Worm Breeder's Gazette 11(5): 23

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.

fem-2, unc-45, and daf-7 - Part II

Dave Pilgrim

The left end of LGIII has at last been sorted out.  The mapping of 
fem-2 and unc-45 onto the contig had been stymied by a lack of 
N2/RW7000 polymorphisms in convenient places.  This was overcome by 
looking for differences between N2 and RC301, a solution that has been 
used successfully by others.  Several new RFLPs were found in the 
region where unc-45 was suspected to reside, and a new set of 
recombinants was constructed between N2 and RC301 over the unc-45 
daf-7 intervals.  Using the results 
from the RFLP analysis, unc-45 was found to be inseparable from one of 
the new polymorphisms (eP97) and injection of an unc-45(r450ts) strain 
with the cosmid that detected the polymorphism (W10B10) was able to 
rescue the Unc phenotype.
The accurate positioning of unc-45 and daf-7 (whose successful 
rescue has been previously reported) allowed the interpolation of a 
position for fem-2 between eP93 and eP64, and a set of overlapping 
cosmids covering this region was injected (one at a time, with pRF4 as 
a positive control).  Two of these cosmids, which overlap for about 
half their length based on restriction mapping, were able to rescue 
the Fem phenotype of fem-2(b245ts).  There are several lambda clones 
in the region, which were isolated for a different reason, but I have 
not had time to test them yet.
Recent genetic mapping suggests that vab-6 lies very close to the 
left of unc-45 (less than 15 kbp, by my calculations) and may be on 
the same cosmid, but this has not yet been tested by rescue.
Finally, daf-7 has been successfully rescued on at least two 
occasions using YAC DNA that has been purified from a pulsed-field gel,
without taking too much care to avoid shearing the purified DNA.  One 
of the two rescued animals transmits the phenotype, although only to a 
fraction of the brood.