The cuticle of nematodes is a complex structure of highly cross-linked proteins and is mainly composed of collagens and cuticulins. Most cuticulin proteins have a signal sequence (SS), a Zona Pelucida (ZP) domain and a transmebrane domain (TM). In screens for
dog-1 induced mutations with a strong visible phenotype, we have identified several alleles of
dpy-1. We have cloned
dpy-1 based on its genetic map position, as determined by SNP mapping, and the presence of a poly Guanine stretch in its 3UTR, as poly-G stretches are known to become unstable in a
dog-1 background. We have determined that we recovered so many
dpy-1 alleles in the
dog-1 screen due to the presence of three unstable sites at this locus. We have sequenced the
dog-1 induced alleles as well as discovering that
dpy-1(
e1) is an early stop mutation. The
dpy-1 cDNA differs in some respects from the predicted M01E10.2. DPY-1 is a large cuticulin protein containing a SS, a long N-terminal low complexity domain, a von Willibrand factor A (vWFA) domain, a ZP domain and a C-terminal TM.
dpy-1 is transcribed in waves just prior to the L2, L3, L4 moults. We have also identified and determined the gene structure of the
dpy-1 ortholog from Pristionchus pacificus and shown that mutations in the
Ppa-dpy-1 gene lead to a strong Dpy phenotype very similar to
Cel-dpy-1. This indicates an evolutionary conservation of DPY-1 function between these species. DPY-1 is most similar to the CUT-6 protein, however, using bioinformatic searches and phylogentic analysis, we have found that
cut-6 and
dpy-1 genes are present in a wide range of nematodes and define two separate and ancient families of proteins. DPY-1 proteins have regions of strong conservation as well as other low complexity regions that are evolving more rapidly and are very specific for a particular species and its close relatives. This suggests that DPY-1 proteins may help to define unique properties of nematode cuticles. We are presently determining better the temporal pattern of
dpy-1 transcription by quantitative PCR and are testing two polyclonal antibodies against
dpy-1 polypeptides in Western blots and in immunofluorescence experiments.