Characterization, Expression And Evolution Of AmphiARF And AmphiApoD Genes From Amphioxus

Amphioxus or lancelet, a cephalochordate, is the extant invertebrate most closely related to the proximate ancestor of vertebrate, and has long been regarded as the model animal for insights into the evolution of vertebrate and the embryonic development. Its genetic information on gene sequence and expression pattern has been widely used for interspecies comparative genome studies and developmental homology analysis. In this paper, it is for the first time to report the cloning, characterization, phylogenetic analysis and expression analysis of AmphiARF and AmphiApoD from the gut cDNA library of amphioxus Branchiostoma becheri tisingtauense.The cDNA AmphiARF, encoding a novel member of ARF family was for the first time identified. It is 1483 bp long and contains an open reading frame encoding 227 amino acids. The deduced amino acid sequence has 64%, 61% and 61% identity to fungus ARF, fruit fly ARFII and human ARF5, respectively. It includes the motifs involved in GTP binding (DVGG, NKQD and SAK) and hydrolysis (GXDXXGK), and a glycine at position 2 for N-terminal myristoylation, which are all characteristic of ARFs. However, AmphiARF has a 46 amino acid C-terminal extension (positions 183-227), which was not found in any ARF family previously described including ARF-like proteins. This suggests that AmphiARF is a new member of ARF family, which was also supported by the phylogenetic analysis. The phylogenetic trees were constructed using the amino acid sequence of AmphiARF excluding the C-terminal 46 amino acids and those of 24 representative full-length ARF proteins, and 8 full-length ARF-like proteins by neighbor-joining and maximum parsimony method respectively. Both two constructed trees reveal that AmphiARF fails clustering with any of the three known classes I, II and III. Instead, it branchedoff the three classes and formed a distinct lineage in the tree. It is highly likely that the extended C-terminal of AmphiARF is a novel addition occurring after cephalochordate/vertebrate split. RT-PCR and Northern blotting analysis both reveal that AmphiARF transcript is present in all tissues examined including hepatic caecum, hind-gut, testis, ovary, gill, muscle, and notochord, consistent with the involvement of ARFs in the fundamental role like vesicular trafficking of proteins.This paper also reports the characterization and expression pattern of the amphioxus apolipoprotein D (AmphiApoD), which represents the first invertebrate member of the apolipoprotein D (ApoD) family. The cDNA is 730 bp long, and contains a 564 bp open reading frame corresponding to a deduced protein of 187 amino acids including a signal peptide of 21 amino acids. It has one potential asparagines-linked glycosylation site and four cysteine residues. Moreover, the two consensus sequences of the lipocalin superfamily are also found in the amphioxus protein and coincide with most conserved regions. The structure of AmphiApoD predicted by protein structure homology-modeling shows that the protein consists of an eight-strand antiparallel 3 -barrel, with a COOH-terminal a -helix. Intramolecular disulfide bond is also found in AmphiApoD between Cysteines 8 and 113. The aforementioned characterizations of the AmphiApoD structure are consistent with those of the ApoDs. Sequence and phylogenetic analysis based on AmphiApoD and 7 known vertebrate ApoDs indicate that AmphiApoD falls at the base of the vertebrate ApoD family. This suggests that AmphiApoD may be the archetype of the ApoD gene. The distribution of ApoD mRNA among amphioxus tissues was determined by Northern blot analysis, the AmphiApoD gene was found express in hind-gut, notochord, ovary and testes at different levels. The semi-quantitative RT-PCR also shows that AmphiApoD mRNA level can be regulated by the hormones. These indicate the role of ApoD as a lipid- or steroid hormone-binding protein. In situ hybridization reveals that AmphiApoDexpression is restricted to notochord, somites and primitive gut in neurulae and larvae, which implies that the expression of ApoD gene probably arose in the endomesoderm during embryogenesis in the last common ancestor of all chordates. This study may facilitate investigating the role of ApoD and the evolutionary origin of ApoD family.