age-1 thermotolerance may be associated with
hsp16 accumulation Gordon J. Lithgow, Tiffany M. White, and Thomas E Johnson. Institute for Behavioral Genetics, Campus Box 447, University of Colorado, Boulder, Colorado, 80309. Place a four-day-old hermaphrodite on an agar plate at 35C 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 35C (1 ) which we have coined increased intrinsic thermotolerance (Itt). These mutations also lead to an extension in life span at 20C (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 30C-35C and after recovery periods of up to three days. Hermaphrodites of N2 and TJ1052 (
age-1) were grown at 20C 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 35C. Larger differences were observed one and two days after a heat shock for 2 hrs at 30C. 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 30C 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.