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

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

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

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

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

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.

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

Wild-type v. Mutant Plants: Did the T-DNA insertion disrupt seed/other development?

No Phenotypic Differences Among Leaves/Plants/Seeds

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

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.

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)