Co-expression of odorant receptor genes in zebrafish

Abstract

Olfactory receptors constitute the largest G-Protein-Coupled Receptor family in humans and in other vertebrates. Olfactory receptor genes are expressed in the olfactory sensory neurons in the olfactory epithelia and are responsible in detection of odorant molecules. The mechanisms that regulate OR gene choice are still largely unknown, but it has been shown through various methods that the majority of olfactory sensory neurons only express a single OR gene. This phenomenon has been named the one receptor-one neuron rule and it has been shown, during development, the OSNs that express more than one are selectively reduced, possibly by apoptosis. In zebrafish, which has a repertoire of 136 intact OR genes, an exception to the one receptor-one neuron rule has been reported. Members of the OR103 family have been shown to be co-expressed and the nature of this co-expression was OR103-1 being co-expressed with at least one of OR103-5 and OR103-2 in every cell OR103-1 is expressed. In this thesis, I designed experiments to find the underlying mechanism of this co-expression. I used 5’-RACE, RT-PCR, and gene-specific RACE to obtain transcript information of several OR103 members, which have revealed a possible single transcript encompassing both OR103-1 and OR103-5 coding sequences, separated by the intergenic region. In order to understand if both OR103-1 and OR103-5 could be translated from this single mRNA, which would require IRES activity by the intergenic sequence, I used tissue specific promoters to express, in vivo, two different fluorescent proteins that have been separated by the intergenic region. Finally, I used Hk-2 cell cultures to quantitatively test for possible IRES activity of the intergenic sequence in vitro.