Worm Breeder's Gazette 11(3): 30
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.
Those of you who have been looking at John and Alan's plots of the contigs on LGIII over the last few months will have noticed a cluster of ePs appearing near the left end. These are as a result of my continuing efforts to clone fem-2. I described the beginnings of this in a poster at the meeting last year. As there are no tagged alleles of fem-2 (or unc-45 or vab-7, which map very close), I have been trying to use N21B0 congenic lines to isolate polymorphisms (pm's) near fem-2, and then walk from those to the gene. I was immediately successful in cloning a Tc1 pm (eP64) which I knew was closely linked to unc-45, and used this to identify a contig from the physical map. Since that time I have been refining the genetic map on the left end of III (mec-12 and daf-7 are in the wrong positions in pre-1990 maps) and trying to order the contig with respect to the genetic map. To make a long story short, after much contig gymnastics, in which linkages moved around a bit, I have now found a total of four N2/B0 RFLPs (eP70, 91,92,93) in addition to eP64 which lie on the contig. These allowed me to orient the contig with respect to unc-45 and dpy-1 as shown below (I was using unc-45 for most of the constructions. fem- 2 has a maternal effect and maps ~0.3 map units from unc-45.) For higher resolution, I made some N2/B0 recombinants between daf-7 and unc-45, and when I looked at the DNA of the recombinant strains with cosmids that detect the RFLDs, I was unable to separate eP70 and daf-7. daf-7 was successfully rescued by injection of C50G1, and subsequently by B0412 and DE9, but not by K02F3 or E03B11. I have transmitting lines from the three cosmids that rescue (ROL-6 was used as a co-injection marker. It works very well), but no integrated lines yet. The recombinant profile to date for the region looks as follows: unc-45 (10/45) eP93 (10/45) eP64 (22/45) eP91 (3/45) [eP92 eP70 daf-7]This predicts that unc-45 is to the left of eP93, possibly in a gap that is spanned by several YACs. I have tried to rescue unc- 45 and fem-2 with a mixture of cosmids from the left of the gap, but with no success. I have not found a RFLD to the left of unc-45 either, which would set a leftward bound to where unc-45 could lie. By the time this comes out, I hope to have filled in the gap with clones from a genomic library in lambda that I got from Alan Coulson, and tried to rescue unc-45 and fem-2 by injecting YACs from the region. [See Figure 1]