Larkin 1

Teddy Larkin
Biology 141 Lab
Meghan Murphy
11/29/12
The Resistance of Saccharomyces cerevisiae (Yeast) Cultures Coated in Different
Sun Protection Factors of Sunscreen to Ultraviolet (UV) Radiation
Abstract
Nested parallel experiments were run on both SPF protected and unprotected
yeast. A UV dose escalation comparison trial was performed on unprotected cells to
measure the genetic liability of the rad1 mutant compared to the RAD1 wild type. In a
second trial, the impact of escalating SPF doses was measured using the same rad1 and
RAD1 yeast by exposing them to 4 seconds of UV radiation and measuring growth on
plates coated with lotion (SPF= 0) and plates coated with SPF= 15, 30, and 50,
respectively. Mean cell counts were compared using the unpaired two-tailed, students t
test. At UV exposure times of 5,10,15, and 20 seconds, the percent survival of RAD1
cells was 22%, 4.4%, 4.4%, 2.2% respectively, which was significantly higher than the
percent survival of the rad1 mutant cells (0.3%, 0.2%, 0.1%, and 0% respectively;
p<0.01). After 4 seconds of UV exposure, RAD1 and rad1 plates coated with SPF=0
lotion survived approximately 64% and 57% (p>0.05). Furthermore, SPF protected
RAD1 cells all survived at rates near 100%, regardless of the SPF strength of the
sunscreen (p >0.05). SPF-protected rad1 cells also survived near 100% when SPF=15 or
30. Genetic susceptibility to UV damage is related to the NER genes (rad1 mutants) but
this vulnerability can be mitigated with even SPF = 15 or 30 sunscreen.

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Introduction
Sunlight wreaks havoc on unprotected human skin by means of ultraviolet (UV)
radiation. There are three main types of UV radiation, UVA, UVB, and UVC, but the
biggest offenders are the first two .Ultraviolet (UV) light falls under the spectrum of light
that has a wavelength shorter than that of visible light and ranges in frequency from
10nm-400nm. UV radiation has been demonstrated to be cytotoxic, leading to mutations
and even death in certain circumstances. (Caitlin R. Russell 2006). UV light targets DNA
and damages the purine-pyrimidine structure of the double helix. By disrupting the
purine-pyrimidine linkages in DNA, UV radiation creates pyrimidine dimers, covalent
bonds between adjacent thymines and cytosines, which form kinks in the DNA double
helix (Freeman et al. 1989). These kinks disrupt the replication process and, in some
cases, can lead to cellular death. The paper, Chemical responses of single yeast cells
studied by fluorescence microspectroscopy under solution-flow conditions, discusses the
increasing importance of the study of the effects UV radiation has on organisms.
Yeast cells are often used to conduct research concerned with mutation and
irradiation because of their availability and easily observable growth. Yeast cells are
eukaryotic and are more similar to human cells than bacteria (Kevin Conant 1993). The
effects of radiation on yeast cells can be correlated to the effects it will have on other
types of cells, including human. Yeast cells share the same basic cellular architecture as
mammalian cells and have many of the same metabolic pathways. Yeast is thus a more
appropriate model for the detection of mutagens and carcinogens. Unlike bacteria, the
cellular mechanisms yeast uses to deal with DNA damage are strikingly similar to those

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in human cells. Yeast cells are also more physically robust than bacteria and can
therefore function in a wider range of environmental conditions, making them a more
versatile tool (Andrew W. Knight 2004).
The pathway responsible for resolving bulky modifications of the DNA resulting
from ultraviolet (UV) light or a variety of environmental chemicals is the nucleotide
excision repair (NER) pathway. Because the NER pathway is so highly conserved,
researchers use model systems to gain a better understanding of its role in humans
(Fagbemi et al. 2011). Saccharomyces cerevisiae is a prime example of one such model
system. S. cerevisiae is an extremely effective model because it is a unicellular eukaryote
with a sexual life cycle similar to human cells, with many pre-existing techniques of gene
manipulation (Manny, T.R. and Manny, M.L. 1993). S. cerevisiaes NER pathway
depends on the gene RAD1 and its job in repairing the UV radiation damage. The WT
strain containing the RAD1 gene codes for a nuclease that is involved in the removal of
the impaired DNA on both sides of a PD, therefore giving the cell a certain level of UV
resistance. Conversely, the mutant strain containing the mutant rad1 gene is incapable of
constructing this nuclease and is unable to repair the damage done to its DNA by UV
radiation (Pannunzio et al. 2010).
In human epithelial cells, UV radiation can cause excessive damage to DNA that
could lead to skin disorders. With the increasing amount of UV radiation coming through
the atmosphere, new methods of protecting ourselves from UV radiation must be
explored. Sunscreens have long been the most widely used protectorate by humans on
their skin (Nichols and Katiyar 2010). The effectiveness of a sunscreen is usually
determined by its sun protection factor (SPF).

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To determine the effectiveness of sunscreens, we prepared a dilution of the yeast
cell culture expressing the WT gene RAD1 and plated uniform samples on YPD plates.
The samples were then exposed to UV light while being covered in Seran wrap coated
evenly by SPF 15, SPF 30, and SPF 50 sunscreens. For the positive control, we exposed
lotion (not SPF) covered plates to UV light without any sunscreen protection. For the
negative control we exposed plates to UV light without any yeast cells plated. We
hypothesized that the sunscreen would protect the S. cerevisiae colonies against UV
radiation, and that samples that were protected would have a higher percentage survival
rate than those that received no protection. Therefore, if the SPF rating of sunscreen
contributed to its ability to absorb UV light, then the S. cerevisiae covered by SPF 50
sunscreen would be most resistant against UV light, those covered by SPF 30 would have
the second most resistance, and those covered by SPF 15 would have the third most
resistance. If our results support our hypothesis and prediction, then we can accept our
hypothesis that sunscreen compliments S. cerevisiae yeast cells resistance against UV
light.

Materials & Methods

Effects of Ultraviolet Light on Wildtype and Mutant Yeast Cultures
Two undiluted cultures of yeast cells, one expressing the wildtype (WT) gene
RAD1 and the other expressing the mutant rad1 gene, were grown at 30 degrees Celsius
for 24 hours. Two dilutions were then made, 1 mL of a 10-5 molar concentration of yeast
cell cultures expressing the WT gene, RAD1 and 1 mL of a 10-3 molar concentration of
yeast cell cultures expressing the mutant gene, rad1. After mixing, the dilutions were

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vortexed and 6-100 L samples of each dilution were plated onto 12 individual YPD
plates. A single YPD plate containing the mutant rad1 strain and single YPD plate
containing the WT RAD1 were exposed to UV light in the hood. The first pair of plates
were not exposed and the remaining 5 pairs were exposed at 5, 10, 15, 20 and 30 second
intervals respectively. Immediately after being exposed the samples were placed in a
dark bag to protect them from degradation. The plates were then incubated for one week
at room temperature. The surviving colonies on each plate were then counted in order to
calculate the total number of viable cells remaining from the original culture. To
calculate the cells/mL in the original culture the colonies on the plates that did not
undergo UV radiation were counted, divided by the amount plated, and multiplied by the
molar concentration of the dilution.
Cells/mL =

# of colonies
(mL plated) x (dilution)

After performing this equation to determine the total number of viable cells that
were originally plated on each YPD plate, we counted the number of colonies of the
plates exposed to UV radiation, which allowed us to calculate the percent survival of
yeast cells.

How Different Levels of Sunscreens Sun Protection Factor Affect Wildtype and
Mutant Yeast Cultures Under Ultraviolet Light Exposure
The impact of escalating SPF doses was measured using the same rad1 and RAD1
yeast by exposing them to 4 seconds of UV radiation and measuring growth on plates
coated with lotion (SPF= 0) and plates coated with SPF= 15, 30, and 50, respectively.

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The WT and rad1 yeasts were diluted to

and

dilutions respectively. The

diluted yeast cells were gently centrifuged immediately before they were plated. The UV
exposure time for all yeast cells was kept constant at 4 seconds. Sunscreen was spread
evenly on one side of the plastic wrap and put over the plates. The remaining plates were
1) treated with lotion under UV light exposure, 2) no protectant under UV light exposure,
and 3) not exposed to any UV light. All plates were placed immediately in the dark at
room temperature. Following a week of exposure to darkness in a brown bag, the yeast
cells were counted. Using the colony counts, the yeast cells/mL and the percent survival
of the yeast cells were calculated for RAD1 and rad. A column graph (Figure 2) was
created based on the percent survival of the yeast cells. Statistical analysis was performed
on the data in the form of an unpaired two tailed t test. This unpaired, two tailed t test was
used to compare mean cell counts between RAD1 and rad1.
Results
Mean cell counts were compared using the unpaired two-tailed, students t test. At
UV exposure times of 5,10,15, and 20 seconds, the percent survival of RAD1 cells was
22%, 4.4%, 4.4%, 2.2% respectively, which was significantly higher than the percent
survival of the rad1 mutant cells (0.3%, 0.2%, 0.1%, and 0% respectively; p<0.01). After
4 seconds of UV exposure, RAD1 and rad1 plates coated with SPF=0 lotion survived
approximately 64% and 57% (p>0.05). Furthermore, SPF protected RAD1 cells all
survived at rates near 100%, regardless of the SPF strength of the sunscreen (p
>0.05). SPF-protected rad1 cells also survived near 100% when SPF=15 or 30. Genetic
susceptibility to UV damage is related to the NER genes (rad1 mutants) but this
vulnerability can be mitigated with even SPF = 15 or 30 sunscreen.

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The data appears to show that the preponderance of yeast cells had little to no
growth when exposed to UV light for more than 25 seconds. RAD1 and rad1 yeast (Fig
1) were exposed to UV light for 4 seconds during the sunscreen experiment. Although the
survival rate of RAD1 yeast treated with SPF 15, 30, and 50 did not differ significantly;
they had significantly more growth than the RAD1 treated with lotion as seen in Figure 2.
RAD1 treated with SPF 15 had a survival rate which was significantly greater (p= 0.0066)
than that of the RAD1 treated with lotion (SPF 0). The survival rates of RAD1 yeast
treated with SPF 30 and 50 also had significantly more growth (p= 0.0223; p=0.0363)
than that of the lotion treatment. Additionally, the survival rate of rad1 yeast treated with
SPF 15, 30, and 50 differed significantly from each other. For the most part they had less
growth than the rad1 treated with lotion also seen in Figure 2. The survival rate of rad1
treated with SPF 30 was significantly greater (p= 0.0002) than the one treated with lotion.
While he survival rates of rad1 yeast treated with SPF 15 and 50 did not have
significantly more growth (p= 0.1245; p= 0.1245) than that of the lotion treatment.

Discussion
In this experiment, different levels of SPFs of sunscreen were tested on RAD1
and rad1 yeast cells to observe if the hypothesis that the higher the SPF is, the more
effective a sunscreen is at protecting the cell against UV damage was true. If the
sunscreen has a high SPF concentration, we predicted that it would protect from the UV
radiation more effectively compared to the sunscreen with low SPF. The hypothesis was

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examined by testing the growth of RAD1 and rad1 yeast cells covered with sunscreens
with 3 different SPFs after the exposure to UV radiation.
Most yeast cells had little to no growth when exposed to UV light for more than
25 seconds. RAD1 and rad1 yeast (Fig 1) were exposed to UV light for 4 seconds during
the sunscreen experiment. The 22% RAD1 at 5 seconds is significantly higher than the
0.3% survival of rad! given the same exposure, confirming the importance of that repair
pathway. Although the survival rate of RAD1 yeast treated with SPF 15, 30, and 50 did
not differ significantly, they had significantly more growth compared to that of RAD1
treated with lotion (Fig 2). The survival rate of RAD1 treated with SPF 15 was
significantly greater (p= 0.0066) than the one treated with lotion. The same trend was
observed for the survival rates of RAD1 yeast treated with SPF 30 and 50. The RAD1
yeasts protected with sunscreen of SPF 30 and 50 had significantly more growth (p=
0.0223; p=0.0363) than lotion treatment. Yeasts that were treated with sunscreen
generally had more growth than the sample that was exposed to UV radiation with no
protectant on. The data, graphs, and results of the t-test allow us to accept our hypothesis
that the higher the SPF is, the more effective a sunscreen is at protecting the cell against
UV exposure was true.
Mean cell counts were compared using the unpaired two-tailed, students t test. At
UV exposure times of 5,10,15, and 20 seconds, the percent survival of RAD1 cells was
22%, 4.4%, 4.4%, 2.2% respectively, which was significantly higher than the percent
survival of the rad1 mutant cells (0.3%, 0.2%, 0.1%, and 0% respectively; p<0.01). After
4 seconds of UV exposure, RAD1 and rad1 plates coated with SPF=0 lotion survived
approximately 64% and 57% (p>0.05). Furthermore, SPF protected RAD1 cells all

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survived at rates near 100%, regardless of the SPF strength of the sunscreen (p
>0.05). SPF-protected rad1 cells also survived near 100% when SPF=15 or 30. Genetic
susceptibility to UV damage is related to the NER genes (rad1 mutants) but this
vulnerability can be mitigated with even SPF = 15 or 30 sunscreen.
A few limitations of our experiment were that the mutant strain we used was
contaminated, the appropriate plates were not all immediately placed in darkness, the
sunscreen was coated on very thickly (an unrealistic amount for someone to have on their
skin), and we only performed three trials for each concentration of SPF. A few plausible
extensions of our experiment include: performing ANOVA statistical analyses to further
help interpret our data, coating the sunscreen on in exact allocated amounts, performing
more than three trials, use pure UVA or pure UVB as we do not know the exact UV
composition of the light source, and using a different model organism than yeast.
Another extension that we could have fixed in the design is to BLIND the person doing
the counting and ensure that multiple raters (counters) are used and that they are blinded
to the study hypothesis and SPF and UV exposure doses. Performing a t-test assumes a
normal distribution and another extension would have rather been to compare the cell
counts between the two rad1 groups using Wilcoxon rank sum or Mann Whitney tests as
these are not parametric, they do not require a normal distribution.

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Works Cited
Chemical responses of single yeast cells studied by fluorescence microspectroscopy
under solution-flow conditions. Kogi O, Kim HB, Kitamura N, Analyst. 2002
Jul;127(7):967-71.
Conant, Kevin. 1993. USING YEAST AS AN ULTRAVIOLET LIGHT
MEASUREMENT TOOL.
Knight AW. (2004). UMIST, Dept Instrumentat & Analyt Sci, POB 88, Manchester M60
1QD, England. Using yeast to shed light on DNA damaging toxins and radiation.
Analyst 129, (10) 866
Manny, T. R. and Manny, M. L. (1991) A Handbook for Using Yeast To Teach Genetics.
Genetics Education Networking and Enhancement Project, Dept. of Physics,
Kansas State University, Manhattan, KS. 66056
Pannunzio NR, et al. (2010) RAD59 and RAD1 cooperate in translocation formation by
single-strand annealing in Saccharomyces cerevisiae. Curr Genet 56(1):87-100
Russell, Caitlin R. "EFFECTS OF ULTRAVIOLET RADIATION ON YEAST." (2006):
1-9. Web.
Sevanan Murugan, Pongiya Uma Devi and Peedikayil Neetu John, 2011. Antimicrobial
Susceptibility Pattern of Biofilm Producing Escherichia coli of Urinary Tract
Infections. Current Research in Bacteriology, 4: 73-80.

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Cell Type
rad1
rad1
rad1
rad1
rad1
rad1
RAD1
RAD1
RAD1
RAD1
RAD1
RAD1

Time (sec) of UV
Exposure
0
5
10
15
20
30
0
5
10
15
20
30

Number of
Colonies
1000
3
2
1
0
0
45
10
2
2
1
0

Number of
Cells/mL
1.0x107
3.0x104
2.0x104
1.0x104
0
0
4.5x107
1.0x107
2.0x106
2.0x106
1.0x106
0

Percent Survival
100%
0.3%
0.2%
0.1%
0%
0%
100%
22.2%
4.44%
4.44%
2.22%
0%

Figure 1 represents the ultraviolet light exposure time (sec), colony counts, number
of cells/mL, and percent survival of RAD1 and rad1. For 0 seconds, RAD1 and rad1
both had 100% survival rate. Starting from 5 seconds, rad1 yeast had less growth
than RAD1 yeast. The rad1 and RAD1 cells both had a 50% survival rate when
exposed to ultraviolet radiation for 4 seconds. In both the rad1 and RAD1 cells there
is a trend of decreasing colonies, cells/mL and percent survival as the time of
exposure is increased. The kill point for both RAD1 and rad1 yeasts when they were
exposed to ultraviolet radiation for 25 seconds or more as there was 0% survival.

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Figure 2 is a representational graph of the percent survival of both rad1 and RAD 1 yeast cells after 4 seconds
in the different experimental groups (no protectant, lotion, no exposure, SPF 15, SPF 30, and SPF 50).
Although the survival rate of RAD1 yeast treated with SPF 15, 30, and 50 did not differ significantly, they had
significantly more growth compared to that of RAD1 treated with lotion. The survival rate of RAD1 treated
with SPF 15 was significantly greater (p= 0.0066) than the one treated with lotion. The same trend was
observed for the survival rates of RAD1 yeast treated with SPF 30 and 50. The RAD1 yeasts protected with
sunscreen of SPF 30 and 50 had significantly more growth (p= 0.0223; p=0.0363) than lotion treatment. Yeasts
that were treated with sunscreen generally had more growth than the sample that was exposed to UV radiation
with no protectant on.