Flashcards in Exam 4: Lecture 9 Deck (13)
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1
Luciferase Assay
-can determine presence and degree of activation potential of DNA binding protein
2
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
3
Determining if So-Eya complex functions as transcriptional activator
-control experiments must accompany actual experiment to determine relevance
4
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
5
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
6
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
7
Experimental Condition (UAS-S0 & UAS-Eya)
-luciferase/renilla ratio much higher than any of controls
-real experimental value of So-Eya transcriptional activation
8
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
9
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
10
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
11
Histidine
-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
12
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
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