1. HIP14 in zebrafish was successfully cloned into a pDrive and sequenced. Alignment analysis was performed by comparing the amino acid sequence in zebrafish to the amino acid sequence in humans. Alignment analysis and phylogenetic tree created using Clustal W2. The Cloning and Characterization of Huntingtin Interacting Protein 14 (HIP14) in Zebrafish Emily Wright and Dr. Wendy Boehmler, Department of Biology, York College of Pennsylvania Conclusions A novel HIP14 gene in zebrafish was identified. Expression of HIP14 has been found to occur in various adult tissues and different developmental time periods. Sequence comparisons and phylogenetic analysis provide strong support that zebrafish possess the HIP14 gene. Future Experiments Perform an in situ hybridization on embryos to determine when and where gene development occurs. Perform a morpholino knockdown of HIP14 to determine if the gene plays a role in neurodevelopment. Acknowledgements I want to thank Dr. Wendy Boehmler for her assistance and guidance throughout this project. Background Information on HIP14 Mutations in HIP14 result in the development of the neurological disorder known as Huntington’s disease in humans. Is localized within the Golgi membranes of human brains. Gene is composed of 5 different domains, all of which combine to make a gene that is 1894 basepairs in length. One domain present is the DHHC domain, which is surrounded by an area rich in cysteine residues. Questions/Objectives Question 1. Is HIP14 expressed in adult zebrafish? Question 2. Is HIP14 expressed solely in the adult brain? Question 3. Is HIP14 expressed embryonically? Methods Compared human and zebrafish amino acid sequences by using the program called BLAST (Basic Local Alignment Search Tool). RT-PCR was conducted using cDNA collected from adult zebrafish, embryo, and various tissues. Figure 1: RT-PCR of HIP14 at different developmental times. Bands appeared at 2KB for adult, 3 and 6 day embryos. Figure 2: RT-PCR of HIP14 gene on various tissues. After running a tissue panel, it was determined that HIP14 was found in the brain, gut, heart, muscle and eye. TCG AGC AGC AAG ATC GCGCTA CAC CAG CTG GTA TCC 3’ 5’ (1894bp) Reasons to Use Zebrafish Mutations in zebrafish phenotypes resemble that of human disorders. Can easily and quickly breed zebrafish. Eggs can be studied and manipulated in order to study gene function. Genome has been fully sequenced. Zebrafish HIP14 Figure 4. A phylogram tree showing the relative similarity of the HIP14 genes among the following organisms. Human Table 1. Pairwise comparisons between Zebrafish/Mouse and Human HIP14 proteins. http://www.3dnucleus.com/ashop/prodimg/52.jpg http://www.funnyphotos.net.au/images/ordinary-zebrafish1.jpg Figure 3. Alignment of various amino acid sequences. Literature Cited Doonley, K. and Zon, L.I. 2000. Zebrafish: a model system for the study of human disease. Genetics & Development 10: 252-256. Goytain, A., Hines, R.M. and Quamme, G.A. 2008. Huntingtin-interacting proteins, HIP14 and HIP14L, Mediate Dual Functions, Palmitoyl Acyltransferase and Mg 2+ Transport. Journal of Biological Chemistry 48 33365- 33374. Grunwald, D.J and Eisen, J.S. 2002. Headwaters of the zebrafish-emergence of a new model vertebrate. Nature Reviews 3: 717-724. Huang, K., Yanai, A., Kang, R., Arstikaitis, P., Singaraja, R.R., Metzler, M., Mullard, A., Haigh, B., Gauthier-Campbell C., Gutekunst, C.A., Hayden, M.R. and El-Husseini, A. 2004. Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in palmitoylation and trafficking of multiple neuronal proteins. Neuron 44: 977-986. Table 2. Chromosomal locations of HIP14 gene. *10 other genes that are syntenic to these chromosome locations. 2 KB Adult 3 Day6 Day BrainGutHeartMuscleEye