Robert E. Savage

Phenotypic expression in chick erythrocyte X rat myoblast hybrids and in chick myoblast X rat myoblast hybrids

Abstract Attempts were made to reprogram chick erythrocyte nuclei to specify the synthesis of chick myosin. Chick erythrocytes were fused with rat myogenic cells with the aid of UV-inactivated Sendai virus. In the heterokaryons and hybrid myotubes which resulted from this fusion, the erythrocyte nuclei resumed RNA synthesis and formed nucleoli. Although some new chick antigens developed in those myotubes which contained fully reactivated chick erythrocyte nuclei, accumulation of chick myosin could not be detected by immunological methods. Neither small heterokaryons nor large hybrid myotubes which were actively synthesizing rat myosin reacted with antibodies directed against chick myosin. A small number of mononucleated cells, believed to be synkaryons formed by mitotic division of heterokaryons, did, however, react strongly with antibodies directed against chick myosin and showed a cross striation typical of skeletal muscle. The frequency of such cells was too low, however, to permit karyological analysis or further characterization of the antigen. Hybrids between chick myoblasts and rat myoblasts produced both chick and rat myosin thus indicating that simultaneous translation of chick and rat mRNA for myosin in a common cytoplasm was possible. In summary the evidence obtained suggested that reprogramming of chick erythrocyte nuclei, if it did occur in the present system, was a rare phenomenon. The possibility that hybrids between chick erythrocytes and rat myoblasts expressed markers typical of an erythroid phenotype was examined by immune staining with antibodies directed against chick haemoglobin. The results suggested that haemoglobin was introduced into hybrid cells by erythrocytes which failed to lyse before fusion. The intensity of this immune fluorescence decreased with increasing time after fusion. The rate at which this decrease occurred was not affected by inhibition of RNA synthesis. Thus, there was no evidence for the accumulation of haemoglobin in the hybrid cells.

Intracellular antigen migration in interspecific myoblast heterokaryons

Abstract Intracellular migration of species-specific nuclear antigens was studied in chick-rat heterokaryons. These cells were produced by virus-induced or spontaneous fusion of different chick cells with rat myoblasts or myotubes. Chick erythrocyte nuclei introduced into rat myogenic cells increased in volume and were reactivated to synthesize RNA. As the chick erythrocyte nuclei enlarged, they rapidly accumulated rat nuclear antigens. Rat nucleolar and nucleoplasmic antigens assumed a distribution in the chick nuclei corresponding to that in rat nuclei. In hybrid myotubes formed by the spontaneous fusion of chick myoblasts and rat myoblasts antigen exchange was at a much lower level. Some exchange of both rat and chick nuclear antigens could, however, be detected also in this system. Thus chick nuclear envelope and nucleolar antigens migrated into the rat myoblast nuclei and assumed an intranuclear localization analogous to that in chick nuclei. On the basis of these results it appears that antigenic nuclear macromolecules are constantly exchanged between the rat and chick nuclear compartments and the cytoplasm of the heterokaryon. During the rapid nuclear swelling which occurs when chick erythrocyte nuclei are activated in rat myoblast heterokaryons, the inward migration of rat nuclear antigens into the chick erythrocyte nucleus is more impressive than the migration of chick antigens into the rat nuclei.

Nucleocytoplasmic Interactions and the Control of Nuclear Activity

Summary The nuclei of mature chick erythrocytes are genetically inactive and do not synthesize RNA, DNA, or protein. In heterokaryons formed by fusing chick red cells with human HeLa cells, the erythrocyte nucleus is reactivated, grows in size, and resumes RNA synthesis. The reactivation process occupies several days after cell fusion and results in the synthesis of new chick specific protein. The signals activating the chick genome appear to operate at several different levels. Early changes in the physicochemical properties of chick deoxyribonucleoprotein appear to be triggered by changes in the ionic environment. Nuclear growth during the first two days after fusion is due mainly to an accumulation in the chick nucleus of human nucleospecific molecules, probably proteins. These proteins are taken up from the surrounding HeLa cytoplasm or directly from HeLa nuclei lying in close contact with the chick nuclei. With increasing numbers of chick erythrocyte nuclei in the cytoplasm, less human nucleospecific proteins accumulate in each chick nucleus and the rate at which RNA synthesis accelerates is slowed down. By reactivating chick erythrocyte nuclei in the cytoplasms of rat and mouse cells representing different lines of cell differentiation, attempts have been made to study how the cytoplasm influences the phenotype expressed by the reactivated chick erythrocyte nucleus. Chick erythrocyte nuclei have been reactivated in rat myoblasts and myotubes at a time when rat myosin synthesis starts. No significant chick myosin synthesis has been demonstrated in spite of the fact that the chick erythrocyte nuclei developed a nucleolus, produced RNA, and expressed other chick genes in the form of chick antigens. Hybrids made by fusing chick myoblasts with rat myoblasts, however, synthesized both chick and rat myosin.