1. The Activating switch of Zn2 + by the mutation of the amino acid Tyrosine to Alanine in the Dopamine Transporter protein possibly impairing cocaine’s effect on locomotion

2. Cocaine in the Nervous System Stimulant Science.education.nih.gov

3. Short term use Increased blood pressure Increased heart rate Risk of cardiac arrest Long term use Changes the chemistry of the brain

4. What’s the significance of Zinc in Dopamine Transporter ? Loland et al

5. Can this be done in C.elegans to further examine if the switch of Zn 2+ by the mutation of Tyrosine to alanine can possibly cocaine’s effect in locomotion ? Biology.syr.edu

6. They are the simplest organisms with a nervous system They have their whole genome sequenced. They are easily accessible. Has short generation time. Cost and time effective.

7. Which gene codes for DAT in C.elegans? Dat-1 gene codes for C.elegans Located on Chromosome III Ncbi.nlm.nih.gov

8. Does the Same Tyrosine used in hDAT mutation exist in C.elegans? EMBOSSEbi.ac.uk C.Elegans DAT Human DAT

9. What mRNA codons are responsible for the production of Tyrosine? Start ORF program. (Star.mit.eu)

10. Possible codons for Tyrosine: UAC UAU Biochem.com

11. UAC UAU Tyrosine GC U GC C GC A GC C Alanine

12. CRISPR/CAS9Method Sander et al

14. Measure effectiveness of mutation by analyzing cocaine’s ability to inhibit dopamine uptake in Y328A DAT both in the presence and absence of Zn 2+

15. Transfection Method Niborlab.com Ibioexperts.com

16. Possible Outcomes Dopamine Uptake percent is going to be greater in the mutated DAT compared to wild type as concentration of cocaine increases. Dopamine Uptake percent of the mutated DAT is going to be identical to wild type as concentration of cocaine increases. Dopamine Uptake percent is less in the mutated DAT compared to wild type as concentration of cocaine increases.

17. Loland et al Cocaine’s ability to inhibit DA uptake has been impaired

18. Loland et al Cocaine’s ability to impair DA uptake has not been affected

19. Loland et al Cocaine’s ability to impair DA uptake has been amplified.

20. Possible Outcomes Dopamine Uptake percent is going to be greater in the mutated DAT compared to wild type as concentration of cocaine increases. Dopamine Uptake percent of the mutated DAT is going to be identical to wild type as concentration of cocaine increases. Dopamine Uptake percent is less in the mutated DAT compared to wild type as concentration of cocaine increases. Further experiments to see whether or not mutated dopamine transport makes C.elegens less susceptible to behavioral effects of cocaine (i.e locomotion)

21. References Loland, C. J., Norregaard, L., Litman, T., & Gether, U. (2002). Generation of an activating Zn2+ switch in the dopamine transporter: Mutation of an intracellular tyrosine constitutively alters the conformational equilibrium of the transport cycle. Proceedings of the National Academy of Sciences of the United States of America, 99(3), 1683–1688. doi:10.1073 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC122251/pdf/pq0302001683.pdf Musselman, H. N., Neal-Beliveau, B., Nass, R., & Engleman, E. (2012). Chemosensory Cue Conditioning with Stimulants in a Caenorhabditis elegans Animal Model of Addiction. Behavioral Neuroscience, 126(3), 445–456. doi:10.1037 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3367381/pdf/nihms368977.pdf Nian-Hang ChenNian-Hang Chen, Maarten E. A. Reith, Michael W. Quick. (2004). Synaptic uptake and beyond: the sodium- and chloride- dependent neurotransmitter transporter family SLC6. Pflugers Arch - Eur J Physiol 447:519–531 DOI 10.1007/Maarten E. A. ReithMichael W. Quick http://link.springer.com/article/10.1007%2Fs00424-003-1064-5#

22. Gether, Ulrik, Andersen, Peter H., Larsson, Orla M, Schousboe, Arne. (2006). Neurotransmitter transporters: molecular function of important drug targets. TRENDS in Pharmacological Sciences Vol.27 No.7. 375-383 http://www.sciencedirect.com/science/article/pii/S0165614706001271 Norregaard, L., Frederiksen, D., Nielsen, E. O. & Gether, U. (1998) EMBO J. 17, 4266–4273. Lo, T.-W., Pickle, C. S., Lin, S., Ralston, E. J., Gurling, M., Schartner, C. M., … Meyer, B. J. (2013). Precise and Heritable Genome Editing in Evolutionarily Diverse Nematodes Using TALENs and CRISPR/Cas9 to Engineer Insertions and Deletions. Genetics, 195(2), 331–348. doi:10.1534 http://www-ncbi-nlm-nih-gov.proxy.library.vcu.edu/pmc/articles/PMC3781963/ Chiu, H., Schwartz, H. T., Antoshechkin, I., & Sternberg, P. W. (2013). Transgene-Free Genome Editing in Caenorhabditis elegans Using CRISPR-Cas. Genetics, 195(3), 1167–1171. doi:10.1534 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3813845/pdf/1167.pdf