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)/sy1animals 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/n1O59),
sem-5 (
n2019 )andlet-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.