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Lecture 19 : Olfaction

Lecture 19 : Olfaction. 11/9/09. Final project requirements. 1. Must consider the senses Compare one sense across organisms Compare multiple senses within one organism Consider just one sense in just one organism. Final project. 2. It must have a molecular component

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Lecture 19 : Olfaction

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  1. Lecture 19 : Olfaction 11/9/09

  2. Final project requirements 1. Must consider the senses Compare one sense across organisms Compare multiple senses within one organism Consider just one sense in just one organism

  3. Final project 2. It must have a molecular component Search databases and compare genes underlying the senses Use literature to learn about the molecular basis of a sense

  4. Final project A. Hand in a formal presentation (80%) 4-5 pg paper News and Views article à la Science or Nature Create web pages Make a movie or video (which could be shown for B) B. Give a brief (3-4 min) oral presentation (20%) Share what you’ve learned

  5. Timeline • Next Wednesday (11/11) • 1 paragraph describing your idea • Idea will be approved • I will help with finding background info • 11/18 • Outline of background material • Outline of methods - what are you going to do

  6. S M T W Th F S

  7. Timeline • Wed 11/25 - Help session (no class) • Wed 12/2 - Some preliminary results • 12/7 and 12/9 • Presentations • All projects due 12/9

  8. Examples from past years • The sequence and mode of visual pigment evolution in mammals: What could our mammalian ancestors see? • Drosophila taste reception • Pain in phantom limbs • TRPC2 and VNO-Dependent Pheromone Sensing • Comparison of cephalopod and human vision • How do salmon migrate? • The Dog Nose • Movie about Daphne Soares’ research on crocodile pressure detectors • Butterfly vision

  9. Olfaction • What are the olfactory receptors? • How many OR’s are there? • How are smells encoded?

  10. 1. Odor receptors • Much our sense of taste comes through odors • Odors can • Trigger memories • Warn of danger • How do we detect 10,000 different odors?

  11. Olfaction • Sensory cells in olfactory epithelium • Neurally connected to olfactory bulb Olfactory epithelium

  12. Vertebrate olfactory epithelium • Ciliated receptors • 12 million receptors • 20 cm2 surface area • Primary neurons • Sustentacular cells • Secrete mucus • Basal cells • Stem cells which replace both cell types

  13. Nobel Prize 2004

  14. What did they know Odor stimulates adenylyl cyclase Increases cAMP Cell depolarizes cyclic nucleotide gated channel Dependent on GTP Likely involves G protein signaling

  15. What can they assume?

  16. Assumptions • Receptors are membrane bound GPCRs • Encoded by multigene family Need diversity to detect so many compounds • Expression should be limited to olfactory epithelium

  17. Methods • Try to amplify GPCRs from olfactory cDNA using degenerate primers TM I II III IV V VI VII

  18. Methods • Try to amplify GPCRs from olfactory cDNA using degenerate primers • Cut PCR product with restriction enzyme • If get simple product - not OR • If get complex product - could be OR • Sequence

  19. PCR products (A) and their digestion (B) PCR cut HinfI cuts at G^ANTC

  20. PCR products (A) and their digestion (B) PCR cut HinfI cuts at G^ANTC

  21. Sequence genes • Use PCR products to probe and screen cDNA library (olfactory epithelium) • Sequence positive clones • Found 18 unique ones

  22. Sequence reveals that receptors have 7 TM

  23. Genes are only expressed in olfactory epithelium

  24. No introns • OR genes do not have any introns! TM

  25. Unique receptors • Many different receptors • Same receptor occurs in only few cells (0.1%) and randomly across olfactory epithelium

  26. One neuron - one receptor

  27. All receptors for same odorant project to the same location

  28. Olfaction

  29. 2. How many OR’s are there??

  30. Comparison of vertebrate ORsSearch genomes of zebrafish, pufferfish, frog, chicken, human and mouse

  31. Methods

  32. 9 classes of OR Zfish - 98 Puffer - 40 Frog - 410 Chicken - 78 Human - 388

  33. 9 classes of OR Zfish - 98 Puffer - 40 Frog - 410 Chicken - 78 Human - 388

  34. Comparison of functional and total gene #

  35. Many pseudogenes Zfish - 98 / 35 Puffer - 40 / 54 Frog - 410 / 478 Chicken - 78 / 476 Human - 388 / 414 Functional genes / Pseudogenes

  36. Conclusions • Fish most diverse OR’s • Unique expansions in birds, amphibs and mammals MRCA=most recent common ancestor

  37. OR’s distributed across the genome Difference between mammals and fish Distributed everywhere How does one gene - one receptor work??????

  38. Species differences • Different surface areas • Dog = 40x human • Different repertoire of receptors • Mouse 1000 genes - 900 active • Humans 640 genes - 330 active

  39. Mammalian OR’s

  40. Mammal OR’s

  41. Compare gene gains / losses

  42. Gene gains / losses

  43. How do 387 OR’s code for 10,000 smells?

  44. Olfactory stimulants

  45. Measure response of isolated mouse olfactory neuronsDecrease in fluorescence = increase in calcium = cell responds to odorant

  46. Single receptor recognizes multiple chemicals

  47. Single odorant is recognized by multiple receptorsUnique set?

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