Temporal and Spatial Expression Patterns of Transgenes Containing Increasing Amounts of theDrosophilaClock Geneperiodand alacZReporter: Mapping Elements of the PER Protein Involved in Circadian Cycling

Abstract

Rhythmic oscillations of the PER protein, the product of the Drosophila period (per) gene, in brain neurons of the adult fly are strongly involved in the control of circadian rhythms. We analyzed temporal and spatial expression patterns of three per-reporter fusion genes, which share the same 4 kb regulatory upstream region but contain increasing amounts of per's coding region fused in frame to the bacterial lacZ gene. The fusion proteins contained either the N-terminal half (SG), the N-terminal-two-thirds (BG), or nearly all (XLG) of the PER protein. All constructs led to reporter signals only in the known per-expressing cell types within the anterior CNS and PNS. Whereas the staining intensity of SG files was constantly high at different Zeitgeber times, the in situ signals in BG and XLG files cycled with approximately 24 hr periodicity in the PER-expressing brain cells in wild-type and per01 loss of function files. Despite the rhythmic fusion-gene expression within the relevant neurons of per01 BG files, their locomotor activity in light/dark cycling conditions and in constant darkness was identical to that of per01 controls, uncoupling protein cycling from rhythmic behavior. The XLG construct restored weak behavioral rhythmicity to (otherwise) per01 files, indicating that the C-terminal third of PER (missing in BG) is necessary to fulfill the biological function of this clock protein.

A Small Conserved Domain of Drosophila PERIOD Is Important for Circadian Phosphorylation, Nuclear Localization, and Transcriptional Repressor Activity

“…Two transgenic lines, containing different truncated PER fusion proteins, suggested that a region of approximately 230 aa might be involved in PER phosphorylation and degradation (10,47). In transgenic per-sg flies, PER-SG (aa 1 to 637 followed by betagalactosidase) showed defective phosphorylation and was highly stable, i.e., exhibited no observable circadian oscillation in PER phosphorylation or PER levels.…”

“…Moreover, Kim and colleagues have observed nuclear localization of their PER⌬ construct in flies (25a). PER-SG, in contrast, does not interfere with behavioral period in an otherwise wild-type background (47), probably because the SG fusion protein lacks a PER CLK-CYC-interacting domain (5). All of these considerations suggest that PER⌬ biological activity in flies reflects the ability to repress CLK-CYC activity.…”

Functional Analysis of Circadian Pacemaker Neurons inDrosophila melanogaster

“…After 2 h fixation, the flies were rinsed three times for 15 min in PB, and their brains were dissected as whole mounts. After blocking in 5% normal goat serum overnight, triple immunostainings were performed on the whole-mount brains with a rabbit anti-PER serum [diluted 1:1000 (Stanewsky et al, 1997)], a rat anti-TIM serum [diluted 1:1000 (Kaneko et al, 1997)], and the monoclonal mouse antibody nb33 (diluted 1:100). The latter recognizes the neuropeptide pigmentdispersing factor (PDF)-positive neurons (Veleri et al, 2003).…”

Moonlight shifts the endogenous clock of Drosophila melanogaster

“…38 with the following alterations: Proteins were transferred to nitrocelluose by using a wet blot chamber (Amersham Biosciences Europe, Freiburg, Germany) for 3 h at 4°C. After transfer, membranes were washed in 1ϫ TBS, blocked for 1 h in Li-COR Biosciences (Bad Homburg, Germany) Odyssey Blocking Buffer and incubated with anti-PER [1:10,000; raised against the entire PER protein (39)] or anti-TIM [1:5,000; raised against GST fusion proteins expressing the residues 222-577 of TIM (40)]. The secondary antibody was fluorochrome coupled [goat anti-rabbit IRdye800 (Rockland, Gilbertsville, PA) at dilution 1:5,000 in 5% dry milk in TBS with 0.1% Tween 20].…”