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) 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 LB 561

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) 5’ UTR --------3’ UTR Exon 1 Intron Exon 2 Intron Exon 3 Intron Exon 4 2 136 250 361 720-742 827 1197 981

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 W/T Homozygous Homozygous Mutants At5g09240 Plants 1-12, WT Homozygous Mutants W/T Homozygous Why are there double bands? lbb1 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 1 2 3 4 Promoter Region TOPO Vector

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)