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Drosophila cDNA linked to Human Genetic Disease of Pseudohypoaldosteronism

Alexis Moyer

Jared Reid

BIO 230W, Section 18

Zach Fuller, Hannah Herriot

October 20, 2016

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Introduction:

There are many linkages that can be found between the proteins of humans and the

cDNA of Drosophila, when comparing the genes of one to the other. These connections can lead

to new ideas and increase understanding of human genetic diseases. In this experiment, the

cDNA came from the model organism of drosophila. There are many reasons that Drosophila

was chosen as the organism used for this experiment, including the fact that they are easily

manipulated and closely linked to humans. Many genetic diseases in humans have homologous

proteins with these types of flies (1). Some of these diseases, include human cancers, Parkinson’s

disease, and epilepsy, all in which discovery of these protein mutations in the model organism

makes the drosophila a good subject for drug screening and better understanding of the diseases

(2). The purpose of this experiment is to identify a protein domain that is homologous to a

disease in humans, which could lead to further discovery.

The proteins that code for diseases in humans are typically mutated and defective. The

homologous part that we are looking at are the protein domains that are similar within the

drosophila cDNA and human DNA. Protein domains can operate independently of the protein

and are repeated throughout all eukaryotes. The protein domains that make up particular proteins

play a main role in human diseases because of the different arrangements of the domains within

each protein that is linked to the disease. The specific arrangement of protein domain makes up

the protein family, which identifies for the genetic disease that is present. (2)

These types of studies are made possible because of the connection of genes between

humans and many model organisms, such as drosophila. DNA libraries allow for analysis of

DNA fragments of specific organisms. In this study, cDNA was used, which is complimentary

DNA that is reverse transcribed from mRNA. This specific type of DNA is a representation of
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the genes found in a particular tissue of the organism. The libraries made up of the cDNA need

to be inserted with plasmids, due to the instability of them by themselves. The specific plasmid

used in this study was pNB40, which was then mixed with the E. coli cells and stored as the

library. (2)

Plasmids, such as pNB40, are circular and must match the restriction site of the cDNA, in

order to form the DNA library. The plasmid used in this study also contains an ampicillin-

resistance gene, which allowed E. coli to continue growing in the culture. Without this gene, the

E. coli bacteria would die, due to the antibiotic ampicillin, and the study would not be able to be

run. Other types of plasmids can contain advantages such as the one used throughout this

experiment.

Isolation and amplification are major steps found within this research. The plasmid DNA

needed to be isolated and amplified, in order to continue with the analysis for homologous

proteins. To isolate the plasmid DNA from the chromosomal DNA, it needs to be lysed, which

releases the plasmid DNA from the rest of the cells. This is then washed and eluted with buffers,

in order to avoid any contamination and lead to better results in the amplification. The plasmid

DNA that was isolated is then amplified, using polymerase chain reactions, to make many more

copies of this particular DNA. In order to complete this process taq polymerase is used to add

monomers to the 3’ end of the growing strand of DNA. Another important part of PCR is DNA

template and primers, which allow for the elongation of DNA strands. The purpose of amplifying

the plasmid DNA is so that it can be analyzed using gel electrophoresis.

Gel electrophoresis is used to determine the size and how successful the isolation and

amplification was on the plasmid DNA. The size is determined by how far the solutions in the

wells move in relation to each other and the DNA ladder. This is measured in terms of how many
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base pairs are found within the DNA in each well. Contamination can be determined because it is

known that the PCR product should move less because it has more base pairs than the regular

plasmid DNA. Also if nothing shows up in the well lane for the PCR product DNA, it can be

concluded that the PCR was not run correctly.

Sequencing of the DNA occurs in order to determine the specific nucleotides that make

up the plasmid DNA. From the sequencing, the coding can be traced and looked at for

homologous protein domains, which can then be used to answer the main research question of

this study, which is whether or not Drosophila is a reliable model organism for better

understanding and research of human genetic diseases. Answering this question requires the

identification of the drosophila protein that was found by sequencing the cDNA. From this

identification of the protein, homologous human protein and protein domains are found and

analyzed for their function. Studying the homology between the two can lead to information on

how to control this protein within humans. (2)

Materials and Methods

This experiment took five weeks to complete and analyze all of the data and all

procedures were followed as given in the lab manual.

The first thing that had to be done for this experiment was that E. coli colonies had to be

inoculated and grown overnight. These E. coli colonies were injected with pNB40 that was

resistant to ampicillin, which was the antibiotic that stopped the other colonies from growing.

After the colonies were grown, the plasmid DNA was isolated from the rest of the culture. This

was done by first using a lysis solution, then a wash solution, and finally an elution solution. By

doing this, the plasmid DNA is captured and separated from the rest of the bacterial DNA.
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This isolated DNA was then amplified, in order to figure out the size of the plasmid

insert. Each plasmid was mixed with the master mix and placed in a PCR machine, which is the

process of elongating and making more copies of the plasmid DNA. The next step of the

experiment was to run a gel electrophoresis, using the two plasmid DNAs and the two plasmid

PCR products. The gel electrophoresis was run to determine the size of the plasmid and to

identify any contamination that could have occurred throughout the whole process. After seeing

the size of the plasmids, the cDNA that was present in the plasmid was sent to a sequencing

facility to be sequenced. With this sequenced cDNA, it was possible to analyze it for

homologous proteins that were linked to human diseases, using programs such as MEGA,

CDART, and BLAST. (2)

Results

The first part of the analysis was to figure out whether the PCR was successful and to

look at the sizes of the products. In figure 1 below is a picture of the gel that was run with the

two plasmid DNAs and two PCR products. The size can be determined by looking at how far the

DNA moved along the gel and comparing it to the other wells and the DNA ladder.

Figure 1:
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Well 6 – negative control

Well 5 – plasmid B PCR product

Well 4 – plasmid B DNA

Well 3 – plasmid A PCR product

Well 2 – plasmid A DNA

Well 1 – DNA ladder

From this photograph it can be seen that the PCR products moved further than the regular

DNA, meaning the regular plasmid DNA was heavier than the PCR product. Also in well 5, the

PCR product does not show up, showing either a contamination or an unsuccessful PCR. After

the gel electrophoresis was run, the plasmid DNA was sequenced to be analyzed by tracing the

coding of the DNA. In figure 2 below is the trace of the sequenced plasmid DNA.

Figure 2: A – green, T – red, G – black, C - blue

Each nucleotide has a different color to make it easy to trace which is the most present at

that time. As seen in figure 2, there are N nucleotides, which mean that they could not be read
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and indicates a messy region of the DNA. The beginning of the trace is considered much better

quality and then starting at 50, it begins to get messy with many overlapping peaks. (2)

By using this trace in the program, called blast, it is easy to find the closest related

mRNA to the sequenced DNA. The closest related protein to this plasmid was Drosophila

Melanogaster longitudinals lacking transcript variant AA with the Reference sequence of

NM_176128.5 (ZCY4XU2P01R)(3) . With this the CDART program is then used to look at the

protein domains found within this specific protein. The only protein domain found in this

specific protein was BTB (4). This protein is then looked at for the closest human linked protein.

With 33% identical amino acids each were the kelch-like protein 3 isoform 1 (NP_059111.2) and

kelch-like protein KLHL3A(AAF20938.1) (ZCYSJX1K01R)(3).

Discussion:

The protein that is closest linked to the plasmid DNA of the drosophila was a kelch-like

protein. These types of proteins main function are ubiquitination, which is a post-translational

modification. Mutations in this protein can be a cause for pseudohypoaldosteronism type IID.

This is a disease that is caused by the increasing levels of a steroid hormone, called aldosterone

(5). It causes humans who have it to lose the salt in their body and go into hypertension. Also

this disease messes with the metabolisms of those that suffer with it (6).

Throughout this research many questions have been answered, including the overarching

question, asking whether Drosophila is a good model to study genetic dieseases in humans. The

answer to this question is yes, it was found that the protein found within the cDNA was

homologous to the KLHL3 protein found in humans, which was then found to lead to a disease,

as explained above. This information could lead to a better understanding of the disease of

pseudohypoaldosteronism. Since, Drosophila was found to have homologous proteins with

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humans, these organisms can help with research on how to prevent and cure the disease. It could

help in the discovery of certain drugs and other ways to stop the disease from getting serious or

even prevent the disease from happening in humans.

The errors found when looking and analyzing the gel that was run could be due to many

human errors. One error could be that the master mix was not made correctly or was

contaminated, making the PCR product in well 5 not show up. Also there was a problem with the

gel rig used because the port that it was plugged into was not working correctly, so the whole gel

rig was moved and could have had an influence on the errors found when reading the gel. These

errors could lead to the sequencing being messy and the analysis of the protein being wrong.

Further research could be done to find other model organisms that could be used to help

in the discovery of more human genetic diseases. Also further study could be done with the

Drosophila to look for cures for the genetic disease found within this research.

Reference

(1) Reiter LT, et. al. (2002, June 6). Using Drosophila melanogaster to uncover human disease

gene function and potential drug target proteins. 387-99.

(2) Nelson, K, Fisher C, Ward AM, and J. Malcos. Biol 230W: Biology of Molecules and Cells

Laboratory Manual. 2016. Department of Biology, The Pennsylvania State University.

(3) Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local

alignment search tool." J. Mol. Biol. 215:403-410. PubMed

(4) Geer L et al. (2002), "CDART: protein homology by domain architecture.", Genome

Res.12(10)1619-23

(5) KLHL3. (2016, July 29). Retrieved October 12, 2016, from Wikipedia
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(6) M J Dillon, e. a. (1980). Pseudohypoaldosteronism. Archives of Disease in Childhood, 427-

434.