At2G37120: A Gene Exploration

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At2G37120 A Gene Exploration
An Arabidopsis Journey Through Experimental
Questioning By Jeremy Ziskind
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What is the structure of AT2G37120?
123257
118631
-201 -180 -53 1
146 173 509 713 855 932
1212 1241
-887 -823

• Expressed in Suspensor of Scarlet Runner Bean
• Chromosome 2 of Arabidopsis
• -932 base pairs in length
• -2 exons
• -5 and 3 UTRs
• What organisms is a potential ortholog found in?
• -Oryza sativa, Japanese rice

Madison Insert
SALK Inserts
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What is the Genes 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!!!

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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

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Where is S1FA mRNA present in Arabidopsis and SRB?
Conclusions S1Fa is affected by lec1 because
expression patterns are different in the absence
of lec1
mRNA accumulation patterns in Scarlet Runner Bean
100 Kb Ladder

• PcL1L Control

S1FA Bands
Primer Dimerazation
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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 1212
1241
DNA Pool 5 with a size of about 1400 base pairs
Sequencing results verify that the insert is in
DNA Pool 5!
DNA Pool Screen
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What is the genotype of Arabidopsis plants with
SALK insert 118631?
Negative Water Control
Wild-type Allele Amplification
Mutant Allele Amplification
WT 9 8 7 6 5 4 3 2 1
Tubulin
Conclusions SALK plants 1-9 are homozygous for
the wild-type allele and have no T-DNA insertions
Extremely faint Tubulin
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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