Transcriptionally inactive chick erythrocyte nuclei were reactivated by Sendal virusinduced fusion of erythrocytes with rat L6J1 myoblasts. We used antibodies to trace the appearance of a specific protein engaged in transcription of a defined class of genes, those coding for rRNA, during reactivation. Using immunofluorescence microscopy, we found increasing amounts of rat RNA polymerase I to appear, during a certain period of time after fusion, in the reforming nucleoli of the chick nuclei. Amounts of rat RNA polymerase I sufficient to be detected by immunofluorescence microscopy had accumulated in the newly developed chick nucleoli 72-190 h after fusion was initiated. This time interval coincides with the time when chick rRNA synthesis can first be detected. The results raise the possibility that during these stages of the reactivation process chick rRNA genes are transcribed by heterologous RNA polymerase I molecules of rat origin.Mature chick erythrocytes are terminally differentiated cells in which the nuclear chromatin is tightly condensed, and in which replication, as well as transcription, have almost completely stopped (l). After fusion with actively transcribing mammalian cells, the nuclei of chick erythrocytes undergo a reactivation process characterized by an increase in nuclear size and protein content and by dispersion of chromatin and development of nucleoli (2-6). This increase of nuclear size is paralleled by a selective uptake of mammalian nucleusspecific proteins (4, 7) as well as by disappearance of some specific components of the chick erythrocyte chromatin (7,8).In the present study we examined the nucleocytoplasmic exchange of specific proteins during the reactivation of chick erythrocyte nuclei in heterokaryons. To this end we used an antiserum against rat RNA polymerase I (9), which does not cross-react with the corresponding enzyme from nonmammalian species (Fig. I b)) We show that rat RNA polymerase I molecules entered the chick nucleus during the reactivation process and accumulated in the reforming nucleolus.
MATERIALS AND METHODSCells: L6Jl, a myogenic subclone of Yaffe's L6 rat myoblast line (10), isolated in our laboratory by John Coleman (see reference I l), was cultured on Dulbecco's minimal essential medium (DME) containing 10% fetal calf serum.Scheer, V., and K. M. Rose, manuscript submitted for publication.Cells were plated on glass hemocytometer cover glasses at a density of approximately 50,000 cells/cm 2 in 20 cm 2 plastic dishes. The following day, cells were treated with 0.2 ~,g/ml of mitomycin C (Sigma Chemical Co., St. Louis, MO) for 16 h and then fused with chick erythrocytes.FIGURE 1 Cultured chick fibroblasts as seen with phase-contrast (a) and epifluorescence (b) optics after staining with antibodies to rat RNA polymerase I. Chick nucleoli are completely negative (denoted by arrows in a). Bar, 10/~m. x 1,000.