Cloning Newt Melanocortin Receptors

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

2. molecular connection HORMONES BEHAVIOR • transcriptional regulation • membrane receptor SLOW RAPID signal response response response cell signal nucleus 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

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

6. 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?

7. Ultimately, test CORT binding to MCRs But first, sequence the receptors Proposal: Clone MC Receptors GCCTAGCTAGAT… ATCGATCGATGA… CORT ATGAATGCTACAA… CTAGATCGATAG… MC3R MC2R MC4R ATGCACTTCGATA… MC1R MC5R

8. Gene Sequence (Clone) Animal Tissue Amplified Gene 1 2 3 4 RNA cDNA Sequence Primers Strategy: Obtaining the Clone

9. Gene Sequence (Clone) Animal Tissue Amplified Gene 1 2 3 4 RNA cDNA Sequence Primers Step 1: RNA Extraction rRNA (GGUCAUAC…) • Tissue expresses gene of interest (e.g. mc2r in kidneys) • Lyse tissue, isolate nucleic acids, digest genomic DNA (AUCGCCAA…) tRNA total RNA mRNA (CUAGGUAC…)

10. Gene Sequence (Clone) Animal Tissue Amplified Gene 1 2 3 4 RNA cDNA Sequence Primers 100s of bases C U A G G U A C A A C U C A G G G C C A U A G G U G G C A A G A T C C A U G T T G A G A C A Reverse Transcriptase Step 2: Reverse Transcription rRNA (GGUCAUAC…) (AUCGCCAA…) cDNA tRNA (GATCCAUG…) mRNA (CUAGGUAC…) • Reverse transcribe RNA to obtain cDNA encoding mc2r

11. Gene Sequence (Clone) Animal Tissue Amplified Gene 1 2 3 4 RNA cDNA Sequence Primers GGAGAAYYTGATBGTCCTBCTGGC Newt mc2r? Mouse mc2r ATTGGCATATTGGAGAACTTGATTGTCCTCCTGGCTGTGATCAAAAATAAAAATCTCCAG Cow mc2r GTTGGGGTTTTGGAGAACCTGATGGTCCTTCTGGCTGTGGCCAAGAATAAGAGTCTTCAG Human mc2r GTTGGAGTTTTGGAGAATCTGATCGTCCTGCTGGCTGTGTTCAAGAATAAGAATCTCCAG Pig mc2r ATTGGGGTTTTGGAGAATCTGATCGTCCTTCTGGCTGTGATCAAGAACAAGAATCTTCAG Step 3a: Degenerate Primers • Design degenerate oligomeric primers to bind cDNA templates conserved across species • Amplify gene through polymerase chain reaction (PCR)

12. Central Services Lab Step 4a: Partial Gene Sequencing Gene Sequence (Clone) Animal Tissue Amplified Gene 1 2 3 4 RNA cDNA Sequence Primers Partial Newt mc2r GGAGAACTTGATGGTCCTGCTGGCTGT • Sequence amplified gene fragment = Partial clone GGTCAAGAATAAG . . . . . . . 100s of bases . . . . . . . . . . . CTGGC AATCGGGGTCTTCATCTTCTGTTGGGC

13. Gene Sequence (Clone) Animal Tissue Amplified Gene 1 2 3 4 RNA cDNA Sequence Primers C A A C C C G forward primer reverse primer C C T C T T G A A C T G T T G G G C Step 3b: Gene-Specific Primers • Deduce gene-specific primers from partial sequence • Amplify entire gene through rapid amplification of cDNA ends (RACE)-PCR G G A G A A C T T G A T G G T C 100s of bases 5’ RACE 3’ RACE

14. Step 4b: Whole Gene Sequencing Gene Sequence (Clone) Animal Tissue Amplified Gene 1 2 3 4 RNA cDNA Sequence Primers • Sequence amplified gene = Clone Central Services Lab

15. Partial mc1r gene ------------------ ??? bases for 5’ UTR ------------------ 1 ---------------- ~185 bases to start codon --------------- 186 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 -------------------

16. 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

17. 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

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