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Figure 1. Isolation of CNRs by Differential Display Method. (A) (a) Schematic diagram of the differential display method using three different stages of ameloblasts: secretory (S), early-maturation (EM), and late-maturation (LM) stages. Panels (b) and (c) show differential displays with reverse primers oligo(dT)15A and oligo(dT)15G, respectively. Arrows represent CNR1 (b) and CNR5 (c) cDNA identified by band sequencing. (B) Schematic structures of cadherins and CNRs. Bold lines under CNRs represent conserved regions in the CNR family. S, signal peptide; EC, extracellular domain; TM, transmembrane domain; CP, cytoplasmic domain. A comparison of rat, mouse, and human C-terminal protein sequences is also shown. The nucleic acid and amino acid sequences for these clones were deposited in GenBank (accession number AB045585 for CNR1 and AB045586 for CNR5). (C) Tissue-specific expression of CNR mRNA in adult (upper) and P1 rats (lower) was analyzed by RT-PCR. Primers from the conserved regions were used. CNRs were expressed in the brain and incisor but not in other tissues. (D) mRNA expression was quantified by real-time PCR in three stages of ameloblasts. The relative percentage in each stage is shown in relation to the highest value as 100%. Statistical analysis was performed with the use of InStat3 software (GraphPad Software, San Diego, CA, USA). All results were expressed as mean ± SE, and P < 0.05 was used for significance. S, secretory stage; EM, early-maturation stage; LM, late-maturation stage.
