DNA Microarray Analysis of Yeast During a Diauxic Shift Leads to the Induction of PCK1 Jennie Alman & Andrea Fletcher Washington & Jefferson College Department of Biology Washington, PA Introduction Materials & Methods Continued Discussion DNA microarray analysis is a technique used dry in a 50 mL conical tube with a kim-wipe • The data provided from DNA in molecular biology to analyze the entire genome in the bottom. DEPC-treated water was microarray analysis showed several of an organism. The use of DNA microarray added to the cDNA sample to make a 25 μL analysis in model organisms can help to understand genes had been induced or repressed the function and molecular biology of more sample. Then 25 mL of 2x formamide-based in S. cerevisiae after the diauxic shift complex organisms (Brown and Botstein, 1999). hybridization buffer was added to the sample (Figure 3). Saccharomyces cerevisiae, also known as yeast, are and incubated for 10 min at 80 °C. The cDNA eukaryotic fungi microorganisms commonly used sample were transferred to the center of the • After the diauxic shift 7 genes were in baking and fermentation processes. They also slide, and the cover slip was placed on the induced by a factor of 8 or more, serve as important model organisms for researchers short edge of the slide and lowered onto the however, the pathway of these genes because of their ability to reproduce quickly and remains unknown. their relatively small genome. sample with a syringe needle. The slide and water were then placed in a 50 mL conical tube • Under aerobic conditions the gene Even in the presence of oxygen yeast display a preference for anaerobic respiration because and incubated at 37 °C overnight. The next code YKR097W was induced by a day, the slide was transferred coverslip down factor of 2.56. YKR097W correlates converting glucose to ethanol is the fastest way with gene PCK1 which codes for for them to generate cellular energy in the form of into a tube containing room temperature 2x Figure 5. Growth curve of yeast grown in the presence of glucose. phosphoenolypyruvate carboxykinase, Saccharomyces cerevisiae were cultured to measure the effect of a diauxic ATP. Aerobic respiration requires oxygen in order to SSC+0.2%SDS until the coverslip slid off of Figure 2. Labeling of microarrays with Genisphere Array 350 Kit. This shift from anaerobic to aerobic respiration. When glucose is abundant, the generate cellular energy; however, an organism may kit allows each type of cDNA to be labeled with a 3DNA Capture Reagent the enzyme responsible for the the slide. The slide was then placed into 55 °C growth of the yeast is exponential. As the yeast use up the glucose they containing fluorescent dyes so the genes can be seen as either induced or conversion of oxaloacetate into use anaerobic respiration if oxygen is not present. S. shift from anaerobic respiration to aerobic respiration. This switch causes 2x SSC +0.2%SDS, incubated for 15 min at repressed on the microarray. The yeast in the aerobic environment were phosphoenolypyruvate (PEP) during a lag in growth as the yeast cells produce the enzymes necessary for the cerevisiae undergo anaerobic respiration in the form labeled with a Cy5 capture reagent appearing red on the computer and 55 °C, placed into 2x SSC, and incubated for metabolism of sugars; this lag is known as a diauxic shift. This graph of fermentation as long as glucose is available. This the yeast in the anaerobic environment were labeled with a Cy3 capture gluconeogenesis (Figure 1). represents the growth of the yeast taken at intervals. preference for anaerobic respiration allows them to 15 min at room temperature. Next, the slide reagent appearing green on the computer. • The induction of PCK1 was expected reproduce more rapidly and gives them a survival Figure 1. Metabolic pathways indicated by gene expression. was transferred into 0.2x SSC, and incubated advantage over other microorganisms. This diagram depicts the metabolic pathways that lead to the for 15 min at room temperature. The second hybridization mix was prepared by thawing to be higher than 2.56 if the diauxic shift had been fully completed. The cells In the presence of high amounts of glucose yeast production of usable energy. The blue box highlights one gene 2x-formamide based hybridization buffer and incubating for 10 min at 55 °C. may have not entered aerobic metabolism and gluconeogenesis when they induced from the microarray data, PCK1. PCK1 codes for phos- were used for analysis and should have been cultured for a longer time period grow exponentially. As the yeast consume all of the phoenolypyruvate carboxykinase, an enzyme that converts ox- The rest of the steps are light sensitive. 25 μL of antifade-treated hyb mix, 10 μL of glucose present, they begin aerobic metabolism. At aloacetate to phosphoenolpyruvate (After DeRisis et al., 1997). (Figure 5). this time the exponential growth of the yeast begins DEPC-water, 2.5 μL of Cy3 capture reagent, and 2.5μL of Cy5 reagent were mixed and incubated for 10 min at 75 °C (Figure 2). 50 μL of the solution was pipetted onto slide • Induction of PCK1 serves as a crucial regulatory switch because it bypasses to level off as they produce the enzymes necessary for the metabolism of sugars (DeRisi et an irreversible step in glycoysis. The conversion of PEP into pyruvate by the al., 1997). This lag in the growth curve of yeast cells due to the switch from anaerobic to using the coverslip protocol. The slide was incubated at 37 °C for 2.5 hrs. The slide was enzyme pyruvate kinase is one of the most highly regulated steps of glycolysis aerobic respiration is known as a diauxic shift. transferred, coverslipside down, into a tube containing room temperature 2x SSC + 0.2% and is diverted during gluconeogensis by phosphoenolpyruvate carboxykinase When and where a gene is expressed provides strong evidence towards its biological SDS +1mM DTT. The slide was sloshed gently until the coverslip came off, transferred into (DeRisi et al., 1997). role (DeRisi et al., 1997). DNA microarrays can be used to measure the change in 55 °C 2x SSC +1mM DTT, and incubated for 15 min at room temperature. The slide was expression levels of an entire genome under different conditions. As S. cerevisiae adapt then transferred into 0.2x SSC + 1mM DTT and incubated for 15 min at room temperature. • The presence of glucose significantly represses the expression of PCK1, to environmental conditions, certain genes are repressed or induced due to the change in and the translation of PEP carboxykinase in S. cerevisiae, as well as other the metabolic pathway from glycolysis and fermentation to gluconeogenesis and aerobic The slide was placed into a 50 mL tube with a kim-wipe at the bottom and spun dry. The tube was stored in a conical tube, covered with foil, and sent to Davidson College for eukaryotic organisms (Leuker et al., 1997). Future experimentation may metabolism (i.e. TCA cycle and oxidative phosphorylation) (Figure 1). include the use of conditions under varying levels of glucose to determine DNA microarrays show great promise in understanding the mechanism of diseases, scanning. The data obtained from the scan were analyzed using Magic Tool software the influence of glucose levels on PCK1. Also the use of other eukaryotic finding new treatments, and learning about the genomic underpinnings of human disorders (GCAT, 2008). organisms such as C. elegans, would be useful in determining the induction (Friend and Stoughton, 2002). DNA microarray analysis of S. cerevisiae will provide and repression of PCK1. insight into the role environmental conditions play in the expression of genes involved in Results metabolic pathways. • In relation to human disorders, type 2 diabetes mellitus and obesity may be associated with the regulation in the expression of PCK1. The disregulation affects the storage and release of fatty acids (Beal et al., 2004). Further DNA Materials & Methods microarray exploration with PCK1 will provide information on the function, S288C S. cerevisiae were cultured to measure the effect of a diauxic shift from regulation, and molecular biology of phosphoenolypyruvate carboxykinase in anaerobic to aerobic respiration. The measurements were monitored by taking samples prevalent human disorders. and plotted on a curve growth of the yeast in the presence of glucose (Figure 5). A colony of S288C yeast was transferred to 5 mL YPD and incubated at 30 °C overnight with shaking. 1 mL of this solution was transferred to 200 mL YPD in a 500 mL flask Literature Cited and incubated with shaking at 30 °C. The Absorbance at 600 nm was taken after eight • Beale, E., Hammer, R., Antoine, B., Forest, C. 2004. Deregulated glyceroneogenesis: hrs to determine if it is the desired value. The volumes of yeast culture were collected PCK1 as a candidate diabetes and obesity gene. Trends in Endocrinology and in separate tubes that correlate with capacity of the RNA isolation kit. The yeast Metabolism. 15(3): 129-135. cultures were spun in a clinical centrifuge for 10 min. The supernatant was poured • Brown, P., Botstein, D. 1999. Exploring the new world of the genome with DNA off and refrigerated for use as the “reference” yeast. The other yeast were grown until microarrays. Nature America. 21: 33-37. the absorbance at 600nm was approximately 6.9 for use as the “experimental” yeast • DeRisi, J.L., Vishwanath, R.I., Brown, P.O. 1997. Exploring the metabolic and genetic (GCAT, 2008). The yeast RNA were prepared from a Ribopure Yeast Kit (Ambion, control of gene expression on a genome scale. Science. 278: 690-278. Austin, TX) by resuspending the pellets with 2 x 108 cells in a mixture of lysis buffer, • Friend, S.H., Stoughton, R.B. 2002. The magic of microarrays. Scientific American. 286 (2): 44-53. SDS, and phenol/chloroform/IAA. The quantity of RNA was checked with a UV • Genome Consortium for Active Teaching (GCAT). 2008. http://www.bio.davidson.edu/projects/GCAT. spectrophotometer at 260 and 280 nm. The quality of the RNA was determined by • Leuker C., Sonneborn, A., Delbrück, Ernst, J. 1997. Sequence and promote regulation of running a 1 μg sample on a 1% agarose gel. The gel was run for 30 min at 75-100 V. the PCK1 gene encoding phosphoenolpyruvate carboxykinase of the fungal 10 μg aliquots of RNA were precipitated to be labeled by the florescent capture tags. Figure 4. Gridding and flagging of DNA microarray data. Using Magic Tool 32 grids pathogen Candida albicans. Gene. 192: 235-240. The cDNA were synthesized from the mRNA using reverse transcriptase and specific per each microarray plate were created, each grid had the dimensions of 20 rows by 22 columns. Manual flagging was done to exclude irregular spotting from the microarray. primers. The mRNA were then degraded by sodium hydroxide. A Genisphere Array 350 The blue “X’s” represent the squares that were flagged and excluded from data analysis. Kit (Genisphere, Hatfield, PA) was used to label the cDNA with a fluorescent capture Acknowledgments Figure 3. Yeast genome microarray. Yeast were grown in the presence of glucose and analyzed after tag that will bind to a dendrimer tag on each type of cDNA. The cDNA were purified the diauxic shift occurred from anaerobic to aerobic respiration. The cDNA of each type was labeled We would like to thank Dr. Alice G. Lee and the Genome Consortium for Active Teaching for help in the using a Qiagen PCR CleanUp Kit (Qiagen, Valencia, CA). The microarray slides were with a fluorescent cDNA probe and prepared from mRNA isolated from yeast cells harvested shortly development of this project. This work was supported in part by Undergraduate Science Program Educa- prehybridized by incubating the slides for an hour in 3x SSC, 0.1% SDS, and 0.1mg/ after inoculation. This image was gathered from the scanning of the slides and visualized using Mag- ic Tool software. Green dots indicate repressed genes, red dots indicate induced genes, yellow dots tion Grant No. 52006323 from the Howard Hughes Medical Institute to Washington & Jefferson College. mL sonicated salmon sperm DNA. The slides were dipped into distilled water and spun indicate no change in gene expression, and black dots indicate the gene was not expressed.