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Genes That Direct Transcription Co-activator Proteins : Do they disrupt/alter seed
development?Gene 1: At5g09250Gene 2: At5g09240
Combiz Richard Abdolrahimi06-09-2005
HC 70AL Gene Discovery LabDr. Bob Goldberg
HHMI Institute/UCLA
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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
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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
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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
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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
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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.
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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 HomozygousWhy are
there double bands?
lbb1 lbb1
Exon 4, AT5g09240
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Wild-type v. Mutant Plants: Did the T-DNA insertion disrupt seed/other development?
No Phenotypic Differences Among Leaves/Plants/Seeds
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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
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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.
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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)