Characterization of Dvilp7 in Drosophila virilisJuliette McGhee & Hunter KelleyBCMB 403 - Neurogenetics Lab

Dept. of Biochemistry & Cellular & Molecular Biology, Univ. of Tennessee, Knoxville, TN 37996

Drosophila melanogaster central nervous system (CNS) image and illustration

The central nervous system (CNS) was extracted from D. melanogaster using a stereo-microscope and very sharp

tweezers. The larva was cut in half and its outer shell removed. The CNS was then extracted from other tissues

and rinsed. Fixative and PBTx were applied and the specimen was stained with X-gal and incubated at 37° C for approximately 30 minutes. The result was mounted

on a slide glass and observed under a 1000x magnification light microscope.

Research Data AbstractGlucose is an important energy source for many organisms and cannot be digested without the help of insulin. In Drosophila, insulin/insulin-like signaling (IIS) is highly conserved¹. Understanding Drosophila insulin-like peptide (Dilp) genes will provide insight into possible mechanisms regulating IIS and how they relate to growth and development, metabolism, reproduction, stress response, and longevity within the Drosophila family of model organisms.1 This study successfully characterized the Dilp7 gene from Drosophila virilis (Dvilp7) through PCR and RACE experiments, E. coli transformation, and preparative and analytical gel electrophoresis analysis. cDNA was constructed from mRNA and target cDNA isolated in 3’-RACE-I and 3’-RACE-II experiments. Target DNA was then purified and transformed using E. coli and sequenced to compare with genomic DNA to determine intron sequences. 5’-RACE-I and 5’-RACE-II experiments were conducted to reconstruct the entire Dvilp7 sequence. Classification of this sequence expands the understanding of genetic mechanisms underlying the Dilp series of genes.

Methods• RNA Purification & Reverse Transcription: D. virilis flies were separated into

heads/bodies, homogenized and mRNA purified. Samples were subjected to reverse transcription (RT) reaction to obtain cDNA template for PCR reactions.

• 3’-RACE-I (PCR): RT cDNA was purified using Qiaquick kit purification columns. 3’-RACE-I PCR was run to amplify all cDNA (target & non-target) (Fig. 1).

• 3’-RACE-II (Nested PCR): cDNA from 3’-RACE-I was subjected to a gene-specific primer for amplification of target cDNA only (Fig. 1).

• DNA Purification and Ligation: Preparatory gel electrophoresis was run to isolate the Dvilp7 cDNA from non-target cDNA (Fig. 7).

• Transformation of E. coli: Purified DNA was ligated using pGEM-Teasy vector system and Taq DNA polymerase, E. coli was then plated with X-gal and IPTG and incubated (Fig. 4).

• Plasmid DNA Purification, Restriction Digestion & Sequencing: Bacterial cells were inoculated, harvested and recombinant DNA was purified using a QIAPrep kit from Qiagen.

• 5’-RACE-I (PCR): QT-primed cDNA was subjected to a poly-A tailing reaction with TdT buffer, dATP and TdT. This was then combined with a QT primer (Dvilp7-r1) (Fig. 2)

• 5’-RACE-II (Nested PCR): PCR was run with Q1 and Dvilp7-r2 primers and diluted 5’-RACE-I template (Fig. 2).

• DNA Purification and Sequencing: DNA was purified and sequenced at the sequencing facility at the University of Tennessee, Knoxville using a Sephadex G-50 gel filtration matrix purification column and a ThermoFisher Scientific 3730 DNA Analyzer.

• Genomic DNA Preparation from D. virilis: Genomic DNA PCR was prepared and sequenced in the same fashion as the 5’-RACE-II PCR.

• Analysis of Sequence Data: Gel electrophoresis and electropherogram examination (Fig 9).

DiscussionRACE experiments are powerful tools used specifically to obtain a full-length cDNA, they allow for cloning of rare target cDNA, such as Dvilp7. The 3’-RACE-I experiment used primers for both the whole cDNA sample (Q0) as well as a Dvilp7 gene specific primer (Dvilp7-f1) to increase the numbers of the target cDNA to an extent. The 3’-RACE-II experiment used a primer specific to Q1 and a primer specific to the Dvilp7 gene (Dvilp7-f2) to amplify only the target cDNA. Taq DNA polymerase adds an A overhang to the cDNA allowing it to be ligated to the pGEM-T vector used in the PCR reactions. Prior to 5’-RACE PCRs, purified cDNA was ligated into a plasmid vector. Recombinant plasmids contained an ampicillin resistance gene (amp’) allowing them to be distinguished from empty plasmids in a blue/white screening. The Dvilp7-r1 primer used for the 5’-RACE-I and II experiments was a reverse primer to amplify the 5’ sequence flanking the known sequence. The result of the 3’-RACE-II and 5’-RACE-II DNA sequencing were electropherograms of the target sequence. These electropherograms were analyzed to determine the cDNA Dvilp7 gene sequence.

ResultsAnalytical gel electrophoresis reactions were run to determine the success of the 3’-RACE-II (Fig. 6) and 5’-RACE-II PCRs (Fig. 7). The electropherograms from the 3’-RACE-II, 5’-RACE-II, and genomic DNA PCRs were examined to find primer boundaries and determine the beginning and end of introns. The 5’-RACE sequence was then converted into its reverse compliment using bioinformantics.org sequence manipulation tool suite². This allowed for the sequences to be assembled into a single full-length Dvilp7 cDNA sequence (Fig. 5). The conceptual translation product was determined by finding the open reading frame using NCBI³ and ExPASy⁴ ORF finders and to determine the longest reading frame of the cDNA sequence (Fig. 3). This gave the amino acid sequence of the Dvilp7 gene (Fig. 5). The signal peptide was determined using the CBS Prediction Servers ProP v1.0 server⁵ (Fig. 5). The first intron was found to be a Phase-1 intron and the second intron a Phase-2 intron.

Acknowledgments Our sincerest thanks to Dr. Jae Park for his scientific guidance, discussions and assistance in the lab. Without your support we would not be graduating. We would also like to thank Moon Ma for her assistance in the lab, Mr. Joe May for sequencing our DNA, and the BCMB Neurogenetics Lab class of Fall 2016 for their collaboration in this research project.

Citations1. Kannan, K., & Fridell, Y. (2013). Functional implications of Drosophila insulin-like peptides in metabolism aging, and dietary restriction. Frontiers In

Physiology, 4, Frontiers In Physiology, 2013, Vol.4. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3797364/)2. Bioinformatics - Https://www.bioinformatics.org/sms/rev_comp.html3. NCBI - https://www.ncbi.nlm.nih.gov/orffinder/4. ExPASy Bioinformatics Resource Portal - http://web.expasy.org/translate/ 5. CBS Prediction Servers ProP v1.0 - http://www.cbs.dtu.dk/services/ProP/6. Gloyn, A. L., Pearson, E. R., Antcliff, J.F., Proks, P., Bruining, G.J., Slingerland, A. S., … & Edghill, E. L. (2004). Activating mutations in the gene encoding

the ATP-sensitive potassium-channel subunit Kir6. 2 and permanent neonatal diabetes. New England Journal of Medicine, 350(18), 1838-1849.7. Owerbach, D., Bell, G.I., Rutter, W.J., Brown, J.A., & Shows, T.B. (1981). The insulin gene is located on the short arm of chromosome 11 in humans.

Diabetes, 30(3), 267-270.

Figure 2. Diagram of typical 5’-RACE procedure

Figure 8. Analytical gel electrophoresis of 5’-RACE-II cDNA with DNA marker

Figure 6. Analytical gel electrophoresis of 3’-RACE-II cDNA with DNA marker

Figure 5. Full length Dvilp7 cDNA sequence and polypeptide protein characterization

Figure 1. Diagram of typical 3’-RACE procedure

Figure 3. Diagram of Dvilp7 putative translation product

Figure 7. Preparative agarose gel of 3’-RACE-II cDNA

Figure 4. Diagram of typical plasmid recombination procedure

Figure 9. Sample electropherogram plot of results from separation done by electrophoresis

Background & Significance• In humans, preproinsulin interacts with the signal recognition molecule in the

cytosol of the beta cell, which then sends proinsulin to the endoplasmic reticulum. Cleavage of the signal sequence allows proinsulin to fold into the ER lumen, leading to the formation of disulfide bridge between chain B and chain A, and on chain A independently between basic amino acids. Cleavage of chain C forms insulin.

• Neonatal diabetes is caused by a mutation in preproinsulin. The signal peptide isn’t allowed to cleave, leading to the alteration of cellular trafficking.6

• Somatic cell hybrids differentiated from restriction endonuclease mapping with different chromosome combinations have allowed researchers to localize the human insulin gene through the use of cloned rat and human cDNA probes, concluding that the human insulin gene is found on chromosome 11.7

• Seven insulin-like genes have been identified in D. virilis. Alteration to these can lead to size deficits and elevated blood sugar levels, characterized by a Dvilp2 gene mutation. 1

• The significance of this study is the isolation and characterization of the Dvilp7 gene, which may be used in future investigations to broaden understanding of the physiological roles and mechanisms underlying the transcriptional regulation of insulin signaling in Drosophila.