Stress, Newts and DNA
OR…
Identification of the cDNA
Sequence of
Prodynorphin
Katie Miles
Frank Moore, Mentor
Nociceptors
Signal
Sense and
respond to pain
in the body
Tissue damage,
and actual or
impending injury
Joe
Cause
Release
of
Opioid peptides are our
natural analgesics
Peptides that
produce an
analgesic (pain
relieving)
response
Pain signals to
the brain via
peripheral nerves
and spinal cord
• Natural analgesics
- Inhibit the perception of pain
- Primitive use of analgesics when Morphine and Codeine didn’t
exist
• Four main types
• Each type of opioid binds selectively to one of four opioid
receptors, with dynorphin binding to the kappa opioid receptor
(KOR).
• 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.
• 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.
• 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
Make cDNA
Obtain
sequence
data
Dynorphin
sequence
Sequence
product
Design
custom
primers
PCR
desired
product
mRNA from Newt brain
AAAAAA
Polymerize with Super Script II (RT)
C
G
AAAAAA
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
• 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
Cow_DYN
Human_DYN
Rat_DYN
Mouse_DYN
Cow_DYN
Human_DYN
Rat_DYN
Mouse_DYN
596F
GGTGAGGTGGGCCATGAGGACCTGTACAAGCGCTACGGGGGATTCTTACGGCGCATCCGT
GATAGCATGGGCCATGAGGACCTGTACAAACGCTATGGGGGCTTCTTGCGGCGCATTCGT
GATCAGGTAGGGCATGAGGACCTGTACAAACGCTATGGGGGCTTCCTGCGGCGCATTCGC
GATCAGGTAGGGCATGAGGACCTGTACAAACGCTATGGGGGCTTCCTGCGGCGCATTCGC
* *
* ** ***************** ***** ***** *** * ******** **
675R
CCCAAGCTCAAGTGGGACAACCAGAAGCGCTATGGTGGCTTCCTCCGGCGTCAGTTCAAG
CCCAAGCTCAAGTGGGACAACCAGAAGCGCTATGGCGGTTTTCTCCGGCGCCAGTTCAAG
CCCAAGCTTAAGTGGGACAACCAGAAACGCTATGGTGGTTTTCTGCGGCGTCAGTTCAAG
CCCAAGCTGAAGTGGGACAACCAGAAGCGCTATGGTGGTTTCCTGCGGCGTCAGTTCAAG
******** ***************** ******** ** ** ** ***** *********
* = completely conserved base
Start
400 bp
300 bp
200 bp
100 bp
• When the desired product is obtained, it will be sequenced by
the Central Services Lab at OSU
An electroferrogram
• 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
• Rapid amplification of cDNA ends (RACE PCR)
- Used to obtain the full length cDNA sequence in the urodele model.
RNA
AAAAA
Reverse Transcription with 3’ RACE adapter
AAAAA
TTTTT-adapter
AAAAA
TTTTT-adapter
PCR
TTTTT-adapter
Gene-specific primer
3’ RACE Primer
•
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
Acknowledgements
Howard Hughes Medical Institute
Sam Bradford
Eliza Walthers
Dr. Frank Moore