1. Identification and Cloning of Two Hyperprolinemia Genes in Danio rerio Abbie Werner, Department of Biology, York College Introduction Hyperprolinemia is an autosomal recessive disorder characterized by increased proline concentrations in the cerebrospinal fluid, plasma and urine (Shivananda et al. 2000). There are two types of hyperprolinemia: Type I is the result of one of sixteen different missense mutations on the proline dehydrogenase gene (Bender et al. 2005). Type II is categorized either by a single missense mutation (S352L) or a frameshift mutation on the Δ 1 -pyrroline 5-carboxylate dehydrogenase gene (Srivastva et al. 2012). Clinical manifestations of hyperprolinemia include mental retardation, infantile seizures and neuropsychological conditions, such as schizophrenia and schizoaffective disorder. Danio rerio are convenient, inexpensive models that can be used in the identification and classification of known and unknown human morbidities, owing to the fact that their genome has been fully sequenced. While hyperprolinemia has been modeled in zebrafish through the administration of high amounts of proline to their water (Savio et al. 2012), the genes associated with the disease have never been identified or characterized in zebrafish. Objectives 1.Determine whether these two hyperprolinemia genes exist in zebrafish 2.Determine the similarity of the genes between humans and zebrafish Methods ZebrafishHuman Ortholog NameMap Position aldh111 rbb4I111 myog111 mdm4111 cyb5r1111 olfml3111 cntn2111 mgc26818111 her2111 pax7a111 cgi115111 irf2bp2111 prodh2122 crkl2122 pcqap2122 prodh21222 pvalbb1222 aco21222 tomm221222 l3mbtl21222 tnrc6b1222 tef1222 Table 1. Syntenic relationships of human and zebrafish genes. The chromosomes in humans and zebrafish containing ALDH share 12 genes. The chromosomes containing PRODH share three, while the PRODH2 chromosomes share seven genes. Literature Cited Bender, H.-U., Almashanu, S., Steel, G., Hu, C.-A., Lin, W.-W., Willis, A., Pulver, A., and Valle, D. 2005. Functional consequences of PRODH missense mutations. The American Journal of Human Genetics. 76: 409-420. Savio, L. E. B., Vuaden, F. C., Piato, A., Bonan, C. D., and Wyse, A. T. S. 2012. Behavioral changes induced by long- term proline exposure are reversed by antipsychotics in zebrafish. Progress in Neuro-Psychopharmacology & Biological Psychiatry. 36: 258-263. Shivananda, Christopher, R., and Kumar, P. 2000. Type I hyperprolinemia. Indian Journal of Pediatrics. 67: 541- 543. Srivastava, D., Singh, R. K., Moxley, M. A., Henzl, M. T., Becker, D. F., and Tanner, J. J. 2012. The three-dimensional structural basis of type II hyperprolinemia. Journal of Molecular Biology. 3: 176-189. Conclusions 1.Hyperprolinemia genes do exist in zebrafish. 2.There is high identity between ALDH (77%) and PRODH (61%) genes. 3.The zebrafish genome went through a genome-wide duplication event leading to two PRODH genes. 4.Based on the analysis of human and zebrafish chromosomes, there are many conserved genes between the two. Future Directions Complete a tissue panel of the zebrafish to determine whether the genes are localized to one area. Do an embryological study to look for a specific stage during development at which the genes are expressed. Complete a chemical library screen to look for potential hyperprolinemia treatments. Acknowledgements I would like to thank Dr. Boehmler for her help in the completion of this project. I would also like to thank the York College Biology Department for their continuing support. Figure 3. Comparison of human and zebrafish ALDH genes. Genes were aligned using Clustal Omega software where identical amino acids are marked with an asterisk. The two genes shared 77% identity. Figure 1. Agarose gel electrophoresis from a PCR of cDNA collected from adult zebrafish. Amplification of the PRODH gene appeared successful according to bands present in all three lanes corresponding to approximately 1,100 base pairs. Other bands correspond to excess primers or nonspecific amplicons. 500 BP 1000 BP http://ngs-brescia.blogspot.com/2013/05/pubmed-highlight-zebrafish-genome.html Figure 2. Comparison of human and zebrafish PRODH genes. Genes were aligned using Clustal Omega software where identical amino acids are marked with an asterisk, while highly conserved regions are marked with a colon. The genes shared 61% identity. BLAST to find zebrafish genes Design primers Find location on chromosome Euthanize fish Extract/purify RNA Identify contigs and ESTs Determine the synteny via Woods table Reverse transcription to form cDNA Verify amplification of genes with gel electrophoresis PCR the cDNA to amplify genes Amino acid alignments with Clustal Omega software