Nuclear Roles in the Post-Conjugant Development of the Ciliate Euplotes aediculatus II. Experimentally Induced Regeneration of Old Macronuclear Fragments1

Following conjugation in ciliates, the usual fate of the old pre-conjugant macronucleus is resorption. In some species, however, old macronuclei, or their fragments, have the ability to reform functional vegetative macronuclei when new macronuclear anlagen are defective. The present work on Euplotes shows that if anlagen are allowed to carry out their essential roles in early exconjugant development, including influence on cortical reorganization such that feeding can resume, they can then be permanently damaged by UV-microbeam irradiation and regeneration of old macronuclear fragments can occur. E. aediculatus exconjugants were anlage-irradiated at 40–60 hr of development and the irradiated cells cultured individually and fed. Squashes revealed enlargement and anteriorward migration of the persistent (posterior) macronuclear fragments. The first post-conjugant fission of such cells was delayed (times ranged 6–43 days) and did not seem to involve the damaged anlagen, which remained rudimentary, did not divide along with the cells, and were subsequently resorbed. It appeared that cell fission was supported by the fragments of the old macronuclei, which either divided or partitioned themselves between the two daughter cells. Mating tests performed on early clones derived from irradiated exconjugants revealed ample conjugation competence; intraclonal conjugation in such clones was also apparent. The absence of the immature period seen in normal exconjugants provides further evidence that the clones arose from cells with regenerated macronuclei.     

Sequence of the Small Subunit Ribosomal RNA Gene from the Hypotrichous Ciliate Euplotes aediculatus1

ABSTRACT. We have determined the complete nucleotide sequence of the coding region of the small subunit rRNA gene of the hypotrichous ciliate Euplotes aediculatus. It is 1882 nucleotides long and contains several inserts not present in the small subunit rRNA genes of the hypotrichs Oxytricha nova and Stylonychia pustulata. A comparison of the sequences suggests that E. aediculatus is much less closely related to these other two hypotrichs than they are to each other. Although the gene sequence of E. aediculatus is drifting more rapidly than those of these other two species, its faster evolutionary clock is not enough to account for the degree of difference between them.     

Cortical Control over the Direction of Macronuclear Elongation in the Heterotrich Ciliate Stentor coeruleus1

Earlier experimental work involving macronuclear implants in Stentor coeruleus has shown that the cytoplasmic cortex of the nuclear site 1) attracts the macronucleus and 2) holds it in place during interphase. Now experiments indicate macronuclei transferred with overlying cortex elongate in the direction of the transferred cortical pigment stripes, whether or not the transferred stripes realign in the direction of the host stentor's stripes. Therefore the third function of the cortex is to determine the direction of elongation and thus assure that both daughter cells at division receive part of the macronucleus.     

New Methods for Cultivating,Harvesting, and Purifying Mass Cultures of the Hypotrich Ciliate Euplotes aediculatus1

A new method is described for mass cultivation of Euplotes aediculatus, a hypotrich ciliate containing omikron-symbionts. The ciliate cultures, continuously aerated in Erlenmeyer flasks (5000 ml) with 4500 ml medium, yield densities of 2300 cells/ml which are four to five times higher than cell densities of cultures grown in unaerated Fernbach flasks. Harvesting such cultures involves the application of 25-μm mesh sieves. Cells so concentrated can be purified by using columns or special chambers in which Euplotes migrates towards the cathodes in an electric field (field strength 7 V/cm).     

Dorsal Kinetosomal Distributions in the Ciliate Genus Euplotes1

The percentage allocations of ciliary units to each ciliary row across the dorsal surface were assessed for seven marine and five freshwater populations, representing the most commonly collected morphotypes of the genus Euplotes. In marine forms, there is a spectrum of dorsal kinetosomal distributions, within which is included the characteristic distribution for the vannus complex of sibling species. In contrast, there are only two basic ciliary patterns on the dorsal surface of the most ubiquitous freshwater “species”. The congruence between the classification of morphotypes based on dorsal ciliary patterns and that based on ventral cirral patterns is remarkable, despite the morphogenetic independence of kinetosomal structures on these two surfaces.     

Some New Phenomena in Macronuclear Development of Euplotes patella Exconjugants

Two new phenomena were observed during macronuclear development in E. patella. During the formation of giant chromosomes, the number of chromosomes decreased while individual chromosomes gradually became longer and thicker. Immediately before macronuclear elongation, ring-like anlagen appeared, which did not contain chromatin at their centers. The course of macronuclear development in Euplotes is reconsidered in light of these findings.     

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.     

Nuclear roles in the postconjugant development of the ciliated protozoan Euplotes aediculatus. 1. Evidence for sequential roles of the differentiating macronucleus in exconjugant development

Thin-section and freeze-fracture data retrieved from developing fetal adrenals of mouse, rabbit, and rat reveal that gap junctions which appear as cortical and medullary cells morphogenetically assort themselves. In the presumptive zona fasciculata and reticularis communicating junctions develop between neighboring cells just prior to the onset of steroidogenesis. In the rodents, gap junctions assemble in large, particle-free formation plaques 1 day before the proliferation of smooth endoplasmic reticulum and the vesiculation of mitochondria (morphological features which are indicative of steroidogenesis). Similar observations have been obtained in fetal rabbits, but here gap junctions apparently develop in the absence of formation plaques. In each species, gap junctions apparently develop 1 or 2 days prior to a surge in corticosteroid production, suggesting that the formation of communicating junctions during the early phases of adrenal cortical differentiation may provide an avenue to coordinate steroidogenesis in the developing fetal cortex.     

Conjugation-Specific Genes in the Ciliate Euplotes crassus: Gene Expression from the Old Macronucleus

ABSTRACT. 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 ( conZ47 ) 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 conZ47 gene indicates that this conjugation-specific gene is transcribed from the old macronucleus.     

Micronuclear and Macronuclear Sequences of a Euplotes crassus Gene Encoding a Putative Nuclear Protein Kinase

The sequences of a 1.8-kbp macronuclear DNA molecule (V3), and the majority of its micronuclear counterpart, are reported. The macronuclear V3 DNA molecule contains an open reading frame that is interrupted by a single intron, while the micronuclear copy is interrupted by four internal eliminated sequences, one of which is located within the intron. The predicted protein product of the macronuclear V3 gene is a 471-amino acid polypeptide that is very similar to a group of protein-serine/threonine kinases from both plant and animal species, some of whose members appear to be involved in cell cycle or growth control.     

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.     

Correlation of Ciliary and Nuclear Development in the Life Cycle of Euplotes

SYNOPSIS. Kinetosomal changes, as indicative of cytoplasmic reorganization in binary fission and during and after conjugation, were followed in a zoochlorellae-bearing species of Euplotes. The preconjugant peristome, designated the first generation peristome, breaks down partially after the conjugants have paired; the basal section, comprising the shorter adoral membranelles and the undulating, membrane, is resorbed. A new peristome, the second generation peristome, arises as a small pit near the left ventral margin, in midline, at the time when the micronuclei are in the first meiotic prophase. By the time of the second meiotic division a single set of new cirri, the second generation cirri, has formed in each conjugant. This second set is not perfect, lacking one of the frontals. Neither the second generation peristome nor cirri develop very far, or migrate, until after separation of the conjugants. Then the new peristome replaces the old one and the new cirri become functional. However, the new peristome lacks an undulating membrane and does not complete its development, bearing only a fraction of the normal number of membranelles. At its posterior termination, at the time of condensation of the macronuclear anlage, another peristome, the third generation peristome, is formed and develops as a granular, and later striated, invagination extending posteriorly. It appears to integrate with its predecessor and, as its constituent membranelles develop, a third generation single set of new cirri arises. These replace the imperfect previous set, all of the cirri being represented. In anticipation of the first postconjugant fission, all of the cirral apparatus is discarded again and two new sets (fourth generation cirri) originate; the old (combination second and third generation) peristome is retained by the proter while a new one is provided for the opisthe. It is evident, therefore, that a rather far-reaching cytoplasmic reorganization accompanies the nuclear changes of conjugation, seeming, for the most part, to follow the nuclear changes. The old macronuclear fragments have been found not to fuse with the macronuclear anlage.     

Ca2+-dependent contractility of isolated and demembranated macronuclei in the hypotrichous ciliate Euplotes aediculatus

The hypotrichous ciliated protozoan Euplotes aediculatus possesses a characteristic C-shaped somatic nucleus (macronucleus) within the cytoplasm, which shows dynamic shape change during the cell cycle. It is shown that isolated macronuclei possess Ca(2+)-dependent contractility. Macronuclei were isolated, stuck fast on the glass surface, and subjected to different concentrations of Ca(2+) in a Ca(2+)-EGTA buffer. The nuclei became expanded at [Ca(2+)]<10(-7)M, and they contracted on subsequent addition of higher concentrations of Ca(2+). Cycles of expansion and contraction of the nucleus could be repeated many times by alternate addition of EGTA and Ca(2+), indicating that the size of isolated nuclei can be regulated by [Ca(2+)] alone. The nuclear contraction was observed in all phases of the cell cycle, but contractility was less evident around replication bands in the S phase. In addition to the hypotrichous ciliate Euplotes, similar Ca(2+)-dependent nuclear contractility was found to exist in other cell types, including protozoans of different taxa (a heliozoon Actinophrys sol and a peniculine ciliate Paramecium bursaria), and also mammalian culture cells (HeLa cells). Our findings suggest a possibility that Ca(2+)-dependent nuclear contractility may be shared among diverse eukaryotic organisms.     

Biochemical Study of Proteins of Cortical Cytoskeleton in the Ciliate Isotricha prostoma1

In Isotricha prostoma, proteins of isolated cortices retaining the ecto-endoplasmic fibrillar boundary (EEB) have been separated by one- and two-dimensional gel electrophoresis. In addition to that of tubulin, four main bands corresponding to polypeptide molecular weights of 185, 35, 23, and 22 kd have been observed on 9% SDS-polyacrylamide gels. Supernumerary major bands within a low molecular weight range (11-19 kd) are evident after separation on 7.5-15% polyacrylamide gradient gels. Two-dimensional analysis discloses - 150 polypeptide spots, most of them focusing within an acidic pl range 4.6-5.3. The cortical fibrillar structures are still distinguishable after incubation in solutions containing 1.0 M K. Cl (12 h) or 0.6 M Kl (8 h). A prolonged KI treatment (52 h), however, results in the removal of EEB filaments (?4 nm in diameter) and of a large part of the kinetid elements, so that the insoluble material is mainly represented by a reticulum of 6-nm filaments interconnecting the proximal regions of kinetosomes. Two proteins with MW of 58 and 63 kd appear to be constituents of this kinetosome-associated filamentous reticulum. Some of the cortical proteins with MW ≤23 kd are the most likely candidates for components of EEB filaments. The absence of decoration with heavy meromyosin confirms that actin is not a major component of Isotricha cortical cytoskeleton.     

Genetic Characterization and Use of a Restriction Fragment Length Variant in the Hypotrichous Ciliate Euplotes crassus1

Two forms of a macronuclear DNA molecule differing in the presence or absence of a restriction endonuclease recognition site have been detected in the hypotrichous ciliate Euplotes crassus. Through a series of genetic crosses the two forms were shown to be allelic, being derived from a single micronuclear genetic locus. This restriction fragment length variant (RFLV) was used as a genetic marker to determine that the migratory and stationary pronuclei generated during mating can be genetically non-identical. In addition, the RFLV was used to investigate the efficiency of processing of the alternate alleles during macronuclear development and their subsequent transmission during vegetative growth. Little or no bias in the processing and/or amplification of the two alleles was observed during macronuclear development. During vegetative growth, however, changes in the relative amounts of the two alleles were observed.     

The Excystment Process in the Ciliate Euplotes muscicola: An Integrated Light and Scanning Electron Microscopic Study1

Resting cysts and the excystment process in the freshwater ciliate Euplotes muscicola were studied by both light and scanning electron microscopy. Groups of distinctly crested resting cysts adhere to the substrate. Silver-stained preparations reveal surface conservation of dorsal kinetosomes and dorsal argyrome while ventral organelles are directed inward. Excystment involves the development of an expanding excystment vacuole concurrent with a localized thinning on the dorsal cyst wall surface. Cells exit through the pre-formed ostiole, mid-dorsal region first, initially by the force of cytoplasmic streaming, but later aided by cirral movement. Newly emerged cells retain the excystment vacuole and show no dorsal ridging. As the cell expels its excystment vacuole and partially unfolds, normal trophont morphology is re-established. Both cyst structure and cyst typology have implications for hypotrich taxonomy.