1. Stress, Newts and DNA OR…

2. Identification of the cDNA Sequence of Prodynorphin Katie Miles Frank Moore, Mentor

3. Nociceptors Sense and respond to pain in the body Signal Tissue damage, and actual or impending injury Transmit Pain signals to the brain via peripheral nerves and spinal cord Cause Release of Peptides that produce an analgesic (pain relieving) response Opioid peptides are our natural analgesics Joe

4. Natural analgesics - Inhibit the perception of pain - Primitive use of analgesics when Morphine and Codeine didn’t exist

5. Four main types Each type of opioid binds selectively to one of four opioid receptors, with dynorphin binding to the kappa opioid receptor (KOR).

6. Prodynorphin – a genetic precursor to dynorphin codes for three different opioid peptides Dynorphin – in a family of neuropeptides known for controlling pain responses and behavioral responses to stress stimuli Other opioids include: Codeine, hydrocodone (Vicodin), Morphine, and Methadone.

7. Opioid peptides modulate pain signals in the body by binding to and activating specific types of opioid receptors. Kappa Opioid Receptor (KOR) plays an important role in the pain pathway The nucleotide sequence for prodynorphin has only been identified in six mammals, two fish, and one amphibian.

8. New sequences will aid in piecing together dynorphin’s evolutionary tree Due to the relative abundance of newts in Western Oregon and their low cost, newts have been chosen as the model animal Before new drugs can be made or dynorphin’s receptors inhibited, one must decipher the sequence of prodynorphin

9. Design custom primers Make cDNA PCR desired product Sequence product Dynorphin sequence Obtain sequence data

10. mRNA from Newt brain AAAAAA TTTTTT First Strand of cDNA is made TTTTTT Removal of RNA with RNase HcDNA that is ready to undergo PCR TTTTTT AAAAAA TTTTTT C G Polymerize with Super Script II (RT) primer

11. Design custom Polymerase Chain Reaction (PCR) primers - Allows for rapid amplification of small amounts of DNA with a specific nucleotide sequence - Designing primers without knowing the exact DNA sequence - Look at sequence alignments

12. Cow_DYN Human_DYN Rat_DYN Mouse_DYN Cow_DYN Human_DYN Rat_DYN Mouse_DYN * = completely conserved base

14. 400 bp 300 bp 200 bp 100 bp Start

15. When the desired product is obtained, it will be sequenced by the Central Services Lab at OSU An electroferrogram

16. Knowing the sequence of the cDNA product will facilitate the design of new primers that are specific to newt prodynorphin - New primers will allow for a more precise determination of the sequence

17. Rapid amplification of cDNA ends (RACE PCR) - Used to obtain the full length cDNA sequence in the urodele model. AAAAA Reverse Transcription with 3’ RACE adapter TTTTT-adapter AAAAA RNA PCR TTTTT-adapter 3’ RACE PrimerGene-specific primer AAAAA TTTTT-adapter

18. At present, no sequence data has been obtained from this project Other avenues for sequence determination are being pursued - Highly degenerate primers - Designing new primers Prodynorphin is probably a low-copy gene in the newt genome

19. Acknowledgements Howard Hughes Medical Institute Sam Bradford Eliza Walthers Dr. Frank Moore