Abstract:
Odorant receptors which partake in the detection of both water-soluble and airborne chemicals constitute the largest family of seven-transmembrane G-Protein-Coupled Receptors in the majority of vertebrates. Since their identification as GPCR proteins, the mechanism of gene regulation in ORs has been extensively studied. It has been shown that ORs in mammals are divided into two major classes, Class I and Class II, and this phenomenon is believed to play an important restrictive role in various aspects of OR gene expression, one of which is the second choice mechanism by which olfactory sensory neurons switch to an alternate OR in case their first attempt fails at the expression of a functional OR. Zebrafish OR repertoire consists of 136 intact OR genes, including only a single Class II OR. The relatively small size of the OR repertoire and the presence of a single Class II gene, OR101-1, makes the zebrafish species an ideal model organism to study class restriction. In order to ascertain its nature as a Class II OR, the phylogenetic background of OR101-1 was analyzed by the construction of two different phylogenetic trees which include zebrafish and human OR gene repertoires. OR101-1 was shown to be much more closely related to human ORs. Olfactory epithelium-specific expression of OR101-1 was shown through RT-PCR analysis of cDNA from 8 different zebrafish tissues. Early developmental expression patterns of OR101-1 was assessed by RT-PCR, qRT-PCR of embryonic cDNA, and expression of OR101-1 as early as 24 hours post-fertilization was observed. In situ hybridization studies using OR101-1-specific antisense RNA riboprobes also corroborated our previous findings. Finally, two transgenic BAC constructs that would allow the visualization of OR101-1 expression were designed and partially cloned: an OR101-1-IRES-Venus tag construct and a YFP deletion construct replacing the OR101-1 coding sequence to study the second choice mechanism in OR101-1 expressing OSNs.
