| Research Abstract |
In order to understand the phylogenetic distribution and expression of amelogenin genes in vertebrates, Southern hybridyzation, in situ hybridyzation and PCR amplification were performed in thirteen vertebrate species selected from seven taxonomic classes. The species used in this study were as follows ; the human, mouse (Mammalia), chicken (Aves), caiman, tortoise, snake (Reptilia), frog (Amphibia), gar-pike, lungfish, eel (Osteichthyes), dogfish (Chondrichthyes) and hagfish, lamprey (Agnatha). The Southern hybridyzation assay revealed that the hybridyzation with the human cDNA non-radioactive probes was obtained in the genomic DNA of all the species used. This demonstrates partial DNA sequence similarities between these species, and may indicates the universal distribution of the amelogenin gene or amelogenin gene-like sequences in vertebrates. Then, the gar-pike was selected and was examined by in situ hybridyzation by using human cDNA non-radioactive probes, because of it's signifi
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cance for the phylogenetical location on the enamel evolutions as revealed by fine-structural and immunocytochemical analyzes. Although the hybridyzation was detected with the human cDNA probes by Southern hybridyzation, the signals of mRNA was not detected in the tooth germs of the gar-pike. Furthermore, in the PCR amplification targeted to the homologous sequences with the exon 6 of human amelogenin gene, the products were obtained in the genomic DNA of the human (400bp), mouse (400bp), chicken (780bp), caiman (400bp), tortoise (750bp) and snake (750bp). However no products were obtained in frogs, fishes and cyclostomes. After performing the sequencing of products in the chicken, caiman, tortoise and snake, the DNA sequences of the caiman only corresponded with the exon 6 of human amelogenin gene by 48.5%. A lower but slight homology were recognized in the products of the chicken, tortoiose and snake. Consequently, the present study demonstrates that the amelogenin gene of amelogenin gene-like sequences may be conserved universally in vertebrates. Less
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