Biochemical analysis of reactivated chick erythrocyte nuclei isolated from chick/HeLa heterokaryons

Sendai virus-induced fusion of chick erythrocytes with human HeLa cells results in the formation of multinucleated hybrid cells (heterokaryons). In these cells the erythrocyte nuclei take up human proteins from the cytoplasm and undergo a general “reactivation” reaction resulting in transcription, and eventually replication of the chick DNA. The early events in this reactivation process were analysed by isolating reactivated chick erythrocyte nuclei from heterokaryons zero to 15 hours post-fusion, at a time when extensive decondensation of the chick chromatin occurs (and transcription of chick DNA is initiated) but very little chick DNA replication is occurring. The isolated chick nuclei were sufficiently free from contaminating HeLa nuclear material to allow an electrophoretic and chromatographic analysis of 3H-labelled proteins accumulating in the chick nuclei. The proteins were labelled by incubating the cultures with 3H-labelled amino acids. Radioactivity in the histone group of nuclear proteins was shown to be composed predominantly of f1 histone. Furthermore, this f1 histone could be identified as being HeLa in origin and its migration into reactivating chick erythrocyte nuclei was correlated with a loss of chick-specific f2c histone. The appearance of radioactivity in the major non-histone group of nuclear proteins was highly preferential relative to the histone group of proteins. Electrophoresis on sodium dodecyl sulphate-poly acry lamide gels showed that many subgroups of non-histone proteins present in HeLa nuclei moved into the reactivated chick erythrocyte nuclei, whereas some other human proteins did not. Furthermore, pulse-chase type experiments showed that similar patterns of labelled proteins appeared in the erythrocyte nuclei irrespective of whether label was administered only to the mammalian cell before fusion or was given to heterokaryon cultures after fusion. The data presented are discussed in relation to changes in nuclear protein metabolism influencing chromatin structure and the availability of DNA for transcription.