Structure of per–lacZ fusion genes analyzed for temporal and spatial expression. In the top panel, the structure of a 13.2 kb genomic DNA fragment containing the per gene is shown. This construct restores rhythmic behavior after transformation intoper01 mutants (Citri et al., 1987). The solid line represents upstream untranscribed regulatory sequences as well as introns and 3′ untranscribed DNA. Thebars reflect exon sequences; solid partsdesignate untranslated and open bars protein-coding DNA sequences. Striped portions represent the PAS domain, which can function as an intermolecular protein dimerization motive (residues 240–496) (Burbach et al., 1992). Shaded areasrepresent the C domain involved in intramolecular protein interactions (residues 524–685, Huang et al., 1995), and checked areas represent the GT repeats, which vary in number among different Drosophila melanogaster strains (e.g., 20 Gly-Thr pairs in the wild-type strain Oregon-R) (Yu et al., 1987). Potential PEST sequences, identified by the program PESTFIND (Rechsteiner et al., 1987) are indicated byasterisks and extend from residues 135 to 162 (exon 3), 652 to 664 (exon 5), 1184 to 1215 (exon 6), and 1242 to 1256 (also showing the highest PEST score, exon 7). The arrowindicates per’s transcription start site (+1). Translation starts at the NcoI site and stops 32 bp 3′ of the NciI site. The differentper–lacZ fusion genes contain various amounts of this genomic per DNA fragment, fused in frame to theE. coli lacZ gene. They all share the same 5′ regulatory region (from the BamHI site at −4200) and transcribed sequences up to the SacI site in exon 5 (including the PAS domain), where the SG fragment is fused to lacZ. SG contains only one complete PEST sequence and is missing parts of the C domain and all of the GT repeat. The BG construct contains additionalper-coding DNA up to the BamHI site in exon 5, including two PEST sequences as well as the complete C domain and GT repeat. XLG extends to the NciI site in exon 8, including two additional PESTs that are missing in BG. Note that this construct does not quite encode the full-length PER protein (see Materials and Methods).
