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Arabidopsis Gene At1G49560 Maria Garcia June 5, 2008
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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
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What’s Upstream and Downstream?
At1G49550 At1G49560 (1377bp) At1G49570 5’ ’ ’ ’ ’ ’ 2894bp bp 3’ 5’ 3’ ’ 3’ ’
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Gene Structure Size of gene: 1377bp Chromosome Number: 1 Nucleotide Positions: bp bp bp 5’ ’ Exon1 175bp Exon2 206bp Exon3 268bp Exon4 77bp Exon5 276bp 3’ ’ Intron Intron Intron Intron 86bp bp bp bp
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Where and When is my gene most active?
RT-PCR 1.5% agarose gel 05/20/08 105v Expected Size ~333bp 100bp Ladder 100bp Ladder Leaf + RT Silique + RT Silique - RT Positive Negative Leaf - RT
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Hypothesized T-DNA Insertion from Tair Website SALK_080209
Expected Sizes: WT= 591bp TDNA= 472bp 5’ ’ T-DNA 3’ ’ LBb1-150bp Fw-20bp 5’ ’ 322bp bp 720bp 66bp 3’ ’ Rv-21bp
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The Results Expected Sizes: WT- 591bp T-DNA- 472bp 100bp ladder
1% Agarose Gel 05/15/08 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
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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?
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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
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Wild Type vs. Knockout Seeds
Wild Type Seeds 25X Knockout Seeds 25X
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Wild Type vs. Knockout Embryos
Wild Type Embryos 64X Knockout Embryos 64X
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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 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/ hr 105V
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Sequencing Results
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Presented By: Maria Garcia and Kristin Gill
Arabidopsis Gene At2G05900 Presented By: Maria Garcia and Kristin Gill
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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
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What is the structure of the gene?
Size of gene: 939bp Chromosome Number: 2 Nucleotide Positions: bp 3’ ’ ’ ’ ’ ’ At2G05890 At2G05900 Exon At2G05910 891bp bp 5’ ’ ’ ’ ’ ’
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Where and When is my gene most active?
RT-PCR 1.5% agarose gel 04/24/08 105V Expected Size~217bp Leaf - RT Leaf + RT Silique +RT Silique - RT 100bp Ladder Positive Negative
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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 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
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Acknowledgements! Anhthu Bui Bekah Charney Min Chen Daisy Robinton Tomo Kawashima Brandon Le Dr. Bob Goldberg
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