DNA Microarray Analysis of Yeast During a Diauxic Shift Leads to the Induction of PCK1

Jennie Alman & Andrea FletcherWashington & Jefferson College Department of Biology

Washington, PA

Introduction Materials & Methods Continued

Results

Discussion

Literature Cited

Acknowledgments

DNA microarray analysis is a technique used in molecular biology to analyze the entire genome of an organism. The use of DNA microarray analysis in model organisms can help to understand the function and molecular biology of more complex organisms (Brown and Botstein, 1999). Saccharomyces cerevisiae, also known as yeast, are eukaryotic fungi microorganisms commonly used in baking and fermentation processes. They also serve as important model organisms for researchers because of their ability to reproduce quickly and their relatively small genome.

Even in the presence of oxygen yeast display a preference for anaerobic respiration because converting glucose to ethanol is the fastest way for them to generate cellular energy in the form of ATP. Aerobic respiration requires oxygen in order to generate cellular energy; however, an organism may use anaerobic respiration if oxygen is not present. S. cerevisiae undergo anaerobic respiration in the form of fermentation as long as glucose is available. This preference for anaerobic respiration allows them to reproduce more rapidly and gives them a survival advantage over other microorganisms.

In the presence of high amounts of glucose yeast grow exponentially. As the yeast consume all of the glucose present, they begin aerobic metabolism. At this time the exponential growth of the yeast begins to level off as they produce the enzymes necessary for the metabolism of sugars (DeRisi et al., 1997). This lag in the growth curve of yeast cells due to the switch from anaerobic to aerobic respiration is known as a diauxic shift.

When and where a gene is expressed provides strong evidence towards its biological role (DeRisi et al., 1997). DNA microarrays can be used to measure the change in expression levels of an entire genome under different conditions. As S. cerevisiae adapt to environmental conditions, certain genes are repressed or induced due to the change in the metabolic pathway from glycolysis and fermentation to gluconeogenesis and aerobic metabolism (i.e. TCA cycle and oxidative phosphorylation) (Figure 1).

DNA microarrays show great promise in understanding the mechanism of diseases, finding new treatments, and learning about the genomic underpinnings of human disorders (Friend and Stoughton, 2002). DNA microarray analysis of S. cerevisiae will provide insight into the role environmental conditions play in the expression of genes involved in metabolic pathways.

The data provided from DNA •microarray analysis showed several genes had been induced or repressed in S. cerevisiae after the diauxic shift (Figure 3).After the diauxic shift 7 genes were •induced by a factor of 8 or more, however, the pathway of these genes remains unknown.

Under aerobic conditions the gene •code YKR097W was induced by a factor of 2.56. YKR097W correlates with gene PCK1 which codes for phosphoenolypyruvate carboxykinase, the enzyme responsible for the conversion of oxaloacetate into phosphoenolypyruvate (PEP) during gluconeogenesis (Figure 1). The induction of PCK1 was expected •to be higher than 2.56 if the diauxic shift had been fully completed. The cells may have not entered aerobic metabolism and gluconeogenesis when they were used for analysis and should have been cultured for a longer time period (Figure 5).Induction of PCK1 serves as a crucial regulatory switch because it bypasses •an irreversible step in glycoysis. The conversion of PEP into pyruvate by the enzyme pyruvate kinase is one of the most highly regulated steps of glycolysis and is diverted during gluconeogensis by phosphoenolpyruvate carboxykinase (DeRisi et al., 1997).The presence of glucose significantly represses the expression of PCK1, •and the translation of PEP carboxykinase in S. cerevisiae, as well as other eukaryotic organisms (Leuker et al., 1997). Future experimentation may include the use of conditions under varying levels of glucose to determine the influence of glucose levels on PCK1. Also the use of other eukaryotic organisms such as C. elegans, would be useful in determining the induction and repression of PCK1.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 microarray exploration with PCK1 will provide information on the function, regulation, and molecular biology of phosphoenolypyruvate carboxykinase in prevalent human disorders.

S288C S. cerevisiae were cultured to measure the effect of a diauxic shift from anaerobic to aerobic respiration. The measurements were monitored by taking samples 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 and incubated with shaking at 30 °C. The Absorbance at 600 nm was taken after eight hrs to determine if it is the desired value. The volumes of yeast culture were collected in separate tubes that correlate with capacity of the RNA isolation kit. The yeast cultures were spun in a clinical centrifuge for 10 min. The supernatant was poured off and refrigerated for use as the “reference” yeast. The other yeast were grown until the absorbance at 600nm was approximately 6.9 for use as the “experimental” yeast (GCAT, 2008). The yeast RNA were prepared from a Ribopure Yeast Kit (Ambion, Austin, TX) by resuspending the pellets with 2 x 108 cells in a mixture of lysis buffer, SDS, and phenol/chloroform/IAA. The quantity of RNA was checked with a UV spectrophotometer at 260 and 280 nm. The quality of the RNA was determined by running a 1 μg sample on a 1% agarose gel. The gel was run for 30 min at 75-100 V. 10 μg aliquots of RNA were precipitated to be labeled by the florescent capture tags. The cDNA were synthesized from the mRNA using reverse transcriptase and specific primers. The mRNA were then degraded by sodium hydroxide. A Genisphere Array 350 Kit (Genisphere, Hatfield, PA) was used to label the cDNA with a fluorescent capture tag that will bind to a dendrimer tag on each type of cDNA. The cDNA were purified using a Qiagen PCR CleanUp Kit (Qiagen, Valencia, CA). The microarray slides were prehybridized by incubating the slides for an hour in 3x SSC, 0.1% SDS, and 0.1mg/mL sonicated salmon sperm DNA. The slides were dipped into distilled water and spun

Beale, E., Hammer, R., Antoine, B., Forest, C. 2004. Deregulated glyceroneogenesis: • PCK1 as a candidate diabetes and obesity gene. Trends in Endocrinology and Metabolism. 15(3): 129-135.

Brown, P., Botstein, D. 1999. Exploring the new world of the genome with DNA • microarrays. Nature America. 21: 33-37.

DeRisi, J.L., Vishwanath, R.I., Brown, P.O. 1997. Exploring the metabolic and genetic • control of gene expression on a genome scale. Science. 278: 690-278.

Friend, S.H., Stoughton, R.B. 2002. The magic of microarrays. • Scientific American. 286 (2): 44-53.Genome Consortium for Active Teaching (GCAT). 2008. http://www.bio.davidson.edu/projects/GCAT.• Leuker C., Sonneborn, A., Delbrück, Ernst, J. 1997. Sequence and promote regulation of •

the PCK1 gene encoding phosphoenolpyruvate carboxykinase of the fungal pathogen Candida albicans. Gene. 192: 235-240.

Figure 5. Growth curve of yeast grown in the presence of glucose. Saccharomyces cerevisiae were cultured to measure the effect of a diauxic shift from anaerobic to aerobic respiration. When glucose is abundant, the growth of the yeast is exponential. As the yeast use up the glucose they shift from anaerobic respiration to aerobic respiration. This switch causes a lag in growth as the yeast cells produce the enzymes necessary for the metabolism of sugars; this lag is known as a diauxic shift. This graph represents the growth of the yeast taken at intervals.

Figure 1. Metabolic pathways indicated by gene expression. This diagram depicts the metabolic pathways that lead to the production of usable energy. The blue box highlights one gene induced from the microarray data, PCK1. PCK1 codes for phos-phoenolypyruvate carboxykinase, an enzyme that converts ox-aloacetate to phosphoenolpyruvate (After DeRisis et al., 1997).

Materials & Methods

dry in a 50 mL conical tube with a kim-wipe in the bottom. DEPC-treated water was added to the cDNA sample to make a 25 μL sample. Then 25 mL of 2x formamide-based hybridization buffer was added to the sample and incubated for 10 min at 80 °C. The cDNA sample were transferred to the center of the slide, and the cover slip was placed on the short edge of the slide and lowered onto the sample with a syringe needle. The slide and water were then placed in a 50 mL conical tube and incubated at 37 °C overnight. The next day, the slide was transferred coverslip down into a tube containing room temperature 2x SSC+0.2%SDS until the coverslip slid off of the slide. The slide was then placed into 55 °C 2x SSC +0.2%SDS, incubated for 15 min at 55 °C, placed into 2x SSC, and incubated for 15 min at room temperature. Next, the slide was transferred into 0.2x SSC, and incubated for 15 min at room temperature. The second hybridization mix was prepared by thawing 2x-formamide based hybridization buffer and incubating for 10 min at 55 °C.

The rest of the steps are light sensitive. 25 μL of antifade-treated hyb mix, 10 μL of 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 using the coverslip protocol. The slide was incubated at 37 °C for 2.5 hrs. The slide was transferred, coverslipside down, into a tube containing room temperature 2x SSC + 0.2% SDS +1mM DTT. The slide was sloshed gently until the coverslip came off, transferred into 55 °C 2x SSC +1mM DTT, and incubated for 15 min at room temperature. The slide was then transferred into 0.2x SSC + 1mM DTT and incubated for 15 min at room temperature. 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 scanning. The data obtained from the scan were analyzed using Magic Tool software (GCAT, 2008).

Figure 4. Gridding and flagging of DNA microarray data. Using Magic Tool 32 grids 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. The blue “X’s” represent the squares that were flagged and excluded from data analysis.

Figure 2. Labeling of microarrays with Genisphere Array 350 Kit. This kit allows each type of cDNA to be labeled with a 3DNA Capture Reagent containing fluorescent dyes so the genes can be seen as either induced or repressed on the microarray. The yeast in the aerobic environment were labeled with a Cy5 capture reagent appearing red on the computer and the yeast in the anaerobic environment were labeled with a Cy3 capture reagent appearing green on the computer.

Figure 3. Yeast genome microarray. Yeast were grown in the presence of glucose and analyzed after the diauxic shift occurred from anaerobic to aerobic respiration. The cDNA of each type was labeled with a fluorescent cDNA probe and prepared from mRNA isolated from yeast cells harvested shortly 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 indicate no change in gene expression, and black dots indicate the gene was not expressed.

We would like to thank Dr. Alice G. Lee and the Genome Consortium for Active Teaching for help in the development of this project. This work was supported in part by Undergraduate Science Program Educa-tion Grant No. 52006323 from the Howard Hughes Medical Institute to Washington & Jefferson College.