Amicronucleate Cell Research Articles

Timing and characteristics of nuclear events during conjugation and genomic exclusion in Paramecium multimicronucleatum

Ciliated protists are ideal material for studying the origin and evolution of sex, because of their nuclear dimorphism (containing both germline micronucleus and somatic macronucleus in the same cytoplasm), special sexual processes (conjugation and autogamy), and high diversity of mating-type systems. However, the study of sexual process is limited to only a few species, due to the difficulties in inducing or observing conjugation. In the present study, we investigate the conjugation process in Paramecium multimicronucleatum: (1) of the three prezygotic divisions, all micronuclei undergo the first two divisions (meiosis I, II), while a variable number of nuclei undergo the third division (mitosis); (2) the synkaryon divides three times after fertilization, giving rise to eight products that differentiate into four macronuclear anlagen and four micronuclei; (3) cells restore the vegetative stage after two successive cell fissions during which the macronuclear anlagen are distributed into daughter cells without division, while micronuclei divide mitotically; (4) the parental macronucleus begins to fragment following the first meiotic division and finally degenerates completely; (5) the entire process takes about 110 h, of which about 85 h are required for macronuclear development. In addition, we describe for the first time the process of genomic exclusion occurring between amicronucleate and micronucleate cells of P. multimicronucleatum, during which the micronucleate cell contributes a pronucleus to the amicronucleate cell, resulting in both exconjugants being homozygotes. These results provide new insights into the diversity of sexual processes and lay an important cytological basis for future in-depth studies of mating systems in ciliates.

Conjugation in Euplotes raikovi (Protista, Ciliophora): New Insights into Nuclear Events and Macronuclear Development from Micronucleate and Amicronucleate Cells.

Ciliates form a distinct group of single-celled eukaryotes that host two types of nuclei (micro and macronucleus) in the same cytoplasm and have a special sexual process known as conjugation, which involves mitosis, meiosis, fertilization, nuclear differentiation, and development. Due to their high species diversity, ciliates have evolved different patterns of nuclear events during conjugation. In the present study, we investigate these events in detail in the marine species Euplotes raikovi. Our results indicate that: (i) conjugation lasts for about 50 h, the longest stage being the development of the new macronucleus (ca. 36 h); (ii) there are three prezygotic micronuclear divisions (mitosis and meiosis I and II) and two postzygotic synkaryon divisions; and (iii) a fragment of the parental macronucleus fuses with the new developing macronucleus. In addition, we describe for the first time conjugation in amicronucleate E. raikovi cells. When two amicronucleate cells mate, they separate after about 4 h without evident nuclear changes; when one amicronucleate cell mates with a micronucleate cell, the micronucleus undergoes regular prezygotic divisions to form migratory and stationary pronuclei, but the two pronuclei fuse in the same cell. In the amicronucleate cell, the parental macronucleus breaks into fragments, which are then recovered to form a new functional macronucleus. These results add new information on the process of conjugation in both micronucleate and amicronucleate Euplotes cells.

Effect of the micronucleus on cellular structures in several hypotrichous ciliates

It is commonly believed that there is a sharp functional demarcation between the two types of nuclei of ciliated protozoa: the micronucleus for reorganization of the nuclear apparatus during sexual reproduction and the macronucleus for controlling somatic functions. This traditional view, however, has been critically re‐examined recently and correlative researches have shown that the micronucleus plays an essential role in the development of some ciliates. Here we report some morphological evidences about the effect of the micronucleus on cellular structures from amicronucleate cell lines of five hypotrichous ciliates (Pseudourostyla cristata, Pseudokeronopsis monilata, Paraurostyla weissei, Uroleptus piscis and Oxytricha faurei). All amicronucleate cell lines were generated by amputation and investigated by protargol staining. These observations manifest that micronucleus is indispensable to vigor and viability for P. weissei, to maintaining the integrity of adoral zone of membranelles (AZM) and/or undulating membranes (UM) for P. cristata, P. monilata and U. piscis, and to keeping regularity of cell shape as well as of the number and the structure of the macronuclei for O. faurei. These results suggest that the manifestations of the somatic function of micronucleus for different ciliates are varied and the close species might have a similar form of expression.This work was supported by a grant from NSFC (No: 30370210) to L. P. JIN.

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.

Somatic function of the micronucleus of Stylonychia mytilus during asexual propagation

The somatic function of the micronucleus during vegetative propagation of the hypotrichous ciliate Stylonychia mytilus was investigated, by generating amicronucleate cell lines with microinjection or amputation. The amicronucleate cell lines exhibited reduction in fission rate and ingestion shortly after the loss of the micronucleus. There was partial recovery of growth rate in the following months of propagation. Amicronucleate cultures carried both normal-looking and abnormal cells. The normal-looking ones underwent binary fission, and also physiological reorganization, in the usual manner. After binary fission, normal-looking amicronucleates gave rise to normal-looking post-dividers, which might then undergo abnormal transformation in the interfission period by developing a bulge on the posterior-right side of the cell (Type A), or on the dorsal surface (Type B). Type A cells could enter binary fission directly, eventually recovering normal cell shape. The hump-back Type B cells went through atypical cortical reorganization(s) to generate spherical cells, which then recover to normal shape by normalizing reorganization(s). The emergence of these two types of abnormalities was correlated with the failure of macronuclear division during binary fission. These observations indicate that the micronucleus possesses morphogenetic function, and that this function is division-related. The micronucleus probably affects cytoskeletal/membranellar development during binary fission, and in its absence the cortical abnormalities exhibited represent a delayed expression of the infirmities incurred during binary fission. In view of the persistence of such abnormalities in amicronucleate cultures for up to a year of culture, the morphogenetic function of the micronucleus appeared not to be replaced; this resembles the situation in the hypotrich Pseudourostyla cristata, but differs from Paramecium where recovery to near-normal is the rule. Chromatin bodies resembling pseudomicronuclei were present in low frequency in the culture, and these appeared to arise in connection with macronuclear development in Type B cells.

Regeneration of amicronucleate fragments in the hypotrichous ciliate Pseudourostyla levis

This work aims to elucidate the micronuclear functions in regeneration of the hypotrichous ciliate Pseudourostyla levis. A micronucleate stock DH-1 and an amicronucleate cell line DH-1a-6 obtained from a cell of the same stock by microsurgical enucleation were used in this work. In the operation, cells at the early stationary phase were transected at their equatorial zone by a fine glass needle. These fragments (promers and opimers) were impregnated by the protargol technique. Developmental processes of the adorai zone of membranelies (AZM), frontal-ventral-transverse (FVT) cirri, marginal cirri (MC) and dorsal kineties were very similar between micro- and amicronucleate fragments. Splitting of old membranelles in the lapel part (LP-membranelles) of the AZM, however, always occurred at stage 5 in micronucleate promers, whereas the same developmental event was observed during stages 2-3 in amicronucleate promers. These observations indicate that the absence of micronuclei does not affect cortical development and cortical organelle assembly during regeneration. At stage 9 a bare area without membranelles in the lapel part of the AZM appeared only in amicronucleate regenerants after the formation of the new AZM. After the appearance of the bare area (stage 9), the mean number of LP-membranelles (22.7 ± 3.8) of regenerants was significantly smaller than that of regenerants (42.0 ± 4.7) at stage 8 (p ≤ 0.01). These findings indicate that a considerable number of LP-membranelles were lost. Therefore, the micronuclei might play some important role in fixing LP-membranelles firmly in the cell cortex.

Repressive function of the micronucleus during asexual reproduction in Paramecium tetraurelia: Experimental analysis with defective-micronucleates generated by laser microbeam irradiation

The present paper reports on the experimental analysis of a novel regulatory function of the micronucleus of Paramecium tetraurelia. Previous studies have made clear that amicronucleate cell lines shortly after their generation generally suffer depression, in exhibiting low viability, slow growth and abnormal oral development in binary fission, but they eventually recover to near-normal. A compensatory mechanism is thus activated in the absence of the micronucleus, to allow recovery of the amicronucleate cell line. Implicit in this conclusion is the role of the micronucleus in repression of the compensatory mechanism. The present study tested this notion by perturbing the micronucleus with laser microbeam irradiation. This operation generated cell lines possessing defective micronuclei; during their asexual propagation, some cells lost the micronucleus and gave rise to amicronucleates. The viability of amicronucleate cell lines derived in this manner was found to be higher, compared to others generated by a different operation involving instantaneous removal of normal micronuclei from the cell with a microinjection needle. Some evidence also suggested that their oral development was less abnormal during the initial depression period. Hence, damage of the micronucleus has apparently facilitated the activation of the compensatory function, and the latter might have occurred even before the loss of the defective micronucleus. The present findings provide support for a regulatory role of the micronucleus during asexual propagation. Previous studies have indicated that the physical basis of the compensatory mechanism resides with the macronucleus. The micronuclear repressive function may be directed against this compensatory mechanism of the macronucleus.

Programming of the macronucleus of Paramecium during asexual and sexual reproduction: A further study with cytidine analogues, dimethylsulfoxide, L-ethionine and N-butyric acid

The control of the function of the macronucleus of Paramecium is studied, in connection with its role in the compensation for the asexual somatic function of the micronucleus. Following removal of the micronuclei, amicronucleate cell lines as a rule suffer a transient period of growth and developmental depression in the initial phase of asexual propagation. But they gradually recover to near-normal. Previous studies of treatment of amicronucleate cells with cytidine analogues have implicated the macronucleus in compensating for the somatic function of the micronucleus following the loss of the micronucleus, and the activation of this macronuclear function probably involves DNA-demethylation. The present study further tests this notion, by treating micronucleate cells with agents known to promote demethylation of 5-methylcytosine. After treatment, the cells were vegetatively propagated, and then enucleated to give rise to amicronucleate cell lines. Treatments with dimethylsulfoxide, L-ethionine, and 5-aza-2'-deoxycytidine promoted recovery in amicronucleate cell lines thus derived. Cells treated with 6-azacytidine did not produce such an effect. Hence, the compensatory mechanism, presumably residing in a repressed state in the macronucleus, can be activated or primed to activate by demethylating agents even before the loss of the micronucleus, and once established the new macronuclear programme perpetuates in succeeding asexual cell generations. This shows that during asexual propagation the macronuclear programme can be altered to 'pre-adapt' the cells for amicronuclearity. Treatment of micronucleate conjugants with 5-azacytidine, when the macronuclear anlagen develop, produced clones that had become similarly pre-adapted. There were also some indication of persistence of such effects of the analogue into the next clonal cycle following autogamy. The notion of macronuclear DNA-demethylation as a basis for the activation and maintenance of the compensatory mechanism is discussed.

The Somatic Function of the Germ Nucleus in Pseudourostyla cris tat a: Asexual Reproduction and Stomatogenesis

ABSTRACTThe micronuclei of the ciliated protozoan Pseudourostyla cristata were eliminated by amputation shortly before binary fusion. The amicronucleate cell lines derived from regenerants were maintained for more than a year. They exhibited a lower viability and reduced vigor in asexual propagation. There was some improvement in the growth of the cell lines 1 mo after operation, but the growth rate remained subnormal even up to 1 yr of culture. The exact cause of the poor growth and survival in the first 3 wk after operation, whether the loss of the micronucleus or operational damage, remains to be determined. It is nevertheless clear that the micronucleus is important for subsequent asexual propagation. The amicronucleate cell lines were permanently crippled in morphogenesis, unlike the situation in Paramecium amicronucleates in which stomatogenesis returned to near‐normal during asexual propagation. They always included some cells with a characteristically defective adoral zone of membranelles, reduced number of frontal‐ventral‐transverse cirri, and reduced body length. They were also reluctant to encyst. It is evident that the micronucleus is important for maintaining normality of the oral apparatus. It is postulated that the permanent stomatogenic crippling of amicronucleates might be related to genomic reduction in the developing macronucleus in sexual reproduction, as exhibited by other hypotrichs. The morphological defects associated with the adoral zone of membranelles may be rationalized as arising from the spreading of a zone of degeneration in the cortex affecting the left edge of the membranelles.

5-azacytidine affects the programming of expression of the somatic nucleus of Paramecium

This report introduces a new system in the study of programming of genomic function during development of the somatic nucleus of Paramecium tetraurelia. Previous works have established a definite, but replaceable, role of the germ nuclei (micronuclei) in oral development in the asexual cycle; their removal from the cell generates viable amicronucleate cell lines, which characteristically suffer a transient period of growth depression marked by abnormal oral development. Such cell lines gradually recover, showing that a compensatory mechanism is activated in the absence of the germ nuclei to bring the cell back to near-normal. To test the notion that the somatic nucleus (macronucleus) is involved in this compensation, cells possessing micronuclei were treated with 5-azacytidine during sexual reproduction when new somatic nuclei develop. These cells were then propagated asexually for a number of fissions in the absence of the drug, and thereafter micronuclei were removed from them. The amicronucleate cell lines generated in this manner clearly did not suffer a depression as severe as the untreated controls did in terms of growth rate and oral development, and they recovered much sooner. This supports the notion that the somatic nucleus is the physical basis of the compensatory mechanism. This study suggests that the stomatogenic sequences in question normally become repressed in the somatic nucleus developing in sexual reproduction, and that 5-azacytidine administered to the cells at this time could alter this programme which then persists during subsequent asexual propagation. The possibility that the somatic nucleus is programmed by methylation of cytosine at the 5' position is discussed.

Cytidine Analogues and Stomatogenic Recovery in Amicronucleate Paramecium tetraurelia and Paramecium jenningsi

ABSTRACTRemoval of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5‐azacytidine, 5‐aza‐2′‐ deoxycytidine and 5‐fluoro‐2′‐deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6‐azacytidine, 2′‐fluoro‐2′‐deoxy‐cytidine and cytosine‐β‐D‐arabinofuranoside did not. These results suggest that (i) 5‐methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.

Stomatogenic function of the micronucleus in Paramecium jenningsi

The stomatogenic function of the micronucleus in Paramecium jenningsi is documented in the present report. Amicronucleate cell lines were generated by removal of the micronuclei with a microinjection needle. Such cell lines exhibited a temporary state of growth depression marked by the development of abnormal oral apparatuses, and they gradually recovered to nearnormal. During sexual reproduction, the amicronucleates failed to develop an oral apparatus but resorbed the pre-existing oral apparatus, resulting in astomy. The types of stomatogenic abnormalities exhibited during asexual and sexual reproduction, apart from some unique features, were largely similar to those observed in P. tetraurelia. Reimplantation of the micronucleus into amicronucleates during the depression period likewise promoted stomatogenic recovery, both in speed and in extent. These similarities between the two species indicate that the control of oral development by the micronucleus in them have much in common, suggesting that the stomatogenic function of the micronucleus is a characteristic of the genus Paramecium. Some of the cells in the renucleated cell lines lost the micronuclei during asexual propagation. Some preliminary observations indicated that such amicronucleates arising in the renucleated cell lines were not as abnormal as those amicronucleates derived by enucleation in the early phase of depression, but were comparable to the latter after the latter had recovered. This suggested that such amicronucleates had suffered only a "minor depression". The genetic implications of this was discussed in the context of the mechanism of stomatogenic recovery in amicronucleates.

Persistence of Oral Structures in the Sexual Process of Amicronucleate Paramecium tetraurelia

ABSTRACT In the sexual process, amicronucleate Paramecium tetraurelia, unlike micronucleates, fail to produce an oral apparatus, but resorb the pre‐existing one. Exceptions were found in some amicronucleate cell lines in which about 1% of the cells possessed oral structures, including pieces of oral membranelles, sometimes complete with buccal cavity, after autogamy or conjugation. By following oral development in the sexual process in some detail, the present study supports the view that these oral structures are derived from the pre‐existing oral apparatus and not newly developed from the oral primordium. The possible involvement of the micronucleus and the pre‐existing oral apparatus in oral resorption is discussed. The possession of a functional oral apparatus after the sexual process may open up a new evolutionary avenue to the amicronucleates.

Stomatogenesis in Paramecium tetraurelia: The roles of the micronucleus and the pre-existing oral apparatus

Removal of the micronuclei from Paramecium generated amicronucleate cell lines which produced oral apparatuses of much reduced length and abnormal oral membranellar pattern during an initial depression period. They subsequently recovered, but only partially. Reimplantation of the amicronucleates with micronuclei generally allowed a greater oral length to be reached in renucleated cell lines, and more normal membranellar pattern in some, but full recovery was frequently not attained even in these. However, the length of the oral apparatuses produced by the renucleated cells during autogamy, when the pre-existing oral apparatus degenerates, were mostly comparable to those of micronucleate controls. These relationships are discussed in terms of the control of development of the oral apparatus by the micronucleus and the preexisting oral apparatus.

Cortical positioning and the fate of oral structures during the sexual process in amicronucleate homopolar tandems of Paramecium

Abstract Paramecium tetraurelia normally resorbs the preexisting oral apparatus (and develops a new one) during sexual reproduction. Violation of this rule was found in amicronucleate cell lines. These cell lines generated chains of two cells (homopolar tandems) at a low frequency, as a result of incomplete binary fission during a transient growth depression period following emicronucleation. In autogamous chains, the proter resorbed the pre-existing oral structures, while some of the opisthes retained them. The oral structures in the opisthes of the chains were unusually close to the opisthes’ anterior end. The ectopic location of these oral structures might account for their retention, formally understood in terms of the theory of positional information. It is suggested that nongenic factors, likely involving components of the rigid cortical matrix, are involved in the fixation of positional values.

Reorganization in Amicronucleates with Defective Mouth of the Ciliate Pseudourostyla levis1

ABSTRACT. The purpose of this work is to examine the reorganization process in amicronucleates with defective mouth of the multimicronuclear ciliate, Pseudourostyla levis. The amicronucleates were derived from fragments obtained by transection of normal micronucleates. The cell size of the amicronucleates was extremely unstable and varied from 57.5 to 276.3 μm long (n = 146), whereas the micronucleates kept rather stable cell lengths with a range from 162.5 to 266.3 μm (mean ± SD = 213.1 ± 19.6 μm, n = 206). The renucleates obtained by transplantation of a micronucleus to an amicronucleate returned to almost normal cell size (mean ± SD = 203.7 ± 16.5 μm long, n = 54). Under the usual culture conditions in the amicronucleate cell line, the number of abnormal cells with defective mouth rapidly increased, up to about 60%, until a stationary phase. Similarly, abnormal cells also appeared in micronucleates although the frequency was always less than 10%. The mean number of membranelles in the normal adoral zone of membranelles (AZM) was 82.6 ± 3.9 (±SD, n = 49) vs. 57.3 ± 7.9 (n = 81) in ciliates with defective mouths. The missing part of the AZM was always the anterior part of the lapel. Cells with defective mouth underwent reorganization (= physiological regeneration) under the usual culture conditions. During reorganization, the lapel part of the old AZM was transformed into a new collar part. The defective mouth was repaired through this developmental process. These results suggest that in P. levis the decrease of food supply often leads to the loss of a specific part of the AZM and that this membranellar loss is suppressed by the existence of micronuclei.

The somatic function of the micronucleus in asexual reproduction of Paramecium: thermosensitivity of amicronucleates

It has been established that following removal of the micronucleus in Paramecium tetraurelia, the amicronucleate cell line enters a depression period, characterized by slow growth rate and oral abnormalities, at normal growth temperature (27°C). Such cell lines gradually recover in growth rate and stomatogenesis. In the present study, 4 recovered amicronucleate cell lines were challenged with high temperatures (35°C, 36°C, and 36.5°C). They exhibited growth rate reduction and abortive cytokinesis at 35°C and 36°C, and died at 36.5°C. In addition, they demonstrated oral defects similar to those observed in the depression period: disruption of the regular oral membranellar pattern, reduction in the length of the oral apparatus, and impaired phagocytosis (food vacuole formation). These high temperature‐induced abnormalities were largely restricted to amicronucleates, and were rare or seen to a much lesser extent in sister micronucleate cell lines. This study demonstrates the participation of the micronucleus in conferring thermotolerance on the cells. It is hypothesized that the micronucleus specifies heat‐shock proteins to maintain the integrity of oral and somatic cytoskeletal elements at high temperature.

The role of the micronucleus in stomatogenesis in sexual reproduction of Paramecium tetraurelia: laser microbeam irradiation of the micronucleus.

Fifteen amicronucleate cell lines and 22 cell lines with defective micronuclei were obtained following selective laser microbeam irradiation of the micronucleus. The amicronucleate cell lines showed reduced growth rate and formed abnormal oral apparatuses in asexual reproduction, and failed to produce any oral apparatus in autogamy, in agreement with previous observations on amicronucleate cells obtained by micropipetting. The 22 cell lines with defective micronucleus exhibited various abnormalities of the oral apparatus newly formed during autogamy. These abnormalities included the arrest of membranelle assembly, reduction in the length of the buccal cavity and oral membranelles, disruption of the organization of the membranelles, quadrulation of the dorsal peniculus, and failure of addition of membranellar basal body rows. Hence the micronucleus plays multiple roles in sexual stomatogenesis, deciding early steps of oral membranelle assembly and affecting their subsequent patterning. Our results agree with the notion that the micronucleus acts during a critical period between the second meiotic division and up to the formation of the zygotic nucleus to control the early stage of oral membranelle assembly. Laser microbeam irradiation might have created recessive mutations and/or chromosomal aberrations, which were expressed during this critical period with the formation of abnormal postmeiotic nuclei.

Nuclear Differentiation in Exconjugants of Paramecium caudatum: Role of Nuclear Division in Differetiation: (conjugation/nuclear differentiation/nuclear division/nuclear transplantation/Paramecium).

It has been known that, immediately after the third division of fertilization nucleus (synkaryon), nuclei localized near the posterior region of exconjugant are to be macronuclear anlagen and those near the anterior region are to be presumptive micronuclei in Paramecium caudatum. One of such posterior nuclei was transplanted into amicronucleate cell at vegetative phase in this work. The implanted nuclei were able to divide at every fission. Their DNA content was nearly equal to or less than ordinary micronuclei during vegetative phase. When conjugation was induced between clones obtained and amicronucleates, macronuclear anlagen developed from the division products of implanted nuclei and thereafter derivative caryonides were true to the marker gene of implanted nuclei. The results indicate that there was no intrinsic difference between nuclei localized anteriorly and those situated posteriorly in exconjugant. Differentiation of nuclei into macronucleus may be irreversible at the stage of anteroposterior localization of the nuclei. The role of nuclear division in differentiation may be only to transport the daughter nuclei into the cytoplasm/cortex differentiated anteroposteriorly.

Micronucleus-specific DNA sequences in an amicronucleate mutant of Tetrahymena.

DNA from the amicronucleate Tetrahymena cell line BI3840 was probed for DNA sequences which are limited to the micronucleus in wild-type cells. Four micronucleus-specific DNA sequences were not detectable in DNA from the amicronucleate cell line. Two of the six micronucleus-specific DNA sequences tested hybridized to DNA from amicronucleate cells. Both the number of fragments homologous to these sequences and the intensity of hybridization were reduced in the DNA from the amicronucleate cells relative to DNA from a wild-type cell line, indicating that less than one micronucleus equivalent of the micronucleus-specific DNA sequences was retained in the amicronucleate cell line. Thus many micronucleus-specific DNA sequences were eliminated from the developing macronucleus of BI3840 as they are in wild-type cells, but in at least two cases the elimination was incomplete. In situ hybridization suggested that the DNA sequences which are limited to the micronucleus in wild-type cells are present in the macronucleus of the amicronucleate cell line. Southern blots of DNA from the amicronucleate cell line were also probed with DNA sequences which are retained in the macronucleus. At least two types of genome rearrangements occurred in the BI3840 macronucleus as they do in wild-type cells. No spurious rearrangements were observed.