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Tol1 is an active DNA-based transposable element residing in the genome of the medaka fish Oryzias latipes. This element belongs to the hAT transposable element family, of which complete copies have relatively long sequences. In addition, we found that Tol1 elements as long as 18 and 20 kb occur in the medaka fish genome. These facts suggest that Tol1 is suitable for carrying large DNA fragments as a gene transfer vector. Focusing on this, we conducted two kinds of manipulations of the element. The first was to eliminate internal regions dispensable for transposition. It was revealed that a Tol1 element consisting of 157-bp left- and 106-bp right-terminal regions could be transposed without a loss of transposition efficiency. Next, we prepared long Tol1 elements by incorporating unrelated DNA fragments into this short Tol1 clone and examined their transposition efficiencies. The transposition frequency decreased as the element size increased. The longest Tol1 element we examined measured 22.1 kb, and its transposition frequency was approximately one fifth that of a 2.1-kb element. However, this frequency was still significantly higher than that of a random integration of DNA into the chromosomes. The element size of 22.1 kb is the longest ever reported for DNA-based elements currently used for mammals. Thus, Tol1 is a superior gene-transfer vector with a large cargo capacity.

DNA-based transposable elements can be used as tools for gene engineering and gene therapy. A great advantage over RNA-mediated elements and retroviruses is the simplicity and safety of usage. The Tol1 element of the medaka fish Oryzias latipes has structural features of DNA-based elements. Although its excision has already been demonstrated, de novo insertion has not been observed, and a transposase has not been hitherto identified. We first cloned, through in silico search alignments and genomic library screenings, a 4.4-kb Tol1 copy carrying open reading frames and then identified, by mRNA analysis, a 2.9-kb transcript coding for 851 amino acids. The protein product of this transcript catalyzed transposition of a nonautonomous Tol1 copy in human and mouse culture cells. This identification of a fully functional Tol1 transposase could lead to the development of new tools for basic and translational molecular biology applications in mammals.

The medaka fish albino mutant, i(1) is one of the Tomita collection of medaka pigmentation mutants which exhibits a complete albino phenotype, because of inactivation of the tyrosinase gene due to insertion of a transposable element, Tol-1. Recently, mosaic black-pigmented i(1) medaka fish have arisen in one of our laboratory breeding populations. Their pigmented cells have been observed in all of the tissues, including the eye and skin, in which melanin is detectable in the wild type. In this study, we analyzed the tyrosinase gene of revertants and showed Tol-1 to have been precisely excised from the gene, suggesting a causal relationship. Mosaic patterns of pigmentation indicate spontaneous somatic excision of the element from the tyrosinase gene. To our knowledge, this is the first transposable element with somatic excision activity demonstrated phenotypically in vertebrates. The pattern of pigmentation in mosaic revertants indicates frequencies of melanin pigments to be consistent with the numbers of melanophores per unit area of body sites, such as the eyes, head and dorsal trunk.

Liver tumor nodules were induced by the administration of diethylnitrosamine [(DENA) CAS: 55-18-5; N-nitrosodiethylamine] to the fish, Oryzias latipes. A histochemical study showed decreased ATPase and glucose 6-phosphatase (Glc-6-Pase) activities in most of the tumor nodules. Increased ATPase staining and, occasionally, Glc-6-Pase staining were also observed. In some nodules, the distribution of bile canaliculi was disordered, indicating positive ATPase activity. Basophilic and eosinophilic nodules could be discriminated by histologic examination. The observations on serial histochemical and histologic sections revealed extreme heterogeneity in the phenotypes of the nodules. Measurement of the enzyme-altered areas indicated that the development of nodules was more prominent in male than in female fish. Experiments with sex-reversed fish, XX males and XY females, suggested that the sex difference in the susceptibility to DENA does not result from the difference in sex chromosomes but from the difference in sexual phenotypes.

When the medakas, Oryzias latipes, were treated with diethylnitrosamine (DENA) for 6-8 weeks, large liver tumors grew in many of them at 11-13 weeks. In the fish with fully developed tumors, some aneuploid hepatic cells, detected by DNA-Feulgen cytofluorometry, were already found after 3-6 weeks of the DENA treatment. The allogeneic transplantation of liver grafts into eye chambers showed that the grafts obtained from DENA-treated or tumor-bearing fish could survive for longer periods than those from untreated fish. Effects of temperature on liver tumorigenesis were examined at 5-8 degrees C and at 22-25 degrees C. When the fish were treated with DENA at a high temperature, small tumor foci appeared during the treatment, and they grew to large tumors if the fish were kept at a high temperature afterward. Most of the fish had no tumors with the treatment given at a low temperature. Only a few tumors were found in the fish treated with DENA at a low temperature and then kept at a higher one. In the measurement of the labeling and mitotic indexes of liver cells, DNA synthesis and cell divisions occurred during the DENA treatment at a high temperature. A close relationship between cell proliferation and appearance of the histological changes in liver tissue was observed. Three to 4 days after a partial hepatectomy was performed on each medaka, mitotic figures of liver cells were frequently observed, and the maximum mitotic index of more than tenfold the control level was obtained at 4 days. The enhancing effect of the partial hepatectomy on the liver tumorigenesis by DENA treatment was examined.