Immune response and myoblasts that express Fas ligand.

; daf-2(e1370) hermaphrodites at the nonper-missive temperature and examining cross progeny for dauer formation. The descendants of the cross progeny were also examined to ensure that the mutations were not unlinked, noncomplementing mutations. We also mapped many of the mutations by testing for linkage with unc-29, which maps near daf-16, or else with daf-16–linked restriction fragment length polymorphisms using PCR (32). 15. One such gene may be daf-18. This gene has been identified by a single mutation that suppresses both dauer formation and the lifespan extension of daf-2 mutants (7, 10, 28). However, many daf-18 individuals show severe morphological abnormalities, suggesting that this gene has other, possibly essential, functions (28). The fact that we did not find any daf-18 alleles supports this hypothesis. In addition, we note that because we screened F 2 progeny of mu-tagenized animals, we would have missed mutants that were maternally rescued. 16. We first attempted to clone daf-16 by positional mapping but found that the gene was located in a gap in the physical map between cosmids AE7 and ZK39. To isolate daf-16::Tc1 insertion mutants, we screened daf-2(sa189); mut-6 animals for spontaneous mutants that did not become dauers when cultured at 20°C. One mutant, mu147, also suppressed dauer formation at 25°C. This mutation failed to complement daf-16(m26) and was closely linked to unc-29, which maps near daf-16. mu147 was subsequently crossed to either unc-29(e1072); daf-2(e1370); him-5(e1490) or daf-2(e1370); him-5(e1490) mutants, and homozygous Daf-16(Ϫ) and Daf-16(ϩ) recombinants were obtained. Genomic DNA was prepared from these recombinants and analyzed by Southern blot hybridization with the 1.6-kb Tc1 sequence as probe. A 6.1-kb Tc1-hybridizing fragment was detected in the Xba I– digested genomic DNA, which was present in 20 of 20 daf-16(Ϫ) recombinants but absent in 15 of 15 daf-16(ϩ) recombinants and also absent in the wild-type strain (N2). DNA from the corresponding region was then extracted from agarose gels and circularized by self-ligation. An inverse PCR strategy was used to identify a Tc1-containing fragment with the expected size of 5.