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Oregon State University. Cloning Newt Melanocortin Receptors. Jeremy Gregory HHMI Summer 2004. Dr. Frank Moore Department of Zoology. molecular connection. HORMONES. BEHAVIOR. transcriptional regulation. membrane receptor. SLOW. RAPID. signal. response. response. response. cell.
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Oregon State University Cloning NewtMelanocortin Receptors Jeremy Gregory HHMI Summer 2004 Dr. Frank Moore Department of Zoology
molecular connection HORMONES BEHAVIOR • transcriptional regulation • membrane receptor SLOW RAPID signal response response response cell signal nucleus General Background Two mechanisms of molecular action:
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
analgesia cardiovascular regulation energy homeostasis exocrine secretion immunomodulation inflammation neuromuscular regeneration pigmentation steroidogenesis sexual function temperature control Background: Melanocortin System 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?
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
Gene Sequence (Clone) Animal Tissue Amplified Gene 1 2 3 4 RNA cDNA Sequence Primers Strategy: Obtaining the Clone
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…)
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
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)
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
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
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
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 -------------------
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