Macronuclear Differentiation Research Articles

Histone rearrangements accompany nuclear differentiation and dedifferentiation in Tetrahymena

Histone synthesis and deposition into specific classes of nuclei has been investigated in starved and conjugating Tetrahymena. During starvation and early stages of conjugation (between 0 and 5 hr after opposite mating types are mixed), micronuclei selectively lose preexisting micronuclear-specific histones α, β, γ, and H3 F. Of these histones, only α appears to accumulate in micronuclear chromatin through active synthesis and deposition during the mating process. Curiously, α is not observed (by stain or label) in young macronuclear anlagen (4C, 10 hr of conjugation). Thus, young macronuclear anlagen are missing all of the histones which are known to be specific to micronuclei of vegetative cells. By 14–16 hr of conjugation, we observe active synthesis and deposition of macronuclear-specific histones, hv1, hv2, and H1, into new macronuclear anlagen (8C). Thus macronuclear differentiation seems well underway by this time of conjugation. It is also in this time period (14–16 hr) that we first detect significant amounts of micronuclear-specific H1-like polypeptides β and γ in micronuclear extracts. These polypeptides do not seem to be synthesized during this period, which suggests that β and γ are derived from a precursor molecule(s). Since these micronuclear-specific histones do not appear in micronuclear chromatin until after other micronuclei have been selected to differentiate as macronuclei, we suspect that micronuclear differentiation is also an important process which occurs in 10–16 hr mating cells. Our results also suggest that proteolytic processing of micronuclear H3 S into H3 F (which occurs in a cell cycle dependent fashion during vegetative growth) is not operative during most if not all of conjugation. Thus micronuclei of mating cells contain only H3 S which also seems consistent with the fact that some micronuclei differentiate into new macronuclei (micronuclear H3 S is indistinguishable from macronuclear H3). Interestingly, the only H3 synthesized and deposited into the former macronucleus of mating cells is the relatively minor macronuclear-specific H3-like variant, hv2. These results demonstrate that significant histone rearrangements occur during conjugation in Tetrahymena in a manner consistent with the fact that during conjugation some micronuclei eventually differentiate into new macronuclei. Our results suggest that selective synthesis and deposition of specific histones (and histone variants) plays an important role in the nuclear differentiation process in Tetrahymena. The disappearance of specific histones also raises the possibility that developmentally regulated proteolytic processing of specific histones plays an important (and previously unsuspected) role in this system.

Elimination of DNA sequences during macronuclear differentiation in Tetrahymena thermophila, as detected by in situ hybridization.

Previous studies have indicated that certain sequences in the micronuclear genome are absent from the somatic macronucleus of Tetrahymena (Yao and Gorovsky, 1974; Yao and Gall, 1979; Yao, submitted). The present study used in situ hybridization to follow the elimination process during the formation of the new macronucleus. Micronuclear-specific DNA cloned in recombinant plasmids was labelled with 3H and hybridized to cytological preparations of T. thermophila at various stages of conjugation. Despite a smaller size and lower DNA content, the micronucleus has more hybridization than the mature macronucleus. Hybridization initially increased in the anlage (newly developing macronucleus) to reach a maximal level right after the old macronuclei has disappeared. The hybridization in the anlage than decreased to a significant extent prior to the first cell division. The results suggest that the micronuclear-specific sequence is first replicated a few rounds before it is eliminated from the anlage, and the elimination process occurs without nuclear division.

Positional control of nuclear differentiation in paramecium

Nuclear reorganization, which results in the differentiation between macronuclear anlagen and micronuclei during autogamy or conjugation in Paramecium tetraurelia, was compared in wild-type cells and in two mutants, mic44 and kin241, which form abnormal numbers of macronuclear anlagen and micronuclei. Our observations show that all macronuclear anlagen derive from the nuclei positioned at the posterior pole of the cell at the second postzygotic division. This posterior localization is transient and correlated with a marked change in cell shape and decrease of cell length. These results suggest that cytoplasmic or cortical factors precisely located in the posterior pole are essential to trigger macronuclear differentiation and that the control of nuclear positioning is dependent upon precise modifications of cell shape.

GENETIC ANALYSIS OF MATING TYPE DIFFERENTIATION IN PARAMECIUM TETRAURELIA. II. ROLE OF THE MICRONUCLEI IN MATING-TYPE DETERMINATION

The two complementary mating types, O and E, of Paramecium tetraurelia are normally inherited cytoplasmically. This property has generally been interpreted to indicate the presence of cytoplasmic factors that determine macronuclear differentiation towards O or E. In these macronuclear-cytoplasmic interactions, the micronuclei were held to be unbiased and the determination to be established in the course of macronuclear development. In order to ascertain whether the micronuclei were actually neutral, amicronucleate clones were needed and a method to produce them was developed. In crosses between amicronucleate clones and normal micronucleate clones, we have observed regular deviations from cytoplasmic inheritance: the commonest deviation is that most O amicronucleate cells become E when they receive a micronucleus from an E partner. The data can be interpreted by assuming that the micronuclei are predetermined and that the apparent "cytoplasmic" inheritance of the two mating types is due, in E cells, to E-determining factors present in the cytoplasm and in the nucleus; and, in O cells, to O-determining factors present only or mainly in the nucleus.

Colchicine and differentiation of macronuclear anlagen in Paramecium caudatum

In exconjugants of Paramecium caudatum, 4 macronuclear anlagen out of 8 postzygotic division products with the same appearance were differentiated. When the postzygotic nuclear division was inhibited by colchicine, normal macronuclear anlagen were developed but number of the anlagen was fewer than normal one. The result suggests a possibility that the role of postzygotic nuclear division in macronuclear differentiation is to produce regular number of anlagen but not to develop them. Moreover, micronuclear differentiation seemed to occur regardless of the inhibition of postzygotic nuclear division. The result suggesting independence of macronuclear differentiation and fragmentation of old macronucleus was also obtained.

Macronuclear differentiation during oral regeneration in Stentor coeruleus.

The moniliform macronucleus of Stentor coeruleus coalesces and renodulates during division, reorganization and regeneration. These nuclear events are spatially and temporally synchronized with oral primordium development occurring at stages six and seven of membranellar morphogenesis. Coalesced, elongating and early renodulating macronuclei at states six and seven contained microtubules within double membrane-bound channels, passing through the nucleus parallel to the long axis. The number of microtubules per channel varied between 4 and 23. Microtubules were also found in the perinuclear cytoplasm at these stages, forming a loose network around the nucleus. The microtubules and channels are absent in control cells and macronuclei of regenerating cells prior to stage six. These transient microtubules and channels appearing in late stage six and stage seven may provide the axial plane on which elongation of the macronucleus proceeds.

Serotype expression and transformation in Tetrahymena pyriformis.

SYNOPSIS. Of the 4 serotypes of Tetrahymena pyriformis, syngen 1, the H (expressed at 20–35 C) and T (expressed at 36–40 C) serotypes and their genetic bases have received the most attention. The present study describes the L and I serotypes, considers their transformations to and from the H serotype, and compares inter‐locus repression with allelic repression.The L serotype occurs below 20 C. No strain differences have been found between L antigens. However, the rate of transformation between H and L varies in the families, which can be ranked according to their preference for the H or L serotype. A3 is strongly L; D1 is strongly H; the other families form a continuum between these extremes. Preference for the H or L serotype is not correlated with the H allele present.Serotype differences may be hereditary, by the “dilution” test for hereditary differences. Altho the environment largely determines which serotype will be expressed, sublines can maintain established H and L serotypes for more than 40 fissions in a common environment. Crosses between H and L cells suggest macronuclear control of serotype determination and a cytoplasmic influence on macronuclear differentiation. Evidence for the cytoplasmic influence comes from both conjugants and nonconjugants.The I serotype is induced from the H serotype at room temperature by treatment with specific H antibodies, which are also necessary for I maintenance. The I serotype consists of a series of unstable serotypes which transform among themselves. Clones go thru an initial I type one day after induction and then diversify to express secondary types. The initial type and the secondary types are characteristic of the family but are not related to the H genotype. Family C1 lacks 2 secondary types found in other families studied.The differences between inter‐locus repression and allelic repression are marked. Altho inter‐locus repression is labile, all efforts to reverse allelic differentiations have failed. H heterozygotes maintained for long periods of time in the I or L state showed no reversal of H allelic repression.

Clonal analysis of nuclear differentiation in Tetrahymena

Nuclear differentiation is responsible for mating type determination in many ciliates. In Tetrahymena pyriformis, syngen 1, the focus of differentiation is the macronuclear subunit. We have analyzed mating type distribution within caryonides of these strains, in order to derive some insight into nuclear events. Cell lineages were established for the first two macronuclear divisions following conjugation. The resulting secondary subcaryonides were expanded 30-fold, and each subline was tested for mating type. Two kinds of caryonides were observed: monotypic caryonides yielded sublines all of the same mating type; polytypic caryonides produced sublines of different mating types. Most polytypic caryonides were ditypes, with one mating type (the majority type) in excess over the other (the minority type). No evidence was found for any systematic relationship between the mating types in a ditypic caryonide, except that mating type II appears primarily as a monotype. Thus, the mating type combinations and some aspects of the quantitative patterns involve indeterminate or random nuclear events. The nuclear events are not entirely random, however. Coordination among subunits being differentiated is indicated by the large numbers of monotypic caryonides, the rarity of high polytypes and the eccentricity of outputs in ditypes. The output ratios suggest that the most likely time for subunit differentiation is the 32-unit stage. The distribution of mating types among sublines indicates that subunit assortment is nonrandom for the first two macronuclear divisions. Finally, reexamination of an aberrant pair which might have indicated an exception to early macronuclear differentiation provides no unequivocal evidence for an exception to the rule of caryonidal inheritance in this syngen of T. pyriformis.

Sequential nuclear differentiation in Tetrahymena.

UCLEAR differentiation is often responsible for phenotypic differences among genically identical lines in ciliates and is demonstrated by patterns of assortment in vegetative pedigrees. The first example of such a nuclear differentiation ( SONNEBORN 1937, 1939) was the “caryonidal inheritance” of mating types in syngen 1 of Paramecium aurelia. In this case, macronuclei usually differentiate in an “all-or-none” fashion early in their development, and mating types are distributed at the cell divisions at which whole new macronuclei are assorted. Macronuclear differentiation is also the basis for mating type determination in the Group B syngens of P. aurelia, which appeared initially to manifest “cytoplasmic inheritance” (SONNEBORN 1954; NANNEY 1957). In syngen 1 of Tetrahymena pyriformis, macronuclear differentiation is again responsible for intraclonal mating type differences, but the diploid subunits of a single compound macronucleus often differentiate to express different mating types and then continue to assort for many cell divisions (NANNEY 1956; ALLEN and NANNEY 1958). Submacronuclear differentiation and vegetative assortment have also been demonstrated to underlie intraclonal variations in the H-immobilization antigens (NANNEY and DUBERT 1960) and the esterase and acid phosphatase isozymes (ALLEN 1960,1961 ; ALLEN, MISCH and MORRISON 1963). The specificity of the H-immobilization antigens is controlled by alleles at the H locus. Within each of the 11 inbred strains of syngen 1, T. pyriformis, one of four alleles (HA, HC, HD, and HE) is found in homozygous condition. Heterozygotes at the H locus initially express both antigenic types, but in the course of cellular proliferation produce sublines expressing only one or the other serotype. This intraclonal differentiation is interpreted as due to the repression or inactivation of one of the two alleles in each subnucleus. The probabilities of repression of the two alleles determine the ratio of the two kinds of pure subclones, which is characteristic of the allelic combination. An analysis of the distribution of this “output ratio” in vegetative pedigrees shows that the H antigenic determination occurs either before or soon after the first macronuclear division, as an uncoordinated event among the subnuclei (NANNEY, NAGEL and TOUCHBERRY 1964). The timing of allelic repression has been studied in three of the possible six heterozygotes at the H locus. The objective of the present study was to extend and complete the analysis of the remaining allelic combinations. The distributions within

Antigenic transformation in relation to nutritional conditions and the interautogamous cycle in Paramecium aurelia

In syngen 4, stock 51, KK paramecin-sensitive, mating type VII Paramecium aurelia, the transformation of antigenic type 51 B, induced by homologous antiserum treatment followed by exposure to 31 °C, was studied in the course of autogamy-immature and autogamy-mature stages of the life cycle under conditions of maximal growth in mass culture. It was found that during the autogamy-immature stage of the life cycle the induced antigenic transformation was exclusively 51 B to 51 A, while during the autogamy-mature stage of the life cycle the transformation 51 B to 51 D as well as 51 B to 51 A was observed. The underlying mechanism of this phenomenon is obscure. However, the author is inclined to support the view that this altered pattern of transformation of serotype during the autogamy-mature stage of the life cycle may be the expression of macronuclear differentiation in the meaning suggested previously by Skaar [12]. In mating type VII the frequency of transformation from 51 B to 51 A was significantly higher in starved animals than in animals kept in conditions of maximal growth. In one series of experiments with mating type VIII, the antigenic transformation from 51 B to 51 A was dominating over the whole period of the life cycle, while transformation from 51 B to 51 D was rare and occurred only during the autogamy-mature stage of the life cycle.

Nuclear differentiation and transitional cellular phenotypes in the life cycle of Paramecium

The life cycle of Paramecium aurelia includes successive stages of sexual immaturity, maturity and senescence reflecting a series of cellular phenotypes which are heritable for brief periods; in the course of vegetative reproduction new cell types are differentiated and the clone has achieved the next life cycle stage. Conjugation transforms a sexually mature cell into a sexually immature exconjugant. The duration of the period of sexual immaturity is shown to be controlled by the action of at least one genetic locus and by the past history of the parental clone. Experimental techniques broadly analogous to nuclear transplantations in amphibia and other microorganisms permit an analysis of the role of the macronucleus in the differentiation of new life cycle stages; the data are consistent with the hypothesis that the macronucleus undergoes age-correlated changes and that these are ultimately responsible for the reappearance of mature cells in an exconjugant clone. Since the parental generation is found to influence the duration of immaturity of its progeny, the prezygotic macronucleus is presumed to control, in some unknown manner, the development of the postzygotic macronucleus. The hypothesis of progressive macronuclear differentiation and internuclear communication appears to account in a satisfactory way for other life cycle phenomena.