Worm Breeder's Gazette 14(1): 56 (October 1, 1995)
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.
Depts. of Genetics & Anesthesiology, Case Western Reserve University, Cleveland, OH-44106.
For the past few years, we have been trying (very hard) to clone the unc-1 gene. unc-1 is one of the suppressors of the altered anesthetic sensitivities of unc-79 and unc-80 mutants. Using a combination of the STS mapping technique as well as Southern blotting, we mapped unc-1 relative to the polymorphic markers stP41 and meP4. In each case, 100 % of the unc1nondpy3 animals were negative for the marker and 100 % of the dpy3nonunc1 animals were positive for it. (We screened 100 animals for the stP41 marker and 32 for the meP4 marker.) This result indicated that unc-1 was very close to each of these markers. Initially, we had focussed our attention on the region around the cosmid F53H8 because F53H8 & F35H12 detected the endpoints of fcDf1 & fcDf2 both of which cover unc-1; CO8A12 detected polymorphisms in some EMS induced unc-1 alleles; & None of the other cosmids in this region detected any significant polymorphic differences between N2 and the different unc-1 alleles. However, despite exhaustively microinjecting cosmids from this region, we did not see any rescue. We therefore had to accept the possibility that fcDf1 & fcDf2 involved multiple breakpoints. We then shifted our attention to the cosmids in the region of meP4. The cosmid CO4D1 rescues the kinked phenotype of an unc-1(e580) mutant. Of 99 F1 rollers, 9 showed strong F1 rescue.Of these 9, 6 remained stably rescued in the F2 generation. Of the other 90 nonrescued F1 rollers,12 gave stable F2 rollers, of which 2 were rescued, giving a total of 8 stably rescued lines. Interestingly, each of the rescued animals also throws a varying proportion of wildtype (ie. rescued nonrolling ) animals. Some of these remain wildtype while others either express the rol-6 marker at a later stage of development &/or give rescued roller progeny. Using CO4D1 as a probe, we are not able to detect any polymorphic differences between N2 and any of the unc-1 EMS induced or spontaneous alleles. However, when the 1.6kb Tc1 fragment was used as a probe, we detected polymorphisms in 6 of the 10 spontaneous unc-1 alleles. We are in the process of characterizing them further. Currently, our efforts are also directed towards cloning the smallest fragment of CO4D1 that can rescue the unc-1(e580) mutant and using that as a probe to isolate a cDNA corresponding to the unc-1 gene.