Arabidopsis Thaliana Gene AT1G05290 and Gene AT1G34355

1
Arabidopsis ThalianaGene AT1G05290andGene
AT1G34355

• By, Krista Templeton

2
What is a Transcription Factor?

• Transcription factors are proteins involved in
  the regulation of gene expression that bind to
  the promoter elements upstream of genes and
  either facilitate or inhibit transcription.
• Through this process they control and regulate
  gene expression

3
AT1G05390 The B-Box Type Zinc Finger
4
What Protein Does AT1G05290 Encode For?

• Arabidopsis Zinc-Finger Proteins Function as
  Transcription Repressors under Drought, Cold, and
  High-Salinity Stress Conditions

5
What is the Structure of AT1G05290?
Exons UTR Introns

• 5 Exons, 4 Introns
• Size 1,394bp without UTR 1,411 with UTR
• Chromosome 1
• Reverse Orientation 3-5
• Nucleotide Position 1,539,151 1,540,561
• Gene encodes 369 amino Acids

TDNA Insert (1,325bp)
6
At1g05290 Genotyping, Homozygousity?
Hemizygous Pairing
Wild Type

• 38 genotyped plants
• 29 Hemizygous
• 9 Wild Type
• Possible seed lethality
• 1.7x10-5 possibility that a homozygous pairing
  still exists

7
Can Nomarski Tell Us Anything?
Younger Seed
Older Seed
Heart Stage
Cotyledons present
Normal Embryo Development
8
No Homozygous Pairings and Normal Embryo
Development?

• What could be happening
• Affects shoot apical meristem?
• Affects germination?
• Affects root developmeny?

9
What Genes Surround AT1G05290?
At1g05290
At1g05291
At1g05280
5
3

• At1g05291 Unknown protein
• At1g05280 Fringe related protein
• Upstream Region 1,626bp
• Downstream Region 785bp

10
Where is At1g05290 Active?
Negative Control Positive Control Seed RT Seed
RT Leaf RT Leaf RT 100bp Ladder

• According to the gel, At1g05290 does not appear
  to be active in the seed or the leaf

RNA Accumulation RT-PCR 105v 4/24/2008
11
AT1G34355Forkhead-Associated/SMAD Domain
12
What protein does At1g34355 encode?

• It encodes a forkhead-associated (FHA) domain
  containing protein
• Forkhead-associated (FHA) domains are the
  hallmark of the family of Rad53-like checkpoint
  protein kinases.

13
What is the function of the Rad53-like checkpoint
protein Kinases?

• Rad53-like Kinases include some tumor suppressor
  proteins
• They play crucial rolls
• in cell cycle arrest
• the activation of repair processes following DNA
  damage
• replication blocks

14
What is the Structure of At1g34355?

• Exons
• Introns
• UTR

• 7 Exons, 6 Introns
• 4,595 Base Pairs
• Chromosome 1
• Nucleotide Position 34,355bp
  38,950bp
• Forward Orientation 5-3

Actual TDNA Insertion Site According to
genotyping and Blastn
Expected TDNA Insertion Site as predicted by SALK
(2381bp)
(2,352bp)
15
What are the surrounding genes?
At1g34350
At1g34355
At1g34360

• Upstream region 401bp
• Downstream region 971bp
• At1g34350 unknown function
• At1g34360 translation initiation factor

16
At1g34355 Genotyping Gel
Hemizygous pairing
Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Plant
6 Plant 21WT Positive Control Negative Control 1
KB Ladder
1 Agarose gel 5/15/08 259-4pm 105V
Homozygous pairing
Expected Wild Type band 1.4kb Expected TDNA
approx. 700bp LBb1 FWD primers
(623pb150bp)
17
What Can we Conclude From the Genotyping Gel?

• 7 plants were genotyped
• Homozygous plants 1,2,3,5
• Hemizygous plants 4,6
• Wild Type 1
• Negative control is clear, so there is no
  contamination
• Unknown band, slightly smaller than TDNA
• Not Seed Lethal
• Possible phenotypic changes

18
Where is At1g34355 Active?
Gene Active in the Seed
Tubulin Primers and Positive Control
Less active in leaf
RNA Accumulation RT-PCR 5/20/08 544-650pm
Negative Control Positive Control Seed RT Seed
RT Leaf RT Lear RT 1KB Ladder
19
Does the RT-PCR Results Match the Genechip Data?

• According to the Micro-array data, gene AT1G34355
  is active in various stages of seed development
  and only slightly active in the seedling.

20
Which Colonies Contain Recombinant Plasmids?
All six colonies have recombinant plasmids
The upstream region was concluded to be a perfect
match once the sequencing results were blasted
Proof of Upstream Region Plasmid size 2.6KB DNA
Insert 0.56KB 1 Agarose gel 5/8/08 105v 413pm
Colony 6 Colony 5 Colony 4 Colony 3 Colony
2 Colony 1 1 KB Ladder
21
What Do Healthy Seeds and Embryos Look Like?
Plant 1 Siliques and Seeds
Healthy Embryo
22
Dead Seeds
Unhealthy Seed Development in Plant 3
Underdeveloped seeds
23
Conclusion

• For AT1G05290 we did not find any homozygous
  pairings but embryo development is normal
• For AT1G34355 homozygous pairings were present.
  There was some trouble with seed development,
  possibly because a similar gene was also inactive
  in some plants.
• No Phenotypic changes in either genes.

24
THE END!
25
Acknowledgments

• THANKS YOU
• DR. G!
• Anthu!
• Daisy!
• Bekah!
• Brandon!
• Ingrid!