Post on 02-Dec-2021
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
Department of Civil and Environmental Engineering
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.
Department of Civil and Environmental Engineering
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
Department of Civil and Environmental Engineering
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
Department of Civil and Environmental Engineering
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
Department of Civil and Environmental Engineering
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
Department of Civil and Environmental Engineering
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
 ⋅=i
ii tAMAreaofMoment0
,
Graphical Depiction of the Moment of Area
Department of Civil and Environmental Engineering
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).
Department of Civil and Environmental Engineering
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.
Department of Civil and Environmental Engineering
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)
Department of Civil and Environmental Engineering
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.
Department of Civil and Environmental Engineering
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
Department of Civil and Environmental Engineering
Proof of Concept: Results
StressPerturbationAsymptoticExpressionPatterns(Percent)Non-asymptoticExpressionPatterns(Percent)Heat Shock 25oto 37o C944TemperatureHeat Shock 37oto 25o C8212Hyper-osmoticShock942OsmoticHypo-osmoticShock829HydrogenPeroxideExposure904OxidativeMenadioneExposure8214DTT Exposure906Protein(Chemical)DiamideExposure944
Department of Civil and Environmental Engineering
Proof of Concept: Results
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
Department of Civil and Environmental Engineering
Proof of Concept: Results
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
Department of Civil and Environmental Engineering
Proof of Concept: Results
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
Department of Civil and Environmental Engineering
Proof of Concept: Results
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
]
Department of Civil and Environmental Engineering
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.
Department of Civil and Environmental Engineering
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
Department of Civil and Environmental Engineering
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
Department of Civil and Environmental Engineering
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.
Department of Civil and Environmental Engineering
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.
Department of Civil and Environmental Engineering
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.
Department of Civil and Environmental Engineering
Acknowledgments
Syed Hashsham
Jim Tiedje
Bruce Dale
Susan Masten
Sean Spellman
Center for Microbial Ecology
Charlene Hardy
THE END