The polypeptides synthesized by mature embryonic erythrocytes prepared from the peripheral blood of 14-to 15-day-old chicken embryos were analyzed by two-dimensional gel electrophoresis. Fewer

Conditions for the preparation, purification, and maintenance of karyoplasts which could regenerate to reform whole viable cells were defined. Results of biochemical analyses of such karyoplasts at various times during regeneration indicated that a reproducible biosynthetic program was followed. Thus, an examination of the polypeptides made during regeneration by two-dimensional gel electrophoresis showed that the pattern of radiolabeled polypeptides synthesized at each time studied was specific and was significantly different from that observed at other times during regeneration. Polypeptides associated with three major cellular fractions-nuclear, cytoskeletal-microtrabecular, and solublewere among the most dramatically regulated molecules. Other polypeptides, such as the major components of microfilaments and intermediate filaments, were synthesized at relatively constant rates and were assembled into structures throughout regeneration. Likewise, microtubules appeared to be reformed throughout regeneration, even in the absence of identifiable centriole-associated organizing centers. Finally, analysis of DNA synthesis by autoradiography showed that, even when prepared from whole cells synchronized at the Gl/S interface, karyoplasts could not begin making DNA until they had regenerated an almost complete complement of cytoplasm.Cytochalasin-induced enucleation of mammalian cells produces primarily two types of cellular fragments: cytoplasts, or enucleated cells, and karyoplasts, or nuclei surrounded by thin shells of cytoplasm enclosed within outer cell membranes (5,18,21