HC70AL Final Presentation

1. HC70ALFinal Presentation Chris McQuilkin June 4th, 2009

2. Gene One: AT4G36540 • A transcription factor involved in DNA binding • A bHLH Gene • “basic Helix-Loop-Helix” refers to the structure of the protein • bHLH genes are found in Eukaryotes and are highly conserved across species

3. AT4G36540What is the Structure of the Gene? T-DNA Insertion Site LB Translational Start Codon Translational Stop Codon Forward Primer Reverse Primer  Intron 1 Intron 2 Intron 3 Intron 4 116 288 85 180 92 207 91 134 91 93 331 Exon 1 Exon 2 Exon 3 Exon 4 Exon 5 Total length= 1,714 base pairs

4. Where is the Gene Active? • Why is there no band in the positive control?

5. RT-PCR Forward Primer Intron Forward Primer

6. Where is the Gene Active?

7. Where is the Gene Active?Promoter Cloning Krista Templeton and Auni Hovanesian, HC70AL 2008

8. Promoter Cloning PCR of AT4G3540 Promoter Region 1 hr 120 volts *Two fragments were amplified by PCR ~1.7 kb= Expected Size of PCR-amplified promoter ~0.8 kb= Unexpected PCR product iProof Polymerase Positive Control 1 kb Ladder

9. AscI-Digested pENTR Plasmid DNA From Six E. coli Colonies Promoter Cloning Expected Plasmid alone= 2.5 kb Expected Plasmid + Promoter= 4.2kb Observed= 2.5kb and 3.5 kb

10. Gene one: Genotyping 1 hr, 120 volts Wild-type Control

11. Gene one: Genotyping 04/16/2009 1 hr, 120 volts ~1000 base pairs Why are there two bands in the Lanes 1 and 3? ~900 base pairs ~250 base pairs Hemizygous Homozygous Hemizygous Hemizygous What is the expected size of the T-DNA band? What is the expected size of the Wild-Type Band? ~250 bases 887 Bases

12. Concatamers T-DNA Insertion Site LB LB  Reverse Primer  Translational Start Codon Translational Start Codon Forward Primer Translational Start Codon Translational Start Codon Intron 1 Intron 1 Intron 2 Intron 2 Intron 3 Intron 3 Intron 4 Intron 4 116 119 288 288 85 85 180 180 92 92 207 207 91 91 134 134 91 91 93 93 331 334 Exon 1 Exon 1 Exon 2 Exon 2 Exon 3 Exon 3 Exon 4 Exon 4 Exon 5 Exon 5

13. Nomarski Observation Mutant Embryo and wild-type embryo show no phenotypic differences

14. Nomarski Observation Mutant seed coat and wild- type seed coat show no phenotypic differences

15. Results • Three hemizygous and one homozygous T-DNA plants were identified • Two bands were observed in each of the lanes containing DNA with a T-DNA insert • Gene AT4G36540 mRNA accumulation was observed in both the leaf and silique of Arabidopsis

16. Results • The promoter did not insert into any of the E. coli plasmids that were screened. • Four of the colonies contained an unidentified insert about 1 kb long • No phenotypic changes were observed in either the hemizygous or homozygous T-DNA Arabidopsis plants using Nomarski Observation

17. Conclusion • AT4G36540 is not lethal, or there may be another DNA sequence that codes for the same protein • Although no phenotypic differences were observed in the seed coat or embryo using Nomarski, there could be differences that are more subtle or appear at different stages of development

18. What next? • Look further for phenotypic changes in Arabidopsis mutants—examine different tissues and more stages of development • Sequence the unidentified DNA fragment taken up by the E. coli C600 plasmids • Repeat PCR of the promoter • Excise the band containing recombined plasmid DNA, and repeat transformation • If a promoter is identified, use GUS to study where the gene is expressed • Grow more plants from the identified mutant lines • Repeat sequencing reaction of region between LB and T-DNA primers to confirm location of the T-DNA insert

19. Gene Two: AT1G75240 • A homeobox gene • Homeobox genes regulate development and cell differentiation • A homeobox is a sequence • They are found in animals, plants, and fungi

20. AT1G75240What Is The Structure of the Gene? Predicted T-DNA Insert LB Reverse Primer Forward Primer 330 77 97 927 254 Translational Start Codon Translational Stop Codon Total Length: 1,685 Base Pairs

21. Where is the Gene Active? AT1G75240 mRNA in Arabidopsis Leaf and Silique 1 hr 120 volts Silique cDNA Leaf cDNA Tubulin mRNA AT1G75240 mRNA Gene is active in the silique but not the leaf

22. Where is the Gene Active?

23. Genotyping Arabidopsis 1 Arabidopsis 2 Arabidopsis 3 Arabidopsis 4 Arabidopsis 5 Arabidopsis 6 Arabidopsis 7 Arabidopsis 8 Arabidopsis 9 Arabidopsis 10 Arabidopsis 11 Arabidopsis 12 WT Control LBb1 Control 100 bp 100 bp

24. Results • All the plants screened were wild-type

25. Conclusion • The chance of getting all wild-type plants is extremely low, so it is possible that the Salk Institute sent the wrong kind of plant

26. Thank you! • Anhthu Bui • Brandon Chen • Bob Goldberg • Daisy Robinton • Ingrid Nelson • Kristin Gill • Min Chen

27. SRB Contig Sequencing Contig Length: 50.7 KB

28. Approach • Contig entered into three online databases that look for predicted genes: FGENESH, GENSCAN, and GeneMark

30. 2,000 1 240 396 - 507 1134 - 1442 1901 - 628- 729 759-846 999-1097 1134-1642 1843- 1324-1428 396-507 999-1092 1901- 459- 572 281- 427 2,001 -2550 3272 4,000 -2550 -2086 2126-2351 2990 2894 3046

31. Approach • Predicted Genes were entered into a BLAST search to see if the predicted gene matched Expressed Sequence Tags (ESTs) found in other species

33. Approach • Whole Contig was searched for DNA repeats

35. GENSCAN Results: GENSCAN

36. Results: GENSCAN

37. Results: FGENESH

38. Results: GeneMark

39. Results: Gene Mark