The histones of the ciliated protozoan Stylonychia mytilus

Histones were extracted from pure macronuclei, micronuclei and macronucleus anlagen and from chromatin which was isolated from these different nuclear fractions. Analysis of these preparations by polyacrylamide gel electrophoresis showed differences in electrophoretic patterns between the histones of these nuclei.     

Chromatin structure in the macronucleus of the ciliate Stylonychia mytilus.

Evidence is presented that macronuclear chromatin in the hypotrichous ciliate Stylonychia mytilus occurs in discrete fragments, each representing at least single genes. The size of these fragments varies between 3 and more than 70 nucleosomes with an average length of about 18 nucleosomes. This observation is discussed with respect to macronuclear structure of hypotrichous ciliates.     

Morphology and development of mirror-image doublets of Stylonychia mytilus

This paper describes the cortical anatomy and development of mirror-image doublets of Stylonychia mytilus, analyzed using the protargol technique. The reversed, or "left-handed" (LH) component of these doublets is a mirror image of the normal or "right handed" (RH) component with regard to the arrangement of cortical structures. The mirror-image patterning is imperfect, however, as the individual ciliary structures of the LH component all are of normal internal asymmetry, and the orientation of membranelles is inverted. Certain structures that would be expected to form near the line of symmetry are absent. During cell division and cortical reorganization, ciliary primordia arise and become arranged in a mirror-image pattern that is more perfect than that exhibited by the mature structures. Deviations from a mirror-image pattern appear at late stages when organelle sets differentiate within ciliary primordia: for example, the membranelle set differentiates within the oral primordium of the LH component in a sequence that is an inversion rather than a mirror image of the corresponding sequence of the RH component. This mixed control of oral development by different cortical "informational systems" accounts for some of the characteristic abnormalities of the mature oral structures of the LH component.     

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.     

The formation of polytene chromosomes during macronuclear development of the hypotrichous ciliate Stylonychia mytilus

The formation of polytene chromosomes during macronuclear development of the ciliate Stylonychia mytilus was examined in spread electron microscopical preparations. The chromatin organization of early macronuclear anlagen closely resembles the organization of micronuclear chromatin. In the course of polytenization 300 A chromatin fibers become organized in loop-like structures laterally attached to a thinner axial fiber. It is suggested that this reorganization of chromatin during polytenization is a necessary event for the subsequent chromatin elimination.     

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.     

Programmed autophagocytosis accompanying conjugation in the ciliate Stylonychia mytilus

Conjugation and postconjugant development in Stylonychia is accompanied by a period of approximately 80 hr during which the cells are unable to ingest food. This period is one of considerable synthetic activity, encompassing important portions of the development of the new macronucleus. Light- and electron-microscopic observations of conjugating pairs and exconjugant cells reveal a process of autophagocytosis that may provide the supplies of energy (and precursors) necessary for postconjugant developmental events. Small autophagosomes (AP) form in conjugants; degenerating mitochondria are prominent among the inclusions observed in these bodies. Soon after separation of pairs, large dense AP appear, apparently by coalescence and condensation of the smaller AP. These “mature” AP give only a slight acid phosphatase reaction. The number of AP slowly declines during postconjugant development; about 60 hr after separation all the AP have been digested. Several observations suggest that this autophagocytosis is part of the developmental program initiated soon after mating begins: (1) The first indications of AP formation occur while conjugating pairs are still able to feed, and thus cannot be attributed to the stress of starvation; (2) formation of large numbers of AP is rather abrupt, whereas their dissolution is very gradual, covering most of the nonfeeding period; and (3) the pattern of AP formation and dissolution is similar in cells whose new macronuclear development has been prevented by brief hydroxyurea treatment.     

RNA and Macronuclear transcription in the ciliate Stylonychia mytilus

Nuclear and cytoplasmic RNA of Stylonychia mytilus were analyzed on denaturing polyacrylamide gels. The molecular weight of rRNA precursor molecules is within a range of 2.1×10⁶ daltons. A comparison between the electrophoretic pattern of nuclear non-ribosomal RNA and cytoplasmic mRNA indicates that a considerable amount of nuclear RNA sequences is of higher molecular weight than cytoplasmic RNA sequences. The molecular weight distribution of cytoplasmic RNA supports the assumption that also in Stylonychia an average sized mRNA molecule contains 1,200–1,500 nucleotides according to a molecular weight of 4×10⁵ to 5×10⁵ daltons. The size of the polyadenylic acid fragment of poly-A⁺ RNA molecules is about 120 nucleotides. The total mass of cytoplasmic RNA is around 7.5/10¹⁰ g/cell, corresponding to 1.2×10⁷ average sized mRNA molecules per cell. RNA excess hybridization experiments show that 60% of the DNA sequences are transcribed into nuclear RNA and that the cytoplasmic mRNA sequences are homologous to about 40% of macronuclear DNA sequences. There is no indication of different frequency classes within the mRNA. The number of different mRNA species in a Stylonychia cell is 1.2–1.5×10⁴. On the average each of them is present about 1,000 times in every cell.     

Histone genes in macronuclear DNA of the ciliate Stylonychia mytilus

DNA in the macronucleus of Stylonychia mytilus exists as discrete gene-sized fragments which are derived from micronuclear DNA through a series of well-defined developmental events. It has been proposed that each of the DNA fragments might represent a gene and its controlling elements. We have investigated this possibility using genes which code for the five histone proteins. Macronuclear DNA fragments were fractionated according to size by agarose gel electrophoresis, the fragments transferred to nitrocellulose filters using the technique of Southern, and the filter-bound DNA hybridized with labeled cloned histone genes of the sea urchin, Psammechinus miliaris. Results indicate, first, that sequences homologous to the five individual histone gene probes are present in discrete macronuclear fragments which appear as bands in the gel hybridization assay. Secondly, for each of the five individual histone gene probes the homologous DNA fragments are several in number, ranging in size from 7.6 Kb (Kilo base pairs) to 0.73 Kb. For example, the largest of six detected fragments hybridizing to the H3 gene probe contains approximately 10 times the amount of DNA required to code for a Stylonychia H3 histone. The smallest detected fragment hybridizing to the H3 probe contains enough DNA to code for approximately two copies of the histone. Finally, in general, no two histone gene probes hybridized to the same macronuclear DNA fragment. This result indicates that genes coding for the five histones in Stylonychia are not located together on the same macronuclear DNA fragments and implies that the five functionally related genes would not be transcribed together as a polycistronic unit.     

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.     

Studies on the histones of the ciliate Stylonychia mytilus

Histones were extracted from pure macronuclei and maoronuolear anlagen and were analyzed by different electrophoretic techniques and by amino acid analysis. Fractionation of the histones on SDS-gels showed that the histone fractions of the macronucleus and the macronuclear anlagen are identical in their molecular weights. Comparison with calf thymus histone fractions showed considerable similarities in the molecular weights. Analysis by polyacrylamide-urea gel electrophoresis and by amino acid analysis showed quantitative differences in some histone fractions between these two types of nuclei.     

An analysis of the development program in relation to RNA metabolism in the ciliate Stylonychia mytilus

In the ciliate Stylonychia during conjugation and the terminal stages of the division cycle, development is dependent upon presynthesized mRNA. The conjugating animals show complex nuclear and cortical changes in a chronological order. Disturbance of the cortical organization or arrest of nuclear divisions in conjugants and exconjugants does not induce the cells to adjust the developmental program; the nuclear and cortical changes proceed without any reference to each other in a defined course. Similarly the terminal stages of the division cycle are not affected by cortical amputations. In contrast, vegetative animals up to mid S phase show constant RNA turnover in relation to the progress of the division cycle. In these animals, cortical amputation leads to an immediate arrest of the cell-cycle events, which are resumed only after regeneration. Nucleocytoplasmic interactions in relation to RNA metabolism of the ciliate are discussed.     

Morphology and development of left-handed singlets derived from mirror-image doublets of Stylonychia mytilus

Mirror-image doublets of Stylonychia mytilus include 2 sets of cortical structures, one with the normal "right-handed" (RH) arrangement, the other with a reversed "left-handed" (LH) arrangement. These sets, however, are incomplete, with certain structures, most notably cirri of the right marginal type, missing near the line of symmetry. When a mirror-image doublet is bisected longitudinally to separate the RH and LH components physically, each fragment undergoes a regeneration process that restores a complete set of cortical structures, including the previously missing cirri of the right marginal type. In the resulting LH cell, all ciliary structures are present in an arrangement that is globally reversed in relation to that found in RH cells; in particular, marginal cirri of the left-marginal type are formed at the cell's right margin, and marginal cirri of the right-marginal type are produced at the cell's left margin. Whereas the regenerated RH fragment always divides and initiates a clone of normal singlets, the LH fragment, though structurally nearly complete, in all cases eventually dies without dividing. The cause of death is starvation due to the formation of an abnormal oral apparatus. In the Discussion, we consider the nature and consequences of a reversal of global positional information.     

A transformation system for the hypotrichous ciliate Stylonychia mytilus

We describe a transformation system for the ciliate Stylonychia mytilus. The neomycin resistance gene from Escherichia coli transposon Tn5, which codes for the enzyme phosphotransferase and confers resistance to the antibiotic G 418, was ligated into macronuclear `gene-size' DNA molecules. Using this recombinant DNA for transformation experiments we show that the gene is replicated and expressed in transformed cells.     

Ciliary protein conservation during development in the ciliated protozoan, Oxytricha

The ciliated protozoan Oxytricha fallax possesses multiple highly localized clusters of basal bodies and cilia, all of which are broken down and rebuilt during prefission morphogenesis-with one major exception. The adoral zone of membranelles (AZM) of the ciliate oral apparatus contains approximately 1,500-2,000 basal bodies and cilia, and it is the only compound ciliary structure that is passed morphologically intact to one daughter cell at each cell division. By labeling all proteins in cells, and then picking the one daughter cell possessing the original labeled AZM, we could then evaluate whether or not the ciliary proteins of the AZM were diluted (i.e., either by degradation to constituent amino acids or by subunit exchange) during cell division. Autoradiographic analysis demonstrated that the label was highly conserved in the AZM (i.e., we saw no evidence of turnover), and electrophoretic data illustrate that at least one of the proteins of the AZM is tubulin. We, therefore, conclude that for at least some of the ciliary and basal body proteins of Oxytricha fallax, AZM morphological conservation is essentially equivalent to molecular conservation.     

Heat shock induced ultrastructural alterations in Stylonychia mytilus cells

Fine structural observations on heat shocked cells of S. mytilus reveal that cell organelles undergo structural alterations. Mitochondria show distorted shapes with disorganized cristae and vacuolar spaces. Pulse heat shock results in dilated rough endoplasmic reticulum, abundant polysomes as well as smooth endoplasmic reticulum. Heat shocked cells show membrane bound bodies containing osmiophilic cores. In macronuclei, dense chromatin breaks up into discrete bodies accompanied by the appearance of bundles of fine filaments and clustering of nuclear pores. The most prominent changes are noticed in nucleoli. Within 15 min of heat shock, nucleoli show hypertrophy and fine fibrillar zone which gradually replaces the granular zone by 120 min giving the nucleoli ring shaped configuration. In S phase cells, macronuclei show the arrested replication band in which the diffused zone (the site of DNA replication) is absent.