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.
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 lesion. 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 follows: 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. elegans.