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.

age-1 Thermotolerance May Be Associated With hsp-16 Accumulation.

Gordon J. Lithgow, Tiffany M. White, 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
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.