Dadna Hartman

Analysis of developmentally regulated genes of the parasite Haemonchus contortus

Differential regulation of gene expression in the development of Haemonchus contortus was analysed using RNA arbitrarily-primed PCR. A study of third-stage larval and adult H. contortus revealed large differences between the two stages; 32 and 30% unique third-stage larval and adult RNA arbitrarily-primed PCR products, respectively. This finding is consistent with a high degree of differential gene expression between these developmental stages. A number of adult products were sequenced, revealing 11 molecules to be similar to deposits within sequence databases. Four other molecules that did not have significant similarity to sequences in the databases may represent developmentally regulated genes specific to H. contortus. Northern analysis of the putative adult-expressed molecules with homologues in the databases confirmed that four were expressed only in adults, while four were expressed in both stages, but had different sized transcripts. This may reflect differential splicing, or expression of closely related but different molecules at different life cycle stages. Two molecules were present in mRNA populations from both stages, suggesting these were false stage-associated molecules. No transcript was detected for one molecule by Northern analysis, probably due to low level of expression. In situ hybridisation analysis was used to localise expression of transcripts in the adult parasite, in particular, to gain some insight into the nature of those molecules with no known predicted function.

HcSTK, a Caenorhabditis elegans PAR-1 homologue from the parasitic nematode, Haemonchus contortus

A putative serine/threonine protein kinase (HcSTK) from the parasitic nematode Haemonchus contortus was characterised at the mRNA and amino acid levels. HcSTK displays a high level of identity (85-93% in the catalytic domain) with proteins of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily, which represent signal transduction molecules involved in establishing and maintaining polarity in proliferating and differentiating cells. The transcript of hcstk is expressed in different developmental stages (second-, third-, fourth-stage larvae and adults) and various organs (muscle, intestine and reproductive) of H. contortus. In addition, there are several isoforms which appear to relate to a single gene. The expression profile of hcstk is similar to that of Caenorhabditis elegans PAR-1, and the level of sequence identity among members of the PAR-1/MARK STK subfamily, representing a range of species of vertebrates (e.g. humans and rodents), invertebrates (e.g. insects and C. elegans) and yeast, suggests that HcSTK may be involved in a conserved signal transduction pathway.

Haemonchus contortus: molecular characterisation of a small heat shock protein

A cDNA encoding a predicted small heat shock protein, HSP20, was isolated from the parasitic nematode Haemonchus contortus. This cDNA encoded a predicted protein of 156 amino acids, which had high sequence identity with other nematode small heat shock proteins. Southern blot analysis of genomic DNA suggested that in H. contortus HSP20 is encoded by a single copy gene. The HSP20 transcript and protein were expressed in the infective larvae (L3), early L4 and adult stages, but expression was not increased by heat shock treatment. In situ hybridisation analysis was used to localise expression of HSP20 mRNA in the adult parasite. Similar HSPs (heat shock protein) were detected by Western blotting in Ancylostoma caninum, Dictyocaulus viviparus, and Toxocara canis, but not in Trichostronglyus colubriformis. The conservation of HSP20 in several different nematode species may reflect its importance to parasites that require mammalian hosts as a part of their development. Index Descriptors and Abbreviations: Haemonchus contortus; nematode; small heat shock protein; L3, infective larvae; xL3, exsheathed L3; eL4, early L4; EST, expressed sequence tag; HSP20, heat shock protein 20; sHSP, small heat shock protein

Isolation and characterization of class II myosin genes from Haemonchus contortus

In this study, cDNAs encoding myosin from the parasitic nematode Haemonchus contortus were isolated and characterized. Several exhibited a considerable degree of sequence variation at the nucleotide and limited divergence at the amino acid levels within the various functional domains. The results suggest that the cDNAs isolated represented a single myosin heavy chain, which, by comparison with a number of other myosins, is inferred to represent a homologue of a muscle myosin (CeMHCA) of the free-living nematode Caenorhabditis elegans. The findings could have implications for investigating cytoskeletal dynamics and/or signalling pathways.