The molecular cloning and characterization ofunc-73: A complex gene that plays a role in axon guidance and cell migration in Caenorhabditis elegans

Mutations in the Caenorhabditis elegans unc-73 gene indicate that it plays an important role in cell and growth cone migrations. The gene was cloned and its expression pattern was analyzed in the attempt to define how unc-73 functions in these complex processes. The unc-73 open reading frame was first located by Tc1 transposon tagging. Additional methods including phenotypic rescue, cDNA isolation, and RT-PCR were used to identify the complete gene which spans 32,429 base pairs of genomic DNA. unc-73 is predicted to encode two proteins, UNC-73A and UNC-73B, that are similar to the human Trio and rat Kalirin proteins, respectively. The 2488 residue UNC-73A protein contains eight spectrin-like repeats, a tandem Db1 homology (DH) and pleckstrin homology (PH) domain combination, an SH3-like domain, a second DH/PH domain combination, an immunoglobulin domain and a fibronectin type III domain. The shorter 1638 residue UNC-73B protein is essentially identical to UNC-73A but terminates just downstream of the SH3-like domain. Interestingly, all of the unc-73 mutants that have been tested, including a putative null mutation, can be rescued by this smaller protein.;Molecular analysis of several unc-73 alleles revealed two particularly informative mutations. A nonsense mutation in exon 4 of unc-73(gm40) suggests that it is a molecular null allele. A missense mutation in unc-73(rh40) alters a highly conserved amino acid located within the first DH domain. This residue may define a structurally important site within the DH domain.;Using unc-73/reporter gene fusion constructs, I found that unc-73 is widely expressed in most cell types throughout development in C. elegans. In several cells a correlation between the onset of expression and the initiation of cell migration or axon outgrowth was observed that is consistent with a proposed role for unc-73 in these processes. The distribution of UNC-73 protein, as determined by indirect immunofluorescence using anti-UNC-73 polyclonal antibodies, appears concentrated in the nervous system, from late embryos through to the adult stage. Together, the unc-73 phenotype, expression pattern and sequence similarity to the Trio and Kalirin proteins suggest that UNC-73 acts in a protein complex that regulates the actin cytoskeleton during cell and growth cone migrations in C. elegans.