At2G37120: A Gene Exploration

An Arabidopsis Journey Through Experimental Questioning

By Jeremy Ziskind

What is the structure of AT2G37120?

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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

123257

-887 -823

112011DNA Pool 5

118631

Madison Insert

SALK Inserts

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

•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

•Comparing AT2G37120 gene expression (protein sequence) in Arabidopsis to Scarlet

Runner Bean expression

Arabidopsis Genechip Data

0

2000

4000

6000

8000

10000

12000

Wild TypeOvules

Wild Type24-Hr Seeds

Wild TypeMid-

Maturation(Cotyledon

Stage) Seeds

Wild TypeMature Green

Seeds

Wild TypePost-

MaturationGreen Seeds

Wild TypeSeedlings

(3DAI)

Wild TypeLeaves

Wild TypeRoots

Wild TypeStem

Wild TypeC24 Leaves

Wild TypeFloral Buds

Organs

Le

vels

of

Act

ivit

y

PcL1L Control

S1FA BandsPrimer Dimerazation

mRNA accumulation patterns in Scarlet Runner Bean

100 Kb Ladder

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

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Is there a knockout in Madison Lines? Rounds 1 and 2

Superpool 28

Control

Superpool 28

DNA Pool 5

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

What is the genotype of Arabidopsis plants with SALK insert #118631?

Extremely faint Tubulin

Negative Water Control

Wild-type Allele Amplification

WT 9 8 7 6 5 4 3 2 1

Tubulin

Mutant Allele Amplification

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