Analysis of ecdysteroid action in Malacosoma disstria cells: Cloning selected regions of E75- and MHR3-like genes

Subba R. Palli, Sardar S. Sohi, Barbara J. Cook, Dave Lambert, Tim R. Ladd, Arthur Retnakaran

Research output: Contribution to journalArticlepeer-review

32 Scopus citations

Abstract

IPRI-MD-66 (MD-66) cells respond to 20-hydroxyecdysone (20E, 4 × 10-6M) in the medium by producing cytoplasmic extensions, clumping and attaching themselves to the substrate. These morphological changes are at a maximum by 6 days post treatment. Degenerate oligonucleotides, designed on the basis of conserved amino acid sequences in the DNA and ligand binding regions of the members of the steroid hormone receptor superfamily, were used in RNA-PCR to isolate two cDNA fragments, Malacosoma disstria hormone receptor 2 (MdHR2) and Malacosoma disstria hormone receptor 3 (MdHR3) from the MD-66 cells. Comparison of deduced amino acid sequences of these cDNA fragments with the members of the steroid hormone receptor superfamily showed that MdHR2 is most closely related to E75 proteins of Manduca sexta, Galleria mellonella and Drosophila melanogaster. The MdHR3 is most closely related to Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Drosophila hormone receptor 3 (DHR3) proteins. At a concentration of 4 × 10-6M, 20E induces the expression of MdHR2 and MdHR3 beginning at 3 h, reaching maximum levels in 12 h and declining in 24 h. MdHR2 binds to a 2.5 kb mRNA, whereas MdHR3 binds to a 4.5 kb mRNA. Based on sequence similarity, RNA size and ecdysone inducibility, we conclude that these cDNA fragments, cloned from MD-66 cells, are regions of E75- (MdHR2) and MHR3- (MdHR3) like genes.

Original languageEnglish
Pages (from-to)697-707
Number of pages11
JournalInsect Biochemistry and Molecular Biology
Volume25
Issue number6
DOIs
StatePublished - Jun 1995

Bibliographical note

Funding Information:
Acknowledgements--We thank Dr W. M. Cheliak for discussions throughout the study and for critical comments on the manuscript, Dr J. Barrett for advice on sequence alignment and two anonymous reviewers for their helpful comments. Supported by Canadian Forest Service and National Biotechnology Strategy Fund.

Keywords

  • Drosophila ecdysone-inducible gene
  • E75
  • Ecdysone
  • IPRI-MD-66 cells
  • Malacosoma disstria
  • Manduca
  • hormone receptor 3 (MHR3)

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Insect Science

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