Exam 4: Lecture 9 Flashcards Preview

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Luciferase Assay

-can determine presence and degree of activation potential of DNA binding protein


Example to determine activation strength of So-Eya complex

-conducted in eukaryotic cell line
-using Drosophila Kc167 cells
-cells simultaneously transformed with number of plasmids all containing components necessary
-mt-GAL4 used to induce expression of genes of interest
-mt enhancer activated by presence of copper sulfate
-UAS-So and UAS-Eya constructs activated in response to presence of GAL4
-enhancer/promoter-luciferase transcriptional reporter contains binding sites for So-Eya complex
-binding of So-Eya to enhancer element leads to transcription of luciferase gene which can then be measured
-UAS-Renilla is also activated in response to GAL4
-used to control for transfection efficiency which can vary from experiment to experiment
-level of Luciferase activity is divided by the amount of Renilla activity
-gives normalized ratio for comparison across samples


Determining if So-Eya complex functions as transcriptional activator

-control experiments must accompany actual experiment to determine relevance


First Control (UAS-GFP)

-GFP not transcription factor and cannot bind to enhancer
-this control provides level of baseline expression that is derived from minimal core promoter


Second Control (UAS-So)

-Ratio of luciferase/renilla is higher than that of GFP control
-provides baseline activity of So
-activity can be intrinsic to So protein itself or due to interactions with proteins that are found in Kc167 cells


Third Control (UAS-Eya)

-luciferase/renilla ratio higher than GFP control and UAS-So
-represents amount of endogenous So expressed in Kc167 cell and can bind to Eya


Experimental Condition (UAS-S0 & UAS-Eya)

-luciferase/renilla ratio much higher than any of controls
-real experimental value of So-Eya transcriptional activation


Measuring Transcriptional Activation Potential

-from luciferase assay clear that So-Eya is strong activator
-expression of So on its own is sufficient to activate transcription too (slightly lower levels)
-begs question can So itself activate transcription


Transcriptional Reporter Assay addressing question if So can activate transcription

-UAS-lacZ reporter construct transformed simultaneously into yeast cells with plasmid that contains Sine Oculis fused to DNA binding of GAL4
-chimeric protein will bind to UAS sites
-if So contains activation domain then it will be able to direct RNA Pol II transcription of lacZ reporter
-as you can see full-length So is capable of activating LacZ expression
-optix protein is used as negative control since it is unable to activate transcription


Determining location of activation domain in assy

-individual portions of So protein removed
-modified proteins then run through same assay
-activation domain identified when loss of individual domain leads to loss of lacZ expression and activity
-case of So two activation domains one in SIX protein-protein interaction motif and one in non-descript carboxy portion of protein



-yeast cells either take up histidine from surrounding material or they can synthesize it in vivo
-remove histidine from media cells can synthesize in vivo
-remove histidine from media and delete one enzyme that catalyzes it (HIS3) cell dies
-yeast HIS3 gene encodes imidazoleglycerol-phosphate dehydratase which catalyzes last step in histidine biosynthesis pathway
-converts imidazoleglycerol phosphate into histidine


Histidine assay

-use biosynthesis of histidine to measure strength of transcriptional activator
-HIS3 gene mutated and cells placed in media that lacks histidine
-normally die
-UAS-HIS3 construct inserted into yeast genome
-construct expressed, cells able to synthesize histidine and will grow
-cells also transformed with plasmid that contains So fused to GAL4 DNA binding domain
-know So can activate transcription, want to determine strenght


Determining Strength of Activation

-to determine add inhibitor of histidine biosynthesis to media
-if transcriptional activator is weak, low levels of HIS3 activation will not be enough to overcome high concentrations of inhibitor
-if strong activator it will activate transcription of HIS3 at high enough levels so high concentrations of inhibitor are overcome
-helps understand relative strength of TF's