Stephen J. Callister

Department of Civil and Environmental Engineering

Quantification of Genomic Response to Environmental Perturbationusing a Modulus of Stability

Stephen J. CallisterDepartment of Civil and Environmental Engineering, Michigan State University, East

Lansing, Michigan

February 27, 2003


Introduction

•The advancement of technology has dramatically increased our ability to studygene expression on a large scale.

•Current techniques used for analysis of gene expression in response toperturbation have proved useful for enlarging our knowledge about emergencyresponse genes, transcriptional regulation, implied function etc...

•Common experiments performed using these technologies are perturbationresponse experiments.

•Relating gene response and perturbation could give insight when comparing:

•Commonly expressed genes across different environmental perturbations.

•Gene expression to different magnitudes of the same environmentalperturbation.

•The similar genes found on different genomes.


Introduction

OBJECTIVE - Quantify the relationship between genomic response andperturbation using a Modulus of Stability.

Definitions:

Stability - The ability of a system to withstand a period of stress and return toequilibrium afterwards. (Source: Harrison, G.W. (1979) American Naturalist 113,659-669)

System - Set of selected genes expressed either upwards or downwards inresponse to an environmental perturbation.

Modulus of Stability - Index that can be used to understand the stress(perturbation) strain (response) relationships of a genomic system.

1. Quantify the perturbation.2. Quantify response - observed patterns of gene expression.3. Relate the two


Quantifying the Perturbation

•Two typical perturbations - pulse and press (Bender et al., 1984; Yodzis, 1988).

duration

ampl

itude

A

duration

ampl

itude

BP(a,d) P(a,d)

Graphical Representation of Pulse and Press Perturbations


What are Typically Observed Patterns of GeneExpression?

time

Rel

ativ

e ge

ne e

xpre

ssio

n

1

1

1 1

1

1

1

I

II

III

VII

VI

V

IV

Generalized Relative Gene Expression Patterns in Response toPerturbation


Quantification of Patterns of Gene Expression

•Stability parameters

Resilience- Rate at which an asymptoticallystable system returns to equilibrium.

Return time- Time required for an asymptotically stable system to return to equilibrium. Inverse of resilience

Reactivity- Rate at which a system departsfrom equilibrium. Complimentary to resilience

Resistence- Maximum deviation from the pre-perturbed equilibrium.

1

Res

iste

nce

ReactivityRel

ativ

e G

ene

Exp

ress

ion

Time

1

1/e

Returntime

Res

iste

nce

ResilienceReactivity

Graphical Depiction of StabilityParameters


Quantification of Patterns of Gene Expression

•Aggregate measure of stability parameters (Hashsham et al., 2000):R

elat

ive

Gen

e E

xpre

ssio

n

11

AmplificationEnvelope

Center ofMass

MomentArm

PerturbationAxis

Duration

Â ⋅=i

ii tAMAreaofMoment0

,

Graphical Depiction of the Moment of Area


Relating the Two

Perturbation as a function of the amplitude and duration.This represents the stress portion of the definition.

This is the strain (response in terms of gene expression) portion ofthe definition quantified in terms of an aggregate measure of thestated stability parameters where n is the number of one type orresponse for m number of characteristic expression pattern.

( )Ú Ú

Ú

= =

≡m

j

n

i

jijiji rlrsk

dap

1 1

,,, ...,

),(stability of modulus

Comparatively smaller values indicate that less stress is required per resulting unitof strain (response).


Modulus of Stability: Application

1. Proof of Concept -Used previously published data and the Modulus of Stabilityto quantify genomic response to perturbation.

2. Current Research - Expand our understanding of the behavior of the Modulus ofStability to perturbation.


Proof of Concept: Introduction

•Raw data was obtained from the Stanford Microarray Database results werepublished by Gasch et al. (2000) Molecular Biology of the Cell 11, 4241- 4257.

•Organism studied - Saccharomyces cerevisiae.

•Eight perturbations were used for proof of concept.

Environmental PerturbationType of Cellular Stress1. Temperature from 25o to 37o CTemperature2. Temperature from 37o to 25oTemperature3. Hyper-osmoticOsmotic4. Hypo-osmoticOsmotic5. Hydrogen peroxideOxidative6. MenadioneOxidative7. DiamideProtein (chemical)8. DithiothreitolProtein (chemical)


Proof of Concept: Calculating Modulus of Stability

Excel

GeneSpring

Mathcad

Raw data downloadedto elucidatesignificantly

expressed genes.

Expressed genesshowing >2-foldexpression across

perturbations

Cluster genes usingK-means clustering

and standardcorrelation.

Sort clustered genesinto characteristic

patterns of expression.

Calculate Moment ofarea

Moment of areaused to calculate

modulus of stability.


Proof of Concept: Results

0 100 200 300 400 5000.00

0.75

0.80

0.85

0.90

0.95

1.00

Hyper-osmotic

Hypo-osmotic

DTT

Diamide

Menadione

Hydrogen peroxide

Time (min)

Con

cent

ratio

n (m

ol l-1

)

3.0x10-6

1.5x10-6

Comparison of Quantified Perturbations



StressPerturbationAsymptoticExpressionPatterns(Percent)Non-asymptoticExpressionPatterns(Percent)Heat Shock 25oto 37o C944TemperatureHeat Shock 37oto 25o C8212Hyper-osmoticShock942OsmoticHypo-osmoticShock829HydrogenPeroxideExposure904OxidativeMenadioneExposure8214DTT Exposure906Protein(Chemical)DiamideExposure944



1 gene

5 genes

20 genes

79 genes

216 genes

433 genes

All Genes

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07H

yper

-osm

otic

Hyp

o-os

mot

ic

Hea

t 37o

to 2

5o C

Hea

t 25o

to 3

7o C

Men

adio

ne

Hyd

roge

n pe

roxi

de

Dith

ioth

riet

ol (

DTT

)

Dia

mid

e

Log

Mom

ent o

f Are

a (m

in)2

1.0E+00

1.0E+01

1.0E+02

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

Hyp

er-o

smot

ic

Hyp

o-os

mot

ic

Hea

t 37o

to 2

5o C

Hea

t 25o

to 3

7o C

Men

adio

ne

Hyd

roge

n pe

roxi

de

Dith

ioth

riet

ol (

DTT

)

Dia

mid

e

Log

Mom

ent o

f Are

a (m

in)2



YDL022W

YER070W

YDL124W

YFL014W

YKL009W

YDR513W

ORF’s with the largest %contribution

Hyp

er-o

smot

ic

Hyp

o-os

mot

ic

Hea

t 37o

to 2

5o C

Hea

t 25o

to 3

7o C

Men

adio

ne

Hyd

roge

n pe

roxi

de

Dith

ioth

riet

ol (

DTT

)

Dia

mid

e0%

20%

40%

60%

80%

100%

Per

cent

Con

trib

utio

n to

Mom

ent o

f Are

aGeneral Categories ->Metabolism->C Compound and Carbohydrate -> Utilization



ORFs

Hyp

er-o

smot

ic

Hyp

o-os

mot

ic

Hea

t 37

to 2

5 C

Hea

t 25

to 3

7 C

Men

adio

ne

Hyd

roge

n P

erox

ide

Dia

mid

e

DT

TBiological Process Molecular Function

YDL022W x x Glycerol Accumulation glycerol-3-phophate dehydrogenase (NAD+)YER070W x x DNA Replication ribonucleoside-diphosphate reductase

YDL124W x x Unknown unknownYFL014W x x x x x Heat, osmotic, oxidative stress heat shock protein

YKL009W x Unknown unknownYDR513W x x Redox homeostasis, oxidative stress thiol-disulfide exchange intermediate



1.0E-10

1.0E-09

1.0E-08

1.0E-07

1.0E-06

1.0E-05

1.0E-04

1.0E-03

1.0E-02

1.0E-01

1.0E+00

Hyp

er-o

smot

ic

Hyp

o-os

mot

ic

*Hea

t 37o

to 2

5o C

*Hea

t 25o

to 3

7o C

Men

adio

ne

Hyd

roge

n pe

roxi

de

Dith

ioth

riet

ol (

DTT

)

Dia

mid

e

Log

Mod

ulus

of S

tabi

lity

[mol

(l m

in)-1

] *[

C(m

in)-1

]


Proof of Concept: Conclusions•Modulus of stability can be applied to the study of gene expression to perturbationto give an understanding of the “stress” and “strain” of a genomic system.

•The genomic system can be made up of a few expressed gene to larger sets ofgenes representing a specific genomic feature, such as the environmental stressresponse.

•Dithiothrietol exposure resulted in the least stability of the environmental stressresponse.

•Environmental stress response exhibited the greatest stability to hypo-osmoticshock.

•Menadione, hydrogen peroxide, diamide, and DTT had the smallest modulus ofstability values indicating that less stress (perturbation) is required per unit strain(gene expression response) compared to the osmotic and temperature perturbations.

•Further testing is needed, including statistical analysis, to give more validity to theconcept.


Current Research: Introduction

•All living organisms have developed mechanisms to maintain water potentialexerted on their cellular components due to natural changes in external osmolytes.

•Saccharomyces cerevisiae is one of a few organisms in which the effects of andresponse to sudden changes in water potential shock have been well studied.

Salt (Hyper-osmotic)

Seconds

Water Outflow

Glycerol Accumulation(minutes to hours)

Growth Arrest

Growth Resumes

Water (Hypo-osmotic)

Seconds

Glycerol Release(minutes to hours)

Water Inflow

Growth Resumes

Growth Arrest

Source: Hohmann S., 1997. Yeast Stress Response. Chapman and Hall Pub.

HIGH OSMOLARITY

INDUCTION

Chr. IV

GPD1

GPD1

Glycerol 3-Phosphate DehydrogenaseGlycerol Production

HOG Pathway


Current Research: Introduction

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0 50 100 150 200 250 300 350 400

Control1.2M0.8M0.4M

Time (min)

OD

600

Perturbation

Resumption ofGrowth

Resumption ofGrowth

Resumption ofGrowth


Current Research: Hypotheses

•Hypothesis 1 A relationship exists between the stability of expressed genes tohyper-osmotic shock and the magnitude of the perturbation

Perturbations

Genomic Responses

Linear Non-linear No Relationship

•Hypothesis 2 The stability of expressed genes to hyper-osmotic shock increases with successive changes in the magnitude of the perturbation compared to non-successive changes.


Current Research: Procedure

•Roughly 200 genes exhibiting significant expression are being studied.

•These genes make up five complete pathways. (Glycerol Lipid Metabolism, CellCycle, Glycolysis/Gluconeogenesis, MAPK signaling pathway)

•Real Time PCR using SYBR I Green will be used to measure gene expression.

•Saccharomyces cerevisiae will be subject to press perturbations of salt resulting infive (0.3 M, 0.6 M, 0.9 M, 1.2 M, and 1.4 M) final concentrations ranging frommild to severe hyper-osmotic shock.

•Selected Real Time PCR over Microarray for the following reasons:

1. Real Time PCR is generally a more accurate measure of mRNAs.

2. For proof of hypotheses a whole genome array is not necessary.

3. Estimated that cost is be less using Real Time PCR for our purposes.


Summary

•The Modulus of Stability quantitatively relates the perturbation to the genomicresponse allowing for several types of comparisons to be made.

•The Moment of Area used to quantify gene expression response and is anaggregate measure of the stability of the genomic system.

•Validation of the Modulus of Stability, beyond proof of concept, is currently ongoing.

•As with all new concepts rigorous testing and criticism is required to ultimatelydetermine the value of such a concept.


Acknowledgments

Syed Hashsham

Jim Tiedje

Bruce Dale

Susan Masten

Sean Spellman

Center for Microbial Ecology

Charlene Hardy

THE END

Stephen J. Callister

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