The duration of the periods of the cell cycle in a culture of human female fibroblasts was determined by tritiated thymidine autoradiography. The S period was 7.5 h, the G2 period was 5 h and the Gt period was 8.5 h. In all the monolayer cultures studied, only about one fourth of the cells were actively proliferating. The remaining cells were in a resting, or G0, stage. If a cell culture was exposed to tritiated thymidine for 30 min, labeled cells were in the S period and unlabeled cells were in the G2, G1 or G0 period. If exposed for 5 h, labeled cells were in the S or G2 period and unlabeled cells were in the G: or G0 period. If exposed for 13.5 h, labeled cells were in the S, G2 or G: period and unlabeled cells were in the G0 period. When the autoradiograms were exposed for only one to three days, the presence or absence of a sex chromatin body or condensed nuclear chromatin in both labeled and unlabeled cells could be determined. In this manner the status of the nuclear chromatin and the nuclear size of cells could be assessed during the various portions of the cell cycle. The following observations are based on the study of more than 12,000 XX, XXXXY and XY fibroblasts. (1) The percentage of sex chromatin positive cells did not vary significantly during any of the periods of the cell cycle. This indicated that the sex chromatin body underwent DNA replication without loss of its heterochromatic coiling and suggested that the inactive X chromosome did not become active during any specific portion of the cell cycle. (2) Non-specific condensed coiling of nuclear chromatin occurred most often in actively proliferating fibroblasts and least often in resting cells. The distinction between non-specific condensed coiling of chromatin and heterochromatin is emphasized. (3) The most significant factors which appeared to affect nuclear size were: (a) following cell division, nuclei tended to be small and have condensed chromatin; and (b) resting cells, which were presumably metabolically hypoactive, tended to have a larger nuclear size. The latter may have been the result of greater distention during fixation. When these two factors were excluded, there was no significant difference in the size of sex chromatin positive and sex chromatin negative nuclei. (4) The area of the sex chromatin body increased significantly following DNA replication, while the area of the nucleus did not. This suggested that the area of the tightly coiled sex chromatin body was closely related to its DNA content, while the area of the nucleus containing loosely coiled chromatin was more closely related to the tertiary folding of the chromatin than its DNA content. The nature of sex chromatin negative cells is discussed. It is proposed that they represent the loss of heterochromatic coiling without the loss of genetic inactivation.
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