Difference between revisions of "Part:BBa K626005"

Latest revision as of 18:45, 21 September 2011

Gal4 - NHR64

This composite part is a nuclear hormone receptor consisting of a Gal4 yeast transcription factor and NHR64 (C. elegans Nuclear Hormone Receptor 64) Ligand Binding domain. This part can be used as a transcription factor regulated by unknown lipid molecules for the regulation of genes having UAS before their protein coding sequence.

NHR 64 LBD

The nhr-64 encodes a conserved nuclear receptor that is a member of the NR2 subfamily of nuclear receptors that contains Drosophila and human HNF4 (mutations in human HNF4A are associated with Type I MODY (maturity-onset diabetes of the young)). By homology, NHR-64 is predicted to function as a transcription factor that may activate or repress transcription in response to a hormonal signal. However, as loss of nhr-64 activity via RNAi does not result in any obvious abnormalities, the precise role of NHR-64 in C. elegans development and/or behavior is not yet known. The nhr-64 is broadly expressed and detected in anterior and posterior neurons, the ventral nerve cord, the pharynx, gut, and hypodermis.

Gal4 DBD

This protein is a positive regulator for the gene expression of the galactose-induced genes such as GAL1, GAL2, GAL7, GAL10, and MEL1 which encode for the enzymes used to convert galactose to glucose. This protein contains a fungal Zn(2)-Cys(6) binuclear cluster domain.

This composite artificial transcription factor will activate any reporter or any gene in general that has a UAS (Upper Activating Sequence) 3' of it's promoter. The usual binding sites of reporters, contain multiple UAS elements. In order to have a POPS output, the LBD has to recruit activators in the cell. This can be initiated by ligand binding or by recruiting a protein that has a fused strong activator like the VP activator.

More on GAL4 DNA binding domain: [http://en.wikipedia.org/wiki/Two-hybrid_screening].

Uniprot GAL4 DNA binding domain: [http://www.uniprot.org/uniprot/P04386].

View GAL4 DNA binding domain crystal structure here: [http://www.rcsb.org/pdb/explore/jmol.do?structureId=3COQ&bionumber=1].

With this system NHR (Nuclear Hormone Receptor) ligands or NHR interacting partners can be screened.

The NHR: cofactor-VP interaction should be also broken by a potential ligand binding, this is why this setup is also suitable for ligand identification. The benefit of the cofactor-VP interaction test is that the dynamic range of the assay is much higher than the dynamic range of the normal Gal4-NHR ligand activation assay.

Gel electrophoresis picture of Gal4-NHR64 fusion part in pSB1C3 and in pCDNA3.1 expression vector.

Activation of Gal-NHR8 and Gal-NHR64 to oil sand extractions.

Sequence and Features