Transcription profiling of Lumbricus rubellus to determine seasonal changes in gene expression
Ontology highlight
ABSTRACT:
ORGANISM(S): synthetic_DNA
SUBMITTER: Peter Kille
PROVIDER: E-MAXD-36 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Publications
Transcriptome profiling of developmental and xenobiotic responses in a keystone soil animal, the oligochaete annelid Lumbricus rubellus.
Owen Jennifer J Hedley B Ann BA Svendsen Claus C Wren Jodie J Jonker Martijs J MJ Hankard Peter K PK Lister Linsey J LJ Stürzenbaum Stephen R SR Morgan A John AJ Spurgeon David J DJ Blaxter Mark L ML Kille Peter P
BMC genomics 20080603
<h4>Background</h4>Natural contamination and anthropogenic pollution of soils are likely to be major determinants of functioning and survival of keystone invertebrate taxa. Soil animals will have both evolutionary adaptation and genetically programmed responses to these toxic chemicals, but mechanistic understanding of such is sparse. The clitellate annelid Lumbricus rubellus is a model organism for soil health testing, but genetic data have been lacking.<h4>Results</h4>We generated a 17,000 seq ...[more]
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Project description: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. 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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. 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