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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