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Homology in Proteins of Model Organism Drosophila Melanogaster Help Further

Understand Disease Mechanisms of Myeloid Leukemia in Homo Sapiens
Elizabeth Kim
Lab Partner: Ashley Braxton
BIOL 230W, Section #902
TA: Alex Campbell, Ravi Patel
12 October 2015

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Introduction
Humans have evolved from simple eukaryotes into very complex organisms. Despite the
evolutionary differences we see in life today, the genetic makeup has not strayed too far.
Humans and monkeys are undoubtedly the most similar phenotypically, portraying several
homologies in anatomy and behavior (1). Scientists have been studying the similarities and
differences among all species to determine the physiology and function to better understand all
biological processes. Due to technological advances like Polymerase Chain Reaction (PCR) and
manipulation of plasmids, researchers have discovered that many organisms have similar protein
domains, sections of protein that serve a specific function and have evolved independently. With
this information, scientists have compiled a large database called a genomic cDNA library that
hold DNA sequences reverse transcribed from messenger RNA, mRNA. This allows anyone to
quickly determine whether protein domains are homologous in other organisms or not (1). For
example, researchers have found a homologue in humans and yeast that both require Cdc14, a
protein required for the regulation of cyclin-dependent kinase activity (CDK) in order for the
cells to leave the mitotic stage (2).
Diseases found in humans are a result of misfolding of proteins due to mutation in the
DNA. In order to find a cure, researchers must discover the source of the problem, but
performing experiments on humans would be unethical. Not only is it difficult to maintain the
participants health, but also the human life span is far too long for it to be a practical model in
lab. The model organism that is able to fulfill this position should be highly homologous,
consisting of domains that are of the same protein families. Specifically, the fruit fly Drosophila
Melanogaster is an excellent model organism to manipulate because it has been used in the field
of genetics for over 100 years. Through these studies, genes have been mapped according to

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chromosome location and function. Time is also saved using these organisms because of their
very short life cycles, allowing for as many trials as necessary. Most importantly, Drosophila
have a great amount of homology with humans, crucial for the study of human diseases.
Researchers have discovered that about 75% of disease-causing genes in Drosophila are
homologous in humans (3). For example, the functional homologue of Alzheimers Disease in
fruit flies is the protein domain APP-like or APPL, and deficiencies demonstrated abnormal
behavior. The same impairment in humans of the APP transgene is known to be involved in this
disease (3). By identifying the similar mechanisms involved in diseases of the model organism
will hopefully bring researchers a step closer to creating a treatment option for current terminal
diseases such as Parkinsons disease, epilepsy, and cancer (1). It should be noted that model
organisms are not perfect and implications may arise. Although the organisms have homologous
proteins as humans, the treatment that may work on the animal may not be processed the same
way as in a human body. The obvious differences between species make it difficult to discover a
cure for human diseases.
Throughout this experiment, the function of proteins sequenced from the cDNA library of
the Drosphila Melanogaster were identified, if any, the homology with cDNA of Drosophila and
human proteins, the function of the human protein was determined, and the function of these
proteins was found in Drosophila can help understand the way human diseases involve them was
explained. The overarching goal was to better understand human diseases by comparing the
human genome database to the cDNA library sequence of Drosophila Melanogaster (1).
With the knowledge that Drosophila contain high level of homology with human
diseases, the cDNA sequenced was involved in the function of the mechanism of a homologous

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protein responsible for a disease. This allowed scientists to further study the specific proteins to
eventually find a cure in humans.
Materials and Methods
All procedures were followed according to the lab manual (1).
Colony Picking
Two colonies were picked from an E. coli culture plate. It was important to practice
sterile technique because contamination would result in growth of extraneous bacteria and less of
the Drosophila cDNA. E. coli vectors were grown in ampicillin to minimize unnecessary growth.
The vectors contained an ampicillin resistance gene, ensuring growth of only the E. coli
containing the plasmid. Drosophila cDNA was inserted in plasmid pNB40 which contained the
ampicillin resistance gene. This allowed for optimal growth of cDNA but not E. coli to create a
cDNA library.
Plasmid Isolation
The QuickLyse Miniprep Plasmid DNA purification system was used to isolate the
plasmid DNA from the E. coli vector. This suspended the cDNA in lysis solution temporarily
then removed the plasmid from the culture. The plasmid was collected onto a membrane in the
spin column, and washed with isopropanol buffer. Elution then released the plasmid. Isolation
was crucial in order to amplify the Drosophila cDNA and ensured that no other bacteria were
involved and prepare for polymerase chain reaction.
Polymerase Chain Reaction
Once the cDNA was isolated, PCR was used to amplify the gene by primers and Taq
polymerase. Primers found the SP6 and T7 promoters that were located at either end of the
cDNA insert. Exponential growth was achieved through a series of denaturation, annealing, and

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elongation of the DNA with heating and cooling. The products were copies of the cDNA, which
allowed for efficiency in replication and created a library, which was necessary for developing
the model for human disease.
Gel Electrophoresis
An agarose gel was made to use as a medium for the cDNA to travel. Each sample of
plasmid, the respective PCR mixes, a negative control, and DNA ladder was pipetted into each
well. Gel electrophoresis showed a visual representation of the plasmid DNA and to determine
whether the isolation was successful or not.
DNA Sequencing and Identification
The plasmid DNA were taken to the Nucleic Acid Facility on campus. The dideoxy
method was used and this process entailed copying DNA. Because they lacked the 3 OH group,
the polymerase failed to add more base pairs, resulting in termination. The termination created
fragments and was then separated by size. The migration patterns across the gel were recorded
and are read from 5 to 3. MEGA was used to edit the trace file by removing any overlaps. This
step prepares the sequence to be observed and compared to homologous human proteins.
CDART was used to determine the conserved protein domains the cDNA sequence had. BLAST
was then used to identify the edited trace file to determine whether the cDNA sample is of the
same protein families as humans.
Results
Colony Growth Observations
Plasmid A: small amount of opaque growth at bottom of tube.
Plasmid B: small amount of opaque growth at bottom of tube (4).
Colonies grew successfully and were able to be used in PCR.

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Figure 1. Gel Electrophoresis Photo Under UV Light

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3
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5
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7
8
9
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Figure 1 Legend:
Well 1- DNA Ladder
Well 2- Negative Control (Of
other partners)
Well 3- 4A PCR
Well 4- 4A DNA
Well 5- 4B PCR
Well 6- 4B DNA
Well 7- Random PCR
Well 8- 3A DNA
Well 9- 3B DNA
Well 10- Missing PCR

Ethidium bromide added to agarose gel to allow for fluorescence under UV light. Bands
that have traveled the farthest (to the left) have fewer base pairs and fewer base pairs result in
lighter weight. In return, bands that traveled the least have the most base pairs, in other words are
the heaviest DNA fragments. Multiple banding in lanes may be a result of more than one plasmid
DNA was isolated at once or contamination by E. coli or other materials occurred.
Note: PCR master mix tubes went missing for partners 3A and 3B. This may be a result of
evaporation and was discarded. A band was present in negative control.

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Figure 2. Measurement of DNA Ladder

This figure shows the average base pairs per banding fragment to use as a template to
compare to the banding of plasmid cDNA.
Table 2. Molecular Size and Weight of cDNA Segments
DNA Segment

Plasmid 3A

Plasmid 3B

Random PCR
A
1500

PCR B

Negative
Control
100

Band Size
>2000
2000
N/A
(bps)
This table shows the average base pairs of each band observed after running gel
electrophoresis.
Sequencing Results

Plasmids A and B were sent for sequencing from the group and successfully created trace
files for both samples. Plasmid A was used in this analysis. MEGA was used to trim Plasmid As
sequence and then searched for identification using BLAST.

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Table 3. Drosophila Melanogaster mRNA Results of Plasmid A Trimmed Sequence (5)
Name of mRNA Sequence
Request ID
Accession
Query Length
Protein ID

Drosophila melanogaster
Myelodysplasia/myeloid leukemia factor
(Mlf), transcript variant H, mRNA
164X970J01R
NM_001259426.2
349
NP_001246355.1

Table 3 portrays information of the mRNA sequence such as request ID, accession
number, and length of the sequence. This sequence was the first hit to appear on the list of
BLAST results. The protein ID is the protein domain that correlated with the found sequence
after CDART analysis.
Table 5. Protein Identification of Homologues (5)
ID/Accession
Name of Protein
Protein Domain(s)

Request ID

Drosophila melanogaster
NP_001246355.1
Myelodysplasia/myeloid
leukemia factor, isoform H
Mlf1Ip Superfamily:
myelodysplasia-myeloid
leukemia factor 1-interacting
protein
164X970J01R

Homo sapien
NP_001182362.1
Myeloid leukemia factor 1
isoform 4
Mlf1Ip Superfamily:
myelodysplasia-myeloid
leukemia factor 1-interacting
protein
165FGKK301R

Table 5 expresses the protein domain with the highest level of homology in Homo
sapiens observed corresponding to the protein domain of the Drosophila. The Homo sapien
homolog had an identity of 49%, the highest value among the rest of the results after BLAST
analysis.
Discussion
According to the data recovered in this experiment, the hypothesis was proven correct.
There was a human protein involved in a disease mechanism homologous to a protein domain in

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Drosophila melanogaster. The only protein domain found between both species was responsible
for regulation of transcription and served many functions in the Drosophila, but mutations in the
protein domain of Homo sapiens resulted in myeloid leukemia (5). If further studies were to be
conducted on this specific protein domain, researchers could become more familiar with how the
disease works and eventually create a treatment option.
To come to this conclusion, plasmid DNA of Drosophila was extracted from E. coli
colonies and amplified using PCR technique. Samples of PCR master mixes were lost throughout
the experiment and did not correlate with other samples. Although confirmation of cDNA
amplification could not be confirmed through gel electrophoresis, the original plasmid DNA
samples were sequenced into trace files. This report focuses on plasmid As DNA sequence
which coded for Drosophila melanogaster Myelodysplasia/myeloid leukemia factor (Mlf),
transcript variant H, mRNA. The only protein domain involved in this sequence was Mlf1Ip
Superfamily: myelodysplasia-myeloid leukemia factor 1-interacting protein. This protein family
is a conserved central region responsible for transcriptional repressors located in the nucleus and
cytoplasm. Mlf1Ip was also regulated by the DREF transcription factor motif, which was
responsible for the regulation of rapid cell growth in Drosophila (5). Additional research has
suggested that immunohistochemical analysis of rat C6 and F98 glioblastoma tumor models
expressed a large amount of Mlf1Ip, deducing that this protein may play a role in cancers (6).
The experiment was not perfect and had plenty of room for error. The negative control
from gel electrophoresis showed banding, which calls for unreliable samples due to
contamination. Because the gel was shared with another group, the negative was from the sample
of the other group, making it difficult to conclude whether plasmid A focused in this report could
be faulty or not. Sources of this error may have resulted from improper sterile techniques

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including failure to change micropipette tips after every use, human waste contact such as saliva
or skin cells, and leaving the sample out in the open air for too long. These mistakes would also
result in poor accession hits when analyzing the cDNA sequence. To improve the experiment it
would be beneficial to repeat steps and carefully follow sterile technique. Although it is not
practical, working in an isolated environment with controlled variables minimizing
contamination would account for these issues.
The human homolog to Mlf1Ip was found through BLAST analysis and corresponded to
Myeloid leukemia factor 1 isoform 4 which contained the same protein domain as the
Drosophila sequence, Mlf1Ip. This domain had 47% homology, the amount of identical amino
acids between human and Drosophila protein (1). The correlation between the two species was
not significant, making this organism less reliable as a model organism. There was homology in
function, but Homo sapien Mlf1Ip specifically regulate the fate of hematopoietic cells that reside
in red bone marrow. This suggests that the human homolog is involved in the disease mechanism
of acute myeloid leukemia (5). Acute myeloid leukemia is a form of cancer that results in
overgrowth of white blood cells in the bone marrow, interfering with growth of normal blood
cells (7).
By the end of this experiment, the Drosophila cDNA sequence extracted and isolated
lead to the discovery of the homologous protein domain of Mlf1Ip Superfamily: myelodysplasiamyeloid leukemia factor 1-interacting protein found in Homo sapiens. This protein domain is
active in the human disease called myeloid leukemia. Manipulation of the model organism
Drosophila melanogaster protein homolog will allow researchers to better understand the
function of the disease by comparing functions in the protein domain of humans. This revelation
eludes that there are many other proteins in Drosophila that are homologous in humans,

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introducing a new realm of possibilities of furthering knowledge in human physiology and other
diseases that need yet to find a cure for. With the knowledge of homologous proteins and
function of both the model organism Drosophila melanogaster and Homo sapiens, conducting
further research could contribute to great advancement in finding a cure for myeloid leukemia.
Further studies could focus on the functionality of Mlf1Ip to determine whether this domain can
be manipulated in order to stop the disease. A similar protocol to this experiment could be
followed by testing on a better model organism with higher homology of protein domains would
be the next step.

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References
(1) Penn State Biology Department. Biology 230 Laboratory Manual. Lab handbook. The
Pennsylvania State University, PA. 2014.
(2) Vzquez-Novelle, M. Dolores, et al. "Functional Homology among Human and Fission Yeast
Cdc14 Phosphatases." Journal of Biological Chemistry 280.32 (2005): 29144-50. Web.
(3) Pandey, UB, and CD Nichols. "Human Disease Models in Drosophila Melanogaster and the
Role of the Fly in Therapeutic Drug Discovery." PHARMACOLOGICAL REVIEWS 63.2
(2011): 411-36. Web.
(4) Ashley Braxton: Penn State Biology Department. Biology 230 Laboratory Manual. Lab
handbook. The Pennsylvania State University, PA. 2014
(5) "National Library of Medicine." National Center for Biotechnology Information.
N.p., n.d. Web. 17 Oct. 2015. <http://www.ncbi.nlm.nih.gov>.
(6) Hanissian, S. H., et al. "Regulation of myeloid leukemia factor-1 interacting
protein (MLF1IP) expression in glioblastoma." NCBI (2005)
(7) "Acute myeloid leukemia." Wikipedia. Web. 12 Oct. 2015.