The Trihelix Transcription Factor Family

1. The Trihelix Transcription Factor Family Heather Hernandez

2. What is a Trihelix Transcription Factor? • Also called GT factors. • DNA binding proteins. • Bind to GT elements in promoters. • Bind to promoter elements required for light responsiveness. • Regulate plant-specific processes. • Specific regulatory functions largely unknown. “Conserved Domain” found in all trihelix proteins.

3. Trihelix Transcription Factor Genes Gene name: At3g14180 Location: Chromosome 3 Gene size: 1736 Base Pairs Protein size: 444 amino acids Protein encoded: expressed protein similar to 6b-interacting protein 1 (found in Tobacco) UTR EXON UTR 5’ 3’ REVERSE ORIENTATION

4. Where in Arabidopsis thaliana is At3g14180 active? RT-PCR 05/23/06 UCLA Genechip Data Ladder INF RT + INF RT- SIL RT + SIL RT - + Control - Control + Control - Control 1.5% agarose gel 1.5 hours @ 90v 10 uL EtBr

5. What genotypes are present in my plants? At3g14180 Genotyping At3g14180 Genotyping Ladder Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Wildtype + Control - Control Ladder Plant 6 Plant 7 Plant 8 Plant 9 Plant 10 Plant 11 Plant 12 Plant 13 Plant 14 Plant 15 Plant 16 Wildtype + Control - Control Wildtype band T-DNA “top” T-DNA “bottom” 1% Agarose gel 1.5 hrs @ 105 v 10 uL EtBr 1% Agarose gel 1.5 hrs @ 105 v 10 uL EtBr Wildtype: 1 Heterozygous mutant: 4 Homozygous knockout: 11

6. Where did the two T-DNA bands insert? My sequencing results: LBb1 LBb1 B T Ladder 5’ 3’ BP 1 830 846 BP 1736 Location from SALK matches my sequencing results. (SALK predicted 844 BP into the gene.)

7. What is the impact of the T-DNA inserts? • No visible phenotypical differences seen in homozygous knockout plants vs. wildtype plants.

8. What is the impact of the T-DNA inserts on seed development? Wildtype fully developed Wildtype heart stage Wildtype late torpedo Mutant heart stage Mutant fully developed Mutant torpedo suspensor

9. Trihelix Transcription Factor Gene 2 • Gene name: At1g54060 • Location: Chromosome 1 • Gene size: 1646 Base Pairs • Protein size: 384 amino acids • Protein encoded: expressed protein similar to 6b-interacting protein 1 (found in Tobacco) Exon (BP 1-1646) UTR 293 BP UTR 200 BP 5’ 3’ Forward Orientation

10. Where in Arabidopsis thaliana is At1g54060 active? RT-PCR At1g54060 Genechip Data Ladder Ladder INF +RT INF -RT SIL RT + SIL RT - + Control - Control 1.5 % Agarose gel 1.5 hrs @ 105 v 10 uL EtBr

11. What genotypes are present in my plants? Genotyping At1g54060 Wildtype: 6 Heterozygous T-DNA: 3 Homozygous knockout: 5 • Control Ladder Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Plant 6 Plant 7 Plant 8 Plant 9 Plant 10 Plant 12 Plant 13 Plant 14 Plant 15 Wildtype + Control 1% Agarose gel 1.5 hrs @ 105 v 10 uL EtBr

12. Where did the T-DNA insert? My sequencing results: LBb1 5’ 3’ BP 635 BP 1 BP 1646 My results matched the SALK institute expected insert site of 640 BP into the gene.

13. What is the impact of the T-DNA inserts on phenotype? Plant 6, Silique 1, Top Plant 6 Silique 1 Plant 6, Silique 1, Dissected Plant 6, Silique 1, Bottom

14. T-DNA impact continued… Plant 6, Silique 1, White seeds Plant 6, Silique 2, 12.5x Plant 6, Silique 2, Dissected Plant 6, Silique 2, Bottom

15. How does upstream regulatory activity show where the protein is translated? EcoRI Digest At1g54060 iProof PCR At3g14180 Ladder Sample 1 Sample 2 Sample 3 Sample 4 Ladder “Promoter Region” + Control - Control 1% Agarose gel 1.5 hrs @ 105 v 10 uL EtBr

16. Acknowledgement s • Dr. Robert Goldberg • Dr. Anthu Bui • Mike Gavino • Jonathan Russell • Garima Yagnik • Tomo Kawashima • Jessica Luke • Dr. Xingjun Wang • Lab members!