2. Aim: To understand how the olfactory transduction system is organized Are there several receptor protein “species” each of which detect a class of odorant molecules, or, like the retina, are there relatively few species that respond to a relatively broad panel of odorants? Humans can discriminate ~10,000 monomolecular odorants Approach: 1.Receptors should be related to similar proteins found elsewhere, based on their functional properties. 2.The broad sensitivity of olfactory systems suggests a multigene family of receptor proteins (unlike retina) that should have 7 transmembrane (7TM) spanning regions. 3.Odor receptor proteins should be unique to the olfactory epithelium (based on comparison of cDNA from different tissues).

3. Overview of the olfactory epithelium 3 major cell types: Olfactory sensory neurons Supporting cells Basal (stem) cells Molecular basis of sensory transduction: 1.G-protein coupled receptors activate 2.Adenylate cyclase production of cAMP 3.cAMP activates ion channels Activation of this pathway results in depolarization of the cell membrane potential. Hypothesis: Olfactory receptors are related to other G-protein coupled receptors.

4. “Digestion of one of these, PCR 13, yielded a large number of fragments whose sizes summed to a value much greater than that of the undigested PCR 13 DNA, indicating that PCR 13 might contain multiple species of DNA that are representatives of a multigene family.” “cDNA prepared from olfactory epithelium RNA was subjected to PCR amplification with a series of different primer oligonucleotides [designed based on other 7TMPs] ; the DNA products of appropriate size [based on other 7TMPs (600-1300bp)] were isolated, further amplified by PCR, and size fractionated on agarose gels.”

5. Northern blot analysis indicates that isolated RNAs are specific to olfactory epithelium. Hence these 7TMPs are specific to the olfactory epithelium.

6. Analysis of DNA nucleotide sequences reveal several distinct 7-TMPs These sequences are also novel among other known 7-TMPs externalcytoplasmic

7. Analysis of TM5 region + flanking loops suggests that these 7TMP receptors can be grouped into subfamilies based on sequence homology external cytoplasmic

8. Analysis of DNA sequences reveal several distinct 7-transmembrane proteins Prototypical 7-TMP highlighting regions of variability (black circles). Remember the primers originally used were for TM regions 2 and 6 hence they should be the least variable The high overall variability of the TM regions suggests that these are binding sites for unique classes of odor external cytoplasmic

9. 70 from 7: Olfactory receptors represent a gene super family! Using 7 primers designed from 7 different sequences (Fig 4) a total of 70 bands emerge on a Southern blot suggesting 70 unique DNA sequences (aka. 70 receptor proteins).