A Nuclear Protein Involved in Apoptotic-like DNA Degradation in Stylonychia: Implications for Similar Mechanisms in Differentiating and Starved Cells

Ciliates are unicellular eukaryotic organisms containing two types of nuclei: macronuclei and micronuclei. After the sexual pathway takes place, a new macronucleus is formed from a zygote nucleus, whereas the old macronucleus is degraded and resorbed. In the course of macronuclear differentiation, polytene chromosomes are synthesized that become degraded again after some hours. Most of the DNA is eliminated, and the remaining DNA is fragmented into small DNA molecules that are amplified to a high copy number in the new macronucleus. The protein Pdd1p (programmed DNA degradation protein 1) from Tetrahymena has been shown to be present in macronuclear anlagen in the DNA degradation stage and also in the old macronuclei, which are resorbed during the formation of the new macronucleus. In this study the identification and localization of a Pdd1p homologous protein in Stylonychia (Spdd1p) is described. Spdd1p is localized in the precursor nuclei in the DNA elimination stage and in the old macronuclei during their degradation, but also in macronuclei and micronuclei of starved cells. In all of these nuclei, apoptotic-like DNA breakdown was detected. These data suggest that Spdd1p is a general factor involved in programmed DNA degradation in Stylonychia.     

HISTONE F1 OF TETRAHYMENA MACRONUCLEI : Unique Electrophoretic Properties and Phosphorylation of F1 in an Amitotic Nucleus

Histone fraction F1 has been isolated and purified from macronuclei of the ciliated protozoan, Tetrahymena pyriformis. In many respects, Tetrahymena F1 is similar to that of other organisms. It is the only Tetrahymena histone soluble in 5% perchloric acid or 5% trichloroacetic acid, has a higher molecular weight than any other Tetrahymena histone, is the histone most easily dissociated from Tetrahymena chromatin, and is susceptible to specific proteolytic cleavage. However, unlike F1 in all other organisms, Tetrahymena F1 is not the slowest-migrating histone fraction when analyzed by polyacrylamide gel electrophoresis at low pH. Tetrahymena F1 also exhibits unusual behavior in sodium dodecyl sulfate-containing polyacrylamide gels, migrating faster than calf thymus F1 at pH 10, and slower than calf thymus F1 at pH 7.6. Tetrahymena F1 was found to be highly phosphorylated in rapidly growing cells, suggesting that the relationship between cell replication and F1 phosphorylation previously observed in mammalian cells may extend to all eukaryotes. The observation that extensive F1 phosphorylation occurs in macronuclei, which divide amitotically, argues against a unique role for F1 phosphorylation in the process of chromosome condensation at mitosis.     

Structural transformation of the nuclear matrix in situ.

A striking difference in structure can be revealed between the nuclear matrix isolated from the macronuclei of normal Tetrahymena cells and the nuclear matrix of cells pretreated with 50 μg/ml actinomycin D for 2 h. The latter matrix-type shows residual giant fusion-nucleoli, which are formed in the macronuclei of actinomycin-treated cells. Both nuclear matrix types, however, exhibit an identical qualitative protein pattern in 15% SDS-gel electrophoretograms, with only minor changes in the staining properties of a few peptides. These data support the view that the nuclear matrix represents the residual equivalent to an in situ existing nuclear protein framework, which must be regarded not as a rigid, but rather as a dynamic flexible framework.     

Traumatic Induction of Early Maturity in Tetrahymena

Exposure of conjugating pairs of Tetrahymena thermophila to high temperature (37°) during macronuclear development causes an abortion of many macronuclei, but it also often induces an early appearance of sexual maturity in clones completing macronuclear development. Lines become mature after about 15 cell divisions rather than after 50 or more cell divisions in untreated pairs. The phenotype resembles that associated with Em (early maturity) mutants but, because it is not transmitted to the progeny in the next generation, it must be considered a phenocopy. The hypothesis is developed that an early genotype-environment incompatibility, whether associated with an abnormal genotype or an unusual environment, activates a shunt mechanism permitting the organisms to undertake quickly an ordinarily forbidden sexual lottery.     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : II. Isolation of Macro- and Micronuclei

This report describes a rapid, efficient method for isolating macronuclei from Tetrahymena. The macronuclear fraction contains only small amounts of micronuclear material and little detectable whole cell or cytoplasmic contamination. A method is also described for preparing a "micronuclear fraction" which contains 20–40 micronuclei for every macronucleus present. Electron microscope observations indicate that the ultrastructure of the nuclei in the macronuclear fraction closely resembles that of nuclei in situ. The presence of ribosomes on the outer membrane of micronuclei and of pores in the micronuclear envelope is also described.     

DNA-Dependent RNA Synthesis in Isolated Macronuclei of Blepharisma intermedium (Ciliata)

SYNOPSIS. Autoradiographic studies on DNA-dependent RNA synthesis in isolated macronuclei of Blepharisma intermedium in vitro are described. Isolated macronuclei incubated in a medium containing RNA precursors incorporated cystidine and uridine into RNA. Uptake of cystidine was greater than that of uridine. Addition of ATP did not appreciably affect the uptake of precursors into RNA. Actinomycin D (5 μg/ml) blocked incorporation of cystidine and uridine. It is concluded that in isolated macronuclei of Blepharisma , RNA precursors are 1st converted into triphosphates and then incorporated into RNA thru DNA-dependent RNA polymerase.     

Influence of activator and inhibitors of Ca2+ channels on proliferative activity in Tetrahymena pyriformis infusoria

It was determined that change in DNA content in macronuclei occurs in the T. pyriformis infusoria under the influence of an activator (caffeine) and inhibitors of Ca²⁺ channels (verapamil), NiCl₂, and CdCl₂. Caffeine (10 mM) stimulates DNA synthesis. Verapamil (5 ??M), CdCl₂ (125 ??M), and NiCl₂ (100 ??M) decrease DNA content in macronuclei by 30 min after proliferative stimulation. By 4 h of incubation, there is, on average, 10% less DNA in macronuclei of Tetrahymena preprocessed with verapamil than in the control cells. The cells preprocessed with CdCl₂ and NiCl₂ differ from the control cells by lower DNA content almost at all studied periods, but they restore the level of nuclear DNA by 4 h. It is assumed that trans-mission of proliferative signals in the T. pyriformis has a Ca²⁺-dependent character.     

Isolation and characterization of chromatin replication bands and macronuclei from Euplotes eurystomus

A method is described for isolating replication bands (RBs) from Euplotes eurystomus in quantities sufficient for biochemical analysis. The method involves the disruption of whole cells in a low ionic strength buffer that maintains RB integrity while dispersing macronuclear chromatin. The RBs are then stabilized with MgCl2 and spermidine phosphate and isolated by gradient centrifugation. Staining with silver nitrate and thiol-specific coumarin maleimide has been demonstrated in the RBs of Euplotes and other hypotrichs; both of these properties were maintained in isolated RBs. A method is also described in this study for isolating highly purified macronuclei. Examination of isolated macronuclei and RBs with electron microscopy (EM) indicates that the morphology of both structures remain essentially intact during purification. We also observe with EM an increase in the number of replicating molecules in RBs compared to macronuclei. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates a consistent but minor enrichment of a 55 kilodalton protein in RBs when compared to macronuclear proteins.     

THE FINE STRUCTURE AND MODE OF ATTACHMENT OF THE SHEATHED FLAGELLUM OF VIBRIO METCHNIKOVII

The sheathed flagellum of Vibrio metchnikovii was chosen for a study of the attachment of the flagellum to the bacterial cell. Normal and autolysed organisms and isolated flagella were studied by electron microscopy using the techniques of thin sectioning and negative staining. The sheath of the flagellum has the same layered structure as the cell wall of the bacterium, and in favourable thin sections it appears that the sheath is a continuation of the cell wall. After autolysis the sheath is usually absent and the core of the flagellum has a diameter of 120 A. Electron micrographs of autolysed bacteria negatively stained with potassium phosphotungstate show that the core ends in a basal disc just inside the plasma membrane. The basal disc is about 350 A in diameter and is thus considerably smaller than the "basal granules" described previously by other workers.     

[6N]METHYL ADENINE IN THE NUCLEAR DNA OF A EUCARYOTE, TETRAHYMENA PYRIFORMIS

DNA isolated from macronuclei of the ciliate, Tetrahymena pyriformis, has been found to contain [⁶N]methyl adenine (MeAde); this represents the first clear demonstration of significant amounts of MeAde in the DNA of a eucaryote. The amounts of macronuclear MeAde differed slightly between different strains of Tetrahymena, with approximately 0.65–0.80% of the adenine bases being methylated. The MeAde content of macronuclear DNA did not seem to vary in different physiological states. The level of MeAde in DNA isolated from micronuclei, on the other hand, was quite low (at least tenfold lower than in macronuclear DNA).     

Ultrastructure of the tuboid graptolite tubotheca

The wall of the tubotheca of the tuboid graptoliteEpigraptus eisenacki Mierzejewski has been studied with TEM. It was found that the tubothecal wall is composed of the cortical tissue which differs only slightly from the same tissue described in Dendroidea andMastigograptus sp. In addition a giant tubotheca inDiscograptus schmidti Wiman has been examined with SEM. The tubothecae studied seem to confirm a non-graptolite nature of the organisms living in them, while providing evidence that the tubotheca wall was a genuine graptolite structure overgrowing these organisms.     

Comparison of the sequences of macro- and micronuclear DNA of Tetrahymena pyriformis

Macro- and micronuclei were isolated from Tetrahymena pyriformis (Syngen 1, strain WH-6) and their DNAs compared by isopycnic centrifugation in neutral and alkaline CsCl, by analysis of thermal denaturation properties and by molecular hybridization. Unlike the situation observed in Stylonychia the buoyant densities and thermal denaturation patterns of Tetrahymena macro- and micronuclear DNAs were virtually identical—the only observable differences bordering on the limits of resolution of these techniques. DNA was isolated from the two nuclei which had been labelled with different radioactive isotopes (i.e. ¹⁴C-thymidine and ³H-thymidine), and the renaturation kinetics of mixtures of macro- and micronuclear DNA were examined using a single-strand specific deoxyribonuclease (S₁). Renaturation kinetics obtained using varying ratios of macro- and micronuclear DNA suggested that 80–90% of the sequences present in micronuclei were present in similar amounts in macronuclei. However, careful analyses of the renaturation kinetics indicate that approximately 10–20% of the sequences found in micronuclei are probably absent in macronuclei, and that most of these sequences are probably moderately repetitive (100 copies per genome or less). These findings place severe constraint on possible models concerning the structure of the Tetrahymena macronucleus, and are very different from the situation observed in Stylonychia where it has been suggested that only a small percentage of the sequences in micronuclei are present in significant amounts in macronuclei. Nonetheless, these results along with those in Stylonychia can be taken as an indication that the loss or under-replication of some DNA sequences accompanies macronuclear differentiation in ciliates.     

MITOCHONDRIA OF PROTOZOA

A study of thin sections of Paramecium multimicronucleatum, Tetrahymena pyriformis, Tokophrya infusionum, and Amoeba proteus shows that the mitochondria in all these protozoa are similar in certain aspects of their fine structure to that described in metazoan cells. As in higher organisms the mitochondrion is surrounded by a double limiting membrane and contains protrusions directed inward from the innermost of the double membranes. There are, however, some differences. In a majority of higher organisms the internal structure of mitochondria consists of ridges or cristae mitochondriales and in a few instances only of finger-like projections, or microvilli. In all protozoa described here and elsewhere microvilli represent the dominant structure. They are characteristic therefore of protozoan mitochondria.     

Isolation and Properties of Macronuclei from Tetrahymena thermophila1

A simple and efficient method is described for the isolation of macronuclei from Tetrahymena thermophila (7B). The steps involved are deciliation and removal of the mucocysts’ contents by dibucaine treatment, digitonin mediated lysis, differential centrifugations, and finally isopyenic sucrose density gradient centrifugation. Judging from the distribution of marker enzymes and electron microscopy, the macronuclei obtained were free of cytoplasmic and paniculate contamination and were highly active in endogenous RNA-synthesis (1.5 pmol UTP incorporation/ng DNA min at 30°C). The ratio of protein: RNA: DNA was 2.0:0.33:1.0 (weight) and each macronucleus contained an average of 17 pg DNA. The average yield of isolation was 50%.     

Caspase-like activity is required for programmed nuclear elimination during conjugation in Tetrahymena

During conjugation in the binucleate ciliate, Tetrahymena thermophila, the old macronucleus is eliminated as new macronuclei and micronuclei are ontogenetically derived from the zygote nucleus. The mechanism of programmed nuclear elimination in ciliates may be related to the mechanism of apoptosis in higher organisms since its chromatin undergoes major condensation, its DNA is digested into nucleosome-sized fragments, and it stains positively for TUNEL. The present study explores whether caspases are involved in programmed macronuclear degradation in Tetrahymena. We show here that caspase-like activity is detectable using two specific colorimetric substrates, and that the activity is reduced with specific caspase inhibitors. In addition, using the fluorigenic substrate PhiPhiLux, active caspase-like activity is detected in living cells, localized to cytoplasmic vesicles; activity is not detected in pre- or post-condensed macronuclei. Finally, three different inhibitors of caspase activity cause a block to macronuclear chromatin condensation and elimination. Therefore, a caspase-like enzyme activity is necessary for regulating macronuclear elimination in Tetrahymena. These data support the possibility that macronuclear elimination is related, evolutionarily, to regulated cell death in multicellular organisms.     

Properties of microtubule proteins in different organelles in Tetrahymena pyriformis

Attempts were made to elucidate whether or not microtubules within cilia, oral apparatus and macronuclei in Tetrahymena pyriformis include common proteins, by making use of antiserum to microtubule proteins of cilia. The microtubule fraction containing two protein components was used as antigen and the antiserum to the microtubule proteins was proved to be specific by analysing electrophoretic patterns in the antigen absorption experiments. The antiserum reacted with the dissolved proteins of isolated oral apparatus or macronuclei, forming precipitin lines common to those of cilia. Furthermore, the two organelles were positively stained with the fluorescein-labelled antiserum. These results offered important clues to understand multifariousness in function and behavior of morphologically identical microtubules; that is, various microtubules in the cell appear to include a common protein(s) one another.     

伪尖毛虫细胞核质关系的实验研究

伪尖毛虫Oxytricha fallax是原生动物中高度进化的类型,核器有显著的分化。我们在不直接触及大核的情况下,通过特定的显微外科手术,得到了不同类型的、可遗传的畸型双体。探讨这一新的整体性状产生、维持和传代的机理是一个饶有兴味的课题,同时,既然双体系由二虫联合而成,其核质关系势必更加复杂,因此,它也是一个研究细胞核质关系的良好的新材料。本文主要从以下三个方面对伪尖毛虫进行了研究:1.人工双体伪尖毛虫的形成及其特征。2.人工背联体伪尖毛虫摘除大核的实验研究。3.人工背联体     

STUDIES ON NUCLEAR STRUCTURE AND FUNCTION IN TETRAHYMENA PYRIFORMIS : I. RNA Synthesis in Macro- and Micronuclei

Tetrahymena in the log phase of growth were pulse labeled with uridine-³H, fixed in acetic-alcohol, extracted with DNase, and embedded in Epon. 0.5-µ sections were cut, coated with Kodak NTB-2 emulsion, and developed after suitable exposures. Grains were counted above macronuclei, above 1000 micronuclei, and above 1000 micronucleus-sized "blanks" which were situated next to micronuclei in the visual field by means of a camera lucida. An analysis of grain counts showed that micronuclei were less than ½₀₀₀ as active as macronuclei on the basis of grains per nucleus. Since micronuclei contained, on the average, about ½₀ as much DNA as macronuclei, micronuclear DNA had less than 1% of the specific activity of macronuclear DNA in RNA synthesis. However, even this small amount of apparent incorporation was not significantly different from zero. Comparisons of the frequency distributions of labeled micronuclei with those of micronuclear "blanks" showed no evidence of a small population of labeled nuclei such as might be expected if micronuclei synthesized RNA for only a brief portion of the cell cycle. We conclude from these studies that there is no detectable RNA synthesis in Tetrahymena micronuclei during vegetative growth and reproduction.     

A Method for the Isolation of Macronuclei from Paramecium aurelia

SYNOPSIS. A method is described for the isolation of macronuclei from Paramecium aurelia. It has also been successfully employed with Didinium nasutum. The yield is up to 50% with P. aurelia. Some results on the DNA, RNA and protein contents are given. The isolated macronuclei of P. aurelia are able to incorporate ATP and UTP into acid-insoluble material, a process which is probably RNA synthesis mediated by RNA polymerase. The macronuclei were thus highly purified, and retained a measure of biosynthetic activity.