Transcript A Novel Multigene Family May Encode Odorant Receptors: A

A Novel Multigene Family May
Encode Odorant Receptors:
A Molecular Basis for Odor
Recognition
Linda Buck and Richard Axel
Published in Cell, Volume 65, 175-187 April 5, 1991
Presented by Adam Warner on Oct. 12, 2004
Linda Buck
1984-1991
•Research Associate
1994-1997
•Assistant Investigator
Linda Buck
•Assistant Professor 1991-1996
•Associate Professor 19962001
•Professor 2001-2002
Linda Buck
Full Member - Division of Basic
Sciences
Fred Hutchinson Cancer Research
Centre, (Seattle) 2002-Current
Richard Axel
1970
M.D. School of Medicine
1978-1984
Professor, Pathology and
Biochemistry
Richard Axel
Investigator 1984 - Current
Professor 1999 - Current
How Do We Smell?
(Stinky Hockey Equipment)
•How do we identify the smells around us?
•Humans can distinguish between over ten
thousand different odours
•Even minute changes in molecular
structure of an odorant can cause a
different perception in its smell
Perception of Smell
Interaction between odourous
ligands and specific olfactory
receptors acknowledged
•Two major models for the
interaction that results in smell being
perceived
Two Models
•few receptors, each
able to interact with a
very large number of
molecules
•large number of
receptors, each able
to interact with only
one, or very few
molecules
Previous Findings
Bronshtein and Minor, 1977
•Removal of the cilia leads to a loss in the
perception of odour
Boekhoff et al., 1990
•cilia is isolated from rat olfactory epithelium
•response to odourous molecules leads to a
rise in cyclic AMP
Nakamura and Gold, 1987
• rise in cyclic AMP leads to depolarization
of olfactory neurons
Pathway
Similarities To Other
Pathways
Similar pathway observed when
neurotransmitter and hormone
receptors are stimulated
•superfamily of transmembrane
receptor proteins
•transmembrane domain spans
the membrane seven times
Hypothesis
Olfactory receptors are part of a large
superfamily of transmembrane receptors
(spanning membrane 7 times)
One odorant molecule can only
stimulate one or very few specific
receptors
•must be very diverse so part
of a multigene family
Olfactory receptors should be
localized only to the olfactory
epithelium
Potential Candidates?
RNA isolated from olfactory epithelium
•prepare cDNA
Primer Design
•5 or 6 nucleotides in tm domains 2 and 7
•conserved in transmembrane superfamily
•should amplify homologous sequences in the
olfactory prep
PCR - second round of amplification
•Digestion with Hinfl restriction (4 base cutter)
•run on gel
PCR Explanation
PCR
No digestion
Digestion
Does molecular weight of restriction fragments = original?
Part of 7-Transmembrane
Superfamily?
•Fragments from lane 13 were cloned
into a plasmid vector
sequenced
•clones showed sequence similarity to
transmembrane superfamily
•clones also showed unique sequence
motifs
•new family of receptors!
Screening
•cDNA libraries were screened to obtain
full length cDNA clones
•used fragments from lane 13
cDNA libraries from olfactory sensory
neurons
•Took hybridizing plaques and used
lane 13 PCR primers, PCR
710
bp fragments were purified (original size
of lane 13 band)
Expression
Northern Blot
•hybridation only
seen in olfactory
epithelium
preparation
•receptors are
restricted to the
olfactory
epithelium
Screening
•Found 18 cDNA clones that encoded
proteins in the same new family
•conserved motifs with the seven
transmembrane superfamily
•new features not seen in this new
multigene family
Protein Structure
White = homologous between clones
Black = variable amino acids
How Many Receptors?
•screened rat genomic libraries
•at least 200 positives per haploid
chromosome
•most likely several hundred genes,
each with multiple subfamilies
With only hundreds of positive clones,
how can we distinguish between over
ten thousand different odorous
molecules?
How do we smell so well?
•Several hundred genes is just the lower limit
of what is actually be present
•reliance on PCR and other techniques
•primer design
•One receptor can recognize a small
number of ligands with different affinities
•multiple ligands in one “smell” can be
processed at once by multiple receptor
types
•perceived as one smell but actually is a
few or many odourous molecule types
Overview
Future Directions
•Better understanding of the diversity of
ligand that one receptor can complex with
•Number of different receptors found in
olfactory epithelium
•Differences between rats, humans, and
other species
•Evidence of DNA rearrangement?
•not observed in this experiment
References
A Novel Multigene Family May Encode Odorant Receptors:
A Molecular Basis for Odor Recognition Linda Buck and Richard
Axel. Cell, Volume 65, 175-187 April 5, 1991
Colorado State University Webpage
http://arbl.cvmbs.colostate.edu
Essential Cell Biology second edition. Garland Science. 2003
University Webpages of Harvard, Columbia, John Hopkins
University
Howard Hughes Medical Institute
http://www.hhmi.org/
The Nobel Foundation
http://nobelprize.org/