Hirst, Karen Ann; (1990) Heat shock-directed gene expression in yeast. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In yeast, as in other organisms, an increase in ambient temperature results in the activation of a number of genes (known as heat shock genes); some of which are also activated on entry to stationary phase. This suggested that the promoters of these genes could provide easily performed induction of heterologous gene expression. The potential use and possible disadvantages of heat shock expression vectors for directing heterologous expression were investigated in three parallel studies. These provided information which should find application in the design of both heat shock and growth phase dependent vectors. In addition they also suggested possible roles for the homologous HSP, HSP90, in both growth regulation and the switch-off of the heat shock response in yeast. They consisted of: (1) A system designed to detect translational errors in yeast: As translational errors could be detrimental to the expression of both homologous and heterologous proteins, a system was designed to detect translational errors. This used a temperature-sensitive histidinyl-tRNA synthetase mutant to starve yeast cells overexpressing the homologous protein, PGK, for HistRNAHis at 38°C. In mammalian and E. coli cells, starvation for specific charged tRNAs results in codon misreading which produces aberrant proteins detectable by 2D electrophoresis (Parker et al., 1976). However no such misreading was detected in yeast although evidence was obtained that yeast may respond to starvation for HistRNAHis by arresting translation at histidine codons in prefernce to misincorporation of an incorrect amino acid. (2) Overproduction of the homologous HSP, HSP90: Originally the aim of this particular study was to investigate if protein overproduction at heat shock temperatures was possible or whether trans-activating factors were limiting. The results obtained were unexpected as although HSP90 overproduction was possible, only limited overproduction was acheived at heat shock temperatures. This was found to be due to suppression of heat shock gene transcription by high levels of HSP90. In addition HSP90 overproduction at non-heat shock temperatures had dramatic effects on both cAMP levels and catabolite repression resulting in altered growth regulation. (3) Evaluation of heat shock expression vectors; 3 high copy yeast-E.coli shuttle vectors were constructed so that the inserted heterologous gene, human inteferon-α-2, was under the control of 1, 2 or 3 upstream heat shock element sequences (HSEs) which direct heat shock induced transcription. Although these vectors were not ideal in that there was some expression of inteferon in unstressed cells and no induction on entry to stationary phase; transcription was increased on heat shock at a level dependent upon the number of HSEs present in the promoter.

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