Worm Breeder's Gazette 10(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.

Progress in Cloning a Mutator

I. Mori, D. Moerman and Bob Waterston

We have previously shown that a mutator, necessary for germ-line 
movement of Tc1, can be mapped as a discrete unit on a chromosome.  
The molecular basis of mutator activity is unknown, but our 
observation that mut-5 II and mut-6 IV arose spontaneously in a mut-4 I 
genetic background suggests that a mutator itself can transpose.
In an attempt to clone a mutator we have correlated the genetic map 
position of mut-5 II with a subset of Tc1 elements.  We have examined 
the Tc1 patterns and mutator activities of 27 rol-1 recombinant lines 
isolated from unc-85 rol-1/+++ heterozygotes, and 
cloned three Tc1's(#40, 55, and 118) which compIetely or nearly 
completely cosegregate with mut-5 activity.  mut-5 genetically maps 
approx.  1 m.u.  right of dpy-10 II.   These rol-1 recombinant lines 
contain only 55-65 copies of Tc1 and genomic digestions with four 
different enzymes (EcoRI, SacI, BglII and BamHI) failed to reveal any 
other Tc1's cosegregating with mut-5.  Southern analysis showed that 
all three Tc1 clones map under the deficiency mnDf88.  Lambda clones 
including the TC1(#40) and Tc1(#55) sites were assigned by A.  Coulson 
and J.  Sulston to the same 400 kb contig near lin-5.  The physical 
map positions of these Tc1's agrees with the genetic map position of 
mut-5.If the mutator is indeed an autonomously active form of Tc1, 
like other eukaryotic element systems, one of the mut-5-linked Tc1's 
could be mut-5.  We are currently testing this hypothesis by 
microinjection.  Each Tc1-containing EcoRI fragment was inserted into 
pAST19a (400 bp sup-7 region + pUC19; constructed by RHW and A.  Fire),
and the resulting plasmids were injected into maternally rescued unc-
22(st136::Tc1) 07am) hermaphrodites.  unc-22(
st136::Tc1) is stable (reversion freq.  <10+E-6) in this strain so 
mutator function can be examined by the reversion of unc-22 in the 
stable transformant lines.  We obtained the first stable line which 
has the integration of the injected Tc1's, along with some 
rearrangement of injected DNA.  We have not seen any unc-22 revertants 
yet in this line, but we are hoping to get more transformants.
More precise mapping of mut-5 activity relative to the Tc1(#40) and 
Tc1(#55) sites is in progress to see if mut-5 activity is separable 
from either or both of the cloned Tc1's.  The ability of a 400 kb 
contig for this region also allows us to search for other elements, 
either Tc1 or non-Tc1, as the molecular basis of mutator.
In a related experiment, we have tried to activate the amber Tc1 
variant in unc-22(st137::Tc1), identified by R.  Plasterk and A.  
Sakker.  Crossing in sup-7 in a stable line does not restore mutator 
activity.  Given the negative nature of the results and all the 
caveats interpreting suppression results, we can only conclude it did 
not work.