lecture 19 : olfaction
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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 - PowerPoint PPT PresentationTRANSCRIPT
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Lecture 19 : OlfactionLecture 19 : Olfaction
11/9/09
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Final project requirementsFinal project requirements
1. Must consider the sensesCompare one sense across organismsCompare multiple senses within one
organismConsider just one sense in just one
organism
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Final projectFinal project
2. It must have a molecular componentSearch databases and compare genes underlying the senses
Use literature to learn about the molecular basis of a sense
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Final projectFinal project
A. Hand in a formal presentation (80%)4-5 pg paperNews and Views article à la Science or NatureCreate web pagesMake 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
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TimelineTimeline
Next Wednesday (11/11)1 paragraph describing your idea Idea will be approvedI will help with finding background info
11/18Outline of background materialOutline of methods - what are you
going to do
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November
4 5 6 7
8 9 Today
10 11Idea
12 13 14
15 16 17 18Outline
19 20 21
22 23 24 25Help (no class)
26Thanks-
27giving
28
29 30 Dec 1 2 Prelim results
3 4 5
6 7Present1
8 9Present 2All due
S M T W Th F S
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TimelineTimeline
Wed 11/25 - Help session (no class)
Wed 12/2 - Some preliminary results
12/7 and 12/9PresentationsAll projects due 12/9
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Examples from past yearsExamples 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
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OlfactionOlfaction
1. What are the olfactory receptors?
2. How many OR’s are there?
3. How are smells encoded?
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1. Odor receptors1. Odor receptors
Much our sense of taste comes through odors
Odors can Trigger memoriesWarn of danger
How do we detect 10,000 different odors?
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OlfactionOlfaction
Sensory cells in olfactory epithelium
Neurally connected to olfactory bulb
Olfactory epithelium
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Vertebrate olfactory Vertebrate olfactory epitheliumepithelium
Ciliated receptors12 million receptors20 cm2 surface areaPrimary neurons
Sustentacular cellsSecrete mucus
Basal cellsStem cells which replace both cell types
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Nobel Prize 2004Nobel Prize 2004
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What did they knowWhat did they know
Odor stimulates adenylyl cyclase Increases cAMP
Cell depolarizes cyclic nucleotide gated channel
Dependent on GTP Likely involves G protein signaling
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What can they assume?What can they assume?
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AssumptionsAssumptions
1. Receptors are membrane bound GPCRs
2. Encoded by multigene familyNeed diversity to detect so many compounds
3. Expression should be limited to olfactory epithelium
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MethodsMethods
Try to amplify GPCRs from olfactory cDNA using degenerate primers
TM I II III IV V VI VII
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MethodsMethods
Try to amplify GPCRs from olfactory cDNA using degenerate primers
Cut PCR product with restriction enzymeIf get simple product - not ORIf get complex product - could be OR
Sequence
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PCR products (A) and their PCR products (A) and their digestion (B)digestion (B)
HinfI cuts at G^ANTC
PCR
cut
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PCR products (A) and their PCR products (A) and their digestion (B)digestion (B)
HinfI cuts at G^ANTC
PCR
cut
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Sequence genesSequence genes
Use PCR products to probe and screen cDNA library (olfactory epithelium)
Sequence positive clonesFound 18 unique ones
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Sequence reveals that receptors Sequence reveals that receptors have 7 TMhave 7 TM
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Genes are only expressed in Genes are only expressed in olfactory epitheliumolfactory epithelium
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No intronsNo introns
OR genes do not have any introns!
TM
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Unique receptorsUnique receptors
Many different receptors
Same receptor occurs in only few cells (0.1%) and randomly across olfactory epithelium
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One neuron - one receptorOne neuron - one receptor
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All receptors for same odorant All receptors for same odorant project to the same locationproject to the same location
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OlfactionOlfaction
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2. How many OR’s are there??
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Comparison of vertebrate Comparison of vertebrate ORsORs
Search genomes of zebrafish, pufferfish, Search genomes of zebrafish, pufferfish, frog, chicken, human and mousefrog, chicken, human and mouse
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MethodsMethods
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Zfish - 98
Puffer - 40
Frog - 410
Chicken - 78
Human - 388
9 classes of OR
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Zfish - 98
Puffer - 40
Frog - 410
Chicken - 78
Human - 388
9 classes of OR
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Comparison of functional Comparison of functional and total gene #and total gene #
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Zfish - 98 / 35
Puffer - 40 / 54
Frog - 410 / 478
Chicken - 78 / 476
Human - 388 / 414
Functional genes / Pseudogenes
Many pseudogenes
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ConclusionsConclusions
Fish most diverse OR’s
Unique expansions in birds, amphibs and mammals
MRCA=most recent common ancestor
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OR’s distributed across the OR’s distributed across the genomegenome
Difference between mammals and fishDistributed everywhereHow does one gene - one receptor work??????
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Species differencesSpecies differences
Different surface areasDog = 40x human
Different repertoire of receptorsMouse 1000 genes - 900 activeHumans 640 genes - 330 active
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Mammalian OR’sMammalian OR’s
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Mammal OR’sMammal OR’s
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Compare gene gains / lossesCompare gene gains / losses
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Gene Gene gains / gains / losseslosses
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How do 387 OR’s code for How do 387 OR’s code for 10,000 smells?10,000 smells?
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Olfactory stimulantsOlfactory stimulants
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Measure Measure response of response of isolated isolated mouse mouse olfactory olfactory neuronsneurons
Decrease in Decrease in fluorescence fluorescence = increase in = increase in calcium = cell calcium = cell responds to responds to odorantodorant
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Single Single receptor receptor recognizes recognizes multiple multiple chemicalschemicals
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Single Single odorant is odorant is recognized recognized by multiple by multiple receptorsreceptors
Unique set?Unique set?
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Receptors work in Receptors work in combination to code combination to code
odorantsodorants
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Receptors Receptors each detect each detect different different features of features of odorantsodorants