Stress, Newts and DNA OR…

1. Stress, Newts and DNA OR…

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

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

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

5. Types of Opioid Neuropeptides • 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. Dynorphin • 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. Importance of Dynorphin and the Kappa Opioid Receptor • 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. Rationale • 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. Overall Approach Obtain sequence data Design custom primers Make cDNA PCR desired product Dynorphin sequence Sequence product

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

11. Designing Primers • 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 Cow_DYN Human_DYN Human_DYN Rat_DYN Rat_DYN Mouse_DYN Mouse_DYN Sequence Data * = completely conserved base

13. PCR

14. 400 bp 300 bp 200 bp 100 bp Gel Electrophoresis Start

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

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

18. Results • 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

20. Questions?