Worm Breeder's Gazette 9(2): 22
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.
At the present time, the structures and functions of the molecules involved in the directed outgrowth of axons are not well understood. Current thinking envisions three possible mechanisms for the perturbation of neuronal outgrowth. Mutations could influence the ability of the growing neuron to detect a normal pathway, possibly by affecting either (1) a membrane-bound receptor protein or (2) the environmental attractant with which it interacts. Alternatively, (3) the cytoskeletal rearrangements which are normally triggered upon this interaction could be defective or absent. Mutants that affect neuronal evidence have previously been identified by filling sensory neurons with fluorescent dyes (Hedgecock et al, 1985). Five genes (unc-33, unc-51, nd to influence the growth of neurons in sensory structures, such as amphids and phasmids. These neurons all share a common defect: They are unable to make proper connections between the peripheral sensory structures and the central nervous system. To clone these genes via transposon tagging, we have created a mutator strain by means of a hybrid dysgenic cross. Progeny from a cross of the Tc1 high-copy strain (EM1002, Bergerac) and a Tc1 lowcopy strain (N2, Bristol) were individually placed on separate petri dishes. Siblings of animals from dishes containing mutant progeny were next transferred singly onto fresh plates. In this manner, the genetic properties which lead to high levels of transposition were expected to be conserved, and a mutator strain produced. Progeny exhibiting phenotypes of interest were serially crossed ten times to the low copy parental strain to reduce the number of extraneous transposons and to stabilize the mutations. The mutations isolated include a novel dumpy (dpy), a small (sma), and several uncoordinated (unc-44 and unc-104 ) alleles. The isolation of two alleles for unc-104 suggests that it is a good target for transposition. That the dpy mutation spontaneously reverts at an very high rate (1/100 to 1/1000) implies either the insertion of an especially unstable transposon or the utilization of an insertion site allowing efficient excision. If the mutations were due to the inactivation of functional genes by Tc1 insertion, restriction fragments containing the inserted transposon could be detected by hybridization to Tc1 DNA. To test this possibility, DNA from the backcrossed strains was digested with Eco RI or Hind III and subjected to Southern analysis. In the case of the dpy, restriction patterns using either Eco RI or Hind III reveal only one obvious band above the usual Bristol number, at around 1kb for the Eco RI and 2kb for the Hind III digests. This result indicates the initial insertion event took place in the Tc1 low copy chromosome. DNA from an EMS-induced revertant of the backcrossed dpy strain shows the loss of these bands, further implicating the unique restriction fragments with the mutant phenotype. Classical genetic analysis has proved this dpy to be a novel gene mapping near dpy-10 on chromosome II. DNA from strains produced by three-factor crosses with flanking markers (a dpy-10 deficiency mnDf31 on the left and unc-5 on the right) is currently being isolated to ensure that the Dpy phenotype cosegregates with the unique restriction fragments. The isolation of one of these unique bands for cloning and sequencing purposes will then be undertaken. In the case of unc-44, the six or more extra bands associated with the mutation suggest that the event which generated the Unc phenotype took place in the high copy level chromosome. In this instance, mapping the adjacent transposons by means of three-factor crosses is required in order to determine which extra Tc1 element has produced the mutant phenotype. Transposon tagging promises to facilitate the cloning of genes from the nematode (Moerman and Waterston, 1984; Eide and Anderson, 1985). From the use of transposon mutagenesis in Drosophila to the first uses of transposon tagging in the worm, it is apparent that this method gives great specificity and simplicity. As with other systems, it is expected that some sites in the C. elegans chromosome will be relatively accessible for insertion whereas others will be refractory. The non-random distribution of mutations in the small sample reported here probably reflects this variation.