a genomic experiment
To understand the daf-2 pathway better, an experiment was performed to identify all
DAF-2 ligands (Pierce et al., 2001). Daf-2 is the only gene in the C. elegans genome
that encodes an insulin receptor like molecule (Finch et al., 2001). Hence, ligands that
bind to DAF-2 should belong to the insulin superfamily genes. Proteins of the insulin
superfamily are synthesised as precursors consisting of four domains, Pre, B, C, and A.
In the experiment, open reading frames (ORF) in the genome of C. elegans encoding at least
a putative signal sequence, B chain, and A chain were identified by using multiple searching
tools, such as Blast. Blast is a search algorithm that takes the input sequence and compares
it to all known genetic sequences in a database. In this case, sequences of known insulin
superfamily genes are compared to the entire genome of C. elegans. Then, proteins encoded by
candidate ORF were examined for the presence of structural elements common to insulin superfamily
proteins. The procedure revealed 37 candidate genes named ins-1 through ins-37. After,
using algorithms based on homology modelling, simulated annealing, and energy minimisation,
the tertiary structures of all the INS proteins were determined. Out of the 37 INS proteins,
INS-1 is most similar to human insulin by both primary sequence comparison and structural
homology models (Pierce et al., 2001).
To confirm the candidate genes actually encode DAF-2 ligands, transgenic animals
with increase ins gene dosage were constructed and the effect on the daf-2 pathway
observed (Pierce et al., 2001). High dosage of either ins-1 or and ins-18 cause dauer
arrest, but ins-1 function is not required for dauer arrest. The results indicate
that ins-1 and ins-18 act as an antagonist to Daf-2. Increased gene dosage of other
ins genes does not affect dauer arrest in wild-type C. elegans.