What is AT5G03500? --Background and Structure-- RV FWD 1 4174 Chromosome 5 of Arabidopsis EXONS --4174 bps INTRONS --12 exons Neighboring genes: AT5G03510--2600 bps upstream (Zinc finger protein) AT5G03490--450 bps downstream (UDP glucoronosyl transferase family) 2 ESTS, 3CDNAS By Cecilia Hernandez Spring 2004

What is the function of AT5G03500? --Transcriptional Co-activator-related, with an F-Box domain --Works with a protein complex to regulate transcription How does AT5G03500 compare to SRB EST database? --PCSC14079 --Embryo region: Suspensor --Protein ID: Transcriptional co-activator-like protein; protein id: AT5G03505

In what SRB and Arabidopsis organs is AT5G03500 active? +RT 14DAP E. +RT Flower +RT CH stem +RT CH Leaf +RT Leaf +RT Ovule +RT 30 DAP Axis +RT Stem - - - - - - - - - Transcriptional Co-activator similar to my gene is active in Leaf, Stem, Ovule, 14DAP Embryo, 30DAP Axis AT5G03500 not on Gene Chip =( ----> No results indicating Expression pattern of AT5G03500 in Arabidopsis

MADISON SCREENING ROUND 1: IS THERE A SUPER POOL CONTAINING A KNOCKOUT IN AT5G03500? FW+JL202 PRIMERS USED RESULTS: Lanes F29 and R16 display bands that may indicate a mutagenized line within their respective superpools. Southern Blot (not shown) unambiguously identifies Superpools F29 and R16 as pools of interest, containing a knockout line Sequencing results (not displayed) reveal T-DNA insertion sites in first exon and sixth intron for F29 and R16 superpools, respectively. Control (gene 03500) Superpool 29 Reveals a band ~1400 bps RV+JL202 PRIMERS USED Control (AT5G03500) Superpool 16 reveals a Band ~400 bps 16R 29F 58

MADISON SCREENING RESULTS ROUND 2: WHICH DNA POOL CONTAINS A MUTANT LINE? DNA POOL 276! …Seed Pools must now Be screened.. PCR product of DNA pool 276 reveals an insert; Band~1400 bps DNA Pool 5 58

* Salk Insertions: Where are they? * 08820=Line chosen to survey 1 047899 2222 FWD * 088220 RV 141728 910 3397 083099 2097 1 4174 3142 044326 Salk insertions 2139 Exon 022666 intron * 08820=Line chosen to survey -insert occurs after nucleotide 910 in fourth intron-

What genotype can we predict for plants 2-8, from SALK LINE 088220, using dual PCR and gel analysis with wild type and T-DNA primers? Plant 8 Plant 2 Plant 3 Plant 2 Plant 4 Plant 5 Plant 6 Plant 7 Plant 8 Plant 9 Plant 3 Plant 4 Plant 5 Plant 6 Plant 7 Plant 9 Genotype Results: Plant 2 : +/+ Plant 3 : T/T Plant 4 : T/T Plant 5 : T/T Plant 6 : T/T Plant 7 : +/T Plant 8 : +/+ 06/07/04 FW+RV 06/04/2004 FW+T-DNA Wild type amplification TDNA amplification ---> my gene is not Embryo lethal =( Plant 9 Plant 8 Plant 7 Plant 6 Plant 5 Plant 4 Plant 3 Plant 2 Plant 2 Plant 3 Plant 4 Plant 5 Plant 6 Plant 7 Plant 8 Plant 9 **BUT, one may infer that these results are NOT unambiguous due to consistently inconsistent data and the skepticism necessarily owed to conclusions drawn from negative results 06/09/04 FW+RV 06/09/2004 FW+T-DNA Wild type amplification TDNA amplification

Now what? --Screen Seed Pool #276 --Observe phenotype of relevant plants; Genotype progeny of SALK plants 2-8