Chromatin structure in somatic nucleus of ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis>

The structural organization of macronuclear chromatin of the ciliate Didinium nasutum was studied. The macronuclear genome of D. nasutum is represented by DNA molecules of subchromosomal size. At interphase, macronuclear chromatin is organized into chromatin of 100–200-nm clumps. Some of these clumps form short, thick fibers that consist of several chromatin clumps. Using the differential staining of nucleic acids on ultrathin sections, we revealed perichromatin fibers and granules on the surface of many chromatin clumps. A 3D model of the spatial distribution of chromatin clumps in the macronucleus was built based on serial ultrathin sections and peculiar features of chromatin spatial organization were studied.     

Relative position of nucleolar chromatin and nucleolar components in ciliate <Emphasis Type="Italic">Didinium nasutum</Emphasis> somatic nuclei

According to our computer modeling data obtained earlier, nucleoli in interphase ciliates Didinium nasutum are complex netlike structures, in which the trabeculumor lamella-shaped fibrillar component is located on the periphery, and the granular component in the central part of the nucleolus. Chromatin bodies connected with nucleoli act as the nucleolar organizers in D. nasutum. In the present work, the arrangement of all chromatin bodies, which could correspond to nucleolar organizers by morphological criteria, is studied by means of a 3D-reconstruction. It is shown that all of these chromatin bodies are localized outside the nucleoli, on the fibrillar component’s periphery. Even those chromatin bodies which appeared to be completely surrounded by the fibrillar nucleolar component on single ultrathin sections are actually settled down in nucleolus cavities open to the nucleoplasm. This proves that the RNA processing in D. nasutum nucleoli is directed toward the center of nucleoli, where the granular component is located. The analysis of the nucleolar chromatin distribution made it possible to conclude that different parts of the complex interfase netlike nucleoli of D. nasutum have approximately the same activity.     

Differential DNA Amplification and Copy Number Control in the Hypotrichous Ciliate Euplotes crassus

ABSTRACT. During macronuclear development in hypotrichous ciliated protozoans, several thousand macronuclear DNA molecules are amplified several-hundred fold. We investigated the regulation of this amplification by determining the copy numbers of three different macronuclear DNA molecules in the hypotrichous ciliate Euplotes crassus. Two of the macronuclear DNA molecules were present in approximately 1,000 copies per cell, while the third was present in approximately 6,500 copies per cell. These reiteration levels were achieved either during macronuclear development, or shortly thereafter, and were maintained during vegetative growth. The most abundant macronuclear DNA molecule is present as a single-copy sequence in the micronuclear genome. Thus, its high copy number results from differential amplification. These results indicate that DNA amplification during macronuclear development is regulated individually for each macronuclear DNA molecule.     

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.     

A thermal denaturation study of macronuclear chromatin in Blepharisma japonicum (Protozoa, Ciliophora, Heterotrichida)

The macronuclear chromatin of the ciliate Blepharisma japonicum, in two starvation states, was studied by thermal denaturation analysis. The behaviour of B. japonicum chromatin, native and reconstituted with Tetrahymena pyriformis H1 histone, was analysed. The data obtained are consistent with the hypothesis that B. japonicum macronuclear chromatin contains a H1-like peptide associated with the linker DNA, although this peptide is reduced in amount and/or chromatin stabilising ability when compared to Tetrahymena macronuclear H1.     

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

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

Characterization of the macronuclear DNA of different species ofTetrahymena

Summary The macronuclear DNAs from 20 different species ofTetrahymena were characterized using alternating Orthogonal Field (AOF) gel electrophoresis. Each species has approximately 300 different macronuclear DNA molecules that range in size from about 100–2000 kb pairs. Although the individual macronuclear DNA molecules are not well resolved on an AOF gel, most species have a unique profile of macronuclear DNA. The sequences that hybridize with histone H4 (Tetrahymena) and ubiquitin (yeast) genes were identified on the separated macronuclear DNA molecules of the different species. All species have 2 histone H4 genes located on macronuclear DNA molecules of different sizes. This is consistent with the duplication of the histone H4 gene prior to the speciation events leading to the various species ofTetrahymena. The number and sizes of the macronuclear DNA molecules that hybridize with the ubiquitin probe vary from species to species. A grouping of the different species ofTetrahymena based on this hybridization pattern paralels groupings of the species based on ribosomal RNA sequences and isoenzymes. Some intraspecific variation among different strains ofTetrahymena thermophila was detected using ubiquitin and 5S ribosomal RNA as probes.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

NUCLEOMORPH KARYOTYPE DIVERSITY IN THE FRESHWATER CRYPTOPHYTE GENUS CRYPTOMONAS1

Cryptophytes are unicellular, biflagellate algae with plastids (chloroplasts) derived from the uptake of a red algal endosymbiont. These organisms are unusual in that the nucleus of the engulfed red alga persists in a highly reduced form called a nucleomorph. Nucleomorph genomes are remarkable in their small size (<1,000 kilobase pairs [kbp]) and high degree of compaction (～1 kbp per gene). Here, we investigated the molecular and karyotypic diversity of nucleomorph genomes in members of the genus Cryptomonas. 18S rDNA genes were amplified, sequenced, and analyzed from C. tetrapyrenoidosa Skuja CCAP979/63, C. erosa Ehrenb. emmend. Hoef‐Emden CCAP979/67, Cryptomonas sp. CCAP979/52, C. lundii Hoef‐Emden et Melkonian CCAP979/69, and C. lucens Skuja CCAP979/35 in the context of a large set of publicly available nucleomorph 18S rDNA sequences. Pulsed‐field gel electrophoresis (PFGE) was used to examine the nucleomorph genome karyotype of each of these strains. Individual chromosomes ranged from ～160 to 280 kbp in size, with total genome sizes estimated to be ～600–655 kbp. Unexpectedly, the nucleomorph karyotype of Cryptomonas sp. CCAP979/52 is significantly different from that of C. tetrapyrenoidosa and C. lucens, despite the fact that their 18S rDNA genes are >99% identical to one another. These results suggest that nucleomorph karyotype similarity is not a reliable indicator of evolutionary affinity and provides a starting point for further investigation of the fine‐scale dynamics of nucleomorph genome evolution within members of the genus Cryptomonas.     

Free genes for rRNAs in the macronuclear genome of the ciliate Stylonychia mytilus

When separated on an agarose gel, macronuclear DNA of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3×10⁶ to 14×10⁶ dalton. Hybridization of 25 S rRNA, 17 S rRNA or 5 S RNA to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear DNA but that the DNA in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the DNA fragment coding for rRNA was found to be 4.5×l0⁶ dalton, the fragment coding for 5 S RNA has a molecular weight of 150,000–250,000 dalton.     

Fine structure of the macronucleus during the cell division cycle of Euplotes eurystomus,a Ciliate Protozoon

This paper reports new observations obtained from a study of macronuclear fine structure throughout various stages of the cell division cycle of Euplotes. Study of the ultrastructural organization of the macronuclear chromatin indicates that much of the chromatin is organized into continuous masses, portions of which appear to be attached to the nuclear envelope. The macronuclear envelope appears unchanged in the region of a replication band, and apparent attachments of the chromatin to the inner membrane of the nuclear envelope are maintained in the reticular and diffuse zones. Intranuclear helices were never observed in the diffuse zone. During macronuclear division, linear elements (fibrils or microtubules) were observed in close association with both chromatin bodies and nucleoli. The ultrastructural data suggest that the intranuclear linear fibrils have two functions: elongation of the dividing nucleus, and attachment of chromatin bodies and nucleoli to the envelope. The significance of these observations for macronuclear division and chromatin segregation is considered.     

Morphology,Morphogenesis, and Molecular Phylogeny of a New Soil Ciliate,Sterkiella multicirrata sp. nov. (Ciliophora,Hypotrichia) from China

The morphology and morphogenesis of a new soil hypotrich ciliate, Sterkiella multicirrata sp. nov., was investigated using live observation and protargol staining. The new species is characterised by: body elliptical, 110–180 × 45–75 μm in vivo, an average of 35 adoral membranelles; usually 19 frontoventral‐transverse cirri, consisting of three frontal, five frontoventral, one buccal, four postoral ventral, two pretransverse, and four transverse cirri; four macronuclear nodules, and 2–5 micronuclei. S. multicirrata sp. nov. differs from its congeners mainly in the number of frontoventral‐transverse cirri and macronuclear nodules. Morphogenesis of the new species is similar to its congeners; the primary difference exists in the segmentation of the frontoventral‐transverse cirral anlagen, which is usually generated in a 1:2:3:3:5:5 pattern. Based on the small subunit ribosomal DNA sequence, the phylogenetic position of the new species is discussed.     

Organization of the Euplotes crassus Micronuclear Genome1

Euplotes crassus, like other hypotrichous ciliated protozoa, eliminates most of its micronuclear chromosomal DNA in the process of forming the small linear DNA molecules that comprise the macronuclear genome. By characterizing randomly selected lambda phage clones of E. crassus micronuclear DNA, we have determined the distribution of repetitive and unique sequences and the arrangement of macronuclear genes relative to eliminated DNA. This allows us to compare the E. crassus micronuclear genome organization to that of another distantly related hypotrichous ciliate, Oxytricha nova. The clones from E. crassus segregate into three prevalent classes: those containing primarily eliminated repetitive DNA (Class I); those containing macronuclear genes in addition to repetitive sequences (Class II); and those containing only eliminated unique sequence DNA (Class III). All of the repetitive sequences in these clones belong to the same highly abundant repetitive element family. Our results demonstrate that the sequence organization of the E. crassus and O. nova micronuclear genomes is related in that the macronuclear genes are clustered together in the micronuclear genome and the eliminated unique sequences occur in long stretches that are uninterrupted by repetitive sequences. In both organisms a single repetitive element family comprises the majority of the eliminated interspersed middle repetitive DNA and appears to be preferentially associated with the macronuclear sequence clusters. The similarities in the sequence organization in these two organisms suggest that clustering of macronuclear genes plays a role in the chromosome fragmentation process.     

Sequence and Properties of Cagein,a Coiled-Coil Scaffold Protein Linking Basal Bodies in the Polykinetids of the Ciliate Euplotes aediculatus

In the hypotrich ciliate Euplotes, many individual basal bodies are grouped together in tightly packed clusters, forming ventral polykinetids. These groups of basal bodies (which produce compound ciliary organelles such as cirri and oral membranelles) are cross-linked into ordered arrays by scaffold structures known as “basal-body cages.” The major protein comprising Euplotes cages has been previously identified and termed “cagein.” Screening a E. aediculatus cDNA expression library with anti-cagein antisera identified a DNA insert containing most of a putative cagein gene; standard PCR techniques were used to complete the sequence. Probes designed from this gene identified a macronuclear “nanochromosome” of ca. 1.5 kb in Southern blots against whole-cell DNA. The protein derived from this sequence (463 residues) is predicted to be hydrophilic and highly charged; however, the native cage structures are highly resistant to salt/detergent extraction. This insolubility could be explained by the coiled-coil regions predicted to extend over much of the length of the derived cagein polypeptide. One frameshift sequence is found within the gene, as well as a short intron. BLAST searches find many ciliates with evident homologues to cagein within their derived genomic sequences.     

Chromatin elimination in the hypotrichous ciliate Stylonychia mytilus

Chromatin elimination in the hypotrichous ciliate Stylonychia mytilus was studied by means of electron microscopy using a microspreading procedure. In the polytene chromosomes of the macronuclear anlagen three organization patterns are observed: Bands of various size composed of 300 Å chromatin fibers, large blocks of 300 Å nucleofilaments which probably represent the heterochromatic regions of the chromosome and axial 120 Å filaments. Those DNA sequences which become eliminated belong to the 300 Å fiber type. The eliminated chromatin occurs in the form of rings of variable size corresponding to a DNA content between 18 and 160 Kb while the axial 120 Å filaments appear to be preserved.     

Electron microscopic studies on the macronuclear development in the ciliate Chilodonella uncinata.

The development of macronuclear anlage in the ciliate Chilodonella uncinata has been studied through electron microscopy. The ultrastructure of macro- and micronuclei is described for comparison. During the first stage of development, when the DNA content of the macronuclear anage increases from 2 C to 32 C, chromosomes are visible as distinct osmiophilic bodies inside the anlage. At the end of the initial polyploidization phase, the chromosomes despiralize, giving rise to long filamentous structures 25 to 50 nm in diameter. The latter show a singular banding pattern, with dense bands 12 to 25 nm thick alternating regularly with less dense interbands. Such an organization has not yet been observed in any other type of nucleus. These filamentous structures have been interpreted as highly despiralized oligotenic chromosomes. During the final stage of macronuclear development, these structures condense into thin chromatin strands and small dense granules; the number of granules increases progressively as the chromatin strands disappear. These small granules very likely fuse amongst themselves to form the chromatin granules of the vegetative macronucleus. No evidence has yet been found for a fragmentation of chromatin in this ciliate, but this problem needs further study. The old macronucleus maintains a normal ultrastructure until a late stage of development of the macronuclear anlage, becoming pycnotic only towards the very end of the latter process.     

Gene-sized macronuclear DNA molecules are clustered in micronuclear chromosomes of the ciliate Oxytricha nova.

Following the sexual phase of its life cycle, the hypotrichous ciliate Oxytricha nova transforms a copy of its chromosomal micronucleus into a macronucleus containing short, linear DNA molecules with an average size of 2.2 kilobase pairs. In addition, more than 90% of the DNA sequences in the micronuclear genome are eliminated during this process. We have examined the organization of macronuclear DNA molecules in the micronuclear chromosomes. Macronuclear DNA molecules were found to be clustered and separated by less than 550 base pairs in two cloned segments of micronuclear DNA. Recombinant clones of two macronuclear DNA molecules that are adjacent in the micronucleus were also isolated and examined by DNA sequencing. The two macronuclear DNA molecules were found to be separated by only 90 base pairs in the micronuclear genome.     

Fine Structure of the Macronuclear Anlage of the Hypotrichous Ciliate Keronopsis rubra

In exconjugants of the hypotrich Keronopsis a large, highly polyploid macronuclear anlage is formed from which condensed chromatin bodies are passed into the cytoplasm where they are thought to give rise to the numerous small macronuclei of the vegetative cell. Electron microscopy shows that the chromatin bodies within the macronuclear anlage are separated from each other by sheets of low contrast lamellar material. The anlage appears therefore as a composite nucleus containing prepacked units which are extruded into the cytoplasm following condensation.