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Transactivation Assay Introduction
Regulation of gene expression at the level of transcription is one of the most
efficient means for cells to change their function and/or respond to changes in their
environment. (How else can cells regulate gene expression?*). Eukaryotic transcription
is regulated by a segment of DNA called an enhancer element. Enhancers usually exist
upstream of the gene they regulate, but sometimes extend into the gene itself. A
combination of transcription factors (activators and/or repressors) binds to the enhancer
and tells the basal transcription machinery if a particular gene needs to be expressed, and
if so, to what extent.
Because the functions of many gene products are difficult to measure
biochemically, scientists often engineer reporter constructs in which the promoter of their
gene of interest (and sometimes the beginning of that gene's sequence) is fused to the
sequence of a well-characterized enzyme such as β-galactosidase or luciferase. This
construct can then be transformed into an appropriate cell line and the function of the
promoter analyzed based on the level of enzyme activity (Figure 8.1).
Your Favorite Promoter
β-galactosidase
Transform
into your
favorite cell
line.
Change desired condition:
-alter promoter
-alter transcription factor(s)
-add hormone or other stimulus
Measure enzyme
activity as a
biochemical readout
of promoter strength
or activation.
Figure 8.1. Transactivation assay used for promoter studies
For example, a researcher may be studying a gene she suspects is regulated by
estrogen. To test this hypothesis she can take this gene's promoter, put it in front of βgalactosidase and transform it into her favorite cell line. Then she can expose these cells
to estrogen and biochemically monitor the extent of β-galactosidase expression. If the
enzymatic activity increases after exposure of the cells to estrogen, then expression of the
original gene is likely to be regulated by estrogen as well. Conversely, one might alter a
transcription factor and look to see what effect this has on a promoter known to be
regulated by it. For more information on transactivation assays and reporter constructs
refer to your textbook.
*One way cells can regulate gene expression is by the regulated breakdown of specific
proteins. Your textbook describes several additional mechanisms for regulating gene
expression in eukaryotes.
During the next two weeks of lab you will employ a reporter construct to further
characterize your HePC resistant mutant. Specifically, your goal is to test the
hypothesis that your HePC resistant strain carries a hypermorphic mutation in
either PDR1 or PDR3 (if you need to review what “hypermorphic” means, refer to your
textbook).
As you learned earlier in the semester, drug resistance is a major problem when
treating a variety of diseases, and drug resistance can be caused by several different
mechanisms. One common mechanism cells use to avoid the toxic effects of drugs is to
reduce intracellular accumulation of drugs by pumping them out of the cell. In yeast this
can be achieved by upregulating expression of a variety of ABC transporters which act as
drug pumps. Many such ABC transporters have a broad specificity in terms of the
substrates they can move across membranes, and thus cells that use this strategy are said
to exhibit pleiotropic drug resistance, because they can tolerate higher doses of many
different drugs compared to wild-type cells. Most pdr strains of yeast have been found to
carry gain-of-function mutations in either the transcription factor PDR1 or PDR3. Such
mutant transcription factors dramatically increase the expression of drug pumps such as
Pdr5p, by binding to enhancer elements upstream of the PDR5 gene (Balzi and Goffeau,
1995).
To test the hypothesis that your mutant strain contains a hypermorphic mutation
in PDR1 or PDR3, you will transform your HePC resistant mutant with a reporter
construct containing the pdr response elements (PDREs) from the PDR5 gene fused to
lacZ (this construct was first described by Katzmann et al. 1994). If your mutant exhibits
significantly higher levels of β-galactosidase than a wild-type control, then its pdr
network has been activated, perhaps by a hypermorphic allele of PDR1 or PDR3. Some
additional pieces of evidence that would back up this hypothesis include (1) if the
mutation in your HePC resistant strain is dominant and (2) if your strain was resistant to
other drugs when you tested for pleiotropy earlier in the semester.
References
Balzi, E. and A. Goffeau (1995) Yeast multidrug resistance: the PDR network. J. Bioenerg. Biomembr. 27:
71-6.
Katzmann, D.J., Burnett, P.E., Golin, J., Mahe, Y. and W.S. Moye-Rowley. (1994) Transcriptional control
of the yeast PDR5 gene by the PDR3 gene product. Mol. Cell Biol. 14: 4653-4661.
Transformation of Yeast with a PDRE-β-gal Reporter Construct
Lithium acetate transformation protcol adapted from Gietz and Woods (2002).
The day before your lab period, you must come in to lab and start a culture of your HePC
resistant strain. The culture should be grown in YPD and placed in the shaking water
bath at 30oC
1. During your lab period, pipette 1ml of this culture into a microfuge tube.
2. Pellet the cells by spinning in a microfuge for one minute at 13,000rpm.
3. Use a pipetman to remove the media without disturbing the cell pellet.
4. Resuspend the cells in 500μl of sterile water by gently pipetting the solution up and
down.
5. Pellet the cells by spinning in a microfuge for one minute at 13,000rpm.
6. Use a pipetman to remove the water without disturbing the cell pellet.
7. Resuspend the cells in 200μl of 100mM lithium acetate.
8. Pellet the cells by spinning in a microfuge for one minute at 13,000rpm.
9. Use a pipetman to remove the lithium acetate solution without disturbing the cell
pellet.
10. Repeat steps 7, 8, and 9.
11. Add the following ingredients on top of your cell pellet in the order listed:
240 μL PEG3350 (50% w/v)
36μL 1.0 M LiAc
25μL carrier DNA (2mg/mL)
50μL plasmid DNA (PDRE-β-gal)
12. Vortex the tube vigorously until the cell pellet has been completely resuspended
(about 1 minute).
13. Incubate at 30oC for 30 minutes.
14. Heat shock in a water bath at 42oC for 20-25 minutes.
15. Pellet cells by spinning in a microfuge for one minute at 13,000rpm and remove the
supernatant.
16. Resuspend pellet in 1ml of sterile water by gently pipetting up and down.
17. Pellet the cells by spinning in a microfuge for one minute at 13,000rpm. Remove
approximately 800μL of water, resuspend the cells in the remaining liquid and plate on
SDC-ura. This plate should be incubated at 30oC.
18. In 3 days, return to the lab and you should find plenty of colonies. Place the plates in
the refrigerator for storage. Then one day before your next lab meeting you will need to
start liquid cultures from three of these colonies (read ahead for details!).