During vulval development, the anchor cell signal normally induces P6.p to express the primary cell fate by activating a conserved receptor tyrosine kinase/Ras/Map Kinase signaling pathway. Here we describe the genetic analysis and the molecular cloning of
gap-1 (previously known as
suv-1; Kim,S.K. and Horvitz,H.R.,WBG 11(3),68), a component of this signaling pathway. The mutant phenotype of
gap-1 suggests that it inhibits vulval induction by the
let-60 ras signaling pathway. Null mutations in
gap-1 (see below) suppress the vulvaless phenotype caused by mutations that reduce but do not eliminate signaling activity at a step upstream of
lin-45 raf. For example,
gap-1(
ga133) suppresses the vulvaless phenotype caused by partial reduction-of-function alleles of
lin-3 which encodes the anchor cell signal,
let-23 which encodes a receptor tyrosine kinase,
sem-5 which encodes a GRB-2 homolog and
let-60 which encodes Ras (Table). In addition,
gap-1(
ga133) suppresses the vulvaless phenotype of null alleles of
lin-2,
lin-7 and
lin-10, three genes required for receptor activation. However,
gap-1(
ga133) does not suppress the vulvaless phenotype of a weak allele of
lin-45, which encodes Raf and acts downstream of
let-60 ras. Finally,
gap-1(
ga133) does not exhibit a vulval phenotype in a single mutant. These results suggest that
gap-1 mutations do not allow vulval induction independently of the anchor cell signal, but rather appear to increase the sensitivity of the Pn.p cells towards the anchor cell signal. We were able to define a 9kb DNA fragment of the cosmid T24C12 that contains
gap-1, first by using RFLP mapping, then by finding DNA rearrangements associated with
gap-1 alleles and finally by transformation rescue experiments. The only predicted open reading frame on this DNA fragment encodes a protein of 629 amino acids that is similar to the Drosophila GTPase activating protein GAP1 (27% identical and 53% similar) and to human GAP1(m) (25% identical and 42% similar). Sequence analysis of strong
gap-1 alleles has identified four potential null alleles. The first (
ga133) is a 334 bp deletion causing a frameshift after amino acid 163 and the others (
n1691,
ga14 and
ga12) are stop mutations at positions 151, 174 and 182, respectively. All four alleles are predicted to express non-functional proteins because they each lack the putative catalytic domain. GAP proteins inhibit the signaling activity of Ras by stimulating its intrinsic GTPase activity, which results in a decrease in the levels of Ras in the active, GTP-bound form. Thus, GAP-1 may directly inhibit the signaling activity of LET-60 RAS during vulval induction. Interestingly, double mutants containing
gap-1(
ga133) in combination with mutations that reduce
let-23 receptor activity such as
let-23(
sy1),
lin-2(
n397),
lin-7(
e1449) or
lin-10(
n1390) cause a hyperinduced phenotype in up to 80% of the animals (a gonad-dependent multivulva phenotype). This phenotype is not observed in double mutants containing
gap-1(
ga133) and mutations in genes that act at other steps in the signaling pathway (Table). These observations are paradoxical since single mutants in either
lin-2,
lin-7,
lin-10 or
let-23 have a vulvaless phenotype due to reduced receptor activity but double mutants containing
gap-1(
ga133) and the same mutations have a hyperinduced phenotype indicating increased signaling activity. One explanation for this reversal of phenotypes, previously proposed by Aroian et al. (Genetics (1991) 128,251), is that
let-23 may have two functions; one function is to activate the
let-60 ras signaling pathway, and the other function might be to inhibit vulval induction through a pathway that is yet to be determined. In single mutants, the vulvaless phenotype is the result of reduced activation by
let-23 whereas in double mutants with
gap-1(
ga133), the hyperinduced phenotype might be due to reduced inhibition by
let-23. genotype % Egl % WT % Hin n
gap-1(
ga133) 0 100 0 280
lin-3(
e1417) 88+/-5 12+/-5 0 189
lin-3(
e1417);
gap-1(
ga133) 42+/-6 8+/-6 0 238
let-23(
sy1) 80+/-4 0+/-4 0 427
let-23(
sy1);
gap-1(
ga133) 3+/-2 4+/-5 73+/-6 238
lin-2(
n397) 93+/-4 7+/-4 0 185
gap-1(
ga133)
lin-2(
n397) 4+/-3 2+/-6 74+/-6 197
lin-7(
e1449) 85+/-4 3+/-4 1+/-1 357
lin-7(
e1449);
gap-1(
ga133) 2+/-2 7+/-5 81+/-5 228
lin-10(
n1390) 94+/-3 6+/-3 0 203
lin-10(
n1390);
gap-1(
ga133) 6+/-3 31+/-7 63+/-7 177
sem-5(
n2019) 91+/-3 9+/-3 0 292
gap-1(
ga133)
sem-5(
n2019) 41+/-6 59+/-6 0 306
let-60(
n1876)* 100 0 0 >200**
let-60(
n1876);
gap-1(
ga133)* 14+/-14 86+/-14 0 20
lin-45(
n2018ts) @14C 76+/-6 24+/-6 0 186
lin-45(
n2018ts);
gap-1(
ga133) 72+/-5 28+/-5 0 341 @14C * the progeny of
n1876/+ animals was scored ** from Beitel et al.,(1990) Nature 348:503