DNA polymerases have been isolated from muscle and melanoma tissues of Xiphophorus, which are similar to retroviral RNA-dependent DNA polymerases as they prefer RNA to DNA templates. They appear to associate with submicroscopic structures which exhibit a density of about 1.13 g/ml after sucrose-density-gradient centrifugation. The RNA-dependent-DNA-polymerase-like enzymes could be separated from the DNA-dependent DNA polymerases by DEAE-cellulose chromatography. Further purification on phosphocellulose revealed that the muscle enzyme eluted at the void volume and at about 0.6 M KCl, whereas most of the melanoma enzyme eluted at 0.1 M KCI.Comparison of the template primer specificities of the muscle and melanoma enzymes with those of known DNA polymerases showed obvious similarities to the RNA-dependent DNA polymerase isolated from Rous sarcoma virus.Since 1928, the Xiphophorus fish system has been employed as an animal model for genetic research of melanoma [l -31. The ability to develop melanoma is inherited by Xiphophorus maculutus through a gene which has been designated 'tumor gene'. If Tu of X . maculutus is introduced into the genome of Xiphophorus helleri, rapidly growing melanomas develop spontaneously (for review, see [4]). During the course of our studies on these tumors, we detected an enzyme exhibiting properties of RNA-dependent DNA polymerase [5, 61.RNA-dependent DNA polymerase was first detected in retroviruses by Temin [7] and Baltimore [S]. The enzyme transcribes RNA into DNA. In the late seventies, RNA-dependent-DNA-polymerase-like activities were found in the placentas of monkeys [9], humans [lo] and rabbits [ l l ] as well as in the embryos of geese [12], Japanese quail [13] and humans [14]. Because of the ubiquity of proviruses in vertebrate genomes, it was difficult to decide if the enzymatic activity was caused by a provirus or by a cellular gene. In the last six years, a plethora of data has lent support to the hypothesis that some RNA-dependent DNA polymerase activity may be independent of retroviruses.
MATERIALS AND METHODS
Fish and tissuesMelanoma-bearing xiphophorine fish were produced continuously by introgressive breeding [4], that is by oncogene transfer from the platyfish to the swordtail genome (Fig. 1). The tissue studied were muscle from the backcross generation 23 hybrids G and H and malignant melanomas from the backcross generation 23 hybrid F. All animals were raised at 2 5 T , with a day/night rhythm of 12 h/12 h and Tetramin as the standard food [26].Template primers (dG),,-18 and Escherichiu coli 5s RNA served as RNA templates. Poly(dC)p(dG)12-18, poly(dA)p(dT)12 -18 and activated salmon sperm DNA [27] served as DNA templates. Terminal transferase was tested with poly(dT)lzPoly(C)p(dG)l2-18, poly(A)p(dT)12-18, poly(mC)p- [28].
DNA polymerase assayThe DNA polymerase reactions were performed at 30°C for 2 h. The reaction mixture comprised a total volume of 0.05 ml and contained 100 mM Tris/HCl pH 8.4,50 mM KCI, 0.8 mM MnCl,, 4 mM dithiothreitol, and 2 pg of the respective...