Complete Genome and Plasmid Sequences of the Psychrotolerant Aureimonas Strain SA4125, Isolated from Antarctic Moss Vegetation.

The complete genome sequences of Aureimonas sp. strain SA4125 and its native plasmid pSA4125 were determined. The genome sequence comprises 4,968,066 bp, with a GC content of 66.0%, and contains 4,691 coding DNA sequences (CDSs), 3 rRNA operons, and 50 tRNAs. The native plasmid comprises 131,777 bp, with a GC content of 62.3%, and contains 138 CDSs.

Gas Exchange Rates Decrease and Leaf Temperature Increases in Nicotiana benthamiana Leaves Transiently Overexpressing Hemagglutinin in an Agrobacterium-Assisted Viral Vector System.

In this study, gas exchange characteristics and temperature of Nicotiana benthamiana leaves transiently overexpressing hemagglutinin (HA), an influenza vaccine antigen, with an Agrobacterium tumefaciens-assisted viral vector were investigated. Inoculation of leaves with an empty viral vector not containing the HA gene decreased the net photosynthetic rate (Pn) and transpiration rate (T) from 2 to 3 days post-infiltration (DPI) in the A. tumefaciens suspension. Expression of HA with the vector decreased Pn and T to much lower levels until 4 DPI. Such significant decreases were not observed in leaves infiltrated with suspension of A. tumefaciens not carrying the viral vector or in uninfiltrated leaves. Thus, viral vector inoculation itself decreased Pn and T to a certain extent and the HA expression further decreased them. The decreases in Pn and T in empty vector-inoculated and HA expression vector-inoculated leaves were associated with decreases in stomatal conductance, suggesting that the reduction of gas exchange rates was caused at least in part by stomatal closure. More detailed gas exchange and chlorophyll fluorescence analyses revealed that in HA vector-inoculated leaves, the capacity of ribulose-1,5-bisphosphate carboxylase/oxygenase to assimilate CO2 and the capacity of photosynthetic electron transport in planta were downregulated, which contributed also to the decrease in Pn. Leaf temperature (LT) increased in viral vector-inoculated leaves, which was associated with the decrease in T. When HA vector-inoculated leaves were grown at air temperatures (ATs) of 21, 23, and 26°C post-infiltration, HA accumulated earlier in leaves and the days required for HA content to attain its peak became shorter, as AT was higher. The highest LT was found 1-2 days earlier than the highest leaf HA content under all post-infiltration AT conditions. This phenomenon could be applicable in a non-destructive technique to detect the optimum harvesting date for individual plants to determine the day when leaf HA content reaches its maximum level, irrespective of spatiotemporal variation of AT, in a plant growth facility.

Effect of temperature post viral vector inoculation on the amount of hemagglutinin transiently expressed in Nicotiana benthamiana leaves.

Transient gene expression in whole plants by using viral vectors is promising as a rapid, mass production system for biopharmaceutical proteins. Recent studies have indicated that plant growth conditions such as air temperature markedly influence the accumulation levels of target proteins. Here, we investigated time course of the amount of recombinant hemagglutinin (HA), a vaccine antigen of influenza virus, in leaves of Nicotiana benthamiana plants grown at 20°C or 25°C post viral vector inoculation. The HA content per unit of leaf biomass increased and decreased from 4 to 6 days post inoculation at 20°C and 25°C, respectively, irrespective of the subcellular localization of HA. The overall HA contents were higher when HA was targeted to the endoplasmic reticulum (ER) rather than the apoplast. Necrosis of leaf tissues was specifically observed in plants inoculated with the ER-targeting vector and grown at 25°C. With the ER-targeting vector, the maximum HA contents at 20°C and 25°C were recorded at 6 and 4 days post inoculation, respectively, and were comparable to each other. HA contents thereafter decreased at both temperatures; the rate of reduction appeared faster at 25°C than at 20°C. From a practical point of view, our results indicate that the strategy of targeting HA to the ER, growing plants at a lower temperature of 20°C, and harvesting leaves at around a week after vector inoculation should be implemented to obtain a high HA yield stably and efficiently.

Effects of plant density on recombinant hemagglutinin yields in an Agrobacterium-mediated transient gene expression system using Nicotiana benthamiana plants.

Agrobacterium-mediated transient expression systems enable plants to rapidly produce a wide range of recombinant proteins. To achieve economically feasible upstream production and downstream processing, it is beneficial to obtain high levels of two yield-related quantities of upstream production: recombinant protein content per fresh mass of harvested biomass (g gFM-1 ) and recombinant protein productivity per unit area-time (g m-2 /month). Here, we report that the density of Nicotiana benthamiana plants during upstream production had significant impacts on the yield-related quantities of recombinant hemagglutinin (HA). The two quantities were smaller at a high plant density of 400 plants m-2 than at a low plant density of 100 plants m-2 . The smaller quantities at the high plant density were attributed to: (i) a lower HA content in young leaves, which usually have high HA accumulation potentials; (ii) a lower biomass allocation to the young leaves; and (iii) a high area-time requirement for plants. Thus, plant density is a key factor for improving upstream production in Agrobacterium-mediated transient expression systems. Biotechnol. Bioeng. 2017;114: 1762-1770. © 2017 Wiley Periodicals, Inc.

Histone H4 lysine 20 acetylation is associated with gene repression in human cells.

Histone acetylation is generally associated with gene activation and chromatin decondensation. Recent mass spectrometry analysis has revealed that histone H4 lysine 20, a major methylation site, can also be acetylated. To understand the function of H4 lysine 20 acetylation (H4K20ac), we have developed a specific monoclonal antibody and performed ChIP-seq analysis using HeLa-S3 cells. H4K20ac was enriched around the transcription start sites (TSSs) of minimally expressed genes and in the gene body of expressed genes, in contrast to most histone acetylation being enriched around the TSSs of expressed genes. The distribution of H4K20ac showed little correlation with known histone modifications, including histone H3 methylations. A motif search in H4K20ac-enriched sequences, together with transcription factor binding profiles based on ENCODE ChIP-seq data, revealed that most transcription activators are excluded from H4K20ac-enriched genes and a transcription repressor NRSF/REST co-localized with H4K20ac. These results suggest that H4K20ac is a unique acetylation mark associated with gene repression.

Genetic complementation analysis showed distinct contributions of the N-terminal tail of H2A.Z to epigenetic regulations.

H2A.Z is one of the most evolutionally conserved histone variants. In vertebrates, this histone variant has two isoforms, H2A.Z.1 and H2A.Z.2, each of which is coded by an individual gene. H2A.Z is involved in multiple epigenetic regulations, and in humans, it also has relevance to carcinogenesis. In this study, we used the H2A.Z DKO cells, in which both H2A.Z isoform genes could be inducibly knocked out, for the functional analysis of H2A.Z by a genetic complementation assay, as the first example of its kind in vertebrates. Ectopically expressed wild-type H2A.Z and two N-terminal mutants, a nonacetylable H2A.Z mutant and a chimera in which the N-terminal tail of H2A.Z.1 was replaced with that of the canonical H2A, complemented the mitotic defects of H2A.Z DKO cells similarly, suggesting that both acetylation and distinctive sequence of the N-terminal tail of H2A.Z are not required for mitotic progression. In contrast, each one of these three forms of H2A.Z complemented the transcriptional defects of H2A.Z DKO cells differently. These results suggest that the N-terminal tail of vertebrate H2A.Z makes distinctively different contributions to these epigenetic events. Our results also imply that this genetic complementation system is a novel and useful tool for the functional analysis of H2A.Z.

Removal of bacterial suspension water occupying the intercellular space of detached leaves after agroinfiltration improves the yield of recombinant hemagglutinin in a Nicotiana benthamiana transient gene expression system.

The use of detached leaves instead of whole plants provides an alternative means for recombinant protein production based on Agrobacterium tumefaciens-mediated transient gene overexpression. However, the process for high-level protein production in detached leaves has not yet been established. In this study, we focused on leaf handling and maintenance conditions immediately after infiltration with Agrobacterium suspension (agroinfiltration) to improve recombinant protein expression in detached Nicotiana benthamiana leaves. We demonstrated that the residual water of bacterial suspension in detached leaves had significant impact on the yield of recombinant influenza hemagglutinin (HA). Immediately after agroinfiltration, detached leaves were stored in a dehumidified chamber to allow bacterial suspension water occupying intercellular space to be removed by transpiration. We varied the duration of this water removal treatment from 0.7 to 4.4 h, which resulted in leaf fresh weights ranging from 0.94 to 1.28 g g(-1) relative to weights measured just before agroinfiltration. We used these relative fresh weights (RFWs) as an indicator of the amount of residual water. The detached leaves were then incubated in humidified chambers for 6 days. We found that the presence of residual water significantly decreased HA yield, with a clear inverse correlation observed between HA yield and RFW. We next compared HA yields in detached leaves with those obtained from intact leaves by whole-plant expression performed at the same time. The maximum HA yield obtained from a detached leaf with a RFW of approximately 1.0, namely, 800 µg gFW(-1), was comparable to the mean HA yield of 846 µg gFW(-1) generated in intact leaves. Our results indicate the necessity of removing bacterial suspension water from agroinfiltrated detached leaves in transient overexpression systems and point to a critical factor enabling the detached-leaf system as a viable recombinant protein factory.

Transient junction anisotropies orient annular cell polarization in the Drosophila airway tubes.

In contrast to planes, three-dimensional (3D) structures such as tubes are physically anisotropic. Tubular organs exhibit a striking orientation of landmarks according to the physical anisotropy of the 3D shape, in addition to planar cell polarization. However, the influence of 3D tissue topography on the constituting cells remains underexplored. Here, we identify a regulatory network polarizing cellular biochemistry according to the physical anisotropy of the 3D tube geometry (tube cell polarization) by a genome-wide, tissue-specific RNAi screen. During Drosophila airway remodelling, each apical cellular junction is equipotent to establish perpendicular actomyosin cables, irrespective of the longitudinal or transverse tube axis. A dynamic transverse enrichment of atypical protein kinase C (aPKC) shifts the balance and transiently targets activated small GTPase RhoA, myosin phosphorylation and Rab11 vesicle trafficking to longitudinal junctions. We propose that the PAR complex translates tube physical anisotropy into longitudinal junctional anisotropy, where cell-cell communication aligns the contractile cytoskeleton of neighbouring cells.

Acetylation of Histone H2AX at Lys 5 by the TIP60 Histone Acetyltransferase Complex Is Essential for the Dynamic Binding of NBS1 to Damaged Chromatin.

The association and dissociation of DNA damage response (DDR) factors with damaged chromatin occurs dynamically, which is crucial for the activation of DDR signaling in a spatiotemporal manner. We previously showed that the TIP60 histone acetyltransferase complex acetylates histone H2AX, to facilitate H2AX exchange at sites of DNA damage. However, it remained unclear how the acetylation of histone H2AX by TIP60 is related to the DDR signaling. We found that the acetylation but not the phosphorylation of H2AX is essential for the turnover of NBS1 on damaged chromatin. The loss of H2AX acetylation at Lys 5 by TIP60 in cells disturbed the accumulation of NBS1 at sites of DNA damage. Although the phosphorylation of H2AX is also reportedly required for the retention of NBS1 at damage sites, our data indicated that the acetylation-dependent NBS1 turnover by TIP60 on damaged chromatin restricts the dispersal of NBS1 foci from the sites of DNA damage. These findings indicate the importance of the acetylation-dependent dynamic binding of NBS1 to damaged chromatin, created by histone H2AX exchange, for the proper accumulation of NBS1 at DNA damage sites.

The intersection of the extrinsic hedgehog and WNT/wingless signals with the intrinsic Hox code underpins branching pattern and tube shape diversity in the drosophila airways.

The tubular networks of the Drosophila respiratory system and our vasculature show distinct branching patterns and tube shapes in different body regions. These local variations are crucial for organ function and organismal fitness. Organotypic patterns and tube geometries in branched networks are typically controlled by variations of extrinsic signaling but the impact of intrinsic factors on branch patterns and shapes is not well explored. Here, we show that the intersection of extrinsic hedgehog(hh) and WNT/wingless (wg) signaling with the tube-intrinsic Hox code of distinct segments specifies the tube pattern and shape of the Drosophila airways. In the cephalic part of the airways, hh signaling induces expression of the transcription factor (TF) knirps (kni) in the anterior dorsal trunk (DTa1). kni represses the expression of another TF spalt major (salm), making DTa1 a narrow and long tube. In DTa branches of more posterior metameres, Bithorax Complex (BX-C) Hox genes autonomously divert hh signaling from inducing kni, thereby allowing DTa branches to develop as salm-dependent thick and short tubes. Moreover, the differential expression of BX-C genes is partly responsible for the anterior-to-posterior gradual increase of the DT tube diameter through regulating the expression level of Salm, a transcriptional target of WNT/wg signaling. Thus, our results highlight how tube intrinsic differential competence can diversify tube morphology without changing availabilities of extrinsic factors.

Nap1 stimulates homologous recombination by RAD51 and RAD54 in higher-ordered chromatin containing histone H1.

Homologous recombination plays essential roles in mitotic DNA double strand break (DSB) repair and meiotic genetic recombination. In eukaryotes, RAD51 promotes the central homologous-pairing step during homologous recombination, but is not sufficient to overcome the reaction barrier imposed by nucleosomes. RAD54, a member of the ATP-dependent nucleosome remodeling factor family, is required to promote the RAD51-mediated homologous pairing in nucleosomal DNA. In higher eukaryotes, most nucleosomes form higher-ordered chromatin containing the linker histone H1. However, the mechanism by which RAD51/RAD54-mediated homologous pairing occurs in higher-ordered chromatin has not been elucidated. In this study, we found that a histone chaperone, Nap1, accumulates on DSB sites in human cells, and DSB repair is substantially decreased in Nap1-knockdown cells. We determined that Nap1 binds to RAD54, enhances the RAD54-mediated nucleosome remodeling by evicting histone H1, and eventually stimulates the RAD51-mediated homologous pairing in higher-ordered chromatin containing histone H1.

Protein quality control systems associated with no-go and nonstop mRNA surveillance in yeast.

Quality control systems eliminate aberrant proteins derived from aberrant mRNAs. Two E3 ubiquitin ligases, Ltn1 and Not4, are involved in proteasomal protein degradation coupled to translation arrest. Here, we evaluated nonstop and translation arrest products degraded in a poly(A) tail-independent manner. Ltn1 was found to degrade aberrant nonstop polypeptides derived from nonstop mRNA lacking a termination codon, but not peptidyl-tRNA, even in the absence of the ribosome dissociation complex Dom34:Hbs1. The receptor for activated C kinase (RACK1/ASC1) was identified as a factor required for nascent peptide-dependent translation arrest as well as Ltn1-dependent protein degradation. Both Not4 and Ltn1 were involved in the degradation of various arrest products in a poly(A) tail-independent manner. Furthermore, carboxyl terminus-truncated degradation intermediates of arrest products were stabilized in a cdc48-3 mutant defective in unfolding or the disassembly related to proteasomal degradation. Thus, we propose that stalled ribosomes may be dissociated into subunits and that peptidyl-tRNA on the 60S subunit is ubiquitinated by Ltn1 and Cdc48 is required for the degradation following release from tRNA.

The actin family member Arp6 and the histone variant H2A.Z are required for spatial positioning of chromatin in chicken cell nuclei.

The spatial organization of chromatin in the nucleus contributes to genome function and is altered during the differentiation of normal and tumorigenic cells. Although nuclear actin-related proteins (Arps) have roles in the local alteration of chromatin structure, it is unclear whether they are involved in the spatial positioning of chromatin. In the interphase nucleus of vertebrate cells, gene-dense and gene-poor chromosome territories (CTs) are located in the center and periphery, respectively. We analyzed chicken DT40 cells in which Arp6 had been knocked out conditionally, and showed that the radial distribution of CTs was impaired in these knockout cells. Arp6 is an essential component of the SRCAP chromatin remodeling complex, which deposits the histone variant H2A.Z into chromatin. The redistribution of CTs was also observed in H2A.Z-deficient cells for gene-rich microchromosomes, but to lesser extent for gene-poor macrochromosomes. These results indicate that Arp6 and H2A.Z contribute to the radial distribution of CTs through different mechanisms. Microarray analysis suggested that the localization of chromatin to the nuclear periphery per se is insufficient for the repression of most genes.

The human actin-related protein hArp5: nucleo-cytoplasmic shuttling and involvement in DNA repair.

Certain actin-related proteins (Arps) of budding yeast are localized in the nucleus, and have essential roles as stoichiometric components of histone acetyltransferase (HAT) and chromatin remodeling complexes. On the other hand, identification of vertebrate nuclear Arps and their functional analyses are just beginning. We show that human Arp5 (hArp5) proteins are localized in the nucleus, and that arp5Delta yeast cells are partially complemented by hArp5. Thus, hArp5 is a novel member of the nuclear Arps of vertebrates, which possess evolutionarily conserved functions from yeast to humans. We show here that hArp5 shuttles between the nucleus and the cytoplasm. Furthermore, after the induction of DNA double strand breaks (DSB), cell growth and the accumulation of phosphorylated histone H2AX (gamma-H2AX) are impaired by hArp5 depletion. Association of hArp5 with the hIno80 chromatin remodeling enzyme and decrease of chromatin-bound hIno80 by hArp5-depletion indicate that hArp5 may have a role in the recruitment of the hINO80 complex to chromatin. Overexpression of hArp5 and hIno80 enhanced gamma-H2AX accumulation. These observations suggest that hArp5 is involved in the process of DSB repair through the regulation of the chromatin remodelling machinery.

Functional subdivision of trunk visceral mesoderm parasegments in Drosophila is required for gut and trachea development.

In Drosophila, trunk visceral mesoderm, a derivative of dorsal mesoderm, gives rise to circular visceral muscles. It has been demonstrated that the trunk visceral mesoderm parasegment is subdivided into at least two domains by connectin expression, which is regulated by Hedgehog and Wingless emanating from the ectoderm. We now extend these findings by examining a greater number of visceral mesodermal genes, including hedgehog and branchless. Each visceral mesodermal parasegment appears to be divided into five or six regions, based on differences in expression patterns of these genes. Ectodermal Hedgehog and Wingless differentially regulate the expression of these metameric targets in trunk visceral mesoderm. hedgehog expression in trunk visceral mesoderm is responsible for maintaining its own expression and con expression. hedgehog expressed in visceral mesoderm parasegment 3 may also be required for normal decapentaplegic expression in this region and normal gastric caecum development. branchless expressed in each trunk visceral mesodermal parasegment serves as a guide for the initial budding of tracheal visceral branches. The metameric pattern of trunk visceral mesoderm, organized in response to ectodermal instructive signals, is thus maintained at a later time via autoregulation, is required for midgut morphogenesis and exerts feedback effect on trachea, ectodermal derivatives.