Gene expression of Saccharomyces cerevisiae cells
exposed to commercial wood preservatives by Microarray Analysis
Madison M. Stevens,1 Gary P. Lutz,2 Consuelo J. Alvarez.1 1Department of Biological and Environmental Sciences,
2Prince Edward County High School, Longwood University, Farmville, Virginia
BACKGROUND. With the ever increasing availability of genomic information, working with technologies such as DNA microarray allows researchers to move away from
traditional genetics research where one gene is studied at a time. Instead, an entire genome can be evaluated simultaneously and potential interactions among genes
can be investigated. To explore potential effects of chemical stressors on gene expression, baker’s yeast, Saccharomyces cerevisiae, is used as a model genetic system
due to its simple structure and regulatory gene functions. Gene expression is the process of transferring information from DNA to mRNA and from mRNA to proteins.
The degree of change in mRNA levels between stressed cells and non-stressed cells is an indication of differential gene expression in an organism. Microarray analysis
has the potential to provide insight into the mode of action of toxic and or carcinogenic agents. Pentachlorophenol (PtCP) (see figure 1A) and creosote (see figure 1B)
are commercial wood preservatives that are known to be toxic to humans and wildlife, and both are still in use today protecting railroad ties, utility poles, and wharf pilings
from insect and water damage (see figure 1C).
Creosote Genes
RESULTS.
Gene Name Log2 Expression
Tables 1 (top) and 2 (bottom) Genes
highlighted are a subset of induced (red) and
repressed (green) genes involved in the cell
division cycle. A subset of unknown genes
are highlighted in blue
Ratio
Fold
Biological Function
Molecular Function
YNL170W
-5.431
0.023
43.143
Unknown
Unknown
Fig. 1B Some phenolic constituents of creosote
Fig. 1C Current uses of pentachlorophenol and creosote
Figs. 6 (on left) A subset of induced and
repressed genes with a foreground of red
and green dye intensities equal to or above
200 and a gene expression fold change of 2
or greater
RESEARCH GOALS.
1) To use microarray analysis techniques to measure changes in gene expression when Saccharomyces cerevisiae cells are exposed to Pentachlorophenol or Creosote.
2) To identify links between the changes in gene expression and the biological, molecular and cellular functions of the effected genes.
METHODS. The yeast cells, Saccharomyces cerevisiae, specifically, strain X21801A, were chosen for this genetic investigation. The cells were exposed to the wood
preservatives pentachlorophenol and creosote at concentrations of 50uM or 50
ng/mL respectively for 5-6 hours. As the control, a genetically identical culture of
cells was raised in an analogous fashion; but, it was not exposed to either
chemical. Cells from both control and experimental conditions were harvested at
the log phase, and mRNA was extracted. A non-denaturing agarose gel confirmed
the integrity of both mRNA samples, which were then subjected to the microarray
procedure (see figure 2).
Control
Experimental
Unknown
YDR097C
-1.115
0.462
2.166
mismatch repair
DNA binding activity
nucleus
YDR461W
-1.749
0.297
3.362
signal transduction during conjugation with cellular fusion
pheromone activity
soluble fraction
YDR507C
-1.355
0.391
2.559
protein amino acid phosphorylation
protein kinase activity
bud neck
YDR509W
-5.318
0.025
39.900
Unknown
Unknown
Unknown
YFL036W
-2.639
0.161
6.229
mitochondrial genome maintenance
DNA-directed RNA polymerase activity
mitochondrial matrix
YFL050C
-3.380
0.096
10.412
di-, tri-valent inorganic cation transport
di-, tri-valent inorganic cation transporter activity
plasma membrane
YFL052W
-6.552
0.011
93.800
Unknown
DNA binding activity
Unknown
YGL239C
-1.700
0.308
3.248
Unknown
Unknown
Unknown
YGR022C
-5.545
0.021
46.688
Unknown
Unknown
Unknown
YGR026W
-2.318
0.201
4.986
Unknown
Unknown
Unknown
YGR027W-A
-1.070
0.476
2.100
transposition, RNA-mediated
RNA binding
retrotransposon nucleocapsid
YHR041C
-1.091
0.469
2.131
transcription from Pol II promoter
RNA polymerase II transcription mediator activity
mediator complex
YHR043C
-1.341
0.395
2.534
response to stress
2-deoxyglucose-6-phosphatase activity
cytoplasm
YAR044W
-1.603
0.329
3.038
steroid biosynthesis
oxysterol binding activity
Golgi trans cisterna
YLR154W-A
-1.727
0.302
3.311
Unknown
Unknown
mitochondrion
YMR077C
1.064
2.091
2.091
protein binding
ESCRT III complex, cytoplasm
YMR081C
1.438
2.710
2.710
aerobic respiration
Unknown
Unknown
YOL105C
1.013
2.018
2.018
cell wall organization and biogenesis
transmembrane receptor activity
membrane fraction
YMR096W
1.185
2.273
2.273
pyridoxine metabolism
protein binding
Unknown
YBR161W
1.084
2.120
2.120
glycosphingolipid biosynthetic process
transferase activity, transferring glycosyl groups
vacuole
YDR210W-C
1.017
2.024
2.024
transposition, RNA-mediated
RNA binding
retrotransposon nucleocapsid
YLR243W
1.872
3.660
3.660
Unknown
signal sequence binding
Unknown
YOR204W
1.013
2.018
2.018
translational initiation
RNA helicase activity
YDR309C
1.818
3.527
3.527
establishment of cell polarity
small GTPase regulatory/interacting protein activity
bud tip
YJL212C
1.117
2.169
2.169
sulfur metabolism
oligopeptide transporter activity
integral to plasma membrane
YOL007C
late endosome to vacuole transport
cytoplasm
1.006
2.008
2.008
Unknown
Unknown
bud neck
YIR034C
1.135
2.196
2.196
lysine biosynthesis, aminoadipic pathway
saccharopine dehydrogenase (NAD+, L-lysine forming) activity
cytoplasm
YLR297W
1.072
2.103
2.103
Unknown
Unknown
vacuole
YOL136C
1.060
2.084
2.084
fructose 2,6-bisphosphate metabolism
6-phosphofructo-2-kinase activity
cytoplasm
YGL013C
1.021
2.029
2.029
regulation of transcription from Pol II promoter
DNA binding activity
nucleus
YOL052C-A
1.139
2.203
2.203
response to stress
Unknown
cytoplasm
YER032W
1.288
2.442
2.442
mRNA polyadenylation
Unknown
YGL063W
1.425
2.686
2.686
tRNA modification
pseudouridylate synthase activity
Unknown
YGL065C
1.033
2.046
2.046
oligosaccharide-lipid intermediate assembly
glycolipid mannosyltransferase activity
endoplasmic reticulum
YNL307C
1.000
2.000
2.000
protein amino acid phosphorylation
glycogen synthase kinase 3 activity
soluble fraction
YPL114W
2.941
7.680
7.680
Unknown
Unknown
Unknown
YPL120W
1.319
2.495
2.495
protein-vacuolar targeting
Unknown
membrane fraction
bud neck
YPR199C
3.139
8.806
8.806
positive regulation of transcription from Pol II promoter
RNA polymerase II transcription factor activity
nucleus
YPR201W
5.797
55.600
55.600
arsenite transport
arsenite transporter activity
integral to plasma membrane
YPR203W
1.641
3.118
3.118
Unknown
Unknown
Unknown
PtCP Genes
Gene Name Log2 Expression
Fig. 1A Pentachlorophenol (PtCP)
Cellular Component
Ratio
Fold
Biological Function
Molecular Function
Cellular Component
YHL047C
1.385
2.612
2.612
iron ion homeostasis
siderochrome-iron transporter activity
plasma membrane
YKL071W
3.667
12.705
12.705
Unknown
Unknown
cytoplasm
YJL116C
1.919
3.782
3.782
mitochondrion organization and biogenesis
Unknown
Unknown
YPL058C
3.767
13.617
13.617
transport
xenobiotic-transporting ATPase activity
plasma membrane
YOR371C
2.340
5.063
5.063
signal transduction
signal transducer activity
cytoplasm, plasma membrane
YNL277W
2.209
4.624
4.624
methionine biosynthesis
homoserine O-acetyltransferase activity
cytoplasm
YNL279W
3.005
8.028
8.028
plasma membrane fusion
Unknown
integral to membrane
YOR382W
1.736
3.331
3.331
siderochrome transport
Unknown
cell wall
YDR011W
3.450
10.931
10.931
response to drug
xenobiotic-transporting ATPase activity
plasma membrane
YGR281W
1.661
3.163
3.163
transport
xenobiotic-transporting ATPase activity
plasma membrane
YLR303W
1.915
3.771
3.771
methionine metabolism
O-acetylhomoserine (thiol)-lyase activity
cytoplasm
YOR383C
2.415
5.333
5.333
siderochrome transport
Unknown
cell wall
YHL040C
2.334
5.043
5.043
iron-siderochrome transport
siderochrome-iron transporter activity
endosome
Unknown
YDR133C
-2.114
0.231
4.329
Unknown
Unknown
YKR080W
-1.368
0.387
2.582
one-carbon compound metabolism
methylenetetrahydrofolate dehydrogenase (NAD+) activity
cytosol
YDR165W
-1.068
0.477
2.097
tRNA methylation
tRNA (guanine-N7-)-methyltransferase activity
nucleus
YJL122W
-1.479
0.359
2.787
ribosomal large subunit biogenesis and assembly
Unknown
cytoplasm, nucleus
YJL198W
-1.502
0.353
2.833
phosphate transport
phosphate transporter activity
membrane
YGL234W
-1.729
0.302
3.315
purine base metabolism
phosphoribosylamine-glycine ligase activity
cytoplasm
YFR015C
-2.307
0.202
4.948
glycogen metabolism
glycogen (starch) synthase activity
cytoplasm, mitochondrion
YOR095C
-1.604
0.329
3.041
pentose-phosphate shunt
ribose-5-phosphate isomerase activity
cytoplasm, nucleus
YLR061W
-2.026
0.246
4.073
protein biosynthesis
structural constituent of ribosome
cytosolic large ribosomal subunit
YFR037C
-1.462
0.363
2.754
chromatin modeling
contributes to DNA-dependent ATPase activity
nucleus
YMR142C
-1.759
0.295
3.385
protein biosynthesis
structural constituent of ribosome
cytosolic large ribosomal subunit
YBR181C
-1.581
0.334
2.993
protein biosynthesis
structural constituent of ribosome
cytoplasm
YMR011W
-1.719
0.304
3.292
hexose transport
glucose transporter activity
plasma membrane
YGR159C
-1.173
0.444
2.255
rRNA processing
RNA binding activity
nucleus
YOR096W
-1.654
0.318
3.147
protein biosynthesis
structural constituent of ribosome
cytosolic small ribosomal subunit
YJR145C
-1.488
0.357
2.804
protein biosynthesis
structural constituent of ribosome
cytoplasm
YKL006W
-1.765
0.294
3.398
protein biosynthesis
structural constituent of ribosome
cytosolic large ribosomal subunit
cytoplasm
YLR058C
-1.978
0.254
3.941
one-carbon compound metabolism
glycine hydroxymethyltransferase activity
YKR081C
-1.373
0.386
2.590
ribosomal large subunit assembly and maintenance
rRNA binding activity
nucleolus
YNL141W
-1.760
0.295
3.388
adenine catabolism
adenine deaminase activity
cytoplasm, nucleus
YAL044C
-1.155
0.449
2.227
one-carbon compound metabolism
glycine dehydrogenase (decarboxylating) activity
mitochondrion
YBR154C
-1.786
0.290
3.448
transcription from Pol II promoter
DNA-directed RNA polymerase activity
DNA-directed RNA polymerase III complex
cytosolic small ribosomal subunit
YLR441C
-1.671
0.314
3.184
protein biosynthesis
structural constituent of ribosome
YBR103C-A
-2.343
0.197
5.072
Unknown
Unknown
Unknown
YGL123W
-1.952
0.258
3.869
protein biosynthesis
structural constituent of ribosome
cytosolic small ribosomal subunit
YGR208W
-1.305
0.405
2.470
serine family amino acid biosynthesis
phosphoserine phosphatase activity
cytoplasm, nucleus
YKL180W
-1.560
0.339
2.948
protein biosynthesis
structural constituent of ribosome
cytoplasm
YLR150W
-1.829
0.281
3.554
telomere maintenance
telomeric DNA binding activity
cytoplasm
YGL239C Score=6881 bits (3471),Expect=0.0 Identities=3600/3602 (99%), Gaps = 0/3602 (0%) Strand=Plus/Plus
Query 163
YLR150W
AATCCTGTGGACTTT………………………………………………………..GAGTAAGC 3764
| | | | | || | | | | | | | |
AATCCTGTGGACTTT………………………………………………………..GAGTAAGC 3602
YHR043C
YOL105C
| | | | | | | |
Sbjct 1
YDR461W
YOR371C
YBR161W
YDR507C
Cytoplasm
Vacuole
Vacuole
YFL036W
Fig. 2 The Microarray Technique
YOL007C
Cytoplasm
Mitochondrion
Mitochondrion
Bud
YLR154W-A
DATA ANALYSIS. The computer program MAGIC Tool (Heyer et al., 2005) was used to obtain the data from the scanned chips (see figure 3 for a PtCP chip). The
program is available from the following web address: “http://www.bio.davidson.edu/projects/magic/magic.html”. MAGIC Tool retrieves the scanned images and the gene list
that contains the order in which the genes are printed on a chip. The data is converted into a color image that ranges from green at one end of the spectrum and passing
through yellow to red at the other end of the spectrum. The Cy3 green fluorescent dye was always used with the wild type RNA and the Cy5 red fluorescent dye was
always used for the stressed sample (exposed to pentachlorophenol or creosote). Therefore, green colors on the chip indicate gene repression, red indicates gene
induction, and yellow indicates nearly equal gene expression from the unexposed and exposed cells (see figure 4). The Genomic Consortium for Active Teaching (GCAT)
has a gene list that provides all the information for each gene on the chip. The gene expression data in Magic Tool can be analyzed with its own filtering tools or it can be
exported to be analyzed by other programs like Microsoft Excel (see figure 5).
Nucleus
YKL071W Score=8407 bits (4241),Expect=0.0 Identities=4328/4328 (100%), Gaps=0/4328 (0%) Strand=Plus/Plus
Query 1
| | | | | || | | | || || | | |
| | | | | | | | || | | || |
Fig. 3B Close up of PtCP microarray chip
Fig. 3A Scanned PtCP microarray chip
Fig. 4 Gene expression color scale
RedFGavg RedBGavg GrnFGavg
0.885906
1 0.865772
1.04698 0.948113
1
1.087248 0.981132 1.060403
0.832215 0.886792 0.704698
1.261745 1.009434 1.33557
1.228188
1 1.013423
1.671141 1.235849 1.422819
1.04698 0.915094 0.919463
1.127517 1.009434 1.040268
1.100671 1.04717 1.033557
1.147651 0.985849 1.208054
1.744966 1.353774 1.711409
1.120805 1.009434 1.053691
1.597315 1.174528 1.932886
1.033557 0.990566 0.939597
1.006711 0.825472 0.704698
0.946309 0.877358 0.724832
1.248322 1.113208 1.107383
1.114094 1.075472 1.09396
0.919463 1.033019 0.744966
0.939597 1.004717 0.885906
1.503356 1.410377 1.463087
1.060403 0.910377 1.09396
1.194631 1.009434 0.993289
2.852349 1.896226 2.738255
1.375839 1.018868 1.214765
1.892617 1.132075 1.503356
1.355705 1.141509 0.95302
0.973154 0.877358 0.765101
1.020134 0.872642 0.818792
1.275168 0.990566 1.033557
0.818792 0.915094 0.85906
1.248322 1.009434 1.194631
1.449664 1.287736 1.825503
GrnBGavg
0.759434
0.825472
0.891509
0.778302
1
0.910377
1.103774
0.910377
0.990566
0.981132
0.95283
1.273585
1.014151
1.509434
0.943396
0.853774
0.735849
0.90566
0.915094
0.896226
0.900943
1.287736
0.834906
1.113208
1.745283
1.066038
1.056604
0.919811
0.787736
0.853774
0.981132
0.773585
1.018868
1.349057
Fig. 5
Creosote expression file in Microsoft excel
REFERENCES.
* Agency for Toxic Substances and Disease Registry (ATSDR). 2001. Toxicological Profile for Pentachlorophenol. Atlanta, GA: U.S. Department of Health and Human Services, Public Health Service.
• ATSDR. 2002. Potential for human exposure. In: Toxicological Profile for Wood Creosote, Coat Tar Creosote, Coal Tar, Coal Tar Pitch, and Coal Tar Pitch Volatiles. Atlanta, GA: U.S. Department of Health and Human Services, Public Health
Service. Available: http://www.atsdr.cdc.gov/toxprofiles/tp85-c1.pdf [accessed 11 Feb 2007].
* Creosote constituents found at this website: www.answers.com/topic/cresol [11 Feb 2007].
* Genome Consortium of Active Teaching (GCAT) website for “MAGIC TOOL program and tutorial”. http://www.bio.davidson.edu/projects/magic/magic.html. [1 May 2005].
* Heyer, L.J., Moskowitz, D.Z., Abele, J.A., Karnik, P., Choi, D., Campbell, A.M., Oldham, E.E. and Akin, B.K. (2005). Gene Expression. MAGIC Tool: integrated microarray data analysis. Bioinformatics. 21, 2114-2115.
* National Center for Biotechnology Information (NCBI) website for BLAST Software. http://www.ncbi.nlm.nih.gov/BLAST. [2 Apr 2007].
* Pollack JR, Iyer VR. 2002. Characterizing the physical genome. Nature Genetics 32: 515-521.
* Structure of pentachlorophenol found at this website: www.intox.org/.../supplem/supp/su[p2.htm [11 Feb 2007].
YDR011W
YOR095C
Sbjct 6785
AAGCTTCCTCATTTCGT………………………………………….. AGAATCTATTTCATA 11112
Fig. 8 Blast results for 2 genes of unknown function
Creo3.exp:Creo3 RedFGtot RedBGtot GrnFGtot GrnBGtot
YBR045C_rep1
132
212
129
161
YBR047W_rep1
156
201
149
175
YBR049C_rep1
162
208
158
189
YBR051W_rep1
124
188
105
165
YBR053C_rep1
188
214
199
212
YBR055C_rep1
183
212
151
193
YBR069C_rep1
249
262
212
234
YBR071W_rep1
156
194
137
193
YBR073W_rep1
168
214
155
210
YBR075W_rep1
164
222
154
208
YBR077C_rep1
171
209
180
202
YBR079C_rep1
260
287
255
270
YBR091C_rep1
167
214
157
215
YBR093C_rep1
238
249
288
320
YBR095C_rep1
154
210
140
200
YBR097W_rep1
150
175
105
181
YBR099C_rep1
141
186
108
156
YBR101C_rep1
186
236
165
192
YBR115C_rep1
166
228
163
194
YBR117C_rep1
137
219
111
190
YBR119W_rep1
140
213
132
191
YBR121C_rep1
224
299
218
273
YBR123C_rep1
158
193
163
177
YBR125C_rep1
178
214
148
236
YCR053W_rep1
425
402
408
370
YCR057C_rep1
205
216
181
226
YCR060W_rep1
282
240
224
224
YCR062W_rep1
202
242
142
195
YCR064C_rep1
145
186
114
167
YCR066W_rep1
152
185
122
181
YCR083W_rep1
190
210
154
208
YCR085W_rep1
122
194
128
164
YCR087C-A_rep1
186
214
178
216
YCR088W_rep1
216
273
272
286
YKR081C
YDR097C
YFR037C
YJL122W
YOL052C-A
YGL234W
AAGCTTCCTCATTTCGT…………………………………………..AGAATCTATTTCATA 4328
YDR309C
Fig. 7 A schematic drawing showing where some induced (red) and
repressed (green) genes are located in the budding yeast cell,
Saccharomyces cerevisisae
CONCLUSIONS. The data that has been collected to date in these experiments provide interesting areas for evaluation. Previous investigators using these wood preservatives
have indicated that these chemicals are carcinogenic, so we expect to see induction and repression of genes involved in the cell cycle division. With the creosote data, there are
indications that the genes that we have identified as being repressed are implicated in mismatch DNA repair, signal transduction during conjugation of cellular fusion, bud neck
formation, and DNA-directed RNA polymerase (see YDR097C, YDR461W, YDR507C, YFL036W genes highlighted in green in table #1and in figure 7). In similar manner, there
are indications that the genes that we have identified as being induced are involved in cell wall organization and biogenesis, bud tip formation, bud neck formation, and stress
response (see YLO105C, YDR309C, YOL007C, YOL052C-A genes highlighted in red in table #1and in figure 7). With the pentachlorophenol data, there are indications that the
genes that we have identified as being repressed are involved in ribosomal large subunit biogenesis and assembly, purine base metabolism, pentose-phosphate shunt,
chromatin modeling, and telomere maintenance (see YJL122W, YKR081C, YGL234W, YOR095C, YFR037C, YLR150W genes highlighted in green in table #2 and in figure 7).
In a similar manner, there are indications that the genes that we have identified as being induced are concerned with mitochondrial organization and biogenesis, signal
transduction, and response to drugs (see YJL116C, YOR371C, YDR011W genes highlighted in red in table #2 and in figure 7). Additionally, “blastn” analysis of some genes of
unknown function (biological, molecular, and cellular) indicate that there are similarities between the “unknown” genes and genes involved in cell division cycle (YGL239C,
YLR154W-A) or genes involved in stress response like YKL071W (see figure 8). Lastly, the pentachlorophenol data indicate that one third of the genes effected during the
exposure to this chemical are involved in protein biosynthesis (14 out of 44).
FUTURE WORK. In order to validate the results obtained in these microarray experiments, real time-polymerase chain reaction (RT-PCR) experiments performed with the
highly induced or highly repressed genes are planned. For genes with currently unknown cellular function, cellular localization will be determined by fluorescence after gene
fusion of green fluorescent protein to the respective gene. In addition, sequences of unknown genes will be evaluated using the “blastn” and “blastp” programs to check for
potential domains that could provide indications of their biological and molecular function.
ACKNOWLEDGMENTS.
Longwood Foundation and CAS Dean’s Funds, Longwood Cormier Citizen Scholarship, Genomic Consortium for Active Teaching (GCAT) and its members, and a special acknowledgment to Dr. Leigh Lunsford, Dr. Phillip
Poplin and Ms. Ashley Swandby in the Department of Mathematics and Computer Science.