Really Cool Catchy Title

1
Arabidopsis thalianaB-Box Type Zinc Finger
Jasmonate-ZIM Domain Protein Families

Auni Hovanesian
HC70AL Research Symposium - June 5, 2008
2

What Is My First Gene?
AT1G05290
5 UTR
Chromosome 1 Reverse Orientation
Nucleotides 1,539,151 - 1,540,561
5
3
Exon
5 UTR
ATG
TGA
1,411 bp 1.4 kb
3
What Protein Does This Gene Encode?

• B-Box Type Zinc Finger

4
What Is a Zinc Finger?

• Large superfamily of proteins that can bind to
  DNA
• Consists of two antiparallel ß strands and an
  alpha helix, plus a zinc ion for stability
• Common to transcription factors and regulatory
  proteins

5
Where Is This Gene Active?
6
What Do RT-PCR Results Show?
100 bp Ladder
Pos. Control
Neg. Control
Silique RT
Silique RT-
Leaf RT-
Leaf RT
475 bp
Tubulin Bands (Expected Length 475 bp
Expected cDNA Band Length 175 bp??
Lack of cDNA bands for gene suggests little to no
activity in sampled areas
7
Does GeneChip Microarray Data Confirm This?
Yes, shows no gene activity in any of tested
areas
8
But Is a Transcription Factor Really Going to be
Inactive?

• Unlikely
• Must turn to knockouts to more directly observe
  role of gene in plant development
• Any phenotypic variation points to clear gene
  activity

9
How Is My T-DNA Insert Designed?

FW
5 UTR
LBb1
T-DNA
5
3
5 UTR
ATG
TGA
RV
FW
10
What Were My Genotyping Results?
100 bp Ladder
Pos. Con.
Neg. Con.
Plant 11
Plant 12
Plant 5
Plant 13
Plant 15
Plant 16
Plant 17
Plant 1
Plant 2
Plant 3
Plant 4
Plant 6
Plant 10
Plant 14
Plant 5
Plant 1
Plant 3
Plant 4
Plant 7
Plant 8
Plant 2
Plant 6
Plant 9
475 bp
275 bp
T-DNA Bands (Expected Size 475 bp)
Wild Type Bands (Expected Size 275 bp)
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What Do These Genotypes Suggest?

• Total Screened 38 SALK Plants
• 29 Hemizygous
• 9 Wild Type
• The absence of homozygous T-DNA plants after this
  much genoytyping suggests there are none
• According to the probability calculation, there
  is a 0.00002 chance of still finding a
  homozygous T-DNA after 38 plants have been
  screened
• Homozygous T-DNA Knockout Most Likely Causes Seed
  Lethality!

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But How Do the Observed Phenotypes Complement the
Genotypes?

• Seed lethality is a phenotype, purely by virtue
  of absence
• But directly observed phenotypes contradict
  genotyping results
• No differences were observed between hemizygous
  and wild type plants
• Siliques did not contain 1/4 dead seeds, as would
  expect from 121 ratio of genotype distribution
• Microscopy shows normal seeds and embryos
• Implications?

13
What Is My Second Gene?
AT1G70700
5 UTR
Chromosome 1
Intron
5 UTR
ATG
3 UTR
TGA
Exon
2, 296 bp 2.3 kb
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What Protein Does This Gene Encode?

• Jasmonate ZIM-Domain Protein 9 (JAZ9/TIFY7)
• JAZ protein family
• Involved in regulation of transcription factors
  vital to plant survival

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What Is The Exact Mechanism of This Regulation?

• Represses transcriptional activators of Jasmonate
  (Jasmonic Acid)
• Jasmonate is a plant hormone that induces defense
  mechanisms in response to wounding
• Small amounts of Jasmonate trigger proteasomal
  degradation of JAZ proteins by ubiquitin protein
  ligase SCFCOI1
• Allows previously repressed transcription factors
  to produce more Jasmonate, so plant can respond
  to attack

Transcribes Jasmonate (JA)
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Where Is This Gene Active?
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What Does RT-PCR Tell Us?
Pos. Control (gDNA)
100 bp Ladder
Leaf RT
Silique RT-
Silique RT
Neg. Control
Leaf RT-
475 bp
Tubulin Bands (Expected Length 475 bp)
200 bp
cDNA Bands (Expected Length 182 bp)
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• Presence of gene-specific cDNA bands in both leaf
  and silique samples suggest mRNA activity and
  gene expression in those areas!

19
What Does GeneChip Microarray Data Tell Us to
Confirm These Results?
Confirms some of cDNA band for Silique
activity
Confirms cDNA band for Leaf activity
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Promoter Activity Analysis
What Do We Know About the Upstream Region of My
Gene?
Chromosome 1
1.1 kb
0.22 kb
0.18 kb


AT1G70690
AT1G70700
5 UTR
3 UTR
5
3
Start
Stop
Forward
Forward
Upstream Region 1.5 kb
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How Many Recombinant Colonies Did I Find?
Colony 10
Colony 1
Colony 11
Colony 12
Colony 2
Colony 3
Colony 4
Colony 5
Colony 7
Colony 8
Colony 9
Colony 6
1 kb Ladder
4.1 kb
2.6 kb
1.5 kb
Recombinant Plasmid
Plasmids without Inserts
Expected Size of Upstream Region
Insert
One Recombinant Colony
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How Does This Gene Affect Seed Development?

• Knock-outs are excellent demonstration of genes
  role (inference from loss of function)
• T-DNA insert interrupts gene function
• Absence of homozygous T-DNA genotype could
  suggest knockout causes seed lethality
• But to genotype, must first ascertain expected
  band lengths resulting from PCR

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How Is the T-DNA Insert Designed?
PCR Product

FW
RV
5 UTR
3 UTR
T-DNA
LBb1
RV
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What Were My Genotyping Results?
Neg. Control
Pos. Control
Wild Type
1 kb Ladder
Plant 6
Plant 1
Plant 2
Plant 4
Plant 5
Plant 7
Plant 8
Plant 9
Plant 3
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What Are the Possible Sources of the Mystery
Bands?

• Primer matching part of T-DNA insert sequence and
  creating another product with Reverse primer
• Multiple T-DNA Insertion Sites
• Concatamers
• T-DNA insert possibly flipped and on other
  strand, so interacts with Forward primer instead,
  creating different-sized product

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How Is the T-DNA Insert Designed?
PCR Product

FW
LBb1
T-DNA
LBb1
RV
5 UTR
3 UTR
FW
RV
Sequencing results for 0.44 kb band were
limited, so cannot conclude which is the T-DNA
band with absolute certainty.
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Same Results in Second Batch of Genotyping
Pos. Control
Neg. Control
Plant 1
Plant 6
Plant 10
Plant 12
Plant 11
Plant 13
Plant 2
Plant 3
Plant 4
Plant 5
Plant 7
Plant 8
Plant 9
100 bp Ladder
1 kb Ladder
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What Do These Genotypes Suggest?

• Total screened 21 SALK Plants
• 19 Hemizygous
• 2 Wild Type
• The absence of homozygous T-DNA plants after this
  much genoytyping suggests there are none
• According to the probability calculation, there
  is a 0.002 chance of still finding a homozygous
  T-DNA after screening 21 plants
• Homozygous T-DNA Knockout Most Likely Causes Seed
  Lethality!

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But Do These Genotypes Cause Any Phenotypic
Variations?

• Seed lethality as one major phenotype (since the
  plant doesnt exist!)
• But are there other phenotypes that are directly
  observable in either the plants as a whole or
  their seeds?
• No noticeable differences between wild type and
  hemizygous plants

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What About the Seeds?
Microscopy shows us
Wild Type - 10x
Hemizygous - 10x
Hemizygous - 20x
Wild Type - 30x
No Difference!
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What Could Account for the Discrepancy Between
The Genotypic and Phenotypic Results?
Normal Embryos!

• T-DNA inserts could be distributed unevenly not
  a random sample
• would skew expected 121 ratio
• Gene could affect germination
• accounts for partial or complete lack of
  homozygous T-DNA seeds in hemizygous silique

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What Conclusions Can We Draw?

• Even though both genes exhibit strange pattern of
  seemingly seed lethal genotypes and normal
  phenotypes, their precise role of importance in
  seed development is yet to be determined
• Study of germination is essential step preceding
  definitive conclusions
• Although contradictory data can be confusing, it
  is important to keep pursuing them
• It is out of precisely there baffling puzzles
  that many scientific discoveries are born!

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Acknowledgements

• Anhthu Bui for his wisdom, experienced eyes,
  and humor
• Bekah Charney and Daisy Robinton for their
  support and answers to my many questions!
• Brandon, Tomo, Min, Chen, Kelli, and Jungim for
  all their help
• Ingrid Nelson for all the delicious dinners!
• Dr. Goldberg for making this all possible and
  inspiring me to step into a lab in the first
  place!