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 geneexpression 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.
The gastrointestinal tract is exposed to environmental insult as a result of food intake or in pathological conditions such as diarrhoea, and is therefore protected by the mucus layer. As part of it, trefoil peptides (TFFs) are able to modify the viscoelastic properties of the mucus, protect against experimental ulceration, and promote repair of the epithelia. We investigated, using transient reporter gene assays and RT-PCR in the gastric carcinoma cell line MKN45 and colon carcinoma cell lines LS174T and HT29, whether ethanol and osmotic changes can modify transcriptional activity of TFFs. In a mild hypotonic environment (200 mosmol/l) all three TFF genes were upregulated by at least a factor of 2. In hypertonic medium (400 mosmol/l), TFF1 and TFF3 were down-regulated, whereas TFF2 was up-regulated by elevated concentrations of sodium or chloride in MKN45. Raising the osmolality by ethanol resulted in an up-regulation of TFF3 in both colon cell lines but not in the gastric cell line. We conclude that alteration in TFF gene expression is a response of gut epithelia to deal with osmotic forces and ethanol.z 1998 Federation of European Biochemical Societies.
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