The use of Pufferfish and Zebrafish as Model Organisms for Genomic Structure Analysis and Identification of Novel Genes

The round-spotted pufferfish (Tetraodon fluviatilis) had the genome size of 380 Mb which is the smallest of any vertebrate. We used this organism for complete determination of the genomic structure and characterization of the promoter region of disease-related genes (ret and Menkes-related genes) and all JAK/STAT genes. All pufferfish JAK kinase genes are composed of 24 exons except JAK1 gene, which has an additional exon. A comparison of the exon-intron organization of these genes reveals that all splice sites of JAK kinase genes are nearly indentical, suggesting that the pufferfish JAK kinase genes may evolve from a common ancestor. The putative promoter regions of four JAK kinase genes were tested either in a carp CF cell line or in zebrafish embryos by using CAT or lacZ as reporter genes. Both assays confirmed the transcriptional activities of these promoters in vitro and in vivo. We have also completely determined the genomic structure of seven STAT genes. Currently, we have constructed the constitutively active form of different STAT cDNAs and are analyzing their DNA binding and transactivation activity in various cell lines. At present, we are trying different approaches such as the generation of transgenic zebrafish that carried the constitutively active STAT or microinjection of constitutively active STAT mRNA into one-cell stage zebrafish embryos to test its effect in vivo. We have also cloned and characterized the mnk gene that encodes the copper transporter of the roundspotted pufferfish. The mnk gene is composed of 23 exons spanning about 12.5 kb comparing to ＞150 Kb in human. The intron-exon junctions are all highly conserved between the round-spotted pufferfish and human. By comparing the amino acid sequence, the pufferfish MNK protein shows 62.1% identity to the corresponding human Menkes disease gene product. In addition, we are working on some novel intron-less protein kinases and one brain-specific protein kinase that may involve in the development of zebrafish nervous system. Some techniques, such as wholemount in situ hybridization, transgenic zebrafish, and microinjection of RNAi/double-stranded RNA into zebrafish embryo have been established and used to study the possible function of these protein kinases.

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