Cloning and characterization of two distinct isoforms of rainbow trout heat shock factor 1. Evidence for heterotrimer formation.
To elucidate the molecular mechanism underlying the heat shock response in cold-water fish species, genes encoding heat shock transcription factors (HSFs) were cloned from RTG-2 cells of the rainbow trout Oncorhynchus mykiss. Consequently, two distinct HSF1 genes, named HSF1a and HSF1b, were identified. The predicted amino acid sequence of HSF1a shows 86.4% identity to that of HSF1b. The two proteins contained the general structural motifs of HSF1, i.e. a DNA-binding domain, hydrophobic heptad repeats and nuclear localization signals. Southern blot analysis showed that each HSF1 is encoded by a distinct gene. The two HSF1 mRNAs were coexpressed in unstressed rainbow trout RTG-2 cells and in various tissues. In an electrophoretic mobility shift assay, each in vitro translated HSF1 bound to the heat shock element. Chemical cross-linking and immunoprecipitation analysis showed that HSF1a and HSF1b form heterotrimers as well as homotrimers. Taken together, these results demonstrate that in rainbow trout cells there are two distinct HSF1 isoforms that can form heterotrimers, suggesting that a unique molecular mechanism underlies the stress response in tetraploid and/or cold-water fish species.
Ojima
Nobuhiko
N
Cell Biology Section, Physiology and Molecular Biology Division, National Research Institute of Fisheries Science, Fisheries Research Agency, Yokohama, Japan. ojima@affrc.go.jp
Yamashita
Michiaki
M
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D003001:Cloning, Molecular; D003080:Cold Temperature; D003432:Cross-Linking Reagents / Q000737:chemistry; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss / Q000378:metabolism*; D010802:Phylogeny; D014176:Protein Biosynthesis; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D020449:Repetitive Sequences, Amino Acid; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014157:Transcription Factors
D003432:Cross-Linking Reagents; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D000076249:Heat Shock Transcription Factors; D006360:Heat-Shock Proteins; D020033:Protein Isoforms; D014157:Transcription Factors; D004247:DNA