First published online September 15, 2004
Journal of Experimental Biology 207, 3649-3656 (2004)
Published by The Company of Biologists 2004
doi: 10.1242/jeb.01219
Regulation of heat shock genes in isolated hepatocytes from an Antarctic fish, Trematomus bernacchii
Bradley A. Buckley*,
Sean P. Place and
Gretchen E. Hofmann
Department of Ecology, Evolution and Marine Biology, University of
California, Santa Barbara, CA 93106-9610, USA
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Fig. 1. McMurdo Sound, Antarctica. McMurdo Station (United States Antarctic
Program) and Cape Evans are marked with red dots; the collection site for
Trematomus bernacchii is marked with a green square. Inset,
Antarctica. The white box represents the area depicted on the larger map.
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Fig. 2. Protein synthesis patterns in isolated hepatocytes from Trematomus
bernacchii exposed to heat and chemical stress. (A–D) Patterns from
four individuals exposed for 1 h to 0–12°C or 100 µmol
l–1 CdCl2 or 100 µmol l–1
MG132 are depicted. Following exposure, newly synthesized proteins were
labeled with 35S-radiolabeled cysteine/methionine for 2 h. Proteins
were resolved on 10% polyacrylamide gels. Dried gels were then exposed to
X-ray film. No visible induction of any size class of protein was
observed.
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Fig. 3. Hsp70 and Hsc71 mRNA expression in hepatocytes of Trematomus
bernacchii, exposed to heat and chemical stress. (A) Northern slot blots
of Hsp70 mRNA in hepatocytes from four individuals (lanes 1–4) exposed
for 1 h to 0–12°C or to 100 µmol l–1
CdCl2 or 100 µmol l–1 MG132. Also depicted are
samples of hepatocytes exposed to DMSO controls (CdCl2 and MG132
were delivered in DMSO solutions; see Materials and methods). Samples were
probed with Hsp70 mRNA probes generated in T. bernacchii. (B) Levels
of Hsp70 and Hsc71 mRNA in the hepatocytes from four individuals. No
significant effect of treatment on the concentration of either message was
observed. Values are means ±
S.E.M.
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Fig. 4. HSF1–HSE complexes in the hepatocytes of Trematomus
bernacchii, visualized via electrophoretic mobility shift assay
(EMSA). A competition assay was run to determine HSE probe specificity. 25
µg of homogenized hepatocyte preparation was incubated with 15 pmol of
32P-labeled HSE probe for 20 min at 0°C and then separated on a
5% non-denaturing polyacrylamide gel. Gels were dried and exposed to a
phosphoscreen. HSF1–HSE complexes were visualized on a phosphoimager.
Lane 1: hepatocyte sample and radiolabeled HSE probe only. Lane 2: identical
to lane 1 except for the addition of a 200 mol l–1 excess of
an unlabeled non-competitor DNA probe (AP2 from Promega). Lane 3: identical to
lane 1 except for addition of a 200 mol l–1 excess of
unlabeled HSE probe.
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Fig. 5. HSF1 activity in Trematomus bernacchii hepatocytes. (A)
Representative electrophoretic mobility shift assay (EMSA) image of
HSF1–HSE complexes in hepatocytes from one individual, exposed for 1 h
to 0–12°C or to 100 µmol l–1 CdCl2 or
100 µmol l–1 MG132. Each sample was run in duplicate in
two lanes. (B) Average (mean ± S.E.M.)
HSF1 activity for four individuals exposed to heat and chemical stressors
outlined above. While HSF1 activity was always detected in these cells, no
significant effect of treatment was observed.
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© The Company of Biologists Ltd 2004
