Circadian regulation of bioluminescence in Gonyaulax involves translational control.

Abstract

A 10-fold circadian variation in the amount of luciferin binding protein (LBP) in the marine dinoflagellate Gonyaulax polyedra is reported. This protein binds and stabilizes luciferin, the bioluminescence substrate. In early night phase, when bioluminescence is increasing and LBP levels are rising in the cell, pulse labeling experiments show that LBP is being rapidly synthesized in vivo. At other times, the rate of LBP synthesis is at least 50 times lower, while the rate of synthesis of most other proteins remains the same. The LBP mRNA levels, as determined by in vitro translations and by RNA (Northern) hybridizations, do not vary over the daily cycle, indicating that circadian control of bioluminescence in this species is mediated by translation.In the unicellular marine alga Gonyaulax polyedra, many properties ofthe cells change from day to night, representing, in effect, a cyclic daily differentiation. Such circadian (daily) rhythms continue under constant conditions (e.g., light and temperature) and occur widely in eukaryotes from humans to microorganisms (1). Although believed to be of fundamental importance in the control of diverse processes, neither the biochemical components nor the cellular organization of the underlying oscillatory mechanism is known in any system. Bioluminescence in G. polyedra is a circadian controlled process, occurring predominately during the organism's night phase (2). The cellular capacity for bioluminescence is directly correlated with the presence of the biochemical components involved in the in vitro light-producing reaction: dinoflagellate luciferin, a fluorescent oxidizable substrate noncovalently bound to a luciferin binding protein (LBP), and an enzyme (luciferase) catalyzing the light-producing reaction. Both the luciferin and the luciferase have been shown to undergo circadian variation in amounts with 10-fold higher concentrations of both present in the middle of the subjective night (3)(4)(5)(6).Gonyaulax is the only system known to date in which a circadian controlled process-namely, bioluminescencehas been shown to correlate with both changes in the activity and absolute levels ofa protein-namely, the luciferase (4, 5). We have extended these observations to LBP with two important additions. First, we show that the increase in LBP levels results from a transient increase in the rate of synthesis of the protein and, second, that these changes in synthetic rate are apparently due to a translational control mechanism.MATERIALS AND METHODS Cell Culture. G. polyedra was grown in f/2 medium under a 12-hr light (150 microeinsteins-m 2sec-1)/12-hr dark (LD) cycle to a cell density of -8000 cells per ml (4, 7) and, for many experiments, was maintained under constant dim light (LL; 15 microeinsteins m-2 sec-1). The beginning of the light period is defined as time zero. For biochemical analysis, cells were harvested by centrifugation at 3000 x g (2 min); cells for in vivo labeling and RNA preparations were isolated by filtration on 25-gtm Nitex (8) and Whatman 541...

Dinoflagellate tandem array gene transcripts are highly conserved and not polycistronic

“…Interestingly, the most abundant DNA-binding domain, the cold-shock domain, is also associated with posttranscriptional regulation in eukaryotes (17) and thus may not function as a DNA-binding protein at all in Lingulodinium. The idea that dinoflagellates favor regulation of gene expression at a posttranscriptional level agrees with studies on circadian regulation of protein synthesis showing extensive translation control (24,25). It also is interesting that although our transcriptome contains all four core histones, as well as a suite of histone-modifying enzymes, the histone RNA levels are low compared with higher plants, and the histone proteins are still below the level of detection using antibodies (16).…”

Post-transcriptional control of circadian rhythms

“…The clock controls the synthesis of these proteins, and both LBP and LCF are rhythmically expressed and peak at night, causing night-time flashing. However, the expression of lbp mRNA is not rhythmic, demonstrating that LBP and LCF expression is posttranscriptionally controlled (Johnson et al, 1984;Morse et al, 1989). Further research showed that an RNA-binding protein, clock controlled translational regulator (CCTR), interacts with a UG-repeat sequence in the lbp 3Ј-UTR and represses lbp translation during the day (Mittag et al, 1994).…”

“…Lingulodinium has a highly developed capacity for bioluminescence and is one of the main species contributing to nocturnal luminescence of the sea. This nocturnal bioluminescence is produced from specialized bioluminescent organelles, the scintillons, which contain three components that are necessary for the chemical reaction: the substrate luciferin, a luciferin-binding protein (LBP) and the enzyme luciferase (LCF) (Morse et al, 1989). The clock controls the synthesis of these proteins, and both LBP and LCF are rhythmically expressed and peak at night, causing night-time flashing.…”

Circadian Changes in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Distribution Inside Individual Chloroplasts Can Account for the Rhythm in Dinoflagellate Carbon Fixation

“…For example, previous work on the bioluminescence rhythm in the dinoflagellate Gonyaulax has shown that both dinoflagellate luciferase (the reaction catalyst) and the luciferin (substrate) binding protein LBP contribute to the RLS of the nightly bioluminescence reaction. Cellular levels of both proteins vary seven-to 10-fold between day and night in phase with the 50-to 100-fold variations in bioluminescence capacity (Johnson et al, 1984;Morse et al, 1989a). In a vertebrate example, N -acetyltransferase catalyzes the RLS in vertebrate melatonin synthesis, and N -acetyltransferase levels correlate with the circulating melatonin rhythm (Gastel et al, 1998).…”