Worm Breeder's Gazette 8(1): 38
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As discussed at the meeting, we've been looking for small collagens in vivo because we believe that the collagens isolated from cuticles ( apparent MWs from 60 to over 200 kd) are cross-linked aggregates of numerous smaller primary gene products varying from about 35 to 50 kd in apparent MW. The collagen genes found by Jim Dramer and Joe Cox are small, the vast majority of worm RNAs that hybridize to a chick collagen cDNA probe are small and collagenase sensitive in vitro translation products obtained from RNA isolated from molting worms are small. Using a Western blotting technique, we have now found small collagens present in worms molting from L4s to adults. In the Western blotting technique that proved useful for us, proteins are electrophoretically separated on an SDS gel, electroblotted to diazotized (DPT) paper and reacted with antibody in situ. Bound antibody is then detected using [125I]-labelled protein A. We screened the sonication supernatant, SDS soluble, and SDS- ME soluble (cuticle) fractions from worms isolated either at the peak of the L4 to adult lethargus or as very young adults. The antiserum used was directed against the soluble fraction of the adult cuticle. This serum contains antibodies that bind to all the adult cuticle collagens (as well as the L4 cuticle collagens) and also cross-reacts to some extent with vertebrate collagen, probably type IV. Small collagens ( identified by their collagenase sensitivity) were most abundant in the sonication supernatant but were also detectable in SDS fractions. These collagens can be concentrated further in a 12,000 X g pellet of the sonication supernatant. We don't understand this yet. The small collagens identified have several properties consistent with their being cuticle precursors. Firstly, they have molecular weights similar to collagens translated in vitro from RNA isolated from molting worms and could be coded for on genes approximately the size of those found by Kramer and Cox. Secondly, they bind to antibody present in an antiserum made to soluble adult cuticle; this cuticle fraction contains the characterized cuticle collagens. Thirdly, and most suggestively, the complement of small collagens present changes during the L4 to adult molt in a way very similar to the collagens translated in vitro from RNA isolated at similar times during this molt. At lethargus peak, small collagens of about 46 to 52 kd predominate, but at the end of the molt, the major small collagens are about 36 and 39 kd in size. This mimics the size difference seen between the collagenase sensitive translation products of RNA isolated near the beginning and end of the molt. We are now further characterizing the small collagens present during the L4 to adult molt to determine if they are, in fact, probable cuticle precursors. If we are looking at primary cuticle gene products, our hope is to begin to screen the precursor population present in morphological cuticle mutants to identify those with possible structural gene mutations.