Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.     

冠突伪尾柱虫（Pseudourostyla cristata）实验再生研究

冠突伪尾柱虫(Pseudvurostyla cristata) 含约70枚大核。我们用显微手术横切G1期细胞,得前后两块相等断片;分别培养。60小时后,断片再生完成。在再生过程中,随细胞体积增大,大核数目也增加。大核的数目和细胞体积存在着一定的均衡关系。在细胞无性分裂过程中,许多大核改组后,融合成一个融合大核。这个融合大核具两个仔虫的大核数目和DNA量。我们用显微手术得到含融合大核的后断片。在后断片再生后恢复的虫体内,我们发现本应分配到两个仔虫中去的大核数目,被限制在一个虫体的大核数目上。这说明了细胞质可以影响和调节大核的数目。并还证明了这种虫体大核DNA量较正常虫的大核DNA量约多一倍。其中大部分虫体分裂时,大核不经改组就开始融合和分裂;从而使DNA量回复正常。同讨还发现小部分虫体通过排出大核多余核物质方式来调节大核DNA量。这些现象说明了细胞核质之间存在着一种调节相对平衡和相互协调的机制。     

Comparison of Singlet and Doublet Paramecium tetraurelia: DNA Content,Protein Content and the Cell Cycle*

SYNOPSIS Doublet Paramecium tetraurelia contain either a single macronucleus which is substantially larger than that in a singlet cell, or 2 smaller macronuclei. Doublets have approximately twice the DNA content and twice the total protein content of singlets. The cell cycle of doublets is 164% as long as that of singlets, but the relative position of the macronuclear DNA synthesis period within the cell cycle is the same as in singlets. The DNA content of doublets is regulated so that differences in the number of macronuclei do not produce corresponding changes in DNA content; bimacronucleate doublets have only 27% more DNA than unimacronucleate doublets.     

Regulation of Macronuclear DNA Content in Paramecium tetraurelia*†

Synopsis.
The amitotic division of the macronucleus of Paramecium tetraurelia produces daughter macronuclei which frequently differ in DNA content. In wild-type cells these differences are small, but can be increased substantially by the action of mutant genes. The variance in macronuclear DNA content would increase continuously if there were no mechanism to regulate it. Paramecium has a very effective regulatory mechanism—all cells synthesize similar amounts of macronuclear DNA, regardless of the number of macronuclei or their prereplication DNA content. DNA synthesis is controlled at the level of macronuclear subunits, and the postreplication macronucleus consists of a mosaic of subunits that have undergone different numbers of replication events during the previous cell cycle. It is evident from experimental results that the amount of DNA synthesized can be influenced by the total size or mass of the cell. Experimental modification of the initial DNA content leads to no change in the amount of DNA synthesized, or in the subsequent protein content of the cells, but modification of cell size causes corresponding changes in the amount of DNA synthesized and in the size of the macronucleus. The implications of these observations for cell growth and the cell cycle are discussed.     

A Nuclear Protein Involved in Apoptotic-like DNA Degradation in Stylonychia: Implications for Similar Mechanisms in Differentiating and Starved Cells

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) from Tetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation in Stylonychia.     

Localization of genes for ribosomal RNA in the nuclei of Oxytricha fallax

The location of ribosomal RNA (rRNA) genes in the nuclei of the ciliated protozoan, Oxytricha fallax, was analysed by in situ hybridization. The micronuclear genome of O. fallax has typical chromosomal DNA organization. Macronuclei, although derived from micronuclei, lack chromosomes and instead contain short pieces of DNA ranging from 500 to 20 000 base pairs in length. In situ hybridization was carried out to determine if specific DNA sequences are limited to certain locations within the macronucleus, or if sequences are randomly arranged. Cells were fixed, squashed and then hybridized with ³H-labelled RNA synthesized in vitro using cloned O. fallax rDNA as a template. After autoradiography, silver grains were found to be distributed uniformly over the entire macronucleus without any detectable localization to specific regions. The uniformity of hybridization indicates that rDNA molecules are randomly dispersed throughout the macronucleus and suggests that the macronuclear genetic apparatus lacks any substantial multimolecular organization. S phase macronuclei also showed a uniform distribution of rDNA molecules, irrespective of the position of the replication band at which DNA synthesis takes place. The micronuclei, in contrast, did not show any hybridization, even in cells in which macronuclei were heavily labelled. Macronuclear anlagen, in which the micronuclear chromosomes are polytenized, also do not hybridize. This absence of hybridization indicates a much lower concentration of rDNA in the micronucleus than in the macronucleus. The change in rDNA concentration of rRNA genes presumably occurs during the complicated process of development of a macronucleus from a micronucleus.     

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 had disappeared. The hybridization in the anlage then 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.     

The development of the macronucleus in the ciliated protozoan Stylonychia mytilus

Ciliated protozoa are characterized by generative micronuclei and vegetative polyploid macronuclei. Micronuclei of Stylonychia mytilus contain 1 600 times as much DNA per haploid genome as E. coli. Most of this DNA is shown to be repetitive. The development of the macronucleus involves, as demonstrated by cytology, only 1/3 of the chromosomes which in a first replication phase are polytenized in probably 5 replication steps and appear as giant chromosomes. At this developmental stage considerable amounts of repetitive DNA are still present in the chromosomes. During the subsequent disintegration phase more than 90% of the DNA are eliminated from the macronucleus anlage. The remainder is further replicated five times and composes the final macronucleus. Since this DNA reassociates with a reaction rate almost identical to an ideal second order reaction its kinetic complexity can be determined by comparison with the kinetic complexity of E. coli DNA. Macronuclear DNA reassociates with a kinetic complexity of 26 times the kinetic complexity of E. coli DNA (corrected for GC content) which indicates that macronuclear DNA sequences exist at a ploidy level of 4 096 C. We assume that macronuclear DNA may be present only once per haploid genome. In this case it represents only 1.6% of the DNA in micronuclei or 10% of the DNA in the giant chromosome stage.     

Nuclear differentiation and nucleic acid synthesis in well-fed exconjugants of Paramecium aurelia

Paramecium aurelia exconjugants contain new macronuclear anlagen and numerous fragments of the old pre-zygotic macronucleus. Macronuclear anlagen develop during the first two cell cycles after conjugation. During this time their volume increases from about 11 m³ to about 3700 m³ and more than 10 doublings of DNA content occur. The rate of DNA synthesis is between two and three times as great as in the vegetative macronucleus. — In macronuclear fragments, however, DNA synthesis is suppressed. The rate of DNA synthesis in macronuclear fragments during the extended first cell cycle after conjugation (11 1/2 hr. vs. 5 1/2 hr. for the vegetative cell cycle) is only about one-third of the rate in vegetative macronuclei and there is only a 65% increase in the mean DNA content of fragments. The rate of fragment DNA synthesis continues to decrease during each of the subsequent two cell cycles. — Unlike the rate of DNA synthesis, the rate of RNA synthesis per unit of DNA is similar in macronuclear anlagen, macronuclear fragments and fully developed macronuclei. Macronuclear fragments continue to synthesize RNA at the normal rate long after the new macronuclei are fully developed. Fragments contribute about 80% of all RNA synthesized during the first two cell cycles after conjugation. RNA synthesis begins very early in the development of macronuclear anlagen and nucleolar material appears during the first half-hour of anlage development. — Chromosome-like structures were never observed during anlage development and there was no evidence of two periods of DNA synthesis separated by a DNA poor stage as has been observed in several hypotrichous Ciliates.     

Chromatin elimination and the genetic organisation of the macronucleus in Tetrahymena thermophila

In exponentially growing Tretrahymena thermophila the DNA content of the following structures was determined by cytophotometry: macronuclei of sister cells immediately after division; micronuclei; extranuclear chromatin in dividing cells and postdividers. Further, the development of macro-nuclear DNA amount in successive cell generations was determined. It was found that chromatin elimination is a frequent process reducing DNA content by about 4% per fission. This chromatin disappears within 20 min after division. The quantity of DNA extruded is highly variable and is different from the micronuclear DNA amount or multiples of it. The frequency of generations with two replication rounds as well as those without replication is estimated to be in the range of 2% each. These findings together with the qualitative difference between micro- and macronuclear DNAs suggest that the macronucleus of Tetrahymena is not entirely composed of complete genomes and that parts of the genetic material must be treated specifically for different sequences either during extrusion or during replication.     

Isolation and Properties of Macronuclei from Tetrahymena thermophila1

A simple and efficient method is described for the isolation of macronuclei from Tetrahymena thermophila (7B). The steps involved are deciliation and removal of the mucocysts’ contents by dibucaine treatment, digitonin mediated lysis, differential centrifugations, and finally isopyenic sucrose density gradient centrifugation. Judging from the distribution of marker enzymes and electron microscopy, the macronuclei obtained were free of cytoplasmic and paniculate contamination and were highly active in endogenous RNA-synthesis (1.5 pmol UTP incorporation/ng DNA min at 30°C). The ratio of protein: RNA: DNA was 2.0:0.33:1.0 (weight) and each macronucleus contained an average of 17 pg DNA. The average yield of isolation was 50%.     

Morphology and development of the macronuclei of the ciliates Stylonychia mytilus and Euplotes aediculatus

Some stages of macronuclear anlagen development, known from earlier investigations (see Fig. 1), were studied in detail. The results are: a) The giant chromosomes of Stylonychia mytilus are not somatically paired, but are connected end-to-end to form one or a few composite chromosomes. When they later disintegrate, the bands become isolated granules. b) Spectrophotometric measurements show that during the DNA-poor stage which follows the disintegration of the chromosomes, the macronuclear anlagen of Euplotes have a DNA content of 21 c, while the syncaryotic (deriving from syncarya) and hemicaryotic (deriving from haploid hemicarya) anlagen of Stylonychia have the DNA content of diploid micronuclei (2c). Nevertheless the syncaryotic anlagen of Stylonychia and Euplotes initially develop two nucleoli at the end of this stage, the hemicaryotic anlagen of Stylonychia only one. From this it is concluded that the genes of one giant chromosome band stay together in one granule, c) Labeled DNA from the giant chromosomes which remains in the anlagen during the DNA-poor stage is distributed approximately equally to the daughter nuclei during the first few fissions of the exconjugants.-Autoradiographic experiments showed that the DNA of the macronuclei of Stylonychia that is duplicated at one time in a replication band is not duplicated simultaneously during the next DNA-duplication. The DNA duplications during the second polyploidization stage of the macronuclear anlagen development are exceptions, because the mixing of the macronuclear DNA which occurs before every fission does not occur during the second polyploidization stage.—The pseudomicronuclei which sometimes are formed from the macronuclei in emicronucleated strains of Stylonychia contain numerous elements which are much smaller than the chromosomes.—The macronucleus of Stylonychia is very insensitive to irradiation with X-rays.—The results lead to the following hypothesis: The macronuclei of the two hypotrich ciliates contain unconnected chromomeres or small aggregates which are distributed at random to the two daughter nuclei during the divisions.Research supported by the Deutsche Forschungsgemeinschaft.     

Elimination of germ-line tandemly repeated sequences from the somatic genome of the ciliate Oxytricha fallax

The ciliated protozoa exhibit nuclear dimorphism. The genome of the somatic macronucleus arises from the germ-line genome of the micronucleus following conjugation. We have studied the fates of highly repetitious sequences in this process. Two cloned, tandemly repeated sequences from the micronucleus of Oxytricha fallax were used as probes in hybridizations to micronuclear and macronuclear DNA. The results of these experiments show: (1) the cloned repeats are members of two apparently unrelated repetitious sequence families, which each appear to comprise a few percent of the micronuclear genome, and (2) the amount of either family in the macronuclei from which our DNA was prepared is about 1/15 that found in an equal number of diploid micronuclei. Most, if not all, of the apparent macronuclear copies of these repeats can be accounted for by micronuclear contamination, which strongly suggests that these sequences are eliminated from the macronuclei and have no vegetiative function.     

Cytochemical studies on the problem of macronuclear subnuclei in tetrahymena

DNA amounts in macronuclei and micronuclei of Tetrahymena pyriformis were measured by Feulgen microspectrophotometry. Assuming that the unreplicated micronucleus is diploid, the unreplicated macronucleus was found to contain approximately 45 times the haploid DNA amount. The relationship of these findings to the 45 independently assorting genetic subunits characterized by Allen and Nanney and their collaborators is discussed. The pattern of DNA synthesis in macro- and micronuclei during the cell cycle is also described.     

Nuclear Features of the Heterotrich Ciliate Blepharisma americanum: Genomic Amplification,Life Cycle,and Nuclear Inclusion

Blepharisma americanum, a member of the understudied ciliate class Heterotrichea, has a moniliform somatic macronucleus that resembles beads on a string. Blepharisma americanum is distinguishable by its pink coloration derived from the autofluorescent pigment blepharismin and tends to have a single somatic macronucleus with 3–6 nodes and multiple germline micronuclei. We used fluorescence confocal microscopy to explore the DNA content and amplification between the somatic and germline nuclei of B. americanum through its life cycle. We estimate that the DNA content of the macronucleus and micronucleus are 43 ± 8 Gbp and 83 ± 16 Mbp respectively. This correlates with an approximate DNA content difference of 500‐fold from micronucleus to macronucleus and a macronuclear ploidy of ~1,100 N as compared to the presumably diploid micronucleus. We also investigate a previously reported macronuclear inclusion, which is present sporadically across all life cycle stages; this inclusion looks as if it contains blepharismin based on its fluorescent properties, but its function remains unknown. We also provide additional detail to our understanding of life cycles changes in B. americanum by analyses of fluorescent images. Overall, the data analyzed here contribute to our understanding of the diversity of nuclear architecture in ciliates by providing details on the highly polyploid somatic macronucleus of B. americanum.     

Downward regulation of macronuclear DNA content in Paramecium tetraurelia : Effects of excess DNA on the subsequent DNA and protein content and the cell growth rate

Paramecium cells were selected which received the entire parental macronucleus at fission and thus started the cell cycle with twice the normal post-fission DNA content. During each of the subsequent two cell cycles the cells synthesized approximately as much DNA as did control cells. The amount of excess macronuclear DNA was consequently halved during each cell cycle. The minimum pre-fission DNA content was just larger than the mean post-replication DNA amount, confirming that a similar amount of DNA, approximately equal to the mean post-fission DNA content of the non-selected population, was synthesized in macronuclei, regardless of the post-fission DNA content. These observations confirm a model for DNA content regulation previously devised for Paramecium and are inconsistent with DNA content regulation schemes proposed for other ciliates. The increased DNA content has no effect either on the subsequent total protein content of pre-fission cells, or on the rate of cell growth. This suggests that the rate of cell growth is limited by the size of the cell when the macronuclear gene-dosage is equal to or greater than that in normal cells. The results also suggest that the amount of DNA synthesized within an interfission period is also limited by the size of the cell and is proportional to the cell mass. Paramecium does not require a fixed nucleocy oplasmic ratio as a pre-condition either for cell division, or, by inference, for initiation of DNA synthesis.     

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."