Program of the 2001 International Worm Meeting - Sternberg Lab ...

721
721. CHARACTERISATION OF
MUTANT ALLELES INVOLVED IN
C.ELEGANS CUTICLE FUNCTION
C Ibáñez Ventoso, IL Johnstone
WCMP, Anderson College, University of
Glasgow, 56 Dumbarton Road, Glasgow G11
6NU, Scotland, UK.
C. elegans hypodermal cells synthesise five
distinct cuticles through the nematode life cycle:
the first one within the egg prior to hatching; the
remainder during each moult. The animal form
is determined by the cytoskeletal organisation of
hypodermal cells during elongation of the
embryo. After the elongation process, the
cytoskeleton becomes disorganised and the
cuticle assumes the role of maintaining the
shape of the nematode. We are interested in
identifying genes essential for synthesis of the
nematode cuticle.
Our lab has performed a forward genetic screen
to isolate embryonic lethal mutants that
complete the elongation process but then
collapse, failing to synthesise the L1 cuticle. We
are currently characterising one mutant allele,
named ij015, generated from this screen.
Homozygous embryos for ij015 express
dpy-7_GFP and col-12_GFP constructs
suggesting that transcription of collagen genes
occurs but an intact cuticle is not assembled. We
have positioned this allele on the genetic map
and rescued the mutant phenotype by a single
cosmid clone. Detection of the coding sequence
contained in the positive clone responsible for
the observed phenotypic rescue is on progress.
In an alternative approach, we have identified a
C. elegans gene predicted to encode a bZIP
transcription factor that with RNAi phenocopies
the elongation then collapse phenotype we
associate with cuticle synthesis failure. This
transcription factor appears to be expressed in
hypodermis of embryos and later stage animals.
We have also found a recessive mutation
causing a similar phenotype to that obtained
with RNAi. This together with the observed
spatial expression pattern indicates this
transcription factor may have a role regulating
expression of genes involved in the formation of
the first cuticle.
722. The structural role and
interactions of the DPY-7 cuticular
collagen in the exoskeleton of
Caenorhabditis elegans.
Martyn Quinn, Iain Johnstone
The University of Glasgow, Wellcome Centre
for Molecular Parasitology, Anderson College,
56 Dumbarton Rd, G11 6NU, Scotland (UK)
722
The C.elegans cuticle (exoskeleton) is a
multi-layered extracellular matrix consisting
predominantly of small collagen-like proteins
that are extensively cross-linked. The cuticle is
synthesised by the underlying hypodermis 5
times throughout the lifecycle of the animal.
Previous investigations have indicated that the
cuticular collagen DPY-7 has an important
structural role within the complex architecture
of the exoskeleton. Using immuno-fluorescence
we have shown that this collagen locates to
circumferential stripes within the cuticle. There
is one DPY-7 containing stripe per cuticular
annulus. We are utilizing a range of techniques
(including immuno-electron microscopy and
scanning electron microscopy) to further
elucidate the precise localisation of this protein
within the cuticular matrix.
Using a mono-clonal antibody specific for
DPY-7, we can detect the protein on western
blots from cuticle extracts. The DPY-7 detected
is much larger than monomer. Cuticle collagens
are crosslinked by non-reducible bonds during
formation of the cuticle. The various high
molecular weight species we detect will be
cross-linked forms of DPY-7. By a combination
of genetic and immuno-fluorescent means we
have shown that DPY-7 physically interacts
with at least three other cuticle collagens. We
are interested to determine if the cross-linking
of DPY-7 we detect by westerns is with the
same collagen molecules we know by other
means DPY-7 interacts with. We are attempting
to purify the cross-linked species with the
intention of identifying cross-linked partners.