Sequence comparisons of fourteen characterized C. elegans cuticle collagens have identified some highly conserved amino acids. These conserved amino acids include the cysteines flanking the Gly-X-Y regions and four short sequences termed homology boxes A-D. Alterations of some of these conseNed residues have been shown to cause morphological changes in the animal. For example, a Cys substitution for the third Arg in homology box A in sqt-l and
rol-6 causes a right roller phenotype while the same mutation in
dpy-10 leads to a left roller phenotype. To investigate the importance of the conseNed amino acids in the cuticle collagens, we have made site- specific mutations of the sqt-l and
rol-6 collagen genes and subsequently introduced them into animals by microinjection. To demonstrate that altered collagens in transgenic arrays produce appropriate phenotypes we have tested cloned mutant alleles of sqt-l and
rol-6. We have found that the recessive alleles produce roller phenotypes in null backgrounds but not in WT, while the dominant alleles produce phenotypes even in the WT background. Thus the transgenic system produces appropriate phenotypes. So far we have made several site-specific mutants. A Cys-to-Ser substitution at the carboxy end of the Gly-X-Y of
rol-6 causes a recessive-like left roller phenotype. The same alteration in sqt-l also causes a recessive left roller phenotype. This is the first left roller mutant of
rol-6. An Arg-to-Cys substitution of the first Arg in homology box A of
rol-6 was found to cause a dominant-like right roller phenotype, again identical to the same mutation in sqt-l. When the second Arg in homology box A of sqt-l was changed to Cys (box A consensus:RxxrRQ, in sqt-l and
rol-6, cys-to-arg changes on 1st and 3rd R are dom. right roller) no abnormal phenotype was detected in the transformants. These results indicate that the presence of a Cys anywhere in Box A is not sufficient to cause a dominant right roller phenotype. In general, the same alterations in sqt-l and
rol-6 produce the same phenotypes indicating that conserved amino acids in these two collagens share similar functions during cuticle construction.