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Studies on an inducible gene system : the heat shock response in trout cells

University of British Columbia

The heat shock phenomenon has been characterized in cultured fibroblasts of the rainbow trout, Salmo gairdnerii. The response was elicited by one of two methods: temperature elevation or sodium arsenite exposure. The stress situations resulted in the rapid expression of a set of novel polypeptides, the heat shock polypeptides (hsps), normally absent in trout cells. At least six hsps have been identified and molecular weights assigned; these are referred to as hsp30, hsp32, hsp42, hsp62, hsp70, and hsp87. Translational control on pre-existing mRNAs was observed in cells under prolonged arsenite exposure. The heat shock response is a reversible process in trout cells. Two cDNAs, THS70.7 and THS70.14, encoding partial information for two distinct species of trout hsp70 were isolated and characterized. These sequences are identical at 73.3% of the nucleotide positions in their regions of overlap, and their degree of sequence conservation at the polypeptide level is 88.1%. The two derived trout hsp70 polypeptide sequences show extensive homology with amino acid sequences for hsp70 from Drosophila and yeast. Southern blot analysis of trout testis DNA reveals a small number of bands hybridizing to the hsp70 genes in this species. The trout hsp70 cDNA sequences cross-hybridize with restriction fragments in genomic DNA from HeLa cells, bovine liver, nematodes, and Drosophila. Northern blot analysis of RNA from arsenite-induced RTG-2 cells (the trout cell line), using the trout hsp70 cDNAs as probes, reveals the presence of three hsp70 mRNA species. Both heat shock and sodium arsenite result in rapid synthesis of trout hsp70 mRNA. Similarly the repression of hsp70 mRNA is very rapid, especially during recovery from a temperature stress. An artifact of cDNA cloning was identified, i.e. an IS-element (named T31) was isolated and characterized as orginating from a trout cDNA library. However, further analysis proved T31 to be a prokaryotic mobile element that had inserted itself into pBR322 during the preparation of the cDNA library.

Text

2010-06-13

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http://hdl.handle.net/2429/25619

Biochemistry and Molecular Biology

University of British Columbia

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