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.Circadian rhythmicity is an ancient form of biological regulation. The ability of an organism to determine the time of day in order to regulate metabolic events is close to ubiquitous within the eukaryotes and, additionally, is found in more than one cyanobacterial species (17). Although the ability to tell time from a molecularly based oscillator is thematically conserved across many phyla, the outputs that the circadian clock regulates are organismally dependent, and in many systems, reporters are used to monitor these rhythms. There is a rich history of using luminescence for this purpose in circadian biology. Studies using circadianly regulated endogenous luciferase in the marine dinoflagellate Gonyaulax polyedra to follow the clock (34) were precursors for a host of experiments using luminescent reporters in plants (35
In vitro bioluminescence components of the dinoflagellates Gonyaulax polyedra, G. tamarensis, Dissodinium lunual, and Pyrocystis noctiluca were studied. The luciferases and luciferins of the four species cross-react in all combinations. All of these species possess high-molecular weight luciferases (200,000-400,000 daltons) with similar pH activity profiles. The active single chains of luciferases from the Gonyaulax species have a MW of 130,000 while those from P. noctiluca and D. lunula have a MW of 60,000. Extractable luciferase activity varies with time of day in the two Gonyaulax species, but not in the other two. A luciferin binding protein (LBP) can easily be extracted from the two Gonyaulax species (MW approximately 120,000 daltons), but none could be detected in extracts of either D. lunula or P. noctiluca. Scintillons are extractable from all four species, but they vary in density and the degree to which activity can be increased by added luciferin. Although the biochemistry of bioluminescence in these dinoflagellates is generally similar, the observations that D. lunula and P. noctiluca apparently lack LBP and have luciferases with low MW single chains require further clarification.
Various temperatures relative to a 25 degrees C control have been applied as phase-resetting agents in release-assay experiments using the conidiation rhythm of the mold Neurospora crassa. The larger the difference in temperature from the 25 degrees C control, the stronger the phase-resetting effects. Phase-resetting curves of the weak type (type 1) are observed for temperatures up to 28 degrees C and down to 22 degrees C, whereas temperatures above 28 degrees C and less than 22 degrees C generally cause phase-resetting curves of the strong type (type 0). Singularity behavior occurs at approximately 22 degrees C and 28 degrees C when 25 degrees C is used as the control temperature. When a different control of 29.5 degrees C is used in a release-assay experiment and the resetting temperature is 25 degrees C, near-singularity behavior is observed.
During measurements of the circadian (approximately 24-hr) rhythms of spontaneous bioluminescence in the marine dinoflagellate Gonyaulax polyedra, the individual cultures in vials were shielded from otherwise constant dim light for 1-3 min every 20-60 min by a photomultiplier housing that was moved from vial to vial. The high-frequency dark pulses caused a small but consistent shortening of the free-running circadian period, but there was no indication that the dark pulses caused entrainment. Hardware and software components of the microcomputer-controlled data collection system are described. A microcomputer controlled the movement of the photomultiplier and acquired the data via an analog-to-digital converter. The algorithms distinguished and separately recorded background glow, intermittent flashes, and total light from populations ranging in number from 10(3) to 10(5) cells in volumes from 1 to 10 ml. Fast video display techniques allowed continuous on-line viewing of incoming data, together with a display of the data recorded over the preceding day or two. Detection of mechanical and software errors coupled with recovery systems maintained high reliability of data collection.
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