Molecular Biology Of Amphioxus: Cloning, Expression And Evolution Of AmphiHMGB,AmphiUbf80 And AmphiPrxV Genes

Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. It has been recognized as the most important model animal to analyze the origin and evolution of vertebrates. Study on the gene structure, function and expression in amphioxus will greatly contribute to the origin and evolution of the vertebrates.In the present study, a cDNA expression library was constructed according to SMART cDNA library construction kit user manual from the adult amphioxus guts using pcDNA3 eukaryotic expression vector. By sequencing random-selected clones from the library and using bioinformatics, 54 novel expressed sequence tags (ESTs) were obtained, in which there were 14 full-length cDNAs, including peroxiredoxin gene, HMG-box gene, ubiquitin gene, signal recognition particle, acetyltransferase gene and lysozyme gene. Functional study on these novel genes is an on-going task in our laboratory.Amphioxus AmphiHMGB encoding a hign-mobility-group (HMG) protein was identified from the gut cDNA library of the amphioxus B. belcheri tsingtauense. Structural analysis suggested that it is a member of HMGB family, but not of HMGN or HMGA family. However, unlike the other vertebrate HMGB proteins which all contain a long acidic C-terminal domain, the AmphiHMGB is lack the acidic C-terminal tail. Molecular phylogenetic analysis shows that AmphiHMGB represents the precursor gene that existed prior to the origin of HMGB1, HMGB2 and HMGB3. Dot blot and northern blot studies revealed that AmphiHMGB expression is restricted mainly to ovaries and guts in adult amphioxus while it is highly expression during embryogenesis. Whole mount in situ hybridization showed that AmphiHMGB transcripts were present ubiquitously in the early embryos from zygote to neurula, but they decreased gradually in mesoderm and endoderm and neural tube with development, and became restricted to epidermal cells in the hatched larva. It appears that HMGB plays a role in the mitosis and differentiation of embryonic cells.Another amphioxus cDNA, AmphiUbf80, encoding ubiquitin/ribosomal fused protein S27a, was isolated from the gut cDNA library of B. belcheri tsingtauense. The deduced N-terminal 76 amino acids of the protein were 100% identical to those ofABSTRACThuman and rat ubiquitin (Ub), and the carboxyl extension protein (CEP) sequences were also highly similar to those of the homologous proteins of human, rat and other species. Alignment data of the CEP sequences from different animals showed that AmphiUbf80 was not only similar to vertebrate Ubf80 but also to invertebrate Ubf80. In addition, its C-terminal sequences, 69-CLTFVYNK-76, shared both the vertebrate-specific Ubf80 consensus (69-CLTYCFNK-76) and the invertebrate counterpart sequences. Phylogenetic analysis based on the amino acids of the ubiquitin/ribosomal fused proteins of different animals well reflected the established phylogeny of the chosen organisms, and amphioxus was branched off from chordates and formed a separate cluster with an intermediate placement between invertebrates and vertebrates in the tree. However, amphioxus Ubf80 also showed some unique characters such as its amino acid residues at sites 39, 54, 63 and 74 in the CEP. It appears that amphioxus not only represents an organism transitional from invertebrates to vertebrates in phylogeny but also a possible diverged form radiated from the chordate ancestor at early chordate evolution. The possibility that some mutations in Ub genes have taken place in different lines early at the divergence of plants and animals is also discussed.The third amphioxus cDNA, AmphiPrxV, encoding peroxiredoxin V, was isolated from the gut cDNA library of B. belcheri tsingtauense. It contained 187 amino acids deduced from the nucleotide sequence, and showed 65% amino acid sequence identity with human AOEB166. The analysis of N- and C-terminal domains revealed amino acid sequences characteristic of features of mitochondrial and peroxisom...