Programmed macromolecular synthesis in regenerating karyoplasts.

Abstract

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

Transient correction of genetic defects in cultured animal cells by introduction of functional proteins.

“…In a second experiment, 84% of the loaded cells were heavily labeled. Since the single thymidine block method of synchrony results in the accumulation of only about 80 to 90% of cells in the S phase of the cell cycle (12), it is likely that even more of the cells in fact received TK during scrape labeling and that they were simply not scored as positive because they were not in the S phase during the labeling period. The scrape loading method has several attributes which should promote its widespread use: it is simple to perform, it requires little skill and no special or expensive equipment, it can be completed in minutes, it is applicable to many different cell types without modification and, most importantly, it yields a large population of cells loaded with a substantial quantity of material.…”

“…and then processed for autoradiography. Ltk-cells were synchronized in the S phase of the cell cycle prior to scrape loading as described previously (12). Magnification, x600.…”