Abstract
The cytochromes P450 are a ubiquitous family of enzymes involved in metabolism of a wide variety of compounds, both naturally occurring and man-made. A number of isozymes of cytochrome P450 are induced by characteristic compounds. Some of these groups of compounds commonly occur in the marine environment as pollutants. It was proposed, by studying the mechanisms of gene regulation, to develop methods of monitoring marine pollution by using levels of certain P450 isozymes in marine animals (in particular the widely used marine mussel Mytilus edulis) as indices of seawater quality. In this project I have: 1) used radiolabelled vertebrate cDNA probes from cytochrome P450 isoenzymes and other proteins to detect homologous sequences in marine invertebrates, particularly in M. edulis. 2) successfully detected mRNA and DNA sequences related to the cDNA probes used in nucleic acid samples purified from mussel and other marine species. 3) performed assays to determine whether the isozymes identified were inducible, comparing mussel with fish for a number of different P450 cDNA probes. 4) performed PCR amplification of mussel DNA using primer pairs derived from published vertebrate P450 sequences. 5) synthesized a mussel genomic DNA library in the vector lambda EMBL3a to facilitate further examination of which P450 genes are present in the molluscan genome. From this work I have determined that sequences homologous to probes from the vertebrate P450I family and P450IV family isozymes are expressed in the mussel, whereas sequences homologous to probes derived from mammalian P450II family isozymes appear to be absent. In comparison with the vertebrate equivalents however, the molluscan P450s appear to be regulated differently: a variety of experimental and field conditions failed to produce clear induction of invertebrate P450, as detected using these probes. Sequences sufficiently similar to fish P450IA1 were present in mussel DNA to allow in vitro amplification using primers derived from the fish P450 sequence. However primers to the rat P450IVA1 did not amplify a fragment in mussel. The gene library synthesized will allow clones encoding mussel P450s to be isolated, following identification by either probes derived from mammalian P450s or probes derived from in vitro amplification of mussel P450 sequences. In the latter case, sequencing of the amplified fragment will first be required. This work complements existing data from studies using other techniques and facilitates additional study to isolate and further analyse the functioning of mussel P450s.