Worm Breeder's Gazette 15(5): 50 (February 1, 1999)

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.

Closing in on a clone of the mut-2 mutator of C. elegans

Queta Boese , John Collins

Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824

We are cloning the elusive mut-2 mutator gene because we believe it will help us to understand how host eukaryotic genomes regulate activity of the transposable elements that they harbor. Transposon activity can result in genetic alterations with significant consequences for the integrity of the genome and the organism itself. Understanding this activity is also very useful for investigators who wish to use transposons as sophisticated molecular tools. Genetic activity of transposons in C. elegans is tissue specific (gem-line vs soma) and dependent on genetic background. Efforts to understand the molecular basis of Tc1 control (very active in the soma but repressed in the germ-line) led to isolation of the mut-2 mutator (defined by mut-2(r459)) (1). Subsequent characterization of the mut-2 strain reveals genome wide control of ALL transposon families in C. elegans as well as effects on genome stability (higher incidence of meiotic non-disjunction - Him). Cloning the mut-2 mutator gene will provide valuable insight into mechanisms that appear to effect genome wide control of chromatin stability
As reported previously, I have localized the mut-2 mutator to a 0.25 mu interval between dpy-14 and sem-4 on LG I corresponding to a physical distance of ~170 kb (2). The completed sequence information for this region identifies 33 open reading frames (ORF’s). For most genes, cloning would be straightforward from this point. However, the mut-2 mutator phenotypes do not lend themselves to standard rescue approaches and as a result I have assayed for other phenotypes that are more suitable for rescue strategies.
The original mut-2 mutator strain, TR674 (mut-2(r459) unc-54::Tc1), was viable but sick at 20oC and inviable at 25oC. Because TR674 is a N2/Bergerac hybrid, it was originally thought that the temperature sensitive (ts) nature was a consequence of the ts behavior associated with Bergerac. However, I determined that this was not the case by showing that the non-mutator parent of TR674, TR445 (unc-54::Tc1), is completely viable at 25oC. Further testing of recombinant mut-2 versus non-mut-2 strains generated in our lab revealed that the ts phenotype segregates with the mut-2 mutator. Mutator embryos laid at 20oC and shifted to 25oC develop into adults that are sterile. When L4 animals are shifted from 20oC to 25oC they develop into adults which produce a significantly reduced brood size (<10 progeny/adult) which in turn are completely sterile.
I have subsequently used a free duplication spanning the mut-2 interval to rescue the sterile phenotype at 25oC. Now that I have identified a rescuable phenotype, I should be able to use traditional methods for cloning by rescue. I am currently in the process of testing other duplications and cosmids that span smaller regions of the mut-2 interval (Fig1). I would also like to note that mut-2 is expressed in the germ-line, therefore issues associated with repression of germ-line specific genes intorduced as transgenes will need to be addressed.

1. Collins et al. 1987. Nature 328:726-728.
2. Boese et al., 1998 East Coast Worm Meeting Abstract, p51.