Worm Breeder's Gazette 13(2): 30 (February 1, 1994)
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.
sa62 was isolated from a screen of EMS-mutagenized worms in Jim Thomas' laboratory. It appeared as a partially penetrant bivulva. As the phenotype was similar to that of lin-18 mutants, Jim kindly sent the strain to us. In the course of backcrossing and mapping we found that the strain contained two linked mutations on chromosome II. One was a recessive lethal (sy 220) that maps close to rol-1 .The other was a semi-dominant Muv ( sa62 )that maps between rol-6 and unc-4 ,as does let-23 .Three-factor crosses indicated that sa62 was within 0.1 map units of let-23 .
To test whether sa62 is an allele of let-23 ,we exploited the observation that animals having sa62 in trans- to the lethal let-23 alleles sy97 , mn23 ,or sy16 are healthy, partially penetrant Muvs. We predicted that if sa62 were in a different gene, it should produce some Muvs whether in cis- or in trans- to let-23 (lethal).If sa62 were a let-23 allele, then a lethal allele of let-23 should be a cis- dominant suppressor of the Muv phenotype of sa62 /+.To obtain lethal alleles of let-23 in cis- to sa62 ,we mutagenized sa62 e120 hermaphrodites with EMS, mated them with let-23 (syl)males and isolated Fl Egl progeny ( let-23 (let)/ sy1 animals are Vul). From 4800 Fl we isolated 3 Egls that produced dead larvae of the let-23 type. Two of these ( sy264 and sy265 )were outcrossed, and shown to be lethal in trans- to let-23 ( sy16 )and Egl when crossed again to sy1 .The third isolate ( sy266 )could not be outcrossed, as males had severely crumpled spicules, and hermaphrodites were highly penetrant Vuls. We constructed a sa62 sy264 e120 /mnClstrain and scored vulval differentiation and P11 /P12fate. All of 44 animals examined were wild type for both phenotypes. This observation that sy264 is a cis-dominant suppressor of sa62 supports the hypothesis that sa62 is a let-23 allele.
Homozygous sa62 worms are scrawny, very slow-growing Muvs. As a heterozygote, the phenotype is variable, with about 5-50% of animals having one or two pseudovulvae. Gonad ablation experiments show that the vulval differentiation observed in sa62 animals is independent of the anchor cell signal.
Epistasis experiments were performed with lin-3 ( n378 / n1 O59), sem-5 ( n2019 )and let-60 ( sy100 ).At the plate level the worms are Vul with sem-S and let-60 . sa62 ; lin-3 worms are Muv only when lin-3 retains some activity. Thus sa62 is epistatic to lin-3 ,hypostatic to sem-S and Iet-60 .This is consistent with interactions proposed on the basis of the architecture of the predicted gene products; in particular, that SEM-5 is expected to bind activated LET-23 via its SH2 domain. sa62 does not rescue the lethality associated with a lin-3 null allele. Epistasis experiments with lin-2 , lin-7 and lin-10 are in progress.
We have so far not detected restriction fragment length polymorphisms on genomic Southern blots of sa62 ,probing five different restriction digests with 10.5 kb of non repetitive DNA from the 5' and 3' ends of the let-23 locus.
The coding region of let-23 in sa62 animals was sequenced. A single point mutation (G-to-A) was detected in the Cysteine-rich domain I, close to the major ligand binding domain. As a result, Cys 359 is converted into Tyr. This result suggests that the Cysteines in this region are important for the correct function of LET-23 .As the sa62 phenotype is gonad independent, this mutation could allow dimerization of the receptor in the absence of signal.