Download 17 - Rutgers Chemistry

Chapter 17: Analysis of Gene Expression
Outline
 Gene expression in eukaryotic cells
 Reporter gene assays
o Chloramphenicol acetyltransferase (CAT) assay
 pCAT3-Basic Vector
o Luciferase assay
 pGL3-Basic Vector
o Secreted alkaline phosphatase (SEAP) assay
 pSEAP-2 Basic Vector
 RNase protection assays
o Multi-probe RNase protection assay
 Gel mobility shift assays
 Conclusion
 References
By Lisa Jablonski
Chemistry 544 –
Chemical Biology
Professor K.Y. Chen
May 3, 2010
Gene expression in eukaryotic cells
Gene expression in eukaryotic cells involves the transcription of a gene into mRNA, the posttranscriptional modification of mRNA, and the translation of mRNA into proteins.1 The control of
gene expression in eukaryotic cells occurs at six different steps, as described Figure 1:2
Figure 1: Six steps at which eukaryotic gene expression can be controlled. 2
The first step, gene transcription, is regulated by transcription factors (trans-acting elements)
that bind to promoter or enhancer sites (cis-acting elements) on DNA.1
1
Reporter gene assays, RNase protection assays and gel mobility shift assays are several
methods used to analyze gene expression.
Reporter gene assays
In reporter gene assays, a target gene is replaced by a reporter gene, which is a gene whose
expression can be monitored by fluorescence or enzymatic activity of its protein product. The
regulatory sequences that control the target gene will now control the reporter gene and since
the reporter gene’s expression can be monitored, the level, timing and cell specificity of the
regulatory sequences can be determined.2 The steps to create a reporter gene assay are
described in Figure 2:
Figure 2: Steps to creating a reporter gene assay.3
With reporter gene assays, different regulatory sequences (enhancers and/or promoters) can be
tested to determine if they control expression of the target gene in different cells. For example,
Figure 3 shows that regulatory sequence 3 activates the target gene in cell B; regulatory
sequence 2 activates the target gene in cells D, E and F; regulatory sequence 1 does not
activate the target gene in any cells; and the combination of regulatory sequence 1 and 2
activates the target gene in cells E and F.2
Figure 3: Using a reporter
gene to determine which
regulatory sequences affect
gene expression in different
cells.2
2
Different commonly used reporter genes are described in Figure 4:
Figure 4: Commonly used reporter genes.3
Chloramphenicol acetyltransferase (CAT) assay
The chloramphenicol acetyltransferase (CAT) gene is a popular reporter gene. This is a
bacterial gene that evolved to protect bacteria against the antibiotic chloramphenicol (CAM).
The gene encodes for a protein, chloramphenicol acetyltransferase (CAT), that can add an
acetyl group (from acetyl CoA) at one or both of the hydroxyl groups on chloramphenicol. This
action prevents chloramphenicol from binding to ribosomes.4
The degree of acetylation of chloramphenicol reflects the activity of the promoter used. The
degree of acetylation can be measured using thin-layer chromatography and autoradiography.3
In the example below (Figure 5a and 5b), CAM is radioactively labeled, so the autoradiograph
will show unused chloramphenicol, mono-acetylated chloramphenicol and di-acetylated
chloramphenicol. The autoradiograph shows that the Cat gene was not expressed in lane 1 but
was expressed in lanes 2 and 3 (it was expressed more strongly in lane 3). Therefore, it can be
concluded that the promoter used in lane 1 doesn’t activate the target gene (or it deactivates the
transcription of the target gene). The promoter in lane 2 activates the transcription of the target
gene somewhat, and the promoter in lane 3 strongly activates the transcription of the target
gene.
3
Figure 5a (top left):
Running a CAT assay.
Figure 5b (bottom left):
Analyzing a CAT assay.
As described on the Promega website, “The CAT gene is not found in eukaryotes, and therefore
eukaryotic cells contain little or no background CAT activity. This characteristic, as well as the
ease and sensitivity of CAT activity assays, has made CAT a widely used reporter for
mammalian gene expression studies.”5 Promega makes the pCAT®3-Basic Vector that
contains the CAT reporter gene (Figure 6). The vector lacks eukaryotic promoter and enhancer
sequences, allowing a promoter or enhancer region of interest to be inserted and tested for
expression.6
Figure 6: Promega
pCAT®3-Basic Vector.6
4
Luciferase assay
The luciferase enzyme from the firefly (Photinus pyralis) is another popular reporter molecule.
The luciferase enzyme has over a 1,000-fold increase in sensitivity compared to CAT. The
activity of the luciferase enzyme is determined by measuring the luminescence it emits; Figure 7
shows the linear relationship between luciferase enzyme concentration and luminescence.3
Figure 7: Enzyme concentration
and luminescence of luciferase.3
Promega makes the pGL3-Basic Vector that contains the luciferase reporter gene (Figure 8).
Figure 8: Promega pGL3Basic Vector.7
Secreted alkaline phosphatase (SEAP) assay
Secreted alkaline phosphatase (SEAP) is also used as a reporter molecule. The secreted
SEAP enzyme can be assayed directly from the culture medium and permits time-course
5
studies not possible with assays that require cells to be lysed. The cells can be used for further
investigations such as RNA or protein studies. SEAP dephosphorylates CSPD, a
chemiluminescent substrate, and the resulting product decomposes and releases light. The
activity of SEAP can be measured using both chemiluminescent and fluorescent detection
(Figure 9).3
Figure 9: Chemiluminescent
and fluorescent detection
assays of SEAP.3
Clontech makes the pSEAP-2 Basic Vector that contains the SEAP reporter gene (Figure 10).
Figure 10: Clontech
pSEAP-2 Basic Vector.8
Clontech also makes the pSEAP2-Control Vector that contains the SV40 promoter and
enhancer; this vector can be used as a positive control or as a reference (Figure 11).3
6
Figure 11: Using the
Clontech pSEAP2-Control
Vector with pSEAP2-Basic
Vector.3
RNase Protection Assay
The RNase protection assay is one of the main assays used to measure gene expression.
This assay detects and quantifies mRNA species.9 It is also referred to as a ribonuclease
protection assay, and is a type of nuclease protection assay (NPA).10 The procedure for running
an RNase protection assay is listed below and is also visualized in Figure 12:3
1) Isolate RNA sample(s) to be examined for target mRNA expression.
2) Create a labeled antisense RNA probe that is complementary to a several-hundred-base
region of the target mRNA.
3) Hybridize the labeled probe to the total RNA sample.
4) Treat the sample with single-strand-specific RNase which will degrade unhybridized probe
and target.
5) Separate the remaining protected probe-target hybrids on a denaturing polyacrylamide gel.
6) Detect/quantify the RNase-resistant protected probe using autoradiography.
Figure 12:
Procedure for
conducting an
RNase Protection
Assay.10
7
Multi-probe RNase protection assay
In the multi-probe RNase protection assay, multiple antisense RNA probes are added to a
total RNA sample to assay for expression of different mRNA transcripts. In this assay, matching
probes and targets act independently of each other, and therefore different mRNA species can
be detected at the same time. In the example in Figure 13, the total RNAs from different mouse
tissues (embryo, spleen, testes and thymus) were hybridized with seven different anti-sense
RNA probes for c-myc, β-actin, p53, Egr I, Jun B, Ras and cyclophilin RNAs. The multi-probe
RNase protection assay shows which mRNA transcripts were present in the different tissues
and at what intensity.10
Figure 13: Simultaneous
quantiation of multiple
mRNAs using a multiprobe RNase protection
assay.10
The quantity of mRNA expressed can be determined by comparing the intensity of probe
fragments on the autoradiograph to an endogenous internal control. This is a relative
quantitation method.10 For example, a probe can be included in the assay for a housekeeping
gene transcript; the quantity of housekeeping mRNA detected acts as a reference value. By
using the same housekeeping mRNA as a reference, cells can be assayed for different mRNAs
at different points in time, and the changes (if any) of mRNA transcript expression can be
determined.3
Gel mobility shift assays
Gel mobility shift assays (GMSAs) are used to detect interactions between a protein and
DNA. GMSAs detect the interaction between a protein and DNA by the lessening of the
electrophoretic mobility of DNA that occurs when it is bound to a protein (Figure 14).3 GMSAs
are also known as electrophoretic mobility shift assays (EMSAs).11
8
Figure 14: Schematic
diagram of a GMSA.3
The example below shows how a GMSA assay was used to identify a binding protein for a
known promoter region (Figure 15). As cells age and become senescent, the expression of the
thymidine kinase (TK) gene is lessened. The TK promoter contains several regions, including
inverted CCAAT boxes at -36 and -67 base pairs and a GC-rich Sp1 site. DNA fragments were
excised that contained the -67 bp CCAAT box or the Sp1 region, and were added to IMR-90
fibroblast cells that were either young (PDL = 22) or old (PDL = 49). GMSA results show that
young IMR-90 cells contain a binding protein that binds to the -67 bp CCAAT box; old IMR-90
cells do not contain this protein. The binding protein is called CBP/tk. The binding activity for
the Sp1 region of the TK promoter was the same in both young and old IMR-90 cells.12
Figure 15: GMSAs
showing interactions
between binding
protein and different
regions of the TK
promoter in young and
old IMR-90 cells.12
Other methods have been developed to conduct GMSA assays, such as the two-color GMSA
assay for detecting both free nucleic acid, bound nucleic acid, free proteins and bound proteins
in cells (Figure 16). This method uses fluorescent staining instead of radioactive labeling; one
dye stains DNA and the other dye stains protein. This method allows for more comprehensive
information about the protein-DNA interaction.11
9
Figure 16:
Comparison of
radioactive GMSA
assay and fluorescent
GMSA assay.11
Conclusion
Reporter gene assays, RNase protection assays and GMSA assays are all methods to analyze
gene expression. Reporter gene assays are used to identify regulatory sequences (promoters
or enhancers) for a known target gene, and are also used to quantify the strength of regulatory
sequences. RNase protection assays are used to detect and quantify mRNA transcripts.
GMSA assays are used to detect and quantify the binding of DNA binding proteins (transcription
factors) with known promoter regions.
References
1
2
3
4
5
6
Berg, Jeremy M. et al. Biochemistry. Fifth Edition. Copyright 2002.
Alberts, Bruce et al. Molecular Biology of The Cell. Fourth Edition. Copyright 2002.
Lectures notes “Analysis of Gene Expression.” Dr K.Y. Chen, Rutgers University, March
2010.
http://www.bio.davidson.edu/courses/genomics/method/CAT.html
http://www.promega.com Products  Reporter Vectors  FAQs  Genetic Reporters
and Transfection  CAT Reporters  Article # 226191: “What is the CAT Enzyme
Assay System?”
http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_122
2
10
7
8
9
10
11
http://www.promega.com/catalog/catalogproducts.aspx?categoryname=productleaf_259
http://www.clontech.com/images/pt/PT3075-5.pdf
http://www.bdbiosciences.ca/canada/downloads/protocols/RPA.pdf
http://www.ambion.com/techlib/basics/npa/index.html
Jing, Debra et al (2003). “A sensitive two-color electrophoretic mobility shift assay for
detecting both nucleic acids and proteins in gel.” Proteomics, 3:1172-1180.
12 Pang, J.H. and Chen, K.Y. (1993). “A Specific CCAAT-binding Protein, CBP/tk, May Be
Involved in the Regulation of Thymidine Kinase Gene Expression in Human IMR-90
Diploid Fibroblasts during Senescence.” The Journal of Biological Chemistry, 268
(4):2909-2916.
11