Do Antarctic Fish Respond to Heat Shock~
113
Physical Analysis of C. hamatus hsp70 Promoter
We have cloned and sequenced hsp70 gene of the Antarctic fish C.
hamatus. The deduced protein sequence revealed a high degree of
homology with most of the heat inducible HSP70 proteins. Moreover, the
functional domains characteristic among nearly all HSP70s are conserved
in the Antarctic protein, particularly in the -NH2 terminal region that
interacts with proteins during the transport or the folding/unfolding
processes, and in the -COOH terminal part, exerting ATPase activity [12].
Special attention was given to the characterization of the promoter
region of hsp 70 gene. In all promoters of eukaryotes, despite the
evolutionary distance between organisms, typical cis-acting elements
(HSE, heat shock element) have been found [13]. HSEs are regions for
specific binding of heat shock transcription factor (HSF) which is essential
to induce transcriptional activation [14]. Most heat shock promoters
contains several HSEs located both proximal and distal to the initiation of
transcription start site. The HSE can be described as a series of pentameric
units arranged as inverted adjacent arrays of the sequence 5 '-nGAAn-3 ,
[15-17].
A functional HSE is composed of a minimum of three pentamers with
additional reiteration resulting in higher affinity interactions between the
HSF and the HSE [18-20].
Distal HSEs can function as enhancer elements and may be crucial for
transcriptional activity [21,22]. Multiple HSEs may have a synergistic
effect on transcriptional activity, while rotational alignment confers higher
transcriptional activity when two HSEs are within 80 bp apart [23,24].
Amin and co-authors [23] have also conducted experiments suggesting
that HSEs does not need to be located at a specific distance from a
downstream sequence such as the TATA box. Figure I summarizes the
results of sequence analysis performed on C. hamatus hsp 70 gene
promoter.
We identified six HSEs, three of which match exactly with two 5 bp
units nGAAn. Moreover, typical upstream elements found in most
eukaryotic promoters of genes transcribed by RNA polymerase II [25] are
present also in hsp70 promoter of C. hamatus. This is the case of the
TAT A box and the CAA T box, usually located respectively at about 20
and 80 nt upstream of the start point of transcription. At the start point
there is a tendency for the first base of mRNA to be A, flanked on either
side by pyrimidines spanning the positions between -3 and +5 [25]. The
promoter under analysis presents all those features, with multiple possible
113
Physical Analysis of C. hamatus hsp70 Promoter
We have cloned and sequenced hsp70 gene of the Antarctic fish C.
hamatus. The deduced protein sequence revealed a high degree of
homology with most of the heat inducible HSP70 proteins. Moreover, the
functional domains characteristic among nearly all HSP70s are conserved
in the Antarctic protein, particularly in the -NH2 terminal region that
interacts with proteins during the transport or the folding/unfolding
processes, and in the -COOH terminal part, exerting ATPase activity [12].
Special attention was given to the characterization of the promoter
region of hsp 70 gene. In all promoters of eukaryotes, despite the
evolutionary distance between organisms, typical cis-acting elements
(HSE, heat shock element) have been found [13]. HSEs are regions for
specific binding of heat shock transcription factor (HSF) which is essential
to induce transcriptional activation [14]. Most heat shock promoters
contains several HSEs located both proximal and distal to the initiation of
transcription start site. The HSE can be described as a series of pentameric
units arranged as inverted adjacent arrays of the sequence 5 '-nGAAn-3 ,
[15-17].
A functional HSE is composed of a minimum of three pentamers with
additional reiteration resulting in higher affinity interactions between the
HSF and the HSE [18-20].
Distal HSEs can function as enhancer elements and may be crucial for
transcriptional activity [21,22]. Multiple HSEs may have a synergistic
effect on transcriptional activity, while rotational alignment confers higher
transcriptional activity when two HSEs are within 80 bp apart [23,24].
Amin and co-authors [23] have also conducted experiments suggesting
that HSEs does not need to be located at a specific distance from a
downstream sequence such as the TATA box. Figure I summarizes the
results of sequence analysis performed on C. hamatus hsp 70 gene
promoter.
We identified six HSEs, three of which match exactly with two 5 bp
units nGAAn. Moreover, typical upstream elements found in most
eukaryotic promoters of genes transcribed by RNA polymerase II [25] are
present also in hsp70 promoter of C. hamatus. This is the case of the
TAT A box and the CAA T box, usually located respectively at about 20
and 80 nt upstream of the start point of transcription. At the start point
there is a tendency for the first base of mRNA to be A, flanked on either
side by pyrimidines spanning the positions between -3 and +5 [25]. The
promoter under analysis presents all those features, with multiple possible