The wide variety of real-time amplification platforms currently available has determined that standardisation of DNA measurements is a fundamental aspect involved in the comparability of results. Statistical analysis of the data arising from three different real-time platforms was conducted in order to assess inter-platform repeatability. On three consecutive days two PCR reaction mixes were used on each of the three amplification platforms - the LightCycler, ABI PRISM 7700 and Rotor Gene 3000. Real-time PCR amplification using a fluorogenic 5' exonuclease assay was performed in triplicate on negative controls and DNA plasmid dilutions of 108-102 copies to give a total of 24 reactions per PCR experiment.

The visual pigment melanopsin is expressed in intrinsically photosensitive retinal ganglion cells (ipRGCs) in the mammalian retina, where it is involved in non-image forming light responses including circadian photoentrainment, pupil constriction, suppression of pineal melatonin synthesis, and direct photic regulation of sleep. It has recently been shown that the melanopsin-based light response in ipRGCs is attenuated by the neurotransmitter dopamine. Here, we use a heterologous expression system to demonstrate that mouse melanopsin can be phosphorylated by protein kinase A, and that phosphorylation can inhibit melanopsin signaling in HEK cells. Site-directed mutagenesis experiments revealed that this inhibitory effect is primarily mediated by phosphorylation of sites T186 and S287 located in the second and third intracellular loops of melanopsin, respectively. Furthermore, we show that this phosphorylation can occur in vivo using an in situ proximity-dependent ligation assay (PLA). Based on these data, we suggest that the attenuation of the melanopsin-based light response by dopamine is mediated by direct PKA phosphorylation of melanopsin, rather than phosphorylation of a downstream component of the signaling cascade.

Light-activated opsins undergo carboxy-terminal phosphorylation, which contributes to the deactivation of their photoresponse. The photopigment melanopsin possesses an unusually long carboxy tail containing 37 serine and threonine sites that are potential sites for phosphorylation by a G-protein dependent kinase (GRK). Here, we show that a small cluster of six to seven sites is sufficient for deactivation of light-activated mouse melanopsin. Surprisingly, these sites are distinct from those that regulate deactivation of rhodopsin. In zebrafish, there are five different melanopsin genes that encode proteins with distinct carboxy-terminal domains. Naturally occurring changes in the same cluster of phosphorylatable amino acids provides diversity in the deactivation kinetics of the zebrafish proteins. These results suggest that variation in phosphorylation sites provides flexibility in the duration and kinetics of melanopsin-mediated light responses.