Genetic interactions with sqt-l suggest that
dpy-2 and
dpy-10 encode important structural components of the cuticle that may physically interact with the sqt-l collagen. For example, in the
dpy-10 (
el28) Dpy background, sqt-ll + animals for four different sqt-l alleles (scl,
scl3,
sc99, sclOl) are DpyLRol. In addition, suppression of
glp1 and mup-l mutants by alleles of both
dpy-2 and
dpy-10 imply a role for these genes in other aspects of development. We have isolated and identified
dpy-2 and
dpy-10; they encode small collagens similar in structure to the other previously studied cuticle collagens. The proximity of the genes (3.5 kb) and high degree of homology of the encoded collagens (40.8%) strongly suggest that a gene duplication event gave rise to the pair. Interestingly, trans-heterozygotes for alleles of
dpy-2 and
dpy-10 fully complement, suggesting functional independence of the collagens. We have sequenced four alleles of
dpy-2 and ten of
dpy-10. Seven of ten point mutations create glycine substitutions within the triple helical portion of the protein. These glycine substitution mutants presumably interfere with the assembly and stability of the triple helix. Substitutions at different positions can cause Dpy or DpyLRol phenotypes; some are temperature sensitive.
dpy-10 (
el28) is a mutation in the 3' splice site of intron 2 that changes the obligate AG to AA. RT-PCR indicates the presence of both unspliced and normally spliced
dpy-10 message in
el28 RNA.
dpy-10 (
cg36) creates an ochre codon near the end of the Gly-X-Y sequence; its DpyLRol phenotype is consistent with other genetic data that suggests DpyLRol is the null phenotype of
dpy-10.
dpy-10 (
cn64) is a dominant left roller that contains a Cys-for-Arg substitution in the A homology block. This mutation occurs at exactly the same position as the sqt-l (
el350) and
rol-6 (sulO06) Cys-for-Arg substitutions which result in dominant right roller phenotypes. Thus the equivalent mutation in different collagens can result in opposite phenotypes. It is significant that each of the dominant cuticle collagen mutants studied thus far is a gain of cysteine; the new cysteines probably allow for the formation of ectopic disulfide bonds within the cuticle. Sequence of two mutator induced
dpy-10 alleles (provided by D. Riddle and T. Schedl) revealed that
m457, a Dpy, and
q323, a DpyLRol, contain Tcls inserted at exactly the same site in the same orientation. The difference in phenotypes appears to reflect different amounts of somatic excision between the two alleles.
q323 excises approximately ten times less frequently than
m457, possibly due to differences in the two Tcl elements.