Worm Breeder's Gazette 10(1): 36

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.

Molecular Cloning of unc-13

I. Maruyama

The unc-13 gene is located 0.025 mu to the right of unc-15 on the 
centre of LG I.  The mutants have a distinctive phenotype; paralysed, 
kinky and small, and show irregular pharyngeal pumping.  More than 30 
alleles, including 5 amber mutants, have been isolated and the genetic 
analysis has been well investigated.  It is curious that unc-13(e309) 
allele is specifically suppressed by sup-6 mutation which is located 
on LG II, and which does not suppress amber or ochre mutation.  In the 
last gazette, we reported, based on an EM serial reconstruction of a 
part of ventral cord, that the largest pair of interneurons in the 
ventral cord of unc-13(e450), AVAL and AVAR, had wrong gap junctions 
to the excitatory motoneurons of the classes VB and DB, as a major 
Towards understanding a molecular mechanism to specify neural 
connectivity, we have cloned the unc-13 gene as a first step.  The 
strategies employed were as 
1)  We identified the location of unc-15 gene on 'the genome map' by 
walking one step both sides of Paramyosin DNA which was isolated by 
Hiroaki Kagawa.
2)  Using Southern hybridization of a set of cosmid clones to a semi-
balanced sDf6/unc-15(e73) genomic DNA, the orientation of the contig 
on the genetic map was determined by locating the sDf6 deletion end 
point which is located in between unc-15 and unc-13 on the genetic map.

3)  By genomic Southern hybridization of a set of cosmids, RFLP's(
Restriction Fragment Length Polymorphism) in 2 Kb EcoRV fragment 
region were found in five independently isolated mutants, four of 
which were isolated from TR679 mutator background and generously 
donated by Roger Hoskin and Jorge Mancillas, and a spontaneous 
revertant, where the RFLP disappeared, was isolated from one of these 
mutants by Jorge Mancillas.  Furthermore, an insertion mutation was 
detected in the same 2 Kb EcoRV fragment in one spontaneously isolated 
revertant, kindly donated by Jonathan Hodgkin, from mab-11(e2008);unc-
13(e51) background.
The mutation has been mapped on the left hand side of canonical unc-
13 mutations, 0.0005 mu away from e51, by isolating intragenic 
recombinants.  The distance between unc-15 and unc-13, about 80 Kb, 
have been measured by constructing the restriction map of a set of 
cosmids encompassing the region, which suggests that one map unit is 
more than 3000 Kb long in this chromosomal region.  An mRNA of 
approximately 3 Kb has been lit up near the RFLP fragment by Northern 
blot analysis, and it seems likely that unc-13 has many close 
relatives comprising of a large family because purified RFLP DNA 
hybridizes to many bands on wild type genomic DNA blot, with a lower 
intensity than that of unc-13 itself.  Further analyses of the gene 
are in progress.
In the course of this experiment, it was found that three 
spontaneous mutants, which display relatively weak phenotypes compared 
to the canonical ones, show the curious behavior moving backwards when 
touched on their tail.  It would be worth trying a serial 
reconstruction of neural circuitry in these mutants to further 
investigate a basic behavior, forward and backward locomotions, in C.