An insulin-like receptor
daf-2 signal pathway regulates both dauer formation and adult life span in C. elegans . A search for genes downstream of
daf-2 was performed by employing the differential display PCR method to compare the expression pattern of
daf-2 mutant and N2 wild-type animals. With the primer sets tested so far, a total of thirty-four unique clones have been isolated from differentially displayed bands and sequenced. Database search for each clone gave the physical map location, the predicted open reading frame and the functional homologies. Eight of the clones are novel proteins. Northern analysis was used to confirm the differential expression pattern shown by DD-PCR. We named these verified genes as dao genes for d af-2 a djusted o ver- or under-expression. The
dao-1 gene showed significantly reduced expression in
daf-2 mutant adults but similarly high levels in N2 adults and
daf-16;
daf-2 mutant adults. Therefore
dao-1 gene is positively regulated by DAF-2 and negatively regulated by downstream signaling component, DAF-16. Northern hybridization of
dao-1 in aged samples indicates
dao-1 expression correlates with wild-type life span. The
dao-2 mRNA displayed a similar pattern as
dao-1 . The
dao-3 gene was over-expressed in N2 young adults but not the
daf-16;
daf-2 mutant adults, suggesting this gene is regulated by the
daf-2 gene but not the
daf-16 gene. The patterns of both
dao-4 and
dao-5 expression were higher in
daf-2 young adults and lower in N2 and
daf-16;
daf-2 double mutant adults. The sixth clone has relatively higher expression level in dauer samples compared to the adult samples and there is no difference in levels among adult samples. This gene is not regulated by
daf-2 signaling and it is a false positive in our DD-PCR assay, though it may contribute to dauer formation or maintenance. Except for
dao-3 , which is either upstream of
daf-16 or in a non-
daf-16 branch, the dao genes are downstream of
daf-16 and these target genes may contribute to the processes of dauer formation and of life span determination.