Gene expression of Saccharomyces cerevisiae cells exposed to commercial wood preservatives by Microarray Analysis Madison M.
Download ReportTranscript Gene expression of Saccharomyces cerevisiae cells exposed to commercial wood preservatives by Microarray Analysis Madison M.
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.