Arabidopsis Gene At1G49560 Maria Garcia June 5, 2008

What does At1G49560 code for? MYB Family Transcription Factor Protein Protein size: 333 amino acids MYB (Myeloblast) consists of a 203 family transcription factor proteins first identified in oncogenes. R1, R2, R3 repeats Subfamilies are classified by number of adjacent repeats (MYBR1, R2R3-type MYB, MYB3) Protein Function: Regulatory role in developmental processes SANT Superfamily Found in regulatory transcriptional represser complexes Regulation in cell shape, secondary metabolisms, and disease resistance, and in response to growth regulators and stresses

What’s Upstream and Downstream? At1G49550 At1G49560 (1377bp) At1G49570 5’ 3’ 5’ 3’ 5’ 3’ 2894bp 2975bp 3’ 5’ 3’ 5’ 3’ 5’

Gene Structure Size of gene: 1377bp Chromosome Number: 1 Nucleotide Positions: 18346222-18348031bp 18346222bp 1834831bp 5’ 3’ Exon1 175bp Exon2 206bp Exon3 268bp Exon4 77bp Exon5 276bp 3’ 5’ Intron Intron Intron Intron 86bp 113bp 93bp 83bp

Where and When is my gene most active? RT-PCR 1.5% agarose gel 05/20/08 5:44pm-6:50pm @ 105v Expected Size ~333bp 100bp Ladder 100bp Ladder Leaf + RT Silique + RT Silique - RT Positive Negative Leaf - RT

Hypothesized T-DNA Insertion from Tair Website SALK_080209 Expected Sizes: WT= 591bp TDNA= 472bp 5’ 3’ T-DNA 3’ 5’ LBb1-150bp Fw-20bp 5’ 3’ 322bp 269bp 720bp 66bp 3’ 5’ Rv-21bp

The Results Expected Sizes: WT- 591bp T-DNA- 472bp 100bp ladder 1% Agarose Gel 05/15/08 2:59-4:00pm @ 105volts Expected Sizes: WT- 591bp T-DNA- 472bp ~700bp ~591bp ~470bp ~350bp 100bp ladder Negative Positive Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Plant 6 Plant 7

How many T-DNA inserts do we see? Plant 4 Plant 5 Positive Negative ~500 >400 100 bp ladder 100 bp ladder Fw + Rv Fw + LBb1 Rv + LBb1 Fw + Rv Fw + LBb1 Rv + LBb1 Does the result show whether there is more than one insert or a concatemer?

Hypothesized T-DNA Insert Based on PCR Results LBb1-150bp LBb1-150bp T-DNA T-DNA T-DNA T-DNA LBb1-150bp LBb1-150bp Fw Fw Rv Rv

Wild Type vs. Knockout Seeds Wild Type Seeds 25X Knockout Seeds 25X

Wild Type vs. Knockout Embryos Wild Type Embryos 64X Knockout Embryos 64X

The Upstream Region Upstream Region Size: 2. 8kb pENTR Vector Size: 2 The Upstream Region Upstream Region Size: 2.8kb pENTR Vector Size: 2.58kb Recombinant Plasmid Size: 5.38kb 1% agarose gel 04/29/08 4:50pm-5:52pm @150V 5.0kb 2.5kb Colony 2 Colony 5a Colony 5b Colony 10 Colony 4 1KB Ladder Negative Positive 1kb Ladder Gene 2 1% Agarose Gel 05/16/08 1hr run @ 105V

Sequencing Results

Presented By: Maria Garcia and Kristin Gill Arabidopsis Gene At2G05900 Presented By: Maria Garcia and Kristin Gill

What Protein does it encode for? SET- Domain Containing Protein Protein Size: 312 amino acids Set Domain are protein lysine methyl enzymes Methylation of histone lysine functions as a post-translational modification Serves as a marker for the recruitment of specific complexes that direct the organization of chromatin Important in the regulation of chromatin and gene expression  

What is the structure of the gene? Size of gene: 939bp Chromosome Number: 2 Nucleotide Positions: 2256967-2257905bp 3’ 5’ 5’ 3’ 3’ 5’ At2G05890 At2G05900 Exon At2G05910 891bp 384bp 5’ 3’ 3’ 5’ 5’ 3’

Where and When is my gene most active? RT-PCR 1.5% agarose gel 04/24/08 6:06pm-7:20pm @ 105V Expected Size~217bp Leaf - RT Leaf + RT Silique +RT Silique - RT 100bp Ladder Positive Negative

Genotyping Results Plant Set #1 Plant Set #2 100bp ladder 100bp ladder 1% agarose gel 04/15/08 1hr run @ 105V 1% agarose gel 04/17/08 1hr run @ 105V Homozygous T-DNA Band ~426bp T-DNA Band~426bp WT ~770bp 100bp ladder 100bp ladder Plant WT Negative Plant 16 Plant 25 Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Positive Plant 18 Plant 21 Plant 23 Plant WT Plant 17 Negative Plant 13 Plant 12 Positive Plants #2,3,4,5 are WT Plant #1 is Hemizygous T-DNA Plants #25,12,21,18,17,23 are WT Plant #13 is Hemizygous T-DNA Plant #16 is Homozygous T-DNA

Acknowledgements! Anhthu Bui Bekah Charney Min Chen Daisy Robinton Tomo Kawashima Brandon Le Dr. Bob Goldberg