Martin Sharratt i7463234

Identifying and experimentally testing mechanisms

behind stress-induced mutagenesis in Drosophila
melanogaster
Dr Paul Hartley has agreed to supervise my independent research project.

Summary
Stress-induced mutagenesis is a function of evolutionary capacitance whereby an organism
responds to higher than normal selection processes by increasing mutation rate in the
genome, and thus providing a higher chance of generating a mutation that can counteract
the selection pressures being applied. The purpose of this study is to identify and
quantitatively study the biological mechanisms behind previously observed stress-induced
mutagenesis induced in Drosophila melanogaster.
In my proposed project, I would be subjecting the common fruit fly, D. melanogaster to
environmental pressures known to induce stress-based mutagenesis in a gradated fashion,
to see if the rate of mutation could be accurately controlled using environmental pressures
rather than by complex genetic manipulation. The main contribution of this work would be
to determine if the mechanisms behind stress-induced mutagenesis could be experimentally
controlled, allowing for more targeted responses for current gene therapy, the ability to
focus the development of future innovations into this field and to explore the ways in which
the human body may regulate the development of mutations and thus evolution in its own
genome.

Research Context
It is well known and basic biology that mutations in the genetic code of the human genome
can cause a change in the phenotype produced by the protein composition described in the
base code of the genome. These in turn can develop into what may be considered
deleterious and thus most probably a medically treated disease (Bernstein et al. 1985), but
could also provide a biological adaptation to the organisms environment.
Not all of these mutations arise from what could be called conventional causes, such as a
failure in the processes of transcription and translation of DNA and RNA, or as a result of
environmental damage from reagents or particles that can react/bond with the DNA
molecule and change its composition (Lynch 2010). Instead of these conventional mutation
vectors, there is well documented evidence to show that the genome of an organism can
self-regulate mutagenesis and increase its frequency when desirable (Bergman and Siegal
2003, Masel 2005).
A problem of increasing the rate of mutations in an organism is that invariably, most non-
neutral mutations are deleterious (Crow 1999), but doing it via stress-induced mutagenesis
is an effective way of doing it because these mechanisms are only activated in the event of
high selection pressures. This means that only beneficial and/or neutral mutations persist in
Martin Sharratt i7463234

the gene line as deleterious mutations will invariably be punished by the highly stressful
environment by causing their bearers to perish before they can pass these mutations to
their offspring. Additionally, once sufficient beneficial mutations accrue such that the
organism is no longer in an environment it would consider to have high selection pressures,
the mechanism works to reduce mutagenesis and create a more stable organism genome.
The specific mechanism that I will be focusing on in this project is the heat-shock protein
Hsp90, which ordinarily acts as a buffer against undesirable phenotypic variation
(Rutherford and Lindquist 1998). It is well documented that in especially stressful
environments, otherwise deleterious mutations can be more beneficial than maintaining
phenotypic stability (Remold and Lenski 2001, Haag et al. 2003) and Hsp90 has been
observed in experimental conditions to either be inhibited by or react to these
environments depending on the specific stress (Jarosz and Lindquist 2010). My proposal is
to expand on these observations by focusing on one known stressor (in this instance,
temperature) and trying to ascertain if there is a reliable correlation between stressful
temperatures e.g. if higher temperatures lead to proportionally higher rates of mutagenesis.
If it is found that one can effectively influence the rate of mutations accrued in a genome by
manipulating these processes, long-term, population based gene therapy and genetic
counselling could be targeted at the pressures that induce mutations in tandem with the
many genes affected by mutation (Muthuswamy 2011), saving time and money on multiple
avoided and potentially invasive treatments (Wirth et al. 2013).
The other key benefit of this research is that it would further research already conducted on
the possibility that the human genome takes steps to self-regulate evolution, and the
mechanisms by which it achieves this (Galhardo et al. 2007). This is clearly a matter of
significant academic importance, and one that deserves time and expertise applied to
explore it.

Objectives
There are two main objectives to my proposal, which cover all aspects of the experimental
process and other vital sections of my proposed project such as data analysis:

Subject groups of Drosophila melanogaster to environmental pressures known to

influence evolutionary capacitance over multiple generations, and measure rates of
mutation within these groups.
Examine the cellular contents of these groups to determine if there is a correlation
between the mechanisms that are known to control evolutionary capacitance and
the selection pressures imposed on the model organisms used.
The overall approach that I have taken for the methods of my proposed project is to ensure
that the results of this project are not merely qualitative but as quantitative as is feasibly
possible. This will involve detailed statistical analysis of all relevant data and especially
ensuring that there is a detailed examination of the degree to which controlled variables
affected rates of mutation, and whether this is statistically significant.
Martin Sharratt i7463234

As the intended method does not involve testing in humans, I have deemed any attempts to
qualitatively examine the effects of the mutations and their resultant phenotypes to be
unreliable when trying to apply them to the phenotypes seen in humans containing the
same mutation in their genome. While this inadequacy is limiting, there are very good
reasons why the approach I have chosen will use my chosen model organism.
Firstly, the ethical basis for human genetic testing when there are readily available non-
human test subjects is obviously non-existent but worth mentioning as a reason for animal
models. Testing on organisms more readily comparable in physiology to humans such as
mice would be preferable due to the ability to record more qualitative data, but the sheer
cost and administration load is unfeasible for a project of this size and scope.
D. melanogaster is the model organism chosen for this project for many reasons, the main
one being because of its ubiquitous use as a model organism (Shuman and Silhavy 2003,
Beckingham et al. 2005, Roberts 2006), which in turn is because of its ease of use compared
to most other model organisms and the quantity and rate at which D. melanogaster breeds,
allowing for easy collecting of a large amount of data for comparison and analysis.

Methods
There are two parts to the method of my proposed project, corresponding to the two
objectives outlined previously in this proposal. Part one describes how I will produce
mutagenesis in D. melanogaster using environmental selection pressures, part two
describes the processing of the mutations that will develop in the fly model, and part two
describes how I will determine if this mutagenesis can be correlated with the imposed
selection pressure.
Part One
The first part of my project is to produce stress-induced mutagenesis in D. melanogaster
using the environmental pressures of heat. I would have 5 groups of D. melanogaster
allowed to live in temperature controlled vivarium, identical but for the constant
temperature that each group is kept at. The optimal incubation temperature that would be
used for the control group of D. melanogaster is (approximately) 22oC. It should be noted
that deviation from this tends to alter the speed of the natural life cycle of D. melanogaster
and so this would be accounted for when managing time.
The two experimental groups would be kept at 22oC for 12 hours a day, and then increase
up to 42oC and 62oC respectively and kept at that temperature for 12 hours, repeated over
the course of ten weeks. As the average life cycle of D. melanogaster from egg to sexually
reproductive adult is approximately 7 days in optimal conditions, this should produce
approximately six to nine generations of organisms that can be analysed for mutations. This
constant subjection to alternating temperatures should induce mutagenesis as seen in other
studies (Masel 2005, Galhardo et al. 2007).
It should be noted that there is a slight possibility that using temperature as a selection
pressure may become unviable if the experimental groups of D. melanogaster respond so
Martin Sharratt i7463234

poorly to temperature variation as to become unviable populations. If this occurs, then

there is the back-up plan of using well documented mutagenic inhibitors (Solit and Chiosis
2008) that specifically target Hsp90 and inhibit its role as an evolutionary buffer. In this
instance, the independent variable would switch from temperature levels to quantities of
mutagen introduced to D. melanogaster colonies.
Part Two
The aspects of mutant physiology that I will be focusing on are ones that can be easily
ascertained by loss of heterozygosity assays changes in body pigmentation, eye
pigmentation and wing structure are common indicators of genomic instability in D.
melanogaster (Rifkin et al. 2003) and if a gene regulating these phenotypes mutates, this
will produce homozygous or trans-heterozygous mutants that would have easily observed
phenotypes such as a modification of wing structure or an alternation in eye pigmentation.
Any additional mutations that arise during the experiment will be noted for completion,
although a thorough examination of its relationship with environmental pressures will only
be conducted in the event that the mutation arises frequently and persistently in the
experimental groups, and does not arise in the control group of D. melanogaster.
Part Three
Once I have produced enough data to accurately determine statistical significance the next
step would be (once remaining D. melanogaster are humanely euthanized and disposed of
safely) to analyse the data gathered and determine if there is a measurable and/or
proportional relationship between the imposed environmental pressures of fluctuating
temperatures and mutagenesis rates in D. melanogaster.

Significance of Research
Following on from points made previously, the main importance of this research is to
determine if one can environmentally influence mutagenesis in an organism, but unlike
what has been done in previous research (Otto and Feldman 1997, West et al. 1998,
Whitlock and Bourguet 2000), this project would seek use selection pressures rather than
more complex experimentation such as genetic transformation or the use of p-transposons
(Hummel and Klmbt 2008).
From this, the other reasons that this research is important follow; gene therapy, as it exists
currently, is close to $1 million in cost in some German hospitals (Morrison 2015) - any way
to reduce the frequency of mutations arising in populations of humans (and potentially
other financially important organisms) by alteration of environmental factors would almost
definitely aid attempts to reduce cost and maximise the amount of benefit per treatment.
One example of further research that could stem from this project would be, assuming my
research is successful in its aims to determine a way to use environmental pressures to
induce mutagenesis and to determine a proportionality between mutagenesis and
environmental stress, to ascertain if the biology of D. melanogaster is regulating the
evolutionary capacitance of its genome by regulation of Hsp90 by other biological
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molecules. Research like this and the proposal I am making would be a massive step forward
in understanding the mechanisms that regulate and control an organisms adaptation to its
environment and biological niches.

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