How do we find a knockout for

AT4G37790 and what is this gene’s

function in seed development?

A Research Talk by Ita Nagy

HC70ALSpring 2004

What do we know about AT4G37790?Common Name: HAT-22Homeobox leucine-zipper protein

Codes for nuclear protein sized at 278 AA

RegulatoryDNA bindingImportant for transcription factor

functionContains 1 homeobox domainLocated on chromosome 4 of

ArabidopsisForward strand

•THE LEUCINE ZIPPER. Dimers result from leucine residues at every other turn of the a-helix.  When the a-helical regions form a leucine zipper, the regions beyond the zipper form a Y-shaped region that grips the DNA in a scissors-like configuration.

Evolutionary Relationships of HAT Protein Sequences

-Alignment of HAT9 and HAT22 reveals 90% identity in the homeodomain-leucine zipper region and 71% identity overall.

-Little homology is seen outside of the oustide the homeodomain-leucine zipper region for other HAT proteins.

Where is HAT-22 active in Arabidopsis Thaliana and in SRB?

• In SRB, the gene was found to be the most active in Sample 1 (Genomic Leaf +RT), Sample 3 (Genomic Stem +RT), and Sample A (Isolated Leaf +RT).

• In Arabidopsis, the gene is most active in lec1 Stem (signal detection 4087.3 P), the WT Stem (3170 P), lec1 Leaves (2678.5 P), and WT Leaves (2491 P).

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What is the structure of AT4G37790?

•1471 bp, Forward Orientation, 3 exons and 2 introns

•5’ and 3’ region UTR

•FW Primer located 100 bp upstream / RV Primer 540 bp downstream

•Adjacent Genes: AT4G37780 8.9 kb upstream / AT4G37800 6.4 kb downstream

•Madison t-DNA insert within gene at 661 bp; SALK t-DNA insert outside gene at –312 bp, perhaps inside the promoter region

Which superpool from the Madison Facility contains a t-DNA insert in AT4G37790?

Superpool 3 contained the correct hit, as shown in the FW autoradiogram above. The RV autoradiogram contained no hits. From the gel, the target segment was sized at ~800 bp.

Below the control ~800 bp

Which DNA pool contains a t-DNA insert in AT4G37790?

• Amplify PCR products from 9 DNA pools belonging to Superpool 3.

• Use FW & JL202 primers and a + control, Superpool 3.

• Can we find a PCR product with the same size as the hit segment from our superpool screening (~800 bp)?

• The primers amplify an ~800 bp segment in DNA Pool 4.

800 bp

How do SALK Lines help us see how AT4G37790 functions in seed development?

• SALK Line Used: 085964.56.00

• T-DNA Insertion at -312 bp

• Complementary Orientation

•Genotype plants through PCR with FW+RV primers, then t-DNA primer+RV primer because orientation is complementary. Plants homozygous for the mutant allele prove that knocking out AT4G37790 is not embryo lethal. We can phenotype these plants to see how they are lacking in development.Salk Professor Joanne

Chory with Salk Plants

What are the genotypes of the SALK plants with respect to the t-DNA

insert? 1 2 3 4 5 6 7 8 9 wt

There are no homozygous mutants (band in PCR1 and band in PCR2).

Plants 1 and 5 are heterozygous for the mutant allele. They have a band in PCR1 but also in PCR2 sized at ~2300 bp which is exactly the segment we want, since there are ~2300 bp between the Salk insert at –312 and the RV primer at 2011.

Where do we go from here?

• SALK LINES: Collect seeds from heterozygous mutant plants and attempt to raise homozygous mutants.

• If homozygous mutants are obtained, knockout is not embryo lethal. However, we can PHENOTYPE the plants to see whether abnormalities give us clues to the importance of AT4G37790 in seed development.

• Salik examination• MADISON FACILITY: Continue to search for

the knockout by narrowing DNA Pool 4 down to a specific seed pool and then down to a specific t-DNA insert line. When this line is found, grow plants and continue to investigate whether knocking out AT4G37790 significantly affects seed development.

•

How do we Study Gene

1. Isolate RNA from different vegetative organs of the SRB.

2. Remove contamination from RNA (such as genomic DNA).

3. Perform RT-PCR to transform mRNA into cDNA, then amplify cDNA using primers for the gene-specific region.

4. RT-PCR Methodology

5. Run products on a gel to see which samples amplified. The organs that provided samples with the most amplification are the ones where the gene is most active.

In the SRB?

Activity