2009 Abstracts - Association for Chemoreception Sciences

#P118 Poster session III: Cortical chemosensory processing/Receptor genomics and molecular biologyCharacterizing Olfactory Sub-genome throughCustom MicroarraysXiaohong Zhang, Florencia Marcucci, Dongjing Zou,Stuart FiresteinDept. of Bio. Sci. Columbia University NewYork, NY, USAThe large number of olfactory receptors in rodents necessitateshigh-throughput methods to reveal their expression patterns. Wedesigned a second-generation high-density oligonucleotide arraycontaining all mouse and rat OR genes with different probecoverage. These arrays were approved to be reliable tools tomonitor OR expression. About 1000 mouse ORs and 900 rat ORswere found to be specifically enriched in the olfactory epithelium.It also enabled us to identify the expression profile for the wholeOR family in different tissues and at successive developmentalages. The onset of OR genes at early age and loss of OR genes atold age were found. The temporal expression profiles of all ORswere classified into five interesting patterns, indicating theirpossible behavior-related functions. We also applied our customarray to certain knockout mice and discovered the OR expressionchange, which is proved to be the most promising application ofthe arrays to explore OR regulation.#P119 Poster session III: Cortical chemosensory processing/Receptor genomics and molecular biologyNext Generation Sequencing as a Tool for ComprehensiveVariation Analyses of Human Olfactory Receptor GenesYehudit Hasin 1 , Tsviya Olender 1 , Miriam Khen 1 , Ifat Keydar 1 ,Hans Lehrach 2 , Marcus Albrecht 2 , Bernd Timmerman 2 , DanielReed 3 , Charles J. Wysocki 3 , Jan Korbel 4 , Doron Lancet 11Dept. Molecular Genetics, Weizmann Institute of ScienceRehovot, Israel, 2 Dept. Vertebrate Genomics, Max PlanckInstitute for Molecular Genetics Berlin, Germany, 3 MonellChemical Senses Center Philadelphia, PA, USA, 4 Gene ExpressionUnit, European Molecular Biology Laboratory Heidelberg,GermanyGenomic variation in olfactory receptor (OR) genes most likelyunderlies odorant-specific phenotypes, as attested by reports ofthe respective linkage of OR7D4 and OR11H7P gene variants tothe perception of androstenone and isovaleric acid (Nature449:468 ‘07; PLoS Biol 5:e284, ‘07). Genetic variations that resultin a complete functional knockout of an OR gene in someindividuals are eminent candidates to underlie thresholdphenotypes, as exemplified by OR segregating OR pseudogenes(Nat Genet 34:143 ‘03) and whole-OR deletion alleles (PLoSGenet 4:e1000249 ‘08). Past work has been based on limitedgenomic sequence data and therefore conveys a partial view of theOR variation landscape. We now use the recently introducedNext-Generation DNA sequencing technologies to augment thelist of deleterious and function-modifying OR variations in thehuman genome. As part of this effort we performed Solexa-Illumina deep sequencing of the entire coding region of 96 intactORs (with 250bp flank at each end), in a pool of 21 HapMapindividuals of different ethnic origin. We achieved averagecoverage of 40X per chromosome, thus accurate identification ofrare variations present in the sample. We find a surprising amountof new polymorphisms, some causing premature stop codons ormutating highly conserved amino acids, thus presumablydeleterious. In parallel, we initiate an exploration of novel entire-OR deletion alleles by generating new sequences and scrutinizingpublic data from large-scale sequencing efforts, e.g. the 1000genomes project. Genotyping of current and future instances ofOR gene inactivation events in >400 individuals that we havescored for odorant thresholds may help identify new genotypephenotypecorrelations in human olfaction.#P120 Poster session III: Cortical chemosensory processing/Receptor genomics and molecular biologyOR5D3P, a pseudogene with a functional activity potentialAlex Veithen, Magali Philippeau, Françoise Wilkin, PierreChatelainTecnoScent S.A. Brussels, BelgiumIn humans, it is assumed that 2/3 of the olfactory receptorsubgenome has succumbed to pseudogenisation. Although someOR genes may still be represented by either unaltered andpseudogenic alleles, others seems to be “old” pseudogene sincethe mutation accounting for their pseudogenisation is alreadyrecorded in the ortholog receptor of other primate species. Thehuman receptor OR5D3P corresponds to this latter situation. Itowes its pseudogene status to the lack of a start codon though noother alteration is observed in the rest of its sequence. This ORhas been cloned into an expression vector in fusion with a tagcorresponding to the 20 first amino acids of bovine rhodopsin toallow its expression into a heterologous cell model Using anautomated calcium imaging-based assay, we identified anisylacetate as a ligand for OR5D3P. The deorphanisation was furtherconfirmed by concentration-response analysis performed withboth a reporter gene-based assay and HTRF-based immunoassayallowing direct measurement of cAMP. These results indicate that,despite its pseudogene status, OR5D3P has not accumulated othermutations that would hamper its functionality and suggests that apositive selection pressure has been acting. A possible explanationfor these observations is that the lack of a start methionine couldbe rescued by an upstream exon. Such an approach has alreadybeen proposed for the corresponding chimp ortholog, OR11-140.Further analysis of OR5D3P in the olfactory epithelium remainsto be performed in order to test this intriguing hypothesis.#P121 Poster session III: Cortical chemosensory processing/Receptor genomics and molecular biologyIdentification and characterisation of a carboxylicacid-responding human ORMagali Philippeau, Alex Veithen, Françoise Wilkin, PierreChatelainTecnoScent S.A. Brussels, BelgiumShort chain carboxylic acids constitute an important source ofsweat malodours. Identifying the human olfactory receptors thatrecognize this class of molecules is therefore of interest.Understanding how the associated odour information is processedcould possibly lead to the identification of receptor antagonistsand/or modulators that could efficiently block the perception ofthese offensive smells. Here we describe the deorphanisation ofOR51E1, a receptor that was initially found to respond toisovaleric acid (IVA). The activation by IVA was confirmed bythree different functional assays (i.e. single cell calcium imaging,64 | AChemS Abstracts 2009