Our potential for dissecting the complex processes involved in eukaryotic DNA replication has been dramatically increased with the recent development of cell‐free systems that recreate many of these processes in vitro. Initial results from these systems have drawn together work on the cell cycle, the enzymology of replication, and the structure of the nucleus.

DNA extraction, playing an irreplaceable role in molecular biology as it is an essential step prior to various downstream biological analyses. Thus, the accuracy and reliability of downstream research outcomes depend largely on upstream DNA extraction methodology. However, with the advancement of downstream DNA detection techniques, the development of corresponding DNA extraction methods is lagging behind. The most innovative DNA extraction techniques are silica‐ or magnetic‐based. Recent studies have demonstrated that plant fiber‐based adsorbents (PF‐BAs) have stronger DNA capturing ability than (...) classic materials. Moreover, magnetic ionic liquid (MIL)‐based DNA extraction has gathered attention lately, and extrachromosomal circular DNA (eccDNA), cell‐free DNA (cfDNA), and microbial community DNA are current research hotspots. These require specific extraction methods, along with constant improvements in the way they are used. This review discusses the significance as well as the direction of innovation of DNA extraction methods to try to provide valuable references including current status and trends for DNA extraction. (shrink)

Mutations in specific genes result in birth defects, cancer, inherited diseases or lethality. The frequency with which DNA damage is converted to mutations increases dramatically when the cellular genome is replicated. Although DNA damage poses special problems to the fidelity of DNA replication, efficient mechanisms exist in mammalian cells which function to replicate their genome despite the presence of many damaged sites. These mechanisms operate in either error‐prone or error‐free modes of DNA synthesis, and frequently involve DNA strand‐pairing reactions. (...) Genetic studies in yeast and other eukaryotes suggest that replication through DNA damage is highly regulated and catalysed by complex biochemical machineries composed of many specialised gene products. Knowledge of the molecular details by which such factors facilitate the replication of damaged DNA in mammalian cells should reveal basic rules about how DNA damage induces mutagenesis and carcinogenesis. (shrink)

Nous étudions dans cet article les différentes formes de prédication seconde détachée en position initiale dans un corpus comparable composé de textes d’économie en anglais et en français. Ce corpus a été annoté sous le logiciel Analec. L’enjeu est de montrer en quoi un même phénomène syntaxique est exploité, sur le plan discursif, de façon divergente dans chacune des deux langues. Une étude qualitative et quantitative des prédications secondes du corpus montre que la prédication seconde prend le plus souvent la (...) forme d’une participiale en –ing en anglais, où elle se spécialise dans un rôle méta-énonciatif de balise textuelle pour le lecteur, ce qui n’est pas le cas en français. (shrink)

The fungal cell wall is a dynamic structure that protects the cell from changes in osmotic pressure and other environmental stresses, while allowing the fungal cell to interact with its environment. The structure and biosynthesis of a fungal cell wall is unique to the fungi, and is therefore an excellent target for the development of anti‐fungal drugs. The structure of the fungal cell wall and the drugs that target its biosynthesis are reviewed. Based on studies (...) in a number of fungi, the cell wall has been shown to be primarily composed of chitin, glucans, mannans and glycoproteins. The biosynthesis of the various components of the fungal cell wall and the importance of the components in the formation of a functional cell wall, as revealed through mutational analyses, are discussed. There is strong evidence that the chitin, glucans and glycoproteins are covalently cross‐linked together and that the cross‐linking is a dynamic process that occurs extracellularly. BioEssays 28: 799–808, 2006. © 2006 Wiley Periodicals, Inc. (shrink)

As the new Cell-free DNA (Cf-DNA) prenatal screening test for Down syndrome was being introduced into the UK’s fetal anomaly screening program, Down syndrome charities had an opportunity to participate. An experience of co-production where we were the minority voice then followed. This paper explores that process and our experience as a charity. Institutional and societal structures meant that it was difficult to be heard and a significant amount of bias was noted within the program. Consequently, our viewpoints (...) were often considered and then dismissed. However, at times we were listened to, and feel that there were some valuable changes made resulting from our involvement. The end product was far from reflective of all that we stand for, and there are still lessons to be learned in England about the need to place a higher value on minority voices of lived experience in a co-production exercise. (shrink)

Oxidised bases, such as 8-oxo-guanine, occur in cellular DNA as a result of attack by oxygen free radicals. The cancer-protective effect of vegetables and fruit is attributed to the ability of antioxidants in them to scavenge free radicals, preventing DNA damage and subsequent mutation. Antioxidant supplements (e.g., β-carotene, vitamin C) increase the resistance of lymphocytes to oxidative damage, and a negative correlation is seen between antioxidant concentrations in tissues and oxidised bases in DNA. Large-scale intervention trials with β-carotene (...) have, however, led to increases in cancer. Recent measurements of the frequency of oxidised DNA bases indicate that earlier estimates were greatly exaggerated; there may be only a few thousand 8-oxo-guanines per cell. Convincing evidence for mutations resulting from oxidative damage, in tumours or cultured cells, is lacking. It seems that efficient antioxidant defences together with DNA repair maintain a steady-state level of damage representing minimal risk to cell or organism. BioEssays 21:238–246, 1999. © 1999 John Wiley & Sons, Inc. (shrink)

Oxidised bases, such as 8-oxo-guanine, occur in cellular DNA as a result of attack by oxygen free radicals. The cancer-protective effect of vegetables and fruit is attributed to the ability of antioxidants in them to scavenge free radicals, preventing DNA damage and subsequent mutation. Antioxidant supplements (e.g., β-carotene, vitamin C) increase the resistance of lymphocytes to oxidative damage, and a negative correlation is seen between antioxidant concentrations in tissues and oxidised bases in DNA. Large-scale intervention trials with β-carotene (...) have, however, led to increases in cancer. Recent measurements of the frequency of oxidised DNA bases indicate that earlier estimates were greatly exaggerated; there may be only a few thousand 8-oxo-guanines per cell. Convincing evidence for mutations resulting from oxidative damage, in tumours or cultured cells, is lacking. It seems that efficient antioxidant defences together with DNA repair maintain a steady-state level of damage representing minimal risk to cell or organism. BioEssays 21:238–246, 1999. © 1999 John Wiley & Sons, Inc. (shrink)

Recent advances in our understanding of the mechanism and regulation of eukaryotic DNA replication have been expedited by the use of cell‐free systems capable of initiation of DNA replication. The system capable of replicating plasmid DNAs containing the SV40 origin of DNA replication in vitro is a paradigm for studies on the replication of other virus DNAs and the replication of cellular chromosomes. This review outlines some of the contemporary issues and developments related to this complex problem.

Despite advances in treatments over the last decades, a uniformly reliable and free of side effects therapy of human cancers remains to be achieved. During chromosome replication, a premature halt of two converging DNA replication forks would cause incomplete replication and a cytotoxic chromosome nondisjunction during mitosis. In contrast to normal cells, most cancer cells bear numerous DNA deletions. A homozygous deletion permanently marks a cell and its descendants. Here, we propose an approach to cancer therapy in which (...) a pair of sequence‐specific roadblocks is placed solely at two cancer‐confined deletion sites that are located ahead of two converging replication forks. We describe this method, termed “replication blocks specific for deletions” (RBSD), and another deletions‐based approach as well. RBSD can be expanded by placing pairs of replication roadblocks on several different chromosomes. The resulting simultaneous nondisjunctions of these chromosomes in cancer cells would further increase the cancer‐specific toxicity of RBSD. (shrink)

New developments in molecular and cell biology during the past three decades have demonstrated that cells use a language of their own, namely"cell language". The essential features of the so-called cell language theory formulated in 1997 are reviewed as a prelude to establishing the concept ofmicrosemiotics'' and "microsemiosis''' the semiotics and sign processes, respectively, on the molecular level. Peircean semiotics is largely concemedwith macroscopic signs and hence is here referred to as macrosemiotics. The possibihty of extending Peircean (...) semiotics from macroscale to microscale was clearly foreseen by Sebeok in 1968 who also intuited the evolutionary, link between macro- and microsemiosis, leading to the formulation of what is here called the "Sebeok doctrine of signs" that contains 5 major testable propositions. The concept of "cosmosemiosis" as the sum of three terms, physiosemiosis, macrosemiosis, and microsemiosis, is defined and related to a theoretical model of the Universe known as the Shillongator, published in 1991, according to which superstrings serve as the primal source of free energy and information and hence as the Universal Sign Processor, similar to DNA acting as the primary biological sign processor in the living cell. Thus, the Shillongator as the physical effector or effectuator of cosmosemiosis appears to provide a coherent theoretical framework to integrate i) the Sebeok doctrine of signs, ii) Deely's theory of physiosemiotics, iii) macro- or Pericean semiotics, iv) the present author's microsemiotics, and v) Merleau-Ponty's ontology ofthe Flesh. (shrink)

Replication protein A (RPA), the major single‐stranded DNA‐binding protein in eukaryotic cells, is required for processing of single‐stranded DNA (ssDNA) intermediates found in replication, repair, and recombination. Recent studies have shown that RPA binding to ssDNA is highly dynamic and that more than high‐affinity binding is needed for function. Analysis of DNA binding mutants identified forms of RPA with reduced affinity for ssDNA that are fully active, and other mutants with higher affinity that are inactive. Single molecule studies showed that (...) while RPA binds ssDNA with high affinity, the RPA complex can rapidly diffuse along ssDNA and be displaced by other proteins that act on ssDNA. Finally, dynamic DNA binding allows RPA to prevent error‐prone repair of double‐stranded breaks and promote error‐free repair. Together, these findings suggest a new paradigm where RPA acts as a first responder at sites with ssDNA, thereby actively coordinating DNA repair and DNA synthesis. (shrink)

Constructivist biosemiotics foundations imply the first-person basis of cognition. CBF are developed by the biology of cognition, relational biology, enactive approach, ecology of mind, second order cybernetics, genetic epistemology, gestalt, ecological perception and affordances, and active inference by minimization of free energy. CBF reject the idea of an objective independent reality to be represented by information processing in order to be the fittest. CBF assumes that perception is the behavioral configuration of an object and objects are tokens for eigen-behaviors. (...) Cognition takes place in the organism-environment structural coupling during the ontogeny and phylogeny of all biological unities including unicellular organisms. Therefore, if exogenous DNA particles are just tokens for the cell signalling eigen-behaviors, if there is no ‘information’ in the DNA sequence, how can we explain that the same virus or trans- sequence is associated with a similar phenotype? We call this ‘exogenomic problem’. With this basic example, but sufficiently generic to the whole biological world, we agree respectively with Autopoiesis, -system, and Gaia theory: i) ‘Information, code and meaning’ in the DNA sequence belong to the domain of the observer’s description. ii) Genetic ‘information’ is not a program or algorithmic software in DNA sequence. Rather it is a microphysical observable mode of eigen-behaviors in biological unities. iii) The transfer and acquisition of DNA particles is a biospheric phenomenon that maintains its homeorhesis, symbiotic and biosemiotic entailment. Based on the theoretical and experimental results of these theories, it is concluded that genetic ‘information’ is not a genomic sequence, nor any kind of information, but for the cell DNA must embody physical forcing. Genetic characters are the effects and not the cause of phenotype and DNA particles do not ‘use or manipulate’ cellular metabolism. Rather, any cellular configuration change that occurred before or during DNA perturbation is determined on the basis of the cellular standpoint. (shrink)

A large number of cellular proteins bind ATP, frequently utilizing the free energy of ATP hydrolysis to drive specific biological reactions. Recently, a family of closely related ATP‐binding proteins has been identified, the members of which share considerable sequence identity. These proteins, from both prokaryotic and eukaryotic sources, presumably had a common evolutionary origin and include the product of the white locus of Drosophila, the P‐glycoprotein which confers multidrug resistance on mammalian tumours, and prokaryotic proteins associated with such diverse (...) processes as membrane transport, cell division, nodulation and DNA repair. A comparison of these various proteins provides valuable insights into the function and evolution of the multicomponent systems with which they are associated. (shrink)

Noninvasive prenatal screening using cell-free DNA, which analyzes placental DNA circulating in maternal blood to provide information about fetal chromosomal disorders early in pregnancy and without risk to the fetus, has been hailed as a potential “paradigm shift” in prenatal genetic screening. Commercial provision of cell-free DNA screening has contributed to a rapid expansion of the tests included in the screening panels. The tests can include screening for sex chromosome anomalies, rare subchromosomal microdeletions and aneuploidies, and (...) most recently, the entire fetal genome. The benefits of this screening tool are generally framed, by both providers and commercial laboratories, as enhancing reproductive autonomy and choice by providing an earlier, simpler, and more accurate screening while potentially reducing the need for invasive follow-up testing. The majority of the literature has explored these issues empirically or conceptually from a European or North American vantage point, one that assumes normative priorities such as individual reproductive autonomy and the clinical availability of maternal health care or prenatal screening programs within which cell-free DNA screening is offered. While its implementation has raised both challenges and opportunities, very little is known about real-world experiences and the implications of the rapid introduction of cell-free DNA screening outside of North America and Europe, especially in low- and middle-income countries. To begin addressing this gap in knowledge, we organized a four-day international workshop to explore the ethical, legal, social, economic, clinical, and practical implications of the global expansion of cell-free DNA screening. We describe eight key insights that arose from the workshop. (shrink)

The quantitative study of regulation of cell growth and proliferation began with the development of the technique for monolayer culture of vertebrate cells in the late 1960s. The basic parameters were defined in the early physiological studies, which continued through the next decade. These included specific and non-specific growth factors and the requirement for continuous exposure to such factors through most of the G1 period for progression to S. In the course of this work, the diversity of biochemical responses (...) and the critical role of increased protein synthesis and accumulation for the onset of DNA synthesis were elucidated. In particular, a central role of free cytosolic Mg2+ in direct regulation of protein synthesis and in ancillary processes as a response to membrane perturbation was established. Eventually, the physiological era was superseded by the molecular era beginning in the 1980s. This work focussed on specific receptors for growth factors that entrained a protein kinase cascade, which terminated in a higher frequency of initiation of protein synthesis. However, the molecular studies virtually ignored the key results of the physiological era. Recent studies of the penultimate molecular steps in the regulatory pathway of protein synthesis, however, have supported a model of growth regulation involving membrane perturbation and MgATP2− concentration, results that integrate the findings of the physiological and molecular eras. The resulting relatively simple “membrane, magnesium mitosis” (MMM) model of proliferation control can explain the seeming paradox of the variety of specific and non-specific growth-enhancing treatments that are mediated by the plasma membrane and which bring about a shared, complex but coordinated growth response that drives cell proliferation. BioEssays 27:311–320, 2005. © 2005 Wiley Periodicals, Inc. (shrink)

Over recent years, evidence has been accumulating in favour of the free radical theory of aging, first proposed by Harman. Despite this, an understanding of the mechanism by which cells might succumb to the effects of free radicals has proved elusive. This paper proposes such a mechanism, based on a previously unexplored hypothesis for the proliferation of mutant mitochondrial DNA: that mitochondria with reduced respiratory function, due to a mutation or deletion affecting the respiratory chain, suffer less frequent (...) lysosomal degradation, because they inflict free radical damage more slowly on their own membranes. Once such a mutation occurs in a mitochondrion of a non‐dividing cell, therefore, mitochondria carrying it will rapidly populate that cell, thereby destroying the cell's respiratory capability. The accumulation of cells that have undergone this transition results in aging at the organismal level. The consistency of the hypothesis with known facts is discussed, and technically feasible tests are suggested, of both the proposed mechanism and its overall contribution to mammalian aging. (shrink)

Apoptosis (programmed cell death) is an evolutionarily conserved cellular process, important for development and homeostasis(1). Most apoptotic cells share a common set of morphological and physiological characteristics that distinguish them from necrotic deaths(2). While genetic studies have indicated that these characteristic changes result from the activation of an endogenous ‘suicide program’(3), little is known about the nature of this program and the molecular events underlying these changes. Two recent papers(4,5) describing cell‐free extracts that reproduce several of the (...) characteristic changes observed in apoptotic cells promise to make these phenomena accessible to biochemical analysis. (shrink)

Analyser av cellefritt DNA fra foster i gravide kvinners blod gir nye muligheter innen fosterdiagnostikk: Testene er bedre enn eksisterende tester, de reduserer risikoen og er billigere. Flere land har tatt i bruk disse testene, og Helsedirektoratet i Norge har mottatt søknad om å ta i bruk en test som erstatter tidlig ultralyd og blodprøver. Likevel nøler norske myndigheter. Hvorfor gjør de det? Ett av svarene er at non-invasive prenatale tester fører med seg en rekke faglige og moralske spørsmål og (...) gir flere grunnleggende etiske utfordringer. Denne artikkelen gjennomgår et bredt knippe av de utfordringene som NIPT reiser. Hensikten er å synliggjøre hvorfor NIPT påkaller etisk refleksjon og å bidra til en åpen debatt og en transparent beslutningsprosess. Artikkelen identifiserer fem sentrale og konkrete spørsmål for vurderingen av NIPT.Nøkkelord: non-invasiv prenatal diagnostikk, testing, fravalg, foster, blodprøve, ekspressivisme, statsliberalt dilemma, dilemma, abort, retten til ikke å viteEnglish summary: Ethical challenges with non-invasive prenatal tests Non-invasive prenatal testing performed with the use of massively parallel sequencing of cell-free DNA in maternal plasma gives extended possibilities in prenatal screening. The tests are claimed to be better than existing alternative tests, they reduce the risk, and it is claimed they are cheaper. They have been used in several countries since 2012, and the University Hospital of North Norway has applied to the Directorate of Health to replace first trimester ultrasound and plasma screening with NIPT. The Directorate of Health is reluctant to reply. Why is this? One of the answers may be that NIPT raises a series of professional and moral questions, and poses profound ethical challenges. This article reviews a series of the challenges with NIPT. The aim is to highlight why NIPT calls for ethical reflection and to contribute to an open debate and a transparent decision-making process. The article identifies five major questions to address in the assessment of NIPT: What are the criteria for screening and removing certain conditions? How certain do we have to be that these conditions do appear in living children? How should we handle information from NIPT for living persons? How can we avoid that persons with the conditions that are selected for screening and removal are insulted, stigmatized, or discriminated? How can we avoid that systematic screening and removal of fetuses with certain conditions develop norms and values that breach with basic social or ethical principles? (shrink)

What general conclusions can be drawn about the reception of zymase, its relation to the larger shift from a protoplasm to an enzyme theory of life, and its status as a social phenomenon?The most striking and to me unexpected pattern is the close correlation between attitude toward zymase and professional background. The disbelief of the fermentation technologists, Will, Delbrück, Wehmer, and even Stavenhagen, was as sharp and unanimous as the enthusiasm of the immunologists and enzymologists, Duclaux, Roux, Fernback, and Bertrand, (...) and of Pfeffer, the experimental plant physiologist. Other skeptics—Voit, Kupffer, and Fischer—were conservatives in traditional fields. In all these cases it seems clear that professional commitments and outlook profoundly influenced the reception of zymase.In some cases there is a correlation with a specific earlier statement favoring the protoplasm theory or predicting a fermentation enzyme. Reynolds Green is a striking case of both. In 1893 he stated his belief that life processes in higher plants and fungi were identical, both being mediated by protoplasm.109 Thus he claimed that digestion was not carried out by enzymes, as in higher animals, but by the whole protoplasm, and so too fermentation in yeast: “All the metabolic processes must be carried out in the unicellular organism in the same mass of protoplasm.”110 But Green also insisted that there was no essential difference between “organized” and “unorganized ferments”; enzymes were simply more highly differentiated and specialized forms of primitive protoplasm. He spoke of “the alcoholic ferment” (that is, enzyme), and of intracellular enzymes—very advanced beliefs for 1893. Thus it is understandable why Green saw no reason to doubt his first negative results with yeast juice, and rushed into print. But it is also understandable that he persisted and soon became an outspoken advocate of Buchner's view; like Wroblewski and Pfeffer, he had already accepted the idea of an intracellular fermentation enzyme.The physiological chemists also fit the pattern in a special way. Physiological chemistry is generally regarded as the source of biochemistry; indeed, many early biochemists were trained in physiological chemistry, and imbibed there a progressive interest in enzymes and metabolism. It is not surprising, therefore, that on the whole Neumeister, Madfadyen, Wroblewski, and Loew accepted cell-free fermentation. But what is surprising, and significant, is the diversity of opinion in this group, from Abeles' living protoplasm to Wroblewski's organized array of active proteins. The feelings of the physiological chemists were mixed, and there was enough of the old protoplasm view in Macfadyen's and even Wroblewski's views to allow Buchner to see them as attacks on zymase. The ideas of physiological chemistry were also changing; the idea of protoplasm was already adjusting to the new interest in enzymes in the early 1890's. But in contrast to immunochemistry, the change in physiological chemistry was gradual, uneven, undramatic, and relatively invisible. For the new biochemistry, the tradition of physiological chemistry was the source of traditional conservatism as well as of new ideas of enzymes.In general, then, I claim that it was not experimental facts that determined attitudes toward zymase so much as previous commitments, experience, and expectations. Initially, at least, zymase was less a determinant of opinion than a touchstone of pre-existing opinion. Like a prism, it revealed the spectrum of existing attitudes toward vital phenomena.Also relevant here is the fact that the experimental evidence was entirely ambiguous; the interpretation of Buchner's experiments with antiseptics or dried yeast depended entirely on how far one was willing to stretch the scope of the terms “protoplasm” and “enzyme”. Likewise, how important one judged the instability of zymase to be, or the low level of in vitro activity, depended largely on one's point of view. There was no crucial experiment, no certain proof. Logically zymase could be regarded as protoplasm. A. Fischer did so in 1900;111 Macfadyen was still wavering in 1907;112 and Beijerinck was holding out as late as 1916.113 There were undoubtedly others. Besides the special case of Reynolds Green and the technologists, there are no recorded cases of conversion.But if there was no evidence to convert a dedicated protoplasmist, there was plenty to encourage a dedicated belief in the enzyme theory, and that, I believe, is precisely what happened. The arguments of Buchner et al in support of zymase had the greatest effect not on the protoplasmists but on those who were already predisposed toward the biochemical view. The zymase debate enabled the emerging group of biochemists to recognizethemselves as a group with a community of out-look and objectives; it brought the issue of protoplasm vs. enzyme into the open and gave it a specific and concrete issue on which to turn. The debate sharpened the biochemists' awareness that their point of view was becoming the new mainstream of biochemical thought.All this of course had an important secondary effect on those who opposed the new view, or who were not immediately concerned with it. After the zymase debate, it would have been almost impossible to ignore the new ideas; the dramatic and wide publicity the debate enjoyed would have made it difficult not to take sides. Buchner's successful defense of zymase itself became an important influence on opinion; the new movement had to be looked at with respect. But the primary effect of the debate, I assert, was on those who were already inclined to the new view. The outcome was not a mass conversion of individuals, nor the ejection of one idea by another. The sides probably did not change much; the real change was in the way each side regarded itself and its place in history. The zymase debate may perhaps best be regarded as a process of selection. Buchner's party became aware of itself as the new progressive point of view, while those holding the traditional view could no longer expound it publicly without appearing obstinate or old-fashioned. Thus the way was clear for the gradually wider establishment of the new ideas, especially among the new generation of biochemists. In this specific sense, the acceptance of zymase was a social phenomenon.This way of looking at the zymase debate also illuminates its coincidence with the emergence of biochemistry as a profession. The new professional trappings and organizations were the social manifestation of the new self-awareness gained from the zymase controversy. One of the most important effects on those disposed toward the new view was the awareness of an intellectual community and of the need to give that community social forms. The almost mythical importance of Buchner's “victory” over the old protoplasm theory that later became current is readily understandable. It was a useful ideology for a group anxious to assert the novelty of their approach, a novelty which they in fact felt quite keenly.But if the primary effect of the zymase controversy was on those predisposed to accept zymase, then obviously the success of zymase depended on the fact that change had already begun to occur. Indeed, one of the most striking, and again unexpected, results of this study is the extent to which the importance of enzymes was already recognized in the early 1890's. The new immunology clearly pointed the way. Macfadyen and Hans Buchner were looking for active proteins inside the cell in 1893. Reynolds Green, Wroblewski, and Pfeffer had all anticipated the discovery of a fermentation enzyme. Other discoveries in enzymology in the 1890's pointed the same way.114 Moreover, it is clear from this study that the old protoplams theory was adapting to the new discoveries concerning the importance of enzymes. The sidechains of the old protoplasm molecule were simply reinterpreted as enzymes, as in Wroblewski's theory, for example, or in Paul Ehrlich's extremely influential “sidechain theory” of antibody formation (1897). Despite the later views of Hopkins et al., the old protoplasm and the new enzyme theories were not irreconcilable; a continuity of development had already begun when zymase came so dramatically on the scene. Buchner himself observed that the difference between the two views had become largely verbal; in retrospect we might say, not verbal, but social and historical. The gap between the continuous change in ideas and the contemporary feeling of sharply discontnuous change is a measure of the elusive but real social and historical implications of the zymase debate, of the demise of an old establishment, and the emergence of a new.In retrospect, then, it is clear that a trend was already under way in various quarters when zymase appeared. But it was still a collection of isolated and dispersed events. The zymase controversy made the trend visible and gave it a dramatic unity. The debate influenced those who had already begun to change. It was so effective because things had begun to change and because it coincided with the direction of that trend. (shrink)

Ribonucleotide reductase (RNR) catalyzes the rate limiting step in the production of deoxyribonucleotides needed for DNA synthesis. In addition to the well documented allosteric regulation, the synthesis of the enzyme is also tightly regulated at the level of transcription. mRNAs for both subunits are cell cycle regulated and inducible by DNA damage in all organisms examined, including E. coli, S. cerevisiae and H. sapiens. This DNA damage regulation is thought to provide a metabolic state that facilitates DNA replicational repair (...) processes. S. cerevisiae also encodes a second large subunit gene, RNR3, that is expressed only in the presence of DNA damage. Genetic analysis of the DNA damage response in S. cerevisiae has shown that RNR expression is under both positive and negative control. Among mutants constitutive for RNR expression are the general transcriptional repression genes, SSN6 and TUP1. Mutations in POL1 and POL3 also activate RNR expression, indicating that the DNA damage sensory network may respond directly to blocks in DNA synthesis. A protein kinase, Dun1, has been identified that controls inducibility of RNR1, RNR2 and RNR3 in response to DNA damage and replication blocks. This result suggests that the RNR genes in S. cerevisiae form a regulon that is coordinately regulated by protein phosphorylation in response to DNA damage. (shrink)

Mild and massive DNA damage are differentially integrated into the cellular signaling networks and, in consequence, provoke different cell fate decisions. After mild damage, the tumor suppressor p53 directs the cellular response to cell cycle arrest, DNA repair, and cell survival, whereas upon severe damage, p53 drives the cell death response. One posttranslational modification of p53, phosphorylation at Serine 46, selectively occurs after severe DNA damage and is envisioned as a marker of the cell death (...) response. However, the molecular mechanism of action of the p53 Ser46 phospho‐isomer, the molecular timing of this phosphorylation event, and its activating effects on apoptosis and ferroptosis still await exploration. In this essay, the current body of evidence on the molecular function of this deadly p53 mark, its evolutionary conservation, and the regulation of the key players of this response, the p53 Serine 46 kinases, are reviewed and dissected. (shrink)

DNA transfer directly from cell to cell (conjugation) is common among prokaryotes, particularly Gram‐negative bacteria like Escherichia coli. The phenomenon invariably requires a set of plasmid genes in the DNA donor cell. In addition, E. coli itself makes limited and specific contributions to the donor activity of strains carrying the conjugative plasmid F. These contributions have yet to be defined biochemically, but it is already clear that the cell envelope is an importan nexus between plasmid‐ and (...) chromosome‐encoded proteins required for the establishment and maintenance of DNA donor activity. (shrink)

The many genetic complementation groups of DNA excision‐repair defective mammalian cells indicate the considerable complexity of the excision repair process. The cloning of several repair genes is taking the field a step closer to mechanistic studies of the actions and interactions of repair proteins. Early biochemical studies of mammalian DNA repair in vitro are now at hand. Repair synthesis in damaged DNA can be monitored by following the incorporation of radiolabelled nucleotides. Synthesis is carried out by mammalian cell extracts (...) and is defective in extracts from cell lines derived from individuals with the excisionrepair disorder xeroderma pigmentosum. Biochemical complementation of the defective extracts can be used to purify repair proteins. Repair of damage caused by agents including ultraviolet irradiation, psoralens, and platinating compounds has been observed. Neutralising antibodies against the human single‐stranded DNA binding protein (HSSB) have demonstrated a requirement for this protein in DNA excision repair as well as in DNA replication. (shrink)

DNA metabolic events such as replication, repair and recombination require the concerted action of several enzymes and cofactors. Nature has provided a set of proteins that support DNA polymerases in performing processive, accurate and rapid DNA synthesis. Two of them, the proliferating cell nuclear antigen and its adapter protein replication factor C, cooperate to form a moving platform that was initially thought of only as an anchor point for DNA polymerases δ and ε. It now appears that proliferating (...) class='Hi'>cell nuclear antigen is also a communication point between a variety of important cellular processes including cell cycle control, DNA replication, nucleotide excision repair, post‐replication mismatch repair, base excision repair and at least one apoptotic pathway. The dynamic movement of proliferating cell nuclear antigen on and off the DNA renders this protein an ideal communicator for a variety of proteins that are essential for DNA metabolic events in eukaryotic cells. (shrink)

This personal account traces a series of studies that led from DNA physical chemistry to anticancer drug mechanisms. Chemical crosslinking as a basis for anticancer drug actions had been suspected since the time of the first clinical reports of the effectiveness of nitrogen mustard in 1946. After the elucidation of the DNA helix‐coil transition, several nearly concurrent findings in the early 1960s established the paradigm of DNA interstrand crosslinking. The DNA filter elution phenomenon was discovered in the early 1970s, and (...) lent itself to the development of practical assays for DNA crosslinks and other DNA lesions in mammalian cells. The assays allowed studies of the effects of DNA damaging agents at pharmacologically or toxicologically relevant doses, and have been widely applied in studies of mutagenic and chemotherapeutic agents. During the period 1979–1986, DNA filter elution studies led to the paradigm of DNA topoisomerases as targets of anticancer drug action, and this has become one of the most active areas of anticancer drug development. (shrink)

There is growing evidence that mutations can arise in non‐dividing cells (both bacterial and mammalian) in the absence of chromosomal replication. The processes that are involved are still largely unknown but may include two separate mechanisms. In the first, DNA lesions resulting from the action of endogenous mutagens may give rise to RNA transcripts with miscoded bases. If these confer the ability to initiate DNA replication, the DNA lesions may have an opportunity to miscode during replication and thus could give (...) rise to apparently ‘adaptive’ mutations. A second mechanism is suggested by recent work in starved bacteria, showing that there is much more turnover of chromosomal DNA than has been previously thought. This could permit polymerase errors to lead to mutations in non‐dividing cells. Such cryptic DNA synthesis, which may essentially replace existing DNA rather than duplicating it, could, in principle, act as an additional source of variability on which selection may act, initially in the absence of cell division. In mammals such processes would undoubtedly have implications for germ cell mutagenesis and carcinogenesis. (shrink)

Developments in Non-Invasive Prenatal Testing (NIPT) and cell-free fetal DNA analysis raise the possibility that antenatal services may soon be able to support couples in non-invasively testing for, and diagnosing, an unprecedented range of genetic disorders and traits coded within their unborn child’s genome. Inevitably, this has prompted debate within the bioethics literature about what screening options should be offered to couples for the purpose of reproductive choice. In relation to this problem, the European Society of Human Genetics (...) (ESHG) and American Society of Human Genetics (ASHG) tentatively recommend that any expansion of this type of screening, as facilitated by NIPT, should be limited to serious congenital and childhood disorders. In support of this recommendation, the ESHG and ASHG cite considerations of distribution justice. Notably, however, an account of justice in the organization and provision of this type of screening which might substantiate this recommendation has yet to be developed. This paper attempts to redress this oversight through an investigation of Norman Daniels’ theory of Just health: meeting health needs fairly. In line with this aim, the paper examines what special moral importance (for Just health) screening for the purpose of reproductive choice might have where concerning serious congenital and childhood disorders in particular. The paper concludes that screening for reproductive choice where concerning serious congenital and childhood disorders may be important for providing women with fair opportunity to protect their health (by either having or not having an affected child). (shrink)

The rise of genomic studies in Africa – not least due to projects funded under H3Africa – is associated with the development of a small number of biorepositories across Africa. For the ultimate success of these biorepositories, the creation of cell lines including those from selected H3Africa samples would be beneficial. In this paper, we map ethical challenges in the creation of cell lines.

The organization of eukaryotic genomes as chromatin provides the framework within which regulated transcription occurs in the nucleus. The association of DNA with chromatin proteins required to package the genome into the nucleus is, in general, inhibitory to transcription, and therefore provides opportunities for regulated transcriptional activation. Granting access to the cis‐acting elements in DNA, a prerequisite for any further action of the trans‐acting factors involved, requires the establishment of local heterogeneity of chromatin and, in some cases, extensive remodeling of (...) nucleosomal structures. Challenging problems relate to the establishment of this heterogeneity at the level of the single nucleosome and to the mechanisms that operate when nucleosomal arrays are reorganized. Recent developments indicate that chromatin reconstitution in cell‐free systems allows the biochemical analysis of the interplay between transcription factors and chromatin components that brings about regulated transcription. (shrink)

International guidelines recommend that prenatal screening for fetal abnormalities should only be offered within a non-directive framework aimed at enabling women in making meaningful reproductive choices. Whilst this position is widely endorsed, developments in cell-free fetal DNA based Non-Invasive Prenatal Testing are now raising questions about its continued suitability for guiding screening policy and practice. This issue is most apparent within debates on the scope of the screening offer. Implied by the aim of enabling meaningful reproductive choices is (...) the idea that screening services should support women in accessing prenatal tests that best enable them to realize the types of reproductive choice that they find important. However, beyond whatever options meet the quality standards required for facilitating an informed decision, the remaining criteria of facilitating autonomous choice is strictly non-directive. As a result, policy makers receive little indication prior to consultation with each individual woman, about what conditions should be prioritized during the offer of screening. In this paper we try to address this issue by using the capabilities approach to further specify the non-directive aim of enabling meaningful reproductive choice. The resulting framework is then used to assess the relative importance of offering prenatal screening where concerning different types of genetic condition. We conclude that greater priority may be ascribed to offering prenatal screening for conditions that more significantly diminish a woman’s central capabilities. It follows that serious congenital and earlier-onset conditions are more likely to fulfill these criteria. (shrink)

Polyploid cells contain multiple copies of all chromosomes. Polyploidization can be developmentally programmed to sustain tissue barrier function or to increase metabolic potential and cell size. Programmed polyploidy is normally associated with terminal differentiation and poor proliferation capacity. Conversely, non‐programmed polyploidy can give rise to cells that retain the ability to proliferate. This can fuel rapid genome rearrangements and lead to diseases like cancer. Here, the mechanisms that generate polyploidy are reviewed and the possible challenges upon polyploid cell (...) division are discussed. The discussion is framed around a recent study showing that asynchronous cell cycle progression (an event that is named “chronocrisis”) of different nuclei from a polyploid cell can generate DNA damage at mitotic entry. The potential mechanisms explaining how mitosis in non‐programmed polyploid cells can generate abnormal karyotypes and genetic instability are highlighted. (shrink)

Under certain conditions, the cell cycle can be arrested for a long period of time. Vertebrate oocytes are arrested at G2 phase, while somatic cells arrest at G0 phase. In both cells, nuclei have lost the ability to initiate DNA synthesis. In a pair of recently published papers,1,2 Méchali and colleagues and Coué and colleagues have clarified how frog oocytes prevent untimely DNA synthesis during the long G2 arrest. Intriguingly, they found only Cdc6 is responsible for the inability of (...) immature oocytes to replicate DNA. Cdc6 is a key component for replication licensing, and for G0 cells to re‐enter the proliferative stage. Strikingly similar strategies for preventing the untimely replication in both cells suggest that the suppression of replication licensing is a universal mechanism for securing the prolonged arrest of the cell cycle. BioEssays 25:313–316, 2003. © 2003 Wiley Periodicals, Inc. (shrink)

International guidelines recommend that prenatal screening for fetal abnormalities should only be offered within a non-directive framework aimed at enabling women in making meaningful reproductive choices. Whilst this position is widely endorsed, developments in cell-free fetal DNA based Non-Invasive Prenatal Testing are now raising questions about its continued suitability for guiding screening policy and practice. This issue is most apparent within debates on the scope of the screening offer. Implied by the aim of enabling meaningful reproductive choices is (...) the idea that screening services should support women in accessing prenatal tests that best enable them to realize the types of reproductive choice that they find important. However, beyond whatever options meet the quality standards required for facilitating an informed decision, the remaining criteria of facilitating autonomous choice is strictly non-directive. As a result, policy makers receive little indication prior to consultation with each individual woman, about what conditions should be prioritized during the offer of screening. In this paper we try to address this issue by using the capabilities approach to further specify the non-directive aim of enabling meaningful reproductive choice. The resulting framework is then used to assess the relative importance of offering prenatal screening where concerning different types of genetic condition. We conclude that greater priority may be ascribed to offering prenatal screening for conditions that more significantly diminish a woman’s central capabilities. It follows that serious congenital and earlier-onset conditions are more likely to fulfill these criteria. (shrink)

Histone acetylation has been recognized as an important post‐translational modification of core nucleosomal histones that changes access to the chromatin to allow gene transcription, DNA replication, and repair. Histone acetyltransferases were initially identified as co‐activators that link DNA‐binding transcription factors to the general transcriptional machinery. Over the years, more chromatin‐binding modes have been discovered suggesting direct interaction of histone acetyltransferases and their protein complex partners with histone proteins. While much progress has been made in characterizing histone acetyltransferase complexes biochemically, (...) class='Hi'>cell‐free activity assay results are often at odds with in‐cell histone acetyltransferase activities. In‐cell studies suggest specific histone lysine targets, but broad recruitment modes, apparently not relying on specific DNA sequences, but on chromatin of a specific functional state. Here we review the evidence for general versus specific roles of individual nuclear lysine acetyltransferases in light of in vivo and in vitro data in the mammalian system. (shrink)

Base pair mismatches in DNA arise from errors in DNA replication, recombination, and biochemical modification of bases. Mismatches are inherently transient. They are resolved passively by DNA replication, or actively by enzymatic removal and resynthesis of one of the bases. The first step in removal is recognition of strand discontinuity by one of the MutS proteins. Mismatches arising from errors in DNA replication are repaired in favor of the base on the template strand, but other mismatches trigger base excision or (...) nucleotide excision repair (NER), or non‐repair pathways such as hypermutation, cell cycle arrest, or apoptosis. We argue that MutL homologues play a key role in determining biologic outcome by recruiting and/or activating effector proteins in response to lesion recognition by MutS. We suggest that the process is regulated by conformational changes in MutL caused by cycles of ATP binding and hydrolysis, and by physiologic changes which influence effector availability. (shrink)

Error‐free genome duplication and accurate cell division are critical for cell survival. In all three domains of life, bacteria, archaea, and eukaryotes, initiator proteins bind replication origins in an ATP‐dependent manner, play critical roles in replisome assembly, and coordinate cell‐cycle regulation. We discuss how the eukaryotic initiator, Origin recognition complex (ORC), coordinates different events during the cell cycle. We propose that ORC is the maestro driving the orchestra to coordinately perform the musical pieces of replication, (...) chromatin organization, and repair. (shrink)

A widely accepted assumption in radiobiology is that ionizing radiation kills cells by inducing forms of damage in DNA structures that lead to the formation of lethal chromosome aberrations. One goal of radiation biology research is the identification of these forms of DNA damage, the characterization of the mechanisms involved in their repair and the elucidation of the processes involved in their transformation to chromosome damage, In recent years, evidence has accumulated implicating DNA double stranded breaks as lesions relevant for (...) cell killing. Here, the available information on this topic is reviewed together with the methods most commonly used to quantitate induction and repair of this type of lesion. The presentation concludes with an outline of present research directions and future goals. (shrink)

The proliferation of mitochondrial DNA (mtDNA) with deletion mutations has been linked to aging and age related neurodegenerative conditions. In this study we model the effect of mtDNA half-life on mtDNA competition and selection. It has been proposed that mutation deletions (mtDNAdel\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{del}$$\end{document}) have a replicative advantage over wild-type (mtDNAwild\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{wild}$$\end{document}) and that this is detrimental to the host cell, especially in (...) post-mitotic cells. An individual cell can be viewed as forming a closed ecosystem containing a large population of independently replicating mtDNA. Within this enclosed environment a selfishly replicating mtDNAdel\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{del}$$\end{document} would compete with the mtDNAwild\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{wild}$$\end{document} for space and resources to the detriment of the host cell. In this paper, we use a computer simulation to model cell survival in an environment where mtDNAwild\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{wild}$$\end{document} compete with mtDNAdel\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{del}$$\end{document} such that the cell expires upon mtDNAwild\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{wild}$$\end{document} extinction. We focus on the survival time for long lived post-mitotic cells, such as neurons. We confirm previous observations that mtDNAdel\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{del}$$\end{document} do have a replicative advantage over mtDNAwild\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{wild}$$\end{document}. As expected, cell survival times diminished with increased mutation probabilities, however, the relationship between survival time and mutation rate was non-linear, that is, a ten-fold increase in mutation probability only halved the survival time. The results of our model also showed that a modest increase in half-life had a profound affect on extending cell survival time, thereby, mitigating the replicative advantage of mtDNAdel\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\text {mtDNA}_{del}$$\end{document}. Given the relevance of mitochondrial dysfunction to various neurodegenerative conditions, we propose that therapies to increase mtDNA half-life could significantly delay their onset. (shrink)

The problem we discuss is whether mammalian cells possess genes whose expression is specifically enhanced by DNA damage in order to cope with the damage. The paradigm is the SOS response in E. coli. We conclude that there is compelling evidence that DNA‐damaging agents do affect gene expression, and that mutation frequencies are increased, but proof that a repair process per se is induced remains elusive. We offer here the hypothesis that recognition of the presence of DNA damage by poly(ADPribose) (...) polymerase effects preprogrammed changes in gene expression. (shrink)

It has been recently demonstrated that yeast cells are able to partially regress chromosome segregation in telophase as a response to DNA double‐strand breaks (DSBs), likely to find a donor sequence for homology‐directed repair (HDR). This regression challenges the traditional concept that establishes anaphase events as irreversible, hence opening a new field of research in cell biology. Here, the nature of this new behavior in yeast is summarized and the underlying mechanisms are speculated about. It is also discussed whether (...) it can be reproduced in other eukaryotes. Overall, this work brings forwards the need of understanding how cells attempt to repair DSBs when transiting the latest stages of mitosis, i.e., anaphase and telophase. (shrink)

In response to DNA‐damage, cells have to decide between different cell fate programmes. Activation of the tumour suppressor HIPK2 specifies the DNA damage response (DDR) and tips the cell fate balance towards an apoptotic response. HIPK2 is activated by the checkpoint kinase ATM, and triggers apoptosis through regulatory phosphorylation of a set of cellular key molecules including the tumour suppressor p53 and the anti‐apoptotic corepressor CtBP. Recent work has identified HIPK2 as a regulator of the ultimate step in (...) cytokinesis: the abscission of the mother and daughter cells. Since proper cytokinesis is essential for genome stability and maintenance of correct ploidy, this finding sheds new light on the tumour suppressor function of HIPK2. Here we highlight the molecular mechanisms coordinating HIPK2 function and discuss its emerging role as a tumour suppressor. (shrink)

In 1956, I decided to apply my experience in microbial genetics to developing analogous systems for human cell lines, including the selection of mutants with either a loss or gain of a biochemical function. For instance, mutants resistant to azahypoxanthine showed a loss of the HPRT enzyme (hypoxanthine phosphoribosyl transferase), whereas gain of the same enzyme was accomplished by blocking de novo purine biosynthesis with aminopterin, while supplying hypoxanthine and thymine (HAT selection). Using HAT selection, we: (i) genetically transformed (...) HPRT− mutant cells to HPRT+ wild type by using DNA extracted from HPRT+ cells, and (ii) selected HPRT+ hybrid cells by fusing HPRT− D98/AH2 cells with skin cells. These approaches, which we dubbed in 1962 as a [first step toward gene therapy], contributed to the later development of (i) cell fusion techniques, (ii) the development of monoclonal antibodies, (iii) routine transformation of mammalian cells with cloned genes, and (iv) methods for creating transgenic organisms. (shrink)

Methylation of DNA plays an important role in the control of gene expression in higher eukaryotes. This is largely achieved by the packaging of methylated DNA into chromatin structures that are inaccessible to transcription factors and other proteins. Methylation involves the addition of a methyl group to the 5‐position of the cytosine base in DNA, a reaction catalysed by a DNA (cytosine‐5) methyltransferase. This reaction occurs in nuclear replication foci where the chromatin structure is loosened for replication, thereby allowing access (...) to methyltransferases. Partly as a result of their recognising the presence of a methylcytosine on the parental strand following replication, these large enzymes are able to maintain the distribution of methyl groups along the DNA of somatic cells and, thereby, maintain tissue‐specific patterns of gene expression. (shrink)