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Lecture 21 Notes.docx

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Western University
Biology 2581B
Susanne Kohalmi

Genetics Lecture 21 NotesAnalyzing the Code from Genome to Phenome Defining the Interactome ProteinProteinTwo Hybrid AssaySeparate the activities of the GAL4 proteinOne component contains GAL4 binding domain and other contains GAL4 activation domainCreate a fusion protein that expresses a protein that consists of the binding domain When proteins dont interactGal 4 binding domain bound to cis acting elementno activation of reporter gene When proteins interactBinding domain in close proximity to activation domainthey can activate expression of reporterHave one budding yeast strain expressing the DNA binding domain to your first protein of interestB for baitHave another haploid strain of opposite typeexpressing activation domain fusing to second protein of interestMix 2 cells togetherforms a diploid induced mating where diploid strain is expressing both fusion proteinsIf 2 proteins dont interact then reporter is offIf you are expressing the gene the cell is able to make His on its ownIf it is off the cell is going to be completely dependent on the researcher adding exogenous His to the mediaPatches of yeast cells grown on an agar plateif interacting then see growthThe Yeast Two Hybrid Assay can be used to define proteinprotein interactions on a genome wide scale through the use of arraysMacro arrayRepresents an agar plate and each circle represents a growing colony of yeast cells but each different colony is expressing a different fusion protein
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