FRA

The present study was undertaken to examine the gene structure, the mRNA expression profiles and the mechanism of transcriptional regulation of fish heat-shock proteins （HSPs）, using the rainbow trout Oncorhynchus mykiss as a model system.

Published studies using teleost cultured cells clearly demonstrate that teleosts can mount a stress response very similar to that observed with other vertebrates. However, HSP genes have only been cloned from a modest number of different fish species, and molecular studies of the fish HSPs are still in their early descriptive phase.

Heat-shock protein 70 （Hsp70） is the major stress-inducible protein in vertebrates and highly conserved throughout evolution. Rainbow trout have evolved by tetraploidization from a diploid ancestor, raising the possibility that multiple Hsp70s exist in the genome. To obtain full-length cDNA clones encoding Hsp70, a plasmid cDNA library was constructed from a heat-shocked juvenile of rainbow trout and screened. Consequently, two Hsp70s, Hsp70a and Hsp70b, were identified and found to have 98.1% identity in their deduced amino acid sequences. Southern blot analysis indicated that the two Hsp70s are encoded by distinct genes in the genome. Northern blot analysis showed that each of Hsp70a and Hsp70b expressed two mRNA species having different sizes by heat stress in rainbow trout RTG-2 cells.

To comprehensively analyze the mRNA expression profile of HSPs, multiple HSPs were isolated from RTG-2 cells and quantitatively compared for their mRNA levels between unstressed and heat-shocked cells using real-time quantitative RT-PCR analysis. To clone multiple HSP genes at once, 200 cDNAs were arbitrarily isolated from the cDNA library constructed from heat-shocked RTG-2 cells and sequenced. Consequently, nine cDNAs encoding HSPs were identified, namely, Hsp90βa, Hsp90βb, Grp78, Hsp70a, Hsc70a, Hsc70b, Cct8, Hsp47, and DnaJ homolog. By Northern blot analysis, single bands were detected for all genes examined except Hsp70s where two mRNA species having different sizes were detected in heat-shocked cells irrespective of Hsp70a and Hsp70b. Quantitative RT-PCR analysis showed that the accumulation levels of Hsp70a and Hsp70b mRNAs were dramatically increased after heat shock, and the mRNA levels in heat-shocked （28℃, 3 h） cells were 480- and 510-fold of those in controls, respectively. The increased levels of Hsc70a, Hsc70b and Hsp47 mRNAs were 1.3-, 2.8- and 1.6-fold, respectively, after heat shock. There were no differences in mRNA levels of the other four HSP genes between control and heat-shocked cells with a significance level of 0.05.

To demonstrate the existence of the heat-shock transcription factor （HSF） in rainbow trout, genes encoding HSF1 were cloned from RTG-2 cells. Consequently, two distinct HSF1s, named HSF1a and HSF1b, were identified. Southern blot analysis showed that each HSF1 is encoded by a distinct gene. The two HSF1 mRNAs were coexpressed in unstressed RTG-2 cells and in various tissues. In electrophoretic mobility shift assay, each in vitro translated HSF1 bound to a cis-acting DNA element, the heat-shock element. Chemical cross-linking and immunoprecipitation analysis showed that HSF1a and HSF1b form heterotrimers as well as homotrimers.

The present study demonstrated that, in rainbow trout, at least some HSP and HSF have duplicate genes attributed to ancestral tetraploidization. This finding suggests that the comprehensive identification of duplicate genes is prerequisite to accurately examining gene expression profiles of rainbow trout cells. Since the mRNA expression profiles of HSPs may differ among stressors, it is interesting to compare the differences of the profiles for utilizing HSPs as a biomarker of various environmental stresses. Further, the present study suggests that a unique molecular mechanism, which functions through two distinct HSF1 isoforms, underlies the stress response in tetraploid and/or cold-water fish species such as rainbow trout. Since the lower activation temperature of rainbow trout HSF1 is a unique feature among vertebrate counterparts, a detailed comparison of rainbow trout and other vertebrate HSF1s will lead to further insight into the activation mechanism of the transcription factor.