Amphioxus Molecular Biology: The Amphisrp19, Amphilysc Amphis19 Cloning, Expression And Evolution

Amphioxus, a cephalochordate, is the closest living relative to vertebrate, and has been widely known as the most important animal to study the origin and evolution of vertebrates. Studies on gene structure, function and expression in amphioxus w ill contribute to the understanding of the origin and evolution of the vertebrates. In this paper, the cloning, expression and phylogenetic analysis of AmphiSRP19, AmphiLysC and AmphiS19 are carried out.AmphiSRP19, encoding the protein SRP19, is isolated from the gut cDNA library of Branchiostoma belcheri tsingtaunese. This is the first SRP19 gene isolated from protochordates. The predicted amino acid sequence of AmphiSRP19 exhibits 52.8%, 54.9%, 42.3%, 35.3%, 28.2% and 19.7%-24.6% similarity to its known counterparts from human, mouse, Drosophila, rice, thale cress and five fungi species, respectively. Like other eukaryotic SRP19 proteins, AmphiSRP19 has the most -conserved amino acid residues located at the sites required for binding to SRP RNA. Northern blotting and in situ hybridization analyses show that AmphiSRP19 is expressed in all the tissues examined, including notochord, gill, muscle, gut and fully-grown testes and ovaries of adult amphioxus, and in all the embryos and larvae ranging from zygote to 2-day larva. These results agree well with the expectation that SRP 19 is an essential house-keeping gene.Study on lysozymes remains open in amphioxus. The existence of c-type lysozyme genes in amphioxus has been found, first such data in the basal chordates including urochordate and cephalochordate. This is in contrast to the absence of c-type lysozyme genes in urochordate. Gene organization analysis reveals that amphioxus c-type lysozyme (AmphiLysC) gene is similar to vertebrate c-type lysozyme genes with respect to the number and sizes of both exons and introns. AmphiLysC possesses main features characteristic of the digestive c-type lysozyme such as lower number of basic amino acids (low pi values) and pH-optimum inacidic range. Moreover, AmphiLysC is predominantly expressed in the gut, and the gut lysozyme exhibits highest activity at pH 5.5. These indicate that AmphiLysC is mainly a digestive c-type enzyme. However, the ubiquitous expression of AmphiLysC in non-digestive tissues such as ovaries, testes, notochord, gill and muscle suggests that it may also play a non-digestive role like antibacterial defense. It is highly likely that AmphiLysC is an enzyme with a combined function of digestion and bacteriolysis. Phylogenetic anlysis shows that AmphiLysC is more related to invertebrate c-type lysozymes than its vertebrate counterparts, and g-type lysozyme is basal to c- and i-type lysozymes, favoring the notion that g-type lysozymes are structurally closest to the lysozyme ancestor.The third amphioxus cDNA, A mphiS19, encoding the ribosomal protein S 19, was isolated from the gut cDNA library of Branchiostoma belcheri tsingtaunese. The cDNA contains a 444 base pairs (bps) open reading frame (ORF), flanked by a 27 bp 5' untranslated region and a 138 bp 3' untranslated region. The ORF encodes a putative 147 amino acid protein with a calculated molecular mass of 16,222 Da. Alignment of the complete amino acid sequences of 14 eukaryotic S19 revealed that AmphiS19 exhibited >71.0% similarity to all known vertebrate homologues and <59.9% to those of the other eukaryotes including invertebrates. The phylogenetic analysis based on the amino acids of invertebrate and vertebrate S19 proteins showed that the amphioxus protein was at the base of a clade of vertebrate SI9 proteins, indicating that amphioxus is not only the sister group of extant vertebrates but also the basal lineage of chordates.