Worm Breeder's Gazette 13(5): 41 (February 1, 1995)
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.
Department of Biological Sciences, University of Alberta, Edmonton,
Alberta CANADA T6G 2E9
We previously reported the rescue and cloning of unc-119, a gene
necessary for locomotion in C. elegans (WBG 13#4, p.59). Our earlier work
suggested that the phenotype of unc-119 mutants is the result of a defect
in the nervous system. We have since sequenced the C. briggsae homolog of
unc-119 from a lambda Charon4 genomic clone that also rescues C. elegans
mutants. There is strong conservation (90% identity) of the ORF in the
presumed exons, but the intron sizes are quite different. In the C.
elegans gene, the average size of the introns is 630 bp, but in C.
briggsae, it is only 90 bp. We have also determined the locations of the
base changes in the EMS-induced alleles ed3 and ed4. Both are nonsense
mutations. The predicted protein shares no similarity with database
sequences, although a strong similarity was picked up with a C. elegans
predicted LGII ORF(C27H5.1).
We made GFP and b-galactosidase fusions using the vectors described
by Martie Chalfie (Science 263: 802-805) and Andy Fire (Gene 93:189-198).
The fusion proteins contain ~100 amino acids of the amino terminal half of
Unc-119. Animals transgenic for the beta-gal fusion show staining
localized to cell bodies of the nervous system. Staining is evident in
some cells well before comma stage, at which point intense anterior
staining is seen. By hatching, and continuing for the remainder of the
life cycle, it appears that staining occurs in most if not all neurons,
but we have yet to confirm this. A diversity of neuron types definitely
stain. Cell bodies in the ventral and dorsal nerve cords, the nerve ring,
pharynx, the HSN's, and amphids are easily visualized. Unfortunately, the
fixing step destroys some of the resolution, a problem that the GFP
construct overcomes.
The GFP fusion enables fluorescence in the same cell bodies as beta-
gal (at least after hatching), but many associated neuronal structures
also appear to fluoresce, consistent with other findings that the nuclear
localizing signal in these vectors is not effective with small fusions (A.
Fire et al, WBG 13#4, pp.30-31). However, this may permit visualization
of the entire nervous system in living animals from embryo to adult.
Under high power, for example, extremely thin commissures can be seen
interconnecting lateral processes in anesthetized adults.
Our current problem is explaining why staining appears in many types
of neurons, and yet all the unc-119 mutants, which are putative nulls,
exhibit only a motility defect. It may be that the loss of function of
unc-119 is only detrimental in motor neurons; alternatively, the fusion
constructs may not contain the necessary regulatory sequences.
Note: The C. briggsae genomic library was a gift from Terry Snutch
and David Baillie.
[The actual article also contains a figure.]