Drosophila as a Model for Human Disease: cDNA isolation and Analysis Drosophila as a Model for Human Disease: cDNA

isolation and Analysis Joshua W. Zollman

1

From the Undergraduate Department of Biology: The Pennsylvania State University

To whom correspondence should be addressed: Joshua W. Zollman, 342 Atherton Hall, University Park, PA 16802, USA. Tel.:(610) 220 2134; E-mail: jwz5153@psu.edu

INTRODUCTION Proteins consist of autonomous folding units called domains (1). Domains have specific functions and are capable of evolving independently (1). Domains can evolve by a variety of mechanisms, including duplications of domain coding sequences, mutations of coding sequences that lead to modified three dimensional structures with useful properties, and gene recombination resulting in new arrangements of domains (2). The presence of domains, along with their three-dimensional structures, have been found to have specific effects on protein function (3). As such, rearrangements and changes in existing domains can lead to variations in overall protein function (1). At times, mutations in the underlying DNA result in defective proteins that are responsible for human disease (1). An increasing number of human diseases are being traced back to a specific protein or protein domain (1). Thus, studying protein domains and their underlying sequences plays a vital role in understanding human disease. Many protein domains are highly conserved among organisms (1). For example, about 90% of protein domains have been found across eukaryotic organisms (3). Certain organisms show significant homology with humans, and have a particularly high number of conserved domains. The fruit fly, Drosophila melanogaster, has a particularly high degree of homology of DNA sequence and significant conserved biology with humans (1). Research has shown significant conserved proteins from mammals to Drosophila. One such study showed that a human synaptic vesicle membrane protein was also present in Drosophila (4). Furthermore, Almost 75% of disease-causing genes in humans are suspected to have homologues in Drosophila (5). In addition, 70% of known human cancer

genes and 60% of known human genetic disorders have Drosophila homologues. Because of this high level of homology to humans and the fact that using humans as subjects for research is problematic, Drosophila has been studied extensively as a model for human disease (1). Many other characteristics of Drosophila also make it a good choice, including its long short life span, its relative large number of offspring, and its ease of genetic manipulation (6). Drosophila has served as a model for human neurodegenerative diseases, such as Alzheimers Parkinsons disease, Huntingtons disease (6), diabetes, and various cancers including glioblastoma, prostate cancer, ovarian cancer, and more (1). The knowledge acquired from Drosophila models has been used to develop treatments (1). Thus, using Drosophila as a model for human disease continues to have potential for both uncovering crucial information about the causes and functions of various diseases and for developing new treatments. In order to compare the Drosophila and human genomes, the DNA of Drosophila must be isolated and amplified so that it can be sequenced. As such, plasmid transformation and PCR techniques are vital in isolating Drosophila cDNA clones (1). After the Drosophila cDNA has been sequenced, it can be compared to the human and Drosophila genome databases using the NCBI databases and BLAST analysis (7). 1BLAST analysis has proven very useful in identifying human disease genes that are also present in Drosophila (7). For example, Retier et al. performed a systematic BLAST analysis of 929 human disease genes and compared it to the genome of Drosophila. 714 different human disease genes, matching up with 548 distinct Drosophila sequences were identified (7). The purpose of this study is to identify potential models in Drosophila that may aid in the

Drosophila as a Model for Human Disease: cDNA isolation and Analysis understanding of human disease. To accomplish this, Drosophila cDNA library clones were sequenced and these cDNA sequences were used to search the human genome for potential homologous human genes, protein domains and disease-related functions. The cDNA sequences were also used to identify associated protein domains in Drosophila and identify their function. Based on research that has identified a large number of Drosophila homologs to human disease-causing genes, such as the research performed by Reiter et al., along with the abundance of studies that have successfully identified conserved protein domains from humans to Drosophila, I anticipated finding homologous human genes and conserved protein domains in the isolated Drosophila cDNA library. From this, I expected the isolated Drosophila protein to serve as an effective model for human disease. Results of the experiment confirmed the hypothesis, as conserved protein domains between Drosophila and humans were revealed. These results demonstrate the potential for Drosophila as model for cancers and other disease. EXPERIMENTAL PROCEDURES 2 An agar plate with individual E. coli colonies each containing a plasmid with a gene for ampicillin resistance and a gene for Drosophila cDNA sequences was produced. Two colonies from the agar plate were randomly selected and inoculated in 3ml of Luria broth plus penicillin. The cultures were grown overnight, with aeration at 37C. The cultures were then stored at 4C for one week. Plasmid DNA was then isolated through use of the QuickLyse Miniprep Plasmid DNA purification system. A small amount of each isolated plasmid DNA was then amplified via the polymerase chain reaction (PCR). A PCR master mix was used, containing buffer, dNTPs, Taq polymerase, and two primers that target the SP6 and T7 phage RNA polymerase promoters in the plasmid. In one tube, 24l of Master Mix was combined with 1l of the first plasmid DNA. In another tube, 24l of Master Mix was combined with the other plasmid DNA. A negative control was then produced by combining 24l of Master Mix with 1l of sterile water. After PCR was complete, the products were frozen for one week. Agarose gel electrophoresis was then used to separate the amplified cDNA by size. The agarose gel was created by combined 250mg of agarose with 25ml of 1XTAE buffer. The mixture was then heated the microwave for 40 seconds. The solution was then allowed to cool for approximately 2 minutes. 1l of ethidium bromide was then added to the molten agarose. The gel was then cast and allowed to harder for 10 minutes. Due to problems sealing the dams of the electrophoresis unit, the gel production and casting was repeated three times until successful. Then, 4l of 6x loading buffer was added to each of the PCR product tubes. For each plasmid sample of plasmid DNA, 2 l of the plasmid DNA was combined with 8l of water and 2l of 6x loading dye. Wells 1-6 of the gel electrophoresis unit were filled with 5 l of 4GenScript PCR DNA Ladder, Plasmid A DNA, Plasmid A PCR product, Plasmid B DNA, Plasmid B PCR product, and the PCR negative control product, respectively. Electrophoresis was carried at 97 volts. Photographs of the gel were then taken through use of a UV box (Figure 1). In our group, this stage was carried out by the lab TA, who was also in charge of determining which plasmids to submit for sequencing. Because sequencing of our plasmids yielded no results, we instead used one of last years sequencing data files (file 03g03_SP6_ER_Nov-16-2011.ab1). The 34Peaks computer software program was then used to examine the sequence data and isolate a high quality region of DNA to use for BLAST analysis. The sequence was cut from base 71 to 1244, and this section was used for analysis. A nucleotide BLAST search was performed on the DNA sequence (query ID: lcl|64333) to find any homologous proteins and conserved domains in humans. A search of the Drosophila genome was then carried out to investigate cDNA function and proteins domains in Drosophila. RESULTS Observation of the liquid cultures showed E. coli growth. No contamination was observed. Agarose gel electrophoresis (Figure 1) showed insert bands for the Plasmid A DNA and Plasmid B DNA. Comparison with the Genscript PCR DNA Ladder revealed that Plasmid A DNA was approximately 1,500 base pairs in length and

Drosophila as a Model for Human Disease: cDNA isolation and Analysis Plasmid B DNA approximately 2000 base pairs in length. The DNA PCR products showed no insert bands in the gel lanes. Because there was no PCR DNA product present, the DNA could not be submitted for sequencing. In order to continue the experiment, cDNA sequencing data from last year (file 03g03_SP6_ER_Nov-16-2011.ab1) was used for BLAST analysis instead. The sequence was 1341 base pairs in length, with relatively high quality until base 1244, after which the 4Peaks software consistently gave a DNA quality reading of 0. The sequence between base pair numbers 77 and 1244 was of relatively high quality, and was used for BLAST analysis. A BLAST search of the cDNA matched with the Drosophila cDNA sequence SD02803. This corresponds with the Drosophila gene locus AY06156 (accession # AY061576.1, 3419 base pairs). Search results of this gene on the NCIB database showed a corresponding protein Drosophila (CG10289, Protein ID: AAL29124.1), however further investigation of the protein using the FlyBase database revealed that the biological processes in which the protein is involved are unknown, and that its molecular function is also unknown. While, the nucleotide blast search using the human genomic plus transcript search set showed no human sequences with considerable homology to the cDNA sequence (query showed 0% convergence) BLAST search using the nucleotide collection revealed a conserved protein domain family, the SAP domain superfamily (cl09325). DISCUSSION The results of agarose gel electrophoresis suggest that PCR was unsuccessful in amplifying the Drosophila plasmid DNA. While bands appeared in the plasmid A and B DNA lanes, no bands appeared in the PCR product lanes. It is unclear why the amplification of the plasmid DNA using PCR failed; however, there are several possible explanations. Likely, the cDNA insert was missing in the plasmid, causing the primers to align directly next each other. This would lead to no DNA being amplified. A mistake in the Quicklyse MiniPrep procedure could have led to the failure of isolating the plasmid DNA. It is also conceivable that some level of DNA contamination occurred, or that degradation of the DNA occurred while the samples were stored over the week. Although the lack of an amplified PCR product meant that we could not submit a cDNA sample of sequencing, BLAST analysis of last years sequencing data yielded significant results. The cDNA sequence revealed the presence of the conserved SAPS superfamily. According the NCBI database and the work of Luke et al., this family includes a conserved region from proteins that are required for G1 cyclin transcription (8). 5 In addition, the Superfamily HMM library and genome assignments server shows that the SAP superfamily plays roles in various biological functions in higher organisms such as humans, including response to DNA damage, regulation of gene expression, mitotic cell checkpoints, and the regulation of interphase. The conserved domain architecture tool of the NCBI database also showed convergence of human zinc finger proteins in homo sapiens with the SAPS superfamily. These zinc finger proteins play significant roles in DNA binding and transcriptional regulation (9). Zollman et al., in a study of Drosophila genes, also observed domains present in zinc finger proteins that were evolutionarily conserved from Drosophila to mammals (10). The findings of this study reveal a zinc finger-encoding proto-oncogene gene that is association with human lymphoma. The role of the SAP superfamily in the regulation of the cell cycle and cell cycle checkpoints as well as its role in DNA regulation suggest that it could play an important role in human cancers. The disruption of any one of these vital functions could potentially lead to disease such as cancer. Furthermore, the role of zinc finger proteins in transcriptional regulation suggests a possible connection to cancer. The work of Zollman et al. confirms this with the finding that the disruption of genes related to zinc finger proteins is associated with a human lymphoma. In this study, the fact that the CG10289 Drosophila proteins biological function is unknown makes it difficult to evaluate its usefulness for understanding human disease involving this particular protein. However, if future studies uncover the function of the CG10289 protein in Drosophila, it may emerge as a useful model for understanding corresponding protein function in humans. Despite that the

Drosophila as a Model for Human Disease: cDNA isolation and Analysis function of this protein is unknown at this time, the fact that protein domains, such as those found in zinc fingers and the SAPS superfamily are conserved from Drosophila to humans, demonstrates the usefulness of Drosophila as a model of human disease. This study, along with countless others, demonstrate that studying the genes responsible for conserved domains in Drosophila may provide insight into the mechanisms controlling human cancers and other diseases, as well as help develop possible therapeutic treatments.

Drosophila as a Model for Human Disease: cDNA isolation and Analysis REFERENCES

1. Department of Biology. (2012, Fall). Bio 230W Laboratory Manual: cDNA Isolation and Analysis: The Pennsylvania State University. 2. Chothia, C., Gough, J., Vogel, C., and Teichmann, A., 2003. Evolution of the Protein Repertoire. Science. 300, 1701-1703. 3. Janin, J., Wodak, S.J., 1983. Structual Domains in Proteins and Their Role in the Dynamics of Protein Function. Prog. Biophys. Molec. Biology, 42, 21-78. 4. Sudhof, T.C., Baurnert, M., Perin, M.S., Jahn, R., 1989. A synaptic vesicle membrane protein is conserved from mammals to Drosophila. Neuron, 2, 1475-1481. 5. Pandey, U.B., Nichols, C.D., 2011. Human Disease Models in Drosophila melanogaster and the Role of the Fly in Therapeutic Drug Discovery. Pharmacological Reviews, 63, 411-436. 6. Bilen, J., Bonini, N.M., 2005. Drosophila as a Model for Human Neurodegenerative Disease, 2005. A Systematic Analysis of Human Disease-Associated Gene Sequences in Drosophila Melanogaster. The Annual Review of Genetics, 39, 153-171. 7. Reiter, L.T., Potocki, L., Chien, S., 2001. Genome Research, 11, 1114-1125. 8. Luke, M.M., Della Seta, F., Di Como, C.J., Sugimoto, H., Kobayashi, R., Arndt, K.T., 1996. Mol Cell Biol, 6, 2744-1755. 9. Laity, J.H., Lee, B.M., Wright, P.E., 2001. Zinc finger proteins: new insights into structural and functional diversity. Current Opinion in Structural Biology, 11.1, 39-46. 10. Zollman, S., Godt, D., Priv, G.G., Couderc, J.L., Laski, F.A, 1994. The BTB domain, found primarily in zinc finger proteins, defines an evolutionary conserved family that includes several developmentally regulated genes in Drosophila. Proceedings of the National Academy of Science, 91, 10712-10721.

FOOTNOTES
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This lab was completed as part of the Biology 230W class in fall 2012. See reference 1 for lab manual citation. 2 Prepared in advance by Lab TA 3 Software can be found at http://www.mekentosj.com/science/4peaks 4 www..genscript.com 5 SUPERFAMILY is a database of structural and functional annotation for all proteins and genomes. http://supfam.cs.bris.ac.uk/SUPERFAMILY/index.html FIGURE LEGENDS FIGURE 1. Photograph of gel electrophoresis of plasmid DNA & PCR products. Well 1: 5 l of PCR DNA Ladder. Well 2: Plasmid A DNA. Well 3: Plasmid A PCR product. Well 4: Plasmid B DNA. Well 5: Plasmid B PCR product. Well 6: PCR negative control product. Agarose gel electrophoresis showed insert bands for the Plasmid A DNA (Well 3, approximately 1500 base pairs in length) and Plasmid B DNA (Well 6, approximately 2,000 base pairs in length). The DNA PCR products showed no bands.

Drosophila as a Model for Human Disease: cDNA isolation and Analysis

Figure 1 Agarose Gel Electrophoresis Lanes: 1 2 3 4 5 6