Worm Breeder's Gazette 10(1): 35

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Molecular Cloning of the unc-6 Gene

N. Ishii, B. Stern, E. Hedgecock and J. Culotti

Figure 1

The unc-6 gene is required for guiding axons and migrating cells 
along the epidermis and for specifying certain epidermal cell fates.  
We have isolated two spontaneous unc-6 mutants, rh1035 and ev453, from 
the TR679 and RW7097 mutator strains, respectively.  Using the 
flanking genes lev-9 and dpy-7 to select recombinants, the rh 1035 
mutation was shown to be genetically inseparable from a Tc1 element 
contained in a 13 kb EcoRI fragment or a 2.8 kb XbaI fragment.  The 
XbaI fragment was cloned into pUC19 and the Tc1 element excised with 
BalI.  The resulting plasmid, pNJ6, retains an 18 bp palindrome 
derived from the terminal inverted repeats of the Tc1 element plus 
approximately 1200 bp of the presumptive unc-6 gene.
When used as a probe for Southern analysis, the pNJ6 insert detects 
a 1.25 kb XbaI fragment in N2 Bristol DNA, a 2.8 kb fragment in both 
rh1035 and ev453 DNA, and a restored 1.25 kb fragment in DNA from 
spontaneous unc(+) revertants of both mutations.  The insert detects 
an 11.5 kb EcoRI fragment in N2 Bristol DNA, a 13 kb fragment in 
rh1035 DNA, and two fragments of 6 and 7 kb in ev453 DNA.  This 
suggests that Tc1(ev453) contains an internal EcoRI site.  Spontaneous 
revertants of both rh1035 and ev453 restore the 11.5 kb EcoRI fragment 
observed in N2 Bristol.
We have begun sequencing the pNJ6 insert.  The site of Tc1(rh1035) 
insertion is 56 bp upstream of a consensus intron sequence suggesting 
that the element is in an unc-6 exon.

Figure 1