Fig. 1. Schematic representation of the human genomic pISSHG5a2 clone. The top of the figure shows the genomic structure of the hPer1 gene with 23 exons and 22 introns. Exons are depicted as open boxes and identified with Roman numerals, and introns are indicated by horizontal continuous lines connecting the open boxes. Repeated sequences are depicted as filled striped boxes. The bottom of the figure shows the cDNA of RIGUI by continuous open boxes. Below this is shown the coding region in the PER1 gene product, with bHLH (black box) and PAS (open box) domains. The probe used to isolate pISSHG5a2 clone, APB8, is depicted as a horizontal bold line.

Fig. 2. FISH mapping of the hPer1 gene. Normal human metaphases hybridized to biotinylated clone pISSHG5a2 and detected with avidin fluorescein isothiocyanate (yellow–green signals). The inset shows a double fluorescence in situ hybridization, in which the centromere of chromosome 17 (yellow) and clone pISSHG5a2 on 17p12 (red) were simultaneously detected. Metaphase chromosomes are counterstained with DAPI.

Fig. 3. Schematic representation of the transcriptional control region of hPer1 gene. Top: map of the 3270 bp region, containing 5′ flanking sequences with potential E-boxes, exon I, intron 1 and exon II harbouring the translation initiation codon, ATG. Bottom: blow-up of the region surrounding the predicted transcription starting point (tsp). The location of the TATA-box and the first nucleotide of RIGUI cDNA are indicated.

Fig. 4. Promoter reporter constructs and expression assays. (A) Genomic regions of different extent (represented by lines) were assayed for promoter activity in a CAT reporter vector. The arrow indicates the putative transcription start site. Vertical lines represent CpG dinucleotides. Triangles in constructs A2 mut and A-TATA mut indicate the position of mutagenized ATG initiator codons of the hPER1 gene product. (B) Bars represent the level of activity of the assayed promoter regions, estimated by measuring the level of CAT enzyme and normalizing relative to β-gal enzyme synthesized from a cotransfected construct. Four to seven transfections experiments were carried out for each construct. In order to compare results from different experiments, CAT activity measured for A2 construct was arbitrarily taken as 1 unit in all experiments and activity of the remaining constructs was calculated relative to that measured for A2 construct.

Table 1. Exon–intron boundaries of the hPer1 genea

Table 2. ESTs matching the hPer1 gene sequencea