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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