Analysis of Drosophila melanogaster cDNA Sequences to Model Human Disease

Cameron Herbst










30 October 2013
Bio 230W Section 006
Lab Partner: Jun Seong Oh
TAs: Carrie Lewis, Marisa Rugino, Alexa Marucci
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Introduction

Proteins are fascinating biological macromolecules that serve to accelerate biological
reactions, regulate gene expression, and much more. Homology exists among proteins between
different species, allowing for comparisons to be drawn between the mechanisms that control the
proteins themselves as well as their biological pathways and functions. Homology of this type
can be seen in what are known as protein domains. Protein domains are regions of a protein that
have been conserved throughout the evolutionary history of an organism and act as autonomous
folding units that have specific functions and can even evolve independently. Mutations to these
protein domains can cause the three-dimensional tertiary structure of the protein to fold
improperly, thus inhibiting function and leading to disease. Also important to note are protein
families, which are groups of related proteins that come from a common ancestor and have
similar structures, sequence, and function
1
.
Armed with this knowledge, we have the tools to assess the overarching research
question addressed in this study. Our goal here was to identify and isolate cDNA sequences in
Drosophila melanogaster that code for homologous proteins which can ultimately act as models
for human disease in other clinical studies. A cDNA sequence is one that is created from an
mRNA template and therefore does not contain any introns, only exons. This synthesis is said to
run anti to the Central Dogma of molecular biology
1
. Once this has been completed, the
sequence can be amplified and submitted to a bioinformatics database (in this case the National
Center for Biotechnology Information) to identify human homologs and their properties. This
problem can be better addressed in the form of the question: what potential models for
understanding human disease emerge when we search the human genome database with cDNA
library sequences from Drosophila melanogaster
2
?
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Drosophila is used commonly as a model organism in laboratory experiments due to its
ease of breeding and culturing, short life cycle, and high levels of genetic homology when
compared to Homo sapiens. In fact, Drosophila homologs exist for ~70% of human cancer genes
and ~60% of known human genetic disorders
2
. With this taken into consideration, it is expected
that the cDNA sequence yielded from Drosophila will have a human homolog that is directly
linked to a genetic disorder and will serve as a desirable model to study human disease.
Contemporary research similar to this is being conducted within the scientific
community. At the University of Glasgow, UK, the online tool FlyLeaf was recently utilized in a
study very similar to this where homologous genes that affect Drosophila and human tissue
development were observed to obtain models for human disease
3
. Similarly, a study was done at
the Yale School of Medicine where Drosophila Toll protein domains were tested to determine
what they and their human homologs have to do with signaling an immune response
4
. Clearly
this area of research is resulting in many practical applications primarily viewed from a
biomedical perspective.

Materials and Methods

All procedures in this study were carried out as indicated in the lab manual
2
. The
procedure was completed over the course of a four week period and was subdivided into
fragments defined by colony picking, plasmid isolation, PCR, agarose gel electrophoresis, and a
bioinformatics search.
Colony Picking: During the first week of experimentation, two colonies of non-pathogenic E.
coli which had been grown on agar plates treated with ampicillin were selected. The E. coli cells
contained plasmids with genes for both ampicillin resistance and Drosophila cDNA sequences.
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The purpose of including ampicillin resistance was to ensure proper uptake of the desired
plasmids. Our tubes were labeled 006A12 and 006B12, indicating our section as six, plasmid
name, and our group number as twelve.
Plasmid Isolation: After incubating for 6 days at 4
o
C, the cDNA was isolated from the
plasmids via a series of steps involving rinsing with buffers and centrifuging the resulting
solution. Every precaution was taken to guarantee that this stage of the experiment was done in a
sterile fashion so that no DNA contamination from any outside source would affect the results of
the remainder of the study. Our group prepared a negative control tube in order to confirm that
there was no DNA contaminating the sample prior to submission to PCR.
Polymerase Chain Reaction (PCR): After isolation, the cDNA sequence was amplified via
subjection to PCR. PCR works through putting the desired DNA sample through thermal
cycling, which breaks the hydrogen bonds and separates the strands so that primers can bind to
replicate the DNA which is then cooled and the process repeated. The PCR master mix contained
buffer, dNTPs, two primers, and Taq polymerase obtained from Thermus aquaticus. Thermus is
an extremophile whose polymerase maintains fidelity at high temperatures, thereby allowing for
more specific binding of primers. It should be noted that steps seven through thirteen of this
section of the lab was completed for us by our teaching assistants.
Agarose Gel Electrophoresis: Now that the desired cDNA sequences had been amplified, they
were be analyzed in a process known as agarose gel electrophoresis. The masses of the amplified
cDNA sequences were found by running them along a charged gel next to a DNA ladder of
known masses to yield approximate mass values for the samples. Typically the smaller the DNA
fragment is the farther it travels and the larger the DNA fragment the shorter it travels. DNA
contains negatively charged phosphate groups, so on the gel the fragments run from the negative
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end to the positive end. It should be noted that our plasmid was lost so we utilized group 16s
plasmid for this step.
Bioinformatics Search: Finished during the fourth week of the lab, the final objective of the
study was to explore a bioinformatics database and find homologous human proteins to the
corresponding Drosophila cDNA sequence we had obtained. The sequence we analyzed was
from a previous year (21-02A_SP6_F10_Oct-3-2012) because our PCR did not yield a viable
sample. The purpose of this section of the lab was to determine if any homologous genes to those
amplified through PCR exist in either humans or Drosophila and if any possible correlations to
disease in humans can be found.

Results

To the left is a picture of the result of the agarose gel
electrophoresis. Read from left to right the lanes contain the
DNA ladder, DNA plasmid A, PCR product A, DNA
plasmid B, PCR product B, and the negative control. The
mass values read from left to right starting with DNA
plasmid A were 1,500bp, N/A, 1,500bp, 1,250bp, and N/A.
In summation, the molecular weight of the cDNA sequence
yielded from PCR of plasmid B was approximately 1,250bp.
Ultimately, our group did not send any plasmids for
sequencing since our plasmids were lost and we used group
16s plasmids. When submitted to the sequencing software META, the trace file our group used
gave a very nice chromatogram. Next, the cDNA sequence from last year that our group used
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was submitted to the NCBI website via the nucleotide BLAST option. Our request ID was
5AK68DJE01R. The NCBI reference sequence for our cDNA was NM_001259587.1 which
yielded Drosophila melanogaster zipper (zip), transcript variant I as the mRNA in question. The
identification percent of this search was 100%. The homologous gene in humans is MYH10, a
protein coding gene with accession number NP_005955.3.
5
Next a protein BLAST was performed which gave a protein ID of NP_001246516.1
which is zipper isoform I in Drosophila. The human homolog was found to be MYH10 isoform
3 which has the accession number NP_001243024.1. The identification percent for this search
was 63%. The protein BLAST reference ID was 5AMUDR2J01R.
5
There were four conserved protein domains observed in our cDNA sequence. These were
the MYSC type II domain, the Myosin N superfamily domain, the MYSc domain, and the
Myosin tail I domain. All four of the conserved protein domains in the human protein were
found to be conserved in the Drosophila homolog.
5

Discussion

As can be seen in the experimental results, there are four homologous domains present in
both the protein derived from the Drosophila cDNA sequence and the corresponding human
homolog. Given this information, it is reasonable to support the hypothesis and conclude that
studying the zipper transcript variant I gene in Drosophila could prove beneficial to our
understanding of human disease.
Both homologous proteins are found to play roles in movement in each respective
organism. The zipper protein in Drosophila functions to mediate cortical contraction and is the
motor for both smooth and skeletal muscle. In humans the homologous protein serves to regulate
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cytokinesis, enable cell motility, and sustain cell polarity. This protein has been shown to be
involved in the pathways of some diseases in previous studies. For instance, in humans MYH10
isoform III is affected when the actin cytoskeleton of cells are rearranged during a Salmonella
infection. Additionally, it plays a role in the pathway of viral myocarditis and mutations have
been associated with the May-Hegglin anomaly, which causes defects in both heart and brain
development.
5

The largest source of error in this experiment ended up being the loss of our groups
plasmid, which ultimately changed our results as we analyzed a different groups results.
Additionally, our group shared a container of pipette tips with neighboring groups that may not
have been as careful about ensuring their technique was sterile as we were. Lastly, loading the
agarose gel proved to be difficult which could have resulted in some variation in our results.
In the future, experiments involving the intentional mutation of the zipper transcript
variant I gene in Drosophila in order to test for whether or not it produces similar genetic
disorders as would be the case for the homologous MYH10 gene in humans would give better
insight into the mechanism behind what factors drive genetic disorders such as the May-Hegglin
anomaly. This could ultimately yield valuable information that could lead to breakthroughs in
treatments, developmental pathways, or simply a more complete understanding of human
physiology as a whole.





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References

(1) Lewis, Carrie. DNA Isolation and Analysis Lab Week 1 of 4" BIOL 230W. 2013. The
Pennsylvania State University, PA.
(2) Biol 230W Laboratory Manual. 2013. cDNA Isolation and Analysis. The Pennsylvania
State University, PA
(3) Venkateswara, Chintapalli R., Jing Wang, and Julian Dow. "Using FlyAtlas to Identify Better
Drosophila Melanogaster Models of Human Disease." Nature.com. Nature Publishing Group, 29
May 2007. Web. 29 Oct. 2013.
(4) Medzhitov, Ruslan, Paula Preston-Hurlburt, and Charles A. Janeway, Jr. "A Human
Homologue of the Drosophila Toll Protein Signals Activation of Adaptive
Immunity."Nature.com. Nature Publishing Group, 24 July 1997. Web. 29 Oct. 2013.
(5) NCBI. U.S. National Library of Medicine, n.d. Web. 16 Oct. 2013.