Characterization, Expression And Phylogenetic Analysis Of SAHH Gene From Amphioxus Branchiostoma Belcheri

S-adenosylhomocysteine hydrolase(SAHH) is an enzyme which catalyzes the reversible conversion of S-adenosylhomocysteine(SAH) to homocysteine(Hcy) and adenosine(Ado).It plays a central role in the regulation of the intracellular levels of methylation.Amphioxus,a cephalochordate,has long been regarded as the ex(?)ant invertebrate most closely related to the proximate ancestor of vertebrates.Studies on the gene structure,function and expression in amphioxus will contribute to the understanding of the origin and evolution of the vertebrates.The cDNA clone encoding an amphioxus S-adenosylhomocysteine hydrolase (AmphiSAHH) protein was isolated from a gut cDNA library of Branchiostoma belcheri tsingtaunese.It contained a 1305 bp open reading frame corresponding to a deduced protein of 434 amino acids with a predicted molecular mass of approximately 47.8 k(?)a. The deduced amphioxus SAHH protein has 67.3-82.1%identity with its homologues from a variety of organisms including microbes,invertebrates and vertebrates.Phylogenetic analysis showed that vertebrate and invertebrate SAHH proteins are each grouped together, with AmphiSAHH falling at the base of vertebrate SAHH clade,suggesting that the divergence of vertebrate and invertebrate SAHH gene probably occurs prior to the split of invertebrate/vertebrate from a common ancestor around 550 million years ago,the AmphiSAHH might be the archetype of the vertebrate SAHH protein.The whole moun(?) in situ hybridization demonstrates a tissue-specific expression pattern of AmphiSAHH in developing embryos and larvae.The genomic DNA sequence of AmphiSAHH contained eight exons and seven introns,which was similar to B.floridae and sea urchin SAHH exon-intron organization.Sequence comparison suggested the evolutionary appearance of the ten-exon-nine-intron organization of SAHH genes after the split of invertebrate/vertebrate,which is highly conserved since then.The adenosine-induced inhibition of SAHH can improve the gene expression level of AmphiSAHH.AmphiSAHH has been successfully expressed in Escherichia coli and purified. Binding constant and enzymatic activity was confirmed by biochemical assay.Western blot and immunohistochemistry analysis confirmed that amphioxus has a native molecu(?)ar mass of approximately 48 kDa,and SAHH was strongly expressed in hepatic caecum,g(?)ll, spermary and ovary of Amphioxus.