Hsp104-dependent remodeling of prion complexes mediates protein-only inheritance

Inheritance of phenotypic traits depends on two key events: replication of the determinant of that trait and partitioning of these copies between mother and daughter cells. Although these processes are well understood for nucleic acid–based genes, the mechanisms by which protein-only or prion-based genetic elements direct phenotypic inheritance are poorly understood. Here, we report a process crucial for inheritance of the Saccharomyces cerevisiae prion [PSI⁺], a self-replicating conformer of the Sup35 protein. By tightly controlling expression of a Sup35-GFP fusion, we directly observe remodeling of existing Sup35^[PSI+] complexes in vivo. This dynamic change in Sup35^[PSI+] is lost when the molecular chaperone Hsp104, a factor essential for propagation of all yeast prions, is functionally impaired. The loss of Sup35^[PSI+] remodeling by Hsp104 decreases the mobility of these complexes in the cytosol, creates a segregation bias that limits their transmission to daughter cells, and consequently diminishes the efficiency of conversion of newly made Sup35 to the prion form. Our observations resolve several seemingly conflicting reports on the mechanism of Hsp104 action and point to a single Hsp104-dependent event in prion propagation.     

Non-random segregation of DNA strands in Escherichia coli B-r

The segregation of DNA strands during growth of Escherichia coli$\text{B}\text{r}$ has been studied under conditions in which the chromosomal configuration and the ancestry of the cells during growth and division were known. Cells containing either one or two replicating chromosomes were pulse-labeled with [³H]thymidine, and the location of the radioactivity within chains of cells formed by growth in methylcellulose was determined by autoradiography. The locations of the radioactive cells within chains obtained after the second, third and fourth divisions were consistent with the co-segregation of only one of the replicating strands of each chromosome and a fixed region of the cell into daughter cells. The attachment of this strand to the region appeared to become permanent at the time the strand was used for the first time as a template. It is concluded that the segregation of DNA molecules into daughter cells is non-random in E. coli B/r.     

Asymmetric division and cosegregation of template DNA strands in adult muscle satellite cells

Satellite cells assure postnatal skeletal muscle growth and repair. Despite extensive studies, their stem cell character remains largely undefined. Using pulse-chase labelling with BrdU to mark the putative stem cell niche, we identify a subpopulation of label-retaining satellite cells during growth and after injury. Strikingly, some of these cells display selective template-DNA strand segregation during mitosis in the muscle fibre in vivo, as well as in culture independent of their niche, indicating that genomic DNA strands are nonequivalent. Furthermore, we demonstrate that the asymmetric cell-fate determinant Numb segregates selectively to one daughter cell during mitosis and before differentiation, suggesting that Numb is associated with self-renewal. Finally, we show that template DNA cosegregates with Numb in label-retaining cells that express the self-renewal marker Pax7. The cosegregation of 'immortal' template DNA strands and their link with the asymmetry apparatus has important implications for stem cell biology and cancer.     

Colonic stem cell data are consistent with the immortal model of stem cell division under non-random strand segregation

Objectives:  Colonic stem cells are thought to reside towards the base of crypts of the colon, but their numbers and proliferation mechanisms are not well characterized. A defining property of stem cells is that they are able to divide asymmetrically, but it is not known whether they always divide asymmetrically (immortal model) or whether there are occasional symmetrical divisions (stochastic model). By measuring diversity of methylation patterns in colon crypt samples, a recent study found evidence in favour of the stochastic model, assuming random segregation of stem cell DNA strands during cell division. Here, the effect of preferential segregation of the template strand is considered to be consistent with the 'immortal strand hypothesis', and explore the effect on conclusions of previously published results.
Materials and methods:  For a sample of crypts, it is shown how, under the immortal model, to calculate mean and variance of the number of unique methylation patterns allowing for non-random strand segregation and compare them with those observed.
Results:  The calculated mean and variance are consistent with an immortal model that incorporates non-random strand segregation for a range of stem cell numbers and levels of preferential strand segregation.
Conclusions:  Allowing for preferential strand segregation considerably alters previously published conclusions relating to stem cell numbers and turnover mechanisms. Evidence in favour of the stochastic model may not be as strong as previously thought.     

Immortal DNA or epigenetic signature ?

During mitosis each daughter cell inherits a full copy of the maternal genomic material. DNA replication, however, is an imprecise process, thus errors can arise resulting in potentially deleterious mutations over extended rounds of cell division and these may lead to cancinogenesis. Over thirty years ago, J. Cairns proposed that a cell could avoid the accumulation of mutations arising from DNA replication if all template DNA strands are inherited in one daughter cell during cell division, thus giving rise to the notion of < immortal > DNA strands. In this model the stem cells would retain the template DNA (older) strands. Proving or disproving this notion experimentally has been challenging. Further, it has recently become apparent that epigenetic regulation of gene expression plays a critical role in governing cell states, self-renewal and differentiation. In light of these data, can the phenomenon on template DNA strand segregation also reflect this epigenetic signature? In this review we explore these notions, discuss the evidence in support of this theory, the implications, and some of the mechanisms which could explain this phenomenon.     

Biosynthesis of selenocysteine on its tRNA in eukaryotes

Selenocysteine (Sec) is cotranslationally inserted into protein in response to UGA codons and is the 21st amino acid in the genetic code. However, the means by which Sec is synthesized in eukaryotes is not known. Herein, comparative genomics and experimental analyses revealed that the mammalian Sec synthase (SecS) is the previously identified pyridoxal phosphate-containing protein known as the soluble liver antigen. SecS required selenophosphate and O-phosphoseryl-tRNA^[Ser]Sec as substrates to generate selenocysteyl-tRNA^[Ser]Sec. Moreover, it was found that Sec was synthesized on the tRNA scaffold from selenide, ATP, and serine using tRNA^[Ser]Sec, seryl-tRNA synthetase, O-phosphoseryl-tRNA^[Ser]Sec kinase, selenophosphate synthetase, and SecS. By identifying the pathway of Sec biosynthesis in mammals, this study not only functionally characterized SecS but also assigned the function of the O-phosphoseryl-tRNA^[Ser]Sec kinase. In addition, we found that selenophosphate synthetase 2 could synthesize monoselenophosphate in vitro but selenophosphate synthetase 1 could not. Conservation of the overall pathway of Sec biosynthesis suggests that this pathway is also active in other eukaryotes and archaea that synthesize selenoproteins.     

Stem cells propagate their DNA by random segregation in the flatworm Macrostomum lignano

Adult stem cells are proposed to have acquired special features to prevent an accumulation of DNA-replication errors. Two such mechanisms, frequently suggested to serve this goal are cellular quiescence, and non-random segregation of DNA strands during stem cell division, a theory designated as the immortal strand hypothesis. To date, it has been difficult to test the in vivo relevance of both mechanisms in stem cell systems. It has been shown that in the flatworm Macrostomum lignano pluripotent stem cells (neoblasts) are present in adult animals. We sought to address by which means M. lignano neoblasts protect themselves against the accumulation of genomic errors, by studying the exact mode of DNA-segregation during their division. In this study, we demonstrated four lines of in vivo evidence in favor of cellular quiescence. Firstly, performing BrdU pulse-chase experiments, we localized 'Label-Retaining Cells' (LRCs). Secondly, EDU pulse-chase combined with Vasa labeling demonstrated the presence of neoblasts among the LRCs, while the majority of LRCs were differentiated cells. We showed that stem cells lose their label at a slow rate, indicating cellular quiescence. Thirdly, CldU/IdU- double labeling studies confirmed that label-retaining stem cells showed low proliferative activity. Finally, the use of the actin inhibitor, cytochalasin D, unequivocally demonstrated random segregation of DNA-strands in LRCs. Altogether, our data unambiguously demonstrated that the majority of neoblasts in M. lignano distribute their DNA randomly during cell division, and that label-retention is a direct result of cellular quiescence, rather than a sign of co-segregation of labeled strands.     

Calcium dynamics of cortical astrocytic networks in vivo

Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca²⁺]_i events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca²⁺]_i events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca²⁺]_i activity in individual cells and a robust coordination of [Ca²⁺]_i signals in neighboring astrocytes. These findings indicate potential neuron–glia communication in the intact brain.     

Citation advantage of open access articles

Open access (OA) to the research literature has the potential to accelerate recognition and dissemination of research findings, but its actual effects are controversial. This was a longitudinal bibliometric analysis of a cohort of OA and non-OA articles published between June 8, 2004, and December 20, 2004, in the same journal (PNAS: Proceedings of the National Academy of Sciences). Article characteristics were extracted, and citation data were compared between the two groups at three different points in time: at “quasi-baseline” (December 2004, 0–6 mo after publication), in April 2005 (4–10 mo after publication), and in October 2005 (10–16 mo after publication). Potentially confounding variables, including number of authors, authors' lifetime publication count and impact, submission track, country of corresponding author, funding organization, and discipline, were adjusted for in logistic and linear multiple regression models. A total of 1,492 original research articles were analyzed: 212 (14.2% of all articles) were OA articles paid by the author, and 1,280 (85.8%) were non-OA articles. In April 2005 (mean 206 d after publication), 627 (49.0%) of the non-OA articles versus 78 (36.8%) of the OA articles were not cited (relative risk = 1.3 [95% Confidence Interval: 1.1–1.6]; p = 0.001). 6 mo later (mean 288 d after publication), non-OA articles were still more likely to be uncited (non-OA: 172 [13.6%], OA: 11 [5.2%]; relative risk = 2.6 [1.4–4.7]; p < 0.001). The average number of citations of OA articles was higher compared to non-OA articles (April 2005: 1.5 [SD = 2.5] versus 1.2 [SD = 2.0]; Z = 3.123; p = 0.002; October 2005: 6.4 [SD = 10.4] versus 4.5 [SD = 4.9]; Z = 4.058; p < 0.001). In a logistic regression model, controlling for potential confounders, OA articles compared to non-OA articles remained twice as likely to be cited (odds ratio = 2.1 [1.5–2.9]) in the first 4–10 mo after publication (April 2005), with the odds ratio increasing to 2.9 (1.5–5.5) 10–16 mo after publication (October 2005). Articles published as an immediate OA article on the journal site have higher impact than self-archived or otherwise openly accessible OA articles. We found strong evidence that, even in a journal that is widely available in research libraries, OA articles are more immediately recognized and cited by peers than non-OA articles published in the same journal. OA is likely to benefit science by accelerating dissemination and uptake of research findings.     

Support for the immortal strand hypothesis: neural stem cells partition DNA asymmetrically in vitro

The immortal strand hypothesis proposes that asymmetrically dividing stem cells (SCs) selectively segregate chromosomes that bear the oldest DNA templates. We investigated cosegregation in neural stem cells (NSCs). After exposure to the thymidine analogue 5-bromo-2-deoxyuridine (BrdU), which labels newly synthesized DNA, a subset of neural precursor cells were shown to retain BrdU signal. It was confirmed that some BrdU-retaining cells divided actively, and that these cells exhibited some characteristics of SCs. This asymmetric partitioning of DNA then was demonstrated during mitosis, and these results were further supported by real time imaging of SC clones, in which older and newly synthesized DNA templates were distributed asymmetrically after DNA synthesis. We demonstrate that NSCs are unique among precursor cells in the uneven partitioning of genetic material during cell divisions.     

Asymmetric Strand Segregation: Epigenetic Costs of Genetic Fidelity?

Asymmetric strand segregation has been proposed as a mechanism to minimize effective mutation rates in epithelial tissues. Under asymmetric strand segregation, the double-stranded molecule that contains the oldest DNA strand is preferentially targeted to the somatic stem cell after each round of DNA replication. This oldest DNA strand is expected to have fewer errors than younger strands because some of the errors that arise on daughter strands during their synthesis fail to be repaired. Empirical findings suggest the possibility of asymmetric strand segregation in a subset of mammalian cell lineages, indicating that it may indeed function to increase genetic fidelity. However, the implications of asymmetric strand segregation for the fidelity of epigenetic information remain unexplored. Here, I explore the impact of strand-segregation dynamics on epigenetic fidelity using a mathematical-modelling approach that draws on the known molecular mechanisms of DNA methylation and existing rate estimates from empirical methylation data. I find that, for a wide range of starting methylation densities, asymmetric—but not symmetric—strand segregation leads to systematic increases in methylation levels if parent strands are subject to de novo methylation events. I found that epigenetic fidelity can be compromised when enhanced genetic fidelity is achieved through asymmetric strand segregation. Strand segregation dynamics could thus explain the increased DNA methylation densities that are observed in structured cellular populations during aging and in disease.     

Template-switching during DNA synthesis by Thermus aquaticus DNA polymerase I.

Recombinant DNA molecules are often generated during the polymerase chain reaction (PCR) when partially homologous templates are available [e.g., see Pääbo et al. (1990) J. Biol. Chem. 265, 4718-4721]. It has been suggested that these recombinant molecules are a consequence of truncated extension products annealing to partially homologous templates on subsequent PCR cycles. However, we demonstrate here that recombinants can be generated during a single round of primer extension in the absence of subsequent heat denaturation, indicating that template-switching produces some of these recombinant molecules. Two types of template-switches were observed: (i) switches to pre-existing templates and (ii) switches to the complementary nascent strand. Recombination is reduced several fold when the complementary template strands are physically separated by attachment to streptavidin magnetic beads. This result supports the hypothesis that either the polymerase or at least one of the two extending strands switches templates during DNA synthesis and that interaction between the complementary template strands is necessary for efficient template-switching.     

Semiconservative quasispecies equations for polysomic genomes: the haploid case

This paper develops the semiconservative quasispecies equations for genomes consisting of an arbitrary number of chromosomes. We assume that the chromosomes are distinguishable, so that we are effectively considering haploid genomes. We derive the quasispecies equations under the assumption of arbitrary lesion repair efficiency, and consider the cases of both random and immortal strand chromosome segregation. We solve the model in the limit of infinite sequence length for the case of the static single fitness peak landscape, where the master genome has a first-order growth rate constant of k>1, and all other genomes have a first-order growth rate constant of 1. If we assume that each chromosome can tolerate an arbitrary number of lesions, so that only one master copy of the strands is necessary for a functional chromosome, then for random chromosome segregation we obtain an equilibrium mean fitness of [equation in text] below the error catastrophe, while for immortal strand co-segregation we obtain kappa (t=infinity)=k[e(-mu(1-lambda/2))+e(-mulambda/2)-1] (N denotes the number of chromosomes, lambda denotes the lesion repair efficiency, and mu is identical with epsilonL, where epsilon is the per base-pair mismatch probability, and L is the total genome length). It follows that immortal strand co-segregation leads to significantly better preservation of the master genome than random segregation when lesion repair is imperfect. Based on this result, we conjecture that certain classes of tumor cells exhibit immortal strand co-segregation.     

Relationships between chromosome segregation, cell shape and temperature in Escherichia coli.

The partitioning of chromosomes into daughter cells during the division of Escherichia coli is non-random. As a result, the chromosome containing the older template DNA strand has a higher probability of segregating toward the old cell pole than toward the new cell pole. The numerical value of this probability is a function of the incubation temperature. It is shown here that a recent model for explaining the physiological basis for non-random chromosome segregation also explains the temperature dependence of the segregation process.     

The segregation of DNA in epithelial stem cells

It has recently been suggested that stem cells may invariably keep, from one division to the next, the daughter DNA molecules that contain the older of the two parental strands—that is, they may retain a complete set of “immortal strands,” through successive cell divisions (Cairns, 1975). We can test this hypothesis by labeling either the old immortal strands at the time the stem cells are created or the newly synthesized strands during subsequent divisions of the stem cells. In the former case, the stem cells should become permanently labeled; in the latter case, they should eliminate their label on their second division.Experiments of this sort have been conducted with the tongue papilla under steady state conditions and with the regenerating small intestinal crypts. The results clearly show that by far most of the multiplying cells in tongue and intestinal epithelium segregate their DNA “randomly” at mitosis. Nevertheless, the results, though far from conclusive, suggest that there are a small number of cells (1–5 in the stem cell region of each crypt and one at the base of each column of cells in the tongue) that selectively segregate their old and new DNA strands in the expected way. Thus in the immortal strand labeling experiments, there are a few labeled cells that retain their label for up to 4 weeks; conversely, in the new strand labeling experiments, a few cells appear to rid themselves of label after intervals equivalent to approximately two cell cycles.     

DNA methyltransferase-3–dependent nonrandom template segregation in differentiating embryonic stem cells

Asymmetry of cell fate is one fundamental property of stem cells, in which one daughter cell self-renews, whereas the other differentiates. Evidence of nonrandom template segregation (NRTS) of chromosomes during asymmetric cell divisions in phylogenetically divergent organisms, such as plants, fungi, and mammals, has already been shown. However, before this current work, asymmetric inheritance of chromatids has never been demonstrated in differentiating embryonic stem cells (ESCs), and its molecular mechanism has remained unknown. Our results unambiguously demonstrate NRTS in asymmetrically dividing, differentiating human and mouse ESCs. Moreover, we show that NRTS is dependent on DNA methylation and on Dnmt3 (DNA methyltransferase-3), indicating a molecular mechanism that regulates this phenomenon. Furthermore, our data support the hypothesis that retention of chromatids with the “old” template DNA preserves the epigenetic memory of cell fate, whereas localization of “new” DNA strands and de novo DNA methyltransferase to the lineage-destined daughter cell facilitates epigenetic adaptation to a new cell fate.     

Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands

It has been postulated that the stem cells of somatic tissues protect themselves from mutation and cancer risk by selective segregation of their template DNA strands. Self-renewing mammary epithelial stem cells that were originated during allometric growth of the mammary ducts in pubertal females were labeled using [3H]-thymidine (3HTdR). After a prolonged chase during which much of the branching duct morphogenesis was completed, 3HTdR-label retaining epithelial cells (LREC) were detected among the epithelium of the maturing glands. Labeling newly synthesized DNA in these glands with a different marker, 5-bromodeoxyuridine (5BrdU), resulted in the appearance of doubly labeled nuclei in a large percentage of the LREC. By contrast, label-retaining cells within the stroma did not incorporate 5BrdU during the pulse, indicating that they were not traversing the cell cycle. Upon chase, the second label (5BrdU) was distributed from the double-labeled LREC to unlabeled mammary cells while 3HTdR was retained. These results demonstrate that mammary LREC selectively retain their 3HTdR-labeled template DNA strands and pass newly synthesized 5BrdU-labeled DNA to their progeny during asymmetric divisions. Similar results were obtained in mammary transplants containing self-renewing, lacZ-positive epithelial cells suggesting that cells capable of expansive self-renewal may repopulate new mammary stem cell niches during the allometric growth of new mammary ducts.     

Process of Infection with Bacteriophage φX174: XXXIII. Templates for the Synthesis of Single-Stranded Deoxyribonucleic Acid

The origin of the templates for the synthesis of X174 progeny single-stranded deoxyribonucleic acid was studied by means of the mutagenic activity associated with the decay of incorporated ³H-labeled 5-cytosine. The results indicate that the single-strand synthesis occurs in an asymmetric semiconservative manner using as template the complementary strands of the pool of replicative from molecules accumulated during the eclipse period. These complementary strands are repeatedly used as templates, and there is no detectable preferential use of complementary strand templates made early in the eclipse versus those made late.