Using Microarrays to Measure Using Microarrays to Measure Sequence Preferences of Sequence Preferences of

Berenil Binding to the DNA Berenil Binding to the DNA Minor GrooveMinor Groove

Adam BrownAdam BrownMissouri Western State UniversityMissouri Western State University

Coauthors: Steven N. Hart, Kelly J. Malloy, Todd T. Eckdahl, Laurie J. Coauthors: Steven N. Hart, Kelly J. Malloy, Todd T. Eckdahl, Laurie J. Heyer, Martha Shott, Laura L. Mays Hoopes, Gloria YiuHeyer, Martha Shott, Laura L. Mays Hoopes, Gloria Yiu

Missouri Western State University Biology Department, Genome Missouri Western State University Biology Department, Genome Consortium for Active Teaching, Davidson College Biology Consortium for Active Teaching, Davidson College Biology

Department, Pomona College Biology Department Department, Pomona College Biology Department

IntroductionIntroduction

Minor Groove Binding Minor Groove Binding DrugsDrugs

Biological ActivityBiological Activity BerenilBerenil

Berenil Sequence PreferencesBerenil Sequence Preferences

Binding sites 5-6 bpBinding sites 5-6 bpA+T rich A+T rich HeteropolymericHeteropolymeric

ATAT > AATT > AAAA ATAT > AATT > AAAA ATATT > AATAT > AATTT > AAAAATATT > AATAT > AATTT > AAAA

Experimental PlanExperimental Plan

Yeast modelYeast model Expose yeast to Expose yeast to

berenilberenil RNA IsolationRNA Isolation Microarray ChipsMicroarray Chips MAGIC ToolMAGIC Tool Real Time PCRReal Time PCR Data AnalysisData Analysis

Indirect Labeling – 3DNAIndirect Labeling – 3DNA

Includes Two Includes Two HybridizationsHybridizations

Reverse Reverse Transcription Transcription occurs without occurs without labelinglabeling

Requires only Requires only 2.0 ug of RNA2.0 ug of RNA

Microarray ImagesMicroarray Images

MAGIC ToolMAGIC Tool

Microarray DataMicroarray Data

Genes Affected by BerenilGenes Affected by Berenil

50 Genes Turned off50 Genes Turned off15 carbohydrate metabolism, cell division, 15 carbohydrate metabolism, cell division,

proteolysis, response to metals, vacuole proteolysis, response to metals, vacuole fusionfusion

5 mitochondrial or respiration5 mitochondrial or respiration16 unassigned function16 unassigned function14 stress-related14 stress-related

2 Genes Turned on2 Genes Turned onPhosphate metabolism, rRNA processingPhosphate metabolism, rRNA processing

Validation by RT PCRValidation by RT PCR

Expression ratios for selected genes Expression ratios for selected genes validated by Real Time RT-PCRvalidated by Real Time RT-PCR

Sequence AnalysisSequence Analysis

54 affected genes compared to 56 unaffected 54 affected genes compared to 56 unaffected genesgenes

200 nt upstream regions of translation start sites200 nt upstream regions of translation start sites Occurrence of all 5-mer and 6-mer sequences Occurrence of all 5-mer and 6-mer sequences

measuredmeasured Ranking criteriaRanking criteria

Diff between percentage of affected and unaffected Diff between percentage of affected and unaffected regions having a sequenceregions having a sequence

Ratio of occurrence of sequence in affected Ratio of occurrence of sequence in affected compared to unaffected regionscompared to unaffected regions

Difference Criterion SequencesDifference Criterion Sequences

Ratio Criterion SequencesRatio Criterion Sequences

Sequence FeaturesSequence Features

The average A+T content of the The average A+T content of the sequences is 90% (65% for all yeast sequences is 90% (65% for all yeast genes)genes)

Of the 8 possible completely Of the 8 possible completely heteropolymeric sequences, 4 appearheteropolymeric sequences, 4 appear

51% of the dinucleotides are AT or TA. 51% of the dinucleotides are AT or TA. Only 18% of dinucleotides in the 200 bp Only 18% of dinucleotides in the 200 bp upstream of all yeast genes are AT or TA. upstream of all yeast genes are AT or TA.

Direct versus Indirect EffectsDirect versus Indirect Effects

Upstream sequences of 54 affected genes Upstream sequences of 54 affected genes were A+T rich, heteropolymeric were A+T rich, heteropolymeric

But, the method cannot distinguish:But, the method cannot distinguish:Genes directly affected by berenilGenes directly affected by berenilGenes indirectly affected by the product of a Genes indirectly affected by the product of a

directly affected genedirectly affected geneAre the stress-related genes indirectly Are the stress-related genes indirectly

affected?affected?Are their upstream sequences different Are their upstream sequences different

from the rest of the affected genes?from the rest of the affected genes?

Difference Criterion - DirectDifference Criterion - Direct

Ratio Criterion - DirectRatio Criterion - Direct

Difference Criterion - IndirectDifference Criterion - Indirect

Ratio Criterion - IndirectRatio Criterion - Indirect

Features Found Upstream Features Found Upstream Directly Affected GenesDirectly Affected Genes

Average of 92% A+TAverage of 92% A+T 100% are at least 80% A+T100% are at least 80% A+T Difference and ratio measures yield 75% Difference and ratio measures yield 75%

shared sequencesshared sequences 52% of dinucleotides are AT and TA, 52% of dinucleotides are AT and TA,

compared to 18% for all yeast genescompared to 18% for all yeast genes Completely A/T heteropolymeric 5- and 6-mers Completely A/T heteropolymeric 5- and 6-mers

occur at 4.4 times the expected rateoccur at 4.4 times the expected rate The high rate of heteropolymeric tracts of 3-6 The high rate of heteropolymeric tracts of 3-6

nt is statistically significantnt is statistically significant

Chi-squared AnalysisChi-squared Analysis

ConclusionsConclusions

Microarray analysis yielded list of yeast genes Microarray analysis yielded list of yeast genes affected by Berenilaffected by Berenil

Gene functions suggested direct and indirect Gene functions suggested direct and indirect effectseffects Direct category had expected sequence featuresDirect category had expected sequence features Indirect category did not display sequence featuresIndirect category did not display sequence features

Results contribute to Results contribute to an understanding of an understanding of in vivo in vivo sequence requirements sequence requirements

for Berenil bindingfor Berenil binding a new approach to analysis of microarray data setsa new approach to analysis of microarray data sets

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AcknowledgementsAcknowledgementsThanks to the Genome Consortium for Active Teaching (GCAT) Thanks to the Genome Consortium for Active Teaching (GCAT)

and Dr. John N. Anderson (Purdue) for advice and discussions. and Dr. John N. Anderson (Purdue) for advice and discussions. This work was supported by the Missouri Western Summer This work was supported by the Missouri Western Summer Research Institute, and NIH AREA grant 1R15CA096723-01.Research Institute, and NIH AREA grant 1R15CA096723-01.