Cloning Newt Melanocortin Receptors

1
Cloning NewtMelanocortin Receptors

• Jeremy Gregory
• HHMI Summer 2004

Dr. Frank Moore Department of Zoology
2
General Background

• Two mechanisms of molecular action

3
General Background

• Why newts?
• (Taricha granulosa)
• amplexus (clasping)
• unambiguous
• manipulable
• hormone-controlled
• mechanisms in higher vertebrates

newts in amplexus
4
Motivation Corticosterone

• Stress raises cellular CORT levels
• CORT steroid signaling molecule that rapidly
  suppresses amplexus in newts
• Steroids typically act at level of transcription
• CORT action is too rapid for this mechanism
• There must be a CORT membrane receptor
• CORT binding site is opioid-like receptor
• Melanocortin receptors are opioid-like receptors
• Hypothesize that CORT binding site is one of the
  melanocortin receptors

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Background Melanocortin System
Many Physiological Roles

• analgesia
• cardiovascular regulation
• energy homeostasis
• exocrine secretion
• immunomodulation
• inflammation

• neuromuscular regeneration
• pigmentation
• steroidogenesis
• sexual function
• temperature control

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Background Melanocortin System

• Five melanocortin receptors (MCRs) in many
  species
• MC1R skin pigmentation
• MC2R glucocorticoid production
• MC3R energy homeostasis
• MC4R energy homeostasis/sexual function
• MC5R exocrine function
• But in newts?

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Proposal Clone MC Receptors

• Ultimately, test CORT binding to MCRs

• But first, sequence the receptors

GCCTAGCTAGAT
ATCGATCGATGA
CORT
ATGAATGCTACAA
CTAGATCGATAG
MC3R
MC2R
MC4R
ATGCACTTCGATA
MC1R
MC5R
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Strategy Obtaining the Clone
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Step 1 RNA Extraction
rRNA
(GGUCAUAC)
tRNA
(AUCGCCAA)
total RNA
mRNA
(CUAGGUAC)

• Tissue expresses gene of interest (e.g. mc2r in
  kidneys)
• Lyse tissue, isolate nucleic acids, digest
  genomic DNA

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Step 2 Reverse Transcription
rRNA
(GGUCAUAC)
tRNA
(AUCGCCAA)
cDNA
(GATCCAUG)
mRNA
(CUAGGUAC)

• Reverse transcribe RNA to obtain cDNA encoding
  mc2r

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Step 3a Degenerate Primers

• Design degenerate oligomeric primers to bind cDNA
  templates conserved across species
• Amplify gene through polymerase chain reaction
  (PCR)

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Step 4a Partial Gene Sequencing
Gene Sequence (Clone)
Animal Tissue
Amplified Gene
1
2
4
3
RNA
cDNA
Sequence Primers
Partial Newt mc2r
GGAGAACTTGATGGTCCTGCTGGCTGT
GGTCAAGAATAAG . . . . . . .
100s of bases
. . . . . . . . . . . CTGGC
AATCGGGGTCTTCATCTTCTGTTGGGC

• Sequence amplified gene fragment Partial clone

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Step 3b Gene-Specific Primers
A
T
T
C
G
G
A
A
G
A
G
G
T
G
T
C
100s of bases
3 RACE
5 RACE

• Deduce gene-specific primers from partial
  sequence
• Amplify entire gene through rapid amplification
  of cDNA ends (RACE)-PCR

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Step 4b Whole Gene Sequencing
Gene Sequence (Clone)
Animal Tissue
Amplified Gene
1
2
4
3
RNA
cDNA
Sequence Primers
Central Services Lab

• Sequence amplified gene Clone

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Partial mc1r gene

• ------------------ ??? bases for 5 UTR
  ------------------
• 1 ---------------- 185 bases to start codon
  ---------------
• 186 CAAGAACCGCAACCTGCACTCGCCTATGTACTTCTTCATCTGCTG
  CCTAGCGGTATCTGA
• 246 CATGCTGGTCAGTGTGAGCCACCTTGTGGAGACCACAGTCATTCT
  CATGCTACAGCATGG
• 306 GGTTGTGGACATACCGCAAAACGCCCTGCGCCAGATGGACAATAT
  CTTTGACATGATGAT
• 366 TTGCAGTTCAGTGGTGTCCTCACTATCCTTCCTCGGGGTGATAGC
  CGTGGACCGCTACAT
• 426 CACCATCTTCTATGCCCTGCGCTACCACAACATCATGACTATCCG
  GCGGGCAGTGATCAT
• 486 CATCATCCTAATTTGGGTGCTCAGCACCATCTCCAGCATTATCTT
  CATCACCTATCACAG
• 546 CAGCAAAGCGGTCATCATCTGCCTCATCAGTTTCTTCCTCTTCAT
  GCTCATTCTGATGGT
• 606 GACACTCTACATCCACATGTTCGCGTTGGCTCGCCAACATGCCAA
  AAGAATCTACAACCT
• 666 CAACAAGAGGAGGTCCACACCTCACAGAACAAGCCTAAAAGGGGC
  CATCACCCTCACCAT
• 726 CCTGCTGGGGATCTTCTTCCTCTGCTGGGCTCCCTTCTTCCT---
  ---------------
• ---------------- 179 bases to stop codon
  -----------------
• ------------------ ?? bases for 3 UTR
  -------------------

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Complications

• MCRs have similar nucleotide sequences and are
  co-expressed in many tissues
• Non-specific primers bind genes for multiple
  receptors
• Less-expressed receptors are difficult to isolate
  in presence of other receptor types
• Genome contains many pseudogenes, necessitating
  use of RNA

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Results

• mc3r and mc5r completely sequenced from brain
• mc1r partially sequenced from brain and kidney
• mc2r degenerate primers return mc1r fragments
  from kidney and spleen
• mc4r degenerate primers newly designed

CORT receptor unidentified
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Acknowledgements

• Howard Hughes Medical Institute
• Dr. Frank Moore
• C. Samuel Bradford
• Eliza A. Walthers