Aiden Soroko Department of Biological Sciences, York College of Pennsylvania

1. Identification of a Homolog for a Potential Sperm Chemoattractant in the Zebrafish, Daniorerio Aiden Soroko Department of Biological Sciences, York College of Pennsylvania Methods Discussion Develop sequence for fusion protein including linking sequence. Generate oligonucleotides using Gene2Oligo Ligase chain reaction (LCR) used to create gene PCR to amplify DNA, Gel Electrophoresis, Extraction & Purification of PCR Product Ligation of PCR product into pDrive cloning vector High efficiency transformation of E. coli Plasmid purification 114 bp linker A linker was successfully created to combine the possible chemoattractant and green fluorescent protein together to increase the proteins size and also allow for proper folding. Completion of the chemoattractant-GFP fusion protein is needed to test the initial hypothesis that the sequence in the zebrafish genome, homologous to that found in a mouse, will possess sperm-attractant properties. Ideally, if the chemoattractant/GFP fusion protein can be synthesized in a eukaryotic cell where glycosylation will take place and cause the protein to fold and become functional, we suspect it will exhibit chemoattractant properties when placed in close proximity to zebrafish sperm. Chemoattractant Green Fluorescent Protein Figure 1. Schematic for fusion protein construction. The bracket indicates the region synthesized by LCR. A linking sequence was an important addition to space the components out to allow room for proper folding and for the protein to achieve functionality. Accumulation of sperm towards an unfertilized egg. (http://images.sciencedaily.com/2010/10/101003205930-large.jpg) Introduction Sperm chemotaxis, the guidance of sperm to an ovulated egg via the development of a chemical gradient, has been widely demonstrated across several different species of invertebrates and is a very important process affecting reproduction (Sliwa 1999, Xiang et al. 2004). Chemoattractants are produced by and secreted from ovulated eggs and create a gradient that causes sperm to accumulate towards the egg. The gradient manipulates the direction the sperm travel in, and also serves to enhance motility or velocity of the sperm (Xiang et al. 2004). The zebrafish, Daniorerio, is an external fertilizer (Kondoh et al. 2008). Since it releases gametes into the environment, the communication that takes place between the gametes is crucial. The roles of chemoattractants in zebrafish fertilization are relatively unexplored. A known chemoattractant in the mouse, Musmusculus, shows high homology to a sequence in the zebrafish that has thus far been uncharacterized(Harrison 2010). Future Research Figure 2. LCR is a DNA amplification technique that is based on the use of oligonucleotide primers. These primers hybridize to a strand of target DNA and are very accurate as they can discriminate from DNA strands differing by a single base pair. If the oligonucleotides are exactly complementary to the target strand, the ligase will covalently join them together. If not, there will be no product. This method helps to prevent false-positive ligation products. The ligated products can then be used for a template resulting in an exponential amplification process (Weidmann, Wilson, Czajka, et al.). (http://berry.engin.umich.edu/gene2oligo/) Create full fusion protein and integrate into expression vector Transfect eukaryotic monkey kidney cos-7 cells with expression vector Protein extraction & purification using nickel-metal ion chromatography Zebrafish sperm chemoattractant bio assay to test effectiveness of protein Results The 114 base pair linking sequence was successfully inserted into the pDrive cloning vector (Figure 3) and used to transform E. coli. White colonies appeared on the selection plate indicating a successful insertion (Figure 4). Literature Cited Harrison, Z. 2010. Identification of a mammalian homolog to amphibian allurin, a sperm chemoattractant. YCP Thesis. Kondoh, E., Komo, A, and Inaba, K., et al. 2008. Valosin-containing protein/p97 interacts with sperm-attracting factor (SAAF) in the ascidian egg and modulates sperm-attracting activity. Development, Growth, and Differentiation 50: 665-673. Weidmann, M., Wilson, W.J., Czajka, J., Luo, J., Barany, F., and Batt, C.A. 1994. Ligaase chain reaction (LCR) – overview and applications. Genomic Research (3): S51-S54. Xiang, X, et al. 2004. The sperm chemoattractant “allurin” is expressed and secreted from the Xenopus oviduct in a hormone-regulated manner. Developmental Biology 275: 343-355. Hypothesis I hypothesize that this homologous sequence in the zebrafish genome encodes for a protein that exhibits chemoattractant properties and plays a role in reproduction. Figure 3. The pDrive cloning vector used in the high efficiency transformation of E. coli. PCR product inserted into vector at multiple cloning site (location U). A successful insertion interrupts the Plac pathway. Figure 4. E. coli was plated on nutrient agar containing IPTG, X-gal, and kanamycin for selection. Transformed bacteria that incorporated the insertion are white in color while bacteria that did not incorporate the insertion are blue in color. Acknowledgement I would like to thank Dr. Thompson for his guidance on this project.