Worm Breeder's Gazette 8(1): 38

These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Preliminary Identification of Cuticle Collagen Precursors in vivo

S. & J. Politz

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.