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Schistosome extracts with heat shock factor activity revealed by the gel shift assay

Published online by Cambridge University Press:  06 April 2009

R. Levy-Holtzman  and
I. Schechter
R. Levy-Holtzman
Affiliation:
Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
I. Schechter
Affiliation:
Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel
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Summary

To understand the regulated expression of stage-specific genes of schistosomes, it is necessary to identify regulatory DNA elements and DNA-binding proteins that control the level of gene transcription. Here we describe the preparation of Schistosoma mansoni extracts with active transcription factors detected by the electrophoretic mobility shift assay (EMSA). We analysed the hsp70 system of S. mansoni because the promoters of the hsp70 gene contain two heat shock elements (HSE) that differ from the consensus sequence (CnnGAAnnTTCnnG) at one (HSEI) or three (HSEII) positions, and it is known that transcriptional activation of hsp70 genes is mediated by interaction of HSE with the heat shock factor (HSF). Analyses of parasite extracts from different developmental stages demonstrate the presence of putative HSF that correlates with the pattern of hsp70 mRNA expression (cercariae-, schistosomula+, adult worms+). Cercarial extracts did not show binding of 32P-labelled HSEI or HSEII. Extracts of schistosomula and of adult worms kept at 37 or 42 °C showed binding of HSEI but not HSEII. The specificity of HSEI-HSF complex formation was ascertained by inhibition experiments. The EMSA experiments and structural features of the hsp70 promoter indicate that HSEI is the major DNA element responsible for transcriptional activation of the hsp70 gene, while the HSEII may be a redundant sequence with minor (if any) regulatory function. The extracts reported here can be used to study transcriptional control of other schistosome genes.

Keywords

heat shock transcription factorheat shock elementhsp70 promotergel shift assaySchistosoma mansoni
Type
Research Article
Information
Parasitology , Volume 108 , Issue 1 , January 1994 , pp. 35 - 42
Copyright
Copyright © Cambridge University Press 1994

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References

REFERENCES

Aronheim, A., Ohlsson, H., Park, C. W., Edlund, T. & Walker, M. D. (1991). Distribution and characterization of helix-loop-helix enhancer-binding protein from pancreatic B cells and lymphocytes. Nucleic Acids Research 19, 3893–9.Google Scholar
Bienz, M. (1985). Transient and developmental activation of heat-shock genes. Trends in Biochemical Sciences 157–61.CrossRefGoogle Scholar
Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry 72, 248–54.Google Scholar
Cereghini, S., Raymondjean, M., Garcia Carranca, A., Herbomel, P. & Yaniv, M. (1987). Factors involved in control of tissue-specific expression of albumin gene. Cell 50, 627–83.Google Scholar
Chen, L. L., Rekosh, D. M. & Loverde, P. T. (1992). Schistosoma mansoni p48 eggshell protein gene: characterization, developmentally regulated expression and comparison to the p14 eggshell protein gene. Molecular and Biochemical Parasitology 52, 39–52.Google Scholar
Clos, J., Westwood, J. T., Becker, P. B., Wilson, S., Lambert, K. & Wu, C. (1990). Molecular cloning and expression of a hexameric Drosophila heat shock factor subject to negative regulation. Cell 63, 1085–97.Google Scholar
Dignam, J. D., Lebowitz, R. M. & Roeder, R. G. (1983). Accurate transcription initiation by polymerase II in a soluble extract from isolated mammalian nuclei. Nucleic Acids Research 11, 1475–89.Google Scholar
Dorn, A., Bollekens, J., Staub, A., Benoist, C. & Mathis, D. (1987). A multiplicity of CCAAT-box binding proteins. Cell 50, 863–72.Google Scholar
Grossman, Z., Ram, D., Markovics, A., Tarrab-Hazdai, R., Lantner, F., Ziv, E. & Schechter, I. (1990). Schistosoma mansoni: stage specific expression of muscle specific genes. Experimental Parasitology 70, 62–71.Google Scholar
Henkle, K. J., Cook, G. A., Foster, L. A., Engman, D. M., Bobek, L. A., Cain, G. D. & Donelson, J. A. (1990). The gene family encoding eggshell proteins of Schistosoma mansoni. Molecular and Biochemical Parasitology 42, 69–82.Google Scholar
Kingston, R. E., Schuetz, T. J. & Larin, Z. (1987). Heat inducible human factor that binds to a human hsp70 promoter. Molecular and Cellular Biology 7, 1530–4.Google ScholarPubMed
Korner, M., Rattner, A., Mauxion, F., Sen, R. & Citri, Y. (1989). A brain specific transcription activator. Neuron 3, 563–72.Google Scholar
Laclette, J. P., Landa, A., Arcos, L., Willms, K., Davis, A. E. & Shoemaker, C. B. (1991). Paramyosin is the Schistosoma mansoni (Trematoda) homologue of antigen B from Taenia solium (Cestoda). Molecular and Biochemical Parasitology 44, 287–96.Google Scholar
Levi-Schaffer, F. & Smolarsky, M. (1981). Schistosoma mansoni: effect of insulin and low molecular weight fraction of serum on schistosomula in chemically defined medium. Experimental Parasitology 52, 378–85.Google Scholar
Maniatis, T., Goodbourn, S. & Fischer, J. A. (1987). Regulation of inducible and tissue-specific gene expression. Science 236, 1237–45.Google Scholar
Mitchell, P. J. & Tjian, R. (1989). Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245, 371–8.Google Scholar
Nagai, Y., Gazzinelli, G., De Moraes, G. W. G. & Pellegrino, J. (1977). Protein synthesis during cercaria-schistosomulum transformation and early development of the Schistosoma mansoni larvae. Comparative and Biochemical Physiology 57B, 2730.Google Scholar
Neumann, S., Ziv, E., Lantner, F. & Schechter, I. (1992). Cloning and sequencing of an hsp70 gene of Schistosoma mansoni. Molecular and Biochemical Parasitology 56, 357–60.CrossRefGoogle ScholarPubMed
Neumann, S., Ziv, E., Lantner, F. & Schechter, I. (1993). Regulation of hsp70 gene expression during the life cycle of the parasitic helminth Schistosoma mansoni. European Journal of Biochemistry 212, 589–96.Google Scholar
Pelham, H. R. B. & Bienz, M. (1982). A Synthetic heat-shock promoter element confers heat-inducibility on the herpes simplex virus thymidine kinase gene. The EMBO Journal 1, 1473–7.Google Scholar
Rabindran, S. K., Giorgi, G., Clos, J. & Wu, C. (1991). Molecular cloning and expression of a human heat shock factor, HSFl. Proceedings of the National Academy of Sciences, USA 88, 6906–10.Google Scholar
Ram, D., Grossman, Z., Markovics, A., Avivi, A., Ziv, E., Lantner, F. & Schechter, I. (1989). Rapid changes in the expression of a gene encoding a calcium-binding protein in Schistosoma mansoni. Molecular and Biochemical Parasitology 34, 167–76.Google Scholar
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning. A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar
Scharf, K. D., Rose, S., Zott, W., Schoff, F. & Nover, L. (1990). Three tomato genes code for heat shock transcription factors with a region of remarkable homology to the DNA-binding domain of the yeast HSF. The EMBO Journal 9, 4495–501.Google Scholar
Schuetz, T. J., Gallo, G. J., Sheldon, L. & Tempst, P. (1991). Isolation of cDNA for HSF2: evidence for two heat shock factor genes in human. Proceedings of the National Academy of Sciences, USA 88, 6911–15.Google Scholar
Sorger, P. K., Lewis, M. J. & Pelham, H. R. B. (1987). Heat shock factor is regulated differently in yeast HeLa cells. Nature, London 329, 81–4.Google Scholar
Sorger, P. K. & Pelham, H. R. B. (1988). Yeast heat shock factor is an essential DNA-binding protein that exhibits temperature-dependent phosphorylation. Cell 54, 855–64.Google Scholar
Stein, L. D. & David, J. R. (1986). Cloning of a developmentally regulated tegument antigen of Schistosoma mansoni. Molecular and Biochemical Parasitology 20, 253–64.Google Scholar
Strauss, F. & Varshavsky, A. (1984). A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome. Cell 37, 889–901.CrossRefGoogle ScholarPubMed
Wu, B. J., Kingston, R. E. & Morimoto, R. I. (1986). Human hsp70 promoter contains at least two distinct regulatory domains. Proceedings of the National Academy of Sciences, USA 83, 629–33.Google Scholar
Xiao, H. & Lis, J. T. (1988). Germline transformation used to define key features of heat-shock response elements. Science 239, 1139–41.Google Scholar
Yuckenberg, P. D., Poupin, F. & Mansour, T. E. (1987). Schistosoma mansoni: protein composition and synthesis during early development; evidence for early synthesis of heat shock proteins. Experimental Parasitology 63, 301–11.CrossRefGoogle ScholarPubMed
Zimarino, V., Tsai, C. & Wu, C. (1990). Complex modes of heat shock factor activation. Molecular and Cellular Biology 10, 752–9.Google Scholar
Zimarino, V. & Wu, C. (1987). Induction of sequence-specific binding of Drosophila heat shock activator protein without protein synthesis. Nature, London 327, 727–30.Google Scholar