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]