Buck and Axel et al

Question 1

Background

Answer 1

● Understanding the mechanisms 
of sensory transduction can 
help us to understand how 
sensory systems function
● At the time (early 90s), olfaction 
was somewhat of a mystery
● Receptors had not been 
identified
● Many receptors specific to 
single ligands, or few receptors 
that bind to many classes?

Question 2

Figure 1

Answer 2

Background on the olfactory system.

● Previous studies suggest that odorants bind to GPCRs
● The resulting cAMP opens excitatory cyclic nucleotide-gated (CNG) ion
channels
● Eventually leads to AP, which propagates to the brain
● At the time, a lot was actually known about GPCRs - paper even states seven
transmembrane domains
● Similar, conserved mechanism to detection of other substances in body (e.g.
hormones, metabotropic NT receptors)

Question 3

Experimental setup

Answer 3

Three assumptions:
1. Odorant receptors are GPCRs
2. Diversity of receptors = multigene family
3. Expression restricted to olfactory epithelium
Thus:
● Amplify (via PCR) GPCR mRNA/cDNA in olfactory epithelium
● Test whether PCR products contain a mixture of sequences
● If there are lots of distinct receptors, restriction digests will have summed MW far
greater than that of undigested product.

Question 4

Figure 2: PCR amplification suggests a large family of

odorant receptors

Answer 4

● A mixture of fragments that sum to weight of original product, and some
fragments that weigh more in total
● PCR product 13 in particular seemed to have many different cDNA variants
● Thus, possible that there are many distinct, but related olfactory receptors
that are GPCRs
● Further cloning showed that PCR product 13 contained five new, related
GPCRs

Question 5

Figure 3: Northern blot analysis with 20 probes

Answer 5

● Use PCR 13 products to clone original genes; yields more than 20 distinct
clones
● These can then be used as probes, etc.
● Confirmation that expression of these cloned olfactory channels is restricted
to olfactory epithelium
● Important that the band is diffuse -> what does this mean?

Question 6

Figure 4: Protein sequences of ten cDNA clones

Answer 6

● High levels of variability within transmembrane domains, despite similarity to
GPCRs in general
● Likely that this leads to the differences in binding sites
● Based on this info, what hypothesis (one odorant, one receptor; many
odorants, one receptor) seems most plausible?

Question 7

Figure 5: Variability in the proteins sequence of the

putative olfactory receptor family

Answer 7

● High levels of variability within transmembrane domains, despite similarity to
GPCRs in general
● Likely that this leads to the differences in binding sites
● Based on this info, what hypothesis (one odorant, one receptor; many
odorants, one receptor) seems most plausible?

Question 8

Figure 6: Subfamilies within the odorant receptor family

Answer 8

For 18 diff cDNA clones, the protein sequences deduced from the nucleotide sequences. This shows there is a lot of divergence in the region that corresponds to the 5th TM domain and the adjacent cytoplasmic loop, within this divergence you can separate the diff subfamilies B, C, D. Between these subfamilies they encode receptors that bind odourants that have diff molecular structures; but within them they can recognised subtle diff in odourant molecules that are kind of similar in terms of structure.

Question 9

Figure 7: Genomic DNA fragment binding assays

Answer 9

● Buck and Axel claim that each DNA fragment in Fig. 7 to which a divergent
clone probe binds to is a unique gene
● Thus, individual cDNA probes are parts of subfamilies that range from single
to tens of members
● Could be at least 70 different genes; more rigorous experiments suggest 200

Question 10

Figure 8: Northern blots with the same 7 cDNA clones

used previously

Answer 10

• Olfactory GPCRs are restricted to olfactory epithelium, specifically the
  olfactory sensory neurons (OSNs)
  ● Based on rough numbers of expression, each neuron has roughly 20-40 transcripts; thus, it is likely that each OSN only express a subset of all hundred of receptors (as one would expect)

Question 11

Conclusions

Answer 11

● A large, multigene family encodes for putative odorant receptors
● These receptors are likely GPCRs
● Significant subfamilies/hierarchy
● Likely that there are hundreds of unique receptors
● Many receptors, that each bind to a few ligands
● Not likely that receptor diversity is driven by rearrangement of DNA