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Micronucleus and macronucleus morphology. View of four micronuclei and one macronucleus. To display the spherical organization of nuclei, a series of equidistant images (optical sections) were collected using lasterscanning-microscopy. (a) Average intensity planar projections (green) from a series of 42 optical sections were displayed onto planes between the XY-, XZ-, YZ-coordinate axes. 3D reconstruction (red) of the nuclear surface was rendered using To-Pro-3 counterstaining. The surface was partly cut to look inside, above the level of the yellow cutting line marked in planes XZ and YZ. (b) One optical section was selected from the middle of a series of equidistant images to display structural details of a macronucleus (M) and four micronuclei (m) stained with To-Pro-3. The nucleoplasmic connection between the ovoid distal parts of the macronucleus is not visible because it makes a turn, out of the single optical section. (Inset b1) Image taken from another mid optical section; magnification 2.5. Red arrowhead indicates a central delimitated zone of stained chromatin within an unstained spherical putative nucleolus. (c) Stylonychia lemnae DNA on an agarose gel. Left, DNA size marker. Middle, macronuclear gene-sized DNA molecules of 0.4-20 Kbp. Arrow marks ribosomal DNA. Right, micronuclear DNA.
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Spatial and temporal replication patterns are used to describe higher-order chromatin organisation from nuclei of early metazoan to mammalian cells. Here we demonstrate evolutionary conserved similarities and differences in replication patterns of micronuclei and macronuclei in the spirotrichous ciliate Stylonychia lemnae. Since this organism posse...
Contexts in source publication
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... morphology of macronuclei and micronuclei in Stylonychia lemnae was examined by confocal microscopy after chromatin had been stained with To-Pro-3. Fig. 1 provides a typical example and shows that Stylonychia lemnae possesses two micronuclei and one elongated macronucleus. To verify that no major changes of nuclear morphology occur during the isolation of nuclei or the fixation with paraformaldehyde, the morphology of nuclei in fixed and unfixed whole cells was examined after staining ...
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... diameter of the spherical micronuclei is 6-8 μm; the size of a mature macronucleus is typically approximately 80-120 μm long and 25-40 μm wide. Two, more or less ovoid, distal parts of a macronucleus that often carry invaginations are linked by a thin nucleoplasmic connection (Figs 1a and 3a,c). Regions of highly condensed chromatin can be distinguished from regions with more dispersed chromatin (Fig. 1). ...
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... size of a mature macronucleus is typically approximately 80-120 μm long and 25-40 μm wide. Two, more or less ovoid, distal parts of a macronucleus that often carry invaginations are linked by a thin nucleoplasmic connection (Figs 1a and 3a,c). Regions of highly condensed chromatin can be distinguished from regions with more dispersed chromatin (Fig. 1). The macronucleus contains numerous, mostly unstained, spherical structures with one or few delimitated spots of stained chromatin that is mostly located in the centre, or with no visible chromatin at all. These structures have been previously described as putative nucleoli (Kloetzel, 1970;Murti, 1973;Murti and Prescott, 2002) (Fig. ...
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... chromatin (Fig. 1). The macronucleus contains numerous, mostly unstained, spherical structures with one or few delimitated spots of stained chromatin that is mostly located in the centre, or with no visible chromatin at all. These structures have been previously described as putative nucleoli (Kloetzel, 1970;Murti, 1973;Murti and Prescott, 2002) (Fig. 1b, inset b1). The macronuclear DNA of Stylonychia consists of about 15,000 different gene-sized molecules with of 0.4-20 kbp (Fig. 1c). Each of these 'nanochromosomes' is amplified to a copy number of several hundred to up to 10 6 , and each is terminated with telomeres at both ends. By contrast, micronuclear DNA of Stylonychia lemnae is organised ...
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... stained chromatin that is mostly located in the centre, or with no visible chromatin at all. These structures have been previously described as putative nucleoli (Kloetzel, 1970;Murti, 1973;Murti and Prescott, 2002) (Fig. 1b, inset b1). The macronuclear DNA of Stylonychia consists of about 15,000 different gene-sized molecules with of 0.4-20 kbp (Fig. 1c). Each of these 'nanochromosomes' is amplified to a copy number of several hundred to up to 10 6 , and each is terminated with telomeres at both ends. By contrast, micronuclear DNA of Stylonychia lemnae is organised into approximately 120 chromosomes, each having an average size of 18 Mbp ( Ammermann, 1970). The chromatin structure of ...
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... is amplified to a copy number of several hundred to up to 10 6 , and each is terminated with telomeres at both ends. By contrast, micronuclear DNA of Stylonychia lemnae is organised into approximately 120 chromosomes, each having an average size of 18 Mbp ( Ammermann, 1970). The chromatin structure of its micronuclei appears very homogenous (Fig. 1b) and they possess no nucleoli (Prescott, ...
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Citations
... 2D and 3D microscopic studies on replication labeled interphase nuclei demonstrated the genome-wide partitioning into discrete structural entities, called replication domains (RDs) with typical replication patterns for early, mid and late replication [19]. These patterns have been evolutionary conserved [20,21] and indicate the tight coupling of replication timing with nuclear architecture (Fig. 1C). ...
Genome replication requires duplication of the complete set of DNA sequences together with nucleosomes and epigenetic signatures. Notwithstanding profound knowledge on mechanistic details of DNA replication, major problems of genome replication have remained unresolved. In this perspective article, we consider the accessibility of replication machines to all DNA sequences in due course, the maintenance of functionally important positional and structural features of chromatid domains during replication, and the rapid transition of CTs into prophase chromosomes with two chromatids. We illustrate this problem with EdU pulse-labeling (10 min) and chase experiments (80 min) performed with mouse myeloblast cells. Following light optical serial sectioning of nuclei with 3D structured illumination microscopy (SIM), seven DNA intensity classes were distinguished as proxies for increasing DNA compaction. In nuclei of cells fixed immediately after the pulse-label, we observed a relative under-representation of EdU-labeled DNA in low DNA density classes, representing the active nuclear compartment (ANC), and an over-representation in high density classes representing the inactive nuclear compartment (INC). Cells fixed after the chase revealed an even more pronounced shift to high DNA intensity classes. This finding contrasts with previous studies of the transcriptional topography demonstrating an under-representation of epigenetic signatures for active chromatin and RNAPII in high DNA intensity classes and their over-representation in low density classes. We discuss these findings in the light of current models viewing CDs either as structural chromatin frameworks or as phase-separated droplets, as well as methodological limitations that currently prevent an integration of this contrasting evidence for the spatial nuclear topography of replication and transcription into a common framework of the dynamic nuclear architecture.
... 8,9 Although nuclear architecture in different cell types can significantly differ in detail, the abovementioned pattern is evolutionarily conserved from the unicellular to multicellular organisms. 7,10 The crucial role that the spatial arrangement of chromatin plays in transcriptional regulation may account for the evolutionary stability of the nuclear architecture. 11,12 In the transcription process, genes at the nuclear periphery are repositioned to the interior where they are transcribed. ...
Purpose:
The mouse retina is considered a remarkable model for studying gene functions. However, variations in genetic background influence phenotypes in the mammalian retina. Therefore this study aimed to investigate the effects of the genetic background on the nuclear architecture of photoreceptor cells and the light-induced behavior in C57BL/6, 129 × 1/svj, and ICR mice.
Methods:
The nuclear architecture of photoreceptor cells was investigated using various staining methods on postnatal day 21 (P21). Murine behavior was observed using a light-dark compartment test.
Results:
The outer nuclear layer and retina were significantly thicker in C57BL/6 mice than in 129 × 1/svj mice. The percentage of photoreceptors with one chromocenter was significantly higher in C57BL/6 mice than in 129 × 1/svj and ICR mice on P21. The numbers of photoreceptor cells in C57BL/6 and ICR mice were significantly higher than those in 129 × 1/svj mice. The behavior test revealed that the walking distance and velocity in the light compartment were increased in C57BL/6 and ICR mice compared to 129 × 1/svj mice.
Conclusions:
Different mouse strains had a distinct nuclear architecture of photoreceptors on P21, and C57BL/6 and ICR mice were more active than 129 × 1/svj mice in response to light-induced stress.
Translational relevance:
This study demonstrates a technique for assessing retinal structures and nuclear architecture in various strains of mice, which are often used to model human retinal disease. Hence, this study may help to elucidate the effect of genetic or disease-induced variance in retinal architecture and the organization of photoreceptor nuclear content on visual function in humans.
... 17,22 At the interface between the FZ and RZ, replication proteins are localized, and DNA replication occurs at hundreds of foci that initially fire from the nuclear envelope before proceeding toward the nuclear interior ( Figure 1B). [23][24][25] So far, the molecular machinery organizing the RB and controlling its orderly translocation is unknown. The altered chromatin states in the FZ and RZ suggest that chromatin modifiers play a role; however, the mechanisms coordinating the motility of the band and its capacity for initiating replication are more enigmatic, perhaps involving signaling cascades. ...
... The altered chromatin states in the FZ and RZ suggest that chromatin modifiers play a role; however, the mechanisms coordinating the motility of the band and its capacity for initiating replication are more enigmatic, perhaps involving signaling cascades. 23,26 Given the vectorial nature of DNA replication in this system, the RB is a unique but so far largely unexplored model for investigating the underlying chromatin dynamics, nuclear changes, and spatiotemporal patterns associated with DNA replication. ...
... In the RB, the DNA replication hub progresses lengthwise along the axis of the macronucleus as replication proceeds radially from the nuclear envelope toward the interior (Figure 1). 23 Accordingly, the replication envelope may reflect a loss of this lengthwise movement along the axis of the macronucleus. Given the relative simplicity of the replication envelope, it is also tempting to speculate that this system could reflect a reversion to the ancestral state of the RB. ...
DNA replication is a ubiquitous and conserved cellular process. However, regulation of DNA replication is only understood in a small fraction of organisms that poorly represent the diversity of genetic systems in nature. Here we used computational and experimental approaches to examine the function and evolution of one such system, the replication band (RB) in spirotrich ciliates, which is a localized, motile hub that traverses the macronucleus while replicating DNA. We show that the RB can take unique forms in different species, from polar bands to a “replication envelope,” where replication initiates at the nuclear periphery before advancing inward. Furthermore, we identify genes involved in cellular transport, including calcium transporters and cytoskeletal regulators, that are associated with the RB and may be involved in its function and translocation. These findings highlight the evolution and diversity of DNA replication systems and provide insights into the regulation of nuclear organization and processes.
... Different foci pattern exist within S phase nuclei among which we can identify small early replication foci predominantly at the centre of the nucleus and late replicating foci that are bigger and usually found at the nuclear or nucleolar periphery (Nakamura et al., 1986). This three dimensional replication foci pattern is highly conserved in eukaryotes among specifies, as it has been observed in plants and other animals as well (Alexandrova et al., 2003;Mayr et al., 2003;Postberg et al., 2005). ii. ...
DNA replication is very well orchestrated in mammalian cells thanks to a tight regulation of the temporal order of replication origin activation, commonly called replication timing (RT). The replication timing of a given replication domain (RD) is very robust and depends on the cell type. Upon low replication stress, replication forks progress slower and it has been shown that some fragile regions are replicated later or even under-replicated. These replication delay leads to DNA damage and genetic instability, a common marker of cancers. Except for these fragile regions, the direct impact of low replication stress on the RT of the whole genome has not been explored yet. The aim of my thesis was to analyse and compare the replication timing of 6 human cell lines from different tissues (healthy or from tumours) in response to mild replication stress. Assessing this question, I have first observed heterogeneous response in between cell lines, some cancer cells were much more impacted by low replication stress. Strikingly, in some cancer cells, specific RD are undergoing a switch from late to early replication in response to replication stress. Very interestingly, this RT alteration was still detected in daughter cells. These findings disclosed a new mechanism mainly used by cancer cells in response to replication stress that brings another proof of their genome plasticity, allowing a quick response and adaptation to stress that, eventually, gives better resistance to genotoxic agents.
... Generally, nascent RNA in vegetative macronuclei of Stylonychia colocalizes with the same nuclear bodies that contain fibrillarin, which is presumably involved in rRNA processing. Therefore, there are no specialized nucleoli, which occur physically separated from other transcription sites [29,30]. In the parental macronuclear fragments fibrillarin is not detectable. ...
In the ciliate Stylonychia, somatic macronuclei differentiate from germline micronuclei during sexual reproduction, accompanied by developmental sequence reduction. Concomitantly, over 95% of micronuclear sequences adopt a heterochromatin structure characterized by the histone variant H3.4 and H3K27me3. RNAi-related genes and histone variants dominate the list of developmentally expressed genes. Simultaneously, 27nt-ncRNAs that match sequences retained in new macronuclei are synthesized and bound by PIWI1. Recently, we proposed a mechanistic model for ‘RNA-induced DNA replication interference’ (RIRI): during polytene chromosome formation PIWI1/27nt-RNA-complexes target macronucleus-destined sequences (MDS) by base-pairing and temporarily cause locally stalled replication. At polytene chromosomal segments with ongoing replication, H3.4K27me3-nucleosomes become selectively deposited, thus dictating the prospective heterochromatin structure of these areas. Consequently, these micronucleus-specific sequences become degraded, whereas 27nt-RNA-covered sites remain protected. However, the biogenesis of the 27nt-RNAs remains unclear. It was proposed earlier that in stichotrichous ciliates 27nt-RNA precursors could derive from telomere-primed bidirectional transcription of nanochromosomes and subsequent Dicer-like (DCL) activity. As a minimalistic explanation, we propose here that the 27nt-RNA precursor could rather be mRNA or pre-mRNA and that the transition of coding RNA from parental macronuclei to non-coding RNAs, which act in premature developing macronuclei, could involve RNA-dependent RNA polymerase (RDRP) activity creating dsRNA intermediates prior to a DCL-dependent pathway. Interestingly, by such mechanism the partition of a parental somatic genome and possibly also the specific nanochromosome copy numbers could be vertically transmitted to the differentiating nuclei of the offspring.
... Nucleic acids labelling (5-fluorouridine [5FU] for nascent RNA and 5-iodo-2′-deoxyuridine [IdU] or 5-chloro-2′deoxyuridine [CldU] for nascent DNA), in situ antibody stainings, poly[A]-RNA FISH and subsequent confocal microscopy were done as reported [20,21]. A list of oligonucleotides used is provided as Supplemental Information (Additional file 1: Table S1). ...
... Accordingly, during developmental chromosome polytenization DNA replication should not occur simultaneously at all sites. In fact, we recognized spatiotemporally regulated replication patterns in the micro-and macronuclei of vegetative Stylonychia previously [20]. To test whether differentially replicated regions exist also in polytene chromosomes, we performed pulse (5 h)-chase (1 h)-pulse (5 h) labelling of newly replicated DNA using the nucleotide analogues iododeoxyuridine (IdU; early pulse) and chlorodeoxyuridine (CldU; late pulse). ...
... Replication bands in Stylonychia macronuclei are disc-shaped accumulations of hundreds of synchronously firing replication foci, which appear at the distal tips of the macronuclei at the onset of S-phase and migrate thereafter to the centre of the macronucleus. Hereby, the motility of macronuclear chromatin is strictly constrained, ensuring that each nanochromosome becomes evenly replicated and its copy number remains preserved (Fig. 6b) [20]. We described above that 27nt-RNAs were absent in vegetative Stylonychia, whereas a low level 21-22nt-RNAs seemed to exist over the entire life cycle (Fig. 2). ...
Background: During sexual reproduction in the unicellular ciliate Stylonychia somatic macronuclei differentiate from germline micronuclei. Thereby, programmed sequence reduction takes place, leading to the elimination of > 95% of germline sequences, which priorly adopt heterochromatin structure via H3K27me3. Simultaneously, 27nt-ncRNAs become synthesized from parental transcripts and are bound by the Argonaute protein PIWI1.
Results: These 27nt-ncRNAs cover sequences destined to the developing macronucleus and are thought to protect them from degradation. We provide evidence and propose that RNA/DNA base-pairing guides PIWI1/27nt-RNA complexes to complementary macronucleus-destined DNA target sequences, hence transiently causing locally stalled replication during polytene chromosome formation. This spatiotemporal delay enables the selective deposition of temporarily available histone H3.4K27me3 nucleosomes at all other sequences being continuously replicated, thus dictating their prospective heterochromatin structure before becoming developmentally eliminated. Concomitantly, 27nt-RNA-covered sites remain protected.
Conclusions: We introduce the concept of ‘RNA-induced DNA replication interference’ and explain how the parental functional genome partition could become transmitted to the progeny.
... To examine whether DNA and RNA are indeed selectively packaged into different ApoBDs by flow cytometry, we first established a method to monitor the distribution of RNA into ApoBDs. Jurkat T cells were stained with SYTO RNAselect to monitor the localization of RNA prior to apoptosis induction 23 . ApoBDs containing a substantial amount of RNA, or ApoBDs containing no or very low amounts of RNA, were identified by the electronic gating strategy as shown in Supplementary Fig. 4. Localization of RNA into membrane blebs and ApoBDs according to SYTO RNAselect staining was also validated by confocal microscopy ( Supplementary Figs 1d and 2c). ...
Over 200 billion cells undergo apoptosis every day in the human body in order to maintain tissue homeostasis. Increased apoptosis can also occur under pathological conditions including infection and autoimmune disease. During apoptosis, cells can fragment into subcellular membrane-bound vesicles known as apoptotic bodies (ApoBDs). We recently developed a flow cytometry-based method to accurately differentiate ApoBDs from other particles (e.g. cells and debris). In the present study, we aim to further characterize subsets of ApoBDs based on intracellular contents and cell type-specific surface markers. Utilizing a flow cytometry-based approach, we demonstrated that intracellular contents including nuclear materials and mitochondria are distributed to some, but not all ApoBDs. Interestingly, the mechanism of ApoBD formation could affect the distribution of intracellular contents into ApoBDs. Furthermore, we also showed that ApoBDs share the same surface markers as their cell of origin, which can be used to distinguish cell type-specific ApoBDs from a mixed culture. These studies demonstrate that ApoBDs are not homogeneous and can be divided into specific subclasses based on intracellular contents and cell surface markers. The described flow cytometry-based method to study ApoBDs could be used in future studies to better understand the function of ApoBDs.
... With the exception of the examination of Blepharisma undulans Stein using TEM (Kennedy 1965), observations have been collected using nuclear stains with light microscopy and results reported with drawings, demonstrating the variable nature of B. americanum in both cell size and changes in macronuclear morphology throughout its life cycle (Giese 1973;Kennedy 1965;McLoughlin 1957;Suzuki 1954;Young 1938). Fluorescent microscopy, the approach used here, allows for detailed observations of nuclear morphology throughout ciliate life cycles (Maurer-Alcal a and Katz 2016; Postberg et al. 2005), as well as estimates of ploidy levels. ...
Blepharisma americanum, a member of the understudied ciliate class Heterotrichea, has a moniliform somatic macronucleus that resembles beads on a string. B. 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 to an approximate DNA content difference of 500-fold from micronucleus to macronucleus and a macronuclear ploidy of ~1100N 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. This article is protected by copyright. All rights reserved.
... A dynamic localization pattern was observed for Pgm relative to large DAPI-poor regions inside the developing MAC: prominent Pgm foci were detected inside these regions at early developmental stages, before splitting into smaller foci and eventually becoming dispersed at late stages, until a diffuse Pgm signal filled the whole compartment (Supplementary Figure S5C). These nuclear DAPI-poor compartments are reminiscent of prominent spherical DNA-poor structures that were described previously in the developing and mature MAC of ciliates (34)(35)(36) and exhibit similar features to those of putative nucleoli (37). The biological significance of Pgm localization in these compartments will have to be explored in future studies. ...
During sexual processes, the ciliate Paramecium eliminates 25-30% of germline DNA from its somatic genome. DNA elimination includes excision of ∼45 000 short, single-copy internal eliminated sequences (IESs) and depends upon PiggyMac (Pgm), a domesticated piggyBac transposase that is essential for DNA cleavage at IES ends. Pgm carries a core transposase region with a putative catalytic domain containing three conserved aspartic acids, and a downstream cysteine-rich (CR) domain. A C-terminal extension of unknown function is predicted to adopt a coiled-coil (CC) structure. To address the role of the three domains, we designed an in vivo complementation assay by expressing wild-type or mutant Pgm-GFP fusions in cells depleted for their endogenous Pgm. The DDD triad and the CR domain are essential for Pgm activity and mutations in either domain have a dominant-negative effect in wild-type cells. A mutant lacking the CC domain is partially active in the presence of limiting Pgm amounts, but inactive when Pgm is completely absent, suggesting that presence of the mutant protein increases the overall number of active complexes. We conclude that IES excision involves multiple Pgm subunits, of which at least a fraction must contain the CC domain.
... Such examples include the variant surface glycoprotein (VSG) genes found on mini-chromosomes in the parasitic trypanosome Trypanosoma brucei (Navarro, et al. 2007), the crystalline chromosomes of dinoflagellates (Bachvaroff, et al. 2014; de la Espina, et al. 2005) and the fragmented and amplified chromosomes found in some ciliates (e.g. Postberg, et al. 2005; Prescott 1994). Despite the presence of unusual chromosomes, Postberg et al. (2005) have suggested that aspects of the CT-IC model also exist in the ciliate Stylonychia lemnae and may be a common eukaryotic nuclear feature. ...
... Postberg, et al. 2005; Prescott 1994). Despite the presence of unusual chromosomes, Postberg et al. (2005) have suggested that aspects of the CT-IC model also exist in the ciliate Stylonychia lemnae and may be a common eukaryotic nuclear feature. The " gene-sized " nanochromosomes in S. lemnae form chromatin dense regions, resembling chromosome territories, surrounded by a diffuse chromatin poor network throughout the somatic macronucleus (Postberg, et al. 2005). ...
... Despite the presence of unusual chromosomes, Postberg et al. (2005) have suggested that aspects of the CT-IC model also exist in the ciliate Stylonychia lemnae and may be a common eukaryotic nuclear feature. The " gene-sized " nanochromosomes in S. lemnae form chromatin dense regions, resembling chromosome territories, surrounded by a diffuse chromatin poor network throughout the somatic macronucleus (Postberg, et al. 2005). Analyses of interactions between nuclear architecture and patterns of molecular evolution (i.e. ...
The relationship between nuclear architecture and patterns of molecular evolution in lineages across the eukaryotic tree of
life is not well understood, partly because molecular evolution is traditionally explored as changes in base pairs along a
linear sequence without considering the context of nuclear position of chromosomes. The ciliate Chilodonella uncinata is an ideal system to address this relationship between nuclear architecture and patterns of molecular evolution as the somatic
macronucleus of this ciliate is comprised of a peripheral DNA rich area (orthomere) and a DNA poor central paramere (i.e.
a heteromeric macronucleus). Moreover, because the somatic chromosomes of C. uncinata are highly processed into "gene-sized" chromosomes (i.e. nanochromosomes), we can assess fine-scale relationships between
location and sequence evolution. By combining fluorescence microscopy and analyses of transcriptome data from C. uncinata, we find that highly expressed genes have the greatest codon usage bias and are enriched in DNA poor regions. In contrast,
genes with less biased sequences tend to be concentrated in DNA abundant areas, at least during vegetative growth. Our analyses
are consistent with recent work in better-studied systems (e.g. plants and animals) where nuclear architecture plays a role
in gene expression. At the same time, the unusual localization of nanochromosomes suggests that the highly structured nucleus
in C. uncinata may create a 'gene bank' that facilitates rapid changes in expression of genes required only in specific life history stages.
By using "non-model" organisms like as C. uncinata, we can also explore the universality of eukaryotic features while also
providing examples of novel properties (i.e. the presence of a 'gene bank') that build from these features.
