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Quantitative studies on the macronuclei of Paramecium aurelia doublets : determination, DNA content and distribution of DNA to daughter macronuclei Morton, Glenn Thomas

Abstract

This study examined the nuclear events in Paramecium aurelia doublets following sexual reorganization to: 1) Determine how loss in number of macronuclei in older clones occurred; 2) Quantitatively analyse the DNA content of Feulgen-stained doublet macronuclei and the distribution of DNA to daughter cells at division; and 3) Quantitatively compare singlet and doublet P. aurelia with respect to DNA content, total protein content, and length of the cell cycle. If the situation was analogous to that in singlets, four macro-nuclear anlagen would be expected in doublets following conjugation or autogamy. Results, however, showed that the number of macronuclear anlagen in doublets varied considerably. Also, a number of "abnormal" anlagen were observed in doublets which appeared to be undergoing macro-nuclear regeneration. Genetic evidence indicated that the cause of variability in the number of anlagen was due to abnormal patterns of determination of the products of the second post-zygotic division of the synkarya, presumably as a consequence of the abnormal morphology of doublet cells. Distribution of macronuclear anlagen at the first cell division was also irregular; only 34.3% of the daughter cells showed the expected 2:2 distribution of anlagen. Loss of the second macronucleus occurred by the fourth to fifth cell cycle. Misdivision (failure to divide) and missegregation of macronuclei, mechanisms which could account for macronuclear loss, were observed to be occurring approximately 15-20% and five-eight percent of the time. Genetic evidence showed that fusion of macronuclei, a third mechanism which could account for loss of macro-nuclei, could not take place more than about 15% of the time, if it in fact occurred at all. The DNA content of doublets varied slightly between stocks, as well as between cells within stocks. DNA content in doublets was not strictly proportional to the number of macronuclei per cell; additional macronuclei increased the DNA content, but not in proportion to the number of macronuclei. Therefore, mechanisms were present which regulated the amount of DNA per cell to a more nearly constant amount than would be expected from the number of macronuclei alone. When daughter cells contained equal numbers of macronuclei, the mean difference in DNA content between sister cells was about five percent of the G₂ DNA content and the intraclass correlation coefficient between sister cell DNA was high (+0.7 or more). In contrast, when unequal numbers of macronuclei were present in daughter cells, the DNA contents were quite unequal; however, this inequality was not strictly proportional to the number of macronuclei per cell. No net regulation of DNA content occurred during the DNA synthesis period of the cell cycle as evidenced by the fact that there was no significant difference in variation between doublet cells having the G₁ versus the G₂ amount of DNA. A comparison of DNA content in P. aurelia singlets and doublets showed that the mean G₁ DNA content of doublets was approximately 192% that of singlets. Further, the mean DNA content of micronuclei in singlet and doublet cells was the same. A similar comparison of total protein content showed that doublets had approximately twice the macromolecular dry mass of singlets. As reported by Kimball (1967), it was found that the ratio between dry mass and DNA content in doublets is constant when both the cell and the macronucleus are doubling in each cell cycle. Finally, it was shown that doublet P. aurelia have a vegetative cell cycle lasting approximately 164% of the length of singlets. It was suggested that the G₁ period in doublets may be shorter with increased cell size as was observed by Killander and Zetterberg (1965a,b) in mammalian cells, thereby allowing more time for growth in the smaller than in the larger cells before DNA synthesis begins.

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