Worm Breeder's Gazette 11(4): 19

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.

Update on the Cloning of unc-73

Rob Steven, Joe Culotti, Alberto Ruiz and Jorge Mancillas

Mutations in unc-73 generally affect the longitudinal positioning of 
axons and the longitudinal migrations of some cells in C.  elegans.  
Axons or cell migrations are either misplaced in a dorsal or ventral 
direction or their path is shortened and they do not reach their final 
destination (Hedgecock et al., Development 100, 365-382, 1987).
We have previously reported the Tc1 tagging of unc-73 (CSH abstracts,
1989, p.60) and the cloning of the 5kb HindIII fragment containing 
the Tc1 insertion (WBG 11:2, p.58).  The Tc1 was excised from this 
fragment and the remaining flanking sequence was used to probe an EMBL-
3 N2 genomic DNA library.  From the 14 positives identified we now 
have approximately 30 kb of cloned DNA spanning the region of the Tc1 
The DNA from two of these phage clones were sent to Alan Coulson and 
John Sulston to identify the cosmid clones spanning the same region.  
In cooperation with Michel Hamelin, transformation experiments with 
these cosmid clones have been carried out in the attempt to rescue unc-
73 animals.  Indeed, injection of one of the cosmid clones, C11B5, 
rescues the unc-73 phenotype.  We are now injecting with smaller 
segments of cloned DNA in order to localize the region of DNA 
containing the whole unc-73 gene.
Sequencing of 3.4 kb of the 5 kb HindIII Tc1 containing fragment is 
near completion.  The removal of the 1.6 kb Tc1 with EcoRV leaves two 
fragments of flanking genomic DNA of 2.6 kb and 0.8 kb.  Four putative 
exons encoding a total of 279 amino acids have been found within the 2.
6 kb fragment, but no significant homology has been seen with any of 
the proteins in the NBRF or Swiss-Prot databases.
A second independent Tc1 insertion into the unc-73 gene has also 
been identified.  Restriction mapping of the phage clones and Southern 
blots have revealed that this second insertion site is 4.8 kb away 
from the first insertion site.  This information along with the cosmid 
rescue confirms that we have cloned the unc-73 gene.