The Drosophila gene numb encodes a novel cell membrane-associated protein that becomes asymmetrically localised in dividing neuroblasts. Normal segregation of Numb protein is required to specify the fate of the daughter cell that inherits it. Numb contains a phosphotyrosine-binding (PTB) domain but the biochemical activity of the protein is not known and the mechanism whereby Numb specifies cell fate is not fully understood. In order to gain further insight into the function of Numb we are characterising its homologue in C. elegans. The C. elegans gene T03D8.1 is predicted to encode a homologue of the Drosophila Numb protein and is denoted
num-1.
num-1 is located on the far right end of chromosome V and generates two splice variants,
num-1A and
num-1B. The
num-1A transcript is predicted to encode a protein that shares 37% identity with Drosophila Numb over its entire length and 70% identity within the PTB domain. The
num-1B transcript is predicted to encode a protein lacking the PTB domain, having instead a novel domain in its N-terminus. This novel domain shows no homology to DrosophilaNumb or to any other protein in available databases. We have generated
num-1A::gfp and
num-1B::gfp reporter constructs in order to determine the cellular and sub-cellular expression pattern of the respective proteins. The NUM-1A::GFP fusion protein is localised to the plasma membrane of several different cell types in the adult worm, including neurons and epithelial cells in the intestine, vulva and seam, as well as pharyngeal cells. Currently we are characterising NUM-1A::GFP expression in the embryo. The NUM-1B::GFP fusion protein also appears to be localised to the plasma membrane; however, NUM-1B::GFP is expressed in only one single cell, the excretory cell, and its tubular processes, the excretory canals. We have generated worms over-expressing NUM-1A/B from its endogenous promoter, by integrating an array containing multiple copies of genomic DNA encoding both NUM-1A and NUM-1B. Adult worms over-expressing NUM-1A/B are viable but display multiple phenotypes. They are Dpy, have large vacuoles in their intestinal cells (visible under a dissecting microscope), are @% Egl and approximately 15% have defects in vulva development. In addition, worms over-expressing NUM-1A/B have an aberrant excretory canal morphology, with regional enlargement, frequent convolutions and occasional branching of the canal. We are currently generating stable knockout of
num-1A/B to determine its lossof-function phenotype. In an effort to identify new genes that may act together with
num-1A/B we have initiated a screen for suppressors of the phenotypes caused by over-expression of the proteins.