Worm Breeder's Gazette 14(3): 19 (June 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Pathology, University of California - San Francisco 4150 Clement Street - 113B, San Francisco, CA 94121.|
|2||Present address: Wellcome Unit of Molecular Parasitology, University of Glasgow, 56 Dumbarton Road, Glasgow G11, UK|
Proteolytic enzmyes play key roles in the development, feeding and survival of free-living and parasitic nematodes. These enzymes are often expressed in a stage- and tissue-specific manner to carry out defined functions. We are interested in determining the spatial and temporal regulatory mechanisms controlling expression of these enzymes. We have been focusing on gcp-1, a C. elegans Cathepsin B-like cysteine protease gene. Northern blot analysis demonstrated that gcp-1 is expresssed in all stages except the embryo and, by in situ hybridisation, was shown to be expressed specifically in the gut. Immunolocalisation studies have now demonstrated the presence of the GCP-1 enzyme in the gut and also around developing embryos in the gonad, suggesting a role for GCP-1 in embryogenesis. We examined the effect of a cysteine protease inhibitor, Mu-Phe-hPhe - vinyl sulphonylphenyl (a gift from David Rasnick, Khepri Pharmaceuticals), on worm development in liquid culture. At an inhibitor concentration of 200 micromolar there was no apparent effect on larval or adult worms. However after 5 days there was a high number of unhatched eggs and a 90% reduction in the number of worms present in the inhibitor-treated cultures compared to untreated controls, supporting a role for cysteine protease activity in embryogenesis. At present we do not know if this effect is due to inhibition solely of gcp-1 or also of other related cysteine proteases. No gcp-1 mutants have been identified and no Tc1 insertions have been found in the gcp-1 gene (Thanks to Ron Plasterk for trying). While the GCP-1 protease has been localised to the gut and to embryos in the gonad, expression of gcp-1/lac Z reporter constructs, like in situ hybridisation for mRNA, is confined specifically to the gut cells of larval and adult worms, with no expression in the gonad or embryos. It is possible, therefore, that GCP-1 is synthesised in the gut and transported to the gonad, in a similar manner to the vitellogenin proteins. To identify regions regulating gcp-1 gut expression we have generated a series of gcp-1/lacZ reporter constructs. Transformation with these has demonstrated that a region around -200 is important for high level gut expression and the effect of mutating a GATA motif in this region is currently being examined. GATA motifs are also present at positions +5, -50 and -150, relative to the transcription start: mutation of these does not affect the level of expression but does alter the exact position of gut cell nuclei staining, resulting in a decrease in the frequency of anterior gut cell staining and an increase in mid-gut cell staining. None of the deletions or mutations tested resulted in expression in embryos or in any other tissues, indicating an absence of repressor binding sites. GATA motifs have been shown to be involved in correct spatial expression of other C. elegans gut-specific genes including ges-1 ( Egan et al., Dev. Biol. (1995) 170: 397) and vit-2 (MacMorris et al. (1992) Mol. Cell. Biol. 12: 1652). We have also identified several copies of the GATA motif in the upstream region of gut-associated cysteine protease genes of the parasitic nemtodes Haemonchus contortus and Ostertagia ostertagi, suggesting that similar mechanisms regulate gut protease gene expression in parasitic nematodes. We are now planning transgenesis studies using parasitic nematode promoters to drive lacZ expression in C. elegans. This will not only be important for examining the expression and regulation of parasite protease genes but can be extended to examine expression of other genes present in both C. elegans and parasitic nematodes.