Worm Breeder's Gazette 12(2): 101 (January 1, 1992)

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.

Paraquat-Resistance Maps Near the age-1 Locus in Multipoint Mapping in Age Recombinant Strains

Anne Jensen, Tom Johnson

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Institute for Behavioral Genetics, Box 447, University of Colorado, Boulder, CO 80309

Over the last few years both Pamela Larsen and Jacques Van Fleteren have reported an increase in the levels of Cu/Zn superoxide dismutase (SOD) and catalase activities in age-1 strains. Each has used a simple drug resistance assay to test if these higher activity levels have a phenotypic effect. Several Age Fer strains show increased resistance to both hydrogen peroxide (Larsen) or paraquat (Van Fleteren) as compared with the control strains BA713 ( fer-15 age-1 )and N2 .

We decided to confirm that these differences in resistance to the free-radical generating agent, paraquat, actually map to the age-1 locus and not to fer-15 or to the structural gene locus for Cu/ZnSOD, which is also on chromosome II near to dpy-10 (Larsen). Recombinants were generated by Ted Hutchinson and Don Lindsay some two years ago and saved. Each recombination event was targeted to the dpy-10 - unc-4 region using a recombination screen in which dpy-10 ( e124 )and unc-4 ( e120 )were in trans and we identified non-Unc non-Dpy recombinants. This novel scheme was necessary so that the recombinants would be free of marker effects of unc-4 and dpy-10 which can interfere with the assay for Age. Since the desired recombinants are not distinguishable from the parental heterozygote, the procedure involved crossing heterozygous males with Dpy Unc hermaphrodites and identifying non-Unc non-Dpy progeny resulting from recombination in the male, which are subsequently made homozygous. One of the parental chromosomes is further marked with Bergerac DNA so that multiple RFLPs are segregating in the recombinants. The crosses were done reciprocally and results have been previously reported (WBG 11.4:74) showing that the Age and Fer phenotypes are separable. Fer is due to fer-15 and age-1 maps near unc-4 .

These same recombinants were assayed for resistance to paraquat (20mM) by daily measure of dead or alive scoring spontaneous movement or response to touch as the test for viability. We have completed five distinct experiments entailing 20 or more segregating strains in each experiment and have completed replications of all recombinant lines at least twice using age-synchronous cultures at three days of age; each assay was performed in triplicate on cultures of 25 worms each (15 to 20 thousand worms total). Deaths by paraquat are noticeably different from normal death because they do not involve immediate tissue degeneration, the worms appearing grossly normal after death.

The results show that Age strains are more resistant to paraquat than are non-Age strains. All replicates confirm increased resistance of Age strains when assayed at either 48 hours or 72 hours after the time of addition of paraquat. Since there are multiple Bergerac RFLPs segregating in the cross, we can apply quantitative-trait locus mapping procedures to this data as well. Paraquat-resistance levels of strains carrying different allelic types of fer-15 , age-1 ,and 7 Tc1 -generatedRFLPs segregating in the crosses are shown in the Table. age-1 is the "best" marker for paraquat resistance (Age was based on two replications of all life spans completed one to two years ago). RFLPs near age-1 also result in quite different levels of resistance to paraquat; the closer a marker is to age-1 ,the higher the level of paraquat resistance. There is no effect of fer-15 or pZ4 ,the marker closest to the physical map position of the SOD. This shows that whatever is causing the increase in paraquat resistance maps to the Age locus and not to the SOD locus. It would be very exciting if this increased paraquat-resistance and SOD activity results from the age-1 mutation and then lead to the increase in life span. It would also argue for a potentially generalizable effect of the age-1 gene. Caveats: these results are only correlative; they show only that paraquat resistance and increased life probably result from the same event(s); they do not show that increased resistance to free radicals causes the increase in life span.

Mean 48-hour survival and standard deviation after 20mM paraquat treatment

[See Figure 1]

Figure 1