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.