Identification of six mRNA sequences of genes related to multixenobiotic resistance (MXR) and biotransformation in mytilus edulis

Abstract

Tissue-specific gene expression of the MXR (multixenobiotic resistance) and biotransformation-related gene homologs for the multidrug resistance protein 1 (Mdr 1), the multidrug resistance protein (Mrp), the major vault protein (Mvp, i.e. the lung resistance protein, Lrp), glutathione S-transferase (Gst pi), heat-shock protein 70 gene (Hsp70), and cytochrome p450 (Cyp4A) was analysed in the blue mussel Mytilus edulis. Additionally, Topoisomerase II (TopoII) and actin were investigated as indicators for mitotic activity and as internal standards. We identified highly conserved regions in the corresponding genes of several other species, and synthesised degenerate olignucleotide primers designed to amplify these regions from M. edulis mRNA. Resulting RT-PCR products were cloned into a T-tailed vector and DNA-sequenced. The results were evaluated by computer-based sequence alignment with the known gene sequences of blueprint species. PCR amplified fragments were used as probes for Northern blot hybridisation to identify transcript sizes. Specific oligonucleotide primers were designed from the M. edulis sequences for each gene and used for semi-quantitative multiplex RT-PCR to investigate tissue-specific gene expression. We have cloned and DNA-sequenced segments of the M. edulis P-gp, Mvp(Lrp), Gst pi, Hsp70, TopoII and actin genes. Semi-quantitative multiplex RT-PCR rapidly identified characteristic differential gene-expression patterns in a range of mussel tissues, and is therefore a promising tool for comprehensive studies of gene regulation in marine invertebrates in adaptation to various physiological conditions including responses to natural and anthropogenic stressors.