apr-1 is the C. elegans ortholog of the human tumor suppressor adenomatous polyposis coli/APC (Rocheleau et al.). Loss of APC, as well as dominant mutations in -catenin, are observed in sporadic and familial cases of colorectal tumors, as well as other tumor types (Polakis). Previous studies in C. elegans have clearly shown that components of the destruction complex,
apr-1/APC (Gleeson et al.) and
pry-1/axin (Korswagen et al.) negatively regulate the activity of
bar-1/-catenin during post-embryonic development. Reduction of
pry-1/axin results in an over-induced VPC phenotype, and the penetrance and expressivity of this defect increases when the function of
apr-1 is attenuated (Gleeson et al.). Animals homozygous for a strong hypomorphic allele of
apr-1 die during embryogenesis (Hoier et al.). In an effort to better understand the post-embryonic role of
apr-1(and Wnt-dependent processes) we sought to identify hypomorphic, viable alleles of
apr-1 by two independent methods. First, we performed an F2
pry-1 enhancer screen by EMS mutagenesis. We picked 66 fertile F2 animals that contained more than two vulval protrusions (a phenotype rarely observed in non-mutaginized
pry-1 adults), and identified four enhancer lines that transmitted the phenotype to their progeny. The over-induced phenotype in the
pry-1; enhancer double mutants were all suppressed by depleting
bar-1 activity. This result suggests that the enhancer genes may normally function to negatively regulate -catenin. Unfortunately, none of these mutants map to the
apr-1 locus (mapping data will be presented). Second, we applied the RMG (Re-seqencing Mutagenized Genomes, see Weinholds et al) approach to isolate weak alleles of
apr-1. We constructed an F1 library containing 3,840 EMS-mutagenized genomes. Descendents (F3s) of each cloned F1 were used to constuct libraries of frozen, viable worms and genomic DNA lysates. The 1.6 Kb fifth exon of
apr-1 was sequenced from these DNA lysates. Eight putative heterozygous
apr-1 mutants (3 silent and 5 missense mutations) were identified (Nickerson et al.). We were able to recover and confirm 3 of the 5 candidate missense mutants (A591T, T661I, D600N) and will present our phenotypic analysis of these mutants. Gleeson et al., (2002) G&D 16: 1281. Hoier et al., (2002) G&D 14: 874. Korswagen et al., (2002) G&D 16: 1291. Polakis (2000) G&D 14: 1837. Rocheleau et al., (1997) Cell 90: 874. Nickerson et al., (1997) NAR 25: 2745. Weinholds et al., (2002) Science 297: 99.