Lecture 19 : Olfaction

Lecture 19 : OlfactionLecture 19 : Olfaction

11/9/09

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

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

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

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

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

TimelineTimeline

Wed 11/25 - Help session (no class)

Wed 12/2 - Some preliminary results

12/7 and 12/9PresentationsAll projects due 12/9

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

OlfactionOlfaction

1. What are the olfactory receptors?

2. How many OR’s are there?

3. How are smells encoded?

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?

OlfactionOlfaction

Sensory cells in olfactory epithelium

Neurally connected to olfactory bulb

Olfactory epithelium

Vertebrate olfactory Vertebrate olfactory epitheliumepithelium

Ciliated receptors12 million receptors20 cm2 surface areaPrimary neurons

Sustentacular cellsSecrete mucus

Basal cellsStem cells which replace both cell types

Nobel Prize 2004Nobel Prize 2004

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

What can they assume?What can they assume?

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

MethodsMethods

Try to amplify GPCRs from olfactory cDNA using degenerate primers

TM I II III IV V VI VII

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

PCR products (A) and their PCR products (A) and their digestion (B)digestion (B)

HinfI cuts at G^ANTC

PCR

cut

PCR products (A) and their PCR products (A) and their digestion (B)digestion (B)

HinfI cuts at G^ANTC

PCR

cut

Sequence genesSequence genes

Use PCR products to probe and screen cDNA library (olfactory epithelium)

Sequence positive clonesFound 18 unique ones

Sequence reveals that receptors Sequence reveals that receptors have 7 TMhave 7 TM

Genes are only expressed in Genes are only expressed in olfactory epitheliumolfactory epithelium

No intronsNo introns

OR genes do not have any introns!

TM

Unique receptorsUnique receptors

Many different receptors

Same receptor occurs in only few cells (0.1%) and randomly across olfactory epithelium

One neuron - one receptorOne neuron - one receptor

All receptors for same odorant All receptors for same odorant project to the same locationproject to the same location

OlfactionOlfaction

2. How many OR’s are there??

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

MethodsMethods

Zfish - 98

Puffer - 40

Frog - 410

Chicken - 78

Human - 388

9 classes of OR

Zfish - 98

Puffer - 40

Frog - 410

Chicken - 78

Human - 388

9 classes of OR

Comparison of functional Comparison of functional and total gene #and total gene #

Zfish - 98 / 35

Puffer - 40 / 54

Frog - 410 / 478

Chicken - 78 / 476

Human - 388 / 414

Functional genes / Pseudogenes

Many pseudogenes

ConclusionsConclusions

Fish most diverse OR’s

Unique expansions in birds, amphibs and mammals

MRCA=most recent common ancestor

OR’s distributed across the OR’s distributed across the genomegenome

Difference between mammals and fishDistributed everywhereHow does one gene - one receptor work??????

Species differencesSpecies differences

Different surface areasDog = 40x human

Different repertoire of receptorsMouse 1000 genes - 900 activeHumans 640 genes - 330 active

Mammalian OR’sMammalian OR’s

Mammal OR’sMammal OR’s

Compare gene gains / lossesCompare gene gains / losses

Gene Gene gains / gains / losseslosses

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

Olfactory stimulantsOlfactory stimulants

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

Single Single receptor receptor recognizes recognizes multiple multiple chemicalschemicals

Single Single odorant is odorant is recognized recognized by multiple by multiple receptorsreceptors

Unique set?Unique set?

Receptors work in Receptors work in combination to code combination to code

odorantsodorants

Receptors Receptors each detect each detect different different features of features of odorantsodorants