ABSTRACT: Yeast cell growth and treatment:; Yeast strains used were L51 (MATa, ura3-52, leu2-3, 112, his4-519, ade1-100, trp1::HisG, hap1::LEU2) and MHY100 (MATa, ura3-52, leu2-3, 112, his4-519, ade1-100, hem1-delta100). L51 was used for studies of oxygen regulation, and MHY100 was used for studies of heme regulation. To avoid variations from the differences accumulated after many generations of growth of strains, we transformed the L51 strain with the HAP1 gene deleted for studies of Hap1 function. Hap1 protein was expressed in L51 cells by transforming an ARS-CEN plasmid bearing the complete HAP1 genomic sequence. For comparison with cells without Hap1 expressed, an empty vector was transformed into L51 cells. The use of Hap1 expression plasmid generated much more reproducible results than the use of different strains. Yeast cells with or without Hap1 expressed grew at similar rates under both anaerobic and aerobic conditions. We chose to use a low oxygen level (~10 ppb) in this study, in order to identify all oxygen-regulated genes. Previous studies have shown that most, if not all, oxygen-regulated genes are affected a low concentrations, but some genes are not affected at higher oxygen levels (for example, > 1 ppm). Anaerobic (~10 ppb O2, measured by using an oxygen monitor and confirmed by CHEMetrics oxygen kits) growth condition was created by using an anaerobic chamber (Coy Laboratory, Inc.) and by filling the chamber with a mixture of 5% H2 and 95% N2 in the presence of palladium catalyst [61]. L51 cells bearing the Hap1 expression or empty vector were grown under normoxic or anaerobic conditions for 1.5 or 6 hours. The UAS1/CYC1-lacZ reporter plasmid was transformed into yeast cells to confirm the expression of Hap1 and the oxygen levels. Cells were grown in yeast synthetic complete media. Co2+-induced cells were grown in the presence of 400 microM cobalt chloride for 6 hours, as described previously. MHY100 cells were grown in medium containing 2.5 microg/ml (heme-deficient) or 250 microg/ml (heme-sufficient) 5-aminolevulinic acid. For RNA preparations, yeast cells were inoculated so that the optical density of yeast cells was in the range of 0.8-1.0 immediately before the collection of cells. RNA preparation and microarray gene expression profiling:; RNA was extracted from yeast cells exactly as previously described. RNA samples were prepared from 8 different experimental conditions: (1) L51 yeast cells bearing the Hap1 expression plasmid maintained under aerobic conditions, (2) L51 yeast cells bearing the empty expression plasmid maintained under aerobic conditions, (3) L51 yeast cells bearing the Hap1 expression plasmid maintained under anaerobic conditions for 1.5 hours, (4) L51 yeast cells bearing the Hap1 expression plasmid maintained under anaerobic conditions for 6 hours, (5) L51 yeast cells bearing the empty expression plasmid maintained under anaerobic conditions for 6 hours, (6) L51 yeast cells bearing the empty expression plasmid in the presence of 400 microM cobalt chloride for 6 hours, (7) MHY100 cells grown in medium containing 250 microg/ml (heme-sufficient) 5-aminolevulinic acid, and (8) MHY100 cells grown in medium containing 2.5 microg/ml (heme-deficient) 5-aminolevulinic acid. For each condition, three replicates were generated by preparing RNA samples from three batches of independently grown cells. Microarray expression analyses were performed by using three batches of replicate RNA samples. The quality of RNA was high as assessed by measuring absorbance at 260 and 280 nm, by gel electrophoresis, and by the quality of microarray data (see below). The synthesis of cDNA and biotin-labeled cRNA were carried out exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual (2000). The yeast Saccharomyces cerevisiae genome 2.0 arrays were purchased from Affymetrix, Inc. Probe hybridization and data collection were carried out by the Columbia University Affymetrix GeneChip processing center. Specifically, the Affymetrix GeneChip Hybridization Oven 640 and the next generation GeneChip Fluidics Station 450 were used for hybridization and chip processing. Chip scanning was performed by using the GeneChip scanner 3000. Initial data acquisition, analysis was performed by using the Affymetrix Microarray suite. By using GCOS1.2 with the advanced PLIER (probe logarithmic intensity error) algorithm, we calculated and examined the parameters reflecting the image quality of the arrays. Arrays with a high background level in any region were discarded and replaced. The average noise or background level was limited to less than 5%. The average intensity for those genes judged to be present was at least 10-fold higher than those judged to be absent. Also, arrays that deviated considerably in the percentage of present and absent genes from the majority of the arrays were replaced. Arrays with a ï¢-actin 3â/5â ratio greater than 2 were replaced. Normalization of microarray data:; For each microarray, we converted the .DAT image files into .CEL files using the Affymetrix GCOS software. These raw .CEL files were further processed into expression values using the RMA express software by Bolstad. This software uses the robust multiarray average method by Irrizary et al., which involves a background correction and a quantile-based normalization scheme. Experiment Overall Design: Yeast cell growth and treatment: Experiment Overall Design: Yeast strains used were L51 (MATa, ura3-52, leu2-3, 112, his4-519, ade1-100, trp1::HisG, hap1::LEU2) and MHY100 (MATa, ura3-52, leu2-3, 112, his4-519, ade1-100, hem1-delta100). L51 was used for studies of oxygen regulation, and MHY100 was used for studies of heme regulation. To avoid variations from the differences accumulated after many generations of growth of strains, we transformed the L51 strain with the HAP1 gene deleted for studies of Hap1 function. Hap1 protein was expressed in L51 cells by transforming an ARS-CEN plasmid bearing the complete HAP1 genomic sequence. For comparison with cells without Hap1 expressed, an empty vector was transformed into L51 cells. The use of Hap1 expression plasmid generated much more reproducible results than the use of different strains. Yeast cells with or without Hap1 expressed grew at similar rates under both anaerobic and aerobic conditions. Experiment Overall Design: We chose to use a low oxygen level (~10 ppb) in this study, in order to identify all oxygen-regulated genes. Previous studies have shown that most, if not all, oxygen-regulated genes are affected a low concentrations, but some genes are not affected at higher oxygen levels (for example, > 1 ppm). Anaerobic (~10 ppb O2, measured by using an oxygen monitor and confirmed by CHEMetrics oxygen kits) growth condition was created by using an anaerobic chamber (Coy Laboratory, Inc.) and by filling the chamber with a mixture of 5% H2 and 95% N2 in the presence of palladium catalyst [61]. L51 cells bearing the Hap1 expression or empty vector were grown under normoxic or anaerobic conditions for 1.5 or 6 hours. The UAS1/CYC1-lacZ reporter plasmid was transformed into yeast cells to confirm the expression of Hap1 and the oxygen levels. Cells were grown in yeast synthetic complete media. Co2+-induced cells were grown in the presence of 400 microM cobalt chloride for 6 hours, as described previously. MHY100 cells were grown in medium containing 2.5 microg/ml (heme-deficient) or 250 microg/ml (heme-sufficient) 5-aminolevulinic acid. For RNA preparations, yeast cells were inoculated so that the optical density of yeast cells was in the range of 0.8-1.0 immediately before the collection of cells. Experiment Overall Design: RNA preparation and microarray gene expression profiling: Experiment Overall Design: RNA was extracted from yeast cells exactly as previously described. RNA samples were prepared from 8 different experimental conditions: (1) L51 yeast cells bearing the Hap1 expression plasmid maintained under aerobic conditions, (2) L51 yeast cells bearing the empty expression plasmid maintained under aerobic conditions, (3) L51 yeast cells bearing the Hap1 expression plasmid maintained under anaerobic conditions for 1.5 hours, (4) L51 yeast cells bearing the Hap1 expression plasmid maintained under anaerobic conditions for 6 hours, (5) L51 yeast cells bearing the empty expression plasmid maintained under anaerobic conditions for 6 hours, (6) L51 yeast cells bearing the Hap1 expression plasmid in the presence of 400 microM cobalt chloride for 6 hours, (7) MHY100 cells grown in medium containing 250 microg/ml (heme-sufficient) 5-aminolevulinic acid, and (8) MHY100 cells grown in medium containing 2.5 microg/ml (heme-deficient) 5-aminolevulinic acid. For each condition, three replicates were generated by preparing RNA samples from three batches of independently grown cells. Microarray expression analyses were performed by using three batches of replicate RNA samples. The quality of RNA was high as assessed by measuring absorbance at 260 and 280 nm, by gel electrophoresis, and by the quality of microarray data (see below). Experiment Overall Design: The synthesis of cDNA and biotin-labeled cRNA were carried out exactly as described in the Affymetrix GeneChip Expression Analysis Technical Manual (2000). The yeast Saccharomyces cerevisiae genome 2.0 arrays were purchased from Affymetrix, Inc. Probe hybridization and data collection were carried out by the Columbia University Affymetrix GeneChip processing center. Specifically, the Affymetrix GeneChip Hybridization Oven 640 and the next generation GeneChip Fluidics Station 450 were used for hybridization and chip processing. Chip scanning was performed by using the GeneChip scanner 3000. Initial data acquisition, analysis was performed by using the Affymetrix Microarray suite. By using GCOS1.2 with the advanced PLIER (probe logarithmic intensity error) algorithm, we calculated and examined the parameters reflecting the image quality of the arrays. Arrays with a high background level in any region were discarded and replaced. The average noise or background level was limited to less than 5%. The average intensity for those genes judged to be present was at least 10-fold higher than those judged to be absent. Also, arrays that deviated considerably in the percentage of present and absent genes from the majority of the arrays were replaced. Arrays with a ï¢-actin 3â/5â ratio greater than 2 were replaced. Experiment Overall Design: Normalization of microarray data: Experiment Overall Design: For each microarray, we converted the .DAT image files into .CEL files using the Affymetrix GCOS software. These raw .CEL files were further processed into expression values using the RMA express software by Bolstad. This software uses the robust multiarray average method by Irrizary et al., which involves a background correction and a quantile-based normalization scheme.
