ABSTRACT
The circadian expressed luciferin binding protein (LBP) gene from the marine bioluminescent alga Gonyaulax polyedra represents the first dinoflagellate gene that has been cloned and sequenced at both cDNA and genomic levels. Starting with a fragment from the 3' end of the LBP cDNA that was found by immunoscreening of a cDNA library, genomic clones were obtained by the inverse PCR technique. Full length cDNA clones were selected by screening a cDNA library by plaque hybridizations and by PCR amplifications. The LBP sequence has a 2004 nucleotide (nt) open reading frame coding for a protein of 668 amino acids (~75 kDa; GenBank accession # L06908). The reading frame and identity of the clone was confirmed by the sequence of an octapeptide obtained from a purified fragment of CNBr treated LBP. A variant LBP cDNA was found to differ in sequence by about 11% at the DNA level (GenBank accession #L06909). The untranslated regions (UTR) of the mRNA are 111 nt (5' UTR) and 158 nt (3' UTR) long, respectively. The LBP gene contains no introns and exhibits certain features not typical for a eukaryotic gene. Its promoter does not include the typical TATA box within ~ 50 nt upstream of the transcription start, and the usual poly A+ signal AAUAAA is not present on the end of the LBP mRNA. The copy number of the gene is very high (~1000 copies per cell). However, the universal genetic code and conserved positions relevant for the translational apparatus are maintained.Gene regulation in the unicellular marine dinoflagellate Gonyaulax polyedra is of particular interest because many physiological and biochemical processes, including bioluminescence, nitrate-reductase activity and cell motility, are under control of the circadian (daily) biological clock (Morse et al, 1990). Such control is widespread in biological systems from microorganisms to humans; the sleep/wake cycle and activity in animals, photosynthesis in plants and nitrogen fixation in cyanobacteria are all examples of processes subject to temporal control by this clock mechanism (Edmunds, 1988; Hastings, et al., 1991), as is photoperiodic timing of reproduction in both plants and animals (Hoffman, 1981; Lumsden, 1991).
In G. polyedra, the enzyme (luciferase) and substrate (luciferin) responsible for light emission, as well as the luciferin binding protein (LBP), which sequesters the luciferin (a tetrapyrrole), have been shown to vary in their amounts in the cells in relation to time of day (Dunlap & Hastings, 1981; Johnson and Hastings, 1986; Morse et al., 1989a; Nakamura et al., 1989). LBP is rapidly synthesized during the early night phase and can reach ~ 1% of the total cellular protein levels. Several hours later it is preferentially degraded by an unknown mechanism.
When the mRNA levels for the LBP were examined, it was found that they remain the same in cells extracted at different times throughout the daily cycle, as judged by Northern blots. Since the protein is synthesized in vivo for only a short time in the early night, it was concluded that the translation of the message is controlled by the circadian clock mechanism (Morse et al, 1989b). In order to search for control sequences important in temporal regulation, we undertook to clone the cDNA and genomic sequences of the LBP.
We have also sought to obtain information concerning the organization of the LBP gene. The dinoflagellate nucleus is unique in both ultrastructural and biochemical aspects (Loeblich, 1976; Taylor, 1980; Spector, 1984). It contains an unusually large amount of DNA (2-200 picograms per nucleus, depending on the species), in which there is a substantial amount of hydroxymethyl uracil replacing thymine (Rae, 1976; Taylor, 1987). The numerous chromosomes, which appear quite similar, are attached to the nuclear envelope during division and remain condensed throughout the cell cycle. They are devoid of typical histones, lack the differential heterochromatic crossbanding that is found in eukaryotic metaphase chromosomes (Haapala and Soyer, 1974; Rizzo, 1991), and undergo atypical meiosis (Himes and Beam, 1975). These and other matters concerning the nature of dinoflagellate chromosomes, as well as their relationship to typical eukaryotic chromosomes and the evolutionary position of the group, remain to be clarified. Thus, it was of interest to study a dinoflagellate gene and its cDNA. LBP provides a good model for this purpose.