Cloning Newt Melanocortin Receptors Jeremy Gregory HHMI Summer 2004 Dr. Frank Moore Department of Zoology Oregon State University

General Background Two mechanisms of molecular action: HORMONESBEHAVIOR molecular connection – transcriptional regulation signal response cell nucleus SLOW response – membrane receptor signal RAPID

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

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

Background: Melanocortin System analgesia cardiovascular regulation energy homeostasis exocrine secretion immunomodulation inflammation neuromuscular regeneration pigmentation steroidogenesis sexual function temperature control Many Physiological Roles

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?

Proposal: Clone MC Receptors Ultimately, test CORT binding to MCRs But first, sequence the receptors MC1R MC3R MC5R MC2RMC4R CORT GCCTAGCTAGAT… ATCGATCGATGA… ATGAATGCTACAA… CTAGATCGATAG… ATGCACTTCGATA…

Strategy: Obtaining the Clone Animal Tissue RNAcDNA Amplified Gene Gene Sequence (Clone) Sequence Primers 1243

Step 1: RNA Extraction Tissue expresses gene of interest (e.g. mc2r in kidneys) Lyse tissue, isolate nucleic acids, digest genomic DNA Animal Tissue RNAcDNA Amplified Gene Gene Sequence (Clone) Sequence Primers 1243 rRNA tRNA mRNA total RNA (CUAGGUAC…) (GGUCAUAC…) (AUCGCCAA…)

Step 2: Reverse Transcription Reverse transcribe RNA to obtain cDNA encoding mc2r Animal Tissue RNAcDNA Amplified Gene Gene Sequence (Clone) Sequence Primers 1243 rRNA tRNA mRNA (CUAGGUAC…) (GGUCAUAC…) (AUCGCCAA…) cDNA (GATCCAUG…) UAGACGUACAUCACCGGGCAUGAGUGCAGA AAGAUCGTTGACTACG Reverse Transcriptase 100s of bases

Step 3a: Degenerate Primers Design degenerate oligomeric primers to bind cDNA templates conserved across species Amplify gene through polymerase chain reaction (PCR) Animal Tissue RNAcDNA Amplified Gene Gene Sequence (Clone) Sequence Primers 1243 Mouse mc2r ATTGGCATATTGGAGAACTTGATTGTCCTCCTGGCTGTGATCAAAAATAAAAATCTCCAG Cow mc2r GTTGGGGTTTTGGAGAACCTGATGGTCCTTCTGGCTGTGGCCAAGAATAAGAGTCTTCAG Human mc2r GTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAATCTCCAG Pig mc2r ATTGGGGTTTTGGAGAATCTGATCGTCCTTCTGGCTGTGATCAAGAACAAGAATCTTCAG GGAGAAYYTGATBGTCCTBCTGGC Newt mc2r?

Step 4a: Partial Gene Sequencing Sequence amplified gene fragment = Partial clone Animal Tissue RNAcDNA Amplified Gene Gene Sequence (Clone) Sequence Primers 1243 GGAGAACTTGATGGTCCTGCTGGCTGT Partial Newt mc2r GGTCAAGAATAAG CTGGC AATCGGGGTCTTCATCTTCTGTTGGGC 100s of bases

Step 3b: Gene-Specific Primers Deduce gene-specific primers from partial sequence Amplify entire gene through rapid amplification of cDNA ends (RACE)-PCR Animal Tissue RNAcDNA Amplified Gene Gene Sequence (Clone) Sequence Primers 1243 ATTCGGAAGAGGTGTCGCTTGGGTC 100s of bases 3’ RACE5’ RACE TCTCTCAGA reverse primer forward primer CACCCAG

Step 4b: Whole Gene Sequencing Sequence amplified gene = Clone Animal Tissue RNAcDNA Amplified Gene Gene Sequence (Clone) Sequence Primers 1243

Partial mc1r gene ??? bases for 5’ UTR ~185 bases to start codon CAAGAACCGCAACCTGCACTCGCCTATGTACTTCTTCATCTGCTGCCTAGCGGTATCTGA 246 CATGCTGGTCAGTGTGAGCCACCTTGTGGAGACCACAGTCATTCTCATGCTACAGCATGG 306 GGTTGTGGACATACCGCAAAACGCCCTGCGCCAGATGGACAATATCTTTGACATGATGAT 366 TTGCAGTTCAGTGGTGTCCTCACTATCCTTCCTCGGGGTGATAGCCGTGGACCGCTACAT 426 CACCATCTTCTATGCCCTGCGCTACCACAACATCATGACTATCCGGCGGGCAGTGATCAT 486 CATCATCCTAATTTGGGTGCTCAGCACCATCTCCAGCATTATCTTCATCACCTATCACAG 546 CAGCAAAGCGGTCATCATCTGCCTCATCAGTTTCTTCCTCTTCATGCTCATTCTGATGGT 606 GACACTCTACATCCACATGTTCGCGTTGGCTCGCCAACATGCCAAAAGAATCTACAACCT 666 CAACAAGAGGAGGTCCACACCTCACAGAACAAGCCTAAAAGGGGCCATCACCCTCACCAT 726 CCTGCTGGGGATCTTCTTCCTCTGCTGGGCTCCCTTCTTCCT ~179 bases to stop codon ?? bases for 3’ UTR

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

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

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