PROTEINS THAT BIND WITH THE LBD OF THE ECDYSONE RECEPTOR RESPONSIBLE FOR GENE REGULATION IN DROSOPHILA
Disciplines
Biochemistry | Molecular Biology
Abstract (300 words maximum)
The Ecdysone Receptor (ECR) is a nuclear receptor in Drosophila regulating gene activity. ECR consists of a ligand binding domain (LBD), which binds the ligand and other proteins, enhancing its ability to regulate gene expression involved in signaling pathways linked to processes such as cell growth, cell differential, and metamorphosis. However, many proteins are still unknown. This research aims to discover new interacting proteins via yeast two-hybrid assay. This assay uses the modular nature of Gal4, a transcription factor, with a DNA-binding domain (BD) and a DNA activation domain (AD). Gal4 AD-prey proteins from a drosophila cDNA library will be screened with Gal4 DNA BD fused to LBD (bait). If bait and any prey proteins interact, the Gal4 transcription factor is reconstituted and reporter genes are activated, as indicated by blue-colored colonies and resistance to antibiotic aureobascidin. So far, yeast transformations, LBD autoactivation, and positive and negative mating controls have been performed. White colonies appeared on all plates; therefore, the yeast transformations were successful. No colonies appeared on the DDO/X/A plate with LBD; therefore, Gal4 DNA BD-LBD does not autoactivate the reporter genes. Blue colonies appeared on the DDO/X/A plate for the positive control, and no colonies appeared for the negative control. All the controls work as expected, so we will proceed with the library screening of potential LBD protein interactors using the yeast two-hybrid assay. Gaining insights into these interactions expands our understanding of the ECR dynamics in Drosophila. This knowledge has potential implications for human health as nuclear receptors are linked to similar cellular activities.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Dr. Joanna Wardwell-Ozgo
PROTEINS THAT BIND WITH THE LBD OF THE ECDYSONE RECEPTOR RESPONSIBLE FOR GENE REGULATION IN DROSOPHILA
The Ecdysone Receptor (ECR) is a nuclear receptor in Drosophila regulating gene activity. ECR consists of a ligand binding domain (LBD), which binds the ligand and other proteins, enhancing its ability to regulate gene expression involved in signaling pathways linked to processes such as cell growth, cell differential, and metamorphosis. However, many proteins are still unknown. This research aims to discover new interacting proteins via yeast two-hybrid assay. This assay uses the modular nature of Gal4, a transcription factor, with a DNA-binding domain (BD) and a DNA activation domain (AD). Gal4 AD-prey proteins from a drosophila cDNA library will be screened with Gal4 DNA BD fused to LBD (bait). If bait and any prey proteins interact, the Gal4 transcription factor is reconstituted and reporter genes are activated, as indicated by blue-colored colonies and resistance to antibiotic aureobascidin. So far, yeast transformations, LBD autoactivation, and positive and negative mating controls have been performed. White colonies appeared on all plates; therefore, the yeast transformations were successful. No colonies appeared on the DDO/X/A plate with LBD; therefore, Gal4 DNA BD-LBD does not autoactivate the reporter genes. Blue colonies appeared on the DDO/X/A plate for the positive control, and no colonies appeared for the negative control. All the controls work as expected, so we will proceed with the library screening of potential LBD protein interactors using the yeast two-hybrid assay. Gaining insights into these interactions expands our understanding of the ECR dynamics in Drosophila. This knowledge has potential implications for human health as nuclear receptors are linked to similar cellular activities.