Fully Codon-optimized luciferase Uncovers Novel Temperature Characteristics of the Neurospora Clock

Division of Science and Mathematics, University of Minnesota—Morris, Morris, MN 56267, and Departments of Biochemistry and Genetics, Dartmouth Medical School, Hanover, New Hampshire 03755

^* To whom correspondence should be addressed. Email: jennifer.loros{at}dartmouth.edu. jay.dunlap{at}dartmouth.edu.

	Abstract

We report the complete reconstruction of the firefly luciferase gene, fully codon-optimized for expression in Neurospora crassa. This reporter enhances light output by approximately four log orders compared to previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activity of many poorly expressed genes. Time-lapse photography of strains growing on race tubes, in which the frq or eas promoter is used to drive luciferase, shows highest levels of luciferase activity near to the growth front and newly formed conidial bands. Further, we have established a sorbose-medium colony assay that will facilitate luciferase-based screens. The signals from sorbose-grown colonies of strains in which the frq promoter drives luciferase exhibit the properties of circadian rhythms and can be tracked for many days to weeks. This reporter now makes it possible to follow the clock in real-time, even in strains or under conditions in which the circadian rhythm in conidial banding is not expressed. This property has been used to discover short, ca. 15 h, period rhythms at high temperatures, where banding becomes difficult to observe on race tubes, and to generate a high-resolution temperature phase response curve.