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Aim: To understand how the olfactory transduction system is organized Are there several receptor protein “species” each of which detect a class of odorant.

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Presentation on theme: "Aim: To understand how the olfactory transduction system is organized Are there several receptor protein “species” each of which detect a class of odorant."— Presentation transcript:

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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).


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