Inside the world of two Arabidopsis Genes AT5G47140 & AT4G17570

1. Inside the world of two Arabidopsis Genes AT5G47140 & AT4G17570 Presented by Thi Nguyen June 8, 2006

2. The C2C2-Zinc Finger GATA-like 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

3. First Gene: AT5G47140

4. 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

5. 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

6. Genotyping of insertion lines from Salk Line.

7. Genotyping of Insertion Lines from Sail Line.

8. TDNA from Sail expected insertion site 5’ Forward Primer Intron Exon UTR UTR 3’ Reverse Primer First Gene: Anatomical Features

9. Second Gene: AT4G17570

10. 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

11. 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

12. Genotyping of Insertion Line from Salk Line.

13. Second Gene: Anatomical Features LBb1 primer TDNA based on sequencing 5’ Forward Primer Intron UTR Exon UTR Reverse Primer TDNA from Salk expected insertion site 3’

14. 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

15. 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

16. First Gene: Activity of Gene RT-PCR:Gene of interest is transcribed in silique and leaf

17. Second Gene: Activity of Gene Gene Chip Data RT-PCR: Second gene of interest is transcribed in inflorescent and silique

18. Phenotype of Insertion Lines Mutant Wildtype

19. Special Thanks to … • The class of HC70al • The TAs: Mike, Ria, and Jonathan • Anhthu • Brandon • Tomo • Jessica • Xingjun • Dr. Goldberg You guys are CRAZYDELICIOUS.