The continuous 300bp±10bp exon sequence nearest to the 3'-end of the gene was selected as a probe. When the longest exon of the gene is shorter than 290bp, the longest exon was used as a probe. The length of each primer is 30±3bp. Wild type L972 strain's genome DNA was used as a temperate of the first rounds of PCR. For further amplification of the PCR products, the PCR products of the first round were used as a temperate. Details about array construction, data acquiring and processing are published in Chikashige et al. 2007.

Target RNA preparation

For RNA from vegetative growing cells, a single colony of S. pombe cells on a YES plate was inoculated into YES liquid medium. Cells were cultured at 30℃ and collected at 5x106cell/ml to isolate RNA. For RNA from meiotic cell, the vegetative growing cells (5x106cell/ml) were washed in EMM2-N (EMM2 depleted of nitrogen sources) for two times, then transferred to EMM2-N with or without 0.5mg/ml synthetic P-factor (Imai and Yamamoto, 1994) and further incubated at 30°C. Strains used were L972 for nitrogen starvation without P-factor, and CRL672 (h- Dsxa2) for nitrogen starvation with P-factor. Cells were collected at each time point after the nitrogen starvation. Total RNA was isolated by acid phenol methods described in http://www.sanger.ac.uk/PostGenomics/S_pombe/. The polyA-RNA was purified by Oligotex-dT30<super>mRNA purification kit (Takara). Details for labeling of RNA, array scanning and data analysis are published in Chikashige et al. 2006.

r': Expression ratio

ExpExpression ratio: r', in a logarithm to the base 2, is a ratio of expression in a vegetative growing phase and in each condition. If the amount of the expression is changeless, r' becomes 0, and if it increase by the twice r' becomes 1. Details of deriving r' are in Chikashige et al., 2007.

E: Number of mRNA in a vegetative growing cell

E is a number of mRNA of each gene when the number of total mRNA in a vegetative growing cell is assumed to be 100,000. Results from two independent experiments are shown here. Details of deriving E are in Hiraoka et al. 2009.

References