Figure 3

Identification of a transcriptional initiation region in Cd74 intron 1. (A) Overview of the number of integrations within the 3897 bp Cd74 intron 1 illustrated in 100 bp windows. The window spanning the first 100 nucleotides in intron 1 is denoted 1 and all other positions are numbered in relation to this. Integrations in co-transcriptional orientation with Cd74 are marked by dark grey bars and integrations in antisense orientation are marked by light grey bars. The position of primer A-E applied in the RT-PCR analysis in (B) is indicated. (B) RT-PCR analysis on RNA from un-infected NMRI spleen with a Cd74-specific exon 5 primer and primer A to E, respectively. (C) Results of RLM-RACE analyses, with RNA from un-infected mice, indicating the identified transcriptional initiation region in intron 1 dispersed over app. 100 nucleotides (sequence marked in grey). The position of the primer applied for RT-PCR analyses (SR2) in conjunction with a linker-specific forward primer is indicated. (D) Quantitative PCR analyses for detection of the canonical or the novel alternative transcript, respectively, was performed and copy numbers of the two were estimated from copy-number standard-curves derived from amplification on plasmid DNA. The samples included RNA from non-treated NMRI control spleens obtained from two animals (dark grey bars) and RNA purified from tumor 01-762 (light grey bar). The relative ratios of canonical and alternative transcripts in the samples are depicted in the diagram that represents three independent experiments.