Figure 2. Transcriptional start sites determined by SMART 5′ RACE.

A. Gel electrophoresis of the mouse (left 2 photographs) 5′ RACE PCR using, mCB2-R301 GSP1 (1°), yielded 3 CB₂ transcripts containing either exon 1a, exon 1b or exon 2 in the splenocyte (S) and B lymphocyte (B) subpopulations, whereas the T lymphocyte (T) subpopulation has 1 CB₂ transcript containing exon 1a. Nested PCR of the B lymphocyte 5′ RACE PCR product using, mCB2-R217 GSP2 (2°), was done to confirm that the primary PCR products were CB₂ transcripts. Human (right photograph) 5′ RACE PCR and nested PCR using, hCB2-R298 (1°) and hCB2-R163 (2°) GSPs, yielded 1 CB₂ transcript containing exon 1 in B lymphocytes isolated from PBMCs of the three different donors. All RACE products were visualized on a 2% agarose gel stained with ethidium bromide. mw, 100 bp DNA ladder molecular weight standard, the 500bp band is labeled. B. Diagrams of the mCnr2 (sense strand) and hCNR2 (anti-sense strand) gene illustrating the location of the TSSs (upward arrows) and transcript exonal usage. Numbers below the arrows represent the TSS’s location relative to position 1 of the Genbank^™ CB₂ first exons; mouse exons 1a & 1b to NM009924 and exon 2 to BC024052; human exon 1 to NM001841. The transcripts are labeled with their corresponding exon along with the number of nucleotides sequenced for each 5′ RACE transcript. Small black arrows mark the location of the GSPs used for the primary (1°) and nested (2°) PCR reactions. C. The 5′UTR exon sequences of the CB₂ transcripts identified by 5′ RACE (GenBank accession nos. FJ357033-6). The TSS nucleotides are underlined. Each sequence line has 60bp. Mouse primary RACE fragment sizes; exon 1b 778–788 bp, exon 2 614 bp, exon 1a 543 bp. Nested sizes; exon 1b 697–707 bp, exon 2 533 bp, exon 1a 459 bp. Human RACE fragment sizes; primary PCR 455 bp, nested PCR 381 bp.