Ed fish. Phylogenetic analysis divided these genes into 3 clusters (Fig. 1). Cluster 1 was composed of human SSAT2, mouse Ssat2, and invertebrate ssat-like genes. Genes in clusterThree Zebrafish ssat1 GenesFigure 2. Primary structures of zebrafish family of Ssat1 proteins and the constructs of chimeric proteins used in this study. (A) The amino acid sequences of human SSAT1 and zebrafish family of Ssat1 proteins were aligned by MegAlign (Lasergene) with the ClustalW method. The conserved Title Loaded From File residues are shaded black. The secondary structures are denoted according to the structure of human SSAT1 [33]. (B) In each chimeric construct, fragments from Ssat1a are labeled in blue and fragments from Ssat1b are in red. Nucleotide positions and the corresponding amino acid residues are labeled on the top and the bottom of each construct, respectively. doi:10.1371/journal.pone.0054017.gwere ssat2 orthologues from ray-finned fish. At least 2 ssat2 homologous genes were found in all ray-finned fish species analyzed in this study. These ssat2 homologues were furtherdivided into 2 sub-groups that suggested an early duplication event at ssat2 in the common ancestor of ray-finned fishes. Cluster 3 included human SSAT1 and its ML 264 web cognate genes from vertebrates.Three Zebrafish ssat1 GenesCompared with the first 2 clusters, the ssat1 orthologues were more closely conserved. No ssat1 orthologue was found in invertebrates and only 1 ssat1 was found in most vertebrates except that there were 3 ssat1 homologues in zebrafish. The encoded amino acid sequences (Fig. 2A) and cDNA sequences (Fig. S2) of zebrafish ssat1 homologous genes were highly similar to each other, and they were clustered together in the phylogenitic analysis (Fig. 1). Human SSAT1 is located on the X chromosome between the genes for peroxiredoxin 4 (PRDX4), acryl-CoA thioesterase 9 (ACOT9), and apolipoprotein O (APOO) (Fig. S1). The Ssat1 genes of evolutionarily distant vertebrates including medaka, stickleback, takifugu, and tetraodon are located between acot9 and apoo (data not shown). One of the zebrafish ssat1-like genes (NM_001093748) is also located between acot9 and apoo on chromosome 24; we therefore named it ssat1a. The other zebrafish genes (NM_001030199 and NM_001002169) are closely clustered together and located next to prdx4 on chromosome 5. We named them ssat1b and ssat1c, respectively. The ssat-like genes of invertebrates and ssat2 homologous genes of vertebrates are not grouped like ssat1 (Fig. 1) and their genomic localization also differ (data not shown).The Expression Pattern of Zebrafish ssat1 Homologous GenesThe expression patterns of zebrafish ssat1 genes were analyzed by RT-PCR. During normal embryogenesis, ssat1c mRNA was the most abundant in every stage and was stably expressed from 12 to 96 hours post fertilization (hpf). The mRNA of ssat1a and ssat1b were not detected until 24 hpf (Fig. 3A, control). A previous study indicated that treatment of human cells with DENSPM, a spermine analog, enhances SSAT1 expression up to 20 fold [31]. Another group of zebrafish embryos were developed with 10 mM DENSPM added immediately after fertilization. All embryos survived and displayed no obvious abnormalities through 96 hpf. Neither the expression nor mRNA abundance of these ssat1 genes was changed (Fig. 3A, DENSPM). The expression profiles of zebrafish ssat1 genes in the major organs of adult fish were also studied. ssat1a mRNA was mainly expressed in the heart, spleen and kidney, and.Ed fish. Phylogenetic analysis divided these genes into 3 clusters (Fig. 1). Cluster 1 was composed of human SSAT2, mouse Ssat2, and invertebrate ssat-like genes. Genes in clusterThree Zebrafish ssat1 GenesFigure 2. Primary structures of zebrafish family of Ssat1 proteins and the constructs of chimeric proteins used in this study. (A) The amino acid sequences of human SSAT1 and zebrafish family of Ssat1 proteins were aligned by MegAlign (Lasergene) with the ClustalW method. The conserved residues are shaded black. The secondary structures are denoted according to the structure of human SSAT1 [33]. (B) In each chimeric construct, fragments from Ssat1a are labeled in blue and fragments from Ssat1b are in red. Nucleotide positions and the corresponding amino acid residues are labeled on the top and the bottom of each construct, respectively. doi:10.1371/journal.pone.0054017.gwere ssat2 orthologues from ray-finned fish. At least 2 ssat2 homologous genes were found in all ray-finned fish species analyzed in this study. These ssat2 homologues were furtherdivided into 2 sub-groups that suggested an early duplication event at ssat2 in the common ancestor of ray-finned fishes. Cluster 3 included human SSAT1 and its cognate genes from vertebrates.Three Zebrafish ssat1 GenesCompared with the first 2 clusters, the ssat1 orthologues were more closely conserved. No ssat1 orthologue was found in invertebrates and only 1 ssat1 was found in most vertebrates except that there were 3 ssat1 homologues in zebrafish. The encoded amino acid sequences (Fig. 2A) and cDNA sequences (Fig. S2) of zebrafish ssat1 homologous genes were highly similar to each other, and they were clustered together in the phylogenitic analysis (Fig. 1). Human SSAT1 is located on the X chromosome between the genes for peroxiredoxin 4 (PRDX4), acryl-CoA thioesterase 9 (ACOT9), and apolipoprotein O (APOO) (Fig. S1). The Ssat1 genes of evolutionarily distant vertebrates including medaka, stickleback, takifugu, and tetraodon are located between acot9 and apoo (data not shown). One of the zebrafish ssat1-like genes (NM_001093748) is also located between acot9 and apoo on chromosome 24; we therefore named it ssat1a. The other zebrafish genes (NM_001030199 and NM_001002169) are closely clustered together and located next to prdx4 on chromosome 5. We named them ssat1b and ssat1c, respectively. The ssat-like genes of invertebrates and ssat2 homologous genes of vertebrates are not grouped like ssat1 (Fig. 1) and their genomic localization also differ (data not shown).The Expression Pattern of Zebrafish ssat1 Homologous GenesThe expression patterns of zebrafish ssat1 genes were analyzed by RT-PCR. During normal embryogenesis, ssat1c mRNA was the most abundant in every stage and was stably expressed from 12 to 96 hours post fertilization (hpf). The mRNA of ssat1a and ssat1b were not detected until 24 hpf (Fig. 3A, control). A previous study indicated that treatment of human cells with DENSPM, a spermine analog, enhances SSAT1 expression up to 20 fold [31]. Another group of zebrafish embryos were developed with 10 mM DENSPM added immediately after fertilization. All embryos survived and displayed no obvious abnormalities through 96 hpf. Neither the expression nor mRNA abundance of these ssat1 genes was changed (Fig. 3A, DENSPM). The expression profiles of zebrafish ssat1 genes in the major organs of adult fish were also studied. ssat1a mRNA was mainly expressed in the heart, spleen and kidney, and.
