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.
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.