Abstract
We have developed a technique which allows specific detection of proteins expressed from cloned genes. The method involves fusion of an oligonucleotide coding for part of the neuropeptide substance P to the 3' end of the gene; the protein can then be detected with a monoclonal antibody that recognises this peptide. We have used this method to determine the properties of deletion mutants of the major Drosophila heat shock protein, hsp70, expressed in monkey COS cells. The results suggest that this protein has two distinct domains. Both are capable of accumulating in the nucleus of unstressed cells, but only the more highly conserved N-terminal domain is able to bind to nucleoli following a heat shock. This implies that nucleolar binding and nuclear migration are distinct properties of the protein, and suggests that the former may be of functional importance. In addition, we observed a novel effect of heat shock on cellular metabolism: protein fragments that are normally rapidly degraded are stabilized. The effect persists for several hours after the heat shock, but does not require expression of heat shock proteins. Together with previously published data, these results suggest an intimate relationship between protein degradation and the heat shock response.
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- Ashburner M., Bonner J. J. The induction of gene activity in drosophilia by heat shock. Cell. 1979 Jun;17(2):241–254. doi: 10.1016/0092-8674(79)90150-8. [DOI] [PubMed] [Google Scholar]
- Bardwell J. C., Craig E. A. Major heat shock gene of Drosophila and the Escherichia coli heat-inducible dnaK gene are homologous. Proc Natl Acad Sci U S A. 1984 Feb;81(3):848–852. doi: 10.1073/pnas.81.3.848. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bienz M. Xenopus hsp 70 genes are constitutively expressed in injected oocytes. EMBO J. 1984 Nov;3(11):2477–2483. doi: 10.1002/j.1460-2075.1984.tb02159.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bonner J. J., Parks C., Parker-Thornburg J., Mortin M. A., Pelham H. R. The use of promoter fusions in Drosophila genetics: isolation of mutations affecting the heat shock response. Cell. 1984 Jul;37(3):979–991. doi: 10.1016/0092-8674(84)90432-x. [DOI] [PubMed] [Google Scholar]
- Ciechanover A., Finley D., Varshavsky A. Ubiquitin dependence of selective protein degradation demonstrated in the mammalian cell cycle mutant ts85. Cell. 1984 May;37(1):57–66. doi: 10.1016/0092-8674(84)90300-3. [DOI] [PubMed] [Google Scholar]
- Cuello A. C., Galfre G., Milstein C. Detection of substance P in the central nervous system by a monoclonal antibody. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3532–3536. doi: 10.1073/pnas.76.7.3532. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dingwall C., Allan J. Accumulation of the isolated carboxy-terminal domain of histone H1 in the Xenopus oocyte nucleus. EMBO J. 1984 Sep;3(9):1933–1937. doi: 10.1002/j.1460-2075.1984.tb02072.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Finley D., Ciechanover A., Varshavsky A. Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell. 1984 May;37(1):43–55. doi: 10.1016/0092-8674(84)90299-x. [DOI] [PubMed] [Google Scholar]
- Gait M. J., Matthes H. W., Singh M., Sproat B. S., Titmas R. C. Rapid synthesis of oligodeoxyribonucleotides. VII. Solid phase synthesis of oligodeoxyribonucleotides by a continuous flow phosphotriester method on a kieselguhr-polyamide support. Nucleic Acids Res. 1982 Oct 25;10(20):6243–6254. doi: 10.1093/nar/10.20.6243. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ingolia T. D., Craig E. A., McCarthy B. J. Sequence of three copies of the gene for the major Drosophila heat shock induced protein and their flanking regions. Cell. 1980 Oct;21(3):669–679. doi: 10.1016/0092-8674(80)90430-4. [DOI] [PubMed] [Google Scholar]
- Karch F., Török I., Tissières A. Extensive regions of homology in front of the two hsp70 heat shock variant genes in Drosophila melanogaster. J Mol Biol. 1981 May 25;148(3):219–230. doi: 10.1016/0022-2836(81)90536-2. [DOI] [PubMed] [Google Scholar]
- Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]