The kinetics of protein uptake by chick erythrocyte nuclei during reactivation in chick-mammalian heterokaryons

Abstract

Dormant chick erythrocyte nuclei can be reactivated in heterokaryons formed by fusion of chick erythrocytes with mammalian tissue cultured cells. In such hybrid cells the erythrocyte nuclei enlarge concomitantly with the resumption of nucleic acid synthesis. The kinetics of protein accumulation in reactivating nuclei was studied with autoradiographic methods. In addition estimates of the overall specific activity of nuclear protein were made to determine the relative amounts of protein accumulating in the reactivating nuclei. The uptake of mammalian protein labelled prior to fusion in pulse-chase type experiments was rapid in the first hours following fusion between chick erythrocyte-mammalian cell heterokaryons. Furthermore, the uptake of newly synthesized protein into reactivating chick erythrocyte nuclei occurred without any detectable lag thus indicating that protein uptake was a very early event in the reactivation process. In experiments utilising chick erythrocytes which were UV-irradiated prior to fusion, it was shown that an intact DNA template and/or active DNA transcription were not prerequisites for protein accumulation into reactivating chick erythrocyte nuclei. The uptake of preformed protein was not inhibited by puromycin. Estimates of the specific activity of general nuclear protein in reactivated chick erythrocyte nuclei as well as of dry mass to DNA ratios indicated that the chick nucleus attained an overall composition resembling that of the mammalian nucleus. Taken together these facts suggested that the protein uptake by reactivating chick erythrocyte nuclei is more likely to be a cause of reactivation rather than a result of the process.