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The development of cadmium resistance in an Indian muntjac cell line has been investigated. The parent cell line is highly sensitive to cadmium ions. Resistance was obtained by continuous growth of cells in low levels of cadmium with stepwise increments. Four cell lines were developed with resistances of between 50- and 200-fold greater than that of the parental line. Early in the development of resistance an unstable cell line displaying extensive chromosomal rearrangement and an elevated sister chromatid exchange frequency was identified. The more stable resistant lines produced from this original cell line have normal karyotypes. Having passed through the initial period of genome rearrangement the resultant cells acquired several characteristics of morphologically transformed cells. It is concluded that long-term exposure to low levels of cadmium can transform cells in vitro concurrently with their acquiring cadmium resistance.

Amoeba proteus was exposed to a low concentration of methyl di-(β-chloroethyl) amine (MBA, a ‘nitrogen mustard’). Subsequent nuclear transfer was used to study the relative sensitivities of nucleus and cytoplasm to MBA. Nuclei were found to be 10 times as sensitive as cytoplasm. Exposure to MBA caused a short delay before the first division of the nucleus and a longer delay before the second division, the delay before the first division being more marked with exposure to LD100 or higher concentrations. The first cytoplasmic division was delayed after treatment but subsequent division showed little change. MBA caused many abnormalities in division. When cytoplasm was injured the nuclei became swollen or, with less severe damage, divided without subsequent cytoplasmic division. When only nuclear damage was present, such abnormalities as incomplete prophase, non-nucleate division, uneven division, and cytoplasmic fragmentation occurred.

The technique of nuclear tranafer waa used to study the extent to which X-rays damage nuclei and cytoplasms of Amoeba proteus. Results showed that cytoplasm was directly damaged by the radiations. Cytoplasmic damage with doses below 280,000 r was reversible, though damaged cytoplasm could lethally damage normal nuclei. Damage with doses above 300,000 r caused lethal damage and death occurred without division in 0-3 days. Damage to nuclei was probably a combination of a direct action of the radiations and exposure to reversibly damaged cytoplasm. Nuclei were 2.4 times so sensitive as cytoplasms, the LD50 doses being approximately 120,000 r for nuclei and 290,000 r for cytoplasm. Death due to nuclear damage generally occurred without division within 3 to 6 weeks. Nuclear damage cannot be interpreted in terms of the ‘target’ theory.

By means of the nuclear transfer technique for amoeba, combinations of nuclei and cytoplasms from all parts of the cell cycle were available for examining the individual roles of the nucleus and cytoplasm in nuclear DNA replication. Neither 5-phase nor division sphere cyto-plasm proved capable of initiating a new round of nuclear DNA synthesis in the G2 nucleus. There was some indication that G2 nuclei which were transferred into early prophase cells, i.e. before the formation of a regular division sphere, did incorporate more [3H]thymidine than control G, nuclei. Positive proof of the induction of DNA synthesis in ‘immature’ nuclei was observed in only two cases. When young G2 nuclei were transplanted into late G2 amoebae, the addition of the donor nucleus generally resulted in the older nucleus being held in a late G2 phase until the younger nucleus passed through its G2. Division of 90 % of heterophasic homokaryons was synchronous, with a subsequent synchrony of DNA synthesis. A study of variance in [3H]thymidine incorporation by S nuclei sharing the same cytoplasm – using binucleate, trinucleate and multinucleate homokaryons — showed that nuclei through the peak-5 period synthesized DNA at approximately similar rates. The large differences in PHjthymidine incorporation by nuclei of amoebae of equal age appear due to differences in endogenous precursor pools. These would vary both with differences in food intake and with the draining of remote precursor pools for simultaneous cellular activities, particularly RNA synthesis. When sharing the same cytoplasm nuclei in peak 5 incorporated similar amounts of [3H]thymidine. Though cytoplasm did not influence the progress of DNA replication by a nucleus, it did influence the use of exogenous [3H]thymidine by the cell, and in so doing caused much of the variation observed in the labelling of nuclei during S. Nuclei sharing the same cytoplasm, and so subject to the same precursor pool changes, incorporated similar amounts of exogenous thymidine. Once DNA synthesis had been initiated it continued to completion regardless of the cyto-plasm which surrounded it. Thus neither the maintenance nor termination of DNA synthesis required a special cytoplasmic state.

The viability of Amoeba proteus cytoplasm was tested at 1-to 2-day intervals throughout the 14 days following enucleation. Viability was demonstrated by renucleating the anucleate cytoplasms and observing whether the resultant amoebae could resume normal activities such as moving, feeding and dividing. Four lines of A. proteus were studied, approximately 200 transfers being made on amoebae of each line. The introduction of a nucleus into anucleate cytoplasm initiated a temporary reactivation followed by a period of relative inactivity before either death by cytolysis or permanent reactivation with feeding, growth and division. Though approximately 70 % of anucleate cytoplasms were still viable 6 days after enucleation, temporary reactivation disappeared about this time and the period of relative inactivity which generally followed it increased in duration from a few days to several weeks. Few amoebae which were able to divide failed to form clones. Anucleate cytoplasms of wheat-cultured amoebae remained viable for longer periods than anucleate cytoplasms of tetrahymena-fed amoebae.

DNA synthesis has been investigated in Amoeba proteus by pulse-labelling cells of known age with [3H]thymidine. Ninety per cent of the DNA so labelled was synthesized during the first quarter of the cell cycle. Synthesis was in two peaks: the first occurring between 0·5 and 4 h after division and the second at about 10 h. All cells labelled at both peaks. The authenticity of the second peak was proved statistically. Considerable variation was observed among amoebae of similar age. In experiments in which daughter amoebae underwent different treatments, differences in the rates of incorporation of [3H]thymidine due to differences in the nutrition of the cells were found to be a predominant cause of variation. Heavy feeding reduced labelling; starvation increased labelling while at the same time reducing variability.

Mutations have been induced in single amoebae by treatment with the powerful mutagen, N-methyI-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.