Worm Breeder's Gazette 8(1): 47

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.

Spontaneous Twitchers and Tc1 - No Correlations Yet

D. Moerman, B. Waterston

We have been investigating the possibility that the spontaneous 
unstable unc-22 alleles induced in the BO strain of C.  elegans may be 
caused by Tc1.  Our conclusion, based on two types of analysis, is 
that Tc1 is probably not the causative agent of unstable twitchers.  
To investigate the causative role of high Tc1 copy number in 
generating twitcher mutations we have tested several strains in 
addition to N2 and BO for the induction of twitcher mutations.  These 
strains include the 'high' copy number strains, FR, BL1, and EPC-4; 
the 'low' copy number strains, Ga-5, PA-1, and PA-2 (Emmons et al., 
Cell 32:55-65, 1983; Liao et al., P.N.A.S.  80:3585-3590, 1983); and 
HA-8 which has not been examined for Tc1 copy number.  Of these nine 
stains, only the BO strain exhibits the high frequency of spontaneous 
induction of twitcher mutations.  These observations suggest that high 
Tc1 copy number per se is not sufficient to induce unstable unc-22 
mutations.  Rather there is something unique in the Bergerac 
background resulting in the high spontaneous twitcher frequency which, 
for example, could be an altered Tc1 or other factor.
Our second approach to this problem consisted of examining Tc1 copy 
number and position in DNA from a number of different genetic strains 
with and without spontaneous twitcher mutations.  The strains used 
were as follows: N2, RW7012 (contains unc-22(st136), a spontaneous 
twitcher isolated on a Bergerac IV chromosome in the BO strain and 
then outcrossed to an N2(male) strain via 8 cross intercross mating 
cycles), RW7018 (contains unc-22(st137), a spontaneous twitcher 
isolated on a Bristol IV chromosome in a BO background and then 
outcrossed as described for RW7012), Rw2370 (a spontaneous revertant 
to wild type from RW7018), RW1461 (a triple containing daf-14(m77) 
136) 66),st136 derived from 
RW7012), RW1462 (an unc-43 ant from daf-14 
136) -43(e408) 
4)), RW1463 (a daf-14 recombinant from daf-14 
1363 -43 )), 
RW1464 (a triple of daf-14 137) 
rived from RW7018), RW1465 (an unc-43 
ant from daf-14 137) 
-43 4)), RW1466 (a daf-14 
recombinant from daf-14 137) -43 
)).  In DNA from the various strains we could 
detect about 30 bands that hybridized to the Tc1 probe after HindIII, 
EcoRI, or XbaI digestion.  However, we could not identify a Tc1 that 
was strictly associated with the mutated state of the unc-22 locus.  
In particular, we saw no difference between the digestion patterns of 
RW7018 and RW2370 for these three restriction enzymes.  The strains 
derived from three factor crosses showed some banding heterogeneity, 
but again, we did not detect a band that was present in those strains 
containing a twitcher mutation (RW1461 and RW1464), and absent in 
those strains that did not (RW1462, RW1463, RW1465, and RW1466).  
These observations are negative in that we failed to observe a 
difference in Tc1 location consistent with Tc1 as the cause of the unc-
22 mutations.  Of course, we cannot rule out the possibility that the 
critical Tc1 element is in a band comigrating with other Tc1 
containing segments.
Although failing to identify a Tc1 at the unc-22 site, our analysis 
of the recombinants has revealed two putative unc-22 linked Tc1 
polymorphisms, one between daf-14 and unc-22 (a 2 m.u.  interval), and 
one between unc-22 and dpy-4 (a 4.5 m.u.  interval).  The putative 
polymorphism to the left of unc-22 is a 2.1Kb HindIII fragment 
containing Tc1 which we have cloned into lambda 590.  At present we 
are trying to isolate a unique sequence segment from this HindIII 
fragment so that we can use it to position the polymorphism with 
greater certainty.  If the fragment is suitably close to unc-22 we may 
be able to use it to walk to the unc-22 gene.  In conclusion, we feel 
that Tc1 is unlikely to be responsible for generating the unc-22 
mutations, but linked Tc1 polymorphisms may be useful in providing us 
with a molecular probe for the region.