Identification And Tissue-specific Expression Of A Promethin-like Homolog In Amphioxus Branchiostoma Belcheri

Amphioxus or lancelet, a cephalochordate, has long been regarded as the living invertebrate most closely related to the proximate invertebrate ancestor of vertebrates. 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, we report the cloning and tissue-specific expression of promethin-like gene from amphioxus Branchiostoma belcheri.The B.belcheri promethin-like cDNA obtained from the gut cDNA library of Branchiostoma belcheri tsingtaunese is 1014 bp long. The longest open reading frames code for 167 amino acids with predicted molecular mass of approximately 18 KDa. B.belcheri promethin-like has no putative conserved domains. Vertebrate promethins had the similar identity (20-29%) to B.belcheri promethin-like at the amino acid level. No particular vertebrate promethins had markedly higher or lower sequence identity to B.belcheri promethin-like. Primary structural examination showed that the deduced promethin-like homolog was a transmembrane protein with three potential transmembrane helices, resembling the vertebrate promethins. Phylogenetic tree analysis showed that B.belcheri promethin-like located at the base of the vertebrate counterparts, and this is in agreement with the notion that amphioxus is the ancestor of the vertebrates. These suggest that B.belcheri promethin-like may represent the archytype of vertebrate promethins, and the vertebrate promethins including human promethin underwent little divergence after the split of invertebrate/vertebrate from a common ancestor around 550 million years ago.Northern blotting revealed that an approximately 1014 bp B.belcheri promethin-like transcript was detected in the gill, hepatic caecum, hind-gut, testis and ovary. To further explore the expression pattern of promethin-like in adult B. belcheri, tissue section in situ hybridization was conducted. It was found that promethin-like transcripts were present in the hepatic caecum, hind-gut, gill, neural tube, testis and ovary, consistent with the Northern blotting results. No signal was observed in the epidermis, muscle and notochord. These show that promethin-like is expressed in a tissue-specific manner, being in line with the tissue-specific expression of the mammalian promethins.Whole-mount in situ hybridization and RT-PCR results showed that no hybridization signals for B.belcheri promethin-like were detected in the early embryos and larvas.