1 4174

FWD

What is AT5G03500? --Background and Structure--

Chromosome 5 of Arabidopsis--4174 bps

--12 exons

RV

Neighboring genes: AT5G03510--2600 bps upstream (Zinc finger protein)

AT5G03490--450 bps downstream (UDP glucoronosyl transferase family)

2 ESTS, 3CDNAS

EXONS

INTRONS

By Cecilia Hernandez Spring 2004

What is the function of AT5G03500?

How does AT5G03500 compare to SRB EST database?

--Transcriptional Co-activator-related, with an F-Box domain

--Works with a protein complex to regulate transcription

--PCSC14079 --Embryo region: Suspensor --Protein ID: Transcriptional co-activator-like protein; protein id: AT5G03505

In what SRB and Arabidopsis organs is AT5G03500 active?

+R

T L

eaf

- +R

T S

tem

- +R

T F

low

er

- +R

T O

vule

- +R

T 1

4DA

P E

.

- +R

T 3

0 D

AP

Axi

s

- +R

T C

H s

tem

+R

T C

H L

eaf

-- -

AT5G03500 not on Gene Chip =( ----> No results indicating Expression pattern of AT5G03500 in Arabidopsis

Transcriptional Co-activator similar to my gene is active in Leaf, Stem, Ovule, 14DAP Embryo, 30DAP Axis

MADISON SCREENING ROUND 1: IS THERE A SUPER POOLCONTAINING A KNOCKOUT IN AT5G03500?

RESULTS:

Lanes F29 and R16 display bands that may indicate a mutagenized line within their respective superpools.

Southern Blot (not shown) unambiguously identifies Superpools F29 and R16 as pools of interest, containing a knockout line

Sequencing results (not displayed) reveal T-DNA insertion sites in first exon and sixth intron for F29 and R16 superpools, respectively.

FW+JL202PRIMERS USED

Superpool 29Reveals a band ~1400 bpsRV+JL202

PRIMERSUSED

Superpool 16 reveals aBand ~400 bps

Control(gene 03500)

Control(AT5G03500)

29F58

16R

MADISON SCREENING RESULTS ROUND 2: WHICH DNA POOL CONTAINS A MUTANT LINE?

PCR product of DNA pool 276reveals an insert;Band~1400 bps

DNA POOL 276!

DNA Pool 5

58

…Seed Pools must now Be screened..

1 4174

FWD

RV088220910 083099

2097

0226662139

0478992222

0443263142

1417283397*

Salk Insertions: Where are they?

Salk insertions

Exon

intron

* 08820=Line chosen to survey-insert occurs after nucleotide 910 in fourth intron-

What genotype can we predict for plants 2-8, from SALK LINE 088220, using dual PCR and gel analysis with wild type and T-DNA primers?

Genotype Results:Plant 2 : +/+Plant 3 : T/TPlant 4 : T/TPlant 5 : T/TPlant 6 : T/TPlant 7 : +/TPlant 8 : +/+

**BUT, one may infer that these results are NOT unambiguousdue to consistently inconsistent dataand the skepticism necessarily owed to conclusions drawn from negative results

06/0

4/20

04

FW

+T

-DN

A

Pla

nt 2

Pla

nt

3P

lan

t 4

Pla

nt 5

Pla

nt

6P

lan

t 7

Pla

nt

8P

lan

t 9

Pla

nt

9

Pla

nt

8P

lan

t 7

Pla

nt

6

Pla

nt 5

Pla

nt

4P

lan

t 3

Pla

nt 2

Pla

nt 2

Pla

nt 2

Pla

nt

3

Pla

nt

3

Pla

nt

4

Pla

nt

4P

lant

5

Pla

nt 5

Pla

nt

6

Pla

nt

6

Pla

nt

7

Pla

nt

7

Pla

nt

8

Pla

nt

8P

lan

t 9

Pla

nt

9

TDNA amplification

TDNA amplification

06/0

9/20

04

FW

+T

-DN

A

06/0

7/04

FW

+R

V06

/09/

04F

W+

RV

Wild type amplification

Wild type amplification

---> my gene is not Embryo lethal =(

Now what?--Screen Seed Pool #276

--Observe phenotype of relevant plants; Genotype progeny of SALK plants 2-8