Arabidopsis Gene At1G49560

1
Arabidopsis Gene At1G49560

• Maria Garcia
• June 5, 2008

2
What does At1G49560 code for?

• MYB Family Transcription Factor Protein
• Protein size 333 amino acids
• MYB (Myeloblast) consists of a 203 family
  transcription factor proteins first identified in
  oncogenes.
• R1, R2, R3 repeats
• Subfamilies are classified by number of adjacent
  repeats (MYBR1, R2R3-type MYB, MYB3)
• Protein Function
• Regulatory role in developmental processes
• SANT Superfamily
• Found in regulatory transcriptional represser
  complexes

3
Whats Upstream and Downstream?
5 3 5
3 5
3

2894bp 2975bp
3 5
3 5
3 5
4
Gene Structure

• Size of gene 1377bp
• Chromosome Number 1
• Nucleotide Positions 18346222-18348031bp

18346222bp
1834831bp
5 3
Exon2 206bp
Exon3 268bp
Exon4 77bp
Exon1 175bp
Exon5 276bp
Intron Intron Intron
Intron 86bp 113bp 93bp
83bp
3 5
5
Where and When is my gene most active?
RT-PCR 1.5 agarose gel 05/20/08 544pm-650pm _at_
105v
Expected Size 333bp
100bp Ladder
100bp Ladder
Leaf RT
Silique RT
Silique - RT
Positive
Negative
Leaf - RT
6
Hypothesized T-DNA Insertion from Tair
WebsiteSALK_080209
Expected Sizes WT 591bp TDNA 472bp
5 3
T-DNA
3 5
LBb1-150bp
Fw-20bp
5 3
322bp
269bp
720bp
66bp
3 5
Rv-21bp
7
The Results
1 Agarose Gel 05/15/08 259-400pm _at_ 105volts
Expected Sizes WT- 591bp T-DNA- 472bp
700bp
591bp
470bp 350bp
100bp ladder
Negative
Positive
Plant 1
Plant 2
Plant 3
Plant 4
Plant 5
Plant 6
Plant 7
8
How many T-DNA inserts do we see?
Plant 4
Plant 5
Positive
Negative

500
gt400
100 bp ladder
Fw Rv
Fw LBb1
Rv LBb1
Fw Rv
Fw LBb1
100 bp ladder
Rv LBb1
Does the result show whether there is more than
one insert or a concatemer?
9
Hypothesized T-DNA Insert Based on PCR Results
LBb1-150bp
LBb1-150bp
T-DNA

• T-DNA

T-DNA
T-DNA
LBb1-150bp
LBb1-150bp
Fw
Fw
Rv
Rv
10
Wild Type vs. Knockout Seeds
Wild Type Seeds 25X
Knockout Seeds 25X
11
Wild Type vs. Knockout Embryos
Wild Type Embryos 64X Knockout
Embryos 64X
12
The Upstream RegionUpstream Region Size
2.8kbpENTR Vector Size 2.58kb Recombinant
Plasmid Size 5.38kb
1 agarose gel 04/29/08 450pm-552pm _at_150V
5.0kb 2.5kb
Colony 2
Colony 5a
Colony 5b
Colony 10
Colony 4
1KB Ladder
Negative
Positive
Gene 2
1kb Ladder
1 Agarose Gel 05/16/08 1hr run _at_ 105V
13
Sequencing Results
14
Arabidopsis GeneAt2G05900

• Presented By
• Maria Garcia
• and
• Kristin Gill

15
What Protein does it encode for?

• SET- Domain Containing Protein
• Protein Size 312 amino acids
• Set Domain are protein lysine methyl enzymes
• Methylation of histone lysine functions as a
  post-translational modification
• Serves as a marker for the recruitment of
  specific complexes that direct the organization
  of chromatin
• Important in the regulation of chromatin and gene
  expression
•  

16
What is the structure of the gene?

• Size of gene 939bp
• Chromosome Number 2
• Nucleotide Positions 2256967-2257905bp

3 5
5 3
3 5
At2G05900 Exon
At2G05910
At2G05890
891bp 384bp
5 3
3 5
5 3
17
Where and When is my gene most active?
RT-PCR 1.5 agarose gel 04/24/08 606pm-720pm _at_
105V
Expected Size217bp
Leaf - RT
Leaf RT
Silique RT
Silique - RT
100bp Ladder
Positive
Negative
18
Genotyping Results

• Plant Set 1

• Plant Set 2

1 agarose gel 04/15/08 1hr run _at_ 105V
1 agarose gel 04/17/08 1hr run _at_ 105V
Homozygous T-DNA Band 426bp
WT 770bp
T-DNA Band426bp
100bp ladder
100bp ladder
Plant WT
Negative
Plant 16
Plant 25
Plant 1
Plant 2
Plant 3
Plant 4
Plant 5
Positive
Plant 18
Plant 21
Plant 23
Plant WT
Plant 17
Negative
Plant 13
Plant 12
Positive
Plants 2,3,4,5 are WT Plant 1 is Hemizygous
T-DNA
Plants 25,12,21,18,17,23 are WT Plant 13 is
Hemizygous T-DNA Plant 16 is Homozygous T-DNA
19
Acknowledgements!

• Anhthu Bui Bekah Charney Min Chen
• Daisy Robinton Tomo Kawashima Brandon Le
• Dr. Bob Goldberg