In addition to a central circadian clock in the suprachiasmatic nucleus (SCN),
zebrafish (Danio rerio) have local clock systems in their peripheral tissues. These
peripheral tissues express a complement of clock genes that can be synchronized
with the 24 h light/dark cycle and thus may be entrained by light. To date, teleost
multiple tissue (tmt) opsin identified from Fugu rubripes and Danio rerio is the only
opsin that has been proposed as a candidate to mediate this cellular
photoentrainment (Moutsaki et al., 2003). Here we report the discovery of a
multigene family of tmt opsins found not only in the teleost fishes, but in vertebrates,including amphibians, birds, reptiles, and some mammals.
Phylogenetic analysis demonstrated that this gene family consists of three
main classes, tmtI, tmtII and tmtIII, with each duplicating further to give two
paralogues in the zebrafish genome. Their predicted amino acid sequences contain
most of the characteristic features for the function of a photopigment opsin, as well
as seven transmembrane segments indicative of a G protein coupled receptor (GPCR)
superfamily. Significantly, reverse transcription polymerase chain reaction (RT-PCR)
reveals that the tmt opsin genes in zebrafish are both temporally and spatially
regulated. To investigate if these tmt photopigments mediate light-activated currents
in cells, each opsin was expressed in vitro and the responses characterised by calcium
imaging, whole-cell patch clamp electrophysiology, UV-Vis spectrophotometric
analysis, and bioluminescence reporter assay. Collectively, these data suggest that
some of the opsin photoproteins signal via Gi-type G protein pathway. Interestingly,
the spectral analysis obtained shows that most tmt opsins tested are UV-sensitive
when reconstituted in vitro with 11-cis and all-trans retinal, indicating an intrinsic
bistable dynamics. Using site directed mutagenesis on one of the tmt opsins, tmt10,
the potential spectral tuning sites involved in UV detection were tested.
As part of this study, tmt opsin cDNAs were isolated from three populations
of Mexican tetra (Astyanax mexicanus): surface, Pachon and Steinhardt. This allowed
for a direct comparison between the tmt opsins present in the dark adapted species
(cavefish) versus those of the light adapted species (zebrafish). It is hoped that the
findings from this project will contribute to our understanding of non-visual light
detection in fish and the evolution of their non-image forming photoreception.This thesis is not currently available in ORA