Worm Breeder's Gazette 11(2): 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.
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]