Do Antarctic Fish Respond to Heat Shock?
115
Total RNA was purified also from spleen excised from fish adapted at
-1.2 °C (Co) and from spleen incubated for 4 h at -1.2 °C in saline solution
(C4). Normalized Northern blots probed with a fragment of the cloned C.
hamatus hsp70 gene revealed an increase of about 15% in the level of
hsp 70 mRNA in C4 compared to Co, suggesting that handling/extraction of
the spleen and the subsequent incubation in saline solution was stressful
enough to induce an increase in hsp70 transcription. After 4 h of
incubation at 5°C, level of transcription increased about 50% compared to
Co and about 10% compared to C4. Possibly, the heat shock response was
masked by an initial induction of stress proteins in response to handling
conditions.
The level of hsp70 transcript from T. bernacchii was compared with
RNA purified from N rossii, a more eurythermal species that experiences
significant seasonal temperature variations. In this fish, the optimal
temperature of induction of HSPs is 8 °C with an increase in the level of
hsp70 transcript of about 100% compared to the sample heat shocked at 5
°C [30]. The reason why the heat shock response is different in these two
species may be due to the difference in the temperature stability of the
environment that they live in. It is possible to reason that fish like T.
bernacchii adapted to a constant water temperature without seasonal
temperature variations have a poor capacity to respond to heat stresses,
while for species like N rossii the ability to induce the heat shock response
is a constraint for survival.
Discussion
Differences in hsp70 mRNA induction in different species of Antarctic
fish open new molecular and evolutionary questions that need to be
addressed. Differences in regulation of expression may reside in
differences in the promoters of hsp 70 genes of these fish species. The lack
of expression of hsp70 in T. bernacchii and the results of the preliminary
experiments of primer extension make it very likely that HSEs present in
the T. bernacchii hsp70 promoter may not be functional. This hypothesis is
confirmed by our experiments of heat shock on spleen and liver of N
rossii in which we found transcription of hsp70 gene. In particular, N
rossii should have functionally active tandem arrays of nGAAn units and
HSF could induce transcription by binding to these cis-elements. To
compare differences in the promoter organization and in their activity, we
cloned N rossii and T. bernacchii hsp70 promoters from genomic DNA
and sequencing is in progress.
Other and more complex levels of regulation of the heat shock gene
expression may be exerted by the HSF itself, in particular the HSF1, that in
115
Total RNA was purified also from spleen excised from fish adapted at
-1.2 °C (Co) and from spleen incubated for 4 h at -1.2 °C in saline solution
(C4). Normalized Northern blots probed with a fragment of the cloned C.
hamatus hsp70 gene revealed an increase of about 15% in the level of
hsp 70 mRNA in C4 compared to Co, suggesting that handling/extraction of
the spleen and the subsequent incubation in saline solution was stressful
enough to induce an increase in hsp70 transcription. After 4 h of
incubation at 5°C, level of transcription increased about 50% compared to
Co and about 10% compared to C4. Possibly, the heat shock response was
masked by an initial induction of stress proteins in response to handling
conditions.
The level of hsp70 transcript from T. bernacchii was compared with
RNA purified from N rossii, a more eurythermal species that experiences
significant seasonal temperature variations. In this fish, the optimal
temperature of induction of HSPs is 8 °C with an increase in the level of
hsp70 transcript of about 100% compared to the sample heat shocked at 5
°C [30]. The reason why the heat shock response is different in these two
species may be due to the difference in the temperature stability of the
environment that they live in. It is possible to reason that fish like T.
bernacchii adapted to a constant water temperature without seasonal
temperature variations have a poor capacity to respond to heat stresses,
while for species like N rossii the ability to induce the heat shock response
is a constraint for survival.
Discussion
Differences in hsp70 mRNA induction in different species of Antarctic
fish open new molecular and evolutionary questions that need to be
addressed. Differences in regulation of expression may reside in
differences in the promoters of hsp 70 genes of these fish species. The lack
of expression of hsp70 in T. bernacchii and the results of the preliminary
experiments of primer extension make it very likely that HSEs present in
the T. bernacchii hsp70 promoter may not be functional. This hypothesis is
confirmed by our experiments of heat shock on spleen and liver of N
rossii in which we found transcription of hsp70 gene. In particular, N
rossii should have functionally active tandem arrays of nGAAn units and
HSF could induce transcription by binding to these cis-elements. To
compare differences in the promoter organization and in their activity, we
cloned N rossii and T. bernacchii hsp70 promoters from genomic DNA
and sequencing is in progress.
Other and more complex levels of regulation of the heat shock gene
expression may be exerted by the HSF itself, in particular the HSF1, that in