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

doi:10.1016/0965-1748(95)00008-J

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 journal › Article › peer-review

33 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 language	English
Pages (from-to)	697-707
Number of pages	11
Journal	Insect Biochemistry and Molecular Biology
Volume	25
Issue number	6
DOIs	https://doi.org/10.1016/0965-1748(95)00008-J
State	Published - 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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10.1016/0965-1748(95)00008-J

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@article{6139dfa8d4ba43a09e57a5216991bc35,

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

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.",

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

author = "Palli, {Subba R.} and Sohi, {Sardar S.} and Cook, {Barbara J.} and Dave Lambert and Ladd, {Tim R.} and Arthur Retnakaran",

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.",

year = "1995",

month = jun,

doi = "10.1016/0965-1748(95)00008-J",

language = "English",

volume = "25",

pages = "697--707",

number = "6",

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T1 - Analysis of ecdysteroid action in Malacosoma disstria cells

T2 - Cloning selected regions of E75- and MHR3-like genes

AU - Palli, Subba R.

AU - Sohi, Sardar S.

AU - Cook, Barbara J.

AU - Lambert, Dave

AU - Ladd, Tim R.

AU - Retnakaran, Arthur

N1 - 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.

PY - 1995/6

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N2 - 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.

AB - 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.

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KW - Malacosoma disstria

KW - Manduca

KW - hormone receptor 3 (MHR3)

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JO - Insect Biochemistry and Molecular Biology

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

Abstract

Bibliographical note

Keywords

ASJC Scopus subject areas

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