The long term goal for this research program is to contribute to our understanding of the regulation of gene expression. The immediate goals for this proposed project are to identify the structural features of messenger RNAs coding for heat shock polypeptides which determine their selection for translation in heat shock and stress conditions. The heat shock response is a reversible general stress response conferring protection on cells from further insults and characterized by dramatic alteration in gene expression at the transcriptional and translational levels. At the translational level, mRNAs coding for the 8-9 polypeptides whose synthesis is induced in heat shock (the heat shock polypeptides) are selected for translation while translation of the still intact population of normal cell mRNAs is shut down. This requires both changes in the translation system and the presence of a structural signal on heat shock mRNAs identifying them for selective translation in heat shock. In the proposed experiments, recombinant DNA technology and a plasmid-directed in vitro transcription system will be used to identify the structural signals on the heat shock mRNA.
The aims are: (1) to determine whether five test genes are or are not recognized as heat shock genes at the translational level following cloning into the plasmid, pSP64(65), and transcription with SP6 RNA polymerase. The assays for selective translation will be a Drosophila in vitro translation system prepared from heat shocked cells. (2) """"""""Hybrid"""""""" genes will then be constructed consisting of leader sequences from one source and coding sequences from another. (3) The fragments of the """"""""hybrid"""""""" genes active in conferring selection as heat shock mRNAs on coding sequences will be further dissected to identify the smallest possible structure having this activity. (4) Fragments of RNA containing the structural signals determining mRNA selection for translation in heat shock will be used to try to identify components of an in vitro translation system which interact with them. In the event that the experiments in (1) above show the in vitro synthesized mRNAs from intact heat shock and control genes do not retain ability to be translated as expected, structures at the transcription start site will be investigated.
| Sanders, M M; Kon, C (1991) Glutamine is a powerful effector of heat shock protein expression in Drosophila Kc cells. J Cell Physiol 146:180-90 |
| Sherwood, A C; John-Alder, K; Biessmann, H et al. (1989) Overexpression of a 123-kDa anion transport inhibitor binding protein and two cytoskeleton proteins in Drosophila Kc cell variants resistant to disulfonic stilbenes. J Biol Chem 264:1829-36 |
| Sherwood, A C; John-Alder, K; Sanders, M M (1988) Characterization of chloride uptake in Drosophila Kc cells. J Cell Physiol 136:500-6 |
