Alterations of PRDM1 and c-myb in the DLBCL cell line OCI-LY3. (A) Diagrammatic representation of chromosomal inversion between the PRDM1 and c-myb loci and alterations in their transcript and protein products. Boxes indicate exons, with coding frame in black. The 5′ exons derived from the alternate promoters of PRDM1 and c-myb are denoted as 1β. The 5′ and 3′ breakpoints are shown by block arrows, and their positions relative to the 3′ ends of PRDM1 exon 2 and c-myb exon 1 are indicated. The expected sizes of the germ line and rearranged EcoRI fragments spanning the breakpoints and detected by Southern blotting (panel B) are marked. The cryptic exon located approximately 21 kb downstream of PRDM1 exon 2, as well as the c-myb cryptic exon 1, are indicated by asterisks. The 2-bp frameshift deletion (del) and sites of premature translation termination (UGA) are shown. Structures of the truncated PRDM1 and c-MYB are depicted with their relevant functional domains. PR indicates PR domain; ZnF, zinc fingers; ++, basic domain; —, acidic domain; S/T, serine/threonine-rich region. (B) Gene rearrangement and junction sequences at the proximal and distal breakpoints. Left: Southern blot analysis of EcoRI-digested genomic DNA detected gene rearrangement using the PRDM1 exon 2 probe (top) and the c-myb intron 1 probe (bottom). The germ line bands detected in U266 are absent in OCI-Ly3, consistent with deletion of the wild-type allele. Right: Junction sequences for the proximal breakpoint (top) and the distal breakpoint (bottom). Putative breakpoints are indicated by arrowheads. The germ line PRDM1 intron 2 and c-myb intron 1 sequences normally present adjacent to the breakpoints also are shown. The putative 5′ end of the PRDM1 intron 2 sequence translocated next to c-myb intron 1 and the putative 3′ end of the c-myb intron 1 sequence translocated next to PRDM1 intron 2 are marked by open and filled boxes, respectively. Nucleotides that appear to be duplicated next to the breakpoints are indicated in italics. The nucleotide at the proximal breakpoint junction that differs from the germ line sequence (g-to-c change) is underlined. (C) PRDM1 RNA expression analysis in OCI-Ly3. Top left: PRDM1α and PRDM1β transcripts were analyzed by RT-PCR using primers specific to exon 2 and exon 4, or exon 1β and exon 4, respectively. In OCI-LY3, no PCR products could be obtained using the exon 2/exon 4 primer pair as a result of chromosomal inversion (panel A), while normal splicing occurs between exon 1β and exon 4. Bottom left: A Northern blot was hybridized with a PRDM1 exon 4 to 7 cDNA probe, which detected both PRDM1α and PRDM1β transcripts in U266, but only PRDM1β mRNA in OCI-Ly3. The smaller (∼3.5 kb) transcripts present in OCI-Ly3 most likely are derived from alternative polyadenylation, as described previously.¹  Right: Sequence of fusion transcripts consisting of PRDM1 exon 2 spliced to a cryptic exon approximately 21 kb downstream using consensus splice donor and acceptor sites (italics). These transcripts code for the first 61 amino acids of PRDM1α followed by a novel 36 amino-acid peptide sequence (italics). The consensus polyadenylation signal (AATAAA) is underlined. (D) Western blot analysis of PRDM1 in OCI-Ly3 and U266 using antibody to the N terminus of PRDM1. PRDM1α was detected in U266 but not in OCI-Ly3. Amido-black staining of the blot served as loading control. (E) Frameshift mutation of PRDM1. The sequence chromatogram of OCI-Ly3 genomic DNA shows a 2-bp deletion that leads to a frameshift and premature translation termination (bold). The numbering of the nucleotides corresponds to the nucleotide number starting from the 5′ end of exon 5.