Transcript Chapter 9: Tools for Analyzing Gene Expression

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
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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:
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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-AspLys
• 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 outof-focus fluorescence.
• Confocal and multiphoton microscopy
have enabled the imaging of discrete
regions of tissues at high resolution.
Fluorescence terminology
• 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.
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 diffractionlimited 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, highresolution, 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-todetect 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 analysis
of proteins
Four methods are commonly used:
• X-ray crystallography
• Nuclear magnetic resonance (NMR)
spectroscopy
• Cryoelectron microscopy
• Atomic force microscopy (AFM)