1. What is AT5G03500? --Background and Structure--RV
FWD 1
4174
Chromosome 5 of Arabidopsis
EXONS
bps
INTRONS
--12 exons
Neighboring genes: AT5G bps upstream
(Zinc finger protein)
AT5G bps downstream
(UDP glucoronosyl transferase family)
2 ESTS, 3CDNAS
By Cecilia Hernandez Spring 2004

2. What is the function of AT5G03500?
--Transcriptional Co-activator-related, with an F-Box domain
--Works with a protein complex to regulate transcription
How does AT5G03500 compare to SRB EST
database?
--PCSC14079
--Embryo region: Suspensor
--Protein ID: Transcriptional co-activator-like protein; protein
id: AT5G03505

3. In what SRB and Arabidopsis organs is AT5G03500 active?
+RT 14DAP E.
+RT Flower
+RT CH stem
+RT CH Leaf
+RT Leaf
+RT Ovule
+RT 30 DAP Axis
+RT Stem - - - - - - - - -
Transcriptional Co-activator similar to my
gene is active in Leaf, Stem, Ovule, 14DAP
Embryo, 30DAP Axis
AT5G03500 not on Gene Chip =( ----> No results indicating
Expression pattern of AT5G03500 in Arabidopsis

4. MADISON SCREENING ROUND 1: IS THERE A SUPER POOL
CONTAINING A KNOCKOUT IN AT5G03500?
FW+JL202
PRIMERS
USED
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.
Control
(gene 03500)
Superpool 29
Reveals a band
~1400 bps
RV+JL202
PRIMERS
USED
Control
(AT5G03500)
Superpool
16 reveals a
Band ~400 bps
16R
29F 58

5. MADISON SCREENING RESULTS ROUND 2:
WHICH DNA POOL CONTAINS A MUTANT LINE?
DNA POOL 276!
…Seed Pools must now
Be screened..
PCR product
of DNA pool 276
reveals an insert;
Band~1400 bps
DNA Pool 5 58

6. * Salk Insertions: Where are they? * 08820=Line chosen to survey 1
047899
2222
FWD *
088220 RV
141728
910
3397
083099
2097 1
4174
3142
044326
Salk insertions
2139
Exon
022666
intron
* 08820=Line chosen to survey
-insert occurs after nucleotide 910 in fourth intron-

7. 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?
Plant 8
Plant 2
Plant 3
Plant 2
Plant 4
Plant 5
Plant 6
Plant 7
Plant 8
Plant 9
Plant 3
Plant 4
Plant 5
Plant 6
Plant 7
Plant 9
Genotype Results:
Plant 2 : +/+
Plant 3 : T/T
Plant 4 : T/T
Plant 5 : T/T
Plant 6 : T/T
Plant 7 : +/T
Plant 8 : +/+
06/07/04
FW+RV
06/04/2004
FW+T-DNA
Wild type amplification
TDNA amplification
---> my gene is not
Embryo lethal =(
Plant 9
Plant 8
Plant 7
Plant 6
Plant 5
Plant 4
Plant 3
Plant 2
Plant 2
Plant 3
Plant 4
Plant 5
Plant 6
Plant 7
Plant 8
Plant 9
**BUT, one may infer that
these results are NOT unambiguous
due to consistently inconsistent data
and the skepticism necessarily owed to conclusions drawn from negative results
06/09/04
FW+RV
06/09/2004
FW+T-DNA
Wild type amplification
TDNA amplification

8. Now what? --Screen Seed Pool #276
--Observe phenotype of relevant plants;
Genotype progeny of SALK plants 2-8