Timing Of Synthesis Research Articles

Hydrogen isotopes of n-alkanes and n-alkanoic acids as tracers of precipitation in a temperate forest and implications for paleorecords

The hydrogen isotopic composition of leaf waxes (δDwax) primarily reflects that of plant source water. Therefore, sedimentary δDwax records are increasingly used to reconstruct the δD of past precipitation (δDp) and to investigate paleohydrologic changes. Such reconstructions rely on estimates of apparent fractionation (εapp) between δDp and the resulting δDwax. However, εapp values are modified by numerous environmental and biological factors during leaf wax production. As a result, εapp can vary widely among plant species and growth forms. This complicates estimation of accurate εapp values and presents a central challenge to quantitative leaf wax paleohydrology. During the 2014 growing season, we examined εapp in the five deciduous angiosperm tree species (Prunus serotina, Acer saccharinum, Quercus rubra, Quercus alba, and Ulmus americana) that dominate the temperate forest at Brown’s Lake Bog, Ohio, USA. We sampled individuals of each species at weekly to monthly intervals from March to October and report δD values of n-C29 alkanes (δDn-C29 alkane) and n-C28 alkanoic acids (δDn-C28 acid), as well as xylem (δDxw) and leaf water (δDlw). n-Alkane synthesis was most intense 2–3weeks after leaf emergence and ceased thereafter, whereas n-alkanoic acid synthesis continued throughout the entire growing season. During bud swell and leaf emergence, δDlw was a primary control on δDn-C29 alkane and δDn-C28 acid values, which stabilized once leaves became fully expanded. Metabolic shifts between young and mature leaves may be an important secondary driver of δDwax changes during leaf development. In mature autumn leaves of all species, the mean εapp for n-C29 alkane (−107‰) was offset by approximately −19‰ from the mean εapp for n-C28 alkanoic acid (−88‰). These results indicate that in temperate settings n-alkanes and n-alkanoic acids from deciduous trees are distinct with respect to their abundance, timing of synthesis, and εapp values.

Low secondary leaf waxn-alkane synthesis on fully mature leaves of C3 grasses grown at controlled environmental conditions and variable humidity

Leaf wax n-alkanes are long-chained aliphatic compounds that are present in the cuticle of terrestrial plant leaves. Their δ2 H values are used for the reconstruction of past environments and for plant ecological investigations. The timing of n-alkane synthesis during leaf development and the rate of synthesis of secondary n-alkanes in fully matured leaves are still a matter of debate. Using a 2 H-labeling approach we estimated secondary leaf wax n-alkane synthesis rates in mature leaf blades of six C3 grass species grown in climate chambers under controlled environmental conditions. We found that mature grass leaves continue the synthesis of leaf wax n-alkanes after leaf maturation. The rate of secondary n-alkanes synthesis was, however, relatively low and varied in response to atmospheric humidity and among species from 0.09 to 1.09% per day. Our investigation provides new evidence on the timing of cuticular wax synthesis in grass leaves and indicates that the majority of n-alkanes are synthesized during the initial development of the leaf. Our study will improve the interpretation of leaf wax n-alkane δ2 H values in environmental and geological studies as it suggests that secondary synthesis of leaf wax n-alkanes in grass leaves contributes only slightly to the geological record. Copyright © 2016 John Wiley & Sons, Ltd.

Self-Regulation and Interplay of Rsm Family Proteins Modulate the Lifestyle of Pseudomonas putida.

In the plant-beneficial bacterium Pseudomonas putida KT2440, three genes have been identified that encode posttranscriptional regulators of the CsrA/RsmA family. Their regulatory roles in the motile and sessile lifestyles of P. putida have been investigated by generating single-, double-, and triple-null mutants and by overexpressing each protein (RsmA, RsmE, and RsmI) in different genetic backgrounds. The rsm triple mutant shows reduced swimming and swarming motilities and increased biofilm formation, whereas overexpression of RsmE or RsmI results in reduced bacterial attachment. However, biofilms formed on glass surfaces by the triple mutant are more labile than those of the wild-type strain and are easily detached from the surface, a phenomenon that is not observed on plastic surfaces. Analysis of the expression of adhesins and exopolysaccharides in the different genetic backgrounds suggests that the biofilm phenotypes are due to alterations in the composition of the extracellular matrix and in the timing of synthesis of its elements. We have also studied the expression patterns of Rsm proteins and obtained data that indicate the existence of autoregulation mechanisms. Proteins of the CsrA/RsmA family function as global regulators in different bacteria. More than one of these proteins is present in certain species. In this study, all of the RsmA homologs in P. putida are characterized and globally taken into account to investigate their roles in controlling bacterial lifestyles and the regulatory interactions among them. The results offer new perspectives on how biofilm formation is modulated in this environmentally relevant bacterium.

Phosphorylation of yeast glycerol 3‐phosphate acyltransferase Gpt2 regulates the timing of TAG synthesis and lipid droplet morphology

Glycerol‐3‐phosphate acyltransferases catalyze the acylation of glycerol‐3‐phosphate at the sn‐1 position yielding 1‐acyl glycerol‐3‐phosphate (lyso‐phosphatidic acid). This is the first and rate limiting reaction in de‐novo synthesis of phosphatidic acid (PA), the key intermediate for the formation of all glycerophospholipids and triacylglycerols. In all eukaryotic organisms glycerol‐3‐phosphate acyltransferases as well as acyltransferases catalyzing the subsequent acylation reaction converting lyso‐PA to PA occur in redundancy. This led to the hypothesis that isoenzymes are forming different PA pools which supply different pathways. In the model organism yeast Saccharomyces cerevisiae two glycerol‐3‐phosphate acyltransferases have been identified, Gpt2 (Gat1) and Sct1 (Gat2). Most recently we demonstrated the important role of Gpt2 (but not Sct1) in cells challenged by oleic acid as the only carbon source. Whereas growth in oleic acid containing medium induced massive lipid particle accumulation in wild‐type cells, cells lacking GPT2 failed to accumulate these storage compartments of triacylglycerols and other non‐polar lipids, exhibited a strong growth defect and cell viability was significantly reduced. We were intrigued by the fact that the intracellular distribution of Gpt2 and its phosphorylation status changed under these growth condition compared to growth on glucose containing medium. We have identified several phosphorylation sites in Gpt2 by mass spectrometry, which are localized to a serine rich region in the carboxy‐terminus of the protein. Serine to alanine or to aspartate mutations as well as deletion of the serine rich region containing these residues, were introduced in the yeast genome to investigate the role of phosphorylation in the regulation of Gpt2. Importantly, all phosphorylation mutants were able to support life of cells lacking Sct1, indicating these mutants are functional. We show that manipulations of Gpt2 phosphorylation status strongly and specifically affect TAG accumulation, but not metabolism of the most abundant glycerophospholipids during late log phase of growth. Importantly, alterations in the TAG content are accompanied by marked changes in lipid droplet morphology in the respective mutants. Altogether our results support a role for Gpt2 phosphorylation in regulating the timing of TAG synthesis and lipid droplet morphology.Support or Funding InformationThis work has been financially supported by the Natural Sciences and Engineering Research Council of Canada (to VZ and BS) and the Austrian Science Fund (FWF; Project P21251 to KA).

Roles of RNA-binding Proteins and Post-transcriptional Regulation in Driving Male Germ Cell Development in the Mouse.

Tissue development and homeostasis are dependent on highly regulated gene expression programs in which cell-specific combinations of regulatory factors determine which genes are expressed and the post-transcriptional fate of the resulting RNA transcripts. Post-transcriptional regulation of gene expression by RNA-binding proteins has critical roles in tissue development-allowing individual genes to generate multiple RNA and protein products, and the timing, location, and abundance of protein synthesis to be finely controlled. Extensive post-transcriptional regulation occurs during mammalian gametogenesis, including high levels of alternative mRNA expression, stage-specific expression of mRNA variants, broad translational repression, and stage-specific activation of mRNA translation. In this chapter, an overview of the roles of RNA-binding proteins and the importance of post-transcriptional regulation in male germ cell development in the mouse is presented.

Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells.

The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell behavior. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence the allocation of cells to muscle, fat, cartilage, and bone, the mechanisms that regulate the Bmp2 gene are crucial. Key early mesodermal events that require precise Bmp2 regulation include heart specification and morphogenesis. Originally named for its osteoinductive properties, healing fractures requires BMP2. The human Bmp2 gene also has been linked to osteoporosis and osteoarthritis. In addition, all forms of pathological calcification in the vasculature and in cardiac valves involve the pro-osteogenic BMP2. The diverse tissues, mechanisms, and diseases influenced by BMP2 are too numerous to list here (see OMIM: 112261). However, in all BMP2-influenced pathologies, changes in the behavior and differentiation of pluripotent mesenchymal cells are a recurring theme. Consequently, much effort has been devoted to identifying the molecules and conditions that influence BMP2 synthesis and the complex mechanisms that control Bmp2 gene expression. This review begins with an overview of the Bmp2 gene's chromosomal neighborhood and then summarizes and evaluates known regulatory mechanisms and inducers.

Influence of salinity on hydrogen isotope fractionation in Rhizophora mangroves from Micronesia

Hydrogen isotope ratios (2H/1H or δ2H) of plant leaf waxes typically covary with those of precipitation, and are therefore used as a proxy for past hydrologic variability. Mangroves present an important exception to this relationship, as salinity can strongly influence 2H fractionation in leaf lipids. To better understand and calibrate this effect, δ2H values of taraxerol and n-alkanes were measured in the leaves of Rhizophora spp. (red mangroves) from three estuaries and four brackish lakes on the Micronesian islands of Pohnpei and Palau, and compared to the δ2H and δ18O values of leaf water, xylem water and surface water. Net 2H discrimination between surface water and taraxerol increased by 0.9±0.2‰ per part per thousand (ppt−1) over a salinity range of 1–34ppt. Xylem water was always depleted in 2H relative to surface water, and the magnitude of this depletion increased with salinity, which is most likely due to a combination of greater 2H discrimination by roots during water uptake and opportunistic use of freshwater. Changes in the 2H content of xylem water can account for up to 43% of the change in net taraxerol fractionation with salinity. Leaf water isotopes were minimally enriched relative to xylem water and there was not significant variability in leaf water enrichment with salinity, which is consistent with a Péclet-modified Craig–Gordon model of leaf water enrichment. As leaf water enrichment is therefore unlikely to be responsible for increased 2H/1H fractionation in mangrove leaf lipids at elevated salinities, the majority of this signal is most likely explained either by changes in biosynthetic fractionation in response to salt stress or by salinity influenced changes in the timing of water uptake and lipid synthesis.

Stella preserves maternal chromosome integrity by inhibiting 5hmC-induced γH2AX accumulation.

In the mouse zygote, Stella/PGC7 protects 5-methylcytosine (5mC) of the maternal genome from Tet3-mediated oxidation to 5-hydroxymethylcytosine (5hmC). Although ablation of Stella causes early embryonic lethality, the underlying molecular mechanisms remain unknown. In this study, we report impaired DNA replication and abnormal chromosome segregation (ACS) of maternal chromosomes in Stella-null embryos. In addition, phosphorylation of H2AX (γH2AX), which has been reported to inhibit DNA replication, accumulates in the maternal chromatin of Stella-null zygotes in a Tet3-dependent manner. Cell culture assays verified that ectopic appearance of 5hmC induces abnormal accumulation of γH2AX and subsequent growth retardation. Thus, Stella protects maternal chromosomes from aberrant epigenetic modifications to ensure early embryogenesis.

Seasonal variation of leaf wax n-alkane production and δ2H values from the evergreen oak tree, Quercus agrifolia

In order to understand the timing of leaf wax synthesis in higher plants, we analysed the variability in leaf wax n-alkane concentration, composition (expressed as average chain length (ACL)), and δ2Hwax values as well as plant source water δ2H values (xylem and leaf water) in the evergreen tree Quercus agrifolia over a period of 9 months, beginning with leaf flush. We identified three distinct periods of leaf development with the first month following leaf flush being characterized by de novo synthesis and possibly removal of n-alkanes. During the following 3 months, n-alkane concentrations increased sevenfold and δ2Hwax and ACL values increased, suggesting this period was the major leaf wax n-alkane formation period. During the remaining 4 months of the experiment, stable values suggest cessation of leaf wax n-alkane formation. We find that n-alkane synthesis in Q. agrifolia takes place over 4 months, substantially longer than that observed for deciduous trees.

Effect of aridity on δ13C and δD values of C3 plant- and C4 graminoid-derived leaf wax lipids from soils along an environmental gradient in Cameroon (Western Central Africa)

The observation that the hydrogen isotope composition (δD) of leaf wax lipids is determined mainly by precipitation δD values, has resulted in the application of these biomarkers to reconstruct paleoclimate from geological records. However, because the δD values of leaf wax lipids are additionally affected by vegetation type and ecosystem evapotranspiration, paleoclimatic reconstruction remains at best semi-quantitative. Here, we used published results for the carbon isotope composition (δ13C) of n-alkanes in common plants along a latitudinal gradient in C3/C4 vegetation and relative humidity in Cameroon and demonstrated that pentacyclic triterpene methyl ethers (PTMEs) and n-C29 and n-C31 in the same soil, derived mainly from C4 graminoids (e.g. grass) and C3 plants (e.g. trees and shrubs), respectively. We found that the δD values of soil n-C27, n-C29 and n-C31, and PTMEs correlated significantly with surface water δD values, supporting previous observations that leaf wax lipid δD values are an effective proxy for reconstructing precipitation δD values even if plant types changed significantly. The apparent fractionation (εapp) between leaf wax lipid and precipitation δD values remained relatively constant for C3-derived long chain n-alkanes, whereas εapp of C4-derived PTMEs decreased by 20‰ along the latitudinal gradient encompassing a relative humidity range from 80% to 45%. Our results indicate that PTME δD values derived from C4 graminoids may be a more reliable paleo-ecohydrological proxy for ecosystem evapotranspiration within tropical and sub-tropical Africa than n-alkane δD values, the latter being a better proxy for surface water δD values. We suggest that vegetation changes associated with different plant water sources and/or difference in timing of leaf wax synthesis between C3 trees of the transitional class and C3 shrubs of the savanna resulted in a D depletion in soil long chain n-alkanes, thereby counteracting the effect of evapotranspiration D enrichment along the gradient. In contrast, evaporative D enrichment of leaf and soil water was significant enough to be recorded in the δD values of PTMEs derived from C4 graminoids, likely because PTMEs recorded the hydrogen isotopic composition of the same vegetation type.

Ecdysis triggering hormone ensures proper timing of juvenile hormone biosynthesis in pharate adult mosquitoes

Juvenile hormones (JHs) are synthesized by the corpora allata (CA) and play a key role in insect development. A decrease of JH titer in the last instar larvae allows pupation and metamorphosis to proceed. As the anti-metamorphic role of JH comes to an end, the CA of the late pupa (or pharate adult) becomes again “competent” to synthesize JH, which would play an essential role orchestrating reproductive maturation. In the present study, we provide evidence that ecdysis triggering hormone (ETH), a key endocrine factor involved in ecdysis control, acts as an allatotropic regulator of JH biosynthesis, controlling the exact timing of CA activation in the pharate adult mosquito. Analysis of the expression of Aedes aegypti ETH receptors (AeaETHRs) revealed that they are present in the CA and the corpora cardiaca (CC), and their expression peaks 4 h before eclosion. In vitro stimulation of the pupal CA glands with ETH resulted in an increase in JH synthesis. Consistent with this finding, silencing AeaETHRs by RNA interference (RNAi) in pupa resulted in reduced JH synthesis by the CA of one day-old adult females. Stimulation with ETH resulted in increases in the activity of juvenile hormone acid methyltransferase (JHAMT), a key JH biosynthetic enzyme. Furthermore, inhibition of IP3R-operated mobilization of endoplasmic reticulum Ca2+ stores prevented the ETH-dependent increases of JH biosynthesis and JHAMT activity. All together these findings provide compelling evidence that ETH acts as a regulatory peptide that ensures proper developmental timing of JH synthesis in pharate adult mosquitoes.

RNA Granule Assembly and Disassembly Modulated by Nuclear Factor Associated with Double-stranded RNA 2 and Nuclear Factor 45

RNA granules are large messenger ribonucleoprotein complexes that regulate translation and mRNA translocation to control the timing and location of protein synthesis. The regulation of RNA granule assembly and disassembly is a structural basis of translational control, and its disorder is implicated in degenerative disease. Here, we used proteomic analysis to identify proteins associated with RNA granule protein 105 (RNG105)/caprin1, an RNA-binding protein in RNA granules. Among the identified proteins, we focused on nuclear factor (NF) 45 and its binding partner, nuclear factor associated with dsRNA 2 (NFAR2), and we demonstrated that NF45 promotes disassembly of RNA granules, whereas NFAR2 enhances the assembly of RNA granules in cultured cells. The GQSY domain of NFAR2 was required to associate with messenger ribonucleoprotein complexes containing RNG105/caprin1, and it was structurally and functionally related to the low complexity sequence domain of the fused in sarcoma protein, which drives the assembly of RNA granules. Another domain of NFAR2, the DZF domain, was dispensable for association with the RNG105 complex, but it was involved in positive and negative regulation of RNA granule assembly by being phosphorylated at double-stranded RNA-activated kinase sites and by association with NF45, respectively. These results suggest a novel molecular mechanism for the modulation of RNA granule assembly and disassembly by NFAR2, NF45, and phosphorylation at double-stranded RNA-activated kinase PKR sites.

Coordination of wing and whole-body development at developmental milestones ensures robustness against environmental and physiological perturbations.

Development produces correctly patterned tissues under a wide range of conditions that alter the rate of development in the whole body. We propose two hypotheses through which tissue patterning could be coordinated with whole-body development to generate this robustness. Our first hypothesis states that tissue patterning is tightly coordinated with whole-body development over time. The second hypothesis is that tissue patterning aligns at developmental milestones. To distinguish between our two hypotheses, we developed a staging scheme for the wing imaginal discs of Drosophila larvae using the expression of canonical patterning genes, linking our scheme to three whole-body developmental events: moulting, larval wandering and pupariation. We used our scheme to explore how the progression of pattern changes when developmental time is altered either by changing temperature or by altering the timing of hormone synthesis that drives developmental progression. We found the expression pattern in the wing disc always aligned at moulting and pupariation, indicating that these key developmental events represent milestones. Between these milestones, the progression of pattern showed greater variability in response to changes in temperature and alterations in physiology. Furthermore, our data showed that discs from wandering larvae showed greater variability in patterning stage. Thus for wing disc patterning, wandering does not appear to be a developmental milestone. Our findings reveal that tissue patterning remains robust against environmental and physiological perturbations by aligning at developmental milestones. Furthermore, our work provides an important glimpse into how the development of individual tissues is coordinated with the body as a whole.

Cell Cycle and Nucleomorph Division in Pyrenomonas helgolandii (Cryptophyta)

The cells of cryptophycean and chlorarachniophycean algae contain a nucleomorph, a vestigial nucleus derived from red and green algal endosymbionts respectively. The origin of the nucleomorph is therefore different from that of cellular organelles such as mitochondria and chloroplasts. In this study, we sought to determine whether cell cycle regulation of the nucleomorph in the cryptophycean alga Pyrenomonas helgolandii is functionally similar to that of the cell nucleus. We performed an ultrastructural analysis of nucleomorph division in cells prepared by rapid freezing fixation - freeze substitution and also carried out BrdU labeling experiments to determine the timing of nucleomorph DNA synthesis in relation to that of the cell nucleus. In cells cultured under 16 hours light: 8 hours dark conditions, BrdU labeling experiments showed that DNA synthesis in the nucleomorph occurred during a limited period from 2 hr to 4 hr after the beginning of the dark period. The S phase in the nucleomorph started just after completion of the nuclear S phase. Thus, DNA synthesis in the nucleomorph occurred at a defined period of the cell cycle. By contrast, our BrdU experiments showed that the nucleoids of mitochondria and chloroplasts could perform DNA synthesis throughout the whole cell cycle.

Periodic mRNA synthesis and degradation co‐operate during cell cycle gene expression

During the cell cycle, the levels of hundreds of mRNAs change in a periodic manner, but how this is achieved by alterations in the rates of mRNA synthesis and degradation has not been studied systematically. Here, we used metabolic RNA labeling and comparative dynamic transcriptome analysis (cDTA) to derive mRNA synthesis and degradation rates every 5min during three cell cycle periods of the yeast Saccharomyces cerevisiae. A novel statistical model identified 479 genes that show periodic changes in mRNA synthesis and generally also periodic changes in their mRNA degradation rates. Peaks of mRNA degradation generally follow peaks of mRNA synthesis, resulting in sharp and high peaks of mRNA levels at defined times during the cell cycle. Whereas the timing of mRNA synthesis is set by upstream DNA motifs and their associated transcription factors (TFs), the synthesis rate of a periodically expressed gene is apparently set by its core promoter.

36 EFFECT OF ACTIVATION METHODS ON DNA SYNTHESIS AND DEVELOPMENT OF CANINE PARTHENOGENETIC EMBRYOS

Artificial activation is an important step for successful somatic cell nuclear transfer. In mammals, different methods of parthenogenesis have been studied to increase the developmental efficiency of cloned embryos. In an attempt to improve the techniques of nuclear transfer in canine species, this study investigated the timing of DNA synthesis and in vivo development of canine parthenotes produced by different activation treatments. For canine parthenotes, in vivo matured oocytes were obtained by flushing (~72 h after ovulation) the oviducts of mixed breed bitches. Denuded oocytes were cultured for 4 min in 10 μM calcium ionophore, and then they were divided into 2 groups: (1) 2DMAP group was cultured for 2 h in 6-DMAP; (2) 4DMAP group was cultured for 4 h in 6-DMAP. The first experiment determined DNA synthesis of parthenotes by 1 h treatment with incorporation and immunofluorescent detection of thymidine analogue 5-bromo-2′-deoxyuridine-5′-triphosphate (BrdU). The primary antibody was mouse anti-BrdU (Sigma, St. Louis, MO, USA), and the secondary antibody was fluorescein (FITC)-conjugated affinity purified goat anti-mouse IgG (Jackson). In order to examine the pronuclear formation and the onset of DNA synthesis in experimental groups, parthenotes derived from 2DMAP and 4DMAP groups were observed by BrdU incorporation at 2, 4, and 12 h post-activation (hpa). Data were analysed using Graph Prism software (GraphPad, San Diego, CA, USa). The next experiment observed in vivo development as follows: parthenotes were surgically transferred to synchronized recipient female dogs. The implantation rate of parthenogenetic fetuses was observed in uterus of recipients on Day 26 of pregnancy. All parthenotes of the 2DMAP group showed BrdU incorporation at 2 hpa, whereas 4DMAP parthenotes showed 96% BrdU incorporation at 2 hpa. Incorporation of BrdU was detected in all parthenotes of both experimental groups after 4 hpa. A total of 98 parthenotes were transferred to 9 surrogate mothers (53 parthenotes into 5 recipients in 2DMAP group and 45 parthenotes into 4 recipients in 4DMAP group). There was no significant difference in pregnancy rate between the 2 groups (2DMAP: 60% v. 4DMAP: 50%), whereas the implantation rates were significantly higher in 2DMAP (24.5%) compared with 4DMAP (4.4%; P < 0.001). The recovered parthenotes were able to develop to the stages of limb-bud formation, but few parthenotes showed the small and degenerating formation. Regardless of treatment group, the implantation site of the fetuses indicated either one side or both of the uterus. In conclusion, the present results demonstrated that the protocols using combined treatment with 10 μM of calcium ionophore (4 min) followed by a 2-h culture with 1.9 mM of DMAP resulted in completing pronuclear formation and enhancing fetal formation. This result could be useful method for improving canine cloned embryos production. This study was supported by IPET (#311062–04–2-SB010), RNL Bio (#550–20130013), RDA (PJ008975022013), and Research Institute for Veterinary Science and Natural Balance.

Leishmania amazonensis Promastigotes Present Two Distinct Modes of Nucleus and Kinetoplast Segregation during Cell Cycle

Here, we show the morphological events associated with organelle segregation and their timing in the cell cycle of a reference strain of Leishmania (L.) amazonensis promastigotes, the main causative agent of Tegumentary leishmaniasis in the Americas. We show evidences that during the cell cycle, L. amazonensis promastigotes present two distinct modes of nucleus and kinetoplast segregation, which occur in different temporal order in different proportions of cells. We used DAPI-staining and EdU-labeling to monitor the segregation of DNA-containing organelles and DNA replication in wild-type parasites. The emergence of a new flagellum was observed using a specific monoclonal antibody. The results show that L. amazonensis cell cycle division is peculiar, with 65% of the dividing cells duplicating the kinetoplast before the nucleus, and the remaining 35% doing the opposite or duplicating both organelles concomitantly. In both cases, the new flagellum appeared during S to G2 phase in 1N1K cells and thus before the segregation of both DNA-containing organelles; however, we could not determine the exact timing of flagellar synthesis. Most of these results were confirmed by the synchronization of parasites using hydroxyurea. Altogether, our data show that during the cell cycle of L. amazonensis promastigotes, similarly to L. donovani, the segregation of nucleus and kinetoplast do not follow a specific order, especially when compared to other trypanosomatids, reinforcing the idea that this characteristic seems to be species-specific and may represent differences in cellular biology among members of the Leishmania genus.

The D10 Decapping Enzyme of Vaccinia Virus Contributes to Decay of Cellular and Viral mRNAs and to Virulence in Mice

Posttranscriptional mechanisms are important for regulation of cellular and viral gene expression. The presence of the 5' cap structure m(7)G(5')ppp(5')Nm is a general feature of mRNAs that provides protection from exoribonuclease digestion and enhances translation. Vaccinia virus and other poxviruses encode enzymes for both cap synthesis and decapping. Decapping is mediated by two related enzymes, D9 and D10, which are synthesized before and after viral DNA replication, respectively. The timing of D10 synthesis correlates better with the shutdown of host gene expression, and deletion of this gene has been shown to cause persistence of host and viral mRNAs in infected cells. Here, we constructed specific mutant viruses in which translation of D10 was prevented by stop codons or activity of D10 was abrogated by catalytic site mutations, without other genomic alterations. Both mutants formed plaques of normal size and replicated to similar extents as the parental virus in monkey epithelial cells and mouse embryonic fibroblasts. The synthesis of viral proteins was slightly delayed, and cellular and viral mRNAs persisted longer in cells infected with the mutants compared to either the parental virus or clonal revertant. Despite the mild effects in vitro, both mutants were more attenuated than the revertants in intranasal and intraperitoneal mouse models, and less infectious virus was recovered from organs. In addition, there was less lung histopathology following intranasal infection with mutant viruses. These data suggest that the D10 decapping enzyme may help restrict antiviral responses by accelerating host mRNA degradation during poxvirus infection.

RNA synthetic mechanisms employed by diverse families of RNA viruses

RNA viruses are ubiquitous in nature, infecting every known organism on the planet. These viruses can also be notorious human pathogens with significant medical and economic burdens. Central to the lifecycle of an RNA virus is the synthesis of new RNA molecules, a process that is mediated by specialized virally encoded enzymes called RNA‐dependent RNA polymerases (RdRps). RdRps directly catalyze phosphodiester bond formation between nucleoside triphosphates in an RNA‐templated manner. These enzymes are strikingly conserved in their structural and functional features, even among diverse RNA viruses belonging to different families. During host cell infection, the activities of viral RdRps are often regulated by viral cofactor proteins. Cofactors can modulate the type and timing of RNA synthesis by directly engaging the RdRp and/or by indirectly affecting its capacity to recognize template RNA. High‐resolution structures of RdRps as apoenzymes, bound to RNA templates, in the midst of catalysis, and/or interacting with regulatory cofactor proteins, have dramatically increased our understanding of viral RNA synthetic mechanisms. Combined with elegant biochemical studies, such structures are providing a scientific platform for the rational design of antiviral agents aimed at preventing and treating RNA virus‐induced diseases. WIREs RNA 2013, 4:351–367. doi: 10.1002/wrna.1164This article is categorized under: 1RNA Interactions with Proteins and Other Molecules > RNA–Protein Complexes2RNA in Disease and Development > RNA in Disease

Chromosome architecture is a key element of bacterial cellular organization

The bacterial chromosome encodes information at multiple levels. Beyond direct protein coding, genomes encode regulatory information required to orchestrate the proper timing and levels of gene expression and protein synthesis, and contain binding sites and regulatory sequences to co-ordinate the activities of proteins involved in chromosome repair and maintenance. In addition, it is becoming increasingly clear that yet another level of information is encoded by the bacterial chromosome - the three-dimensional packaging of the chromosomal DNA molecule itself and its positioning relative to the cell. This vast structural blueprint of specific positional information is manifested in various ways, directing chromosome compaction, accessibility, attachment to the cell envelope, supercoiling, and general architecture and arrangement of the chromosome relative to the cell body. Recent studies have begun to identify and characterize novel systems that utilize the three-dimensional spatial information encoded by chromosomal architecture to co-ordinate and direct fundamental cellular processes within the cytoplasm, providing large-scale order within the complex clutter of the cytoplasmic compartment.