Fully codon-optimized luciferase uncovers novel temperature characteristics of the Neurospora clock.
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 4 log orders over that with previously available versions, now producing light that is visible to the naked eye and sufficient for monitoring the activities of many poorly expressed genes. Time lapse photography of strains growing in race tubes, in which the frq or eas/ccg-2 promoter is used to drive luciferase, shows the highest levels of luciferase activity near 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, at which banding becomes difficult to observe in race tubes, and to generate a high-resolution temperature phase-response curve.
Gooch
Van D
VD
Department of Biochemistry, Dartmouth Medical School, Hanover, NH 03755, USA.
Mehra
Arun
A
Larrondo
Luis F
LF
Fox
Julie
J
Touroutoutoudis
Melissa
M
Loros
Jennifer J
JJ
Dunlap
Jay C
JC
D001683:Biological Clocks; D003062:Codon / Q000235:genetics*; D049454:Firefly Luciferin / Q000378:metabolism; D015966:Gene Expression Regulation, Fungal / Q000502:physiology*; D008027:Light; D008156:Luciferases / Q000235:genetics*; D009492:Neurospora crassa / Q000235:genetics*; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids; D011401:Promoter Regions, Genetic; D013013:Sorbose / Q000378:metabolism; D013696:Temperature
D003062:Codon; D049454:Firefly Luciferin; D008156:Luciferases; D013013:Sorbose; D010100:Oxygen