Title: Eukaryotic Gene Expression
Purpose: To perform Galactosidase assays on yeast strains containing plasmids with different promoters to determine the activity of these promoter fragments.
Introduction- Transcription:
In this experiment, you will learn one technique used in studying the regulation of gene expression. How much RNA is made from each gene, and when, is a question of life and death for every organism. Fortunately, the outcome is left to the reliable interactions between genes and proteins. The transcription apparatus is so basic to the functioning of all life that some of its features were "optimized" very early on and subsequently carried along with relatively little change throughout evolution. Otherfeatures of that apparatus, however, were modified independently within eachevolutionary lineage as complexity and diversification increased.
We will concentrate here on the regulation of eukaryotic transcription. The corestructure of a gene includes a transcribed portion of DNA coding for the gene product
("structural region") and one, immediately adjacent, regulating the timing and amount oftranscription ("promoter region"). Sequenceelements that control transcription from a distance are called "enhancers" because theyfrequently, but not always, stimulate transcription. The regulatory features characteristic of any given gene depend on the particularcombination of promoter and enhancer elements associated with it.
We will be examining the promoter of one gene in yeast, diagramed in figure 1. I have made several constructs which contain different sections of this promoter fused to a reporter gene (lac Z, found in E. coli, not in yeast, so that any signal obtained is due to the transcription from OUR promoter). I transformed these constructs into yeast where they are stably maintained at 1 copy/cell and are replicated just like normal chromosomes. Your job is to determine how much galactosidase activity is in each cell culture.
Figure One: Schematic of Reporter constructs 2, 3 and 4.
Figure 2: Schematic for Reporter Constructs 5, 6, 7 and 8.
Protocol:
galactosidase Liquid Assays (you will work in pairs)
You have 8 tubes (labeled 1-8) which contain 0.2 mL each of the yeast strains listed in Table I. At the beginning of your lab session, I will tell you the A600 for each strain, to enter into your Table I.
1. Vortex the cultures to mix the cells..
2. Add 0.8 ml Z buffer to each tube (made last week). (P-1000 set to 080)
3. Add 50ul chloroform to each tube (P-200 set to 050)
4. Add 20ul 0.1% SDS to each tube (P-20 set to 200)
5. Vortex each tube for 10"
6. Add 200 ul ONPG (4mg/ml in Z+BME, also made last week). Vortex the tubes to mix.
7. Time reaction at Room temperature (30 minutes from time added to tube 1)
8. Stop with 0.5ml 1M Na2CO3.
9. Pellet 5 minutes in eppendorf centrifuge
10. Carefully transfer upper layer to a cuvette (chloroform will “etch” or dissolve the cuvettes)
11. Read the Absorbance at 420 nm (blank with a tube that says Blank))
12. Calculate activity using the following formula:
Activity Units= Absorbance at 420 nm x 1000 = Velocity/mL
A600 x 30 minutes x 0.2 mL (200uL)
Table I
Cell culture / Absorbance at 600 / Volume (mls) / Time / Absorbance at 420 / Activity Units- No Promoter
2. Wild Type Promoter / 0.2 / 30 / .
3. Deletion DNA Element A / 0.2 / 30 / .
4. Deletion
DNA Element B / 0.2 / 30
5. CYC1 vector alone / 0.2 / 30 / .
6. CYC1 +
2 copies element C / 0.2 / 30
7. CYC1 +
3 copies element C / 0.2 / 30
8. CYC1 +
4 copies element C / 0.2 / 30
Write-up/ Conclusions
Construct ONE graph (bar graph) with the Activity Units for each construct on the Y axis and the names of the promoter constructs on the X axis. Use this graph to answer the following questions.
Questions:
1. What fold difference is there in -galactosidase activity between sample 1 (empty vector) and sample 2 (full length promoter)?
2. Do you think the DNA sequence Element A is a positive acting element or a negative acting element based on the difference between sample 2 (full length) and sample 3 (deletion of Element A)? Explain your rationale.
3. Do you think the DNA sequence Element B is a positive acting element or a negative acting element based on the difference between sample 2 (full length) and sample 4 (deletion of Element B)? Explain your rationale.
4. Samples 5-8 are constructs in which I inserted sequences from the TBP promoter into a DIFFERENT promoter (CYC1) to see their effect in a heterologous system. Based on the differences between samples 6,7, and 8 (2, 3 and 4 copies of Element C) compared to sample 5 (CYC1 alone), is the sequence Element C a positive or negative acting element? Explain your rationale
5. Based on your answers above, what do you think would happen if we put the sequences Element A from the TBP promoter into the CYC1 promoter? Based on the activity of the CYC1 promoter alone, do you think we could really measure a difference?
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