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.
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.