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Drop That Fly Swatter! Fruit Flies Could Save Human Lives

Exploring proteins in Drosophila melanogaster that are homologous to human proteins and what
they implicate about the mechanisms of Parkinsons disease
















Alaina Weinheimer

Partner: Nicole McKay

BIOL 230W, Section 012

TAs: Saima Shahid, Carrie Lewis, Caitlin Clearie

Due date: November 1, 2013
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BACKGROUND

A chimp and a human do not exactly look like blood-related cousins, but humans share a
shocking 98.8% of DNA with chimps and a surprising amount of DNA with yeast, as well (1).
Many living organisms share DNA because many life processes are carried out the same way
and thus have similar, or homologous, proteins. Recent genetic sequencing has enabled
scientists to identify similar protein domains between different species (2). For instance, in
2001, scientists discovered a DNA-binding protein Ku in the bacteria S. coelicolor that has
eukaryotic homolog of DNA primase (3). Many more of these cases of homology are being
discovered in recent time.
Proteins have a three dimensional structure that is dictated by DNA. Often, diseases in
humans are caused by mutations in DNA that lead to protein misfolding and subsequent
malfunctioning. Monitoring diseases that are caused by protein misfolding in humans not always
feasible. Complications can arise from observing disease in humans that can be risky to human
health; also, human lifespans are too long to determine a specific gene as the cause of disease
from observation of generations. To cope with these constraints, scientists utilize the knowledge
of homology between proteins in other organisms to understand their function in the orgainsim
and relate it to the human homolog function in humans. Proteins conserved through evolution
are categorized into protein families; proteins in the same family have similar function(s).
Relatively recently, scientists have found that proteins contain different subunits, or domains,
that contribute different functions to the protein. Thus, homologous proteins are typically in the
same protein family and share similar protein domains. Scientists identify proteins of the same
family and homologous protein domains in other organisms to understand disease mechanisms in
humans. The organisms scientists use containing these homologous proteins use are called
model organisms. Model organisms have DNA closely related to humans, relatively short life
spans, and are easy to experiment with (2).
A particularly effective model organism is the Drosohila melanogaster, or the fruit fly.
The fruit fly shares 75% of disease causing DNA with humans (4). Fruit flies also have short life
spans, are easy to culture, and proliferate to large numbers, making experimentation faster and
enable results to be observed over many generations (5). Several homologous protein domains
that cause disease in humans have been discovered already. For instance, mutations in the
protein domain LRRK2 of the fruit fly, which has homology to the DJ-1 protein domain in
humans, has been found to contribute to Parkinsons disease in humans (4). Some other diseases
that have been understood through fruit fly homologous mechanisms include cancer,
Huntingtons disease, and Leukemia (2).
In this experiment, homologous protein domains between fruit flies and humans were
identified to elucidate mechanisms in human disease. To do so, fruit fly plasmid DNA was
copied through reverse transcriptase to make cDNA or complementary DNA and was isolated
from an E. coli bacteria colony. This plasmid was then amplified via polymerase chain reaction.
Gel electrophoresis was used to separate the cDNA from the rest of the DNA. This cDNA was
then be sequenced. Next, a section of protein-coding DNA was entered into a CDART, which
stands for Conserved Domain Architectural Retrieval Tool, search engine to identify the protein,
its domains, and function in the fruit fly. Next a BLAST search was used to identify human
homologous proteins and domains and their possible function in human disease (6).
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Due to the significant similarity in human disease causing DNA in fruit flies, it is
predicted that the isolate fruit fly cDNA will code for a homologous protein in humans that
causes disease. Finding protein homology between fruit flies and humans will continue to help
scientists understand disease mechanisms, furthering the potential of finding a cure for these
diseases.


MATERIALS AND METHODS

The procedure for this experiment are written in the Biol 230W Laboratory Manual.
2013. cDNA Isolation and Analysis. The Pennsylvania State University, PA.
.
Plasmid cDNA isolation:

A bacterial colony was isolated from E. coli and cultured in a broth. This E. coli
contained the plasmid DNA of the fruit fly. A second E. coli colony was isolated from a plate
that contained ampicillin. Ampicillin-resistant E. coli tend to express the plasmid better because
they dont have to spend as much time expressing genes to fight ampicillin. The two colonies
were cultured in a glass tube containing a Luria Broth plus ampicillin. After allotting a week for
growth, the plasmid DNA was isolated using QuickLyse Miniprep Plasmid DNA purification.
After chemical preparation, the plasmid DNA was ready for PCR, polymerase chain reaction.

Polymerase Chain Reaction:

The isolated plasmid DNA was taken through a polymerase chain reaction to determine
the size of the cDNA insert of the plasmid DNA. During the PCR, the DNA was denatured and
the primers targeted the SP6 and T7 regions of the plasmid cDNA, which lie on the ends of the
single-stranded DNA that needed to be amplified. The enzyme Taq polymerase was then used to
synthesize the complementary stretch of the desired DNA. One tube contained the Plasmid A
cDNA with PCR master mix and the other tube contained Plasmid B cDNA with PCR master
mix. A negative control was made using water instead of DNA and PCR master mix to show
whether or not cross-contamination had occurred.

Gel Electrophoresis:

The isolated regions of DNA were then separated by gel electrophoresis. The gel
electrophoresis separated the cDNA from the plasmid DNA based on size. The gel was prepared
using agarose and 1XTAE buffer. The dye used was ethidium bromide dye. The DNA loading
mix was made by mixing the prepared samples with water and 6X bromophenol blue loading
dye. The sample loaded into the gel included: plasmid A DNA, the plasmid A DNA PCR
product, plasmid B DNA, and the plasmid B DNA PCR product. Each plasmid loading mix was
loaded into the wells of the gel next to the DNA ladder, which was used to measure the size of
each DNA. The gel electrodes were added to the apparatus and then the power supply was
turned on at 100 volts until the DNA dyes appeared to have moved halfway through the gel. The
gel was then photographed under a UV light in a UV box. A picture was taken over the hood of
the UV box.
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Sequencing and Identification:

The plasmids that had successfully traveled in the gel were then sent for sequencing. The
dideoxy method was used to sequence the DNA. Once the DNA was sequenced, the bases were
trimmed after a sequence of cysteine base pairs at one end and before a series of unidentifiable
nucleotides appeared at the other end to result in a protein-coding sequence, or exon. This
sequence was exported as a FASTA file, which was then opened in the NCBI BLAST page for a
nucleotide BLAST. The databse used was others and the selectivity chosen was somewhat
similar sequences (blastn). The result chosen for exploration had a high identity, low E value,
and the accession began with NM. The protein that this DNA coded for was found on the page
of the accession and then conserved domains in humans was found running a BLASTp, choosing
homo sapiens as the organism. The functions of the homologous domains were found on this
website, as well.


RESULTS

Bacterial Growth

Plasmid A: low growth, particles clumped at bottom, no apparent contamination
Plasmid B: low growth, particles clustered at bottom, no sign of contamination
These results show that the E. coli colonies successfully grew in their broths and the fruit fly
plasmid could be amplified.

Figure 1. Gel Electrophoresis Picture Under UV Light






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Table 1. Content of Gel Lanes

Lane # 1 2 3 4 5 6
Content DNA
ladder
Plasmid
A DNA
Plasmid A
PCR product
Plasmid
B DNA
Plasmid B
PCR product
PCR negative
control


Table 2. DNA Segment Size Data

DNA segment Band size (base pairs) Molecular weight (g)
Plasmid A DNA 2,000 21580000
Plasmid A PCR product 2,000 21580000
Plasmid B DNA > 2,000 > 21580000
Plasmid B PCR product n/a n/a
PCR negative control n/a n/a

Molecular weight calculation: band size of DNA X average molecular weight of a base pair

Molecular weight: 2,000 X (650 X 1.66 X 10 ng/uL) = 21580000 g

Figure 1, Table 1, and Table 2 description. It appeared that the Plasmid A DNA and the
Plasmid B DNA successfully traveled in the gel. It looks like DNA is in the Plasmid PCR
product lane; however, this DNA is most likely the Plasmid A DNA, not the cDNA from the
PCR product due to size. It appears that the PCR product of plasmid A is the same size as the
plasmid DNA. The PCR product should be much smaller than the plasmid DNA and should
appear farther in the gel than the plasmid DNA. Thus, the PCR product lane of plasmid A is
likely to have been contaminated with the plasmid A DNA. There is also a second band in the
plasmid A DNA lane. Explanation for multiple bands in the gel include the formation of primer
dimers on cDNA or remaining template DNA in the PCR product, but this is not the case
because this lane contains the template DNA and the band appears to be larger, not smaller, than
the template DNA so primer dimers could not have formed and cross-contaminated into this
lane. The plasmid B DNA successfully traveled in the gel and is slightly larger than the Plasmid
A DNA. The plasmid B PCR product shows no DNA in its lane. It is possible that the Plasmid
B PCR was supercoiled into a smaller product, ran through the gel faster than the DNA in the
other lanes, and therefore is not present in the gel photograph. It is also possible that there were
errors in reaction mixture preparation or loading issues into the gel (7). The plasmid A DNA
appears to have a weight of 2,1580,000, g or 2,000 base pairs.

Sequencing Results

Based on the gel results, plasmid A DNA and plasmid B DNA successfully travelled in
the gel, and thus, both were sent for sequencing. Because the cDNA of both plasmid A and
plasmid B failed to separate from the template DNA, the PCR product DNA from both plasmids
were not sent for sequencing.
The sequencing resulted in trace files. The trace file used in this analysis was from the plasmid
A DNA. The trimmed plasmid A DNA sequnece through the BLAST search engine on the
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NCBI website (citations 8-10) to identify the protein on a plasmid and its domains and functions
that the selection of sequences.


Table 2. Drosophila melanogaster mRNA results from plasmid A DNA trimmed sequence (8)

Name of mRNA
sequence
Drosophila melanogaster lin-19-like (lin19) transcript variant D,
mRNA
Request ID 5FW1NUUA01R
Accession NM_078931.3
Length 3341
Total Score 825
Identity 100%
Query Cover 100%
E value 0.0
Protein ID NP_523655.1

Table 2 description The top hit for mRNA with the highest score, identity, and query cover
that matches the sequence submitted is the D. melanogaster lin-19-like (lin19) transcript variant
D. The accession NM_07893.1 codes for the protein with the ID NP_523655.1 (9).

Table 3 Protein Identification Data

D. melanogaster Homo sapien homolog
Protein ID/Accession NP_523655.1/NM_078931.3 1LDJ_A
Name Lin-19-like, isoform D (D.
melanogaster)
Chain A, structure of the Cul1-
rbx-skp1-fBoxskp2
Protein domain(s) Cullin_Nedd8 Cullin_Nedd8
Function Ubiquitin protein ligase Ubiquitin protein ligase
Request Record ID 5FW1NUUA01R 5FXGFY5601R

Table 3 description This table shows the name of the protein the mRNA coded for, its domains
and functions, as well as those characteristics of the Homo sapien homolog(9, 10). Both of these
proteins only domain is the Cullin_Nedd8, which functions in ubiquitin proteolysis. The
ubiquitin protein ligase is involved in cellular processes, such as metabolism, the cell cycle, and
apoptosis. In regard to disease, this protein can cause tumors (11). Specifically, in humans, this
domain is responsible for the accumulation of Lewy body build-up in the neocortex of the brain
that causes Parkinsons disease (12).


DISCUSSION

Based on the results of this experiment, the hypothesis is supported. The plasmid DNA
sequence from an mRNA in Drosophila melanogaster coded for a protein that has a human
homolog involved in human mechanisms and disease. Thus, by understanding the mechanisms
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of this protein in fruit flies, scientists will better understand the homologous proteins role in
human physiology and disease.
In this experiment, plasmid DNA from D. melanogaster was extracted and sent through
PCR. Although the cDNA in the plasmid did not successfully amplify, according to the gel
electrophoresis, the original plasmid DNA samples were sent for sequencing. In this report, the
plasmid A DNA sequencing results were analyzed. This DNA sequence was mRNA Drosophila
melanogaster lin-19-like (lin19) transcript variant D. This sequence coded for the protein Lin-
19-like, isoform D in D. melanogaster. The only domain in this protein is Cullin_Nedd8.
Cullin_Nedd8 neddylates Cullin family proteins that function in ubiquitin-dependent proteolysis.
This proteolysis is involved in the cell cycle, cellular metabolism, and apoptosis. Cullin_Nedd8
neddylates Cullin family proteins by binding onto a conserved Cullin lysine residue. Also,
Cullin_Nedd8 assembles the ROC1/RDX1 RING-based E3 ubiquitin ligase, which plays a direct
role in tumorogenesis (11).
A BLAST search indicated the human homolog to the protein Lin-19-like, isoform D is
Chain A, structure of the Cul1-rbx-skp1-fBoxskp2. This protein contains the exact same
domain, Cullin_Nedd8 (10). In addition to maintaining the same physiological functions as in
the fly, such as its role in metabolism and apoptosis, Cullin_Nedd8 is responsible for the onset of
Parkinsons disease (12). Parkinsons disease is a neurological disorder that causes loss of
function in the nervous system, which results in lack of control of body motion. Cullin_Nedd8 is
directly involved in Parkinsons disease. It induces the ubiquination of of the alpha-synuclein,
which abnormally builds up to Lewy bodies. These Lewy bodies eventually accumulate into the
neocortex of the brain, which is the main place of neuronal degeneration (14). Because the
domain Cullin_Nedd8 also exists in D. melanogaster, understanding the function of this domain
in the fruit fly could aid in the development of a cure and/or treatment of Parkinsons disease.
As taxes and death are inevitable in life, error is always a possibility in scientific
experiments. As seen in the gel electrophoresis, the PCR failed to amplify the cDNA in the
plasmids. Other error could have occurred in the trimming of the trace file of the sequenced
plasmid DNA. If the trimmed sequence was trimmed too short or too long in certain areas, the
sequence could have resulted in different matches on the BLAST search, which may or may not
have led to human homologs. Other sources of error could have occurred in which result was
chosen as the best match for the sequence. Some of the matches had high max scores, which
indicates the level of similarity between the search and the results, and low error values, but did
not fit the accession format of NP_.... that actually led to a protein. Conversely, a proper
accession hit could have been chosen, but the error value and max score of the hit showed
considerable mismatch (15). If possible, this experiment should be repeated at certain sections to
improve accuracy.
In summary, DNA extracted in this experiment coded for a protein in Drosophila
melanogaster, the fruit fly, which has a homologous protein domain, Cullin_Nedd8, in humans.
The homologous domain in humans is involved in cellular processes, as well as the induction of
Parkinsons disease. Thus, understanding the function of this protein domain in Drosophila
melanogaster will help scientists understand the function of the human homolog in the
mechanisms of Parkinsons disease and human physiology. The discovery of this homologous
protein in fruit flies suggests that there are additional proteins in fruit flies that have human
homologs involved in human physiology and disease. Further sequencing and analysis of the
fruit fly genome may lead to understanding additional diseases and cellular processes in humans.

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