Phosphorylation of histone H3 at serine 10 is correlated with chromosome condensation during mitosis and meiosis in Tetrahymena

H3 phosphorylation has been correlated with mitosis temporally in mammalian cells and spatially in ciliated protozoa. In logarithmically growing Tetrahymena thermophila cells, for example, H3 phosphorylation can be detected in germline micronuclei that divide mitotically but not in somatic macronuclei that divide amitotically. Here, we demonstrate that micronuclear H3 phosphorylation occurs at a single site (Ser-10) in the amino-terminal domain of histone H3, the same site phosphorylated during mitosis in mammalian cells. Using an antibody specific for Ser-10 phosphorylated H3, we show that, in Tetrahymena, this modification is correlated with mitotic and meiotic divisions of micronuclei in a fashion that closely coincides with chromosome condensation. Our data suggest that H3 phosphorylation at Ser-10 is a highly conserved event among eukaryotes and is likely involved in both mitotic and meiotic chromosome condensation.

Resistance to DNA fragmentation and chromatin condensation in mice lacking the DNA fragmentation factor 45

The DNA fragmentation factor 45 (DFF45) is a subunit of a heterodimeric nuclease complex critical for the induction of DNA fragmentation in vitro. To understand the in vivo role of DFF45 in programmed cell death, we generated DFF45 mutant mice. DNA fragmentation activity is completely abolished in cell extracts from DFF45 mutant tissues. In response to apoptotic stimuli, splenocytes, thymocytes, and granulocytes from DFF45 mutant mice are resistant to DNA fragmentation, and splenocytes and thymocytes are also resistant to chromatin condensation. Nevertheless, development of the immune system in the DFF45 mutant mice is normal. These results demonstrate that DFF45 is critical for the induction of DNA fragmentation and chromatin condensation in vivo, but is not required for normal immune system development.

Measurement of community metabolism and significance in the coral reef CO2 source-sink debate

Two methods are commonly used to measure the community metabolism (primary production, respiration, and calcification) of shallow-water marine communities and infer air–sea CO2 fluxes: the pH-total alkalinity and pH-O2 techniques. The underlying assumptions of each technique are examined to assess the recent claim that the most widely used technique in coral reefs (pH-total alkalinity), may have provided spurious results in the past because of high rates of nitrification and release of phosphoric acid in the water column [Chisholm, J. R. M. & Barnes, D. J. (1998) Proc. Natl. Acad. Sci. USA 95, 6566–6569]. At least three lines of evidence suggest that this claim is not founded. First, the rate of nitrification required to explain the discrepancy between the two methods recently reported is not realistic as it is much higher than the rates measured in another reef system and greater than the highest rate measured in a marine environment. Second, fluxes of ammonium, nitrate, and phosphorus are not consistent with high rates of nitrification and release of phosphoric acid. Third, the consistency of the metabolic parameters obtained by using the two techniques is in good agreement in two sites recently investigated. The pH-total alkalinity technique therefore appears to be applicable in most coral reef systems. Consequently, the conclusion that most coral reef flats are sources of CO2 to the atmosphere does not need revision. Furthermore, we provide geochemical evidence that calcification in coral reefs, as well as in other calcifying ecosystems, is a long-term source of CO2 for the atmosphere.

The Role of Topoisomerase II in Meiotic Chromosome Condensation and Segregation in Schizosaccharomyces pombe

Topoisomerase II is able to break and rejoin double-strand DNA. It controls the topological state and forms and resolves knots and catenanes. Not much is known about the relation between the chromosome segregation and condensation defects as found in yeast top2 mutants and the role of topoisomerase II in meiosis. We studied meiosis in a heat-sensitive top2 mutant of Schizosaccharomyces pombe. Topoisomerase II is not required until shortly before meiosis I. The enzyme is necessary for condensation shortly before the first meiotic division but not for early meiotic prophase condensation. DNA replication, prophase morphology, and dynamics of the linear elements are normal in the top2 mutant. The top2 cells are not able to perform meiosis I. Arrested cells have four spindle pole bodies and two spindles but only one nucleus, suggesting that the arrest is nonregulatory. Finally, we show that the arrest is partly solved in a top2 rec7 double mutant, indicating that topoisomerase II functions in the segregation of recombined chromosomes. We suggest that the inability to decatenate the replicated DNA is the primary defect in top2. This leads to a loss of chromatin condensation shortly before meiosis I, failure of sister chromatid separation, and a nonregulatory arrest.

Molecular mechanism for silencing virally transduced genes involves histone deacetylation and chromatin condensation

Virally transduced genes are often silenced after integration into the host genome. Chromatin immunoprecipitation and nuclease sensitivity experiments now demonstrate that silencing of the transgene is characterized by deacetylation of histone H4 lysines and chromatin condensation. Trichostatin A treatment results in dramatic reactivation of gene expression that is preceded by histone acetylation and chromatin decondensation. Analysis of individual histone H4 lysines demonstrate that chromatin domain opening is coincident with rapid acetylation of histone H4 K5, K12, and K16 and that maintenance of the open domain is correlated with acetylation of histone H4 K8. Removal of trichostatin A results in rapid deacetylation of histone H4 K8, chromatin condensation, and transcription silencing. The results suggest that deacetylation of histone H4 lysines and coincident chromatin condensation are critically involved in the silencing of virally transduced genes.

Anomalies in coral reef community metabolism and their potential importance in the reef CO2 source-sink debate

It is not certain whether coral reefs are sources of or sinks for atmospheric CO2. Air–sea exchange of CO2 over reefs has been measured directly and inferred from changes in the seawater carbonate equilibrium. Such measurements have provided conflicting results. We provide community metabolic data that indicate that large changes in CO2 concentration can occur in coral reef waters via biogeochemical processes not directly associated with photosynthesis, respiration, calcification, and CaCO3 dissolution. These processes can significantly distort estimates of reef calcification and net productivity and obscure the contribution of coral reefs to global air–sea exchange of CO2. They may, nonetheless, explain apparent anomalies in the metabolic performance of reefs close to land and reconcile the differing experimental findings that have given rise to the CO2 debate.

The reversible condensation and expansion of the rotavirus genome

Understanding the structural organization of the genome is particularly relevant in segmented double-stranded RNA viruses, which exhibit endogenous transcription activity. These viruses are molecular machines capable of repeated cycles of transcription within the intact capsid. Rotavirus, a major cause of infantile gastroenteritis, is a prototypical segmented double-stranded RNA virus. From our three-dimensional structural analyses of rotavirus examined under various chemical conditions using electron cryomicroscopy, we show here that the viral genome exhibits a remarkable conformational flexibility by reversibly changing its packaging density. In the presence of ammonium ions at high pH, the genome condenses to a radius of ≈180 Å from ≈220 Å. Upon returning to physiological conditions, the genome re-expands and fully maintains its transcriptional properties. These studies provide further insights into the genome organization and suggest that the observed isometric and concentric nature of the condensation is due to strong interactions between the genome core and the transcription enzymes anchored to the capsid inner surface. The ability of the genome to condense beyond what is normally observed in the native virus indicates that the negative charges on the RNA in the native state may be only partially neutralized. Partial neutralization may be required to maintain appropriate interstrand spacing for templates to move around the enzyme complexes during transcription. Genome condensation was not observed either with increased cation concentrations at normal pH or at high pH without ammonium ions. This finding indicates that the observed genome condensation is a synergistic effect of hydroxyl and ammonium ions involving disruption of protein–RNA interactions that perhaps facilitate further charge neutralization and consequent reduction in the interstrand spacing.

Characterization of Source- and Sink-Specific Sucrose/H+ Symporters from Carrot

To understand how sucrose (Suc) is transported from source leaves to developing tap roots of carrot (Daucus carota L.), we cloned two cDNAs (DcSUT1 and DcSUT2) for proteins with homologies to plant Suc/H+ symporters. The deduced polypeptide sequences are 52% identical and have 12 predicted membrane-spanning domains each. Transport activities were confirmed by expression of the clones in yeast cells. Both transporters had optimal activity below pH 5.0 and Michaelis constant values of 0.5 mm. Suc uptake was inhibited by protonophores, suggesting that Suc transport is linked to the proton electrochemical potential across the plasma membrane. DcSUT1 and DcSUT2 had markedly different expression patterns. Transcripts of DcSUT1 were found only in the green parts of plants, with highest levels in the lamina of source leaves, indicating that DcSUT1 is required for the loading of Suc into the phloem. In leaf lamina expression was diurnally regulated, suggesting that Suc export from the leaves is higher during the day than during the night. The mRNA of DcSUT2 was found mainly in sink organs, and no diurnal expression pattern was detected in the storage root. Here, expression was not restricted to the phloem but was much higher in storage parenchyma tissues of phloem and xylem. The close relationship of DcSUT2 with a Suc/H+ symporter from fava bean, which facilitates Suc uptake into the cotyledons of developing seeds, indicates that this carrot Suc transporter may be involved in loading Suc into storage parenchyma cells.

Specific Soybean Lipoxygenases Localize to Discrete Subcellular Compartments and Their mRNAs Are Differentially Regulated by Source-Sink Status1

Members of the lipoxygenase multigene family, found widely in eukaryotes, have been proposed to function in nitrogen partitioning and storage in plants. Lipoxygenase gene responses to source-sink manipulations in mature soybean (Glycine max [L.] Merr.) leaves were examined using gene-specific riboprobes to the five vegetative lipoxygenases (vlxA–vlxE). Steady-state levels of all vlx mRNAs responded strongly to sink limitation, but specific transcripts exhibited differential patterns of response as well. During reproductive sink limitation, vlxA and vlxB messages accumulated to high levels, whereas vlxC and vlxD transcript levels were modest. Immunolocalization using peptide-specific antibodies demonstrated that under control conditions, VLXB was present in the cytosol of the paraveinal mesophyll and with pod removal accumulated additionally in the bundle-sheath and adjacent cells. With sink limitation VLXD accumulated to apparent high levels in the vacuoles of the same cells. Segregation of gene products at the cellular and subcellular levels may thus permit complex patterns of differential regulation within the same cell type. Specific lipoxygenase isoforms may have a role in short-term nitrogen storage (VLXC/D), whereas others may simultaneously function in assimilate partitioning as active enzymes (VLXA/B).

Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST–Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST–Z2 is able to condense 130–150 kb bacterial artificial chromosomes (BACs) into protein–DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein–BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR– cells by GST–Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST–Z2-mediated gene transfer. Because DNA condensation by GST–Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.

Involvement of p38 in Apoptosis-associated Membrane Blebbing and Nuclear Condensation

The stress-activated protein kinase p38 is often induced by cytotoxic agents, but its contribution to cell death is ill defined. In Rat-1 cells, we found a strong correlation between activation of p38 and induction of c-Myc–dependent apoptosis. In cells with deregulated c-Myc expression but not in control cells, cis-diamminedichloroplatinum induced p38 activity and typical features of apoptosis, including internucleosomal DNA degradation, induction of caspase activities, and both nuclear (nuclear condensation and fragmentation) and extranuclear (cell blebbing) morphological alterations. The pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone did not block p38 activation and the p38 inhibitor SB203580 had no detectable effect on the activation of caspases or the in vivo cleavage of several caspase substrates, suggesting that p38 and caspase activation can contribute distinct features of apoptosis. Accordingly, we found that cell blebbing was independent of caspase activity and, rather, depended on p38-sensitive changes in microfilament dynamics likely mediated by heat shock protein 27 phosphorylation. Furthermore, p38 activity contributed to both caspase-dependent and caspase-independent nuclear condensation and fragmentation, suggesting a role in an early event triggering both mechanisms of apoptosis or sensitizing the cells to the action of both types of apoptosis executioners. Inhibiting p38 also resulted in a significant enhancement in cell survival estimated by colony formation. This capacity to modulate the sensitivity to apoptosis in cells with deregulated c-Myc expression suggests an important role for p38 in tumor cell killing by chemotherapeutic agents.

Dressed polyions, counterion condensation, and adsorption excess in polyelectrolyte solutions.

The phenomenon of Manning-Oosawa counterion condensation is given an explicit statistical mechanical and qualitative basis via a dressed polyelectrolyte formalism in connection with the topology of the electrostatic free-energy surface and is derived explicitly in terms of the adsorption excess of ions about the polyion via the nonlinear Poisson-Boltzmann equation. The approach is closely analogous to the theory of ion binding in micelles. Our results not only elucidate a Poisson-Boltzmann analysis, which shows that a fraction of the counterions lie within a finite volume around the polyion even if the volume of the system tends towards infinity, but also provide a direct link between Manning's theta-the number of condensed counterions for each polyion site-and a statistical thermodynamic quantity, namely, the adsorption excess per monomer.

Premature translation of protamine 1 mRNA causes precocious nuclear condensation and arrests spermatid differentiation in mice.

Translational control is a major form of regulating gene expression during gametogenesis and early development in many organisms. We sought to determine whether the translational repression of the protamine 1 (Prm1) mRNA is necessary for normal spermatid differentiation in mice. To accomplish this we generated transgenic animals that carry a Prm1 transgene lacking its normal 3' untranslated region. Premature translation of Prm1 mRNA caused precocious condensation of spermatid nuclear DNA, abnormal head morphogenesis, and incomplete processing of Prm2 protein. Premature accumulation of Prm1 within syncytial spermatids in mice hemizygous for the transgene caused dominant male sterility, which in some cases was accompanied by a complete arrest in spermatid differentiation. These results demonstrate that correct temporal synthesis of Prm1 is necessary for the transition from nucleohistones to nucleoprotamines.