Understanding gene function

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• Understanding gene function
• We now know how to clone genes, but how can we
  learn more about the gene and the gene product?
• Obviously it is helpful if there is a mutant
  phenotype available. We can make inferences
  about the gene function because the gene of
  interest helps prevent the mutant phenotype.
• We know this gene is involved in fruit ripening,
  because if the gene is mutated, the fruits never
  mature.
• Most of the time there is no mutant phenotype
  available, though. So what do we do then?
• Some approaches include
• Northern analysis/ quantitative RT-PCR
• Cell fractionation followed by functional assays
  or Western analysis
• In situ hybridization
• Immunolocalization
• Promoter fusions

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Northern analysis RNA is isolated from one or
more tissues, separated on an agarose gel and
blotted on a nylon membrane. The membrane is
placed in a tube or container with a
hybridization buffer and a probe (radioactive or
chemically labeled). The probe is specific for
the gene of interest (so the gene must have been
cloned). The probe will hybridize to the mRNA on
the blot that corresponds to the gene. After
washing off unbound probe, the labeled probe is
detected via autoradiography (with the
radioactive probe) or via chemiluminescence (with
a chemically labeled probe). In both cases this
results in the formation of a dark band on an
X-ray film. We can now compare expression of the
gene among different samples based on the
intensity of the signal on the film. Advantages
of this technique Relatively fast Relatively
sensitive Oftentimes similar results can be
obtained with quantitative RT-PCR. The
quantitative refers to the relationship between
the PCR product and the amount of template. In
this case you need to have suitable primers and
an optimized PCR protocol.
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Cell fractionation The tissue(s) in which the
protein of interest is expected to be present is
ground and through a number of centrifuge steps
we can obtain different fractions representing
various cell organelles. Out of these different
fractions we can extract proteins and determine
the activity of the protein through an assay. If
that is not possible we can detect the presence
of the protein via a western analysis. Western
analysis The proteins are extracted from one or
more samples, separated on a polyacrylamide gel
and then transferred to a membrane. The membrane
is incubated with antibodies against the protein
of interest (so in this case we must have had
purified protein available). Typically the
antibodies themselves are detected with
antibodies. These secondary antibodies are
typically conjugated with an enzyme that allows
the formation of a colored precipitate. How do
we get the antibodies? Protein purification (to
the point where there are no other proteins
present, other then the protein of interest).
The purified protein is then injected in a lab
animal, usually a rabbit, mouse or chicken.
After a few weeks antibodies are isolated out of
the blood of the animal (or the egg if it was a
chicken).
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In situ hybridization In this case we detect
mRNA inside cells. The main advantage over
northern analyses is that you get more detailed
information about where the gene of interest is
expressed, and you get a better representation of
the expression level. When a gene is expressed
in very specific cells and you isolate RNA out of
the whole tissue, there will be a dilution of the
mRNA, which during a developmental time series
(enlargement of the tissue) could be interpreted
as a reduction in gene expression. In situ
hybridizations are performed on sections of
tissue that has been fixed. The sections are
placed on a microscope slide. A radioactive (or
chemically labeled) probe is placed on the
slide After washing the slide to remove unbound
probe, the slide is coated with a liquid
film Exposure of the film results in silver
grains being deposited in the places where the
label (and thus the mRNA) was present. The probe
is typically an antisense RNA probe, but the
sense RNA probe is often used as a negative
control. This probe is synthesized by in vitro
transcription with RNA polymerase. Several
commercially available plasmids contain promoters
for RNA polymerases. Depending on the
orientation of the cloned cDNA you will get
either the sense or the antisense strand. In
situ hybridizations allow you to look at
tissue-specific expression or at the localization
of mRNAs within the cell. An example of both
can be found in the article by Vermerris et al
(2001) Plant Phys. Biochem 39 161-166.
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The figure was deleted to reduce the file
size. Please see Figure 1 of the article that
was handed out in class.
From Vermerris et al. 2001
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Dark field microscopy dark background silver
grains representing the mRNA of interest show up
as white signal. Bright field microscopy white
background silver grains representing the mRNA
of interest show up as black dots. The article
describes gene expression experiments with
developing potato tubers (yes, just like in the
cDNA-AFLP article!). The expression of specific
genes could be monitored as the tubers
developed. It was also apparent that certain
mRNAs were specifically targeted to starch
grains. This is evidence for an mRNA
localization mechanism. The 3UTR of the mRNA
contains a ZIP code. This is (probably) a
secondary structure that is recognized by
specific RNA-binding proteins. These proteins
also interact with the cytoskeleton and can thus
mediate the transport of mRNAs to certain
locations within the cell. These mechanisms have
also been reported in animals. mRNA localization
provides an extra level of regulation.
Results from in situ hybridizations are
oftentimes very beautiful, so make sure you check
out the original article to see the color photos.
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Immuno-localization This is to western blots
what in situ hybridizations are to northern
blots. In this case we incubate fixed tissue on
a microscope slide with antibodies against the
protein of interest. The antibodies are detected
with secondary antibodies conjugated with an
enzyme that allows the formation of a colored
precipitate. Alternatively, fluoresecent labels
can be used. The presence of the colored
precipitate reveals the presence of the protein.
Immunolocalization can also be used to detect
various chemical present in the cells. The term
tissue printing refers to the pressing of a cut
section of the plant (typically a stem) onto the
nylon membrane. Proteins from the cut surface
will adhere to the membrane. The membrane is now
treated as a western blot. This is much less
labor-intensive than the traditional
immuno-localizations.
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• Promoter fusions
• This is an alternative to in situ hybridizations.
  In this case we use transgenic plants that
  express a reporter gene under control of the
  promoter from the gene of interest.
• Several reporter genes can be used
• GUS (beta-glucuronidase), a bacterial gene. The
  GUS enzyme is typically assayed by immersing the
  tissue in a colorless substrate that turns blue
  upon cleavage by the enzyme.
• Luciferase the gene that enoces the protein
  that allows fire flies to produce light. A
  substrate, luciferin, needs to be supplied to see
  the activity of this reporter gene. You see the
  tissues expressing the gene give light.
• GFP - green fluorescent protein from jellyfish.
  This is a protein that fluoresces when it is
  excited with UV light. The nice thing is that it
  does not require expensive substrates.
• Some considerations when using promoter fusions
• this requires the ability to make transgenic
  plants
• You need to have the promoter from the gene of
  interest, and a big enough piece of it to ensure
  you have all the regulatory elements.
• There is the risk of position effects (i.e.
  unusual expression patterns as a result of the
  surrounding DNA)
• Some of these reporter gene mRNAs and/or their
  proteins are very stable over time. You cannot
  automatically extrapolate the results from the
  reporter genes to the native gene.
• The main advantage is the fact that it is much
  easier and more cost-effective to analyze gene
  expression in the entire plant, relative to the
  microscope slides of fixed tissue necessary for
  in situs and immunolocalizations.