Age-associated change in macronuclear DNA content in Paramecium caudatum

0 YOSHIOMI TAKAGI AND NAOKO KANAZAWA Department of Biology, Nara Women's University , Nara 630 , Japan SUMMARY Macronuclear DNA content in Paramedum caudatwn was found to be almost unchanged with a mean of about 400C during the earlier two-thirds of the life span in terms of the number of fissions and then it dropped rapidly to about one-fifth of the initial content. The age when rapid DNA decline occurred corresponded to that when the characteristics of senescence appeared. This decreasing pattern of macronuclear DNA content contrasted with earlier observations in P. tetraurelia, P. bursaria and Tetrahymena thermophila. The data suggested that in P. caudatum the distribution pattern of macronuclear DNA to daughter cells changed from an almost equal distribution in younger cells to an unequal distribution in older cells, while the relative volume of the macronucleus to the whole cell remained almost constant throughout the life cycle. - This paper is dedicated to the memory of Tracy M. Sonneborn. also known in Tetrahymena thermophila (Doerder & DeBault, 1978) (for species nomenclature, see Nanney & McCoy, 1976). The present investigation addresses the question whether the initial drop in macronuclear DNA is a common phenomenon in ciliates, by the study of changes in the amount of macronuclear DNA in Paramecium caudatum. The cycle of clonal ageing in P. caudatum was recently clarified: the life-span was 527 fissions, characteristics of senescence appeared at about 350 fissions. And nearly the same change in life-cycle, as a function of fission, was reproduced in subclones after they had been kept as stock cultures for more than a year (Takagi & Yoshida, 1980). Also studied in relation to the change in macronuclear DNA content were the macronuclear ploidy level, change in distribution of macronuclear DNA to daughter cells, and change in relative volume of the macronucleus to the whole cell. The results were reported in an abstract (Takagi & Kanazawa, 1980). MATERIALS AND METHODS Cells of known ages were available from cultures of the stock Mi8a, mating type V of P. caudatum syngen 3 (Takagi & Yo8hida, 1980). A single cell was isolated from the stock culture of a given age and allowed to divide about 13 fissions to give a test-tube culture of about 14 ml. Also used were Fj clones, denoted NK clones, from a cross between Yt3G of mating type V and St37 of mating type VI, both of which were kindly provided by Dr K. Hiwatashi of Tohoku University. These clones were cultivated in daily isolation lines and surplus cultures were used for DNA measurements and for the test of sexual immaturity. The surplus culture of a given age was allowed to grow to a test-tube culture of about 14 ml. All of the test-tube cultures were starved for 3 days to exhaust food vacuoles before using them for measurement of DNA. This procedure was useful especially for old cells in which food vacuoles were often irregularly positioned. In other experiments, when distribution of DNA to daughter cells was determined, cells with food vacuoles were used. Culture conditions The culture medium was 2 % lettuce juice in 2 mM-sodium phosphate buffer solution (pH 6-8), inoculated i day before use with KUbsiella aerogenes. The temperature was about 25 C throughout the study except for the stock cultures, which were kept at 17 C. Feulgen microspectrophotometry A cell suspension was placed on a coverslip, hot-air dried with a drier for 10 min, and fixed with 83:1 mixture of ethanol/acetic acid for 10 min. Hydrolysis in 1 M-HC1 (60 C) for 10 min was followed by a rinse with i M-HC1 (o C) for i h since this procedure was useful for bringing about bacterial discoloration without affecting the colour of the macronucleus. The macronuclei were stained as described by Shibatani & Naora (1952), except that the Schiff's reagent was diluted to 1/16 concentration to obtain an optimal range of the optical density; o.D. = 0-2-0-7. Cells were stained in this solution for 1 h, then bleached in dilute sulphurous acid, rinsed in distilled water, dehydrated by passing through ethanol series and xylols, and mounted in Eukit (O. Kindler Ltd). Microspectrophotometric determinations were made by scanning the whole macronucleus with a spot-light of 2 fim in diameter at 565 nm using the Olympus MMSP. The DNA content of the micronucleus, which usually lies in a concavity of the macronucleus, could not be measured separately from the macronuclear DNA content except for some unusual cases when both nuclei existed apart from each other. Such measurements were used to estimate the degree of macronuclear polyploidy. The total extinction value for each measurement was corrected for background. Macronuclear DNA content Macronuclear DNA content was measured in 13 subclones (Table 1). Cells 127-330 fissions old showed a relative DNA content, E%6&, from 150 to 169-6; cells 367-425 fissions old, 81-7-107-8; and cells 468-507 fissions old, 34-1-44-3. A slight decrease in DNA content during two-thirds of the life cycle was followed by a sharp drop during the last one-third of the life cycle (open circles in Fig. 1). The DNA content of cells 330 fissions old was significantly different from (Z-test, P < 0-05) but still 9I-3 % of that of cells 127fissionsold, and the DNA content of cells 507 fissions old was 20-1 % of that of cells 127 fissions old. However, DNA decline might have occurred before the age of 127 fissions. This was examined with young NK clones derived from a new cross, since cells younger than 127fissionswere not available from the stock Mi8a. Among 13 newly produced clones, NK-J, NK-K, NK-L and NK-M showed shorter life spans; they died at 52, 58, 183 and 228fissionsold, respectively. The remaining 9 clones lived for more than 300fissions.Mean macronuclear DNA contents of these 9 clones at the average age of 16, 59, 99 and 152fissionswere shown in Table 2. Although the difference in mean DNA contents among groups of different ages was statistically significant (Z-test, P < 0-05), the average value at 152 fissions was still 90-3 % of that of cells 16 fissions old. In those clones with shorter life-spans, however, the averages at the last measurements were 62-5% (NK-K), 68-2% (NK-J), 69-3% (NK-L) and 84-2% (NK-M) of that of cells 16 fissions old. Thus we conclude that, in clones with long life spans, Age in fissions Fig. 1. Change in macronuclear DNA content with age in an Mi8a clone (O) and 9NK clones ( ) . Data represent the mean total extinction at 565 nm standard deviation (n = 100 for Mi8a, n = 30 for each NK clone) for Feulgen-positive material in the macronuclei of cells starved for 3 days. decrease in macronuclear DNA content is also slight during the period younger than 127 fissions. Distribution of macronuclear DNA to daughter cells Macronuclear DNA contents in two division products were compared between cells of two different fission ages, young cells 136fissionsold and old cells 436 fissions old. In Table 3 the (...truncated)