To investigate the accuracy of retroviral in vitro DNA replication we have examined with two fidelity assays the reverse transcriptases (RTs) from SIVagm, HIV-1, MoMLV as well for comparison the Klenow fragment from E. coli and DNA polymerase a from calf-thymus. These forward mutation assays measured the loss of bacteriophage M13 lacZa gene function by mutations. In the EnvlacZa assay frameshift mutations occurring during polymerisation of a 176 b long simian immunodeficiency virus (SIV) envelope (env) sequence were phenotypically detected by blue/white-plaque screening. To measure in addition substitutions, a 116 b long M13 lacZa gene DNA template was used as the mutational target (LacZa assay). With the SIVagm env gene DNA template, we observed similar levels of frameshift fidelity for all three RTs. Nevertheless, the SIVagm RT was slightly more accurate than the other RTs and nearly all frameshifts were observed at two homopolymeric runs of its homologous template. Measuring also substitution errors at the lacZa template the mutation frequency of the SIVagm RT increased 2.5 fold and that of the HIV-1 RT was enhanced by a factor of 3.

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...

Eingegangen am 9. Februar 1962 2.4-Diacetyl-phloroglucin liefert mit Natriummethylat/Methyljodid bis zu 35 % d. Th. 3.5-Diacetyl-filicinsaure, die durch Wasserdampfdestillation abgetrennt wird. Der Abbau mit verd. Salzsaure ergibt Filicinsaure mit einer Ausbeute von 25 % (bez. auf Phloroglucin).Filicinsaure (I), ein Baustein der Filixkorper3-4), ist nach mehreren Verfahrens-7) aus aliphatischem Material synthetisiert worden. Praparativ ergiebiger war die Kern-

The reverse transcriptase (RT) was partially purified by a newly developed procedure from the simian immunodeficiency virus TYO-7 isolated from an African green monkey (SIVagmTYO-7). The method comprised lysis of the virus with nonionic detergent followed by two centrifugations in isopycnic sucrose density gradients and one velocity sedimentation in a glycerol gradient. The enzyme exhibited a purity of 70-80% and showed an exceptional high specific activity of 135 nmol incorporation of dTMP per milligram of protein in 1 h with poly(rA).oligo(dT) as template-primer (TP). The molecular weight of the native enzyme was estimated by velocity sedimentation analysis as 120K-130K. Investigation of the RT by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that the active enzyme is a heterodimer composed of a 64- and a 50-kDa subunit. The two subunits were identified to be RT specific by Western blot analysis. In activity gels, both subunits exhibited enzymatic activity, whereby the 64-kDa subunit showed the predominant activity. The RT preferred the TP poly(rA).oligo(dT) over poly(rC).oligo(dG). With poly(rCm).oligo(dG), only marginal activity was detected, and no activity was measured with poly(dA).oligo(dT). The TP specificity was influenced by the reaction temperature. The highest activity was measured around the melting temperature of the TP used. Furthermore, the enzyme activity was more thermolabile when measured with poly(rA).oligo(dT) than with poly(rC).oligo(dG). To compare the specificity of RT inhibitors, their inhibition efficiency (IE) was defined as the ratio of the 50% inhibiting concentration (ID50) obtained with the RT in viral lysates to the ID50 of purified RT.