Chapter 9: Tools for Analyzing Gene Expression. In the post-genomic era, researchers need a tool...
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Transcript of Chapter 9: Tools for Analyzing Gene Expression. In the post-genomic era, researchers need a tool...
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Chapter 9:
Tools for Analyzing Gene Expression
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In the post-genomic era, researchers need a tool that enables the direct visualization of biological functions and GFP has turned out to be that tool.
Atsushi Miyawaki, Cell 135 (2008), p. 987
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9.1 Introduction
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After a new gene is cloned, the next steps are to determine:
• The structure of the gene.
• How its expression is regulated.
• The biological functions of the encoded gene product.
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• Gene expression is the production of a functional protein or RNA from the genetic information encoded in the genes.
• The term encompasses both transcription and translation.
• Often, gene expression is used to refer to the process of transcription only.
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• Overview of tools for analyzing gene regulation and function.
• Resource for when tools become relevant for understanding experiments referred to in subsequent or previous chapters.
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Model organisms
• Each model organism is distinctively suited, as a simplified model, to the study of particular complex aspects of biology.
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General attributes of model organisms
• Relatively cheap and plentiful.• Inexpensive to house.• Straightforward to propagate.• Short gestation periods that produce large
numbers of offspring.• Easy to manipulate in the lab.• Some have a fairly small and relatively
uncomplicated genome.
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Classic model organisms for molecular biology
• Bacteriophage lambda ()
• The bacterium Escherichia coli
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Some widely used eukaryotic model organisms
Slime mold: Dictyostelium discoideum
Ciliate: Tetrahymena thermophila
Yeast: Saccharomyces cerevisiae and Schizosaccharomyces pombe
Worm: Caenorhabditis elegans
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Fly: Drosophila melanogaster
Fish: Danio rerio
Plant: Arabidopsis thaliana
Mouse: Mus musculus
Frog: Xenopus laevis and Xenopus tropicalis
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9.2 Transient and stable transfection assays
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• Transfection: the introduction of DNA into eukaryotic cells.
• Plasmid DNA remains extrachromosomal.
• Plasmid DNA is not replicated in mammalian cells and is eventually lost by degradation and by dilution as cells divide.
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• Transient transfection: the introduction of DNA into cells for a short duration.
• Stable transfection: Cells that have stably integrated the plasmid into a chromosome are selected for by drug resistance.
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9.3 Reporter genes
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• A reporter gene is a known gene whose RNA or protein levels can be measured easily and accurately.
• Often used to replace other coding regions whose protein products are difficult to measure quantitatively.
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Some applications of reporter genes:
• The activity of the regulatory regions from another gene in different tissues or developmental stages.
• The efficiency of gene delivery systems.• The intracellular fate of a gene product.• Protein-protein interactions.• DNA-protein interactions.• The success of molecular cloning efforts.
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Commonly used reporter genes
• Generally code for proteins with enzymatic activities or fluorescent properties not typically found in the cells of most eukaryotes.
• The choice of reporter gene depends on the cell system being used, the sensitivity required, and the desired method of analysis.
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CAT reporter gene assay
• Chloramphenical acetyltransferase (CAT) catalyzes the acetylation of chloramphenicol, with acetyl group donated by acetyl CoA.
• Acetylated chloramphenicol can be monitored by:– Autoradiography following thin-layer
chromatography– Enzyme-linked immunosorbent assay (ELISA)
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Analysis of gene expression
Example:
• Activation of reporter gene expression by overexpression of a transcription factor using a cotransfection assay.
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Purification and detection tags: fusion proteins
• Reporter genes can be attached to other sequences so that the reporter protein is synthesized fused to another protein.
• Often a short peptide sequence that serves as an affinity or epitope tag (antigenic determinant) is used.
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Fusion proteins are used for studies of:
• Protein localization.
• DNA-protein interactions.
• Protein-protein interactions.
• To make large quantities of protein for structural studies.
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Commonly used purification and detection tags
Protein or peptide affinity tags:
• Histidine (His) tag: 6-histidine
• GST tag: glutathione-S-transferase
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Immunotags:
• c-Myc: a transcription factor
• FLAG: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys
• HA: influenza A virus haemagglutinin
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Fluorescent protein tags
Green fluorescent protein
• Originally isolated from the jellyfish Aequorea victoria.
• The fluorescence of GFP can be detected directly in living cells.
• GFP can artificially be expressed effectively in every cell type and organism tested so far.
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Properties of green fluorescent protein
• GFP fluorophore is buried in the center of a cylinder formed by an 11-stranded -barrel.
• A fluorophore is a group of atoms in a molecule responsible for absorbing light energy and producing the color of the compound.
• GFP fluorophore arises from an autocatalytic post-translational modification of GFP.
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Fluorescent proteins with different spectra
• Mutant forms of GFP– Enhanced GFP (EGFP): Red-shifted variant – Yellow fluorescent protein (YFP)– Cyan fluorescent protein (CFP)
• Red fluorescent protein from a tropical coral, Discosoma striata (RFP or DsRed)
• Variants of DsRed: fruit fluorescent proteins– mCherry, pmBanana, tdTomato, etc.
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Examples of use of fluorescent fusion proteins
• Tracking the intracellular localization of a protein of interest.
• Multiple labeling of different organelles or structures within the same cells or different tissues of cells in the same organism.
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Production of recombinant protein
• Over-expression of recombinant proteins in bacteria.
• Over-expression of recombinant proteins in eukaryotic cells.
• In vitro translation of recombinant proteins.
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Fluorescence, confocal, and multiphoton microscopy
• Imaging of either fixed or living tissues that have been labeled with one or more fluorescent probes.
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• When samples thicker than 2 m are imaged using conventional fluorescence microscopy, resolution is poor due to out-of-focus fluorescence.
• Confocal and multiphoton microscopy have enabled the imaging of discrete regions of tissues at high resolution.
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• A fluorochrome is a natural or synthetic dye or molecule that can exhibit fluorescence.
e.g. fluorescein isothiocyanate (FITC)
• A fluorophore is a group of atoms in a molecule responsible for absorbing light energy and producing the color of the compound.
• These words tend to get used interchangeably in the scientific literature.
Fluorescence terminology
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Confocal microscopy
• IIlumination is achieved by scanning one or more focused beams of light from a laser across the specimen.
• IIluminated light is focused to a diffraction-limited spot.
• The signal photons are focused onto a detector pinhole that rejects scattered and out-of-focus light.
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• By collecting a series of “optical sections” (Z series) researchers can create, with the help of sophisticated computer algorithms, high-resolution, three-dimensional images of a sample.
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Multiphoton microscopy• Also known as two-photon microscopy.
• The sensitivity of detection is much higher than for confocal microscopy.
• Multiphoton excitation is limited to the plane of focus, thus reducing photobleaching and photodamage of samples.
• Particularly useful for live cell analysis in thick tissues.
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9.4 In vitro mutagenesis
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Three main types of in vitro mutagenesis
• Deletion mutagenesis by PCR removes segments of DNA from a gene clone.
• Linker scanning mutagenesis is the systematic replacement of each part of a gene clone to determine its function.
• Site-directed mutagenesis is the introduction of specific base substitutions or small insertions at defined sites in a cloned DNA molecule.
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9.5 Analysis at the level of gene transcription: RNA expression and
localization
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• Constitutive expression: the gene is expressed at all times.
• Spatial expression: the gene is only expressed in specific tissues in an organism.
• Temporal expression: the gene is only expressed during a specific time in development.
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Techniques for monitoring mRNA levels
• Northern blot
• In situ hybridization
• RNase protection assay (RPA)
• Reverse transcription-PCR
• Quantitative real-time PCR (Q-PCR)
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9.6 Analysis at the level of translation: protein expression
and localization
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• Protein expression can be analyzed in a variety of ways using protein gel electrophoresis and the tools of immunology.
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Protein gel electrophoresis
• Polyacrylamide is used as a gel matrix instead of agarose because it gives better resolution.
• The carbon backbone of protein molecules is not negatively charged.
• Negative charge is provided by including the anionic detergent sodium dodecyl sulfate (SDS) in the loading, gel, and electrophoresis buffers.
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• The amount of SDS bound to each protein is proportional to its molecular weight.
• The rate of migration through the gel is inversely proportional to the logarithm of molecular weight.
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• Gel electrophoresis allows determination of important properties of a protein such as its isoelectric point and approximate molecular weight.
• A protein’s isoelectric point or pI is the pH at which the protein has an equal number of positive and negative charges.
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One-dimensional (1D) SDS-PAGE
• Separates proteins by size
Two-dimensional (2D) PAGE
• Separates proteins by both charge and size.
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Techniques for monitoring protein levels
• Western blot.
• In situ analyses.– e.g. indirect immunofluorescence assay
• Enzyme-linked immunosorbent assay (ELISA).
• Constructing fusion proteins with an easy-to-detect tag.
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Antibody production
• Antibodies are used extensively as tools for molecular biology research.
• They are proteins made by B cells of the immune system.
• An antibody is composed of two heavy chains and two light chains that form antigen binding sites.
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• An antigen is a substance that will induce an immune response.
• An epitope is the region on an antigen to which an antibody can bind.
• One antibody recognizes and binds to one and only one epitope.
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Primary antibodies
Polyclonal antibodies
• When an antigen such as a protein is injected into an animal, a mixture of antibodies is produced and isolated.
• Each antibody in the mixture recognizes a different, specific epitope within the protein.
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Monoclonal antibodies
• Identical antibodies to a specific epitope of a protein.
• Produced by a clone originating from one cell.
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Secondary antibodies
• A second set of antibodies created to target the Fc fragment (constant region) of the primary antibody.– e.g. a FITC-conjugated anti-rabbit secondary
antibody made in goat
• Conjugated to a fluorochrome or to an enzyme for colorimetric or chemiluminescent detection.
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Advantages to using secondary antibodies
• Provide an additional step for signal amplification, increasing overall sensitivity of the assay.
• Can be used with a wide variety of primary antibodies.
• Commercially available.
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9.7 Antisense technology
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Antisense-mediated inhibition of gene expression methods include:
• Antisense oligonucleotides
• RNA interference (RNAi)
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Antisense oligonucleotides
• 15 to 25 nt antisense oligonucleotides bind to a specific mRNA by complementary base-pairing.
• The hybrid duplex is cleaved by RNase H or translation arrest is mediated by blocking read-through by the ribosome.
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Modified antisense oligonucleotides
• Morpholino oligonucleotides are modified DNA analogs with an altered backbone linkage that lacks a negative charge.
• Not substrates for RNaseH.
• Morpholinos are usually targeted to the 5′ UTR or start codon of a target mRNA.
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RNA interference (RNAi)
• A sequence-specific gene-silencing process that occurs at the post-transcriptional level.
• Triggered by double-stranded RNA (dsRNA) molecules.
• dsRNA is processed into short RNAs of ~21-26 nt in length called small interfering RNAs (siRNAs).
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• siRNA triggers a special RNA-induced silencing complex (RISC) to recognize and cleave a complementary RNA.
• The target RNA is then rapidly degraded.
• Silencing of a gene by RNAi “knockdown” allows testing of the role of the gene product in a cell.
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9.8 Analysis of DNA-protein interactions
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Three methods are commonly used:
• Electrophoretic mobility shift assay (EMSA)
• Deoxyribonuclease I (DNase I) footprinting
• Chromatin immunoprecipitation (ChIP) assay
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9.9 Analysis of protein-protein interactions
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Four methods are commonly used:
• Pull-down assay
• Yeast two-hybrid assay
• Coimmunoprecipitation assay
• Fluorescence resonance energy transfer (FRET)
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• What is a key feature of transcription factors that makes the yeast two-hybrid assay possible?
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9.10 Structural analysisof proteins
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Four methods are commonly used:
• X-ray crystallography
• Nuclear magnetic resonance (NMR) spectroscopy
• Cryoelectron microscopy
• Atomic force microscopy (AFM)