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Welcome to the world of
two Arabidopsis genes:
At4g14770 and At3g22760
Presented by:
Matt Emmer
HC70AL
Spring 2006
At4g14770 and At3g22760:
Tesmin/TSO1-like CXC Domain Proteins
• TSO1
– involved in cellular expansion and cytokinesis, as well
as in the development of ovules and microspores
– Contains two cysteine-rich regions similar to CXC
domains involved in chromosome segregation
• Tesmin
– a member of the CXC family, and a testis-specific
protein
Structure of At4g14770
Line)
Gene Size
3452 bp
# of Exons
8
# of Introns
7
Protein Encoded
Tesmin/TSO1-like
CXC Domain
# of Amino Acids
659
T-DNA Location
First Intron
st
(1
Structure of At3g22760
Line)
?
Gene Size
3076 bp
# of Exons
10
# of Introns
9
Protein Encoded
Tesmin/TSO1-like
CXC Domain
# of Amino Acids
610
T-DNA Location
5’ UTR
nd
(2
Where is At4g14770 Expressed?
Where is At3g22760 Expressed?
Were there Mutants in the 1st Line?
WT
Mut
Expected Size
9 Homozygous
Mutants Identified
in First Line
Wild Type
802 bp
T-DNA
644 bp
1 Heterozygous Mutant
What
DNA Concentrations?
Were were
therethe
Mutants
in the 2nd Line?
Plant
Concentration
Plant
Concentration
Plant 3
5 ng/μL
Plant 15
2 ng/μL
Plant 41,624 bp
15 ng/μL
Plant 16
4 ng/μL
Plant 101,095 bp
T-DNA
4 ng/μL
Plant 18
2 ng/μL
Plant 12
1.5 ng/μL
Wild Type
0.5 ng/μL
Plant 13
4.5 ng/μL
Expected Size
Wild Type
Genotyping with separate primers
Seeds and Silique
Embryos
Mutant
vs.
Wild Type
Any Difference?
NO
What’s Upstream of At4g14770?
1918 bp
3237 bp
1319 bp
I-proof Pcr
1918 + 1319 = 3237
Ecori Digest
Single EcoRI Site in Upstream Region
Upstream Bioinformatics
Primers
Eco RI Site
How accurate was the SP6 and
T7 Sequencing Data?
• Only a single mismatch in the SP6 sequencing data
Actual Sequence: AAAGTAACGGACATCAGTTTTTTTTTTCTTTTCCCTTTTTTCTCTTTTTTG
What’s Upstream of At3g22760?
2911 bp
I-proof Pcr
Ecori Digest
Did the T-DNA insert actually knock
out At4g14770?
Did the T-DNA insert actually knock
out At4g14770?
• Two Possibilities:
– Intron was spliced out
– T-DNA was not spliced
out, resulting in a longer
mRNA strand
• Follow up experiment:
Conclusions
• 1st Line mutants were attained
– No mutant phenotype observed
– Evidence to suggest that gene knockout was not successful
• Limited information obtained for 2nd line
– No mutant plants attained
– SALK indicates a T-DNA insert is in 5’ UTR, so gene may
not have been knocked out
• Future Experiments
– Double/Triple Knock Outs to overcome duplication:
• At4g14770, At3g22760, At3g22780
Acknowledgements
The TA’s
• Mike Gaviño
• Ria Yagnik
• Jonathan Russell
The Big Shots
• Dr. Bob Goldberg
• Dr. Anhthu Bui
• Dr. Xingjun Wang
Big Shots To Be
• Tomokazu Kawashima
• Brandon Le
• Jessica Luke
HC70AL
Jordan, Thi, Jennifer,
Brian, Jordan, Jason,
Daisy, Bekah, and
Heather