Download PS401-Mar. 17

Mutagenesis
MUPGRET Workshop
Mutation
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Heritable change in the DNA sequence.
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Naturally occurring
Induced
Types of mutations
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Chromosomal
Point
Insertion/Deletion
DNA repair
Mutagens
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Environmental
Chemical
Mutations as a tool
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Associating a phenotype with a gene.
Understanding gene function.
Studying protein interactions.
Understanding cell lineage and organ
development.
Associating a phenotype with
a gene
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Changes in the DNA sequence that nonfunctional or reduced function proteins
often cause a visible change in the
appearance of the organism.
Some changes do not give visible
phenotypes.
Often identified as an “off-type” in plant
species.
Associating a phenotype #2
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This is often the first step towards
understanding the function of a gene or
to dissecting a biochemical pathway.
The mutation can be either a naturally
occurring one or an induced one.
Can be targeted or random.
Understanding gene function
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“You don’t know how something really
works until you have to fix it.”
Disruptions of the gene can be either
non-functional or “leaky”.
Often the “leaky” phenotypes will really
help you understand how to gene
works.
Understanding gene function
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In the case of targeted mutagenesis
where you know what the other genes
in that would/could be co-regulated
with the mutant are you can
understand the pathway better by
looking at expression of the coregulated genes.
Understanding gene function
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In the case of site directed mutagenesis
where you can target particular
sequences, you can dissect the part of
the protein that is important for
function.
Can help to ID the catalytic site or a
site involved in protein-protein
interactions or a site involved in
transport, etc.
Studying protein-protein
interaction
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Often use a series of alleles that have
defects in different parts of the gene to
identify the site that is required for
protein-protein interaction.
The series can be insertions, deletions,
or point mutations and may come from
nature or be induced or a combination
of the two.
Understanding cell lineage
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Usually used with transposon mutagenesis.
Transposons are mobile pieces of DNA that
can insert into a gene and disrupt its
function.
Insertion can happen throughout
development and can be used to track where
cells came from with visible marker.
Ac/Ds in Maize
Corn example of cell lineage
Methods for detection
mutations
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Alteration in electrophoretic mobility
Mismatch cleavage methods
Mismatch recognition methods
Sequencing
Protein trunctation test
Blazing a Genetic Trail
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It tells the story of how mutations are
involved in several different diseases.
http://www.hhmi.org/genetictrail/
Association Genetics
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Usually used for medical genetics.
Recently applied to plant genetics.
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Which genes were involved in
domestication?
Is this gene responsible for part of the
difference we see in a particular trait such
as plant height?
Dwarf 8
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Mutagenesis and
trait analysis
suggested that d8
might influence
flowering time and
plant height.
D8 study
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Sequenced D8 in many ~100 maize
lines.
Measured flowering time and plant
height in the same material.
Compare DNA sequence to flowering
time and plant height.
D8 summary
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Found several polymorphisms that are
associated with changes in flowering
time.
Data also indicate that D8 has
undergone selection.
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Compare synonymous vs. nonsynonymous
substitutions.