Mature Macronucleus Research Articles

: a database of ciliate genome rearrangements.

Ciliated protists exhibit nuclear dimorphism through the presence of somatic macronuclei (MAC) and germline micronuclei (MIC). In some ciliates, DNA from precursor segments in the MIC genome rearranges to form transcriptionally active genes in the mature MAC genome, making these ciliates model organisms to study the process of somatic genome rearrangement. Similar broad scale, somatic rearrangement events occur in many eukaryotic cells and tumors. The <mds_ies_db> (http://oxytricha.princeton.edu/mds_ies_db) is a database of genome recombination and rearrangement annotations, and it provides tools for visualization and comparative analysis of precursor and product genomes. The database currently contains annotations for two completely sequenced ciliate genomes: Oxytricha trifallax and Tetrahymena thermophila.

Differential expression of histone H3 genes and selective association of the variant H3.7 with a specific sequence class in Stylonychia macronuclear development

BackgroundRegulation of chromatin structure involves deposition of selective histone variants into nucleosome arrays. Numerous histone H3 variants become differentially expressed by individual nanochromosomes in the course of macronuclear differentiation in the spirotrichous ciliate Stylonychia lemnae. Their biological relevance remains to be elucidated.ResultsWe show that the differential assembly of H3 variants into chromatin is strongly correlated with the functional separation of chromatin structures in developing macronuclei during sexual reproduction in Stylonychia, thus probably determining the fate of specific sequences. Specific H3 variants approximately 15 kDa or 20 kDa in length are selectively targeted by post-translational modifications. We found that only the 15 kDa H3 variants including H3.3 and H3.5, accumulate in the early developing macronucleus, and these also occur in mature macronuclei. H3.7 is a 20 kDa variant that specifically becomes enriched in macronuclear anlagen during chromosome polytenization. H3.7, acetylated at lysine-32 (probably equivalent to lysine-36 of most H3 variants), is specifically associated with a sequence class that is retained in the mature macronucleus and therefore does not undergo developmental DNA elimination. H3.8 is another 20 kDa variant that is restricted to the micronucleus. H3.8 is selectively targeted by lysine methylation and by serine or threonine phosphorylation. Intriguingly, the expression and chromatin localization of the histone variant H3.3 was impaired during macronuclear differentiation after RNA interference knock-down of Piwi expression.ConclusionsDifferential deposition of H3 variants into chromatin strongly correlates with the functional distinction of genomic sequence classes on the chromatin level, thus helping to determine the fate of specific DNA sequences during sexual reproduction in Stylonychia. Consequently, H3 variants are selectively targeted by post-translational modifications, possibly as a result of deviations within the recognition motifs, which allow binding of effector proteins. We propose that differential assembly of histone variants into chromatin of various nuclear types could contribute to nuclear identity, for example, during differential development of either new micronuclei or a macronuclear anlage from mitosis products of the zygote nucleus (synkaryon). The observation that the Piwi-non-coding RNA (ncRNA) pathway influences the expression and deposition of H3.3 in macronuclear anlagen indicates for the first time that selective histone variant assembly into chromatin might possibly depend on ncRNA.

Spatial and temporal plasticity of chromatin during programmed DNA-reorganization in Stylonychia macronuclear development

Background:In this study we exploit the unique genome organization of ciliates to characterize the biological function of histone modification patterns and chromatin plasticity for the processing of specific DNA sequences during a nuclear differentiation process. Ciliates are single-cell eukaryotes containing two morphologically and functionally specialized types of nuclei, the somatic macronucleus and the germline micronucleus. In the course of sexual reproduction a new macronucleus develops from a micronuclear derivative. During this process specific DNA sequences are eliminated from the genome, while sequences that will be transcribed in the mature macronucleus are retained.Results:We show by immunofluorescence microscopy, Western analyses and chromatin immunoprecipitation (ChIP) experiments that each nuclear type establishes its specific histone modification signature. Our analyses reveal that the early macronuclear anlage adopts a permissive chromatin state immediately after the fusion of two heterochromatic germline micronuclei. As macronuclear development progresses, repressive histone modifications that specify sequences to be eliminated are introduced de novo. ChIP analyses demonstrate that permissive histone modifications are associated with sequences that will be retained in the new macronucleus. Furthermore, our data support the hypothesis that a PIWI-family protein is involved in a transnuclear cross-talk and in the RNAi-dependent control of developmental chromatin reorganization.Conclusion:Based on these data we present a comprehensive analysis of the spatial and temporal pattern of histone modifications during this nuclear differentiation process. Results obtained in this study may also be relevant for our understanding of chromatin plasticity during metazoan embryogenesis.

The Pathway to Detangle a Scrambled Gene

BackgroundProgrammed DNA elimination and reorganization frequently occur during cellular differentiation. Development of the somatic macronucleus in some ciliates presents an extreme case, involving excision of internal eliminated sequences (IESs) that interrupt coding DNA segments (macronuclear destined sequences, MDSs), as well as removal of transposon-like elements and extensive genome fragmentation, leading to 98% genome reduction in Stylonychia lemnae. Approximately 20–30% of the genes are estimated to be scrambled in the germline micronucleus, with coding segment order permuted and present in either orientation on micronuclear chromosomes. Massive genome rearrangements are therefore critical for development.Methodology/Principal FindingsTo understand the process of DNA deletion and reorganization during macronuclear development, we examined the population of DNA molecules during assembly of different scrambled genes in two related organisms in a developmental time-course by PCR. The data suggest that removal of conventional IESs usually occurs first, accompanied by a surprising level of error at this step. The complex events of inversion and translocation seem to occur after repair and excision of all conventional IESs and via multiple pathways.Conclusions/Significance This study reveals a temporal order of DNA rearrangements during the processing of a scrambled gene, with simpler events usually preceding more complex ones. The surprising observation of a hidden layer of errors, absent from the mature macronucleus but present during development, also underscores the need for repair or screening of incorrectly-assembled DNA molecules.

Synthesis of pre-rRNA and mRNA is directed to a chromatin-poor compartment in the macronucleus of the spirotrichous ciliate Stylonychia lemnae

In contrast to the chromosomal genome organization common to most eukaryotes, DNA in the macronucleus of spirotrichous ciliates like Stylonychia lemnae is organized into small gene-sized nanochromosomes. We intended to elucidate whether a spatial organization of nucleoli similar to other eukaryotes can be found in absence of typical chromosomes. Whereas micronuclei of Stylonychia exhibit homogenously stained heterochromatin and possess no nucleoli, macronuclear chromatin is compartmentalized and contains numerous putative nucleoli. Since the identity of these spherical structures has never been unequivocally demonstrated to date, we applied immunofluorescence techniques together with confocal laser scanning microscopy to identify nucleolar bodies in the macronucleus of Stylonychia and to analyse their spatial organization. We found that multiple spherical bodies, which fulfil nucleolar function, occupy a peripheral localization in mature macronuclei. Using fibrillarin/Nop1p as a nucleolar marker, we monitored the assembly of such nucleolar bodies during macronuclear differentiation. 3D-FISH experiments revealed that rRNA genes are mostly concentrated adjacent to but not inside of fibrillarin/Nop1p-containing bodies. We further showed that transcription sites for rRNA synthesis but also for mRNA synthesis occur predominantly at surfaces of nucleolar bodies and chromatin-poor spaces bordering condensed chromatin. Our data suggest that transcription of rRNA genes in the macronucleus of Stylonychia does not rely on a classical nucleolus-type organization. We assume that vectorial synthesis and processing of rRNA and mRNA is directed to a functional interchromatin compartment.

De novo cytosine methylation in the differentiating macronucleus of the stichotrichous ciliate Stylonychia lemnae.

Dramatic DNA reorganization and elimination processes occur during macronuclear differentiation in ciliates. In this study we analyzed whether cytosine methylation of specific sequences plays a functional role during DNA rearrangement. Three classes of sequences, macronuclear-destined sequences (MDSs, pCE7), members from a large family of transposon-like elements and micronuclear-specific sequences (pLJ01), differing in their structure and future destiny during nuclear differentiation, were studied in the micronucleus, the developing macronucleus and, when present, in the mature macronucleus. While the MDSs become processed to a 1.1 and 1.3 kb gene-sized macronuclear DNA molecule, the family of transposon-like elements represented by MaA81 becomes removed late in the course of polytene chromosome formation. The micronuclear-specific sequence pLJ01 is eliminated together with bulk micronuclear DNA during degradation of polytene chromosomes. No methylated cytosine could be detected in the vegetative macronucleus and no difference in methylation pattern was observed either between micronucleus and developing macronucleus in MDSs or in a micronuclear-specific sequence. However, a significant percentage of the cytosines contained in the transposon-like element becomes methylated de novo in the course of macronuclear differentiation. This is the first demonstration that cytosine methylation in specific sequences occurs during macronuclear differentiation and may provide a first step towards understanding epigenetic factors involved in DNA processing.

Separation of Micronuclear DNA of Stylonychia lemnae by Pulsed-Field Electrophoresis and Identification of a DNA Molecule with a High Copy Number

DNA from the hypotrichous ciliatae Stylonychia lemnae was separated by PFGE. In addition to the separation of the macronuclear DNA molecules with a size up to approximately 40 kb, we were able to separate the micronuclear DNA with a size between approximately 90 kb and 2 Mb. One very prominent 90-kb DNA band appeared on the pulsed-field gels. We propose that this 90-kb DNA fragment represents a linear plasmid residing in the micronucleus in a very high copy number. About 10% of the micronuclear DNA consists of the 90-kb DNA molecule. It appears in the micronucleus as well as in the macronuclear anlagen during macronuclear development but not in the mature macronucleus. Thus, the multicopy DNA is eliminated during fragmentation of the macronuclear anlagen DNA in the course of macronuclear development. Therefore, this 90-kb DNA molecule might serve as an excellent tool to study the recognition and elimination of DNA during nuclear differentiation of hypotrichous ciliates.

Evolution of DNA organization in hypotrichous ciliates.

Profound changes have been introduced into the germline (micronuclear) and somatic (macronuclear) genomes of hypotrichous ciliates during evolution. First, multiple, short, unique, noncoding sequences, called IESs, have been inserted into micronuclear genes. IESs are spliced out of each gene, and the gene segments, called MDSs, are ligated during conversion of the micronuclear genome to a macronuclear genome after cell mating. The IESs in a particular gene can change dramatically in number, position, length, and sequence during speciation. Once inserted, IESs can shift along the DNA of a gene 1 or 2 bp at a time by a mutational mechanism that does not alter the coding sequence of the gene. Second, the MDSs in the same genes have been rearranged into random or nonrandom, scrambled disorder. The origin of nonrandom scrambling patterns can be explained by a model of simultaneous insertion of multiple IESs into a germline gene during evolution. Subsequent recombinations among the IESs may change the MDS arrangement from a nonrandomly scrambled to a randomly scrambled pattern, including inversions of MDSs. Third, during the conversion of a micronucleus to a macronucleus after cell mating, MDSs are ligated in the unscrambled, orthodox order in association with IES excision, and the genes are then removed from the chromosomes as individual, short DNA molecules. These gene-size molecules are amplified many-fold to produce a mature macronucleus. All of these phenomena attest to a remarkable fluidity of the hypotrich genome both over evolutionary time and in the conversion of a germline genome to a somatic genome. The significance of this fluidity for the life and evolution of these organisms is still obscure. Recombination among IESs could shuffle MDSs and facilitate faster evolution of new genes.

Conjugation-specific genes in the ciliate Euplotes crassus: gene expression from the old macronucleus.

Following mating or conjugation, the hypotrichous ciliate Euplotes crassus undergoes a massive genome reorganization process. While the nature of the rearrangement events has been well studied, little is known concerning proteins that carry out such processes. As a means of identifying such proteins, differential screening of a developmental cDNA library, as well as construction of a cDNA subtraction library, was used to isolate genes expressed only during sexual reproduction. Five different conjugation-specific genes have been identified that are maximally expressed early in conjugation, during the period of micronuclear meiosis, which is just prior to macronuclear development and the DNA rearrangement process. All five genes are retained in the mature macronucleus. Micronuclear, macronuclear, and cDNA clones of one gene (conZA7) have been sequenced, and the results indicate that the gene encodes a putative DNA binding protein. In addition, the presence of an internal eliminated sequence in the micronuclear copy of the conZA7 gene indicates that this conjugation-specific gene is transcribed from the old macronucleus.

Elucidation of nucleus-cytoplasm interaction: change in ability of the nucleus to express sexuality according to clonal age in Paramecium.

The expression of sexual activity in Paramecium caudatum is repressed for about 50 fissions after conjugation. The ability of the macronucleus to affect the expression of sexual activity according to the age of clonal development was investigated using the macronuclear fusion-reorganization method. When a mature macronucleus was transplanted into an immature cell and fused with a macronucleus in the immature cell, the clones derived from the recipient showed sexual immaturity. In a reverse experiment, an immature macronucleus was transplanted into a mature cell, and the clones also showed sexual immaturity. The ability of the macronucleus to transform mature cells to immature cells was clonal age-dependent. The characteristics of the immature-mature hybrid macronucleus indicate that the immature macronucleus is dominant over the mature macronucleus with respect to the ability to express sexual activity. On the other hand, in cells of the early immaturity period, the micronucleus, known as the germ nucleus, shows the ability to undergo meiosis and eventually to produce progeny under control of the mature macronucleus. The expression of sexual activity is thought to be governed by the clonal age of the macronucleus and not by the clonal age of the micronucleus or cytoplasm. The macronucleus seems to determine the ability to express sexual activity by counting post-conjugation divisions and keeping track of the age of the clone.

The processing of macronuclear-destined DNA sequences microinjected into the macronuclear anlagen of the hypotrichous ciliate Stylonchia lemnae

We describe the construction of a vector carrying the micronuclear versions of two macronuclear DNA molecules, one of which was modified by the insertion of a polylinker sequence. This vector was injected into the polytene chromosomes of the developing macronucleus of Stylonychia and its processing during further macronuclear development and its fate in the mature macronucleus were analyzed. In up to 30% of injected cells the modified macronuclear DNA sequence could be detected. While the internal eliminated sequences (IES) present in the macronuclear precursor DNA sequence are still retained in the mature macronucleus, the modified macronuclear DNA sequence is correctly cut out from the vector, telomeres are added de novo and it is stably retained in the macronucleus during vegetative growth of the cells. This vector system represents an experimental system that allows the identification of DNA sequences involved in the processing of macronuclear DNA sequences during macronuclear development.

Ultrastructure of the nuclear apparatus of the marine psammobiotic ciliate Trachelocerca multinucleataDragesco (karyorelictida)

Summary The nuclear apparatus of Trachelocerca multinucleata comprises more than 20 macronuclei and about 10 micronuclei arranged in a longitudinal row. The macronuclei vary in fine structure. Young macronuclei contain a large particulate chromocenter and a prominent nucleolus. Mature macronuclei have a giant spongy chromocenter surrounded by several small nucleoli which are partly segregated into fibrillar and granular ones, and a finely fibrillar sphere. Both the nucleoli and the sphere closely contact the material of the chromocenter, which also presents foci of local decondensation. The micronuclei possess a conventional envelope with occasional outgrowths; they lack both nucleoli and spheres. The micronuclear chromatin is fully condensed except of some small regions.

Analysis of nuclei from exponentially growing and conjugated Tetrahymena thermophila using the flow microfluorimeter

Isolated nuclei of Tetrahymena thermophila from both exponentially growing cultures and from cells following conjugation have been analysed using a flow microfluorimeter. The macronuclei from a culture in exponential growth display a single broad distribution of DNA contents, without bimodal character. The micronuclei are virtually all in G2 phase (4C). The mean of the macronuclear DNA distribution is about 12.4 times the micronuclear mean (50C). When cells are starved in preparation for conjugation, the macronuclei DNA content is decreased about 30 %, but the distribution remains similar to that of nuclei from a culture in exponential growth. Following conjugation, the macronuclear anlagen develop through a set of relatively synchronous endoreplications. At 12 h after the initiation of conjugation the anlagen are at a 4C stage and at 18 h they are virtually all at a 8C stage. If the culture is refed, anlagen development progresses to a 16C and 32C, but the synchrony is poorly conserved. Cells that are not refed are arrested at the 8C stage and only a fraction of the population ever become mature macronuclei. In general we do not observe distinct peaks of anlagen with DNA contents in excess of 32C. The amitotic division of macronuclei may obscure any endoreplications producing anlagen stages with higher DNA content.

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

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

Alteration of the Tetrahymena Genome During Nuclear Differentiation*†

Synopsis.The DNA of the macro‐ and the micronucleus of Tetrahymena thermophila has been compared by various biochemical methods. It became evident from their thermal denaturation temperatures and buoyant densities that the 2 DNAs were very similar in overall composition. Small differences were detected when the sequence complexities of these DNAs were compared by DNA renaturation studies. The studies suggested that ˜ 10% of the micronuclear genome was lost or underrepresented in the macronucleus. Comparison of individual gene levels revealed further differences. By using the technic of gene cloning a micronuclear sequence was isolated which hybridized only with micronuclear, but not with macronuclear DNA. These results indicated the occurrence of elimination or underreplication of this sequence in the macronucleus. Gene amplification was also shown to occur. In the micronucleus only a single copy of rDNA was found integrated into the chromosome. During macro‐nuclear development, amplification was observed to occur, and the amount of rDNA to increase, until there were ˜ 200 copies per haploid genome in the mature macronucleus. all of them extrachromosomal and palindromic. The 3rd case of alteration involved a simple repeated sequence, (CCCCAA)n, present in the termini of rDNA and also in many other locations of the genome. Restriction endonuclease digestion studies revealed drastic differences in the organization of the repeats between macro‐and micronucleus. These differences may be interpreted as the results of chromosome fragmentation which occurs at every cluster of the repeats during macronuclear development. The relationship between this event and gene amplification and elimination is discussed.

Polytene chromosomes of Oxytricha: biochemical and morphological changes during macronuclear development in a ciliated protozoan.

After conjugation in the ciliated protozoan, Oxytricha, polytene chromosomes are formed during the development of a macronucleus from a micronucleus. Here we report a microscopic study of these chromosomes and an analysis of their DNA. The polytene chromosomes of Oxytricha bear a strong morphological resemblance to the polytene chromosomes of the Dipteran salivary gland. The nucleus of a developing macronuclear anlage contains 120 +/- 2 polytene chromosomes and each chromosome has an average of 81 hands; a total of about 10,000 bands per nucleus. At a later stage in development, the number of bands per chromosome is reduced by a factor of four, presumably due to fusion of adjacent bands. The polytene chromosomes then break up into their constituent bands, each of which is encased in a vesicle. There are about 2,700 vesicles per nucleus.--During the growth of polytene chromosomes, there is a change in the relative proportion of sequences in the DNA. The DNA from polytene nuclei has a buoyant density of 1.695 g/cc, significantly lighter than the density of the original micronuclear DNA (1.698 G/cc to 1.702 g/cc). We interpret this buoyant density change to be the result of differential replication of DNA sequences during polytene chromosome growth. A second change in DNA composition occurs after the polytene stage of development, shown by a shift in buoyant density to 1.701 g/cc in the DNA of the mature macronucleus. During this second process, the molecular weight of the DNA is reduced from greater than 50 x 10(6) daltons to about 2 x 10(6) daltons.

Electron-microscopic observations on the macronuclear development of Stylonychia mytilus and Tetrahymena pyriformis (Ciliophora-Protozoa).

ABSTRACT This report describes an ultrastructural investigation of macronuclear development follow-ing conjugation in Stylonycltia mytilus (a spirotrichous ciliate) and Tetrahymena pyriformis (a holotrichous ciliate). In S. mytilus, polytene chromosomes are formed in the young macronucleus (macronuclear Anlage). They are subsequently broken between the bands by ‘membranous’ partitions; the assembly of the membranes appears to be concomitant with the formation of the polytene chromosomes. The membranes in the Anlage appear to originate from fibrous material seen in the early Anlage. This fibrous material in the earlier stages is seen concentrated at several points along the border of the inner nuclear membrane. In the later stages it is seen in the interior of the Anlage, outlining the developing polytene chromosomes. As the chromosomes reach the maximum degree of polyteny, the fibrous material condenses to acquire a mem-branous appearance and extends into the interband regions. The Anlage throughout this period shows a progressive increase in size. Subsequently, the membranes enclose individually each band plus portions of the 2 adjacent interband regions of the polytene chromosomes to form a large number of vesicles. After vesicle formation the Anlage shrinks, and the chromatin inside the vesicles shows degradative changes. Finally, the vesicles disappear, the membrane degradation products appear at the nuclear membrane, and the Anlage now contains nucleoli. The Anlage increases its DNA content by multiple roundsof replication to become a mature macronucleus. The ultra-structural changes observed in the Anlage support the idea of genetic diminution (i.e. extensive DNA synthesis, elimination of many DNA nucleotide sequences, and amplification of the remaining DNA nucleotide sequences in a second period of DNA synthesis) proposed earlier on the basis of cytochemical, biochemical, and limited electron-microscope studies. In T. pyriformis, the macronuclear development differs substantially from that of Stylonychia. Features such as the formation and degradation of polytene chromosomes are absent in the macronuclear development of Tetrahymena; the young macronucleus in this cell becomes a mature macronucleus by progressive increment in size and chromatin content with no apparent genetic diminution. These observations agree with cytochemical studies on the macronuclear development of Tetrahymena.

Studies on the Macronucleus of Paramecium aurelia. II. Development of Macronuclear Anlagen

SYNOPSIS. The development of the macronuclear Anlagen of Paramecium aurelia was studied by means of electron, light and ultra‐violet microscopy of timed stages following conjugation. In the youngest Anlagen, no differentiated structures could be made out, and staining reactions gave little or no indications of the presence of DNA or RNA. As development proceeds, a number of conspicuous “sponge‐like” RNA‐containing bodies surrounded by a “matrix” containing DNA can be seen. Eventually these RNA bodies develop DNA centres and apparently disintegrate, yielding the “large bodies” characteristic of the mature macronucleus, and the “small bodies” then also appear. The relation of these observations to interpretations of the structural elements in the macronucleus is discussed.