Arabidopsis Gene At 1 G 49560 Maria Garcia

Arabidopsis Gene At 1 G 49560 Maria Garcia June 5, 2008

What does At 1 G 49560 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. – R 1, R 2, R 3 repeats – Subfamilies are classified by number of adjacent repeats (MYBR 1, R 2 R 3 -type MYB, MYB 3) – Protein Function: • Regulatory role in developmental processes • SANT Superfamily – Found in regulatory transcriptional represser complexes

What’s Upstream and Downstream? 5’ 3’ 5’ At 1 G 49550 2894 bp 3’ 5’ 3’ 5’ At 1 G 49560 (1377 bp) 3’ 5’ 3’ At 1 G 49570 2975 bp 3’ 5’

Gene Structure 5’ 3’ Exon 1 Exon 2 175 bp 3’ Exon 3 206 bp Exon 4 268 bp Exon 5 77 bp 276 bp Intron 86 bp 113 bp 93 bp 83 bp 5’ 1834831 bp 18346222 bp Size of gene: 1377 bp Chromosome Number: 1 Nucleotide Positions: 18346222 -18348031 bp

Where and When is my gene most active? RT-PCR 1. 5% agarose gel 05/20/08 5: 44 pm-6: 50 pm @ 105 v 100 bp Ladder Negative Positive Silique - RT Silique + RT Leaf - RT Leaf + RT 100 bp Ladder Expected Size ~333 bp

Hypothesized T-DNA Insertion from Tair Website SALK_080209 Expected Sizes: WT= 591 bp TDNA= 472 bp 5’ 3’ T-DNA 3’ 5’ LBb 1 -150 bp Fw-20 bp 3’ 5’ 720 bp 322 bp 269 bp 66 bp 3’ 5’ Rv-21 bp

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

How many T-DNA inserts do we see? Plant 4 Plant 5 Positive Negative 100 bp ladder Rv + LBb 1 Fw + Rv Fw + LBb 1 Rv + LBb 1 Fw + Rv 100 bp ladder ~500 >400 Does the result show whethere is more than one insert or a concatemer?

Hypothesized T-DNA Insert Based on PCR Results LBb 1 -150 bp T-DNA LBb 1 -150 bp Fw Fw Rv Rv

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

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

The Upstream Region Size: 2. 8 kb p. ENTR Vector Size: 2. 58 kb Recombinant Plasmid Size: 5. 38 kb 1% agarose gel 04/29/08 4: 50 pm-5: 52 pm @150 V Colony 10 Colony Gene 2 5 b Positive Negative Colony 5 a 1 KB Ladder Colony 4 Colony 2 1 kb Ladder 5. 0 kb 1% Agarose Gel 05/16/08 1 hr run @ 105 V 2. 5 kb

Sequencing Results

Arabidopsis Gene At 2 G 05900 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: 939 bp Chromosome Number: 2 Nucleotide Positions: 2256967 -2257905 bp 3’ 5’ 5’ 891 bp 3’ 3’ At 2 G 05900 At 2 G 05890 5’ 3’ 3’ 384 bp Exon 5’ 5’ 5’ At 2 G 05910 3’

Where and When is my gene most active? RT-PCR 1. 5% agarose gel 04/24/08 6: 06 pm-7: 20 pm @ 105 V Leaf - RT Silique +RT Silique - RT Positive Negative Leaf + RT 100 bp Ladder Expected Size~217 bp

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