Temporal and spatial expression patterns of transgenes containing increasing amounts of the Drosophila clock gene period and a lacZ reporter: mapping elements of the PER protein involved in circadian cycling.
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.
Stanewsky
R
R
Department of Biology, Brandeis University, Waltham, Massachusetts 02254, USA.
Frisch
B
B
Brandes
C
C
Hamblen-Coyle
M J
MJ
Rosbash
M
M
Hall
J C
JC
D000818:Animals; D030801:Animals, Genetically Modified; D001522:Behavior, Animal / Q000528:radiation effects; D001921:Brain / Q000378:metabolism*; D002940:Circadian Rhythm / Q000235:genetics* / Q000502:physiology; D003624:Darkness; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics* / Q000502:physiology; D005123:Eye / Q000378:metabolism; D005786:Gene Expression Regulation / Q000528:radiation effects; D017930:Genes, Reporter; D005813:Genes, Synthetic; D007763:Lac Operon; D008027:Light; D008124:Locomotion / Q000528:radiation effects; D008297:Male; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009474:Neurons / Q000378:metabolism; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics; D009899:Optic Lobe, Nonmammalian / Q000378:metabolism; D009928:Organ Specificity; D056950:Period Circadian Proteins; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D019076:Transgenes
D029721:Drosophila Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; C539040:PER protein, Drosophila; D056950:Period Circadian Proteins; D011993:Recombinant Fusion Proteins