The chemicals 9,10-dimethylbenzanthracene (DMBA), ethionine, daunorubicin, actinomycin D, l-(2-chloroethyl-1)-nitrosourea (CCNU), steroids, croton oil and dimethylsulfoxide (DMSO) were used in order to correlate their effect on the in vitro synthesis of normal and cancer DNA, on DNA strand separation and on accelerated in vivo multiplication of cancer cells. All of the compounds tested strongly stimulate the synthesis of cancer DNA in vitro catalyzed by DNA-dependent DNA polymerase I and measured as an acid-precipitable labeled product. Under the same conditions, the synthesis of DNA originating from healthy tissues is only slightly enhanced, except in the case of croton oil and DMSO. These substances are almost equally active on cancer and normal DNA. Although both cancer and normal DNA contain a large amount of double-stranded regions, the extent of DNA strand separation measured by the increase in UV absorbance (hyperchromicity) in the presence of each compound tested is much higher for all cancer DNA than for corresponding normal DNA. In contrast, DMSO and croton oil do not appear to distinguish cancer DNA from normal DNA. Additive and differential effects of various compounds on cancer DNA strand separation can be observed. Small doses of DMBA and CCNU stimulate the multiplication of Ehrlich ascites tumor cells in vivo in mice. There is thus a possible correlation between DNA strand separation, DNA synthesis, multiplication and differentiation of cancer cells in the presence of the above compunds, which is different from the response of normal cells to these compounds.

In a mutant of Escherichia coli resistant to showdomycin, both the 50S and 30S ribosomal subunits contain RNA species in which the purine concentration greatly exceeds that of pyrimidines. The same is true for total rapidly-labeled RNA. The modified ribosomal RNA hybridizes poorly with homologous DNA, which is apparently unchanged in base composition. Acrylamide gel electrophoresis of mutant ribosomal proteins shows a highly altered protein pattern for both ribosomal subunits, although the activity of these ribosomes is not decreased.In bacteria, the ribosomes play an important role in the translation mechanism for protein synthesis (1) and constitute the primary site of action of several antibiotics (2-6). Each ribosomal subunit, 50S and 30S, has a specific functional role essentially linked to a group of proteins (7,8), whose synthesis is under genetic control (9-12). Structural genes for ribosomal proteins may be the loci conferring sensitivity (12, 13) to and dependence (14) on antibiotics. Thus, it has been shown that certain antibiotics provoke the alteration exclusively of a single protein in 50S ribosomal subunits, while others affect a single protein of the 30S subunits. In a mutant of E. coli resistant to erythromycin or lincomycin, a single protein associated with 50S ribosomal subunits seems to be altered (4,15), while in the case of resistance to streptomycin (11,14) and spectinomycin (5) a single protein of the 30S subunits is functionally modified. The biological activity of ribosomes seems to depend on the presence of the correct ribosomal proteins while the ribosomal RNA is needed for the assembly of these proteins (16).We have previously shown that showdomycin, a naturally occurring "nucleoside" (17, 18), rapidly provokes in E. coli (19,20) Ribosomal RNA (23S, 16S, and 5S) was isolated by the phenol method and separated as described (20). After alkaline hydrolysis (KOH, 0.5 N, 18 hr at 370C), the nucleotides were analyzed using a Dowex 1 X 2 column, -200-400 mesh (20). For base-ratio analysis, 1 mg of each type of RNA was used.

Transforming RNA excreted by s howdomycin-resistant Escherichia coli induces a persistent, heritable, and spectacular change in Agrobacterium tumefaciens B6, a bacterium that carries the oncogenic principle for tumor induction in plants. Transformants possessing new physiological and biochemical properties have completely or partially lost the capacity for tumor induction. They synthetize new ribosomes whose components are profoundly modified. On the basis of biological and biochemical characteristics, one is inclined to consider the completely transformed Agrobacterium tumefaciens as a new species".The production by Escherichia coli of several RNA species in which the concentration of purines exceeds that of pyrimidines is promoted by showdomycin (1, 2). Mutants resistant to this antibiotic synthesize altered RNAs that are no longer complementary to DNA (2). In these mutants ribosomal proteins and several enzymes have been shown to be greatly modified (1, 2). Some possible mechanisms have been proposed (2) to account for sudden appearance of altered RNAs in the presence of showdomycin. One of these predicted the existence in E. coli of an "RNA episome" having purine nucleotides in excess (2, 3). Further investigations confirmed the existence of such an "RNA episome" that is associated with DNA (3). While in wild-type E. coli, this "RNA episome" is apparently not functional and does not dissociate from DNA under normal physiological conditions, it appears to dissociate in the showdomycin resistant (Shor) mutant, since this strain excretes a transforming RNA (3). In brief communications, we have demonstrated the transforming capacity of extracellularly released RNA from E. coli Shor and that of the RNA episome from wild-type bacteria (3). In the present report, we describe the transformation of Agrobacterium tumefaciens by excreted transforming RNA from E coli Shor, accompanied by drastic alteration of the physiological, biochemical, and oncogenic properties of A. tumefaciens. EXPERIMENTAL AND RESULTSIsolation and purification of transforming RNA from E. coliMutants of E. coli ML 30 Shor were obtained as described for E. coli Hfr H M500 Shor (2). The transforming RNA excreted by this mutant was isolated and purified from growth medium as described (2). Among excreted RNA fractions, only those having S values between 5.5 and 6.5 possess the transforming capacity (Fig. 1)

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Carcinogens: 7,12-dimethylbenz(a)anthracene (DMBA), 2-acetylaminofluorene (AAF), 3-methylcholanthrene (MC), daunorubicin, thio-TEPA, cyclophosphamide, l-(2-chloroethyl)2-cyclohexyl-1 -nitrosourea (CCNU) and mitomycin C, which mutate Salmonella typhimurium tester strains in the Salmonella/’microsome mutagenicity test, strongly stimulate the in vitro synthesis of DNA isolated from His” mutants (TA-1538, TA-1537, TA-1535), and induce in vitro His^- DNA strand separation. Four other carcinogens, ethionine, actinomycin D, bleomycin and reserpine, which were not known as mutagens in the Ames test, also strongly stimulate His^– DNA synthesis and His^– DNA strand separation in local areas. Those substances which are neither carcinogenic nor mutagenic: d-lactose, fluorene, cholesterol, and saccharin, do not stimulate either His^– DNA (or His⁺ DNA) synthesis or DNA in vitro strand separation. Steroid hormones, which are carcinogenic only for steroid target tissues, do not react with bacterials DNA.