The
unc-51 gene is required for axonal elongation in C. elegans and encodes a predicted serine / threonine kinase of 856 amino acids ( Ogura et al., Genes Dev. 8, 2389-2400, 1994 ). Recently, we obtained 16 cDNA clones whose products can interact with UNC-51 in a yeast two-hybrid system. One of them was an
unc-51 C-terminal cDNA, and four others encoding a novel protein of 665 amino acids possibly corresponded to the
unc-14 gene ( WBG, 14, No.1, 51, 1995 ) We have now extensively examined the ability of various cosmids and their subfragments to rescue
unc-14(
e57) mutation. The results revealed that the rescuing activity resides in the 5.2kb Hind III - Sal I fragment that encodes the N-terminal half of the novel protein. Furthermore, the mutation sites of the six
unc-14 mutants were determined, and all of them were found to have nonsense mutations near the N-terminus of the protein. All these mutations are located in the 5.2 kb Hind III - Sal I fragment. Therefore, we conclude that this novel protein is UNC-14. The C-terminal half of the protein does not seem to be required for the rescue of an
unc-14 mutation. The genomic sequence analysis revealed that the
unc-14 gene has ten exons. To analyze the expression pattern of
unc-14, we constructed an
unc-14::lacZ fusion gene which was capable of rescuing
unc-14(
e57). This fusion gene is expressed in almost all neurons at all stages, especially in cell bodies and axons. The expression pattern is very similar to that of an
unc-51::lac Z fusion gene, suggesting that UNC-14 directly interacts with UNC-51 in neurons, possibly in axons. We are also examining the expression of an
unc-14::GFP fusion. Does UNC-14 interact with UNC-51 as a substrate or a regulator ? To examine these possibilities, the interacting sites were analyzed by using a yeast two-hybrid system. A region near the N-terminus of UNC-14 ( amino acid residues 200 - 381 ) interacts with a C-terminal region of UNC-51 ( 455 - 856, outside of the kinase domain ) in the two-hybrid system. The same C-terminal region of UNC-51 interacts with itself. We think that UNC-51 acts as a homodimer and that UNC-14 is a regulator of UNC-51 kinase. We plan to examine the direct interactions between UNC-14 and UNC-51, or UNC-51 and UNC-51 by using recombinant proteins produced by E. coli., and the effect of an
unc-51 mutation for the binding.