Is the nuclear matrix the site of DNA replication in eukaryotic cells?

Four types of experiment were carried out to test the recently proposed model of matrix-bound replication in eukaryotic cells. In experiments with pulse-labelling we found preferential association of newly replicated DNA with the matrix only when the procedure for isolation includes first high-salt treatment of isolated nuclei and then digestion with nucleases, or when prior to digestion the nuclei have been stored for a prolonged time. In both cases, however, evidence was found that this preferential association is due to a secondary, artifactual binding of the newly replicated chromatin region to the matrix elements. Pulse-chase experiments and experiments with continuous labelling were carried out to answer the question whether during replication the DNA is reeled through the replication complexes, i.e., whether newly replicated DNA is temporarily or permanently associated with the matrix. The results showed that at that time the matrix DNA does not move from its site of attachment. Since, according to the model of matrix-bound replication, the forks are assumed to be firmly anchored to high-salt resistant proteinaceous matrix structures, the chromatin fragments isolated with endonuclease not recognizing newly replicated DNA and purified by sucrose gradient centrifugation should be free of replication intermediates. The electronmicroscopic analysis of such fragments revealed the existence of intact replication micro-bubbles. Moreover, the fragments with replication configurations appeared as smooth chromatin fibres not attached to elements characteristic for the matrix. All these experiments suggest that the nuclear skeleton is not a native site of DNA replication in eukaryotic cells.     

What regulates secretion of non-stored proteins by eukaryotic cells?

Protein secretion is conventionally viewed as taking place by either of two cellular routes, a regulated pathway, involving external stimuli and secretory granules, and a presumptive ‘constitutive’ pathway, which does not involve hormonal or neuronal stimuli or the production of secretory granules. The evidence reviewed here strongly suggests that there are post-synthesis rate-limiting steps for many proteins released by the ‘constitutive’ pathway and, hence, that regulation in some sense is involved here too. The nature of these rate-limiting determinants and events is discussed.     

The regulation of autophagy in eukaryotic cells: do all roads pass through Atg1?

The induction of autophagy appears to be tightly controlled in all eukaryotic cells. This highly conserved, degradative process is induced by a variety of signals, including nutrient deprivation, and is generally thought to be incompatible with rapid cell growth. Recent work in the budding yeast, Saccharomyces cerevisiae, has suggested that the Atg1 protein kinase is at the center of this control. Atg1, and its associated proteins, appear to be directly targeted by multiple signaling pathways important for the control of both autophagy and cell growth. These pathways involve the small GTP-binding Ras proteins, the Tor protein kinases and the AMP-activated protein kinase, Snf1, respectively. A key question that remains is whether this regulatory paradigm has been evolutionarily conserved. In other words, is Atg1 the primary target of those signaling pathways responsible for coordinating growth with environmental influences in other eukaryotes? Here, we suggest that Atg1 is very likely to fulfill this role but that a truly definitive answer will require that we develop a better understanding of this protein kinase and its targets in all eukaryotes.     

Holliday junctions in the eukaryotic nucleus: resolution in sight?

The Holliday junction is a key recombination intermediate whose resolution generates crossovers. Interplay between recombination, repair and replication has moved the Holliday junction to the center stage of nuclear DNA metabolism. Holliday junction resolvases in the eukaryotic nucleus have long eluded identification. The endonucleases Mus81/Mms4-Eme1 and XPF-MEI-9/MUS312 are structurally related to the archaeal resolvase Hjc and were found to be involved in crossover formation in budding yeast and flies, respectively. Although these endonucleases might represent one class of eukaryotic resolvases, their substrate preference opens up the possibility that junctions other than classical Holliday junctions might contribute to crossovers. Holliday junction resolution to non-crossover products can also be achieved topologically, for example, by the action of RecQ-like DNA helicases combined with topoisomerase III.     

The first eukaryotic cells — Acid hot-spring algae

The Cyanidiophyceae members (PreRhodophyta) may serve as a transitional algal group bridging the cyanobacteria and the unicellular Rhodophyta. This thermoacidic algal group is composed of three genera containing several species. We suggested placing these algae in progressively evolutionary steps: (Cyanidioschyzon Cyanidium Galdieria). This evolutional ladder is based upon various areas of research like biochemistry, amount of nuclear genome and shape of chloroplast nucleoid, ultrastructure and ecological aspects. The first alga —Cyanidioschyzon — is the cornerstone of this succession; it shows mixed features between cyanobacterium and archaebacteria(Thermoplasma-like cell). It demonstrates simple eukaryotic cellular features and has the smallest amount of nuclear and chloroplast DNA. The intermediate alga in this line,Cyanidium, is also a simple cell, but shows more progressive characterizations than theCyanidioschyzon. The third taxon,Galdieria, is already very close to the unicellular rhodophytes (red algae) and indicates typical advanced eukaryotic characterization. We propose thatCyanidioschyzon (considered to be the simplest eukaryote) may have evolved from an association betweenThermoplasma-like archaebacterium and a thermophilic cyanobacterium. Autogenous (non-symbiotic) compartmental steps may have taken place fromCyanidioschyzon toCyanidium and then toGaldieria, and from this alga (group) towards the other unicellular red algae.Dedicated toDr. Jerome F. Fredrick, an enthusiast of our favorite algaCyanidium, on his retirement from directorship of Dodge Chemical Laboratories in Bronx, NYC.     

Origin of eukaryotic cells: 40 years on

The year 1970 saw the publication of Origin of Eukaryotic Cells by Lynn Margulis. This influential book brought the exciting and weighty problems of cellular evolution to the scientific mainstream, simultaneously breaking new ground and ‘re-discovering’ the decades-old ideas of German and Russian biologists. In this commemorative review, I discuss the 40 years that have elapsed since this landmark publication, with a focus on the ‘molecular era’: how DNA sequencing and comparative genomics have proven beyond all doubt the central tenets of the endosymbiont hypothesis for the origin of mitochondria and plastids, and, at the same time, revealed a genetic and genomic complexity in modern-day eukaryotes that could not have been imagined in decades past.     

Cell cycle controls in higher eukaryotic cells: Resting state or a prolonged G1 period?

We express the viewpoint that control over cell growth in higher eukaryotes is achieved predominantly by regular transition of cells from proliferation to rest and vice versa as a result of coordinated interrelationship between intracellular growth inhibitors and extracellular growth factors. The resting state is considered as a special physiological state of a cell where the prereplicative reactions necessary for the onset of DNA synthesis are inhibited. Cells pass into a resting state at each successive cell cycle, with regard to the next cycle, once the threshold level of growth inhibitors has been attained. Cellular rest may thus initiate and proceed in parallel with conventional periods of the cell cycle but in a hidden way. Its termination strictly depends on the appropriate concentration of extracellular growth factors. In the absence of growth factors cells, after completing mitosis, pass into an overt state of rest metabolically different from any period of the cell cycle including G1.     

The Schaechter-Bentzon-Maaløe experiment and the analysis of cell cycle events in eukaryotic cells

The Schaechter–Bentzon–Maaløe (SBM) experiment, performed more than 40 years ago, provides an important lesson for the analysis of the eukaryotic cell cycle. Before this experiment, temperature shifts had been used to synchronize bacteria and determine the pattern of DNA synthesis during the bacterial division cycle. These experiments indicated that DNA replication occurred during a fraction of the division cycle with gaps before and after DNA synthesis, a pattern similar to the eukaryotic division cycle. The SBM experiment studied DNA replication during the division cycle by labeling an unperturbed culture with a short pulse of tritiated thymidine. All cells were found to be labeled, indicating that unperturbed cells synthesize DNA throughout the division cycle. Thus, the SBM experiment was a control experiment demonstrating that artifacts can be introduced by synchronization methods. The idea of an control experiment under unperturbed conditions is proposed for the analysis of data on cell-cycle-specific gene expression in yeast and mammalian cells.     

Why is CpG suppressed in the genomes of virtually all small eukaryotic viruses but not in those of large eukaryotic viruses?

Dinucleotide over- and underrepresentation is evaluated in all available completely sequenced DNA or RNA viral genomes, ranging in size from 3 to 250 kb (available RNA viruses fall into the small-virus category). The dinucleotide CpG is statistically underrepresented (suppressed) in all but four of the small viruses (more than 75 with lengths of < 30 kb) but has normal relative abundances in most large viruses (> or = 30 kb). Most retrotransposons in eukaryotic species also show low CpG relative abundances. Interpretations, especially in some cases of DNA viruses or viruses with a DNA intermediate, might relate to methylation effects and modes of viral integration and excision. Other possible contributing factors relate to dinucleotide stacking energies, special mutation mechanisms, and evolutionary events.     

Epithelial cells in culture: injured or differentiated cells?

Isolation of epithelial cells for cell culture is based on destruction of epithelial integrity. The consequences are manifold: cell polarity and specific cell functions are lost; cells acquire non‐epithelial characteristics and start to proliferate. This situation may also occur in situ when parts of the epithelium are lost, either by apoptosis or necrosis by organ or tissue injury. During recovery from this injury, surviving epithelial cells proliferate and may restore epithelial integrity and finally re‐differentiate into functional epithelial cells. In vitro, this re‐differentiation is mostly not complete due to sub‐optimal culture conditions. Therefore cultured epithelial cells resemble wounded or injured epithelia rather than healthy and well differentiated epithelia. The value of an in vitro cell model is the extent to which it helps to understand the function of the cells in situ. A variety of parameters influence the state of differentiation of cultured cells in vitro. Although each of these parameters had been studied, the picture how they co‐ordinately influence the state of differentiation of epithelial cells in vitro is incomplete. Therefore we discuss the influence of the isolation method and cell culture on epithelial cells, and outline strategies to achieve highly differentiated epithelial cells for the use as an in vitro model.