1. HC70AL Final Presentation Chris McQuilkin June 4 th, 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. AT4G36540 What is the Structure of the Gene? 1162888518092207911349193331 Exon 1Exon 5Exon 4Exon 3Exon 2 Translational Start Codon Translational Stop Codon Intron 1Intron 2Intron 3Intron 4 Total length= 1,714 base pairs T-DNA Insertion Site Forward Primer  Reverse Primer  LB 

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

5. RT-PCR Forward Primer Forward Primer Intron

6. Where is the Gene Active?

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

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

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

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

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

12. Concatamers 1192888518092207911349193334 Exon 1Exon 5Exon 4Exon 3Exon 2 Translational Start Codon Intron 1Intron 2Intron 3Intron 4 1162888518092207911349193331 Exon 1Exon 5Exon 4Exon 3Exon 2 Translational Start Codon Intron 1Intron 2Intron 3Intron 4 T-DNA Insertion Site Forward Primer  Reverse Primer   LB LB 

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. 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 Results

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. AT1G75240 What Is The Structure of the Gene? 3307797927254 Translational Start Codon Translational Stop Codon Forward Primer   Reverse Primer Total Length: 1,685 Base Pairs Predicted T-DNA Insert  LB

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

22. Where is the Gene Active?

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

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. 396-507999-1092 1324-1428 -20862126-23512990 1901- 281- 427 459- 572 1 2,000 2,001 4,000 628- 729759-846999-10971134-1642 1843- 2894 3046 -2550 240 396 - 5071134 - 14421901 - -2550 3272

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. Whole Contig was searched for DNA repeats Approach

35. Results: GENSCAN GENSCAN Predicted Gene (BLASTN) Accession NumberE-valueDescription 1BP033723.11e-110Lotus Japonicus 2FN014331.11e-52Petunia axillaris subsp. axillaris pool of root and petal tissue 3GO2594673e-26Tissue from peanut Arachis hypogaea 4EX522455.10.0Trichome from stem of Medicago sativa 5FK023593.18e-77cDNA from Glycine max (soy bean)

36. Results: GENSCAN Predicted Protein (tBLASTn)Accession NumberE-valueDescription 1Y181695e-70Pisum sativum mitochondrial ccb248 gene and partial rps7 gene. 2FM1793802e-10Vitis vinifera complete mitochondrial genome, cultivar Pinot noir clone ENTAV115 3L408163e-35 4DQ0087915e-60Thottea tomentosa large subunit ribosomal RNA gene, partial sequence; mitochondrial 5AC1444064e-14Medicago truncatula clone mth2-5h18, complete sequence. Predicted Protein (BLASTp) 4 YP_1733522e-41hypothetical protein NitaMp002 [Nicotiana tabacum]. 5No significant match

37. Results: FGENESH Predicted Gene (BLASTn) Accession Number E-valueDescription 1EX5276260.0MTGland_A031_2007-05-22/MTGlandA031_B09_039_1 Medicago truncatula A17 glandular trichome Medicago truncatula cDNA, mRNA sequence 2CD7212242e-25Chardonnay Vitis vinifera 3GD5446551e-40Scarlet Runner Bean globular-stage suspensor region 4 5FN0143319e-88Pool of root and petal tissue of Petunia axillaris 6FF554629.19e-143Vigna Unguiculata Predicted Protein (tBLASTn) 1Y1816918e-118Pisum sativum mitochondrial ccb248 gene and partial rps7 gene. 6AC1444068e-18Medicago truncatula clone mth2-5h18, complete sequence Predicted Protein (BLASTp) 1CAB430242e-118cytochrome c biogenesis protein [Pisum sativum]

38. Results: GeneMark Predicted Protein (tBLASTn) Accession Number (of Lowest E-Value) E-valueDescription 1 AC192958 1e -13 Medicago truncatula chromosome 2 BAC clone mte1-45m19, complete sequence 2 AC147537 1e -23 Medicago truncatula clone mth2-133k2, complete sequence. 3 EX527626 3e -113 Glandular trichome Medicago truncatula 13 BA000042 1e-18 Nicotiana tabacum mitochondrial DNA, complete genomeLength=430597 15 L40816 5e-25 Glycine max mitochondrion polymorphic marker DNA sequence. 16 L40816 7e-46 Glycine max mitochondrion polymorphic marker DNA sequence. 17 AC145156 1e-50 Medicago truncatula clone mth2-7h6, complete sequence. 18 DQ647831 1e-108 Chlorokybus atmophyticus large subunit ribosomal RNA gene, partial sequence; mitochondrial.

39. Results: Gene Mark 19 BA000042 1e-41 Nicotiana tabacum mitochondrial DNA, complete genome. 20 AP004975 1e-26 Lotus japonicus genomic DNA, chromosome 5, clone: LjT21J12, TM0158, complete sequence. 21 AP004975 7e-17 Lotus japonicus genomic DNA, chromosome 5, clone: LjT21J12, TM0158, complete sequence. 23 XM_002336124 2e-12 Populus trichocarpa predicted protein, mRNA. 32 FM179380 5e-29 Vitis vinifera complete mitochondrial genome, cultivar Pinot noir clone ENTAV115. 33 AC144406 7e-29 Medicago truncatula clone mth2-5h18, complete sequence.