Movement of Cytoplasmic Proteins into Nuclei Induced to Enlarge and Initiate DNA or RNA Synthesis

In other studies it has been shown that somatic cell nuclei, which normally do not divide, are induced to enlarge and synthesize DNA when introduced into the cytoplasm of egg cells of Xenopus laevis. Introduction of such nuclei into the cytoplasm of large oocytes, however, causes nuclei to enlarge in a different way and to synthesize RNA but not DNA. In this study the proteins of eggs and oocytes were labelled with radioactive amino acids. Brain or blastula nuclei were then injected into cells containing labelled proteins under conditions in which protein synthesis was inhibited. Movement of cytoplasmic proteins was studied by observing the increase in acid-insoluble label over the injected nuclei by quantitative autoradiography. To prevent nuclear protein synthesis during the experiments, puromycin was injected with the nuclei. An estimation of the size of the labelled amino acid pool, a demonstration of the inhibitory effects of puromycin, and comparison of the amount of labelled material with and without puromycin all showed that protein synthesis in the nuclei played an insignificant role during the course of the experiments. A movement of acid-insoluble label from cytoplasm of egg cells into injected brain nuclei was noted even before they had begun to swell or synthesize DNA. In the initial period of nuclear enlargement there was a disappearance of the heterochromatic clumps characteristic of brain nuclei. This period coincided with a very rapid uptake of label to concentrations about twice that of the surrounding cytoplasm. A subsequent phase of nuclear swelling was characterized by a dilution of stainable nuclear material, loss of basophilia, and establishment of acidophilia of the nuclear contents. Cytoplasmic proteins continued to enter nuclei during this phase, but at a slower rate. Extraction of the soluble materials of labelled eggs with 0.01 M NaCl at pH 7.8 and a subsequent fractionation of the extract showed that there were many radioactive compounds present with molecular weights greater than 5000. The synthesis of DNA was initiated even before nuclear swelling could be detected, proceeding at least through the early stages of swelling. The induction of enlargement in blastula nuclei by oocyte cytoplasm containing labelled proteins was also accompanied by an uptake of label from the cytoplasm. In this case, although the uptake of unlabelled acidophilic material was much greater, the uptake of labelled proteins was much slower than that observed in the egg. The results are discussed in terms of chromosomal changes which occur during nuclear enlargement and during concomitant changes in nucleic acid metabolism.