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Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila

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Abstract

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.

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Acknowledgements

We thank Beth Ryder for her initial work on developmental expression of Sciara EcR. Grant funding from National Institutes of Health to SAG (GM35929) is gratefully acknowledged. GML was supported by a student fellowship from the Agency for Science, Technology and Research (A*STAR), Singapore.

Ethical standards

The experiments in this manuscript comply with current laws in the USA.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Gerald M. Liew

    Present address: Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305, USA

  2. Fyodor D. Urnov

    Present address: Sangamo BioSciences, Inc, 501 Canal Blvd. Suite A100, Richmond, CA, 94804, USA

Authors and Affiliations

  1. Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI, 02912, USA

    Michael S. Foulk, John M. Waggener, Janell M. Johnson, Yutaka Yamamoto, Gerald M. Liew, Fyodor D. Urnov, Yuki Young, Genee Lee, Heidi S. Smith & Susan A. Gerbi

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  1. Michael S. Foulk
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  2. John M. Waggener
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  4. Yutaka Yamamoto
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  5. Gerald M. Liew
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  6. Fyodor D. Urnov
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  7. Yuki Young
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  8. Genee Lee
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  9. Heidi S. Smith
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  10. Susan A. Gerbi
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Corresponding author

Correspondence to Susan A. Gerbi.

Additional information

This paper is dedicated to Michael Ashburner whose pioneering work on the induction of Drosophila polytene chromosome puffs by ecdysone established the foundation for our understanding of transcriptional regulation by steroid hormone receptors.

Michael S. Foulk John M. Waggener, and Janell M. Johnson contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Online Resource 1

EcR phylogenetic tree. Phylogenetic tree derived from Clustal W multiple protein sequence alignment of ecdysone receptors from several insect species. Sciara EcRs are highlighted in yellow. (JPEG 109 kb)

High Resolution Image (TIFF 1020 kb)

Online Resource 2

USP phylogenetic tree. Phylogenetic tree derived from Clustal W multiple protein sequence alignment of ultraspiracle proteins from several insect species. Sciara USP is highlighted in yellow. (JPEG 108 kb)

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a No ScEcR antibody cross-reactivity. GST-ScEcR-A (∼44 kDa) and GST-ScEcR-B (∼36 kDa) N-termini fusion proteins expressed in E. coli and ScEcR-A (∼19 kDa) and ScEcR-B (∼11 kDa) N-termini purified from chitin bead columns after intein cleavage were run out on SDS-PAGE and blotted to nitrocellulose membranes and probed with the antisera indicated (top panel = αScEcR-A; bottom panel = αScEcR-B). There are GST fusion protein degradation products in lanes 1 and 2. b ScEcR-B salivary gland developmental western blot. Salivary gland protein extracts (125 μg) from the stages indicated run on SDS-PAGE and blotted to nitrocellulose. As a positive control, purified GST-EcR-B was run on the same gel. The blot was probed with an affinity purified αScEcR-B antibody. The position where ScEcR-B should run (∼61 kDa) is indicated. c ScEcR-B in situ hybridization to polytene chromosomes. DAPI-stained chromosomes, ScEcR-B in situ hybridization, and the merged images are shown. (JPEG 80 kb)

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Foulk, M.S., Waggener, J.M., Johnson, J.M. et al. Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila . Chromosoma 122, 103–119 (2013). https://doi.org/10.1007/s00412-012-0395-4

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  • DOI: https://doi.org/10.1007/s00412-012-0395-4

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