1. +
Differential gene expression in Saccharomyces
cerevisiae as a result of exposure to
(-)-epicatechin, a flavanol antioxidant found in cocoa
Gen Selden
Pine Crest School

2. +
Background
 Free radicals, or reactive oxygen species (ROS) are constantly
produced from oxygen metabolism in cells
 Examples of free radicals: OH, O2
-, H2O2
 Oxidative stress – cumulative effect of ROS causing
degradation of DNA, lipids, and proteins and damaging the
cells
 Oxidative stress has been seen to play a role in certain neurodegenerative
diseases and cancer
 Antioxidants react with free radicals to reduce oxidative stress
and improve longevity
 Flavanols are a specific type of antioxidant and are thought to
be the main antioxidants present in cocoa
 Predominant antioxidant properties of cocoa are thought to be
mainly a result of (-)-epicatechin

3. +
Background
 (-)-Epicatechin:
 A specific flavanol antioxidant found in cocoa
 May have beneficial effects on certain aspects of health including aging
and disease
 Found in higher concentrations in cocoa than other flavanols such as (+)-
catechin and quercetin
 Found in higher concentrations in cocoa than in other commonly known
sources of antioxidants such as fruits and vegetables
 Saccharomyces cerevisiae – Yeast
 Simple and rapid reproduction
 Easily cultured
 Model organism for humans
 31% homology between genomes
 Humans and yeast share many homologous genes

4. +
Background
 Longevity genes in yeast
 RAS2 - involved in cell proliferation
 IDH2 – associated with the oxidative decarboxylation of the citric
acid cycle and is necessary for cellular respiration
 HST2 – transcriptional silencing, DNA repair, genomic stability, and
overall longevity
 DNM1 – programmed cell death
 Oxidative stress genes in yeast
 CTA1 – codes for the enzyme catalase A, which catalyzes the
breakdown of H2O2 and other oxidative molecules
 GSH1 – codes for GSH, a radical scavenger protein

5. +
Purpose
 To test the effects of varying concentrations of (-)-
epicatechin on the gene expression of the longevity
genes RAS2, IDH2, HST2, and DNM1 and the
oxidative stress genes CTA1 and GSH1 in
Saccharomyces cerevisiae, a model organism for
humans

6. +
Hypothesis
Gene Function Human
Homolog
% Homology Hypothesis
RAS2 Longevity KRAS 54% Upregulated
IDH2 Longevity IDH3A 56% Upregulated
HST2 Longevity SIRT2 61% Upregulated
DNM1 Longevity DNM1L 55% Downregulated
CTA1 Oxidative stress CAT 54% Downregulated
GSH1 Oxidative stress GCLC 53% Downregulated
 RAS2, IDH2, and HST2 are expected to be upregulated because they are
involved in cellular processes necessary for longevity
 DNM1 is expected to be downregulated because of its involvement in
programmed cell death
 CTA1 and GSH1 are expected to be downregulated because exposure to the
antioxidant molecule (-)-epicatechin may reduce the need for cellular
protection against oxidative stress by radical-scavenging proteins

7. +
Materials and Methods
 Yeast was cultured in YPD media at 25oC while rotating for 13.5
hours and exposed to 6 different concentrations of (-)-epicatechin
for 36.5 hours following growth
 0 uM, 50 uM, 100 uM, 250 uM, 500 uM, and 750 uM
 Total RNA isolated using RiboPure Yeast protocol from Ambion
Life Technologies
 RNA presence in each sample confirmed using gel electrophoresis
 cDNA synthesized from extracted RNA using ProtoScript First
Strand cDNA Synthesis Kit
 Primers for PCR were designed using www.operon.com
 PCR was performed to amplify genes of interest
 Gel electrophoresis used to analyze differential gene expression

8. +
Confirmation of the presence of total
RNA
 Presence of total RNA in each
sample was confirmed using
gel electrophoresis
 The 28S and 18S bands
confirm RNA – standard
bands of total isolated RNA
28S
18S
DNA
ladder
0 uM 50 uM 100 uM250 uM500 uM750 uM

9. +
Gel Electrophoresis of HST2
 An increase in
expression was seen in
the 500 uM and 750 uM
samples
 Increase in expression
signified by more intense
bands
 Supports hypothesis that
HST2 would be
upregulated in response
to (-)-epicatechin
DNA
ladder
0 uM 50 uM 100 uM 250 uM 500 uM 750 uM NC DNA
ladder

10. +
Gel Electrophoresis of GSH1
 A decrease in expression
was seen in the 500 uM
and 750 uM samples
 Decrease in expression
signified by less intense
bands
 Supports the hypothesis
that exposure to (-)-
epicatechin leads to a
downregulation of GSH1
DNA
ladder
0 uM 50 uM 100 uM250 uM500 uM750 uM NC DNA
ladder

11. +
Conclusion
 HST2 is expressed under normal cellular conditions and seems
to be upregulated by relatively high concentrations of (-)-
epicatechin
 Suggests that (-)-epicatechin improves longevity and upregulates
genes necessary for longevity
 GSH1 is expressed under normal cellular conditions and
expression seems to be downregulated by (-)-epicatechin at
relatively high concentrations
 Suggests that (-)-epicatechin combats oxidative stress by reducing
free radicals, subsequently downregulating oxidative stress genes
that are necessary to reduce normal cellular oxidative stress

12. +
Future Research
 Tests to confirm differential gene expression of the genes that
have already been tested (GSH1, HST2)
 Test for differential gene expression of remaining genes (RAS1,
IDH2, DNM1, CTA1)
 Including patterns of expression between the longevity genes and
patterns of expression between the oxidative stress genes
 Once patterns of expression in the two gene categories have
been observed, I will expose yeast to a higher concentration
range of (-)-epicatechin (500 uM to 1000 uM)
 Because (-)-epicatechin seemed to cause differential gene
expression in the samples exposed to the two highest
concentrations, 500 uM and 750 uM, in the first trial

13. +
Future Research
 Certain mitochondrial genes such as COQ3 have also been
shown to play a significant role in oxidative stress and the rate
of cell aging
 Determining differential gene expression of these genes may give a
more complete representation of the effects of (-)-epicatechin on
longevity and oxidative stress in yeast
 Concurrent exposure of yeast to (-)-epicatechin and oxidative
stress, in the form of H2O2, may provide a more complete
understanding of the effects of (-)-epicatechin on oxidative
stress and subsequent regulation of gene expression

14. +
References
1. Mecocci P., Fanó G., Fulle S., MacGarvey U., Shinobu L., Polidori MC., Cherubini A., Vecchiet J., Senin U., Beal MF., (1999) Age-dependent increases in oxidative damage to
DNA, lipids, and proteins in human skeletal muscle. Free Radic Biol Med. 26(3-4): 303-8
2. Coyle JT., Puttfarcken P., (1993) Oxidative stress, glutamate, and neurodegenerative disorders. Science. 262(5134): 689-695
3. Valko M., Rhodes CJ., Moncol J., Izakovic M., Mazur M., (2006) Free radicals, metals and antioxidants in oxidative stress-induced cancer. PubMed. 160(1):1-40
4. Keen CL., Holt RR., Oteiza PI., Fraga CG., Schmitz HH., (2005) Cocoa antioxidants and cardiovascular health. The American Journal of Clinical Nutrition. 81(1):2985-3035
5. Schroeter H., Heiss C., Balzer J., Kleinbongard P., Keen CL., Hollenberg NK., Sies H., Kwik-Uribe C., Schmitz HH., Kelm M., (2005) (-)-Epicatechin mediates beneficial effects of
flavanol-rich cocoa on vascular function in humans. PNAS. 103(4): 1024-1029
6. Nutrient Data Laboratory US Department of Agriculture. USDA Database for the Flavonoid Content of Selected Foods. US Department of Agriculture, Agricultural Research
Service, 2007.
7. Wang JF., Schramm DD., Holt RR., Ensunsa JL., Fraga CG., Schmitz HH., Keen CL., (2000) A Dose-Response Effect from Chocolate Consumption on Plasma and Oxidative
Damage. The Journal of Nutrition. 130(8): 21155-21195
8. Spencer JPE., Schroeter H., Kuhnle G., Srai SKS., Tyrrell RM., Hahn U., Rice-Evans C., (2001) Epicatechin and its in vivo metabolite, 3’-O-methyl epicatechin, protect human
fibroblasts from oxidative-stress-induced cell death involving caspase-3 activation. Biochemical Journal 354: 493-500
9. Sun J. et al. (1994) Divergent roles of RAS1 and RAS2 in yeast longevity. The Journal of Biological Chemistry. 269: 18638-18645
10. Kataoka T., Powers S., McGill C., Fasano O., Strathern J., Broach J., Wigler M., (1984) Genetic analysis of yeast RAS1 and RAS2 genes. ScienceDirect. 37(2): 437-445
11. Si H., Fu Z., Babu PVA., Zhen W., LeRoith T., Meaney MP., Voelker KA., Jia Z., Grange RW., Liu D., (2011) Dietary Epicatechin Promotes Survival of Obese Diabetic Mice and
Drosophila melanogaster. The Journal of Nutrition. 141(6): 1095-1100
12. Kim SJ., Han D., Ahn BH., Rhee JS., (1997) Effect of Glutathione, Catechin, and Epicatechin on the Survival of Drosophila melanogaster under Paraquat Treatment. Biosci
Biotech Biochem. 61(2):225-229
13. Delaney JR., Murakami CJ., Olsen B., Kennedy BK., Kaeberlein M., (2011) Quantitative evidence for early life fitness defects from 32 longevity-associated alleles in yeast.
Landes Bioscience. 10(1): 156-165
14. Lamming DW., Latorre-Esteves M., Medvedik O., Wong SN., Tsang FA., Wang C., Lin S., Sinclair DA., (2005) HST2 Mediates SIR2-Independent Life-Span Extension by Calorie
Restriction. Science. 309(5742):1861-1864
15. Cupp JR., McAlister-Henn L., (1991) NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces
cerevisiae. J Biol Chem. 266(33): 22199-22205
16. Zhao K., Chai X., Clements A., Marmorstein R., (2003) Structure and autoregulation of the yeast Hst2 homolog of Sir2. Nature. 10: 864-871
17. Fannjiang Y., Cheng WC., Lee SJ., Qi B., Pevsner J., McCaffery JM., Hill RB., Basñez G., Hardwick JM., (2004) Mitochondrial fission proteins regulated programmed cell death in
yeast. Genes & Development. 18:2785-2797
18. Jamieson DJ., (1998) Oxidative Stress Responses of the Yeast Saccharomyces cerevisiae. Yeast. 14(16):1511-1527
19. Grant CM., Perrone G., Dawes IW., (1998) Glutathione and Catalase Provide Overlapping Defenses Protection against Hydrogen Peroxide in the Yeast Saccharomyces
cerevisiae. ScienceDirect. 253(3): 893-898
20. Grant CM., MacIver FH., Dawes IW., (1996) Glutathione is an essential metabolite required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae. Current
Genetics. 29(6): 511-515
21. Schroeder P., Klotz LO., Sies H., (2003) Amphiphilic properties of (-)-epicatechin and their significance for protection of cells against peroxynitrite. ScienceDirect. 307(1):69-73
22. Thermo Scientific., Assessment of Nucleic Acid Purity. NanoDrop Spectrophotometers, T042 Technical Bulletin. http://www.nanodrop.com/Library/T042-NanoDrop-
Spectrophotometers-Nucleic-Acid-Purity-Ratios.pdf
23. Sastre, J. et al. (2003) The role of mitochondrial oxidative stress in aging. PubMed. 35(1):1-8
24. Lee HC., Wei YH., (2012) Mitochondria and aging. Adv Exp Med Biol. 942:311-327
25. Grant CM., MacIver FH., Dawes IW., (1997) Mitochondrial function is required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae. FEBS. 410(2-3): 219-222
26. Protoscript First Strand cDNA Synthesis Kit, New England BioLabs Inc., https://www.neb.com/products/e6300-protoscript-first-strand-cdna-synthesis-kit, February 6, 2014.