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Identification and characterization of ameloblastin gene in an amphibian, Xenopus laevis.
Ameloblastin (AMBN) is an enamel sheath protein that presumably has a role in determining the prismatic structure of growing enamel crystals. To investigate the relationship between the molecular evolution of the AMBN gene and development of enamel prismatic structures, it is considered to be of great significance in the identification of homologues of the AMBN genes in nonmammals whose teeth lack an enamel prismatic structure. Several clones containing AMBN cDNA were isolated from an African clawed toad tooth cDNA library by screening with a polymerase chain reaction (PCR) method. Sequence analysis of the clones revealed that they were derived from different genes (toad-A and toad-B), which were found to contain ORFs encoding 408- and 352-amino-acid proteins, respectively. The N-terminal part of the toad AMBN proteins and the phosphorylation motif for casein kinase II, as well as several features, were found to be highly conserved throughout the evolution of tetrapods. Exon-intron boundaries were shared by toad and caiman genes with the exception of exons 6, 7 and 10 while human and caiman genes shared them exclusive of exons 8 and 9 which have been found only in the human. As for exon 7, it was absent in both toad genes. Moreover, the AMBN genes were transcribed only in the upper jaw, presumably in teeth. These results may provide useful information for investigation of the evolution of enamel.
Nucleotide and translated amino acid sequences of toad AMBN cDNA clones. Amino acid residues are given in the IUPAC-IUB single-letter code and the signal peptide is underlined with a dotted line. The stop codon is indicated by an asterisk (*) and the polyadenylation signals are underlined with a solid line. Primer positions and orientations are indicated by lines with arrowheads above their locations in the sequence. Vertical lines show the positions of the exon boundaries. (a) toad-A gene; (b) toad-B gene. The sequences can be accessed in the GenBank database under accession nos. AY181985 and AY181986.
Southern blot analysis of the toad AMBN genes. Toad genomic DNA was digested with BamHI(B), EcoRV(E), HindIII(H) and PstI(P) restriction enzymes, and hybridized with the toad AMBN cDNA probe.
Amino acid sequence alignment of AMBNs from human, cattle, pig, rat, mouse, caiman and toad specimens. Amino acid residues are given in the IUPAC-IUB single-letter code. Numbering shows residue positions. Identity with the simple majority consensus sequence at the top is indicated by a dash (–) and deletions introduced for optimal alignment by asterisks (*). Potential phosphorylation residues (PK C, protein kinase C; TK, tyrosine kinase; CK 2, casein kinase II) are indicated by arrowheads. Vertical lines indicate exon borders of the human, caiman and toad AMBN genes. Parts completely identical in tetrapods are indicated by shaded boxes.
Dot-plot of toad-A amino acid sequence (ordinate), as compared to human and caiman AMBN sequences (abscissa). A single dot indicates four matching residues in a window of size 7.
Comparison of the amino acid composition of the deduced toad AMBN proteins with that of human, mouse and caiman AMBN proteins. Analysis for the secreted proteins was conducted after elimination of the signal peptides.
Exon–intron organization of AMBN genes in human, caiman and toad specimens. Shaded and open boxes indicate untranslated and translated exon regions, respectively. Coding lengths of exons with the number of amino acids are shown beneath the boxes. The number enclosed in parentheses beneath exons 1 and 2 in the schema of the human AMBN gene represent coding length, with the second Mets regarded as the start codon.
Neighbor-joining tree of amino acid sequences of AMBN proteins. The scale bar represents the distance based on the proportion of amino acid differences. Gapped sites were removed from all sequences before distance estimates were made. Numbers on the nodes indicate the percentage recovery of that node in 1000 bootstrap replications.
RT-PCR analysis of toad tissues. (A) cDNA was amplified from upper jaw, lower jaw, tibia, heart and liver specimens using AMBN cDNA-specific primer sets AMBX7 and AMBX40 for toad-A (lane a), and AMBX15 and AMBX36 for toad-B (lane b). (B) cDNA was amplified with primers GAPDH1 and GAPDH2 used as a positive control.
Nucleotide sequence alignment of 5′ regions of AMBN genes from various mammalian species, as well as caiman and toad specimens. The conserved start codons (ATGs) are boxed and the nucleotides in the −3 position are indicated by shaded boxes. The stop codons existing in the upstream regions of the start codons are underlined with a solid line.
Neighbor-joining tree of nucleotide sequences of the 3′UTR of the AMBNs. Numbers on the nodes indicate the percentage recovery of that node in 1000 bootstrap replications.