Figure 1

Human ADAM15 gene structure, phylogenetic footprinting, promoter and transcription start site analysis. The panels (A), (B), (C), and the left part of the panel (D) are in register respective to the nucleotide numbering shown under the panel (C). The symbol key for the panels (A) – (C) is shown at bottom right. (A) ADAM15 exon-intron structure is depicted with black and grey boxes representing the constitutively and alternatively used exons, respectively. Exon numbering is indicated under the boxes. The alternative exon 20a/b and 21a/b 5'- and 3'-ends are indicated with pale grey. The green boxes represent the last two exons (exons 15 and 16) of the upstream gene FLJ32785. The spaces separating the exons are proportional to the intron lengths. The solid lines connecting the ADAM15 exons represent the most frequently observed splice pattern corresponding to the ADAM15 mRNA variant 2. The dashed lines connecting the exons 18–22 represent the alternative splicing producing the ADAM15 variant with the longest open reading frame (variant 6). The dashed line between the FLJ32785 exon 16 and the ADAM15 exon 2 depicts the observed fusion splicing between the two genes. The panel (B) shows the repetitive element locations. Conservation of repetitive elements between human and mouse is indicated by the letter c. (C) The upper part shows the sequence-similarity between the mouse and human genes, indicated by the identity percentage on the vertical axis; the mVista sequence-identity plot is shown with pink shading in the intronic regions and with blue or green shading in the exonic regions. The PiP-maker identity blocks are shown with black horizontal lines. CpG islands are indicated with white and grey boxes (see the symbol key). The lower part of the panel C shows the locations of predicted transcription factor binding sequences with the relative strength of prediction on the vertical axis; the Match-program was used to predict the conventional binding motifs (solid orange squares) and the Consite for phylogenetically conserved binding motifs (open circles); predictions with scores ranging from 0.90 to 1.00 are shown for Match and from 5 to 15 for Consite. The location relative to the ADAM15 translation start ATG is indicated by the nucleotide numbering under the panel. (D) ADAM15 promoter analysis with luciferase-reporter expression. The left side of the panel D shows the ADAM15 upstream regions examined in the luciferase reporter experiments. The light blue shading indicates the conserved regions outside of the repetitive elements. The nucleotide location relative to ADAM15 translation start ATG, in register with the panel (C), is shown under the panel. The right side of the panel (D) shows the relative promoter activity of the reporter constructs in comparison to the pGL2-Basic control plasmid and to the SV40 promoter. The black and grey bars indicate the promoter activity measured in SK-BR-3 and HCC-1954 cells, respectively. (E) The transcription start site analysis using RT-PCR described in the Materials and Methods. The position of the upstream primer relative to ADAM15 translation start ATG in each PCR is indicated under the electrophoresis lanes. The upper gel shows the products of 30 PCR cycles and the lower gel those of 35 PCR-cycles.