par-5 is a maternally acting gene with roles in both early cytoplasmic localization and the determination of particular cell fates (especially intestinal and germ line). In our approach to cloning
par-5, we have been taking advantage of the correlation between the genetic and physical maps in the
unc-22 cluster on chromosome IV. Genetic mapping by Diane Morton positioned
par-5 between
dpy-20 and the left breakpoint of sDf7. Recent work in D. Baillie's laboratory provided molecular limits of the
par-5 region; cloning of
dpy-20 by D. Clark delimited the left most cosmid, and S. Prasad's cloning of a Tc1 polymorphism deleted by sDf7 provided a limit on the right. In our transformation experiments, we have been coinjecting oocytes in N2 worms with PD10.46 ( A. Fire's
unc-22 antisense plasmid) and individual cosmids from the region. We select transformants on the basis of the dominant twitching phenotype, establish lines, and then cross twitching hermaphrodites to
dpy-20, 5) males. F2 Dpy twitchers are then tested for rescue of
par-5.To date heritable coinjected twitcher lines have been established for all the cosmids in the region with the following rescue results: 1) No rescue has been seen for the cosmids C35H3 (1 line), ZC2 (1 line), T28C1 (2 lines), C01E4 (6 lines), or T21A4 (6 lines). The Dpy s scored produce 100% dead eggs. 2) Of 3 lines from C02C4, one exhibited a partial suppression of the
par-5 phenotype 5/30 Dpy twitchers produced between 3 and 30 sterile progeny which exhibit the same gonadal defects seen in the occasional progeny produced by
par-5 mothers. In contrast,
dpy-20 others produce at most one or two sterile progeny at 20 C. Thus the presence of the cosmid is acting to partially suppress the embryonic lethality but not the sterility defects associated with
par-5 mutations. 3) Of 10 lines containing C38H7, two exhibit a partial suppression of
par-5; 6/44 and 58/110 of the Dpy s produced sterile broods ranging from 3 to 30. However, one line exhibits complete rescue of the
par-5 phenotype and can be maintained as a fertile line. The rescue is not allele specific; it rescues both
it55 and
it121. I have established a stable line in which all the twitching
dpy-20 are fertile and their non- twitching progeny are par. Southern blot analysis of this line reveals that C38H7 is present. Based on these results,
par-5 appears to lie in the overlap region between C02C4 and C38H7. To localize
par-5 further, I will be testing subclones of C38H7 as well as additional overlapping cosmids. [See Figure 1]