Discovery Of A Novel Nucleotide
Sequence In Taricha granulosa
David J. Stanley
Mentor: Frank L. Moore
Department of Zoology
Cell Signaling : Genomic vs Non-Genomic
Genomic: Gene
transcription
promoted in nucleus;
minutes to hours
Non-Genomic:
Entire signaling
process takes place
outside the nucleus,
no transcription
takes place;
seconds to minutes
Arginine Vasotocin (AVT)
• Major physiological actions: stress
response, water retention.
• 3 distinct receptor isoforms : V1aR, V1bR
(stress signaling), and V2R (anti-diuretic
properties).
• Pituitary, kidney and bladder tissues used.
WhyTaricha?
• V1bR has not been cloned in an amphibian. V2R has, but
only in Hyla japonica (Japanese tree frog).
• Simple, predictable behavior to stimulus.
• Relative simplicity of brain allows for analysis of neuronal
mechanisms.
• Similarity of Taricha brain to human limbic system.
• Abundant in our local environment.
Investigative Strategy
RNA extraction
cDNA synthesis by
Reverse transcription
Sequence PCR product
Design primers
Amplification of desired gene
by PCR
Analysis of PCR products
mRNA 4 cDNA
Random hexamers (
)
SuperScript II reverse transcriptase
Application of RNAase H
Single cDNA strand
Primer Design
Sequences aligned by ClustalW program:
Forward 4
Mouse V2R
Human V2R
Cow V2R
Rat V2R
CATGTATGCCTCCTCCTACATGATCCTGGCCATGACGCTGGACCGCCACC
CATGTATGCCTCTTCCTACATGATCCTGGCCATGACACTAGACCGCCATC
CATGTATGCCTCCTCCTACATGATCCTGGCCATGACACTAGACCGCCATC
CATGTATGCCTCCTCCTACATGATCCTGGCCATGACACTAGACCGCCATC
************ *********************** ** ******** *
3Reverse
Mouse V2R
Human V2R
Cow V2R
Rat V2R
GTGCCATCTGCCGTCCCATGCTGGCGTACCGCCATGGAAGTGGGGCTCAC
GCGCCATCTGCCGCCCTATGCTGGCATACCGCCATGGAGGTGGGGCTCGC
GTGCCATCTGCCGCCCTATGCTAGCATACCGCCATGGAGGTGGGGCTCGC
GTGCCATCTGCCGCCCCATGCTGGCACACCGCCATGGGGGTGGCACTCAT
* *********** ** ***** ** ********** **** ***
* = conserved nucleotide
Considerations:
- Melting or annealing temperature, length of primer.
- GC Clamps.
- Self-annealing should be avoided.
- Position of any degeneracies.
- % GC content.
Primer Use / RT-PCR
Primer
cDNA
Template
3’
5’
Primers
:Forward
Reverse:
N = 2^0
N = 2^1
N = 2^2
N = 2^3
PCR Analysis
Thermal Gradient PCR
Bands expected at 320 bp
Bands expected at 420 bp
Screening of ligated products
Justification
• Sequences can provide clues leading to an
understanding of the structure, pharmacological profile,
distribution of expression, and regulation of a protein.
• Phylogenetics – Evolutionary relationships can be
inferred based on sequence comparisons.
• Proteins of a known nucleotide sequence can be
expressed in any quantity desired.
Further Investigations
• Primers based on Hyla V2R sequence
could be more effective.
Acknowledgements
Howard Hughes Medical Institute
National Science Foundation
Dr. Frank L. Moore
Sam Bradford
Eliza Walthers
And last but not least…The Newts