2331
The Journal of Experimental Biology 203, 2331–2339 (2000)
Printed in Great Britain © The Company of Biologists Limited 2000
JEB2715
HEAT-SHOCK PROTEIN EXPRESSION IS ABSENT IN THE ANTARCTIC FISH
TREMATOMUS BERNACCHII (FAMILY NOTOTHENIIDAE)
GRETCHEN E. HOFMANN1,*, BRADLEY A. BUCKLEY2,‡, SUSANNA AIRAKSINEN3, JOHN E. KEEN2
AND GEORGE N. SOMERO2
1Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA, 2Hopkins Marine Station,
Stanford University, Pacific Grove, CA 93950-3094, USA and 3Department of Biology, University of Turku,
FIN-20014, Finland
*Author for correspondence (e-mail: ghofmann@asu.edu)
‡Present address: Department of Biology, Arizona State University, Tempe, Arizona 85287-1501, USA
Accepted 10 May; published on WWW 10 July 2000
Summary
The heat-shock response, the enhanced expression of one
chaperones could not be detected under any of the
or more classes of molecular chaperones termed heat-shock
experimental condition used, solid-phase antibody
proteins (hsps) in response to stress induced by high
(western) analysis revealed that a constitutively expressed
temperatures, is commonly viewed as a ‘universal’
70 kDa chaperone was present in this species, as predicted
characteristic of organisms. We examined the occurrence
on the basis of requirements for chaperoning during
of the heat-shock response in a highly cold-adapted,
protein synthesis. Amounts of the constitutively expressed
stenothermal Antarctic teleost fish, Trematomus bernacchii,
70 kDa chaperone increased in brain, but not in gill, during
to determine whether this response has persisted in a
22 days of acclimation to 5 °C. The apparent absence of a
lineage that has encountered very low and stable
heat-shock response in this highly stenothermal species is
temperatures for at least the past 14–25 million years. The
interpreted as an indication that a physiological capacity
patterns of protein synthesis observed in in vivo metabolic
observed in almost all other organisms has been lost as a
labelling experiments that involved injection of 35S-labelled
result of the absence of positive selection during evolution
methionine and cysteine into whole fish previously
at stable sub-zero temperatures. Whether the loss of the
subjected to a heat stress of 10 °C yielded no evidence for
heat-shock response is due to dysfunctional genes for
synthesis of any size class of heat-shock protein. Parallel in
inducible hsps (loss of open reading frames or functional
vivo labelling experiments with isolated hepatocytes
regulatory regions), unstable messenger RNAs, the absence
similarly showed significant amounts of protein synthesis,
of a functional heat-shock factor or some other lesion
but no indication of enhanced expression of any class of
remains to be determined.
hsp. The heavy metal cadmium, which is known to induce
synthesis of hsps, also failed to alter the pattern of proteins
Key words: heat-shock protein, Antarctic fish, teleost, Trematomus
synthesized in hepatocytes. Although stress-induced
bernacchii, hepatocyte.
Introduction
Antarctic fish of the teleost suborder Notothenioidei are
extreme stenotherms that live in the cold, thermally stable
waters of coastal Antarctica, where temperatures range from
+0.3 °C to −1.86 °C (Eastman, 1993). Although the precise
geological time at which the Southern Ocean reached its
current temperature range remains under debate, it is certain
that these highly endemic fish have been isolated in extremely
cold water for many millions of years (Eastman, 1993).
Extensive cooling of Antarctic waters probably began when the
Drake Passage opened approximately 25 million years ago,
allowing circumpolar circulation of deep water and preventing
mixing of Antarctic waters with the warmer waters of the
temperate regions (Denton et al., 1991). Current estimates
suggest that the Antarctic notothenioid stock has evolved in
an extremely cold and stable thermal environment for
approximately 14–25 million years (Eastman, 1993; Clarke
and Johnston, 1996).
Given this extensive period of isolation, Antarctic
notothenioids provide a unique opportunity to study
evolutionary adaptation to stable, subzero temperatures. As a
consequence of having undergone 14–25 million years of
evolution in extreme cold, notothenioids are distinguished by
a number of physiological adaptations to low temperatures,
including antifreeze glycoproteins that prevent freezing of
tissues in the subzero, ice-filled waters (DeVries, 1988),
tubulin molecules that polymerize into microtubules at low
temperatures (Detrich, 1991) and enzymes with unusually high
catalytic efficiencies that offset the effects of low temperature
2332 G. E. HOFMANN AND OTHERS
on metabolic rates (Fields and Somero, 1998). In addition to
gaining novel physiological attributes for coping with low
temperatures, some Antarctic notothenioids have been shown
to have lost traits during evolution at subzero temperatures, for
instance, the ability of fishes of the family Channichthyidae to
express hemoglobin and myoglobin proteins (Cocca et al.,
1997; Sidell et al., 1997; Somero et al., 1998). The loss of these
respiratory proteins may reflect the absence of positive
selection for their functions because of the high solubility of
oxygen at low temperature and the relatively sluggish
swimming behavior of channichthyid fishes.
Evolution at the low and stable temperatures of the Southern
Ocean might also permit the loss of traits whose function is to
increase heat tolerance or to facilitate acclimatization to shortterm changes in temperature. One such trait is the heat-shock
response, a cellular process in which organisms subjected to a
heat stress increase the synthesis of a set of molecular
chaperones termed heat-shock proteins (hsps). Under normal
non-stressful conditions, molecular chaperones assist in the
routine folding and compartmentation of newly synthesized
proteins (Ellis, 1990; Hartl, 1996; Fink, 1999). During thermal
stress, heat-induced chaperones, hsps, bind to thermally
denatured proteins, thereby preventing their aggregation and
providing an opportunity for them to re-fold into native,
functional states following restoration of normal body
temperatures. The heat-shock response is thought to be nearly
universal among organisms, in only one case has the heatshock response not been detected (Hydra oligactis: Bosch et
al., 1988), and hsps themselves are highly evolutionarily
conserved in sequence across phyla (Lindquist, 1986; Parsell
and Lindquist, 1993; Feder and Hofmann, 1999). Although
the temperatures that trigger expression of hsps vary among
species according to their adaptation and acclimation
temperatures (Hofmann and Somero, 1996; Feder and
Hofmann, 1999; Tomanek and Somero, 1999), patterns of
expression of hsps show striking levels of conservation among
species.
To determine whether the capacity to elicit the heat-shock
response has remained in Antarctic notothenioid fishes, we
studied populations of Trematomus bernacchii (Family
Nototheniidae) from McMurdo Sound, the southernmost
region of the world ocean, where water temperatures yearround remain within a few hundredths of a degree Celsius of
the freezing point of sea water, −1.86 °C (Eastman, 1993). We
employed in vivo metabolic labelling of whole fish (injection
of 35S-labelled methionine and cysteine during heat stress) to
determine whether sub-lethal heat stress triggered the synthesis
of hsps. We performed metabolic labelling studies with
isolated hepatocytes to determine the effects on patterns of
protein synthesis after heat stress and exposure to the heavy
metal cadmium, which is known to induce hsp synthesis. We
used solid-phase antibody (western) analysis of molecular
chaperones of the 70 kDa class (hsp70 isoforms) to determine
whether stress-induced or constitutively expressed chaperones
of this class were present and, if so, whether acclimation of
fish to 5 °C, the highest temperature to which T. bernacchii can
be acclimated, changed the level of expression of hsp70
isoforms. Although constitutively expressed isoforms of hsp70
were present in T. bernacchii, neither heat nor cadmium stress
led to the induction of a heat-shock response.
Materials and methods
Specimen collection and acclimation conditions
Trematomus bernacchii (Boulenger) were collected in
McMurdo Sound, Antarctica (77°53′S, 166°40′E), in January
1995, 1996 and 1999 using hand lines and baited fish traps.
Specimens were either held in flow-through seawater aquaria
maintained at the ambient seawater temperature (−1.86 °C) or
placed into heated non-flowthrough aquaria for acclimation at
5 °C. Specimens maintained at −1.86 °C ranged in mass from
22.5 to 300 g. Specimens used in the acclimation studies
ranged in mass from approximately 30 to 150 g. No attempt
was made to distinguish gender before use; approximately
equal numbers of male and female specimens were employed.
In the acclimation study, fish (N=3 at each time point) were
killed after 5, 11, 15 and 22 days of acclimation. Fish were
killed by cervical transection and anesthetized with MS-222
dissolved in filtered sea water at −1 °C.
Heat-shock protein induction experiments: in vivo metabolic
labelling with 35S-labelled amino acids
The induction of hsps was tested in whole fish using in vivo
metabolic labelling following the protocol of Dietz and Somero
(1992, 1993). Fish were maintained at −1.86 °C for 3–5 days
prior to the induction experiments to minimize the effects of
capture and handling stress. For the induction experiments,
individual fish were injected intraperitoneally with a 35Slabelled methionine/cysteine amino acid mixture (NEN; 35S
Express label) at 4×106 Bq g−1 body mass. Smaller fish, ranging
in body mass from 22.5 to 50.0 g, were used to economize on
amounts of isotope. The amino acid mixture was diluted 1:1
with nototheniid Ringer’s solution (570 mosmol l−1) composed
of 311 mmol l−1 NaCl, 5 mmol l−1 KCl, 2.5 mmol l−1 MgCl2,
3.0 mmol l−1 CaCl2, 2.5 mmol l−1 NaHCO3, 2.0 mmol l−1
NaH2PO4 and 5.0 mmol l−1 glucose (J. Eastman, personal
communication) prior to injection. The fish were maintained in
ambient temperature sea water during the injection procedure
to avoid prematurely heat-shocking the specimens. Following
the injection, fish were transferred to 4 l vessels containing
continuously aerated sea water equilibrated to the desired
exposure temperature. For the heat-shock treatment, fish (N=4)
were exposed to 10 °C for 2 h and then transferred to −1.5 °C
for a 6 h recovery period. The median survival time of T.
bernacchii at 10 °C is approximately 140 min (Somero and
DeVries, 1967), so the 2 h heat shock at this temperature was
predicted to be severe, but not rapidly lethal. Fish in the control
group (N=3) were maintained at −1.5 °C for 8 h. For heat shock
and control groups, fish were placed directly into water at the
exposure temperature after the injection of 35S-labelled amino
acids. At the end of the 8 h temperature exposure period, the
fish were killed, and samples of brain, heart, liver, white
Lack of heat-shock response in Antarctic fish 2333
skeletal muscle, gill and spleen were dissected (note that
insufficient label was incorporated into white muscle to allow
autoradiographic analysis). Tissues were immediately frozen
on dry ice and stored at −80 °C. Because of restrictions on
modes of shipment of radiolabelled materials from Antarctica,
tissue samples were returned to the USA aboard ship (transit
time, approximately 3 months), where electrophoresis and
autoradiography were performed.
In preparation for SDS–PAGE, tissue samples were
homogenized with a Teflon pellet pestle in 200 µl of SDS
homogenization medium (32 mmol l−1 Tris-HCl, 2 % SDS,
1 mmol l−1 phenylmethylsulfonylfluoride, pH 6.8). Following
homogenization, tissue extracts were first boiled for 5 min, then
centrifuged for 15 min at 16 000 g, and the resulting supernatant
was removed and stored at −20 °C prior to electrophoresis.
Suspension culture of hepatocytes
Hepatocytes were isolated according to the two-step
collagenase perfusion method of Seglen (1976), as modified by
Råbergh et al. (1992), with additional adjustments as follows.
Briefly, fish were anesthetized in MS-222 at −1 °C, and the
portal vein was cannulated to perfuse the blood from the liver.
Both fish and perfusion solution (290 mmol l−1 NaCl, 2 mmol l−1
KCl, 10 mmol l−1 Hepes, 0.5 mmol l−1 EGTA, 25 mmol l−1
Tricine, pH 7.8) were kept on ice during the procedure. The
liver was transferred to a Petri dish, and the gall bladder was
removed. Then, the liver was cut into small pieces and digested
with suspension buffer (292.5 mmol l−1 NaCl, 5.0 mmol l−1
KCl, 2.5 mmol l−1 MgCl2, 3.0 mmol l−1 CaCl2, 2.0 mmol l−1
NaHCO3, 2.0 mmol l−1 NaH2PO4, 5 mmol l−1 glucose,
50 mmol l−1 Hepes, pH 7.8) containing 5 units ml−1 collagenase
type 1A, type IV or type L (Sigma Chemical Co.) for 1 h at 4 °C.
Dispersed hepatocytes were filtered through a nylon filter
(100 µm) and washed twice with the perfusion buffer. Cells
were resuspended in the suspension buffer at a density of
2×106 cells ml−1 and allowed to recover with constant shaking
at 0 °C for 30 min prior to use in experiments. Viability was
measured by Trypan Blue exclusion (0.4 % w/v) before, during
and after the exposures. Viability exceeded 85 % in all
temperature-treated samples; cadmium treatment appeared to
lower the viability (to 62–97 %).
Metabolic labelling of hepatocytes and gill cells
For the heat-shock experiments, cell suspensions (3×106 per
tube) were routinely exposed to the treatment temperature for
1 h followed by a 4 h metabolic labelling step at 0 °C. Cells were
incubated in suspension buffer containing 4×106 Bq of 35Slabelled methionine/cysteine amino acid mixture (NEN 35S
Express Label). During experiments employing cadmium, cells
were incubated for 1 h at 0 °C in cadmium chloride
concentrations ranging from 5 to 150 µmol l−1. Cells were
washed twice to remove the cadmium, and metabolic labelling
in cadmium-free buffer was carried out as above.
Gill cells were isolated from the temperate, eurythermal
goby fish Gillichthys mirabilis following the protocol given in
Kültz and Somero (1995).
Electrophoresis, fluorography and analysis of protein
expression patterns
Patterns of protein synthesis were examined using
SDS–polyacrylamide gel electrophoresis in combination with
fluorography, as described by Hofmann and Somero (1996).
Prior to electrophoresis, tissue extracts were analyzed using
liquid scintillation counting. Samples from each tissue extract
were then diluted into SDS sample buffer (62.5 mmol l−1 TrisHCl, pH 6.8, 10 % glycerol, 2 % SDS, 5 % 2-mercaptoethanol
and 0.01 % Bromphenol Blue) and loaded to equivalent
amounts of radioactivity per lane. The standardized amount of
radioactivity loaded per lane varied for each tissue to ensure
sufficient radioactivity for autoradiography while not
overloading the gel with respect to protein. Protein samples
were subjected to electrophoresis on a 10 % polyacrylamide gel
for 3.5 h at 20 mA. Following electrophoresis, the gels were
treated with EN3HANCE, an autoradiographic enhancer,
according to the manufacturer’s instructions (DuPont NEN).
Fluor-impregnated gels were dried at 60 °C for 2.5 h, exposed
to X-ray film (Kodak X-OMAT AR5) at −70 °C for an
empirically determined period and developed. For tissues from
the whole fish experiments returned by ship to the USA, the
decay of 35S label during the 3–4 month period following
initial labelling required lengthy exposure times of X-ray film,
approximately 4–6 weeks, depending on the intensity of the
residual radioactivity.
Immunochemical analysis of hsp70 in T. bernacchii tissue
Western blot analysis and scanning densitometry were used
to compare the levels of hsp70 in T. bernacchii brain and gill
at sampling intervals during the 5 °C acclimation experiment,
according to the methods described by Hofmann and Somero
(1995). Equal amounts of protein (25 µg) were separated on
7 % gels and transferred to nitrocellulose using semi-dry
electrophoretic transfer; the resulting blots were stained using
an enhanced chemiluminescence (ECL) protocol as the final
detection step. The primary antibody used was an anti-hsp70
rat monoclonal antibody (Affinity BioReagents; MA3-001)
that recognizes both cognate and heat-inducible forms of
hsp70. Protein determinations were made on the tissue extracts
using a Coomassie Plus protein assay (Pierce Chemical Co.).
After the ECL western blot procedure had been completed,
scanning densitometry was used to determine the relative
absorbance of each band. Data are expressed as optical
density×area (mm2). The amount of protein used in each lane
of the gel was optimized to be within the linear range of
detection for the X-ray film.
Results
Whole-organism induction experiments
The fluorograms of radiolabelled proteins in tissues of T.
bernacchii sampled after in vivo metabolic labelling of whole
fish provided no evidence for a heat-shock response (Fig. 1).
After a 2 h heat-shock treatment at 10 °C followed by 6 h of
recovery at −1.5 °C, no size class of protein exhibited enhanced
2334 G. E. HOFMANN AND OTHERS
C
MW
HS
C
HS
HS
C
HS
HS
C
C
212
97.4
68
43
29
Spleen
MW
C
Liver
HS
C
HS
Gill
C
HS
C
HS
212
97.4
68
43
29
Brain
Heart
Fig. 1. Whole-organism heat-shock induction experiments on the Antarctic notothenioid Trematomus bernacchii. The fluorograms show the
protein synthesis pattern in five different tissues from control (C) and heat-shocked (HS) fish. Individual fish were injected with
4×106 Bq g−1 body mass and exposed to either a control or heat-shock treatment (see Materials and methods for experimental protocol). Proteins
were separated on 10 % SDS–polyacrylamide gels; individual lanes were loaded with equivalent amounts of radioactivity, and individual
samples were run in duplicate. 14C-labelled protein molecular mass markers (MW) are shown in kDa.
synthesis in any of the five tissues examined. However,
adequate labelling of proteins was observed, indicating that the
radiolabelled amino acids were efficiently incorporated into
protein during the labelling period despite exposure to a
temperature that is lethal after a period of 2–3 h (Somero and
DeVries, 1967).
Metabolic labelling of isolated hepatocytes
The patterns of protein synthesis observed in isolated
hepatocytes stressed with high temperature (Fig. 2A) or
cadmium treatment (Fig. 3) also provided no evidence of a
heat-shock response. After 1 h incubations at 5, 8 or 10 °C,
followed by metabolic labelling at 0 °C, heat-shocked cells
were viable and capable of synthesizing proteins (Fig. 2A).
Visual inspection of the autoradiograms demonstrated that the
translational capacity of heat-shocked cells was equivalent to
that of control cells (Fig. 2A). The pattern of protein
expression in heat-shocked cells was essentially identical to
that observed in control cells. Although translation started to
be reduced at 15 °C (data not shown), in all experiments
conducted on hepatocytes (N=7 individual cell preparations
from seven livers), there was no detectable induction of hsps,
Lack of heat-shock response in Antarctic fish 2335
A
0
Temperature (°C)
5
8
10
B
C
HS
200 kDa
116 kDa
100 kDa
90 kDa
97 kDa
70 kDa
60 kDa
66 kDa
40 kDa (LMW)
20 kDa (LMW)
45 kDa
31 kDa
21 kDa
Fig. 2. (A) Effects of temperature on protein synthesis patterns of hepatocytes isolated from Trematomus bernacchii. Hepatocytes were
exposed to the temperature indicated above each lane for 1 h. Following metabolic labelling for 4 h at 0 °C, proteins in cell lysates were
separated using SDS–polyacrylamide gel electrophoresis; equivalent counts (300 000 cts min−1) were applied to each lane. Positions of protein
molecular mass standards are shown on the left. (B) Heat-shock induction experiment with isolated gill cells from the eurythermal goby
Gillichthys mirabilis. Control (C) and heat-shocked (HS) cells were incubated for 1 h at 23 °C and 37 °C, respectively, in 2×106 Bq of 35Slabelled methionine/cysteine amino acid mixture. Arrows indicate the molecular masses of the different classes of heat-inducible heat-shock
proteins. LMW, low molecular weight chaperone.
despite heat-shock temperatures that exceeded the ecologically
relevant temperature of T. bernacchii by over 15 °C.
For purposes of comparison and to validate the protocol used
with the hepatocytes of T. bernacchii, Fig. 2B shows an
autoradiogram from an experiment on isolated gill cells from
the eurythermal goby Gillichthys mirabilis. These data show
the classical heat-shock response of cells exposed to elevated
temperatures, using the same protocols employed to study
hepatocytes of T. bernacchii.
Concentrations of cadmium known to induce hsps in other
cell types did not result in hsp synthesis in T. bernacchii
hepatocytes (Fig. 3). In all cases, the hepatocytes exhibited
high levels of protein synthesis; thus, the lack of hsp induction
could not be attributed to poor incorporation of radiolabelled
amino acids into newly synthesized proteins.
Detection of 70 kDa hsp isoforms in T. bernacchii tissue
Constitutively
expressed
70 kDa
hsps
were
immunochemically detected in tissues of T. bernacchii
caught in water at −1.86 °C and never exposed to elevated
temperatures (Fig. 4). Using western analysis, isoforms of the
70 kDa hsp family were detected in gill (Fig. 4) and brain
(data not shown) of freshly caught, non-heat-shocked
individuals. One-dimensional gel electrophoresis revealed
2336 G. E. HOFMANN AND OTHERS
[Cadmium] (µmol l-1)
0
15
25
6
50
100
Band intensity
5
200 kDa
116 kDa
Brain
Gill
4
3
2
1
97 kDa
0
Control
Day 5
Day 11
Day 15
Day 22
Fig. 5. Effects of acclimation to 5 °C on endogenous concentrations
of 70 kDa heat-shock proteins (hsps) in gill and brain of Trematomus
bernacchii. The bar graphs show the amount of 70 kDa hsp isoforms
detected relative to a standard amount of bovine brain heat-shock
cognate 70 (0.1 µg) run on each gel. Each column represents the
mean + S.E.M. for N=3 individual fish (error bars lie within the
column for control brain data). Analysis of variance revealed a
significant interaction between acclimation time and band intensity
(optical density×area) in brain (P<0.0080).
66 kDa
45 kDa
one predominant band with an apparent molecular mass of
72 kDa.
31 kDa
21 kDa
Fig. 3. Effects of cadmium on heat-shock protein induction in
isolated hepatocytes. Cells were incubated in the indicated
concentration of cadmium chloride for 1 h at 0 °C. Following the
cadmium treatment, cells were washed twice with cadmium-free
suspension buffer and then radiolabelled at 0 °C as described in
Materials and methods. The positions of protein molecular mass
standards are shown on the left.
1
2
3
4
5
6
70 kDa
Fig. 4. Western-blot detection of constitutively expressed isoforms of
the 70 kDa heat-shock protein (hsp) gene family in gill tissue from
field-acclimatized Trematomus bernacchii. Samples of gill from five
different fish were screened for 70 kDa hsp isoforms using a rat
monoclonal anti-hsp70 antibody. Each lane contains 25 µg of total
protein; the standard shown on the left (lane 1) is bovine brain heatshock cognate 70 (hsc70) (from StressGen). Lanes contained the
following samples: lane 1, bovine brain hsc70 (0.1 µg); lanes 2–6,
gill tissue from individual specimens of T. bernacchii.
Effect of acclimation on cellular concentrations of 70 kDa
hsps
Gill and brain tissues were analyzed for changes in levels of
70 kDa hsp isoforms during a 22-day acclimation period
(Fig. 5). Acclimation to 5 °C led to an increase in the levels of
constitutively expressed forms of the 70 kDa hsp family in
brain (Fig. 5; ANOVA, P<0.0080) but, in accordance with the
results of the metabolic labelling studies, there was no
indication of induction of heat-induced isoforms. Unlike brain,
gill tissue did not exhibit a statistically significant increase in
hsp70 levels during acclimation to 5 °C (Fig. 5).
Discussion
To determine whether a heat-shock response has been
retained in Antarctic notothenioid fishes during their 14–25
million years of evolution in a cold and thermally stable
environment, we employed well-established methods that have
been used to study the induction of hsps in other organisms.
Metabolic labelling protocols using whole fish (Dietz and
Somero, 1992, 1993) and isolated hepatocytes (Koban et al.,
1987; Airaksinen et al., 1998) have proved to be highly
effective means for detecting the induction of new synthesis of
hsps following heat stress. Although metabolic labelling
yielded clear evidence for induction of hsps in gill cells from
Gillichthys mirabilis (Fig. 2B), none of the five tissues of T.
bernacchii examined in metabolic labelling experiments with
whole fish heat-shocked at 10 °C (Fig. 1) and in hepatocytes
Lack of heat-shock response in Antarctic fish 2337
(Fig. 2A) subjected to heat-shock at temperatures up to 10 °C
showed any indication of induction of synthesis of hsps.
Therefore, we conclude that T. bernacchii has lost the heatshock response.
Because hsps can be induced by a number of physical and
chemical factors that have in common the ability to denature
proteins, the failure of heat stress to induce synthesis of hsps
does not, in and of itself, prove that all types of stress-induced
induction of hsps has been lost in these fishes. Thus, we tested
the effects of a well-established chemical inducer of hsps, the
heavy metal cadmium, on patterns of protein synthesis (Fig. 3).
As in the case of heat stress, no changes were observed in the
patterns of proteins newly synthesized in cadmium-stressed
cells. We conclude, then, that the ability to induce hsps of all
size classes following either physical (temperature) or
chemical stress is absent in this species.
The loss of ability to induce synthesis of stress-induced
chaperones in T. bernacchii represents an additional example
of how the absence of positive selection has allowed
physiological capacities of notothenioid fishes to be lost during
their radiation in the Southern Ocean. The loss of an ability to
synthesize hemoglobin in all species of the Family
Channichthyidae (‘icefishes’; Cocca et al., 1997) and the
absence of myoglobin in several icefishes (Sidell et al., 1997)
may reflect in part the high concentrations of dissolved oxygen
in sea water and body fluids at temperatures near 0 °C. The loss
of the heat-shock response, which is more generally termed the
‘stress response’ to indicate that hsps are induced by a suite of
protein-denaturing physical and chemical factors (Hightower,
1980), may reflect the absence of heat stress and the low levels
of chemical stressors such as heavy metals found in Antarctic
waters.
While extremely unusual, the absence of a heat-shock
response has been observed previously. The freshwater
cnidarian Hydra oligactis was unable to synthesize hsps of any
size class in response to thermal stress, even though a
congener, Hydra vulgaris, synthesized hsps of molecular
masses 23, 70 and 80 kDa (Bosch et al., 1988). Hydra oligactis
is found exclusively in cold, thermally stable environments,
but it is not known whether the loss of the heat-shock response
occurred during evolution under these thermal conditions or,
alternatively, whether the loss of the heat-shock response
relegated this species to habitats with stable low temperatures.
To our knowledge, Hydra oligactis is the only species other
than T. bernacchii in which the heat-shock response has not
been detected.
The apparent lack of induction in T. bernacchii of any size
class of hsp in response to heat stress or exposure to cadmium
suggests that one or more types of lesion have occurred either
in the hsp-encoding genes themselves or in the complex
regulatory mechanisms that govern hsp synthesis. It is
conceivable that the reading frames of all hsp-encoding genes
are disrupted, such that even if transcription of these genes
were to occur, no functional message would result and no
translation of hsps would occur. It is also possible that the hspencoding genes persist as intact reading frames, but mutations
in gene regulatory regions block transcription (see below).
Another lesion leading to the loss of expression of hsps might
involve an unstable hsp-encoding mRNA. Petersen and
Lindquist (1989) have identified a number of factors that affect
the stability of hsp-encoding mRNAs, and variation in the 5′
untranslated region of hsp70 mRNA has been shown to inhibit
translation (Hess and Duncan, 1996). In Hydra oligactis,
failure to translate mRNA is conjectured to account for the
absence of hsp70 synthesis (Gellner et al., 1992).
The complete failure to induce synthesis of any type of hsp
in T. bernacchii might be explained most economically by a
mechanism involving one or more transcription factors that
control the expression of all classes of hsp-encoding genes. An
essential step in the induction of synthesis of hsps is the
interaction between a transcription factor, the heat-shock factor
(HSF), and highly conserved regulatory DNA sequences
termed heat-shock elements (HSE) found in the promoter
regions of heat-shock genes (Morimoto, 1998). Different
transcriptional factors control the activation of inducible and
constitutively expressed hsp genes (for reviews, see Wu, 1995;
Morimoto, 1998), and these distinct HSFs are responsive to
different cellular cues (Morimoto, 1998). In eukaryotes, only
one HSF, designated HSF1, is sensitive to thermal stress and
induces hsp expression in response to heat shock (Morimoto,
1993, 1998; Jedlicka et al., 1997; Zhong et al., 1998). The
actions of the different HSFs are so specific that the activity of
HSF2, the factor that controls constitutive expression, will not
rescue the function of HSF1 when HSF1 has been deleted from
the genome (McMillan et al., 1998; Jedlicka et al., 1997). The
mechanisms by which HSF1 is activated and binds to HSE
regions is not fully understood, but direct temperature control
of HSF1 activity (Zhong et al., 1998) and control by
phosphorylation and several regulatory molecules including
molecular chaperones have all been proposed (Morimoto,
1998; Shi et al., 1998, Zou et al., 1998; Bharadwaj et al., 1999).
If one or more of these mechanisms governing HSF1 function
is dysfunctional in notothenioid fishes, then expression of hsps
could be eliminated. Mutations involving the activity of HSF1
seem a more likely basis for explaining the loss of expression
of multiple size classes of hsps than, for instance, the loss of
hsp synthesis through mutations in the regulatory regions or
reading frames of all individual hsp genes. The detection of
abundant levels of a constitutively expressed chaperone
belonging to the 70 kDa hsp family (Fig. 4; Carpenter and
Hofmann, 1999) indicates that the regulatory factors
controlling expression of the constitutive isoforms of this
family have remained intact during the evolution of this
lineage, as would of course be predicted on the basis of the
need for molecular chaperones in protein biosynthesis under
all physiological conditions. The occurrence of an increased
level of constitutively expressed hsp70 in brain (but not in gill)
following acclimation to 5 °C (Fig. 5) may simply be a
reflection of enhancement of protein synthesis at elevated
temperatures in the warm-acclimated specimens. Because the
heat-shock response is an acute response that occurs within
minutes of heat stress (Lindquist, 1986), the gradual and
2338 G. E. HOFMANN AND OTHERS
continuing rise in chaperone concentrations found in brain are
not indicative of a true heat-shock response.
To increase our understanding of the evolution of the heatshock response in notothenioids, it will be important to
examine other Antarctic notothenioids to establish whether the
loss of this response is found throughout the suborder and to
determine whether the cold-temperate members of this
suborder possess a heat-shock response. If the ancestors of
contemporary cold-temperate notothenioids were themselves
cold-temperate species, then we predict that the heat-shock
response will be found in non-Antarctic notothenioids. We
base this assumption on the observation of a heat-shock
response in all cold-temperate fishes examined to date (see
Dietz and Somero, 1993). However, if cold-temperate
notothenioids are found to lack the heat-shock response, this
would be evidence that their ancestors were Antarctic species
that successfully re-colonized cold-temperate habitats.
We acknowledge the support of the National Science
Foundation through the Office of Polar Programs for funding
the Antarctic Biology Course during which the majority of
these studies were conducted (NSF 98-12707 to Dr Donal
Manahan of the University of Southern California). We
especially thank Dr Donal Manahan for his efforts in
organizing and leading the course and the Crary Laboratory
personnel of Antarctic Support Associates for their excellent
logistical and technical assistance. We also gratefully
acknowledge the ice-fishing skills of Drs. Jonathon Stillman
and Joshua Rosenthal. Additional research was supported by
National Science Foundation grants IBN-9727721 to G.N.S.
and IBN-9723063 to G.E.H.
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