Exam 4: Lecture 9

Question 1

Luciferase Assay

Answer 1

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

Question 2

Example to determine activation strength of So-Eya complex

Answer 2

• 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

Question 3

Determining if So-Eya complex functions as transcriptional activator

Answer 3

-control experiments must accompany actual experiment to determine relevance

Question 4

First Control (UAS-GFP)

Answer 4

• GFP not transcription factor and cannot bind to enhancer

- this control provides level of baseline expression that is derived from minimal core promoter

Question 5

Second Control (UAS-So)

Answer 5

• 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

Question 6

Third Control (UAS-Eya)

Answer 6

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

Question 7

Experimental Condition (UAS-S0 & UAS-Eya)

Answer 7

• luciferase/renilla ratio much higher than any of controls

- real experimental value of So-Eya transcriptional activation

Question 8

Measuring Transcriptional Activation Potential

Answer 8

• 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

Question 9

Transcriptional Reporter Assay addressing question if So can activate transcription

Answer 9

• 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

Question 10

Determining location of activation domain in assy

Answer 10

• 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

Question 11

Histidine

Answer 11

• 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

Question 12

Histidine assay

Answer 12

• 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

Question 13

Determining Strength of Activation

Answer 13

• 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