Identification of key regulated events early in the life of hybrid animal cells constructed by nuclear transplantation.

Reconstituted cells were constructed by fusion of cytoplasts from the human diploid fibroblast cell strain Detroit 532 and karyoplasts from the mouse fibroblast cell line A9. Several cellular properties were examined during the first 48 hr after nuclear transplantation. (i) The overall morphology of the cells originally resembled that of the cytoplasmic donor, Detroit 532, but rapidly changed to approximate that of the nuclear donor, A9. However, definitive changes in the microfilament structure of the reconstituted cells were not seen until 24-48 hr after fusion. These observations support the idea that the presence or absence of an ordered array of microfilament bundles is not the sole determinant of cell shape. (ii) Although cytoplasts and karyoplasts were prepared from cultures of randomly growing cells, the first division of reconstituted cells occurred in a synchronous manner. However, the initiation of DNA synthesis was not synchronized. It thus appeared that, in their first cell cycle, the cells had a G2 period of variable length. The results further suggest that the cytoplasm of interphase fibroblasts contains the material necessary to initiate or support DNA synthesis in a transplanted nucleus but not entry into mitosis. (iii) A two-dimensional gel electrophoretic analysis of polypeptide synthesis in reconstituted cell cultures showed that synthesis directed by transplanted mouse nuclei could be detected as early as 3-6 hr after fusion. Some of the mouse polypeptides detected at the earliest time points studied were not among the major polypeptides synthesized by the parental A9 cells. By about 48 hr after fusion, the pattern of polypeptides produced by reconstituted cells was almost indistinguishable from that of the nuclear donor parent cells.