Mutations induced in amoeba proteus by the carcinogen N-methyl-N-nitrosourethane

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Abstract
Mutations have been induced in single amoebae by treatment with the powerful mutagen, N-methyl-N-nitrosourethane. Three mutants, which differ visibly from the parent strain to the extent that individual amoebae are recognizable and change of characteristics from normal to mutant or mutant to normal can be followed in single cells, are described. These mutant strains have been cultured in the laboratory, using Tetrahymena feeding, without loss of mutant characteristics for over 2 years, undergoing some 100 or more cell cycles. Hybrids made between mutants and the parent strain, using the nuclear transfer technique, are viable and stable. They showed that in all cases crosses between normal and mutant amoebae retained the characteristics of the mutant only if the mutant nucleus was present. Though amoebae from 2 of the mutant strains may revert to normal form with increase in temperature or change to a wheat culturing technique, these ‘apparent normal’ amoebae resume the mutant form on a return to the temperature and feeding conditions normally used. The interaction between mutant and control amoebae was observed in heterokaryons over periods of up to 6 weeks by cutting off pieces of the heterokaryon cytoplasm at regular intervals to prevent division while allowing growth. In general the presence of a control nucleus led to the development of normal characteristics whether the heterologous nuclei were contained in control or mutant cytoplasm. No antagonistic action occurred between mutant and control nuclei when sharing the same cytoplasm. Study of the clones formed by the offspring of heterokaryon divisions showed the mutant and control nuclei unaltered by long periods of close contact. These experiments indicate the chromatin as the site of action of the mutagen. A possible mode of action of the mutagen in amoebae, relating to the functional state of the DNA, is suggested.