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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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