Cytology of an Indian Race of Blepharisma undulans (Stein)

SYNOPSIS. The Indian race of Blepharisma undulans described in this paper measures 150 μ in length. The macronucleus consists of 5–7 nodes, all of equal size. During binary fission, condensation of macronucleus is followed by its elongation and a thinning of the middle region which breaks with the division of the animal. It later attains the typical vegetative form.
During conjugation 7 or 8 micronuclei pass through the first pregamic division, 5 to 7 through the second pregamic division and one product of the second division takes part in the third division. The rest degenerate. At the same time, the macronucleus also starts degenerating. After the synkaryon has divided twice, the conjugating pairs separate. Of the 4 products, 3 become macronuclear anlagen and one, micronuclear anlage.
The micronuclei divide asynchronously both during binary fission and during conjugation. There is apparently considerable diversity in the structure and behaviour of the macronucleus and micronuclei in the different races of Blepharisma undulans.     

Repetitive Micronuclear Divisions in the Absence of the Macronucleus During Conjugation of Paramecium caudatum

ABSTRACT. The germinal micronucleus divides six times during conjugation of Paramecium caudatum : this includes two meiotic divisions and one mitosis of haploid nuclei during mating, and three mitoses of a fertilization nucleus (synkaryon). Microsurgical removal of the macronucleus showed that micronuclei were able to divide repeatedly in the absence of the macronucleus, after metaphase of meiosis I of the micronucleus and also after synkaryon formation. When the macronucleus was removed after the first division of synkaryon, in an extreme case the synkaryon divided five times and produced 32 nuclei, compared to three divisions and eight nuclei produced in the presence of the macronucleus. Treatment with actinomycin D (100 μ /ml) inhibited the morphological changes of the macronucleus during conjugation and induced a multimicronucleate state in exconjugants. However, in other cells, it induced production of a few giant micronuclei. We conclude that the micronucleus is able to undergo repeated divisions at any stage of conjugation in the absence of the macronucleus once the factor(s) for induction of the micronuclear division has been produced by the macronucleus. The macronucleus may also produce a regulatory factor required to stop micronucler division.     

Nuclear functions in postconjugational development of Paramecium caudatum: Ability to form food vacuoles analyzed by nuclear elimination and implantation

Paramecium caudatum loses the ability to form food vacuoles at the crescent stage of the micronucleus from 5 to 6 hr after the initiation of conjugation and regains it immediately after the third division of the zygotic nucleus. To assess the micronuclear function in the development of the oral apparatus after coniugation, prezygotic micronuclei was removed from cells at various stages of conjugation, and their ability to form food vacuoles were examined. (1) When all of the prezygotic micronuclear derivatives were eliminated before the stage of formation of the zygotic nucleus, the exconjugant did not regain its ability. (2) When a zygotic nucleus or postzygotic nuclei were removed, in some cases the cell formed as many food vacuoles as did nonoperated cells after conjugation, while in other operated cells the number of food vacuoles was subnormal. (3) When a micronucleus from a cell at vegetative phase (G1) was transplanted into a cell of an amicronucleate mating pair at the stage between 8 and 9 hr after the initiation of conjugation, the implanted cell regained the ability to form food vacuoles. However, no cell regained the ability when the implantation was carried out within 1 hr after the separation of the mates. The results show that the micronucleus plays an indispensable role in the development of the oral apparatus at the stages of exchange of gametic nuclei and fertilization and that the micronucleus transplanted from asexual cells can fulfill this function. On the other hand, removal of the macronucleus from exconjugants showed that the maternal macronucleus also has an indispensable function in regaining the ability to form food Vacuoles. © 1992 Wiley-Liss, Inc.     

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.     

Causal relations among cell cycle processes in Tetrahymena pyriformis. An analysis employing temperature-sensitive mutants

Utilization of temperature-sensitive mutants of Tetrahymena pyriformis affected in cell division or developmental pathway selection has permitted elucidation of causal dependencies interrelating micronuclear and macronuclear replication and division, oral development, and cytokinesis. In those mutants in which cell division is specifically blocked at restrictive temperatures, micronuclear division proceeds with somewhat accelerated periodicity but maintains normal coupling to predivision oral development. Macronuclear division is almost totally suppressed in an early acting mutant (mola) that prevents formation of the fission zone, and is variably affected in other mutants (such as mo3) that allow the fission zone to form but arrest constriction. However, macronuclear DNA synthesis can proceed for about four cycles in the nondividing mutant cells. A second class of mutants (psm) undergoes a switch of developmental pathway such that cells fail to enter division but instead repeatedly carry out an unusual type of oral replacement while growing in nutrient medium at the restrictive temperature. Under these circumstances no nuclei divide, yet macronuclear DNA accumulation continues. These results suggest that (a) macronuclear division is stringently affected by restriction of cell division, (b) micronuclear division and replication can continue in cells that are undergoing the type of oral development that is characteristic of division cycles, and (c) macronuclear DNA synthesis can continue in growing cells regardless of their developmental status. The observed relationships among events are consistent with the further suggestion that the cell cycle in this organism may consist of separate clusters of events. with a varying degree of coupling among clusters. A minimal model of the Tetrahymena cell cycle that takes these phenomena into account is suggested.     

Nuclear Differentiation in Paramecium caudatum: Analysis by the Monoclonal Antibody against a Micronuclear Antigen

I obtained the monoclonal antibody 93A against a micronuclear antigen of the ciliate Paramecium caudatum . Immunocytochemical observations showed that the antigen 93A appeared in some portion of the micronucleus in every stage of life cycle. In dividing micronuclei, the antigen appeared mainly in their both poles and in fibrous structures between the poles. These results suggest that the micronuclear antigen 93A may be a component of microtubule organizing center and spindles. During nuclear differentiation in P. caudatum , four among eight postzygotic micronuclei differentiate new macronuclear anlagen and one becomes a new micronucleus and the remaining three degenerate. The micronuclear antigen 93A appeared in all of the eight nuclei in the early stage of macronuclear differentiation but then disappeared in the four macronuclear anlagen and eventually persisted only in the new micronucleus, showing that the newly developing macronuclear anlagen lose the micronuclear antigen 93A during their differentiation.     

Elimination of the germ nuclei of Paramecium tetraurelia by laser microbeam irradiation

The germ nuclei (micronuclei) of Paramecium tetraurelia can be eliminated successfully by irradiating the micronucleus with an argon-ion laser microbeam after sensitization with the dye acridine orange. No immediate cytological damage of the irradiated micronuclei is visible, but they are lost before they enter the next division. This method produces cell lines lacking micronuclei (i.e., amicronucleates). These amicronucleates provide favorable materials for the study of micronuclear functions as well as intra-and inter-specific nucleocytoplasmic interactions. Some preliminary observations show that the micronucleus is not required for macronuclear fragmentation and macronuclear regeneration during sexual reproduction, but suggest that the micronucleus might participate in some asexual cellular function in addition to their gametic role.     

Ultrastructure of the micronucleus of the infusorian Didinium nasutum during meiosis]

Six stages can be distinguished in the micronuclear first maturation division prophase of D. nasutum. Nucleolus-like structures of fibrillar nature, connected with micronuclear chromosomes seem to develop at the late leptotene. At zygotene-pachytene, the chromosomes condense, forming irregular loops. This coincides with formation of classically structured synaptinemal complexes in the micronuclei. At diplotene-diakinesis, chromosomal bivalents are uniformly scattered throughout the micronucleus. They aggregate into a net equatorial plate in the first division metaphase; chromosomes show prominent kinetochores with attached chromosomal microtubule bundles. The second maturation division starts immediately after the completion of the first division and is morphologically similar to agamic mitosis of the micronuclei of D. nasutum. During the 2th maturation division prophase, the compact chromosomes form a dense group and show no spreading inside the nucleus. They are interspaced by an amorphous material being possibly involved in the formation of spindle microtubules. The telophase spindle of the 2nd division likely as that of the Ist division divides into three parts, the two daughter nuclei and the separation spindle containing a material of depolymerized microtubules. Only one of the 2nd division derivatives enters the third maturation division. A short telophasic third division spindle is perpendicular to the surface of the contact between the conjugants and produces two pronuclei. The envelopes of the daughter micronuclei are formed from parts of the original nuclear envelope surrounding the entire spindle.     

Observations on the Life-cycle of a New Race of Blepharisma undulans from India

SYNOPSIS. A full account of the nuclear changes during binary fission and conjugation in a local race of Blepharisma is presented in this paper. The macronucleus consists of 2 nodes connected by a strand. Number of micronuclei varies from 6 to 18. During binary fission, condensation of macronucleus is followed by elongation and thinning of the middle region which finally breaks. Daughter nuclei later attain the typical vegetative form. Notably, during binary fission some micronuclei appear to complete their mitoses by the time the macronucleus attains the condensed form, while others lag behind and exhibit practically every stage of mitosis.
During conjugation, from 6 to 10 micronuclei undergo the first pregamic division, the same number through the second division, and two products of the second division take part in the third division. The rest degenerate. Division products of the nuclei in the paraoral region take part in synkaryon formation. The synkaryon undergoes either 2 or 3 divisions. In the former case, of the 4 products, 2 become the macronuclear anlagen, one the micronucleus and the fourth degenerates. In the latter case, of the 8 products, 3 to 4 become the macronuclear anlagen and the rest become micronuclei. Chromatin elimination has been observed during the division of the macronuclear anlage, followed by an extra metagamic fission of the cell.
Comparison with two other races from India and an American race indicates considerable diversity in the structure and behaviour of the nuclear apparatus in different races of Blepharisma undulans.     

Evolution of Amitosis of the Ciliate Macronucleus: Gain of the Capacity to Divide

Ciliates exhibit nuclear dimorphism, i.e. they have a germline micronucleus and a somatic macronucleus. Macronuclei are differentiated from mitotic sisters of micronuclei. The macronuclei of "higher ciliates" are polyploid and divide acentromerically ("amitotically"); they differentiate once per life cycle. By contrast, Karyorelict (KR) ciliate macronuclei are nearly diploid and cannot divide; they must differentiate at every cell cycle. Diverse lines of evidence are presented to support the hypothesis that ancestral ciliate macronuclei were incapable of division (as in living karyorelict ciliates) and that higher ciliates gained, perhaps independently more than once, the ability to divide the macronucleus. Selective pressures that could have driven the evolution and macronuclear division and two plausible step-wise pathways for the evolution of macronuclear division are proposed. These hypotheses are relevant to our understanding of amitosis mechanisms, evolution of nuclear dimorphism, and phylogenetic classification of ciliates.     

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.     

Nuclear envelope defects impede a proper response to micronuclear DNA lesions

When damage is inflicted in nuclear DNA, cells activate a hierarchical plethora of proteins that constitute the DNA damage response machinery. In contrast to the cell nucleus, the ability of micronuclear DNA lesions to activate this complex network is controversial. In order to determine whether the DNA contained in micronuclei is protected by the cellular damage response system, we studied the recruitment of excision repair factors to photolesions inflicted in the DNA of radiation-induced micronuclei. To perform this analysis, primary human dermal fibroblasts were exposed to UV-C light to induce photolesions in nuclear and micronuclear DNA. By means of immunofluorescence techniques, we observed that most micronuclei were devoid of NER factors. We conclude that UV photoproducts in micronuclei are mostly unable to generate an effective DNA damage response. We observed that the micronuclear envelope structure is a determinant factor that influences the repair of the DNA lesions inside micronuclei. Therefore, our results allow us to conclude that photolesions in radiation-induced micronuclei are poorly processed because the repair factors are unable to reach the micronuclear chromatin when a micronucleus is formed or after a genotoxic insult.     

Delayed degradation of parental macronuclear DNA in programmed nuclear death of Paramecium caudatum

In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, are discussed.     

The Nuclear Apparatus of the Ditransversal Ciliate Homalozoon Vermiculare (Ciliophora, Rhabdophora) During Interphase and Division Ii. the Micronuclei and Some Observations On Conjugation

ABSTRACT. The nuclear apparatus of H. vermiculare consists of a single moniliform macronucleus and about 25 micronuclei. the micronuclei are about 3 μm in diameter and characterized by a meshwork of thick condensed chromatin. Mitosis is intranuclear and acentric as in all other ciliates. In metaphase, interpolar and chromosomal microtubules are abundant and the length of the micronuclei increases to about 5 μm. In late anaphase, interzonal microtubules become prominent and the spindle elongates to about 50 μ. In meta- and anaphase, the microtubules of the spindle are attached to the polar vesicles, and in anaphase, chromosomes become attached to it. In contrast to most other eukaryotes, micronuclear mitosis is not strictly bound to cell division in H. vermiculare. While most of the micronuclei divide prior to cytokinesis, others retain their interphasic shape or degenerate. In addition, some micronuclei divide in the interdivision period, i.e. between two successive divisions of the cell and macronucleus. Mating cells of H. vermiculare become joined to each other in the cilia-free region covering the cytostome. In the course of conjugation, the cell membranes and the underlying oral filamentous sheaths of both cells fuse, thus uniting the endoplasm of both cells in the mouth region. Synaptonemal complexes in the meiotic chromosomes are more distinct in H. vermiculare than in most other dilates. the micrographs presented here depict dearly the central filament, transverse elements, and other substructures.     

Micronucleus induction in mammalian cell cultures treated with ionizing radiations

Summary Exponentially growing and plateau phase cultures of Ehrlich ascites tumor cells (suspension strain) were treated with either fast electrons, X-rays, fast neutrons or Am-241-alpha-particles in a dose range from about 0.02 Gy to 1 Gy and for comparison also at higher doses. After the first post-irradiation division, cells were scored for the presence of micronuclei and the micronucleus fraction as well as the number of micronuclei/cell was determined. Micronuclei were counted using the DNA specific stain H 33258 in a fluorescence microscope. A comparison with cytofluorometric measurements established that microscopic detection accounted for up to 90% of all micronuclei present within a sample, the rest probably being hidden in direct observation by the main nucleus.Dose response curves based on the micronucleus fraction as well as on the number of micronuclei/cell were found to be linear in the whole dose range tested at low and at high ionization density. Linearity was maintained also when repair of primary lesions was promoted or suppressed. The RBE of alpha-particles compared with X-rays was dependent on the time of fixation and was at a maximum immediately after the first division (RBE = 4.8 ± 0.5). Micronucleus distribution showed overdispersion relative to Poissonian statistics with every radiation quality used, in accordance with earlier observations on the distribution of acentric fragments in irradiated cultures.     

The Dynamics of Micronuclear Mitosis in the Living Ciliate Spirostomum teres as Revealed by Polarization Microscopy1

ABSTRACT. Micronuclear mitosis in living Spirostomum teres has been studied by sensitive polarization microscopy, and the dynamic aspects of micronuclear division are described. The small, spherical, interphase micronuclei lie in form-fitting depressions in the macronuclear surface. Nuclear division begins with the rounding and slight swelling of the macronucleus and, coincidentally, the micronuclei move out of the depressions and away from the macronucleus, increase in size, and become weakly birefringent. As mitosis proceeds, the micronuclei increase in uniaxial birefringence and elongate to form irregular ovoids that convert to angular structures displaying principal axes of positive birefringence so divergent as to appear oriented at a right angle to one another. Micronuclei maintain this appearance for as long as 60 min and then abruptly change to rectangular-shaped structures, increase in uniaxial birefringence, and begin anaphase elongation. The somewhat dumbbell-shaped micronuclei lengthen at the constant rate of 2.0 μm/min to reach lengths >70 μm. It appears that little half-spindle shortening occurs during spindle elongation. Accompanying the changes in micronuclear spindle length are changes in birefringence. Just before elongation begins, presumably metaphase, the micronucleus is uniformly and intensely birefringent. At the magnifications employed, a chromosome plate is not clearly visible as a region of reduced birefringence. As elongation begins, the putative half-spindles are more birefringent than is the interzone, a condition that is maintained until the spindles have achieved ～30% elongation, at which time a region of increased birefringence develops at the center of the interzone. This pattern persists for a very short time, then gives way to a uniform birefringence of the entire separation spindle that is maintained until elongation is completed. The rate of micronuclear spindle elongation, changes in micronuclear dimensions, and corresponding changes in birefringence are discussed with respect to possible mechanisms of mitosis.     

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."     

Electron Microscopy of the Nuclear Events During Binary Fission in Paramecium multimicronucleatum

SYNOPSIS. From the interphase to the early stage of binary fission in Paramecium multimicronucleatum , when the micro-nuclei are situated close to the macronucleus, the microtubules in the cytoplasm seem to connect the nuclear pores of macro- and micronuclei. During the 1st half of macronuclear division, the microtubules are formed outside the macronucleus, while during the latter half of division, numerous microtubules appear inside it. Chromatin bodies and nucleoli remain unchanged during macro-nuclear division, but the latter show temporory irregularity in shape. In late prophase of micronuclear division, spindle micro-tubules are formed, and a polar structure, composed of randomly dispersed twisting filaments, is formed at each pole of the micro-nucleus at anaphase. Spindle microtubules terminate on the surface of this structure. The nuclear envolope of the macro-and micronuclei remains intact thruout division. The envelope of the daughter micronuclei is derived from the pre-existing one.     

Commitment to the First Cortical Reorganization During Conjugation in Stylonychia mytilus: An Argument for Homology with Cortical Development During Binary Fission

The asexual nature of the first cortical reorganization of conjugation in Stylonychia was analyzed by comparing the effect of amputation performed at different stages of early conjugation to that performed on vegetative cells at different stages of the cell cycle. Amputation of vegetative cells delineated a point of commitment to binary fission at 0.51–0.57 of the cell cycle. Cells amputated before this point were induced to undergo the regenerative mode of asexual development, but those amputated after this point continued with binary fission. In parallel, during conjugation a similar commitment was made around the time of formation of tight mating-pairs: early conjugants amputated around this time might undergo regeneration, and those operated on after this stage continued with the first cortical reorganization as in typical conjugants. The two mates of a pair might differ in their response to amputation, suggesting that the timing of commitment to the first cortical reorganization is not related to the events of conjugation, but rather is individually determined in the vegetative cycle of the cells before they pair up in mating. These observations provide support for the notion that the first cortical reorganization of conjugants is homologous to the asexual mode of cortical development in dividers, according to the theory of developmental heterochrony in the sexual reproduction of hypotrichs. The timing of commitment to the first cortical reorganization was found to temporally correlate with the entrance of the micronuclei into meiosis. Since the first cortical reorganization can proceed without the micronucleus, this raises the possibility that initiation of micronuclear meiosis is closely coupled with, and may be determined by, the commitment to the first cortical reorganization.     

Proteolytic processing of micronuclear H3 and histone phosphorylation during conjugation in Tetrahymena thermophila

During vegetative growth, micronuclei of the ciliated protozoan Tetrahymena thermophila contain two electrophoretically distinct forms of H3, H3S and H3F [4, 5]. Of these two forms, H3F is unique to micronuclear chromatin and is derived from H3S by a physiologically regulated proteolytic processing event [5]. While the function of this processing event is not clear, several lines of evidence [2, 5] suggest that it may be related to chromatin condensation during mitosis. In this report pulse-chase experiments have been used to study the processing of H3S into H3F during the sexual phase of the life cycle, conjugation. Our results demonstrate that even though micronuclei divide mitotically (and meiotically) several times during the mating process, processing of H3S into H3F does not occur. Failure of H3S to be converted into H3F during these divisions causes a significant increase in the amount of H3S (relative to H3F) as conjugation proceeds. By 10 h of conjugation, essentially all of the micronuclear H3 is in the form of H3S (also see [3]). As long as mating cells are maintained under starvation conditions, processing of H3S into H3F does not occur. However, if exconjugants are returned to food and allowed to proceed through the first true cell division following exconjugation, processing of H3S into H3F occurs. Thus, the return of the processing of H3(3) into H3F following conjugation seems to be tightly coupled to a division which is part of a cell division cycle (as appears to be the case with vegetatively growing cells). The relevancy of these results to the differentiation of new macro- and micronuclei is discussed. H3F is specifically phosphorylated in growing cells, and it has been suggested that this phosphorylation event may be related to chromatin condensation during mitosis [2]. Since in mating cells H3S becomes the more predominant form of H3, the pattern of histone phosphorylation was examined during stages of conjugation where micronuclei are active in mitotic division (6-7 h). While a low level of phosphate label is observed over H3S in mating cells, more phosphate label is associated with the small amount of H3F which remains in micronuclei at this stage of conjugation. We also observe significant amounts of phosphate label associated with micronuclear H2A, H2B, and H4 and each of the micronuclear H1-like molecules, alpha, beta and gamma.