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A secreted luciferase from the marine ostracod, Vargula hilgendorfii, is a useful tool for gene expression assays in living mammalian cells. We have cloned the cDNA of a new secreted luciferase from the ostracod Cypridina noctiluca, which inhabits the coast of Japan. C. noctiluca luciferase consists of 553 amino acid residues with a molecular mass of 61,415 Da, as deduced from the nucleotide sequence. The homologies of nucleotide and amino acid sequences with V. hilgendorfii luciferase are 79.2% and 83.1%, respectively. C. noctiluca luciferase can expressed in and secreted from cultured mammalian cells. The characteristic properties of expressed C. noctiluca luciferase are similar to those of V. hilgendorfii luciferase. However, the activity of C. noctiluca luciferase in culture medium is much higher than that of V. hilgendorfii luciferase, suggesting that C. noctiluca luciferase is a highly potent reporter enzyme for real-time and continuous monitoring of gene expression in living cells.

Reporter assays that use luciferase are widely employed for monitoring cellular events associated with gene expression. In general, firefly luciferase and Renilla luciferase are used for monitoring single gene expression. However, the expression of more than one gene cannot be monitored simultaneously by this system because one of the two reporting luciferases must be used as an internal control. We have developed a novel reporter assay system in which three luciferases that emit green, orange, and red light with a single substrate are used as reporter genes. The activities of the luciferases can be measured simultaneously and quantitatively with optical filters. This system enables us to simply and rapidly monitor multiple gene expressions in a one-step reaction.

We have developed a bio/chemiluminescence spectrometer with a cooled charge-coupled-device (CCD) detector to obtain a quantitative luminescence spectrum as the absolute number of all emitted photons at each wavelength. The integrated area of the spectrum divided by the number of reacted substrate molecules gives the quantum yield. Calibration of the absolute sensitivity of the CCD-spectrometer system was performed by using lasers and a tungsten lamp with calibrated powers as primary light standards, and calibration of the light-collection efficiency of the spectrometer with several kinds of cells for liquid samples was achieved by introducing a simple reference double-plate cell. The reference cell is not convenient for final bio/chemiluminescence measurements but is useful for the calibration because it has well-defined angular dependence of light emission, allowing accurate calculation of the light-collection efficiency. Using this CCD-spectrometer system, we re-examined the quantum yield of aqueous luminol chemiluminescence with H2O2 catalyzed by horseradish peroxidase. The quantum yield was constant for a wide range of luminol concentrations, whereas it changed and had an optimum against H2O2 concentrations. The optimum quantum yield was 1.23(+/-0.20)%, which is in good agreement with previously reported values.

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