We have made a detailed study of the X-chromosome replication pattern during the period when X-inactivation is occurring in the mouse embryo. Our observations show unequivocal regionalization of the embryo with respect to the temporal X-chromosome. The switch from isocyclic to allocyclic replication occurs in the embryonic ectoderm at approximately 6 days of development and is random with respect to parental origin of the X-chromosome. In the extra-embryonic tissues, however, the switch to allocyclic replication has apparently occurred prior to 5.3 days of development and almost exclusively involves the paternally-derived X-chromosome. Since these findings are consistent with results obtained in biochemical studies of X-chromosome activity in female embryos, we conclude that there is a close temporal relationship between the cytogenetic and biochemical manifestations of the X-inactivation process. In addition, we have observed a pattern of early paternal X-chromosome replication, transitory in some cases, that is unique to extra-embryonic tissues. These results suggest that there may be some differences in the mechanism by which X-inactivation occurs in the extra-embryonic tissues as compared with the embryonic ectoderm.

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The control of cellular senescence by specific human chromosomes was examined in interspecies cell hybrids between diploid human fibroblasts and an immortal, Syrian hamster cell line. Most such hybrids exhibited a limited life span comparable to that of the human fibroblasts, indicating that cellular senescence is dominant in these hybrids. Karyotypic analyses of the hybrid clones that did not senesce revealed that all these clones had lost both copies of human chromosome 1, whereas all other human chromosomes were observed in at least some of the immortal hybrids. The application of selective pressure for retention of human chromosome 1 to the cell hybrids resulted in an increased percentage of hybrids that senesced. Further, the introduction of a single copy of human chromosome 1 to the hamster cells by microcell fusion caused typical signs of cellular senescence. Transfer of chromosome 11 had no effect on the growth of the cells. These findings indicate that human chromosome 1 may participate in the control of cellular senescence and further support a genetic basis for cellular senescence.

By means of a cytological method involving BrdU incorporation and acridine orange fluorescence staining in combination with embryo manipulation, we studied X-chromosome activity in female preimplantation mouse embryos with special reference to the correlation between X-chromosome inactivation and cell differentiation. There was no sign of asynchronous replication between the two X chromosomes from the one-cell to intermediate blastocyst stage. The allocyclic X chromosome, first detected in late blastocysts, was paternal in origin, mostly replicating early in the S phase and limited to the trophectoderm. Subsequent X-chromosome inactivation occurring in the primary endoderm was also characterized by the involvement of the paternal X and early replication. Both X chromosomes continued to replicate synchronously in the embryonic ectoderm or epiblast at this stage. It was evident that overt cell differentiation preceded the appearance of the asynchronously replicating X chromosome in the trophectoderm and primary endoderm. This finding seems to support the view that cell differentiation is an important correlate of X-chromosome inactivation.