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.

Progress in Cloning unc-44 and Other Genes Using Transposon Tagging

V.I. Wheaton, A.J. Otsuka, and E.M. Hedgecock

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.