Transcript The C2C2-Zinc Finger GATA

Inside the world of two Arabidopsis Genes
AT5G47140 & AT4G17570
Presented by Thi Nguyen
June 8, 2006
The C2C2-Zinc Finger GATAlike transcription factor family
•The family can be divided into several types of zinc
finger proteins, such as C2H2, C2HC, C2C2,
C2HCC2C2, C2C2C2C2 etc, based on numbers and
positions of Cystine and Histidine residues.
•Zinc finger domain regulates gene expression in the
eukaryotic organisms mainly by specifically interacting
with target DNA sequence
•The transcription factor has highly conserved domains
that enables those proteins to bind to this GATA-like
DNA sequence
First Gene:
AT5G47140
First Gene: General Gene
Information
• Located on the chromosome 5
• Size in base pairs: 2802 bp
• Encodes for Zinc Finger GATA protein
– Size of protein: 471 amino acids
• Forward orientation in respect to genome
First Gene: Genotype of
Insertion Lines
• From Salk Line
– 16 plants genotyped with 1 wildtype
– All homozygous wildtype
– Results do not agree with SALK results
• From Sail Line
– Sail_387_F11
– 11 plants genotyped with 1 wildtype
– 3 plants homozygous mutant, 4 plants heterozygous,
4 plants homozygous wildtype
Genotyping of insertion lines from Salk Line.
Genotyping of Insertion Lines from Sail Line.
First Gene: Anatomical
Features
TDNA
from Sail
expected
insertion
site
5’
Forward
Primer
UTR
Intron
Exon
Reverse
Primer
UTR
3’
Second Gene:
AT4G17570
Second Gene: General Gene
Information
• Located on the chromosome 4
• Size in base pairs: 3537bp
• Encodes for Zinc Finger GATA protein
– Size of protein: 511 amino acids
• Reverse orientation in respect to genome
Second Gene: Genotype of
Insertion Lines
• From Salk Line
– 11 plants genotyped with 1 wildtype
– 7 plants homozygous mutant, 4 plants
homozygous wildtype
– TDNA insert is in reverse orientation with
respect to the gene
Genotyping of Insertion Line from Salk Line.
Second Gene: Anatomical
Features
LBb1 primer
5’
UTR
Forward
Primer
Intron
Exon
TDNA
based on
sequencing
UTR
Reverse
Primer
TDNA
from Salk
expected
insertion
site
3’
First Gene: Promoter Cloning
• I expected …
- vector to be 3.5kb
- fragment to be 3kb
- no EcoR1 site within my
upstream region
•Base on sequencing reactions, I
found that the 5’ end of my
upstream region is at the T7 side
of the vector
Plasmid Digest
Second Gene: Promoter Cloning
•I expected …
- vector to be 3.5kb
- insert to be cut into two
fragments due to one
EcoR1site
- top fragment to be
1.889kb
- bottom fragment to be
.817kb
•Future experiments: fuse insert to
GFP and transform plants so that you
can see where it is expressed
Plasmid Digest
First Gene: Activity of Gene
RT-PCR: Gene of interest is transcribed in silique and leaf
Second Gene: Activity of Gene
Gene Chip Data
RT-PCR: Second gene of interest is
transcribed in inflorescent and silique
Phenotype of Insertion Lines
Mutant
Wildtype
Special Thanks to …
•
•
•
•
•
•
•
•
The class of HC70al
The TAs: Mike, Ria, and Jonathan
Anhthu
Brandon
Tomo
Jessica
Xingjun
You guys are CRAZY DELICIOUS.
Dr. Goldberg