Age-Correlated Changes in Expression of Micronuclxar Damage and Repair in Paramecium Tetraurelia

AGE-CORRELATED CHANGES IN EXPRESSION OF MICRONUCLXAR DAMAGE AND REPAIR IN PARAMECIUM TETRAURELIA

STEVEN R. RODERMELI AND JOAN SMITH-SONNEBORN

Department of Zoology and Physiology, The University of Wyoming, Laramie, Wyoming 82071 Manuscript received December 8, 1976 Revised copy received May 31, 1977

ABSTRACT In Paramecium, age is defined as the number of mitotic divisions which have elapsed since the previous cross-fertilization (conjugation) or self-fertilization (autogamy). As the mitotic interval between fertilizations increases, the percentage of nonviable progeny clones increases. In the current study, resolution of conflicting previous reports on the pattern of increase of death and reduced viability in progeny from aging parent cells is found. Some exautogamous clones exhibit a high mortality at young clonal ages, others show no mortality throughout their life span, but most (73%) show an abrupt increase in the percent death and reduced viability in progeny from cells 50-80 fissions old. Ultraviolet-irradiation-induced micronuclear mutations, repairable by photoreactivation, increased with increased clonal age when monitored by percent death and reduced viability of exautogamous progeny of irradiated cells. Loss of dark repair is considered a contributor to the increased expression of micronuclear mutations with increased clonal age.

ARAMECIA have been used as a model system of cellular aging since SONNE- 'BORN (1954a) established the reality of clonal aging in these protozoa. Normal human cells in tissue culture also age and die and are used for cellular aging studies (HAYFLICKand MOORHEAD1961 ; HAYFLICK1965) ; many similarities have been noted between human cells in culture and aging paramecia (HAY- FLICK 1975). The life cycle of paramecia begins with fertilization. Progeny clones exhibit cellular development and aging as a function of the number of cell divisions since the previous sexual process, passing seriatim from the immaturity stage to maturity, senescence, and finally death if a new fertilization does not reinitiate the next cycle (MAUPAS1888, 1889; JENNINGS1944a,b; SONNE- BORN 1954a; SIEGEL1961, 1967). The paramecia used in this study were Paramecium tetraurelia, which contain one macronucleus and two micronuclei. The polygenic macronucleus is responsible for the phenotype of the cell ( SONNE- BORN 1947; SIEGEL1961, 1963) and is comparable to somatic cell nuclei of higher organisms. The micronucleus is considered the repository of the genetic

Present addiess Harvard University, The Biological Laboratorirs, 1G Di%inityAvenue, Cambridge, Mass. 03138.

Genetics 87 : 259-274 October, 1977. 260 S. R. RODERMEL AND J. SMITH-SONNEBORN information, functioning very little with respect to the phenotype of the cell (SONNEBORN1946, 1954b; PASTERNAK1967); it is comparable to germ cell nuclei of higher organisms. Fertilization in paramecia can be by conjugation (cross-fertilization) or autogamy (self-fertilization) . During either of these processes the macronucleus degenerates and the micronuclear zygote develops the new micronuclei and macronucleus for the progeny (DILLER1936). Genetic analyses (SONNEBORN 1939) have also shown that autogamy renders all loci homozygous in one generation, thereby bringing any recessive mutations to phenotypic expression. The advantage of paramecia yielding homozygosity was utilized in the present study to assay for the occurrence of micronuclear mutations as a function of increasing clonal age. RAFFEL(1932) was the first to suggest that death after autogamy is due to lethal mutations occurring in the micronuclei. He was able to demonstrate that, whereas conjugation of young parents yielded a low mortality, the degree of mortality found after conjugation of old cells varied widely from one exconjugant line to the other. PIERSON(1938) and GELBER(1938) also demonstrated increased mortality after self-fertilization (autogamy) as clonal age increased. An inexplicable doubling in mortality after autogamy (from 32-63%) between the twenty-first and twenty-third day after the origin of the clone implied an abrupt increase in mortality. JENNINGS(1944~) also noted an increase in mortality among exconjugants as the age of the parents increased in his studies using Paramecium bursaria. The importance of death after autogamy with respect to cellular aging was first recognized by SONNEBORNand SCHNELLER(1960a), who reported that lethal micronuclear mutations are not detected after autogamy in Paramecium until 80 fissions after the previous fertilization. Mortality after autogamy then increases until an age in reached (220 fissions) at which autogamy yields no viable offspring. SONNEBORNand SCHNELLERconcluded that mutations do not occur at a uniform rate with either time or number of fissions since the previous sexual event, but are specifically “induced” only in old cells. And, based upon evidence obtained from merogones (old cells whose own nuclei fail to undergo meiosis, but into which nuclei from young cells can be introduced), they argued that the agent of induction was old cytoplasm. Unfortunately, these important findings were only briefly reported, and the complete study has never been published. Whereas SONNEBORNand SCHNELLERcontended that lethal micronuclear mutations are induced at a certain time in the life span of a clone, FUKUSHIMA (1975) has recently argued that they occur randomly as a function of age. Like RAFFEL,he presented evidence showing that lethal micronuclear mutations are detected earlier than 80 fissions, and that in the history of a clone the pattern of their occurrence is “stepwise”, in accordance with theoretical expectations of lethality based upon chromosome and lethal mutation numbers (i.e., one lethal mutation on one chromosome of either micronucleus would be expected to yield AGE AND NUCLEAR DAMAGE 26 1 25% death after autogamy; two lethal mutations on different chromosomes in the same micronucleus would be expected to yield 37.5% death, etc.). FUKU- SHIMA, however, based his conclusions upon a small sample size. The first purpose of this study, then, was to reconcile the conflicting claims of SONNEBORNand SCHNELLER(1960a) and FUKUSHIMA(1975) : do mutations occw at a constant rate or are they accelerated as clonal age increases? It should be noted, however, that mutation rate is contingent both on the rate of the occurrence of the mutational event and the ability of the cell to repair the damage, error-free. If the relation between the mutation rate and the error-free repair rate remains constant as clonal age increases, there should be no abrupt increase in mutation rate throughout the life span of the clone. However, an increased difference in mutation rate and repair capacity should be reflected in an increased frequency of mutations that are detected; the increased frequency could be a result of either an increased mutation rate, a decreased repair capacity, or both. The repair capacity of aging clones was thus assayed by determining the percent mortality and reduced viability of progeny after autogamy from (1) UV- irradiated and (2) UV-irradiated, then photoreactivated, clones. The values were corrected by subtracting the percent mortality and reduced viability found for untreated clones at the same ages. The results indicate that “age-induced” micronuclear mutations begin at 50 to 80 fissions as reported by SONNEBORNand SCHNELLER(1960a), though some young clones do exhibit mutations, as claimed by FUKUSHIMA(1975). Loss of error-free repair in the micronuclei of aging clones is considered a contributor to “age-induced“ mutations. The data, however, do not exclude the possibility that the mutation rate increases after 80 fissions; increased mutations and decreased repair could both dramatically change the frequency of mutations detected with age. The results in this study do not imply that vegetative aging in Paramecium is due to the occurrence of “age-induced” micronuclear mutations, since the micronuclei do not function with respect to the phenotype of the cell. In order to extend these findings to a more general aging hypothesis, which includes the vegetative portion of the life cycle, similar results would have to be obtained with respect to the macronucleus; SMITH-SONNEBORN(1971) did provide evidence for loss of error-free repair in the macronucleus of aging cells.

MATERIALS AND METHODS

1. Standard culture conditions Cells of Paramecium tetraurelia, stock 51, mating type VII, were used. These have been kindly supplied by DR. T. M. SONNEBORN,Indiana University; these cells were used since autogamy can be induced by starvation when the cells are about 20 fissions old, and autogamy rarely occurs in daily isolation lines grown under favorable conditions (SONNEBORN1954a). The culture medium was Cerophyl medium (Cerophyl Laboratories. Kansas City, Kansas) inoculated 24 hours before use with Klebsiella aerogenes, incubated at 27”, and adjusted to pH 6.7 immediately before use. 2. Maintenslnce of aging cell lines The procedure of SONNEBORN(1954a) was used for conducting daily isolation lines to obtain aging cells. Our selective procedures differed from those of SONNEBORN(1954a) and are 262 S. R. RODERMEL AND J. SMITH-SONNEBORN DAILY ISOLATION LINES

Cells in Autogamy ///I \\\\ Clones ABCDEFGH Day I 00000000 Day 2 00000000 Day 3 00000000 Day 4 00000000 11111111 Day 40 0 0 0 0 0 0 0 0 O=depression detailed below. Single cells were maintained in plastic disposable spot plates (96 depressions per plate). The spot plates were sterilized with ultraviolet light before use and covered with Saran wrap. Autogamy was taken to indicate zero time and was routinely asceitained using acridine orange (0.3 mg/ml) staining and fluorescence microscopy to identify the macronuclear changes typical of autogamous cells (SMITH-SONNEBORN1974). The age of parent cells used to initiate isolation lines by autogamous progeny was 30 fissions since parental age was found to affect progeny life span (SMITH-SONNEBORN,KLASS and COTTON1974). When 100% of an observed sample of approximately 30 cells exhibited autogamy, eight unstained sister cells were isolated from that depression with a micropipette under a dissecting microscope and removed to a depression containing fresh food as single isolates on day 1 as diagrammed below. On day 2, the number of cells per depression derived from the single cell was deteimined, and a single cell from each depression was reisolated and given fresh food. Cells not transferred were permitted to starve and undergo autogamy, and served as a source both for initiating new progeny lines and for determining percentage death and reduced viability after autogamy (described below). The procedure of counting and reisolation of cells was repeated daily. The log, of the number of cells derived from a single cell is the daily fission rate. The number of fissions per day divided into 24 hours represents an estimate of the time taken for one interfission period. The sum of the number of fissions per day from the day of origin at autogamy to a given day is the fission age of that 'clone. The total number of fissions from the origin of the clone to the death of an isolated cell is the life span of the clone. Ten isolation lines with eight clones per isolation line were used (80 clones). At given fission ages, autogamous cells were assayed for percent death and reduced viability using a minimum of 2-3 different isolation lines at the source of cells. Three isolation lines were expanded to obtain sister cells for a comparison of inter- us intra-clonal variations and the expansion is diagrammed below.

EXPANSION OF DAILY ISOLATION LINES TO OBTAIN SISTER CELLS

Cells in Autogamy Cells in Autogamy / / \ \\\ /// I \\\\ Clones: 0'd 000000 00000000 00000000 00000000 AAAAAAAA AAAAAAAA 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 Sister cell sublines AGE AND NUCLEAR DAMAGE 263 Cells in Autogamy /// f \ \\\ Clones: 0 0 0 0 0 0 0 0 00000000 00000000 00000000 AAAAAAAA 000 000 000 000 000 000 000 000 O=depression

Thus, 24 clones were expanded into 56 sister sublines. Of these 24, only 17 clones with 48 sister sublines could be used for analysis as the other sublines underwent autogamy despite the presence of excess food. This judgment that the cells underwent autogamy is described as follows. If any clone exhibited a drop in daily fission rate followed by a sharp increase in fission rate on a subsequent day, that line was discarded: this pattern is typical of autogamy occurring in the presence of excess food to give rise to a new generation. In addition, if cells in a depression suspected as having been through autogramy were allowed to starve, no autogamy should have been observed in that depression if the cells had actually been through autogamy, since it takes cells an average of only 10 fissions to become sufficiently starved to undergo autogamy, and the immaturity period is usually 20 fissions. Therefore, a change in fission rate and the inability to obtain autogamy in starved depressions were used as criteria before a particular clone was discarded. Although these procedures are useful, an illicit autogamy could have occurred and remained undetected; SONNEBORN(1954a) showed that aged clones did not always have a youthfully high fission rate after autogamy, and this has been confirmed in our laboratory. 3. Bioassay for percent death and reduced viability after autogamy When 95-100% of a sample of cells of a desired fission age was observed to be in autogamy, either 24 or 96 of the unstained cells from that depression were placed as single isolates in spot depressions containing 0.2ml fresh food. At least 25 autogamous depressions were used as a source of 24 or 96 isolates per depression and generated a minimum of 1248 progeny cells as single isolates at each fission age examined. After four days incubation at 27" the isolated cells were then examined and scored as either nonviable, viable, or of reduced viability. The nonviable depressions were those which contained zero (the isolated died and lysed), or less than five living cells (SMITH-SONNEBORN1971). The viable depressions were those in which the exautogamous cells had cleared the medium (no turbidity), and the reduced viability ones those in which the medium had not been cleared after four days incubation post-autogamy. (That such cells were indeed of abnormal viability is confirmed by the finding that of 40 cells taken from depressions classified as reduced viable and reisolated into a fresh supply of food, all 40 died after undergoing from 0 to 20 fissions in the new medium.) A period of only four days incubation was used before assays were done because it was found upon analysis of 520 depressions scoied as reduced viable after this period of time that given two more days of incubation, 86% of those depressions were still of reduced viability, while 10% had died and only 4% had become viable. 4. Determination of intra- vs. inter-clonal variation The 17 clones expanded into sister sublines were used to determine the percentage of death and reduced viability after autogamy. Since at any given age there is a percentage difference between sublines of the same clone in their frequency of death and reduced viability after autogamy, the question was asked: how often can one clone be paired with another clone at a given age and the difference be the same as that found between sister cells? This question was examined statistically by deriving the mean of the differences between the sister lines of the 17 clones at a given age (i.e., the mean of IA,-A,/ + IB,-B,j + /Cl-C21+ ID,-D,I . . . .) and 264 S. R. RODERMEL AND J. SMITEI-SONNEBORN comparing this mean by the student’s t-test (SOKALand ROHLF 1969) with the mean of the differences between five randoinly selected lines and all other lines at that same age (i.e., the mean of IA,-B,I 4- IA,-B,I -I- IA,-C,I -I- IA,-C,I . . . 4- IDZ-A,~-t /Dz-A21f IDZ-BII 3- IDz-BzI . . . f IGZ-AII -I- IGZ-AZI 4- IGz-BlI f IGZ-BzI 4- IGZ-CII . . .). 5. Determinaiion of autogamy death and reduced viability after ultraviolet irradLation and photoreactivation. The percent death and reduced viability after autogamy with cells of a given fission age was determined when samples of these cells: (1) were exposed to 90 se: of UV irradiation and then allowed to undergo autogamy; (2) were exposed to 90 sec of UV, immediately placed under a black light bulb for one hour, and then allowed to undergo autogamy; and (3) were allowed to undergo autogamy untreated. Autogamous untreated cells of a desired fission age were obtained from back depressions in the daily isolation lines and assayed for death and reduced viability after autogamy as described above. Cells of the same age to be irradiated were taken randomly from that day’s daily isolation depressions, quickly taken through three washes in Dryl’s salt solution (DRYL1959), and irradiated for 90 sec in 0.2 ml of this same solution (which is transparent to UV). A germicidal lamp emitting mainly at a wavelength of 253.7 nm supplied the UV irradiation of 1800 ergs/mmZ/min as measured by a YSI Kettering radiometer. A time of 90 sec was used as a dosage which yielded few deaths after irradiation until cells reached an age of 150 fissions, and yet provided a mutational load such that death after autogamy could be observed at all ages. After irradiation, individual cells were either immediately transferred in the dark into separate depressions containing fresh food under a microscope equipped with a red filter to reduce illumination, or exposed to one hour of illumination under a G.E. black light bulb emitting at a rate of 10,000 ergs/mmZ/sec prior to transfer into separate depressions. A glass plate between the animals and the black light prevented transmission of wavelengths below about 300 nm. Both groups were then cotered with aluminum foil and incubated at 27” until the depressions were observed to be in autogamy (usually 4-6 days later). Autogamous depressions were assayed for death and reduced viability as described above. In one experiment, cells were irradiated during division or midway through their cell cycle. This was accomplished by selecting dividing cells from the daily isolation lines and immediately irradiating some, while letting the others progress 2% hours into their interfission period and then irradiating them. For both groups of cells, procedures as described previously were used to assay cells for death and reduced viability following autogamy after UV treatment.

RESULTS Figure 1 summarizes the results from ten isolation lines (309 autogamies and 1.8 x lo4progeny) in which death and reduced viability after autogamy were monitored as a function of clonal age. A very low level of death and reduced viability was found in cells under 80 fissions old; at about 50 to 80 fissions there occurred an abrupt increase in percent death and reduced viability, after which the pattern of increase became linear. There is a possibility of the occurrence of undetected autogamies in sublines, thereby initiating a new generation, which cannot be excluded. Such occurrences contribute to (1) the absence of mortality at “advanced” ages since the cells would be younger than presumed and, (2) an increased variability between sublines. Figure 2 shows representative samples of the variation found between individual clones in their percent death and reduced viability in progeny from parents after autogamy as the clonal age of the parents was presumed to increase, and a comparison of sister sublines within each clone (Clones A-F) . Of the 17 clones with sublines of sister cells (48 sublines) studied, 73% exhibited the pattern AGE AND NUCLEAR DAMAGE 265

0- 0 30 60 90 120 150 180

AGE (Fissions) FIGURE1.-Death and reduced viability after autogamy as a function of clonal age. Each point is the mean of a minimum of 25 autogamous depressions, except the 150 and 180 fission time points, which are the mean of 9 and 11 autogamous depressions, respectively. The mean +- 2>

of increased death and reduced viability only after parental age of 50-80 fissions as seen in Figure 2A; 6% showed a high mortality and reduced viability at an early age and no increase as clonal age increased (Figure 2D): 15% showed no increase in mortality and reduced viability throughout their life span (Figure 2E) and 6% showed an increase in mortality in progeny from young parents which dropped to 0 when the clone was older (Figure 2F,). Intraclonal variation was also observed (Figure 2B and C). Whereas sublines B, and B, showed an 266 S. R. RODERMEL AND J. SMITH-SONNEBORN A 6 loo - loo100 - t f B1 7s - 7s -

so - so -

2s -

20 40 60 80 100 120 140 HK) MO k 4- C D loo t .-t n 0 j- XI- U X -- g 2s- 3 U 2s t111*111111 B 20 40 60 60 100 120 I40 I60 180 20 40 60 80 100 120 140 160 I80 U c O look E

3 .- 20 40 60 80 100 120 140 160 BO 20 40 60 80 100 120 140 160 160

AGE (Fissions) FIGURE2.-The pattern of occurrence of death and reduced viability after autogamy as clonal age increased in individual clones. A-F represents 6 of the 17 clones studied to demonstrate the variation found between clones and within clones, though most of the lines exhibited the pattern of death and reduced viability seen in clone A. Clones were separated into sister lines at 8 or 23 fissions after the origin of the clone at autogamy. Each point represents the mortality and reduced viability of 24 exautogamous progeny from a parental cell at the clonal age indicated. increase in mortality and reduced viability in progeny from aging parents, sub- line B, showed no increased mortality in progeny from parent cells as age increased. Line C, showed an increase in death and reduced viability in progeny 80 fissions earlier than the sister C,. Striking similarities were, however, seen AGE AND NUCLEAR DAMAGE 267 between sister sublines A, and A,, D, and D,, and E,, E, and E,. Whether intraclonal variation was as great as interclonal variation was examined statistically. At given ages, the mean difference between percent death and reduced viability within clones was compared by the student’s t-test (SOKALand ROHLF1969) with the mean difference between a given sister line (using a total of five randomly selected sister sublines) and all other lines. A significant difference would indicate more similarity within clones than between clones. Table 1 shows an indication of similarity within clones at young ages, but not after 8 fissions; i.e., the differences within clones became larger. In addition, since sister/nonsister mean differences were always subtracted from sister/sister mean differences, the negative t-statistic indicates that the interclonal means were more variable than the intraclonal means. The data indicate that intraclonal variation is as great as interclonal variation with respect to the increase in death and reduced viability in progeny from aging parent cells. Micronuclear lethal mutations are likely contributors to increased death and reduced viability in progeny from autogamy as parental age increases ( SONNEBORNand SCHNELLER1960a). Because the detection of mutations is a function not only of their rate of occurrence but also of the rate of their error-free repair, we determined what happens to dark repair as a function of age. Figure 3 summarizes the results of 166 autogamies in which cells of a given age were allowed to undergo autogamy both with and without prior exposure to UV radiation. When values of percent autogamy death and reduced viability after UV treatment are corrected with values obtained after autogamy from untreated cells of the same age (to give the probable percentage of deaths due solely to the loss of dark repair), the data indicate that percent death and reduced viability increases linearly with increased clonal age (the coefficient of correlation is r = 0.97). An alternative nonparametric analysis of this data (to circumvent the necessity of use of a transformation on percent death in order to stablize the variance) reveals a Spearman’s rank correlation coefficient of rs = 1.0 (ZAR1974). The probability of obtaining rs = 1.0 if there is no positive correlation between loss of dark repair and increased fission age

TABLE 1

Analysis of intraclonal vs. interclonal variations

Age’ ut tj: PI 33 30 -1.36 > 0.1 45 54 -2.52 < 0.02 61 78 -1.62 > 0.1 80 70 $0.002 > 0.9 108 24 -1.11 > 0.2 128 30 -0.45 > 0.9

* In fissions. +A conservative estimate of the degrees of freedom (df = 2n,-2) was used (LYMAN MCDONALD,personal communication). $ t = t-statlstic. P = probability of observing a t value as large or larger than the observed when in fact there is no significant difference intraclonally us. interclonally. 268 S. R. RODERMEL AND J. SMITH-SONNEBORN

*b 501 IC U f U 40 E 30 E" U OI 20 0 t a 10 $ 0 0 20 40 60 80 100 120 140 AGE (Fissions) FIGURE3.-Loss of dark repair as a function of clonal age. The ordinate represents values obtained after subtracting values of autogamy death and reduced viability of untreated cells from values of autogamy death and reduced viability of UV-treated cells. Each point represents a minimum of 13 autogamies and 1248 progeny. A total of 166 autogamies and 1.4 X IO4 progeny were examined.

is less than 0.05; hence, it can be concluded that there is, indeed, a positive correlation. That the autogamy deaths after UV treatment were due to lethal micronuclear mutations, and not to some other factor(s), is indicated by the finding that UV- induced damage was repaired at all ages by an alternative method, viz. photoreactivation. Moreover, the data in Table 2 shows that loss of photoreactivation repair, as reflected in percent death and reduced viability after photoreactivation, was not correlated with increasing fission age at the UV dose used. If young cells are as sensitive ta UV as old cells, but can repair the damage, death and reduced viability after UV treatment should be as high with these

TABLE 2 Average percent death and reduced viability after autogamy from photoreactivated (P.R.) cells

Age (fissions) 30 43 63 93 145 Average percent death and reduced viability after autogamy and P.R.* 5 7 17 3 13

*These percentage values are corrected for the percent death and reduced viability after autogamy found in untreated control cells; i.e., control values were subtracted from the P.R. treated cells' values. Each value represents an average of a minimum of 13 autogamous depressions and 1248 progeny. The treatment included UV irradiation for 90 sec (2700 ergs/mmZ/min) and P.R. for 1 hour using a BLB light. Linear increase in post-autogamy mortality and reduced viability as fission age increased was not found after P.R. in contrast to the significant correlation found when these cells were subjected to UV only (Figure 3). AGE AND NUCLEAR DAMAGE 269 cells as with aged cells provided that they are not allowed to repair the damage. Young cells (30 fissions old) were thus irradiated at a time in the cell cycle allowing subsequent repair (from division to just prior to the start of micronuclear s) and also at a time allowing no repair (from just prior to and including the start of micronuclear S) (KIMBALL1969). When these cells were contrasted, it was indeed found that at the dose used throughout these experiments, cells irradiated during division showed only 17% death, whereas cells irradiated 2% hours into their interfission period showed 87% death. Thus, the percent death and reduced viability of young clones can mimic that of old clones if irradiation occurs at a time when repair ability is reduced.

DISCUSSION Data have been presented to show that as age increases there is both a linear loss of dark repair and a generalized abrupt increase in lethal micronuclear damage, as evidenced by an abrupt increase in death and reduced viability after autogamy. The question has been raised, however, whether or not death after autogamy is due solely to the occurrence of lethal micronuclear mutations (MITCHISON1955). The discussion, therefore, deals both with the latter question and with an interpretation of the primary findings of this paper. MITCHISON(1955) questioned whether death after autogamy is due solely to the lethal micronuclear mutations, and suggested a possible role for the cytoplasm and/or macronuclear fragments. His three main arguments in support of this hypothesis are, however, highly speculative: 1. In crosses of wild type cells with genetically marked cells derived vegeta- tively from UV-irradiated cells, the exconjugants of irradiated descent had a higher probability of death than exconjugant clones of normal descent. Since both presumably had the same genotype after conjugation, he argued that the cytoplasm and/or macronuclear fragments of the exconjugant of irradiated ancestry caused these cells to die. However, further results tended to support the conclusion that cytogamy (no exchange of gamete nuclei between mates, but a fusion of two nuclei from within the same animal), rather than conjugation, occurred: it was found that when the exconjugants of irradiated ancestry died, the exconjugants of normal descent never showed segregation after autogamy. If conjugation had indeed occurred, the exconjugants of normal descent should have shown segregation of alleles. It is possible that as yet unknown mechanisms are responsible for the aberrant szgregation ratios. 2. Clones with abnormally long inter-autogamous intervals (200-250 fissions) underwent approximately 160/;, macronuclear regeneration, and these cells showed as high a mortality (100%) as cells this age which underwent normal autogamy. Since the cells with nuclei derived from the old macronuclear fragments (macronuclear regenerates) died as frequently as cells with nuclei derived from the meiotic product of the micronuclei, he argued that the expression of lethal micronuclear mutations could not be the sole cause of death after autogamy. It should be noted, however, that cells 200-250 fissions old are extremely 270 S. R. RODERMEL AND J. SMITH-SONNEBORN old and fall into the age-group where the probability is very high that a cell will not give rise to a viable cell at the next division. Thus, it would not be totally unexpected if the post-macronuclear regeneration cell died having developed from the old macronucleus. 3. There was no phenotypic lag in the expression of death after autogamy. If one assumed that only the mature macronucleus was capable of transcription, this death could not be attributed to the gene products of the still immature macronucleus. However, BERGER’S( 1973) recent data show that transcription of the new genome begins while the two anlage are still differentiating into mature macronuclei, well before the first post-autogamous division when MITCH- ISON observed the first autogamous deaths occurring. Therefore, these results need not exclude the expression of the new genome. Evidence presented in this study that death after autogamy can be a result of lethal micronuclear mutations is provided by the finding that photoreactivation was capable of reversing the effect of UV treatment at all ages examined. If the principal effects of photoreactivation is the repair of cyclo-butane type pyrimidine dimers known to be produced by UV light (WACKER1963; SMITH 1964; SETLOW1966; SUTHERLAND,CARRIER and SETLOW1967, 1968), then the increased autogamy death and reduced viability seen in aging clones after UV treatment was in fact due to an increase in lethal micronuclear mutations. Mor- tality after autogamy need not be directly correlated with the number of mutations present. The available data, however, are consistent with a major role for lethal mutations in death after autogamy. One of the major findings of the present study (confirming SONNEBORNand SCHNELLER1960a), was an abrupt increase in percent death and reduced viability around 50-80 fissions. The findings also confirm FUKUSHIMA’S(1975) observation that death and reduced viability after autogamy can be detected earlier than 80 fissions and in some old clones, not at all. If FUKUSHIMAhad used a larger sample of cells, the more general pattern of an abrupt increase in death and reduced viability after autogamy would probably have emerged; his study included 11 autogamies, the present study, 309 autogamies. SONNEBORNand SCHNELLER(1960a) and SONNEBORN(1975) interpreted the abrupt increase in death after autogamy at 80 fissions as an effect of aged cytoplasm since young nuclei in old cytoplasm (merogones) did not normally survive. Aged cytoplasm, therefore, could be actively inducing mutations in the micronuclei. In light of the data in this study showing reduced repair capacity in aged cells, however. another interpretation can be considered. Since the observation of mutations is dependent both on the induction of the mutation and the ability of cells to error-free repair these mutations, loss of repair in aged cells could account for an abrupt increase in mutations at a certain age, i.e.. when the mutation rate exceeded repair capacity. Since the majority of the clones showed few or no mutations before 50-80 fissions, and most clones showed mutations after 80 fissions. either there is an abrupt increase in mutation rate coupled with an inability to repair the increased number of mutations, or at all ages spontaneous mutations are occurring at a AGE AND NUCLEAR DAMAGE 271 very high rate (as shown by the high proportion of cells after 80 fissions with lethal or detrimental mutations), but these mutations are usually repaired before fixation in the genome. The present study cannot distinguish between these alternatives. The data do, however, provide evidence that dark repair ability is declining with increased clonal age in micronuclear DNA. SMITH-SONNEBORN (1971) presented evidence of declining dark repair of macronuclear DNA after UV irradiation as clonal age increased. Decline in the ability to carry out photoreactivation could be detected in aged cells when higher UV doses were used. It should be noted that DIPPELL(1955) and SONNEBORNand SCHNELLER (1960a,b) found an accumulation of chromosomal aberrations in micronuclei of aging cells. All of the age-correlated increased mortality and reduced viability in progeny from aging parents could be due to (1) age correlated increase in chromosomal aberrations, (2) increased mutation rate, (3) other causes and/or (4)loss of a repair system other than the dark repair system. Regardless of the cause of the abrupt increase in death and reduced viability in the untreated aging cells, the increased death after UV as clonal age increases d-oes appear to be due to loss of repair. KIMBALL(1969) has found that if young cells are irradiated just prior to and at the beginning of the micronuclear S phase (or synthesis of DNA) they are most sensitive to UV. He interprets this sensitivity as a time when the cells are refractory to dark repair (such cells when photoreactivated are rescued). The present study confirmed the results of KIM- BALL and found that at the dose used, 87% of the young cells were killed when repair was disallowed and only 17% when repair could take place. Likewise, if random spontaneous mutations were to occur in young cells just prior to micronuclear s, such mutations would be expected to become fixed; therefore the occurrence of mutations in some young clones could be explained. Thus, young cells can mimic old cells if repair is not possible. The most reasonable interpretation of the increased sensitivity of aged cells to UV, considering (1) that photoreactivation can reverse the UV effect and (2) that young cells can be affected as severely as old cells if irradiated when dark repair ability is minimal, is that old cells have lost the ability to error-free repair their DNA damage. Analysis of inter- uersus intraclonal variation in the percent death and reduced viability in progeny from parents at given fission ages indicated that intraclonal variation increased after 50-80 fissions. The intraclonal similarity at early ages could be attributed to the fact that most clones did not show reduced viability after autogamy at the early ages, and those which showed high mortality may have had the defect before separation of the sublines within the clone, thereby skewing the data in favor of more similarity within than between clones. The fact that clones diverged in their expression of death and reduced viability after 80 fissions indicated that random events were occurring. Such a conclusion of randomness need not exclude a contribution from a programmed process. If loss of repair was continuous, and at 80 fissions random damage became irrepar- able, damage would be expressed after this time in the life span. It should be emphasized that clones which expressed no lethal mutations after autogamy throughout their life span could have been cells which underwent 2 72 S. R. RODERMEL AND J. SMITH-SONNEBORN undetected autogamy in the isolation lines. Such an explanation for the failure of clone D to express an increase in death and reduced viability as the parent cells aged is unlikely since a high level of death and mortality was observed throughout the life span of the parent cells. However, no increase in the percent death and reduced viability need not imply a constant number of mutations; it cannot be said with certainty the number of mutations associated with a given percentage of death and reduced viability after autogamy (e.g.,more than one lethal mutation on the same chromosome would not necessarily increase the percent death observed). Clone D may also have been initiated with chromosomal aberrations. Mutations, when they increased, could have been masked by these aberrations. The simplest explanation of the above study would be that stochastic “hits” mutate the micronucleus and the organism is programmed to lose error-free repair. Whether spontaneous mutations are simply unrepaired or a hostile cytoplasm induces the “hits” after 80 fissions is not known. Other studies in our laboratory ( SMITH-SONNEBORNand RODERMEL1976) indicate that aged cells (160 fissions old) can be starving in the presence of excess food (starvation could lead to loss of available precursors to repair damage). However, cells 80 fissions old have an average of 14 food vacuoles and are not, at least superficially, “starving”, unless digestion within food vacuoles is impaired at this age. On the other hand, age-induced error-prone repair (TROSKOand HART1976) or increased chromosomal aberrations could also account for the observed results: aging human cells have a 100-fold increase in their rate of mutation, producing cells incapable of glucose-6-phospho-dehydrogenasesynthesis (HOLLIDAY1972), and increased chromosomal aberrations have been observed in vitro in both mammalian WI-38 and MRC-5 senescent cell lines (SAKSELAand MOORHEAD 1963; THOMPSONand HOLLIDAY 1975; BENN1976). This study does not imply that vegetative aging in paramecia is due to loss of repair of micronuclear mutations since the micronucleus docs not control the phenotype of the cell, only the genotype for the progeny generation. To extend these findings to a more general aging hypothesis. one would have to assume that the micronuclei have a function in survival not yet uncovered, or that similar changes are occurring in the macronucleus--i.e., random mutations that are unrepaired. SMITH-SONNEBORN(1971) did provide evidence for a similar loss of repair with respect to the macronucleus; thus, the accumulation of somatic mutations would be expected to contribute to the demise of the cell. HARTand SETLOW(1974) have shown that life span is correlated to repair ability, and it seems likely that repair ability is a major variable in cellular aging in paramecia. Loss of error-free repair may not be the primary cause of aging, but it will surely contribute to cell death. This work was supported in part from National Science Foundation grant BMS 7501165, PCM 77-04315 and the Arts and Sciences Division of Basic Research, University of Wyoming. The authors wish to express thanks to DH. LYMANMCDONALD for his statistical consultation. Gratitude is also expressed to DR. T. M. SONNEBORNfor his helpful suggestions in the prepara- tion of this manuscript. AGE AND NUCLEAR DAMAGE 273

One of the authors, STEVENR. RODERMEL,submitted this study in partial fulfillment of the requirements for the Master of Arts Degree in the Department of Zoology and Physiology, the University of Wyoming.

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