Worm Breeder's Gazette 11(2): 29
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.
A multivulva phenotype can require the presence of mutations in two separate genes, despite the fact that each mutation results in an apparently wild-type phenotype by itself (Ferguson and Horvitz 123:109121, 1989). Mutations that can cause this synthetic multivulva (syn Muv) phenotype have been grouped into two classes, class A and class B, such that a double mutant carrying one mutation in each class will have the syn Muv phenotype. lin-9(n112) III is a class B mutation that results in an apparently wild-type phenotype by itself. lin-9 maps 0.01 m.u. to the left of unc-32 III, which has been rescued in germline transformation experiments by John Sulston (Hope et al., WBG 10(2) p. 97). Using the gonadal syncytial injection method developed by Craig Mello et al. (WBG 11(1) p. 18), we microinjected cosmids from the unc-32 region along with a dominant rol- 6 marker into lin-9(n111); lin-15(n433) animals, which are temperature- sensitive for the Muv phenotype and had been raised at the permissive temperature. A ts strain was used to generate animals sufficiently healthy for efficient microinjection, as well as to potentially minimize the amount of rescue activity required from the injected cosmid. The lin-15 allele used, n433, is a class A syn Muv mutation that results in a wild-type phenotype in the absence of a class B syn Muv mutation. The major approach we have used to test cosmids near unc-32 is to raise the F1 progeny of the injected animals at the permissive temperature, clone F1 rollers, raise their progeny at the restrictive temperature and then score the F2 progeny for rescue of the Muv phenotype and the presence of rollers. We chose this approach based on observations that some genes show only F2 rescue in gonadal syncytial injection experiments (Craig Mello and Gian Garriga, personal communications). The cosmid ZK637, which rescues unc-32, also rescues lin-9. Using ZK637, 14 out of 24 lines that transmit rollers show rescue of the Muv phenotype. Interestingly, both the rol-6 and lin-9 rescue activities seem to be incompletely penetrant, because animals from rescued lines with any of the phenotypes Rol non-Muv, Rol Muv, non-Rol Muv or non- Rol non-Muv can produce progeny with the Rol non-Muv rescued phenotype. A similar phenomenon has been previously observed by Craig Mello for rol-6 and several other genes (see article in this issue). In preliminary experiments testing for F1 rescue by injecting ZK637 and raising the F1 progeny at the restrictive temperature, approximately 10% of the F1 rollers have been non-Muv. However, Muv Rol animals ruptured at the vulva less frequently and survived to adulthood more often than did Muv Rol animals generated with just rol- 6 DNA, suggesting that partial rescue might have occurred. In several cases, cloned F1 non-Muv non-Rol animals subsequently produced Rol non- Muv animals in the F2. The only overlapping cosmid left of ZK637 is K01F9. Injection of K01F9 has not rescued lin-9 in seven lines of rollers. We are attempting to localize lin-9 on ZK637 both by testing other overlapping cosmids and by subcloning ZK637.