The Nobel Prize in Physiology or The Nobel Prize in Physiology or Medicine 2004Medicine 2004
Discoveries of odorant Discoveries of odorant receptors and the receptors and the
organization of the olfactory organization of the olfactory system system
醫學一 B9602084 潘 岳 B9602091 謝仁峰
B9602086 鄭尉佐 B9602098 羅緯麟
B9602088 盧柏嘉
Discoveries of odorant receptors and the Discoveries of odorant receptors and the organization of the olfactory system organization of the olfactory system
Richard AxelRichard Axel Linda B.BuckLinda B.Buck
Columbia University New York, NY, USA
Fred Hutchinson Cancer Research Center Seattle, WA, USA
Three assumptionsThree assumptions
Receptors are G-protein-coupled receptors. Receptors are G-protein-coupled receptors. (GPCRs) (GPCRs)
Diverse receptors encoded by multigene famiDiverse receptors encoded by multigene familyly
Receptor expression restricted to olfactory eReceptor expression restricted to olfactory epitheliumpithelium
Homogenize the epithelium cellsHomogenize the epithelium cells
‧RNA
Many double-stranded cDNAsAdd RNA and reverse transcriptase
Reverse transcriptase Reverse transcriptase cDNAcDNA
Specific primerSpecific primer GPCRs familyGPCRs family PCRPCR primer
II VII
Add primers anneal to conserved regions(II VII) of the GPCR family
Different species cDNA exist
M
3600
600
1700
900
1200
1500
300
ElectrophoresisElectrophoresis :: initiationinitiation
M
3600
600
1700
900
1200
1500
300
ElectrophoresisElectrophoresis :: resultresult
M
3600
600
1700
900
1200
1500
300
Simple cDNAs Mixed-up cDNAs
Add restriction enzyme
Northern blotNorthern blot
MaterialRNA from epithlium/brain/spleenlabeled PCR13 products as probeResult Brain & Spleen :no hybridization
Expression is restricted to olfactory epithelium Demonstrate assumption 3Obtain full-length c-DNA
Protein sequenceProtein sequence
N-terminus
C-terminus
various
Related to ligand recognition
Experiment summaryExperiment summary
DNA sequencingDNA sequencing
Assumption 1 :GPCRAssumption 1 :GPCR
Assumption 2 :Multigene→various receptorAssumption 2 :Multigene→various receptor
Northern blottingNorthern blotting
Assumption 3 :Expression locationAssumption 3 :Expression location
protein sequenceprotein sequence
The family of olfactory The family of olfactory genes in humangenes in human
1.1. Over 1000 genesOver 1000 genes
2.2. About 400 genes functionAbout 400 genes function
; 600 genes not function; 600 genes not function
Olfactory receptor genesOlfactory receptor genes
Olfactory sensory
neuron
Species differencesSpecies differences
Sensitivity
High Low
Olfactory Sensory Neuron(OSN)Olfactory Sensory Neuron(OSN)
Olfactory
Sensory neuron
A large family of odorant receptors A large family of odorant receptors One neuron cell expresses
only one type of receptor
7 transmenbrane domains
Nitrite group in extracellular space
Conformational change
Olfactory
Sensory
neuron
Combinatorial receptor codesCombinatorial receptor codes
Sensory transductionSensory transduction
SensorySensory transductiontransduction
Olfactory BulbOlfactory Bulb
Olfactory Receptor Olfactory Receptor V.S.V.S. Olfactory glomerulus Olfactory glomerulus
Map of Olfactory BulbMap of Olfactory Bulb
前 後
QQ :: Human has about 400 types of receptHuman has about 400 types of receptors, but how do they discriminate about teors, but how do they discriminate about ten thousands odorants?n thousands odorants?
Combinatorial receptor codes experimentCombinatorial receptor codes experiment
100
Fura-2
10
1
Combinatorial receptor codesCombinatorial receptor codes
Multigene family
Receptor
express
Functional genes: pseudogenes
400: 600
Combination
Different ordor
recognized
Thank YouThank You !!
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