The characterization of the genetic region at the left end of LGIII, and the corresponding region of the physical map, has allowed the isolation of cosmids containing the
daf-7,
fem-2 and
unc-45 genes. (The current state of the
daf-7 gene will be described in another poster). The project initially began as a search for
fem-2, using the linked RFLD approach, since there were no molecular footholds in the region at that time. As in most cloning projects, most of the problems were unforeseen. The genetic map was wrong in a big way, which led to a brief side- track. Finally, a linked Tcl was cloned which was flanked by unique sequences, and they allowed me to identify a previously unlinked contig in John and Alan's database of ~2 Mb, I have managed to sort out the genetic map of (almost) all the identified genes in the region, and identify N2/BO RFLD's on the contig. The mapping of
fem-2 and unc- 45 onto the contig had been initially stymied by a lack of N2/RW7000 polymorphisms in convenient places. This was overcome by looking for differences between N2 and other wild isolates, a solution that has been used successfully by others. These have proven to be the most useful tool that I have had for narrowing my search. They have allowed me to show that not only
fem-2, but
daf-7,
unc-45 and
vab-6 are all on the contig. The one last hurdle to a complete physical map of the contig is to fill in the gaps that are bridged only by YACs. The recombination data for
fem-2 and
unc-45 suggested that the position of the gaps may be a problem for cloning these genes. Initial results from screening a genomic library in lambda for clones that lie in the gap have not been encouraging, indicating that filling these gaps may be may be one of the most difficult parts of completing the physical map. I have since shown that none of the genes lie in the gaps, adding strength to the idea that these 'YAC gaps' are annoying, but are unlikely to contain genes. 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
unc45(r450ts) strain with the cosmid that detected the polymorphism (WlOB10) was able to rescue the Unc phenotype. The accurate positioning of
unc-45 and
daf-7 allowed the interpolation of a position for
fem-2 between eP93 and eP64, and a set of overlapping cosmids covering this region was injected. Two of these cosmids, which overlap for about half their length based on restriction mapping, were able to rescue the Fem phenotype of km-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.for their ability to rescue
fem-2. Restriction mapping of the lambda clones and cosmids, and cross hybridization to C. briggsae DNA have narrowed down the region that must contain km-2.