Worm Breeder's Gazette 13(5): 81 (February 1, 1995)
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.
Institute for Behavioral Genetics, Campus Box 447, University of Colorado, Boulder, Colorado, 80309.
Place a four-day-old hermaphrodite on an agar plate at 35¡C and it will on average last about 10 hrs before it ceases to respond to touch. That is unless it happens to carry a mutation at the age-1, daf-2 or spe-26 locus. These mutations result in increased survival at 35¡C (1 ) which we have coined increased intrinsic thermotolerance (Itt). These mutations also lead to an extension in life span at 20¡C (2-4, Age). We have begun to investigate the molecular basis of thermotolerance in age-1 strains with the idea that there may be a causal relationship between the Age and Itt phenotypes (see accompanying article). We were prompted by extensive studies of thermotolerance in Drosophila and mammalian ceils to investigate heat shock protein synthesis. We utilized an polyclonal hsp16 antibody (5) kindly provided by Dr. E. Peter M. Candido (Univ. British Columbia, Vancouver, Canada) and a monoclonal hsp70 antibody (Affinity Bioreagents Inc.) to examine hsp accumulation during heat shock at 30¡C-35¡C and after recovery periods of up to three days. Hermaphrodites of N2 and TJ1052 (age-1) were grown at 20¡C to four-days of age, heat shocked on agar plates, washed off and quickly frozen with liquid N2 . Protein extract was prepared by boiling in SDS-PAGE loading dye and extract equivalent to 30 worms electrophoresed. Western blot analysis, followed by densitometry, revealed a reproducible difference in hsp16 accumulation. TJ1052(age-1) accumulated 50-300percent more hsp16 after 2-4 hr heat shocks at 30 and 35¡C. Larger differences were observed one and two days after a heat shock for 2 hrs at 30¡C. Material detected with the monoclonal antibody raised against human hsp70, does not differ extensively between strains. We pursued hsp16 expression differences with transgenic strains containing the hsp16-41::1acZconstruct (6). The construct was integrated and backcrossed five times to N2 and to TJ1 052(age-1) to produce TJ830(rol-6) and TJ831 (age-1 rol-6). We have determined a number of heat shock and recovery conditions in which TJ831 worms show extensively more intense staining than TJ830. For example, after 24 hr at 30¡C there is almost no staining in wild-type worms whereas age-1 worms have stained eggs and pharynx. This system may allow for a dissection of age-1-specific over-expression of hsp1 6. It appears that age-1 thermotolerance is correlated to an increase of expression of at least one class of hsp genes. We wish to know whether expression of this and other stress response genes is involved in determining the life span at nommal temperatures. (1 ) Lithgow et al. J. Geront. B.S.49, B270,1994. (2) Friedman and Johnson, J. Geront. B.S. 4, B102.1988. (3) Van Voorhies, Nature. 360,456.1992. (4) Kenyon et al. Nature, 366,461, 1993. (5) Hockertz etal., FEBS, 280,375,1991 (6) Fire etal, Gene 93,189,1990.