Identification of circadian-clock-regulated enhancers and genes of Drosophila melanogaster by transposon mobilization and luciferase reporting of cyclical gene expression.
A new way was developed to isolate rhythmically expressed genes in Drosophila by modifying the classic enhancer-trap method. We constructed a P element containing sequences that encode firefly luciferase as a reporter for oscillating gene expression in live flies. After generation of 1176 autosomal insertion lines, bioluminescence screening revealed rhythmic reporter-gene activity in 6% of these strains. Rhythmically fluctuating reporter levels were shown to be altered by clock mutations in genes that specify various circadian transcription factors or repressors. Intriguingly, rhythmic luminescence in certain lines was affected by only a subset of the pacemaker mutations. By isolating genes near 13 of the transposon insertions and determining their temporal mRNA expression pattern, we found that four of the loci adjacent to the trapped enhancers are rhythmically expressed. Therefore, this approach is suitable for identifying genetic loci regulated by the circadian clock. One transposon insert caused a mutation in the rhythmically expressed gene numb. This novel numb allele, as well as previously described ones, was shown to affect the fly's rhythm of locomotor activity. In addition to its known role in cell fate determination, this gene and the phosphotyrosine-binding protein it encodes are likely to function in the circadian system.
Stempfl
Thomas
T
Institut für Zoologie, Universität Regensburg, Lehrstuhl für Entwicklungsbiologie, 93040 Regensburg, Germany.
Vogel
Marion
M
Szabo
Gisela
G
Wülbeck
Corinna
C
Liu
Jian
J
Hall
Jeffrey C
JC
Stanewsky
Ralf
R
D000818:Animals; D002940:Circadian Rhythm / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics*; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics*; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D007605:Juvenile Hormones / Q000235:genetics*; D008156:Luciferases / Q000235:genetics
D004251:DNA Transposable Elements; D029721:Drosophila Proteins; D007605:Juvenile Hormones; C060606:numb protein, Drosophila; D008156:Luciferases