lin-25 gene activity is required for vulval development (Ferguson and Horvitz, 1985. Genetics 110 17-72). We are particularly interested in this gene because it may provide a link between two binary decisions during vulval precursor cell (VPC) fate specification: the vulva/non-vulval decision, controlled by let-60 activity (Han and Sternberg, 1990. Genetics 126. 899-913; Beitel et al., 1990. Nature; 348, in press) and the 2 /non-2 decision, controlled by lin-12 activity (Greenwald et al., 1983. Cell 34, 435- 444). Two observations support this idea. Firstly, in lin-25(e1446) hermaphrodites no VPCs adopt the 2 fate: P.6p undergoes the 1 fate and the other VPCs adopt the 3 fate (Ferguson et al., 1987. Nature 326. 259-267). Secondly, double mutants carrying lin-25 loss of function mutations and either let-60 gain of function mutations or lin- 12 gain of function mutations suggest that lin-25 acts downstream of let-60 but upstream of lin-12 (P. Sternberg, personal communication and our own observations). We have obtained evidence that lin-25(e1446) is a null allele. We generated a deficiency in the region, arDf1, which fails to complement markers from vab-8 to lin-25 and used it in genetic dosage studies with lin-25(e1446), 45ts) and a new allele, lin-25( ar90), which we isolated in a complementation screen. The phenotype of lin-25(e1446) is similar both to that of lin-25(ar90) and to another lin-25 mutation, lin-25(n1063). We plan to examine the null phenotype of lin-25 and the interactions of lin-25 null mutations with other mutations affecting vulval development in more detail. We have performed temperature shift experiments on individual lin-25( n545ts) hermaphrodites in which the developmental stage at the time of shifting was determined by Nomarski microscopy. Our results extend those of Ferguson et al. (1987) who performed temperature shift experiments on synchronized populations of lin-25(n545ts) hermaphrodites and concluded that the temperature-sensitive period ( TSP) for lin-25 synthesis or activity (required for egg-laying) extends from the mid-L2 stage to the mid-L3 stage. lin-25(n545ts) animals are essentially 100% vulvaless at 25 C but are (>90%) egg- laying competent at 15 C. We found that some lin-25(n545ts) hermaphrodites shifted from 25 C to 15 C even as late as early L4 stage (well after the VPCs had divided) were Egl+. These results suggest that lin-25 activity is required not only for proper VPC fate specification, but also for another (as yet unknown) event that occurs during the L4 stage. Interestingly, temperature shift experiments have revealed that lin-12 activity is also required in the early L4 stage for egg-laying competence in addition to its earlier role in VPC fate specification (Meera Sundaram, personal communication). In our efforts to clone lin-25 we have mapped Tc1 induced RFLPs in congenic strains kindly supplied by A. Telfer. These strains contain BO chromosomal DNA in the rol-4 to par-1 interval (in which lin-25 maps) in an N2 background. The RFLP closest to lin-25, which we have named arP1, maps 0.05mu to the right. We have cloned the DNA flanking arP1 by inverse PCR and used it to isolate two lambda clones. These clones were fingerprinted by Alan Coulson and John Sulston to the cosmid C06C1. We have established a metric for the region of 44 cosmid lengths/map unit by comparing the physical and genetic distances between arP1 and a second cloned polymorphism present in the congenic strains, TCPAR1. Using the metric we estimate that lin-25 lies on one of the cosmids immediately to the left of C06C1. We are presently attempting to rescue the lin-25 mutant phenotype by microinjection experiments with these cosmids.