Worm Breeder's Gazette 7(1): 61

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.

Identification of Putative C. elegans Collagen mRNAs By Hybridization and in vitro Translation

J.C. Politz

I am using the recombinant plasmid, pCg45, which contains a chick 
collagen type I(2) cDNA insert, as a hybridization probe to detect 
nematode collagen mRNA in synchronized populations of worms.  We hope 
this work will lead to the identification and characterization of the 
cuticle collagen mRNAs, eventually at different developmental stages.
In order to ascertain the probe's usefulness, I am first determining 
whether the probe hybridizes to mRNA which can be translated into 
collagenase sensitive product.  My data so far support this conjecture.
Using the Northern blotting technique, I found the chick insert 
hybridizes to two poly A-containing RNA size classes, a major one of 
~1.1 kb and a minor one of ~5.5 kb.  This is true when the RNA is from 
L4s at lethargus onset, adults who have just molted, or from a mixture 
of unsynchronized L4s and adults.  pCg54 insert, a type I(alpha-1) 
chick collagen cDNA, also hybridized to these RNA size classes but not 
as well as pCg45 insert.  When young adult poly A-containing RNA is 
translated in vitro, the products treated with collagenase and 
compared to undigested controls on SDS gels, two protein bands of 34K 
and 39K are digested.  These same bands also label heavily with [3H]-
proline but barely show up with [35S]-methionine (cuticle collagens 
have very little methionine).  The MWs of these collagens are 
consistent with their being encoded by RNAs in the ~1.1 kb RNA size 
class which hybridizes to the chick probe.
The MWs of these in vitro translated collagens are much lower than 
those of the cuticle collagens we isolate from the adult nematode.  
However, no higher MW collagenase sensitive in vitro translation 
products were seen in these experiments.  Either the in vitro 
translated collagen is not cuticle collagen or the cuticle proteins we 
isolate from cuticles are crosslinked aggregates of smaller proteins.