Genes That Direct Transcription Co-activator Proteins : Do they disrupt/alter seed development?

1. Genes That Direct Transcription Co-activator Proteins : Do they disrupt/alter seed development? Gene 1: At5g09250 Gene 2: At5g09240 Combiz Richard Abdolrahimi 06-09-2005 HC 70AL Gene Discovery Lab Dr. Bob Goldberg HHMI Institute/UCLA

2. Arabidopsis thaliana: What are some characteristics of Genes that Direct Transcription Co-activator Proteins (At5g09250, At5g09240)? • Gene 1: At5g09250 • Chromosome 5, opposite orientation & Reverse (for T-DNA and Gene Primer) • 1344 bp • Protein: 107 aa • Exon(s): 5 • Intron(s): 4 • Gene 2: At5g09240 • Chromosome 5, opposite orientation & Reverse (for T-DNA and Gene primer) • 1197 bp • Protein: 110 aa • Exon(s): 4 • Intron(s): 3

3. Where is the location of the genes and the T-DNA Insert(s)? • AT5G09250-Transcriptional Co-activator • Salk_068658 • WT PCR Size: ~1.6kbp • T-DNA Size: FW+LB: .6kbp • Direction of T-DNA Insert (5’ – 3’) (RV) LB 561 5’ UTR --------3’ UTR Exon 1 Intron Exon 2 Intron Exon 3 Intron Exon 4 Intron Exon 5 1 96 199 342 495 600 972-994 1096 1344

4. Gene 2: Where is the location of the gene and size/orientation of T-DNA Insert? • AT5G09240 • Salk_042140 • WT PCR Size: .8kbp • T-DNA Size: FW+LB=~.6kbp (or 577bp) • Orientation of T-DNA Insert: (5’-3’) (FW) 981 5’ UTR --------3’ UTR Exon 1 Intron Exon 2 Intron Exon 3 Intron Exon 4 2 136 250 361 720-742 827 1197

5. Where is the gene active in the Arabidopsis thaliana Plant? Do your results agree? RT-PCR Gel for: AT5G09250 Transcription Co-activator Protein of At5g09250 should be highly active in post-mature green stage seed; Semi-high levels should be expected in ovule, 24-hr seed, cotyledon-stage seed, and mature-green-stage seed

6. Where is the gene active in the Arabidopsis thaliana Plant? Do your results agree? RT-PCR Gel for: At5g09240 Transcription Co-activator Protein, At5g09240, should be highly active in ovule and 24-hr seed; Semi-high levels should be expected in all the rest except for post-mature stages in the development of the plant.

7. What does genotyping tell you about the tagged plants? At5g09250 Plants 7-13, WT 14 At5g09240 Plants 1-12, WT Why are there double bands? W/T Homozygous W/T Homozygous lbb1 lbb1 Homozygous Mutants Homozygous Mutants Exon 4, AT5g09240

8. Wild-type v. Mutant Plants: Did the T-DNA insertion disrupt seed/other development? No Phenotypic Differences Among Leaves/Plants/Seeds

9. What is the importance of promoter cloning? • No restriction sites within my promoter region for both genes • Size of Promoter Region, At5g09250 = 428 bp • Size of Promoter Region, At5g09240 = 643 bp • The following two diagrams which show fractionating of DNA on a gel indicate that the top picture (At5g09250) had incomplete digestion by EcoRI; while for the bottom picture (At5g09240), digestion correctly shows the plasmid vector to be at 3.5kbp and my promoter region at 643 bp TOPO Vector 1 2 3 4 Promoter Region

10. Summary & What’s Next? • Apparent concatamer in Gene 2 T-DNA Insertion site • No apparent phenotypical differences among the homozygous mutant plants and normal/wild-type plants • Knocking out Genes At5g09250, At5g09240 do not cause an embryo lethal mutation or any other visible abnormalities…Need further research • What to do next? • Genotype more plants to verify existence of concatamer in plants of knocked-out gene At5g09240, Salk_042140 • Cross plants with knockouts in other transcription co-activator protein genes and see if seeds develop with those multiple genes knocked out.

11. A Special Thanks To… • Dr. Goldberg • Dr. Anhthu Bui • Jessica Luke • Brittan Scales • Mike Gavino • Tomo Kawashima • Brandon Le • Dr. Xingjun Wang • HHMI • Fellow Classmates of this class: (Eric, Ria, Jonathan, Rena, Yuya, Yosuke, Mike, Garen, Tim, Emily, and Joanna)