Molecular Cloning And Evolution Of The AmphiS28 Gene From Amphioxus Branchiostoma Belcheri Tsingtauense

In this paper, the complete nucleotide sequence of the cDNA clone encoding an amphioxus (Branchiostoma belcheri tsingtauense) ribosomal protein S28, AmphiS28, has been determined. The full-length cDNA of AmphiS28 is 460 bps in length and it contains a 207bp open reading frame (ORF), flanked by a 31bp 5' untranslated region and a 222bp 3' untranslated region, respectively. The ORF encodes a 68 amino acid protein with a calculated molecular mass of 7,694 Da and its isoelectric point (pI) is 10.44. AmphiS28 contains a total of 14 basic and acidic amino acid residues. Neither basic nor acidic amino acid residues in AmphiS28 were clustered together. The protein is quite hydrophilic (23 of 69 residues aie charged), which is identical to the rat ribosomal protein S28. At the amino acid level, AmphiS28 shares more than 84.8% identity with its vertebrate homologues such as ruman S28 and mouse S28, less than 80.0% with S28 family members of other eukaryotes including the invertebrates, which seem to demonstrate that amphioxus is closer to the vertebrates. The phylogenetic analysis based on the amino acids of the S28 proteins from different species shows that the AmphiS28 gene is intermediate between the S28 members in vertebrates and those in invertebrates, and it pojsibly represents the archetype of the vertebrate S28 genes. This is in line with the notion that amphioxus is an organism transitional from invertebrate to vertebrate. Within the chordate clade, the AmphiS28 branched out first, forming an out group of the vertebrate S28. Moreover, no particular S28 proteins in the vertebrate or the invertebrates show consistently higher sequence similarity to AmphiS28 at the amino ac.ds level. All these results support the notion that amphioxus is not only the sister group of the existing vertebrates but also the basal lineage of chordates.In the Southern blot analysis, hybridization of the DIG labeling probe to digests of genome DNA suggests that there are 3-4 copies of the AmphiS28. The result is compared with the gene copy number of the rat ribosomal protein S28, which demonstrate that the housing protein gene-S28 gene is also duplicated in the origin and evolution of the vertebrates.