Stress, Newts and DNA

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Stress, Newts and DNA OR
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Identification of the cDNA Sequence of
Prodynorphin
Katie Miles Frank Moore, Mentor
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The Body's Response to Pain
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Endogenous Opioids

• Natural analgesics
• - Inhibit the perception of pain
• - Primitive use of analgesics when Morphine
  and Codeine didnt exist

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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).

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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.

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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.

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Rationale

• New sequences will aid in piecing together
  dynorphins 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 dynorphins
  receptors inhibited, one must decipher the
  sequence of prodynorphin

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Overall Approach
Obtain sequence data
Design custom primers
Make cDNA
PCR desired product
Dynorphin sequence
Sequence product
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RT-PCR to make cDNA
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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

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Sequence Data
completely conserved base
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PCR
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Gel Electrophoresis
Start
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Sequencing

• When the desired product is obtained, it will
  be sequenced by the Central Services Lab
  at OSU

An electroferrogram
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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

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Extending the Sequence

• Rapid amplification of cDNA ends (RACE PCR)
  - Used to obtain the full length cDNA sequence
  in the urodele model.

RNA
AAAAA
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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

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Acknowledgements

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

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Questions?