European Union and American legislations require that contaminated soils are tested for the presence of toxic compounds to assess human health risks. Earthworms of the Lumbricus genus are ideal bio-monitors for heavy metal toxicity testing of soil. However, until recently, no information has been available concerning the molecular genetic responses of earthworms to heavy metals. Furthermore, although it is well established that earthworms can adapt to toxic heavy metal polluted environments, no information concerning this adaptation process has been forthcoming. Therefore, the underlying rationale of this longstanding project is to understand not only the molecular responses to acute heavy metal exposure but also to understand the molecular mechanisms invoked that allow earthworms to adapt to heavy metal contaminated environments.
Numerous novel techniques including several differential and subtractive approaches as well as fully quantitative PCR were applied to identify and evaluate metal responsive genes in the earthworm. Highest priority has been given to the identification and functional characterization of major metal binding proteins such as metallothionein (MT). Earthworm metallothioneins are characteristically high in cysteine residues and possess no significant aromatic residues. Metal-responsiveness was confirmed by determining metallothionein-specific expression profiles in earthworms exposed to soils of differing heavy metal concentrations. These findings have now been substantiated by the production of recombinant MT (both isoforms), which has since allowed detailed studies on metal stoichiometry, metal stability as well as MT localization. The latter analyses were performed using polyclonal antibodies raised against the recombinant proteins and subsequently used in Light- and Electron-Microscopy. Overall, the results provide concrete evidence that the two isoforms have different sub-cellular distributions and functions, namely the homeostasis and detoxification of essential and non-essential metals, respectively.
In addition, the earthworm Expressed Sequence Tag (EST) project was initiated in the year 2000 and now contains over 17000 sequences from libraries synthesised from dissected late cocoon embryos, juveniles, adults, anterior sections, and adult earthworms exposed to atrazine, cadmium, copper, fluoranthene or lead. Following the masking of poly A tails and the removal of vector sequences, high quality sequences were submitted to the EMBL/GenBank databases. All sequences were clustered, annotated and integrated into LumbriBASE (see http://www.earthworms.org), a relational database designed to facilitate searches by key words and/or sequence information within the entire dataset or limited to specific libraries. In addition, predicted protein translations with domain and signal peptide identification, pI, molecular weight and putative cellular locations were included with a dedicated Peptide Mass Fingerprint (PMF) search function. In total, over 7500 unique gene objects were identified and have allowed the creation of earthworm micro-arrays, a resource that has recently been exploited in numerous gene expression experiments.
In collaboration with Dr Kille / Dr Morgan (Cardiff University), Prof Blaxter (Edinburgh University) and Dr Spurgeon / Dr Svendsen (CEH Wallingford) we are currently generating several gigabases of raw sequence data using two massively parallel sequencing approaches based at the GenePool in Edinburgh. Using a single earthworm we aim to generate 4 runs on the Roche 454 (~1.5 fold coverage of the genome) and 2 runs on the Illumina SOLEXA (~10 fold coverage of the genome). In addition, the ovotestes have been shipped to Prof Shinobu (Kyorin University, Japan) to construct a Bacterial Artifical Chromosome (BAC) library for subsequent gap filling and contig confirmation. The data will be assembled to yield the first draft genome sequence for the earthworm L. rubellus which will soon be integrated into LumbriBASE.
King's College London
- Faculty of Life Sciences & Medicine
- Analytical and Environmental Science Division
- 150 Stamford Street
- SE1 9NH
Over the past 10 years we have collaborated on numerous projects which have all focused on C.elegans toxicological and ecological genomics. Stephen has played an instrumental role in our collaborative projects...