Worm Breeder's Gazette 14(3): 57 (June 1, 1996)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
|1||Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Higashi-ku, Fukuoka 812-81, Japan|
|2||Department of Biology, McGill University, Montreal H3A 1B1, PQ Canada|
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.