1. The Effects on Differential Methylation Patterns in Volvox Carteri Following Extended Exposure to
Bisphenol A (BPA) and 5-Azacytidine
Amer Nazar
Abstract:
DNA methylation is a crucial process that can have a dominating effect on the phenotypic traits of an
organism. Methylation is the process of adding a methyl group to particular cystines within or in proximity
to the gene (11). In an organismal system, DNA is methylated with the use of enzymes. A gene that is
methylated, normally will not be expressed. Therefore these methylation patterns can control the expression
of specific genes in a system. The organism on which this experiment will be performed on is Volvox
Carteri. Volvox is a type of algae that can usually be found in a variety of fresh water systems. Volvox is an
ideal organism to perform this experiment on because it consists ofonly two different types of cells , which
are somatic cells and asexual reproductive cells (6). Somatic cells being the ones that form the body of the
organism, whereas the asexual reproductive cells’ purpose is growth and reproduction. In an organism this
small it will be easier to spot any phenotypic effects that may result. However, changes in these patterns
can also occur naturally during development, and perhaps any changes we see may not be the result of
toxins. The first toxin is Bispenol A. More commonly known as BPA, it is an organic compound used in
the manufacturing of plastics. The other is 5-Azacytidine. 5-Azacytidine is a synthetic structure,similar to
cytidine, which is a nucleoside present in both DNA and RNA. Both of these are compounds that will
potentially de-methylate the volvox DNA for our experiment. Following exposure we will oberve our
samples and look for phenotypic changes. Results from this and similar experiments could helps us greater
understand the ramifications of methylation vs.de-methylation in DNA research. In Epigenetics there is a
great potential in using research like this to help us better understand gene regulation and how personal
environmental choices could effect ourselves,surrounding organisms and our future offspring.
Research Question:
Are there differences in whole genomic methylation patterns before and after treatment with BPA and 5-
Azacytidine in Volvox carteri? If so,what are they? Are there any noticeable phenotypes? If there is a
noticeable phenotypic change and we are able to isolate that specific gene,does it still experience
differential methylation patterns after exposure to BPA?
Introduction:
DNA methylation is a process that has a profound impact on the development and genetic growth of an
organism. Occurring on the number 6 nitrogen of the adenine purine ring or the number 5 position of the
cytosine pyrimidine ring, methylation is the addition of a methyl group to the structure.Normally the
methylation of a particular gene causes it to be suppressed. These can be genes that may not contribute to
the function of that particular cell, or they can also be genes that maybe detrimental to the development of
the cell/organism. With this process being held in such a delicate balance, it becomes clear that if even one
segment of a sequence is affected, then it could have a dominating effect on the development of the
cell/organism. The model organism for understanding DNA methylation in plants was Arabidopsis
thaliana.
Volvox cateri is green alga that normally develops in a spherical shape (looking at it undermagnification
will reveal a little green sphere with dark reproductive cells visible within. The organism will be spherical
in shape and green in color. It consists of two types of cells. The first type is somatic cells and the second
type is an asexual reproductive cell. Volvox gives us an example of two main features of all multi cellular
organisms, that its cells become specialized and that they cooperate with each other. Through out history
scientists have been commenting on the simplistic way that Volvox displays these characteristics (6). Even
with this interest in Volvox, no real research was made into the reproduction and development patterns of
this organism until the 1960s when scientists discovered how to maintain a culture of Volvox and cultivate
it. A man by the name of William Darden who was at the time only a graduate student headed this research
(6).

2. The objective of our experiment is to look for phenotypicalchanges.Some characteristics of this organism
that I think will be beneficial to this experiment are that, being asexual reproducers, their development can
be adjusted with a light vs.dark cycle that lasts approximately 48 hours (6). This is will allow us the ability
to develop our cultures at a consistent schedule.“ However there is one trait that could be problematic. W e
are going to be introducing toxins to the Volvox organisms to induce demethylation and to hopefully see a
phenotype that is unusual. But even though it is a very simplistic organism, It can develop mutations of its
own accord (6). This will complicate the second stage of our experiment. Assuming that we see a
phenotype develop,we will then have to see whether the change that was observed occurred due to
introduction to a toxin, or was it a mutation that occurred because of natural development. This is where the
tedious part of the experiment would be. We would have to be very careful in our analysis that we don’t
end up assuming that any phenotypes that we see were induced by the toxins. It could be that they
happened randomly of the cell’s own accord.
One gene that plays a major part in Volvox development is regA. Not just in development however, it plays
a major role in gene regulation. A regA transcription particle is responsible for generating a nucleotide
mRNA that is seen at the beginning of somatic cell differentiation (9). If regA were the gene that becomes
demethylated as a result of our experiment, there could be a plethora of mutations that could occur.
Mutations such as deletions from the sequence could produce a phenotype that may not occur naturally.
For the purposes of our experiment, we will be using two different toxins. The first one is 5-Azacytidine.
5-Azacytidine is not a naturally occurring substance.It is a synthetic analogue of the nucleotide Cytidine.
Cytidine is a nucleotide that naturally appears in both RNA and DNA. Originally made about 40 years ago,
it is now sold in the United States under the commercial names “Mylosar” and “Vidaza” (5). The way it
works is that upon introduction to a cell it is absorbed and incorporated into the DNA of an organism where
it inhibits DNA methyltransferase, which in turn does not allow DNA methylation to occur.
The other toxin that we will use is Bisphenol A, also known as BPA. Also not a naturally occurring
substance,BPA is an organic compound that is used in the manufacturing of plastics. It also is found in a
lot of organic solvents. It became a topic of concern when studies showed that it presented hormone like
properties. In 2008, a study demonstrated that BPA was mimicking estrogen in rats. BPA has also been
linked to many dominating health concerns such as obesity and cancer. In this experiment, we hope to
produce phenotypic changes in Volvox by exposing the organism to extended doses of both of these toxins.
For our experiment, we will take colonies of Volvox, working under sterile conditions; we will expose them
to these toxins. There will be multiple trials of this experiment to ensure an accurate conclusion.After a
preset incubation period, the Volvox colonies will be examined thoroughly to see if any phenotypes did
develop. If there is no change then that would be the end of the experiment. However if we do see a
change,then we will move on and our next step would be one or both of the following procedures. We
would have to determine whether or not the new phenotype was naturally occurring or was it induced due
to the introduction of the toxins. The first step would be that we would perform a restriction enzyme
analysis of the whole genome. This will involve isolation of DNA, followed by a gel electrophoresis with
set controls. Then the next step would be to isolate a specific gene that we know has been affected by the
methylation changes due to BPA. This process will involve isolation of RNA, utilization of primers to
make cDNA, a gel electrophoresis to ensure that there is no contamination, and then an RT-PCR to
determine transcription levels.
Experimental Design:
A. Toxicity Screen for BPA and 5-Azacytidine
a. BPA Toxicity Screen1
i. Concentrations: 0, 2, 4, 6, 8, 10, 12 mg/l
ii. Controls:
1. Negative Control: No BPA (0 mg/l)
2. Negative Control: No Volvox carteri
iii. Exposure for one full asexual life cycle (48 hrs.)
iv. Qualitative analysis of varying green concentration

3. 1. No BPA: the darkest green that we will see
2. No Volvox carteri: no color should be seen
v. Do in triplicate.
b. 5-Azacytidine2
i. Concentration: 0, 0.2, 0.4, 0.6, 0.8, 1.0 mM
ii. Controls:
1. Negative Control: No 5-Azacytidine (0.0 mM)
2. Negatinve Control: No Volvox carteri
iii. Exposure for one full asexual life cycle (48 hrs.)
iv. Qualitative analysis of varying green concentration
1. No 5-Azacytidine: the darkest green that we will see
2. No Volvox carteri: no color should be seen
v. Do in triplicate
B. Exposure of BPA and 5-azacytidine to wildtype Volvox carteri
a. Expose BPA and 5-azacytidine to wildtype Volvox carteri with the concentrations found in
the toxicity screen for varying hours in the first 9 hrs of development
i. Exposure for 0, 1, 3, 6, 9 hrs (9th hour is the start of cell differentiation)
1. This corresponds 9 AM, 12 AM, 3 PM, and 6 PM with the cycle starting at
8 AM.
ii. After the exposure is finished spin down the culture into a pellet. Remove as much as
the experimental supernate.
iii. Re-suspend the culture pellet in standard Volvox medium (SVM) and allow
development to continue.
b. Do in triplicates
c. Controls:
i. No BPA/5-Azacytidine at 0, 1, 3, 6, 9 hrs of development
ii. No Volvox carteri
C. Mutant screen of exposed Volvox carteri
a. After 48 hours of growth, isolate phenotypic mutants from each treatment under a stereoscope
and allow to grow into a culture
D. Isolation of DNA
a. Isolate and purify the DNA of each mutant and wildtype with instructions as per manufacture
b. A260/A280 should be obtained to ensure no protein contaminates
E. Global Restriction Enzyme Assay forDifferential Methylation
a. Whole genomic to determine if methylation has decreased in mutants and follow instructions
as per manufacture
b. Methyl sensitive and non-methyl sensitive restriction enzymes
i. Treatments:
1. Wildtype and Mutants
a. –HpaII, -MspI
b. +HpaII, -MspI
c. +HpaII, +MspI
d. –HpaII, +MspI
F. RT-PCR for regA (and Lag, maybe)
a. Isolate RNA from mutants and wildtype as per manufacture instructions
b. Obtain primers specific for regA (and Lag) to make cDNA
i. Primer efficiency?
c. Run an agarose gel to ensure no contamination from proteins and do a A260/A280

4. d. Run the RT-PCR to determine transcription levels against the control of a housekeeping gene,
possibly actin
i. Run on agarose gel to compare wildtype and mutant patterns
G. Bisulfiite Sequencing of regA (and Lag)
a. As per manufacture to see specific cystines that are unmethylated in mutants as compared to
wildtype in regA (and Lag).
This experiment could end up with a plethora of different outcomes.The first is that we go ahead and
conduct the experiment (multiple trials) and there is no change what so ever to the organisms. This would
be a rapid end to our experiment and would give us the conclusion that neither of these toxins had any
effect on the organism. The next possibility is that we do notice several phenotypic changes.This would be
well and good,but then the would come the task of analyzing the samples and isolating bits of DNA and
running a gel electrophoresis on them to see if in fact the mutations that we noticed were induced by the
toxins. This is necessary because,as I mentioned earlier, this organism has the ability to spontaneously
develop mutations on its own. The final result that we could have is that the organisms are killed and we
have to declare an end to the experiment. This experiment requires sterility and caution. The slightest error
by any of us,could potentially have a catastrophic effect on the Volvox carteri. I look forward to
performing these procedures with the utmost caution and succeeding with our goal to discover the effects
that these two toxins may have on differentiation and phenotypic changes in Volvox carteri.

5. Sources:
1) M Bianco, et al. "Differential Accumulation Of BPA In Some Tissues Of Offspring Of Balb-C Mice
Exposed To Different BPA Doses."Environmental Toxicology And Pharmacology 33.1 (2012): 9-
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Effects Of Bisphenol A." Brain, Behavior, And Immunity 25.6 (2011): 1084-1093. PsycINFO. Web.12
Apr. 2012.
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patterns in plants and animals." NIHPA Author Manusripts.(2010): n. page. Web. 12 Apr. 2012.
<http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3034103/?tool=pmcentrez>.
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<http://www.cancer.gov/drugdictionary?cdrid=39153>.
6) Kirk, David L. "Germ-Soma Differentiation in Volvox."Academic Press. (2001): 213-223. Print.
7) Schuettengruber, Bernd, Daniel Chourrout, Michel Vervoort, Benjamin Leblanc, and Giacomo Cavalli.
"Genome Regulation by Polycomb and Trithorax Protiens." Leading Edge Review. cell 128. (2007): 735-
745. Print.
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trithorax group factor to regulate cell fate in plants." CSH Press. (2009): 2723-2728.
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Sources for Experiment Procedure:
10) Li, Rui, et al. Physiological responses ofthe alga Cyclotella caspia to bisphenol A exposure. Botanica
Marina 51, 360-369 (2008)
11) Babinger, Patrick, et al. A link between DNA methylation and epigenetic silencing in transgenetic
Volvox carteri. Nucleic Acids Research 29, 1261-1271 (2001)