Micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase activity which is highly specific for free histone H4

Salt extracts prepared from purified micronuclei and the cytoplasm of growing Tetrahymena contain a histone acetylase (also referred to as histone acetyltransferase) activity which is highly specific for H4 when tested as a free histone. With both extracts, H4 is acetylated first at position 4 (monoacetylated) or positions 4 and 11 (diacetylated), sites diagnostic of deposition-related acetylation of newly synthesized H4 in vivo. As the concentration of cytosolic extract is decreased in the in vitro reactions, acetylation of H3 is also observed. Neither activity acetylates histone in a chromatin form. These activities are distinct from a macronuclear acetylase which acetylates H3 and H4 (macro- or micronuclear) equally well as free histones and which acetylates all four core histones when mononucleosomes are used as substrate. As well, the micronuclear and cytoplasmic activities give similar thermal-inactivation profiles which are different from that of the macronuclear activity. In situ enzyme assays demonstrate a macronuclear-specific activity which acetylates endogenous macronuclear chromatin and an independent micronuclear-cytosolic activity which is able to act upon exogenously added free H4. These results argue strongly that an identical acetylase is responsible for the micronuclear and cytoplasmic activity which is either modified or altogether distinct from that in macronuclei.     

The formation of polytene chromosomes during macronuclear development of the hypotrichous ciliate Stylonychia mytilus

The formation of polytene chromosomes during macronuclear development of the ciliate Stylonychia mytilus was examined in spread electron microscopical preparations. The chromatin organization of early macronuclear anlagen closely resembles the organization of micronuclear chromatin. In the course of polytenization 300 A chromatin fibers become organized in loop-like structures laterally attached to a thinner axial fiber. It is suggested that this reorganization of chromatin during polytenization is a necessary event for the subsequent chromatin elimination.     

Induction of reinitiation of meiosis in amphibian Bufo oocytes by injection of ciliate Paramecium extracts

Germinal vesicle breakdown (GVBD) of amphibian Bufo oocytes can be induced if Paramecium extracts were injected into them. The activity of meiosis-reinitiation-inducing factor (MRIF) appeared in premeiotic G1 cells, then the activity fluctuates according to the degrees of micronuclear chromatin condensation in meiosis. Proliferating micronucleate and amicronucleate cells also showed the same activity. MRIF differed from MPF (M phase promoting factor), because MRIF appeared not only in M phase cells but also in premeiotic interphase cells and its action on the induction of GVBD was inhibited by cycloheximide. Preliminary experiments showed that MRIF was a heat-labile soluble protein.     

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.     

Abnormal micronuclear telomeres lead to an unusual cell cycle checkpoint and defects in Tetrahymena oral morphogenesis

Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a “dynamic pause” in the program of cortical development, which may reveal an unusual cell cycle checkpoint.     

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.     

THE FINE STRUCTURE OF THE NUCLEI OF TETRAHYMENA PYRIFORMIS THROUGHOUT THE CELL CYCLE

The fine structure of the nuclei of logarithmically growing Tetrahymena pyriformis, strain HSM, was studied at 30-minute intervals throughout the cell cycle. Organisms were selected at similar stages of cytokinesis by means of a braking pipette, incubated, fixed in OsO₄, and embedded in agar to facilitate subsequent preparation for electron microscopy. Aggregates of micronuclear chromatin underwent a decrease in density and number with a concomitant increase in size throughout interphase. There were no impressive changes in macronuclear morphology. It was found possible to estimate a cell's progress through interphase by observation of micronuclear morphology, but attempts to correlate changes in fine structure with periods of DNA synthesis were unsuccessful.     

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.     

The effect of trifluoroperazine on microtubules,nuclear division and the nuclear membranes of the ciliate Nyctotherus ovalis

Exposure of the dividing ciliate Nyctotherus ovalis to the tranquilizer trifluoroperazine (TFP; 10 M) leads to the complete disassembly of kinetochore microtubules in the metaphase micronucleus. Interpolar microtubules located underneath the micronuclear envelope at anaphase and telophase stembody microtubules are more resistant to TFP. However, stembodies of drug-exposed ciliates are much shorter than in the controls. In their centre they contain only a reduced number of widely separated microtubules, indicating that assembly of new tubules or elongation of existing microtubules at this site, which appears essential for further separation of the future daughter nuclei, is blocked by TFP. Although microtubules polymerized in the macronucleus during its elongation include a set of tubules made up of more than 13 protofilaments, comparable to the micronuclear stembody microtubules, they are much more sensitive towards drug treatment. Macronuclear tubules become completely depolymerized resulting in failure of nuclear stretching. Already elongated macronuclei can still become constricted in their centre which suggests that microtubules are not involved in this process. Disassembly and higher sensitivity of macronuclear compared with micronuclear microtubules may be explained by a different composition and behaviour of nuclear membranes towards TFP in the two types of nuclei. While the micronuclear envelope may be only partially destroyed where it is facing the macronucleus, the inner membrane of the macronuclear envelope is severely affected by drug treatment. It shows a multitude of infoldings accompanied by attachment of chromatin to it. Cytoplasmic microtubules which proved resistant to other depolymerizing drugs become partly disassembled during TFP treatment.     

The ultrastructure of nuclear division in a suctorian,Tokophrya infusionum

Summary Ultrastructural changes in the micro- and macronucleus throughout division were followed in synchronized cultures of the suctorian, Tokophrya infusionum. After an initial swelling, the micronucleus elongates enormously; microtubules within the micronucleus proliferate and lengthen as the micronucleus elongates. Changes in the macronucleus become visible only after micronuclear division is well underway. The chromatin bodies fuse into long chromatin strands, and the large bundles of microtubules present in the resting macronucleus break up into small groups which parallel the chromatin strands. Colchicine, which prevents reproduction in Tokophrya, seems to block division at a very early stage. The macronucleus appears the same as the resting nucleus of untreated organisms, with numerous microtubules and distinct chromatin bodies. The chromatin in the micronucleus aggregates into large clumps, however, and proliferation of microtubules does not occur.Supported by a Graduate Fellowship at The Rockefeller University.Supported by Grant A1-01407-12 USPHS and Grant A1-08989-01 USPHS.     

Micronuclear and antitumor activities of two newly synthesized pyrimidine derivatives

Micronuclear and antitumor activities of two newly synthesized iodides of pyrazolo[1,5a]pyrimidines were investigated using mice model. It has been shown that both compounds are slightly toxic and have no antitumor activity in mice with Ehrlich's ascites carcinoma. Only one of them has weak micronuclear activity. Both compounds increase substantially micronuclear as well as antitumor activities of cyclophosphamide.     

Fine structure of division in ciliate protozoa. I. Micronuclear mitosis in Blepharisma

The mitotic, micronuclear division of the heterotrichous genus Blepharisma has been studied by electron microscopy. Dividing ciliates were selected from clone-derived mass cultures and fixed for electron microscopy by exposure to the vapor of 2% osmium tetroxide; individual Blepharisma were encapsulated and sectioned. Distinctive features of the mitosis are the presence of an intact nuclear envelope during the entire process and the absence of centrioles at the polar ends of the micronuclear figures. Spindle microtubules (SMT) first appear in advance of chromosome alignment, become more numerous and precisely aligned by metaphase, lengthen greatly in anaphase, and persist through telophase. Distinct chromosomal and continuous SMT are present. At telophase, daughter nuclei are separated by a spindle elongation of more than 40 µ, and a new nuclear envelope is formed in close apposition to the chromatin mass of each daughter nucleus and excludes the great amount of spindle material formed during division. The original nuclear envelope which has remained structurally intact then becomes discontinuous and releases the newly formed nucleus into the cytoplasm. The micronuclear envelope seems to lack the conspicuous pores that are typical of nuclear envelopes. The morphology, size, formation, and function of SMT and the nature of micronuclear division are discussed.     

Micronuclear DNA of Oxytricha nova contains sequences with autonomously replicating activity in Saccharomyces cerevisiae.

Oxytricha nova is a hypotrichous ciliate with micronuclei and macronuclei. Micronuclei, which contain large, chromosomal-sized DNA, are genetically inert but undergo meiosis and exchange during cell mating. Macronuclei, which contain only small, gene-sized DNA molecules, provide all of the nuclear RNA needed to run the cell. After cell mating the macronucleus is derived from a micronucleus, a derivation that includes excision of the genes from chromosomes and elimination of the remaining DNA. The eliminated DNA includes all of the repetitious sequences and approximately 95% of the unique sequences. We cloned large restriction fragments from the micronucleus that confer replication ability on a replication-deficient plasmid in Saccharomyces cerevisiae. Sequences that confer replication ability are called autonomously replicating sequences. The frequency and effectiveness of autonomously replicating sequences in micronuclear DNA are similar to those reported for DNAs of other organisms introduced into yeast cells. Of the 12 micronuclear fragments with autonomously replicating sequence activity, 9 also showed homology to macronuclear DNA, indicating that they contain a macronuclear gene sequence. We conclude from this that autonomously replicating sequence activity is nonrandomly distributed throughout micronuclear DNA and is preferentially associated with those regions of micronuclear DNA that contain genes.     

Amitotic division of the macronucleus in Tetrahymena thermophila: DNA distribution by genomic unit

The macronucleus of the ciliate Tetrahymena cell contains euchromatin and numerous heterochromatins called chromatin bodies. During cell division, a chromatin aggregate larger than chromatin body appears in the macronucleus. We observed chromatin aggregates in the dividing macronucleus in a living T. thermophila cell, and found that these were globular in morphology and homogeneous in size. To observe globular chromatin clearly, optimal conditions for making it compact were studied. Addition of Mg ion, benomyl and oryzalin, microtubule inhibitors, to cell suspension was effective. Globular chromatin appeared when the micronuclear anaphase began at the cell cortex, and disappeared long after cell separation. Using living cells with a small macronucleus at early log phase, we counted the number of globular chromatin per nucleus and measured the DNA content of globular chromatin in the macronucleus which was stained with Hoechst 33342 by using ImageJ. The number of globular chromatin per nucleus was reduced by half after division, indicating the globular chromatin is a distribution unit of DNA. A globular chromatin contained similar DNA content as that of the macronuclear genome. We developed methods for inducing and isolating a cell with an extremely small macronucleus with a DNA amount of one globular chromatin. These cells grew, divided, and give clones, suggesting that the macronuclear genome is not dispersed within the macronucleus and the globular chromatin may be a macronuclear genome. We named this globular chromatin "macronuclear genome unit" (MGU).     

Genomic Organization and Developmental Fate of Adjacent Repeated Sequences in a Foldback DNA Clone of Tetrahymena thermophila

DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago. However, the significance and mechanism of chromatin elimination are not understood. DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila. In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones. All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus. The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy. DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance. This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate. Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA. We found that all subregions defined repeated sequence families in the micronuclear genome. A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated. The inverted repeat family is retained with little rearrangement. Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames. A fourth family occurs in the "loop" region and is rearranged extensively during development. The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes. The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes. The significance of retained inverted repeats to the process of elimination is discussed.     

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.