Development of sexual maturity in the ciliate Euplotes crassus: Sources of variation in the timing of maturity

The life styles of ciliated protists are particularly suitable for experimental analyses of certain aspects of developmental and genetic biology. The progression from sexual immaturity tomaturity to senescence represents one of the most intriguing aspects of developmental programs. The extent to which progeny clones, their subclones, and testers used in the assay result in different lengths of immaturity has been investigated in Euplotes crassus. Six subclones from each of 12 progeny clones from a cross between stocks EC1 and EC2 were tested for maturity with stocks EC3, EC4, and EC5 on every transfer. Analysis of variance was used to partition thetotal variation in fissions to maturity into parts due to clones, subclones, and testers and the interactions between these levels. The error, interaction of subclones and testers, corresponds to a standard deviation of only 4.1 fissions, while the within clone within tester means range from 15.2 to 46.7 fissions; all levels except testers contribute significantly to the total variation. Most of the variability is attributable to clones (66%), the next most to error (16%), the next most to interaction of clones by testers (13%), and the least to subclones (5%). An a posteriori analysis examined whether the differences among clones were due to the cytoplasm of the clone ancestor (exconjugant), its mat (mating-type) locus genotype, or the mated pair it came from. None of these characteristics was able to interpret simply the large variability among clones. These results provide evidence that the transition from immaturity to maturity is quantitative and complex rather than a jump from one well-defined state to another. © 1992 Wiley-Liss, Inc.     

Adolescence and the Reversibility of Maturity in Euplotes crassus

ABSTRACT. The clonal life history of ciliated protists is characterized by a sequence of phenotypes; sexual immaturity, maturity, and senescence. The distinctiveness of immaturity and maturity has been investigated. Standard assays of the onset of maturity of progeny clones from a cross between stocks EC1 and EC2 of Euplotes crassus demonstrated significant differences among clones and among testers within clones. They also revealed that the first positive test(s) of a progeny subclone were typically followed by at least one negative test. Special protocols were devised to investigate if maturity was reversible at the cellular level. In these experiments, the first mating pair of a progeny subclone was split before the consummation of mating. From these two cells as well as from control progeny and tester cells, subclones were established and every leftover cell was tested for maturity after each transfer. Both standard and split-pair progeny subclones had immature and slow- to-mate cells. The number of fissions before progeny exhibited sexual behavior indistinguishable from the testers was more than twice that to the first mating reaction of a subclone. At the first sign of maturity, progeny lines are a heterogeneous population of cells able and not able to mate, but remarkably, clonal descendants of those able to mate may become unable to mate. The development of maturity is progressive, quantitative and non-monotonic rather than an instantaneous switch.     

Methods for Inducing Selfing, Selfing and Its Role in the Life Cycle of Euplotes woodruffi syngen 3 (Ciliophora)

Methods for inducing selfing, and the relation between selfing and the life cycle of Euplotes woodruffi syngen 3 are reported. Three intercrossing stocks were used in this experiment. Selfing was induced with several treatments as follows: cell-free fluid from the cultures of complementary mating types; intact cells of GI or S phase in the cell cycle; heat-killed cells, and lysed cells of GI-, S-, and D-phase cells which were prepared by freeze-thawing. Stock SJ-27 was used as a parental stock from which Fl clones were originated through selfing. The other two stocks, SJ-8 and SJ-19, were used as testers. The period of immaturity varied from clone to clone. The heterotypic conjugation of clones with cells of stock SJ-8 seems to occur earlier in the life cycle than with cells of stock SJ-19. This result shows that this syngen has an adolescent period in the life cycle. The length of selfing immaturity seems to be different from that of crossing immaturity, and selfing appeared slightly later than crossing with testers. But the clones in which selfing 1st occurred are considered to be in adolescence or maturity, not in senility. Once selfing appeared in any clone, the clone continued to produce selfing pairs till just before clonal death. The viability of selfing and of outcrossing were compared and found not significantly different. Inbreeding depression took place in some of the F2 clones by successive selfing. The viability of F2 clones from young parents was significantly higher than that from old parents (220 to 230 fissions) both in selfing and outcrossing. The total life spans which were studied in three F1 clones were 168 to 264 fissions.     

The immaturity interval in Tetrahymena: Genetic and environmental sources of variation

The development of sexual maturity has been studied in Tetrahymena hegewischi. Progeny lines do not typically change from immaturity to mating with all different mating types during a single test interval, but about 30% do mature abruptly. Some testers are more likely than others to participate in the earliest mating reactions of progeny lines which do not mature abruptly. Subcaryonidal vegetative pedigrees of 10 pairs from 4 crosses revealed considerable intrapair variation in the time, measured in fissions, of maturity. The average intrapair coefficient of variation was 20%. A nested ANOVA revealed significant genomic effects on the immaturity interval, but no significant cytoplasmic or caryonidal effects; 56% of the total variation was non-genomic. Growth in different environments had highly significant effects on the immaturity interval. Subclones grown at 27°C with alternate day transfers took on the average 2 to 3 times as many fissions to mature as sister subclones grown at 27°C with daily transfers. Subclones grown at 18°C or 34°C and transferred on alternate days had intermediate maturation times. The greatest range in the immaturity interval among lines of the same genotype was from 34 to 143 fissions. The development of maturity in this species involves genetic control of timing, but the genetic differences are obscured by a large amount of intraclonal variation and sensitivity to the environment.     

Suicide is not the inevitable outcome of "perpetual" selfing in tetrahymenines collected from natural habitats.

A significant fraction of the Tetrahymena clones isolated from natural habitats self (mating occurs within a clone). Early attempts to study such clones failed because stable subclones were rarely, if ever, observed, and isolated pairs all died. Isozyme analysis revealed that these wild selfers were a diverse group; some were very similar to T. australis, a species with synclonal mating type determination and to T. elliotti, shown recently to have a karyonidal mating type system. One originally stable clone of T. australis included some selfing clones after a few years in our laboratory. Other clones manifested unique zymograms. Subclones isolated from 18 selfer strains were heterogeneous. All subclones of several selfers mated massively at each transfer through 100 fissions. Selfing among subclones of other selfers was highly variable or not observed. Although 77% of the pairs isolated died, and 9% of the pair cultures selfed, 15 selfers yielded some viable nonselfing "immature" progeny. Additional immature progeny were obtained by isolating pairs from macronuclear retention synclones. Although some "immature" progeny eventually selfed, most remained stable. Giemsa staining revealed macronuclear anlagen in nearly all mating pairs and some anomalies. Crosses among the F1 progeny clones of the T. elliotti selfers yield viability data comparable to those from crosses among normal strains. Perhaps perpetual selfing is a mechanism of getting rid of deleterious combinations of genes and uncovering better combinations in homozygous state by playing genetic roulette.     

Suicide is not the inevitable outcome of “perpetual” selfing in tetrahymenines collected from natural habitats

A significant fraction of the Tetrahymena clones isolated from natural habitats self (mating occurs within a clone). Early attempts to study such clones failed because stable subclones were rarely, if ever, observed, and isolated pairs all died. Isozyme analysis revealed that these wild selfers were a diverse group; some were very similar to T. australis, a species with synclonal mating type determination and to T. elliotti, shown recently to have a karyonidal mating type system. One originally stable clone of T. australis included some selfing clones after a few years in our laboratory. Other clones manifested unique zymograms. Subclones isolated from 18 selfer strains were heterogeneous. All subclones of several selfers mated massively at each transfer through 100 fissions. Selfing among subclones of other selfers was highly variable or not observed. Although 77% of the pairs isolated died, and 9% of the pair cultures selfed, 15 selfers yielded some viable nonselfing “immature” progeny. Additional immature progeny were obtained by isolating pairs from macronuclear retention synclones. Although some “immature” progeny eventually selfed, most remained stable. Giemsa staining revealed macronuclear anlagen in nearly all mating pairs and some anomalies. Crosses among the F1 progeny clones of the T. elliotti selfers yield viability data comparable to those from crosses among normal strains. Perhaps perpetual selfing is a mechanism of getting rid of deleterious combinations of genes and uncovering better combinations in homozygous state by playing genetic roulette. © 1992 Wiley-Liss, Inc.     

Life Cycle of Autogamous Strains of Euplotes minuta*

SYNOPSIS. The life cycles of 5 autogamous strains of Euplotes minuta are reported. Interautogamic intervals (measured as number of fissions) are quite variable among clones belonging to the same strain, while their variability is much reduced (15 fissions) among sublines of the same clone. By selecting the clone with the shortest immature period to start successive autogamous generations, it has been found that all clones undergo autogamy almost synchronously and have a very short period of immaturity at the 5th autogamous generation. Both conjugation and autogamy, however, are consistently followed by immature periods in all autogamous strains examined. Mating capacity as well as competence for autogamy are reached almost simultaneously in all clones of the strains studied with the exception of about 1/3 of the A-31 clones in which autogamy occurs significantly earlier than conjugation. The results are discussed from the genetic point of view and in relation to the sexual mechanisms operating in nature within different populations of the species.     

Mating-Type Inheritance and Maturity Times in Crosses between Subspecies of TETRAHYMENA PIGMENTOSA

Subspecies 6 and 8 of T. pigmentosa (formerly syngens 6 and 8 of T. pyriformis) share a mating-type system controlled by three alleles with "peck-order" dominance at a single locus. The system is apparently closed and limited to three mating types that are homologous, but not identical, in the subspecies. These relationships are reflected in new mating-type designations.—The viability in some intersyngenic crosses is excellent, and the inheritance of major mating types in first-generation hybrids and their progeny follows the pattern of subspecies 8.—The period of immaturity is shorter than that previously reported for subspecies 8, with 50% of the subclones maturing between 46 and 100 fissions after conjugation. Maturity curves are generally sigmoid, but some are apparently biphasic. The onset of maturity in triplicate sublines from the same synclone is usually highly correlated.     

Variability of autogamy-maturation pattern in genetically identical populations of Paramecium tetraurelia

Autogamy in Paramecium tetraurelia is a form of sexual reproduction in a single cell that results in homozygosity in every genetic locus. Autogamy becomes inducible by natural starvation several fissions after the previous autogamy, and percent autogamy increases gradually with clonal age to reach 100%. We here report the degree of variability of the autogamy-maturation pattern, and how it is inherited through autogamous generations. We assessed the autogamy-maturation pattern by monitoring percent autogamy at the ages of 9, 18 and 27 fissions in the wild-type stock 51. To determine how the autogamy-maturation pattern is inherited, clones that showed the lowest and the highest percent autogamy at age 18 in a given autogamous generation (Gn) were examined for their percent autogamy in the next autogamous generation (Gn+1). This procedure was repeated through successive autogamous generations. We found that percent autogamy at ages 9 and 27 was rather stable (low and high, respectively), while it was extremely variable at age 18 ranging from 3% to 100%. We also found that percent autogamy at age 18 in the progeny clones was variable irrespective of percent autogamy at age 18 in the parental clones; there was no regular rule such as producing progeny with higher (or lower) percent autogamy from parents with lower (or higher) percent autogamy.     

Mutants of Sexual Maturity in PARAMECIUM CAUDATUM Selected by Erythromycin Resistance

Cells of Paramecium caudatum, syngen 3 usually become sexually mature about 50 fissions after conjugation. In order to study the genetic mechanisms that control fission-dependent expression of maturity, an attempt was made to obtain early mature mutants by treatment with N-methyl-N'-nitro-N-nitro-soguanidine. A new cytoplasmic marker, erythromycin resistance, was used to eliminate nonconjugant and macronuclear regeneration clones. Twenty early mature clones were obtained from five different mutagenized cultures. Three of them were genetically analyzed by crosses to wild-type stocks. The results show all three mutants to be controlled by incompletely dominant genes, i.e., the homozygotes became mature 20-25 fissions and the heterozygotes 15 fissions earlier than the wild-type clones. At least two different loci are suggested for the early maturity.     

Correlation of the Period Length of Circadian Rhythms with the Length of Immaturity in Paramecium bursaria

The circadian photoaccumulation rhythm of thirty strains of Paramecium bursaria collected at different places in Japan and China were measured with a microcomputer assisted data collection apparatus. Although most strains showed a period of 23-26 hours in LL, we found two strains of conspicuously different periods; a short period strain (UK1, 21.8 hr) and a long period strain (T316, 28.7 hr). F1 progeny from a cross between the short and the long period strains showed an intermediate period of about 24.7 hours (range 22.5-25.8 hr). The character was not distributed in a Mendelian ratio among the F1 progeny. We isolated a mutant (E2) with short period (21.8 hr) from the stock strain Kz1 by treatment with nitrosoguanidine (MNNG). The progeny of crosses between E2 and UK1, and between E2 and T316 exhibited the short period and the normal period phenotype respectively. Moreover, the progeny from a cross between E2 and a wild type strain (Sj2w) became sexually mature about 25 fissions after conjugation. This length of immaturity is much shorter than that of the progeny from wild type strains (about 50 fissions). This early maturation character was inherited to progeny in a Mendelian ratio. Homozygotes for the early maturation allele (EM2) exhibited mating ability about 15 fissions after conjugation. These data suggest that there is a correlation between the period length of the circadian rhythm and the length of immaturity after conjugation in Paramecium bursaria.     

Actinomycin D-induced change of mating type in the ciliate Dileptus anser

The effects of actinomycin D on the expression and inheritance of mating types (MTs) were studied in mature laboratory clones of the ciliate Dileptus anser. In these ciliates, each mature clone isolated from the natural population belongs to one of three complementary MTs, i.e., MT I, MT II, or MT III. In the course of further cultivation of the clone under laboratory conditions, in a series of vegetative generations, its MT remained unchanged. However, treatment with actinomycin D (15 μg/ml, 3 days) causes these clones to transition to a state that is hereditarily unstable for their MTs. At weekly testing for MT for at least 15 weeks after this treatment (which corresponds to more than 100 cell divisions), many subclones of the treated clone were observed to reversibly exchange their MT for another one; a temporary state of immaturity and/or partial maturity was also revealed. These data confirm our hypothesis about epigenetic MT determination in D. anser. Taking into account that actinomycin D also induces the inheritable destabilization of some characters in amoebas Amoeba proteus, which obviously has an epigenetic nature, this antibiotic might be considered an epimutagen.     

Spontaneous Conversion of Nontransformed Avian Sarcoma Virus-Infected Rat Cells to the Transformed Phenotype

Normal rat kidney (NRK) fibroblasts were infected with the Schmidt-Ruppin strain (SR-D) of avian sarcoma virus (ASV) and cloned 20 h after infection without selection for the transformed phenotype. Most infected clones initially exhibited the flat, nontransformed morphology that is characteristic of uninfected NRK cells. In long-term culture, however, the majority of the SR-D NRK clones began segregating typical ASV-transformed cells. Transforming ASV could be rescued by fusion with chicken embryo fibroblasts from most of the infected clones tested. Three predominantly flat, independently infected clones were further analyzed by subcloning 8 to 10 weeks after infection. Most flat progeny subclones derived at random from two of these "parental" SR-D NRK clonal lines did not yield virus upon fusion with chicken embryo fibroblasts, although a nondefective transforming ASV was repeatedly recovered from the parental clones. This observation suggested that most, but not all, daughter cells in these SR-D NRK clones lost the ASV provirus after cloning. The progeny of the third independent parental cell clone, c17, gave rise to both flat and transformed subclones that carried ASV. In this case, ASV recovery by fusion and transfection from the progeny subclones was equally efficient regardless of the transformation phenotype of the cells. The 60,000-dalton phosphoprotein product of the ASV src gene was, however, expressed at high level only in the transformed variants. The results of a Luria-Delbruck fluctuation analysis and of Newcombe's respreading test indicated that the event leading to the spontaneous conversion to the transformed state occurred at random in dividing cultures of these flat ASV NRK cells at a rate predicted for somatic mutation.     

Analysis of germinal aging in Paramecium caudatum by micronuclear transplantation

The role of the micronucleus in the age-dependent increase in mortality after conjugation in Paramecium has been investigated using micronuclear transplantation. The clone of Paramecium caudatum used for this study had a lifespan of about 750 fissions. In this clone, the fission rate began to decrease about 450 fissions after conjugation. Mortality after selfing conjugation also began to appear at about 450 fissions and gradually increased with clonal age. Cells at about 650 fissions showed 10–70% survival after selfing conjugation but when their micronuclei were transplanted into amicronucleate cells of about 450 fissions, the progeny survival increased to 70–90%. When micronuclei from cells 700–750 fissions old were transplanted into amicronucleate cells of 100–150 fissions, however, increase in progeny survival was very rare. The results indicate that micronuclei in cells up to the age of 650 fissions can function normally if the cytoplasmic environment is young.     

Novel features of computer-simulated clonal life of Paramecium caudatum

The clone of the ciliated protozoan Paramecium caudatum has the immaturity period of about 60 fissions and the lifespan of about 600 fissions. These life cycle figures have been depicted through laboratory experiments that allow continuous cell divisions for hundreds, which never occur in nature. We here constructed the nature-mimicking model culture that alternated the log- and stationary phases to allow conjugation, and computer-simulated the age structure modifying parameters such as cell distributions to start the culture, fission rates, death rates, immaturity periods, probabilities of conjugation, proportions of transplantation and so on. The average and maximum ages in the culture after thousands of alternations were converged to 43±2 and 140±5 fissions, respectively, when parameters for the immaturity period and the maximum clonal lifespan were set at 60 and 600 fissions. This result explains why cells collected in nature are usually young and vigorous. The average and maximum ages proportionally prolonged as the immaturity period was prolonged, as reported true for species of the ciliate. These results indicate the validity of our simulation. The average and maximum ages remained unchanged when the initial condition for starting the culture was changed from two complementary mating-type cells to a population with a quadratic-function distribution, and when the fission rate at the log-phase and the death rate at the stationary phase were modified for older ages. The average and maximum ages changed slightly when either the conjugation rate or the proportion of transplantation was somewhat lowered. Although they changed considerably when such parameters as the immaturity period, conjugation rate and death rate were extremely modified, no clones with the age over 230 fissions appeared in any simulations. These results indicate the robustness of the model, which provides us with fresh insight into the structural system of the clonal lifespan of P. caudatum in nature.     

Age-Correlated Changes in Expression of Micronuclear Damage and Repair in PARAMECIUM TETRAURELIA

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.

     

Interactions between newly developed macronuclei and maternal macronuclei in sexually immature multinucleate exconjugants of Paramecium caudatum

The macronucleus of Paramecium caudatum controls most cellular activities, including sexual immaturity after conjugation. Exconjugant cells have two macronuclear forms: (1) fragments of the maternal macronucleus, and (2) the new macronuclei that develop from the division products of a fertilization micronucleus. The fragments are distributed into daughter cells without nuclear division and persist for at least eight cell cycles after conjugation. Conjugation between heterokaryons revealed that the fragmented maternal macronuclei continued to express genetic information for up to eight cell cycles. When the newly developed macronucleus was removed artificially within four cell cycles after conjugation, the clones regenerated the macronuclear fragments (macronuclear regeneration; MR) and showed mating reactivity, because they were sexually mature. However, when the new macronucleus was removed during later stages, many MR clones did not show mating reactivity. In some extreme cases, immaturity continued for more than 50 fissions after conjugation, as seen with normal clones that had new macronuclei derived from a fertilization micronucleus. These results indicate that the immaturity determined by the new macronucleus is not annulled by the regenerated maternal macronucleus. Mature macronuclear fragments may be "reprogrammed" in the presence of the new macronucleus, resulting in their expression of "immaturity."     

Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure

Recombinant Chinese hamster ovary (rCHO) cells expressing a high level of chimeric antibody were obtained by cotransfection of heavy- and light-chain cDNA expression vectors into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level up to 1.0 microM. To determine the clonal variability within the amplified cell population in regard to antibody production stability, 20 subclones were randomly isolated from the amplified cell population at 1.0 microM MTX (CS13-1.0 cells). Clonal analysis showed that CS13-1.0 cells were heterogeneous with regard to specific growth rate (mu) and specific antibody productivity (qAb), although they were derived from a single clone. The mu and qAb of 20 subclones were in the range of 0.51 to 0.72 day-1 and 10.9 to 19.1 microgram/10(6) cells/day, respectively. During 8 weeks of cultivation in the absence of selective pressure, the mu of most subclones did not change significantly. On the other hand, their qAb decreased significantly. Furthermore, the relative decrease in qAb varied among subclones, ranging from 30% to 80%. Southern and Northern blot analyses showed that this decreased qAb resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. For the sake of screening convenience, an attempted was made to correlate the initial properties of subclones (such as mu, qAb, and Ig gene copies) with their antibody production stability during long-term culture. Among these initial properties examined, only qAb of subclones could help to predict their stability to some extent. The subclones with high qAb were relatively stable with regard to antibody production during long-term culture in the absence of selective pressure (P < 0. 005, ANOVA). Taken together, the clonal heterogeneity in an amplified CHO cell population necessitates clonal analysis for screening stable clones with high qAb.     

Levels and stability of DNA methylation in random surviving cell clones derived from a Chinese hamster cell line after prolonged treatment with 5-aza-2'-deoxycytidine

SCC30 cells (derived from a single cell from the Chinese hamster ovary CHO-K1 cell line, selected on the basis of a stable chromosome complement) were used to select cell variants with hypomethylated DNA. Cells were treated with 5-aza-2'-deoxycytidine (5azadCyd) at 0.1, 1, or 5 microM for two weeks with the medium and drug renewed twice weekly. From the few surviving cells, 25 random single cell-derived clones were grown for freezing cell stocks, and for DNA isolation for 5-methyldeoxycytidine (5medCyd) estimations. After a minimum of one month's recovery from the drug, these cells showed a continuum of 5medCyd levels ranging from ones with the same as the parental clone (2.93%) to ones having lost almost 50% of their DNA methylation. The modal value corresponded to a loss of one third to one quarter of methylated sites. Five subclones with hypomethylated DNA were grown from the frozen stocks. These cells were shown not to be 5azaCyd-resistant cell variants. By the time sufficient cells had been grown to determine DNA methylation levels, the average percentage of 5medCyd had increased to 76% of the SCC30 value compared to 67% at the time of freezing cell stocks. However, this level of DNA hypomethylation remained constant over two months of continuous culture. Cells of one of these hypomethylated subclones were subjected to a second cycle of 5azaCyd treatment. Six random clones from the survivors showed a further decrease averaging 11% in the level of DNA methylation but, by two months in continuous culture, 5medCyd levels had returned to that present before the second cycle of selection. Hence, cell variants can be readily obtained which have lost some 8-10 million methylated sites (pairs of methylated deoxycytidines), and this loss does not compromise cell viability in in vitro culture. This is consistent with mammalian genomes containing a high level of background methylation in non-essential sites. The usefulness of such single cell-derived clones with stably hypomethylated genomes is discussed in relation to understanding the functions of deoxycytidine methylation in mammalian DNA.     

Clonal Variation in Paramecium. I. Persistent Unstable Clones

Clones of Paramecium of identical serotype when cultured in test tubes may differ in their ability to give rise to subclones of this serotype. Characteristically, stable clones yield progeny indistinguishable from their parents, while from unstable clones diverse subclones with new serotypes can be isolated repeatedly. Stable lines are resistant to changes in culture medium and also are unaffected by most sera. In contrast, the numbers and kinds of serotypes displayed among subclones derived from unstable lines are often affected by these same agents. Stable and unstable clones are interconvertible when the medium from individual cultures is repeatedly and frequently replaced by fresh culture fluid. This effect is very likely a result of the removal of the initial exhausted medium with any cell products rather than the addition of fresh nutrient.