title: Application of SSH and a macroarray to investigate altered gene expression in Mytilus edulis in response to exposure to benzo[a]pyrene.
abstract: The lack of genomic resources for aquatic invertebrates restricts their use as sentinel species in coastal environments. It is known that where genomic data are not available, suppression subtractive hybridisation (SSH) can generate cDNA libraries representative of pollutant-responsive gene transcription in aquatic vertebrates. To assess whether the approach was equally suited to aquatic invertebrates, altered gene expression in digestive gland of the mussel, Mytilus edulis, in response to exposure to benzo[a]pyrene (BaP) (1 mg/l) was investigated with SSH and a nylon macroarray. Screening of the subtracted libraries showed 112/250 up-regulated and 25/55 down-regulated clones were positive for differential expression and characterisation of these identified 87 with unique sequence suitable for array on a nylon membrane. The transcripts isolated were from a diverse range of genes involved in general stress, oxidative stress, cell adhesion, transcriptional and translational regulation, transport mechanisms, energy metabolism, cell metabolism, lipid metabolism, protein turnover and activation, lysosomal activity and 22 cryptic clones. Subsequent use of the clones in macroarray format to analyse expression of BaP-responsive genes (0 vs 4 day exposed) showed 0-100-fold increased levels of the forward-subtracted probes and between 0 and 0.1-fold down-regulation of the reverse-subtracted probes. Only 15% of the clones showed less than 2-fold change in expression. The gene ontology of the transcripts isolated demonstrates that BaP elicits a multitude of responses with a major feature being disruption of cellular redox status. The results indicate that the use of SSH and a macroarray is a robust method to discover novel pollutant-responsive genes in aquatic invertebrates.
authors.affiliation: Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow, Scotland, UK. m.a.brown@gcal.ac.uk
authors.forename: M
authors.initials: M
authors.lastname: Brown
authors.forename: I M
authors.initials: IM
authors.lastname: Davies
authors.forename: C F
authors.initials: CF
authors.lastname: Moffat
authors.forename: J A
authors.initials: JA
authors.lastname: Craft
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity*; D018076:DNA, Complementary / Q000145:classification / Q000302:isolation & purification; D004064:Digestive System / Q000187:drug effects; D004781:Environmental Exposure; D005260:Female; D015870:Gene Expression / Q000187:drug effects*; D015723:Gene Library; D008297:Male; D049880:Mytilus edulis / Q000187:drug effects* / Q000502:physiology; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D013997:Time Factors; D014874:Water Pollutants, Chemical / Q000633:toxicity*
chemical_list: D018076:DNA, Complementary; D014874:Water Pollutants, Chemical; D001564:Benzo(a)pyrene