Comparative genomics of nematodes: Caenorhabditis elegans as a tool to study the Haemonchus contortus genome

The genome of the Trichostrongylid nematode parasite of small ruminants Haemonchus contortus is being sequenced at the Pathogen Sequencing Unit of the Wellcome Trust Sanger Institute, Cambridge, UK. Currently, in excess of 800 Mb of genomic sequence is available for this on-going project (http://www.sanger.ac.uk/Projects/H_contortus/). Once available, the fully sequenced and assembled genome of H. contortus will be an extremely valuable resource for both novel drug discovery and biological research into this important pathogen. H. contortus resides in the same Clade (Clade V) of the phylum Nematoda as the free-living model organism Caenorhabditis elegans. Therefore, it is ideally placed to extrapolate the wealth of genomic and biological data available for C. elegans. The extent to which such data can be applied to parasitic nematode research was a major focus of this project. I have concentrated on two well documented and important gene classes: those comprising the β-tubulin gene family and those of the RNA-interference (RNAi) pathway. Control methods for H. contortus are becoming increasingly restricted due to the rise in resistance to current anthelmintic drugs. Benzimidazoles (BZ) are a class of anthelmintic to which there is widespread resistance. Mutations and deletions in both of the β-tubulin genes previously identified from H. contortus, isotypes-1 and 2, have been shown to correlate with BZ resistance. I have identified an additional two β-tubulin loci within the H. contortus genome, which now gives a total of four genes for this family. Using C. elegans as a surrogate expression system together with antibody immunolocalisation in H. contortus I have investigated the expression pattern of three of these H. contortus β-tubulin genes and encoded proteins, and compared these with those of the C. elegans β-tubulin gene family. In addition, I have characterised the phylogenetic relationships of all available Trichostrongylid β-tubulin polypeptide sequences. This has allowed the determination of the evolution of this gene family, and possible association of isotypes-1 and 2 with BZ resistance, across these nematodes. RNAi is a well established technique used in C. elegans to silence gene expression using double stranded RNA (dsRNA). However, RNAi is far less effective and repeatable in parasitic nematode species. Using gene searching techniques I have examined whether genes required for RNAi in C. elegans are present and conserved in the H. contortus genome. Although I identified putative homologues of Dicer (dcr-1) and several other RNAi genes, no sequence homologous to the C. elegans rde-4 gene could be found. This gene is essential for the generation of small inhibitory RNAs (siRNAs) in the C. elegans RNAi pathway. Furthermore, no H. contortus genomic sequence encoding a homologue of Ce-SID-2 was identified. SID-2 is essential for dsRNA uptake from the environment, and sequence differences between C. elegans and C. briggsae SID-2 are responsible for the lack of environmental RNAi in the latter. I have also searched the available genomic sequence databases of Pristionchus pacificus and Brugia malayi for RNAi pathway genes and concluded that components of the RNAi pathway may not be conserved across the phylum Nematoda, although full genome sequences will be required to confirm these findings