Chapter 9:

Tools for Analyzing Gene Expression

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

9.1 Introduction

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.

• 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.

• 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.

Model organisms

• Each model organism is distinctively suited, as a simplified model, to the study of particular complex aspects of biology.

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.

Classic model organisms for molecular biology

• Bacteriophage lambda ()

• The bacterium Escherichia coli

Some widely used eukaryotic model organisms

Slime mold: Dictyostelium discoideum

Ciliate: Tetrahymena thermophila

Yeast: Saccharomyces cerevisiae and Schizosaccharomyces pombe

Worm: Caenorhabditis elegans

Fly: Drosophila melanogaster

Fish: Danio rerio

Plant: Arabidopsis thaliana

Mouse: Mus musculus

Frog: Xenopus laevis and Xenopus tropicalis

9.2 Transient and stable transfection assays

• 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.

• 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.

9.3 Reporter genes

• 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.

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.

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.

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)

Analysis of gene expression

Example:

• Activation of reporter gene expression by overexpression of a transcription factor using a cotransfection assay.

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.

Fusion proteins are used for studies of:

• Protein localization.• DNA-protein interactions.• Protein-protein interactions.• To make large quantities of protein for

structural studies.

Commonly used purification and detection tags

Protein or peptide affinity tags:

• Histidine (His) tag: 6-histidine

• GST tag: glutathione-S-transferase

Immunotags:

• c-Myc: a transcription factor

• FLAG: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys

• HA: influenza A virus haemagglutinin

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.

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.

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.

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.

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.

Fluorescence, confocal, and multiphoton microscopy

• Imaging of either fixed or living tissues that have been labeled with one or more fluorescent probes.

• 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.

• 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

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.

• 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.

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.

9.4 In vitro mutagenesis

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.

9.5 Analysis at the level of gene transcription: RNA expression and

localization

• 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.

Techniques for monitoring mRNA levels

• Northern blot

• In situ hybridization

• RNase protection assay (RPA)

• Reverse transcription-PCR

• Quantitative real-time PCR (Q-PCR)

9.6 Analysis at the level of translation: protein expression

and localization

• Protein expression can be analyzed in a variety of ways using protein gel electrophoresis and the tools of immunology.

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.

• 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.

• 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.

One-dimensional (1D) SDS-PAGE

• Separates proteins by size

Two-dimensional (2D) PAGE

• Separates proteins by both charge and size.

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.

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.

• 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.

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.

Monoclonal antibodies

• Identical antibodies to a specific epitope of a protein.

• Produced by a clone originating from one cell.

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.

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.

9.7 Antisense technology

Antisense-mediated inhibition of gene expression methods include:

• Antisense oligonucleotides

• RNA interference (RNAi)

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.

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.

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).

• 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.

9.8 Analysis of DNA-protein interactions

Three methods are commonly used:

• Electrophoretic mobility shift assay (EMSA)

• Deoxyribonuclease I (DNase I) footprinting

• Chromatin immunoprecipitation (ChIP) assay

9.9 Analysis of protein-protein interactions

Four methods are commonly used:

• Pull-down assay

• Yeast two-hybrid assay

• Coimmunoprecipitation assay

• Fluorescence resonance energy transfer (FRET)

• What is a key feature of transcription factors that makes the yeast two-hybrid assay possible?

9.10 Structural analysisof proteins

Four methods are commonly used:

• X-ray crystallography

• Nuclear magnetic resonance (NMR) spectroscopy

• Cryoelectron microscopy

• Atomic force microscopy (AFM)