A directed nucleotide-sequencing approach for single-stranded vectors based on recloning intermediates of a progressive DNA synthesis reaction

A simple method for site-directed nucleotide sequencing is presented that uses a novel procedure for generating nested 'deletions' within inserts of single-stranded clones. In this method, single-stranded template, sequencing primer, and the Klenow fragment of Escherichia coli DNA polymerase I are used to initiate progressive DNA synthesis of the entire insert of the clone. By time-dependent sampling and pooling of intermediates from the synthesis reaction a series of nested double-stranded DNA subfragments of the insert can be created. Nested subclones are then produced by S1-endonuclease treatment and oriented subcloning methods. First, smaller quantities of template DNA can be used, equivalent to a fraction of a small DNA sequencing prep. Second, it works with single-stranded M13 phage DNA rather than requiring the preparation of double-stranded replicative form DNA as in ExoIII-based methods. Third, the 'deletions' it generates can span areas of simple nucleotide sequence or secondary structure that often halt digestion in the single-stranded exonuclease-based method. Last, the method is adaptable to a larger variety of insert cloning sites than the ExoIII-based method. The main disadvantage of the method is that, due to the lower efficiency of subcloning larger DNA fragments, subclone inserts larger than 3 kb are generated only infrequently.     

The interaction between mobile DNAs and their hosts in a fluctuating environment

The interaction between mobile DNA sequences and their hosts raises important questions in the context of hosts which reproduce clonally with only rare horizontal transmission between clones. The activity of some mobile DNAs as reversible mutators of genes raises the possibility that, in a fluctuating environment, cells may gain an advantage if they have mobile DNAs which mutate genes whose inactivation is favoured in one of the environments that the population encounters. Here we analyse a model of this process and ask what would be the optimal rate of transposition in a population whose elements are maintained by this mechanism. We also examine the impact of horizontal transfer on such a population. With movement of elements between cells, we can imagine elements with differing rates of transposition and host cells with differing rates of transposition. We find that evolution in the population of elements favours a rapid rate of transposition, and evolution of the host cells favours cells in which this rapid rate of element-dependent transposition results in an optimal rate of transposition per cell. However, when horizontal transfer rates are high, some unexpected features of the model are observed. In particular, a polymorphism between cell types (some with an optimal rate of transposition and some with no transposition at all from endogenous elements) can be stably maintained. We consider the possible biological predictions of this analysis.     

Distribution of unique red feather pigments in parrots

In many birds, red, orange and yellow feathers are coloured by carotenoid pigments, but parrots are an exception. For over a century, biochemists have known that parrots use an unusual set of pigments to produce their rainbow of plumage colours, but their biochemical identity has remained elusive until recently. Here, we use high-performance liquid chromatography to survey the pigments present in the red feathers of 44 species of parrots representing each of the three psittaciform families. We found that all species used the same suite of five polyenal lipochromes (or psittacofulvins) to colour their plumage red, indicating that this unique system of pigmentation is remarkably conserved evolutionarily in parrots. Species with redder feathers had higher concentrations of psittacofulvins in their plumage, but neither feather colouration nor historical relatedness predicted the ratios in which the different pigments appeared. These polyenes were absent from blood at the time when birds were replacing their colourful feathers, suggesting that parrots do not acquire red plumage pigments from the diet, but instead manufacture them endogenously at growing feathers.     

Dietary variation and food hardness in sooty mangabeys (Cercocebus atys): Implications for fallback foods and dental adaptation

We present information on food hardness and monthly dietary changes in female sooty mangabeys (Cercocebus atys) in Tai Forest, Ivory Coast to reassess the hypothesis that thick molar enamel is parsimoniously interpreted as a response to consumption of hard foods during fallback periods. We demonstrate that the diet of sooty mangabeys varies seasonally, but that one food—Sacoglottis gabonensis—is the most frequently consumed food every month and year round. This food is the hardest item in the sooty diet. Given that this species has among the thickest enamel within the primate order, a plausible conclusion is that thick enamel in this taxon evolved not in response to seasonally critical function or fallback foods, but rather to the habitual, year round processing of a mechanically protected foodstuff. These data serve as a caution against de rigueur interpretations that reliance on fallback foods during lean periods primarily explains the evolution of thick enamel in primates. Am J Phys Anthropol 154:413–423, 2014. © 2014 Wiley Periodicals, Inc.     

Reciprocal Regulation of Endocytosis and Metabolism

The cellular uptake of many nutrients and micronutrients governs both their cellular availability and their systemic homeostasis. The cellular rate of nutrient or ion uptake (e.g., glucose, Fe³⁺, K⁺) or efflux (e.g., Na⁺) is governed by a complement of membrane transporters and receptors that show dynamic localization at both the plasma membrane and defined intracellular membrane compartments. Regulation of the rate and mechanism of endocytosis controls the amounts of these proteins on the cell surface, which in many cases determines nutrient uptake or secretion. Moreover, the metabolic action of diverse hormones is initiated upon binding to surface receptors that then undergo regulated endocytosis and show distinct signaling patterns once internalized. Here, we examine how the endocytosis of nutrient transporters and carriers as well as signaling receptors governs cellular metabolism and thereby systemic (whole-body) metabolite homeostasis.Interactions between the cell and its environment obligatorily involve events at the plasma membrane. Cell-surface proteins mediate nutrient uptake, product release, and the sensing of environmental changes, including signals from other cells. Appropriate sensing and response to extracellular cues is essential for the individual cell’s survival and for the coordinated cellular behavior in multicellular organisms. Accordingly, maintenance and dynamics of membrane proteins are fundamental mechanisms of cellular homeostasis and survival.Most plasma membrane proteins are in defined equilibria with intracellular endosomal compartments, such that the amount of a given protein at the plasma membrane is determined by the balance of its endocytosis and its recycling back to the cell surface from endosomes and other intracellular compartments (Fig. 1). Changes in the kinetics of membrane protein traffic acutely affect the levels of individual proteins at the cell surface and thereby impact how cells intake nutrients, sense the environment, and respond to external cues.Open in a separate windowFigure 1.Dynamic regulation of the cell-surface content of membrane proteins. Integral membrane proteins found at the cell surface are dynamically localized to the plasma membrane. The amount of any of these proteins at the cell surface is the result of the balance of exocytosis or recycling of vesicles containing that protein from intracellular membrane compartments and the endocytosis of the protein from the cell surface. Regulation of either the rate of exocytosis or endocytosis results in alteration of the cell-surface content of a given protein.Selective molecular mechanisms trigger traffic of plasma membrane proteins through endomembranes. Among them, ubiquitination and phosphorylation stand out as they can directly target the cargo proteins. Ubiquitination is the covalent attachment of the 76-amino acid polypeptide ubiquitin to the ε-amino group of specific lysine residues (reviewed by Miranda and Sorkin 2007; and see also Piper et al. 2014). Ubiquitination of cell-surface proteins is the principal mechanism of control of endocytosis in yeast (MacGurn et al. 2012), whereas in mammals, additional molecular mechanisms regulate the endocytosis of cell-surface proteins, including alterations in conformation that impact interaction with other proteins, and as mentioned, phosphorylation. Each of these modifications can either enhance or reduce the rates internalization, recycling, or degradation of specific proteins, highlighting the complexity of the regulation of endomembrane traffic. The intricate mechanisms that underlie the reciprocal regulation of endocytosis and metabolism are beginning to be understood. Here we discuss the endocytosis mechanisms in the regulation of cellular intake or efflux of iron, cholesterol, Na⁺, and glucose, and in the regulation of receptor signaling relevant to metabolism.     

Spatial analysis of Listronotus maculicollis immature stages demonstrates strong associations with conspecifics and turfgrass damage but not with optimal hosts on golf course fairways

For insects that develop on few hosts and/or have immobile immature stages, optimal oviposition theory suggests that females should seek high‐quality hosts that maximize larval development and reduce competition from conspecifics. However, there is a growing amount of evidence that suggests female choice may often be at odds with their offspring's development. Listronotus maculicollis (Kirby) (Coleoptera: Curculionidae) is a serious pest of golf course turfgrass in eastern North America. The weevil develops on few hosts and demonstrates improved fitness traits when developing on Poa annua L. (Poaceae). However, previous population studies observed either weak or no correlations between the spatial dispersion of larval populations and P. annua in the field. In this study, populations on three golf course fairways were monitored over a 4‐year period (2009–2012) to determine whether the lack of spatial associations between preferred hosts and immatures was a result of spatial scale or the density and distribution of conspecifics. Spatial Analysis by Distance IndicEs (SADIE) was used to characterize the spatial dispersion of populations of individual stages (larvae and pupae), P. annua, and turfgrass damage. Life stages were aggregated in each observation, independent of population density or the spatial dispersion of hosts. The distribution of consecutive and non‐consecutive immature stages was found to be correlated in all years, suggesting that females do not avoid patches already occupied by conspecific eggs. Surprisingly, significant spatial associations were not found between larvae and P. annua when the host plant was relatively abundant. Hence, multiple mechanisms may drive L. maculicollis oviposition site‐selection behavior, and a flexible strategy may allow the weevil to persist in areas where P. annua is not the dominant species. Future studies are required to determine what other factors (e.g., natural enemy‐free space, egg or time limitations) influence oviposition behavior.     

Anterograde Spread of Herpes Simplex Virus Type 1 Requires Glycoprotein E and Glycoprotein I but Not Us9

Anterograde neuronal spread (i.e., spread from the neuron cell body toward the axon terminus) is a critical component of the alphaherpesvirus life cycle. Three viral proteins, gE, gI, and Us9, have been implicated in alphaherpesvirus anterograde spread in several animal models and neuron culture systems. We sought to better define the roles of gE, gI, and Us9 in herpes simplex virus type 1 (HSV-1) anterograde spread using a compartmentalized primary neuron culture system. We found that no anterograde spread occurred in the absence of gE or gI, indicating that these proteins are essential for HSV-1 anterograde spread. However, we did detect anterograde spread in the absence of Us9 using two independent Us9-deleted viruses. We confirmed the Us9 finding in different murine models of neuronal spread. We examined viral transport into the optic nerve and spread to the brain after retinal infection; the production of zosteriform disease after flank inoculation; and viral spread to the spinal cord after flank inoculation. In all models, anterograde spread occurred in the absence of Us9, although in some cases at reduced levels. This finding contrasts with gE- and gI-deleted viruses, which displayed no anterograde spread in any animal model. Thus, gE and gI are essential for HSV-1 anterograde spread, while Us9 is dispensable.Alphaherpesviruses are parasites of the peripheral nervous system. In their natural hosts, alphaherpesviruses establish lifelong persistent infections in sensory ganglia and periodically return by axonal transport to the periphery, where they cause recurrent disease. This life cycle requires viral transport along axons in two directions. Axonal transport in the retrograde direction (toward the neuron cell body) occurs during neuroinvasion and is required for the establishment of latency, while transport in the anterograde direction (away from the neuron cell body) occurs after reactivation and is required for viral spread to the periphery to cause recurrent disease. In addition to anterograde and retrograde axonal transport within neurons, alphaherpesviruses spread between synaptically connected neurons and between neurons and epithelial cells at the periphery (19, 22).The alphaherpesvirus subfamily includes the human pathogens herpes simplex virus type 1 (HSV-1), HSV-2, and varicella-zoster virus (VZV), as well as numerous veterinary pathogens such as pseudorabies virus (PRV) and bovine herpesviruses 1 and 5 (BHV-1 and BHV-5). The molecular mechanisms that mediate alphaherpesvirus anterograde axonal transport, anterograde spread, and cell-to-cell spread remain unclear. However, many studies of several alphaherpesviruses have indicated that anterograde transport or anterograde spread involves the viral proteins glycoprotein E (gE), glycoprotein I (gI), and Us9 (2, 5, 7, 9, 11, 13, 16, 26, 30, 31, 41, 46, 51, 52).Glycoproteins E and I are type I membrane proteins that form a heterodimer in the virion membrane and on the surface of infected cells. Although dispensable for the entry of extracellular virus, gE and gI mediate the epithelial cell-to-cell spread of numerous alphaherpesviruses (1, 3, 15, 20, 34, 38, 49, 53, 54). Us9 is a type II nonglycosylated membrane protein with no described biological activity apart from its role in neuronal transport (4, 18, 32). Here, we used several model systems to better characterize the roles of gE, gI, and Us9 in HSV-1 neuronal spread.Animal models to assess alphaherpesvirus neuronal transport (viral movement within a neuron) and spread (viral movement between cells) include the mouse flank and mouse retina models of infection. In the mouse flank model (Fig. ​(Fig.1A),1A), virus is scratch inoculated onto the depilated flank, where it infects the skin and spreads to innervating sensory neurons. The virus travels to the dorsal root ganglia (DRG) in the spinal cord (retrograde direction) and then returns to an entire dermatome of skin (anterograde spread). The virus also is transported in an anterograde direction from the DRG to the dorsal horn of the spinal cord and subsequently spreads to synaptically connected neurons. The production of zosteriform lesions and the presence of viral antigens in the dorsal horn of the spinal cord both are indicators of anterograde spread in this system. PRV gE and Us9 are required for the production of zosteriform disease, while gI is dispensable (7). In the absence of gE, HSV-1 also fails to cause zosteriform disease. However, unlike PRV, HSV-1 gE is required for retrograde spread to the DRG, so the role of gE in HSV-1 anterograde spread could not be evaluated in the mouse flank model (8, 36, 42).Open in a separate windowFIG. 1.Model systems to study HSV-1 neuronal spread. (A) Mouse flank model. Virus was scratch inoculated onto the skin, where it replicates, spreads to innervating neurons, and travels in a retrograde direction to the neuron cell body in the DRG. After replicating in the DRG, the virus travels in an anterograde direction back to the skin and into the dorsal horn of the spinal cord. Motor neurons also innervate the skin, allowing virus to reach the ventral horn of the spinal cord by retrograde transport. (B) Mouse retina model. Virus is injected into the vitreous body, from which it infects the retina as well as other structures of the eye, including the ciliary body, iris, and skeletal muscles of the orbit. From the retina, the virus is transported into the optic nerve and optic tract (OT) (anterograde direction) and then to the brain along visual pathways. Anterograde spread is detected in the lateral geniculate nucleus (LGN) and superior colliculus (SC). From the infected ciliary body, iris, and skeletal muscle, the virus spreads in a retrograde direction along motor and parasympathetic neurons and is detected in the oculomotor and Edinger-Westphal nuclei (OMN/EWN). Only first-order sites of spread to the brain are indicated. (Brain images were modified and reproduced from reference 47 with permission from of the publisher. Copyright Elsevier 1992.) (C) Campenot chamber system. Campenot chambers consist of a Teflon ring that divides the culture into three separate compartments. Neurons are seeded into the S chamber and extend their axons into the M and N chambers. Vero cells are seeded into the N chamber 1 day before infection. Virus is added to the S chamber and detected in the N chamber, a measure of anterograde spread.The mouse retina infection model (Fig. ​(Fig.1B)1B) has the advantage of allowing anterograde and retrograde spread to be studied independently of one another. Virus is delivered to the vitreous body, from which it infects the retina and other structures of the eye. The cell bodies of retinal neurons form the innermost layer of the retina; therefore, the virus infects these neurons directly, and spread from the retina along visual pathways to the brain occurs in an exclusively anterograde direction. In addition, the virus infects the anterior uveal layer of the eye (ciliary body and iris) and skeletal muscles in the orbit. From these tissues, the virus infects innervating parasympathetic and motor neurons and spreads to the brain in a retrograde direction. The localization of viral antigens in specific brain sites indicates whether the virus traveled to the brain along an anterograde or retrograde pathway (21, 25, 26, 39, 44, 51). PRV gE, gI, and Us9 each are essential for anterograde spread to the brain yet are dispensable for retrograde spread (5, 11, 25, 52). Even a strain of PRV lacking all three of these proteins retains retrograde neuronal spread activity (12, 40, 44). In contrast, in the absence of gE, HSV-1 fails to spread to the brain by either the anterograde or retrograde pathway (51).The Campenot chamber system (Fig. ​(Fig.1C)1C) has the advantage of allowing quantitative measurement of anterograde spread. Sympathetic neurons are cultured in a tripartite ring in which neuron cell bodies are contained in a separate compartment from their neurites. Virus is added to neuron cell bodies in one chamber, and anterograde spread to a separate chamber is measured by viral titers (13, 29, 30, 39, 43). Using this system, gEnull, gInull, and Us9null PRV each were shown to have only a partial defect in anterograde spread, while a virus lacking all three proteins was totally defective (13).We sought to quantify the anterograde spread activity of gEnull, gInull, and Us9null HSV-1 using the Campenot chamber system. While gEnull and gInull viruses were completely defective at anterograde spread, we found that a Us9null virus retained wild-type (WT) anterograde spread activity in this system. This observation was unexpected, since others previously had reported that Us9 is required for efficient HSV-1 anterograde transport or spread (26, 41, 46). Therefore, we further characterized the neuronal spread properties of two independent Us9-deleted viruses in the mouse retina and mouse flank models of infection. Our results indicate that gE and gI are essential for HSV-1 anterograde spread, whereas Us9 is dispensable.     

Micropropagation by tissue culture triggers differential expression of infectious endogenous Banana streak virus sequences (eBSV) present in the B genome of natural and synthetic interspecific banana plantains

The genome of Musa balbisiana spp. contains several infectious endogenous sequences of Banana streak virus (eBSV). We have shown previously that in vitro micropropagation triggers the activation of infectious eBSOLV (endogenous sequences of Banana streak Obino l'Ewai virus ) in the synthetic tetraploid interspecific hybrid FHIA21 (AAAB). In this work, we show that another synthetic tetraploid (AAAB) hybrid and two natural triploid (AAB) plantains are equally prone to the activation of infectious eBSOLV during tissue culture. These results are a strong indication that such activation is a general phenomenon in interspecific Musa cultivars, whether synthetic or natural. We also report the first in-depth study of the correlation between the duration of tissue culture and the level of activation of infectious eBSOLV, and show that specific and common activation patterns exist in these banana plants. We hypothesize that these patterns result from the concomitant activation of infectious eBSOLV and a decrease in the virus titre in neoformed plantlets, resulting from cell multiplication outcompeting virus replication. We provide experimental data supporting this hypothesis. No activation of infectious eBSGFV (endogenous sequences of Banana streak Goldfinger virus) by tissue culture was observed in the two natural AAB plantain cultivars studied here, whereas such activation occurred in the AAAB synthetic hybrid studied. We demonstrate that this differential activation does not result from differences in the structure of eBSGFV, as all banana genomes harbour eaBSGFV-7.