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At2G37120: A Gene Exploration An Arabidopsis Journey Through Experimental Questioning By Jeremy Ziskind What is the structure of AT2G37120? 123257 112011 DNA Pool 5 118631 -887 -823 -201 -180 -53 1 146 173 509 713 855 932 1212 1241 Expressed in Suspensor of Scarlet Runner Bean Madison Insert Chromosome 2 of Arabidopsis SALK Inserts -932 base pairs in length -2 exons -5’ and 3’ UTRs What organisms is a potential ortholog found in? -Oryza sativa, Japanese rice What is the Gene’s Possible Function? • Codes for S1Fa which is most likely a nuclear DNA binding protein •S1Fa important for the binding specificity of a small protein that binds to DNA •Protein only has 70 amino acids (Smallest DNA binding protein ever reported) •Has no homology to any known DNA binding modules Conclusion: S1Fa belong to a NOVEL CLASS OF DNA BINDING PROTEINS!!! SRB EST vs Arabidopsis •Comparing AT2G37120 gene expression (protein sequence) in Arabidopsis to Scarlet Runner Bean expression •EST: PCSC16872 (42125) Length = 408 Score = 39.6 bits (80), Expect(3) = 2e-09 Identities = 15/21 (71%), Positives = 15/21 (71%) Frame = +2 / -2 Query: 353 MRNVTSRPPITSSTINPGFNP 415 MRNV S PP TS TI PG NP Sbjct: 146 MRNVNSNPPTTSRTIKPGLNP 84 •CONCLUSIONS: SRB EST has 71% region of amino acid homology to Arabidopsis protein sequence Where is S1FA mRNA present in Arabidopsis and SRB? Arabidopsis Genechip Data 12000 10000 Levels of Activity 8000 6000 4000 2000 0 Wild Type Ovules Wild Type 24-Hr Seeds Wild Type Wild Type Wild Type MidMature Green PostMaturation Seeds Maturation (Cotyledon Green Seeds Stage) Seeds Wild Type Seedlings (3DAI) Wild Type Leaves Wild Type Roots Wild Type Stem Wild Type C24 Leaves Wild Type Floral Buds Conclusions: S1Fa is affected by lec1 because expression patterns are different in the absence of lec1 Organs mRNA accumulation patterns in Scarlet Runner Bean 100 Kb Ladder PcL1L Control S1FA Bands Primer Dimerazation Is there a knockout in Madison Lines? Rounds 1 and 2 Superpool 28 DNA Pool 5 Superpool 28 Control -201 -180 -53 1 146 173 509 713 855 932 DNA Pool #5 with a size of about 1400 base pairs DNA Pool Screen Sequencing results verify that the insert is in DNA Pool #5! 1212 1241 What is the genotype of Arabidopsis plants with SALK insert #118631? Negative Water Control WT Wild-type Allele Amplification 9 8 7 6 5 4 3 2 1 Mutant Allele Amplification Tubulin Extremely faint Tubulin Conclusions: SALK plants 1-9 are homozygous for the wild-type allele and have no T-DNA insertions Further Experiments •Further SALK genotyping must be performed to determine whether At2G37120 is important for seed development •Phenotyping must be done on SALK #118631 plants to determine if the knockouts affect seed development or any other aspect of plant development •DNA Pool #5 must be narrowed down to find the individual line with a knockout in At2G37120