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.

The unc-14 gene is expressed in neurons with a pattern similar to that of the unc-51 gene.

Ken-ichi Ogura1, Megumi Shirakawa1, Tom Barnes2, Siegfried Hekimi2, Yasumi Ohshima1

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.