Worm Breeder's Gazette 10(1): 34

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.

Molecular Cloning of the unc-5 Gene

C.J. Leung-Hagesteijn, B. Stern, E. Hedgecock and J. Culotti

Unc-5 is one of three identified genes that act in combination to 
guide mesodermal cell and axon migrations along the dorsal/ventral 
coordinate of the body wall.  We isolated 11 spontaneous unc-5 
mutations, two in TR679 and nine in RW7097.  Both TR679 isolates were 
made congenic with N2 Bristol by ten cycles of outcrossing.  The unc-5(
rh1034) congenic strain did not contain a Tc1 that mapped to unc-5.  
The unc-5(ev435) congenic strain contained nearly 20 additional Tc1's 
not present in N2 DNA.  Using flanking dpy-13 and unc-44 markers we 
mapped these Tc1's.  One Tc1 of about 2.2 Kb was not separated from 
unc-5(ev435) by nine recombination events between unc-5 and unc-44.  
This Tc1 was not present in either of two independent spontaneous 
revertants of unc-5(ev435), so we believe it is responsible for the 
unc-5 mutation.
We cloned the 2.2 Kb EcoRI fragment containing this Tc1 in lambda 
gt10, subcloned to puc-19, then removed the Tc1 by cutting with EcoRV 
and religating.  The remaining 0.6 Kb of flanking sequence (containing 
38 bp's of Tc1's inverted repeats) should contain at least part of the 
unc-5 gene or regulatory sequence.  A 2.2 Kb EcoRI fragment was 
labelled on Southern blots of DNA from unc-5(ev435) probed with the 0.
6 Kb fragment, whereas a 0.6 Kb EcoRI fragment was labelled in blots 
of DNA from the following strains:  N2, the two spontaneous revertants 
of unc-5(ev435) and three unc-5 mutations independently derived from 
Don Moerman's mutator strains RW7097.  No detectable hybridization was 
seen on blots of DNA from two other spontaneous unc-5 mutations (
rh1036 and rh1037 isolated in RW7097), suggesting that these may have 
deleted the 0.6 Kb fragment.
We used the 0.6 Kb fragment to probe Chris Link's N2 genomic EMBL-4 
library and pulled out several positive plaques.  One of these 
contains the expected 0.6 Kb EcoRI fragment which hybridizes to the 0.
6 Kb probe.  We will use this genomic clone to examine whether several 
other unc-5 mutations isolated in mutator strains have DNA 
polymorphisms in the cloned region.  We hope that this clone will 
allow the identification of cosmids (A. Coulson and J. Sulston) that 
cover unc-5 and perhaps neighboring genes.