title: Authentic temperature-regulation of a heat shock gene inserted into yeast on a high copy number vector. Influences of overexpression of HSP90 protein on high temperature growth and thermotolerance.
abstract: Heat shock protein HSP90 is relatively abundant in eukaryotic cells even in the absence of heat shock. Its precise function is still unclear, although it is apparently required in higher levels for growth at high temperatures. In this study Saccharomyces cerevisiae transformants were constructed with 50-150 copies of the homologous heat-inducible gene for HSP90 (HSP82) present on a high copy number episomal vector. These transformants were then used to demonstrate: (i) that this heat shock gene displays essentially normal regulation when present in yeast at high copy numbers; (ii) that yeast is an expression host suitable for the high level synthesis of HSP90; and (iii) that increasing normal cellular levels of HSP90 affects a number of physiological properties. The HSP82 gene is normally single-copy in the haploid yeast genome, yet even at 50 to 150 copies per cell it displayed almost normal basal and heat shock-induced levels of expression. Proper regulation of the heat shock element sequence controlling HSP82 is therefore not lost at high gene copy levels. In unstressed cultures in exponential growth at 25 degrees C the low basal expression of the multiple HSP82 gene copies caused a 3 to 7-fold HSP90 overproduction, but HSP90 levels increased 10-fold to 30-40% of total cell protein following temperature upshift to 39 degrees C for 75 min. Heat induction of the chromosomal genes for other heat shock proteins in the same cells was not suppressed relative to cells which were isogenic but for the possession of just a single HSP82 gene, this constituting further evidence that yeast can authentically regulate a large number of heat shock genes. HSP90 overproduction was not protective against heat killing, causing strain-dependent reductions in growth at 37.5 degrees C and in thermotolerance.
authors.affiliation: Department of Biochemistry and Molecular Biology, University College London, UK.
authors.forename: L
authors.initials: L
authors.lastname: Cheng
authors.forename: K
authors.initials: K
authors.lastname: Hirst
authors.forename: P W
authors.initials: PW
authors.lastname: Piper
mesh_terms: D001345:Autoradiography; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D006238:Haploidy; D006360:Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D010957:Plasmids; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D013696:Temperature; D014316:Tritium
chemical_list: D006360:Heat-Shock Proteins; D014316:Tritium; D007930:Leucine