HC70AL Final Presentation Chris McQuilkin June 4th, 2009

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

AT4G36540 What 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

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

RT-PCR Forward Primer Intron Forward Primer

Where is the Gene Active? A DNA microarray is a small chip containing thousands of microscopic wells. Each of the wells contains picomoles of a specific fragment of DNA. This DNA acts as a probe

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

Promoter Cloning *Two fragments were amplified by PCR 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

Promoter Cloning Expected Plasmid alone= 2.5 kb Expected Plasmid + Promoter= 4.2kb Observed= 2.5kb and 3.5 kb AscI-Digested pENTR Plasmid DNA From Six E. coli Colonies TALK ABOUT PLASMID LIGATION AND COLONIES and refer to earlier pic

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

Gene one: Genotyping What is the expected size of the T-DNA band? 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

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 4 119 116 288 288 85 85 180 180 92 92 207 207 91 91 134 134 91 91 93 93 334 331 Exon 1 Exon 1 Exon 2 Exon 2 Exon 3 Exon 3 Exon 4 Exon 4 Exon 5 Exon 5

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

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

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

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

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

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

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

AT1G75240 What 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

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

Where is the Gene Active?

Genotyping Arabidopsis 1 Arabidopsis 2 Arabidopsis 3 Arabidopsis 4 WT Control LBb1 Control 100 bp 100 bp

Results All the plants screened were wild-type

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

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

SRB Contig Sequencing Contig Length: 50.7 KB

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

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

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

Approach Whole Contig was searched for DNA repeats

Results: GENSCAN GENSCAN Predicted Gene (BLASTN) Accession Number E-value Description 1 BP033723.1 1e-110 Lotus Japonicus 2 FN014331.1 1e-52 Petunia axillaris subsp. axillaris pool of root and petal tissue 3 GO259467 3e-26 Tissue from peanut Arachis hypogaea 4 EX522455.1 0.0 Trichome from stem of Medicago sativa 5 FK023593.1 8e-77 cDNA from Glycine max (soy bean)

Results: GENSCAN Predicted Protein (tBLASTn) Accession Number E-value Description 1 Y18169 5e-70 Pisum sativum mitochondrial ccb248 gene and partial rps7 gene. 2 FM179380 2e-10 Vitis vinifera complete mitochondrial genome, cultivar Pinot noir clone ENTAV115 3 L40816 3e-35 4 DQ008791 5e-60 Thottea tomentosa large subunit ribosomal RNA gene, partial sequence; mitochondrial 5 AC144406 4e-14 Medicago truncatula clone mth2-5h18, complete sequence. Predicted Protein (BLASTp) YP_173352 2e-41 hypothetical protein NitaMp002 [Nicotiana tabacum]. No significant match

Results: FGENESH Predicted Gene (BLASTn) Accession Number E-value Description 1 EX527626 0.0 MTGland_A031_2007-05-22/MTGlandA031_B09_039_1 Medicago truncatula A17 glandular trichome Medicago truncatula cDNA, mRNA sequence 2 CD721224 2e-25 Chardonnay Vitis vinifera 3 GD544655 1e-40 Scarlet Runner Bean globular-stage suspensor region 4 5 FN014331 9e-88 Pool of root and petal tissue of Petunia axillaris 6 FF554629.1 9e-143 Vigna Unguiculata Predicted Protein (tBLASTn) Y18169 18e-118 Pisum sativum mitochondrial ccb248 gene and partial rps7 gene. AC144406 8e-18 Medicago truncatula clone mth2-5h18, complete sequence Predicted Protein (BLASTp) CAB43024 2e-118 cytochrome c biogenesis protein [Pisum sativum]

Results: GeneMark Predicted Protein (tBLASTn) Accession Number (of Lowest E-Value) E-value Description 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 7e-46 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.

Results: Gene Mark 19 1e-41 20 1e-26 21 7e-17 23 2e-12 32 5e-29 33 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 7e-17 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.