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 Search databases and compare genes underlying the senses Use literature to learn about the molecular basis of a sense

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

Timeline Next Wednesday (11/11) 11/18 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

S M T W Th F S November 4 5 6 7 8 9 Today 10 11 Idea 12 13 14 15 16 17 18 Outline 19 20 21 22 23 24 25 Help (no class) 26 Thanks- 27 giving 28 29 30 Dec 1 2 Prelim results 3 Present1 Present 2 All due

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

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

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

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?

Olfaction Sensory cells in olfactory epithelium Neurally connected to olfactory bulb http://www.life.uiuc.edu/hing/research/introfig1.html Olfactory epithelium

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 Fain 7.7

Nobel Prize 2004

What did they know Odor stimulates adenylyl cyclase Increases cAMP Cell depolarizes cyclic nucleotide gated channel Dependent on GTP Likely involves G protein signaling Buck and Axel fig 1

What can they assume? How many receptors? Are they all alike or different? Where are they expressed?

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

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

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

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

PCR products (A) and their digestion (B) cut Cut fragment sizes had to add up to more than initial PCR size for there to be multiple products HinfI cuts at G^ANTC

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

Sequence reveals that receptors have 7 TM Black balls show more variable sites. These are in TM III, IV and V. This is likely where ligand binds and where specificity of different receptors lies. This GPCR is “upside down” from rhodopsins Identify key sites know from GPCRs 7 hydrophobic regions Lots of diversity within membrane region Cysteines for disulfide bonds

Genes are only expressed in olfactory epithelium

No introns OR genes do not have any introns! TM

Unique receptors Many different receptors Same receptor occurs in only few cells (0.1%) and randomly across olfactory epithelium They determine this by seeing how many cells express a given OR - not many 0.1%

One neuron - one receptor http://nobelprize.org/nobel_prizes/medicine/laureates/2004/illpres/4_gene.html Using single cell qPCR they find just one OR is present

All receptors for same odorant project to the same location http://nobelprize.org/nobel_prizes/medicine/laureates/2004/illpres/5_cortex.html

Olfaction http://nobelprize.org/nobel_prizes/medicine/laureates/2004/illpres/2_olfactory.html There are about 2000 glomeruli in nose which receive input from 10^6 neurons

2. How many OR’s are there??

Comparison of vertebrate ORs Search genomes of zebrafish, pufferfish, frog, chicken, human and mouse Fish have fewer genes than mammals. What is the evolutionary history?

Methods

9 classes of OR Zfish - 98 Puffer - 40 Frog - 410 Chicken - 78 Human - 388 Nimura and Nei 2005 Fall in 9 classes. Class gamma is greatly expanded in certain groups. These gene duplications are within an organisms so specialized OR’s evolve within certain animals.

9 classes of OR Zfish - 98 Puffer - 40 Frog - 410 Chicken - 78 Human - 388 Nimura and Nei 2005

Comparison of functional and total gene #

Many pseudogenes Zfish - 98 / 35 Puffer - 40 / 54 Frog - 410 / 478 Chicken - 78 / 476 Human - 388 / 414 Nimura and Nei 2005 Functional genes / Pseudogenes

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

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

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

Mammalian OR’s

Mammal OR’s

Compare gene gains / losses

Gene gains / losses

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

Olfactory stimulants Fall in 4 groups with increasing length of the carbon chain

Measure response of isolated mouse olfactory neurons Decrease in fluorescence = increase in calcium = cell responds to odorant Isolate neurons - put on slide and load with fura2 - Calcium sensitive dye

Single receptor recognizes multiple chemicals

Single odorant is recognized by multiple receptors Unique set?

Receptors work in combination to code odorants

Receptors each detect different features of odorants