We propose to use the following approaches to explore the stress response (heat shock response) in human cells: 1. Cloning of the genes and cDNAs. These will be used (i) as tools for analyzing the mechanisms involved in the response, (ii) for elucidation of (possibly common) regulatory regions and effector molecule(s) which bind to them, and (iii) for genetic manipulations and introduction into cells to determine whether different inducers act by the same mechanisms. 2. Transcriptional control is of two types, which we have described previously. Through experiments carried out in vivo and in vitro we will dissect the mechanism further, in particular, (i) to discover the nature of the regulation of transcription of the stress response genes, (ii) to distinguish between various explanations for the decreased production of normal mRNAs and non-coding RNAs, and (iii) to develop cell-free systems as a means of identifying and purifying regulatory components. 3. Translational control can occur at the levels of initiation and elongation in different kinds of stress. We will explore the mechanisms of these processes, and test specific models to explain the unusual dependence of protein synthesis on continuous RNA sysnthesis. Cell-free systems will be developed to allow fractionation and purification of regulatory components responsible for the different types of control. 4. Cell mutants deficient in the responses to various inducing treatments will be isolated to afford a genetic approach to the nature of the lesion(s).
