Transcript Arabidopsis is Cool

The Effect of Overexpressed
AtOgg1 on Spontaneous
Guanine-to-Thymine Mutation
Colin Tominey
Dr. John Hays
Oregon State University
June 27, 2008
Background
• All organisms experience a certain
number of mutations due to chemical and
environmental interactions with their DNA.
• Point mutations have two general
causes:
– Chemical modification of a DNA base
– DNA replication error
Arabidopsis as a Model
Organism
• Arabidopsis thaliana is often used
as a model differentiating
organism.
• Advantages:
– Short generation times (~6 weeks)
– Small, well-documented genome
– Transgenic varieties are easily
created
– Survives multiple gene knockouts
Arabidopsis and Mutation
Research
• Insertion of modified GUS genes into
Arabidopsis created mutation-reporting
plants.
• Each line is designed to report one class
of point mutation (e.g., G→T)
• Reversion of the designated base
reactivates the GUS gene and produces
blue spots on that plant.
GUS Mutation Reporters
wt GUS Codon 11: Active Protein
-C G T-G A A-A T C-G C A-C T T-T A GGUS G→T Reporter Codon 11: Inactive Protein
-C G T-G C A-A T C-G C A-C G T-T A G• When a mutation causes the reporter codon to
revert to the wt codon, active protein is
produced and plant cells are stained blue.
Unexpected Observation
• Spontaneous reversion in the GUS
G  T lines was much higher than that
in other GUS plants:
Spontaneous Reversions
Reversion Type
Spontaneous Reversions
(Events / Plant)
G→T
0.2 – 2.1
C→T
0.007 – 0.03
T→C
0 – 0.01
T→G
0 – 0.03
G→C
0.03-0.04
Potential Sources of G → T
Mutation
• 8-oxoguanine has been implicated in
G → T mutation:
– Pairs strongly with adenine, causing
mutation during replication
– Generated by reactive oxygen species
attacking guanine in the DNA strand
GUS G→T Mutation Reporter
Reversion
-GCA-CGTO
-GCA-CGT-
Top strand retains the
pre-existing codon.
REPLICATION*
X
O
8
O-
Bottom strand forms
a pre-mutagenic
lesion.
-GAA-CTT-
REPLICATION*
Top strand results in fixation of
mutation (reversion to wt) and
production of active GUS
protein.
-GAA-CGTO
X
O
8
* Assumes replication without repair of the lesion.
Defending DNA
• AtOgg1, AtFpg, and AtMyh all code
for proteins that protect Arabidopsis.
• Many bacteria and eukaryotes
(including humans) have these same
kinds of proteins.
• Are these defenses sufficient?
Hypothesis
• High levels of 8-oxoguanine have
overwhelmed the plant’s natural
defenses (OGG1, FPG, and MYH),
thus causing the unexpectedly-high
rate of spontaneous G → T
mutation.
Prediction
• Plants with enhanced AtOgg1
expression will show significantly
less spontaneous G → T mutation
than their siblings with normal
AtOgg1 expression.
The Experiment
• Create a line of Arabidopsis plants that
overexpress the AtOgg1 gene.
• Cross these mutants with existing GUS
G → T reporter lines.
• Compare spontaneous GUS reversion in
new mutants with non-overexpressing
siblings.
Acknowledgments
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Howard Hughes Medical Institute
Oregon State University URISC
Dr. John Hays
Dr. Marc Curtis
Dr. Stephanie Bollmann
Peter Hoffman
Dr. Kevin Ahern