12] Fusions to imidazopyrazinone-type luciferases and aequorin as reporters

Abstract

This chapter discusses the fusion of genes to imidazopyrazinone-type luciferases and aequorin as reporters. In bioluminescent marine organisms, an imidazopyrazinone (3,7-dihydroimidazopyrazin-3-one) compound is used for the luciferase reaction. Two imidazopyrazinone-type luciferins, coelenterazine luciferin and Cypridina luciferin, have been isolated. These two luciferins are similar with a central imidazolepyrazine nucleus in their structures. Coelenterazine is also known as “Watasenia preluciferin,” “Oplophorus luciferin,” and “Renilla luciferin.” Among coelenterazine-type luciferases, Renilla luciferase and Oplophorus luciferase have been isolated and their biochemical properties characterized. Coelenterazine serves as the chromogenic compound of photoproteins—such as aequorin, phialidin, halistaurin, and obelin. In the field of molecular biology, several reporter genes are used for monitoring the gene expression in whole organisms, as well as in single cells. Luciferases, including firefly luciferase and photoproteins, are useful reporter molecules with various advantages for the bioluminescent system: they are sensitive, rapid, and harmless. Moreover, the detection of photon produced by luciferase permits the real-time imaging of gene expression and of the dynamic changes of target protein in living cells. The luminescence reaction of imidazopyrazinone-type luciferases is simpler in required components than that of firefly luciferase, because the imidazopyrazinone-type luciferase systems do not need any cofactors such as ATP and Mg 2+ .