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Background Systemic capillary leak syndrome (SCLS) is a rare disorder characterized by hypotension, hemoconcentration, and hypoalbuminemia associated with increased capillary endothelium permeability. Patients with a chronic form of SCLS present with persistent and progressive generalized edema. However, there have been no reports of chronic SCLS in patients undergoing hemodialysis. Herein, we report a case of chronic SCLS associated with an intravascular large B-cell lymphoma (IVLBCL) in a patient undergoing hemodialysis. Case presentation A 71-year-old male had been on hemodialysis for five years due to diabetic nephropathy. Difficulty in body fluid removal was observed during hemodialysis, and the patient was admitted to our hospital due to exacerbated weight gain and lower limb edema. He had elevated serum lactate dehydrogenase (LDH) levels and thrombocytopenia. His blood pressure was low, and his serum brain natriuretic peptide level was relatively low, despite the increase in body fluid volume. His clinical characteristics suggested a chronic form of SCLS. Random skin biopsy revealed IVLBCL; however, the association between IVLBCL and chronic SCLS remained unclear. He underwent chemotherapy with cyclophosphamide, doxorubicin, vincristine, and prednisolone, followed by rituximab. After the treatment, his serum LDH level decreased, and the difficulty in body fluid removal during hemodialysis improved. The patient’s chronic SCLS seemed to be complicated by IVLBCL. Conclusions Patients with chronic SCLS who are undergoing hemodialysis seem to present with difficulties in fluid removal. The frequency of SCLS complications in cases with malignant lymphomas, including IVLBCL, is considered to be extremely low. However, clinicians should be aware of SCLS as a complication of malignant lymphomas.

Chemolithoautotrophic bacterium Ralstonia eutropha is a versatile host for production of various useful compounds including polyhydroxyalkanoates (PHAs) under both heterotrophic and autotrophic conditions. In this bacterium, Calvin–Benson–Bassham (CBB) cycle is functional even under heterotrophic conditions on sugars and reutilizes CO2 emitted through sugar metabolisms into PHA, leading to increase in yield of the storage polyester. This study focused on isopropanol production from glucose by engineered strains of R. eutropha. The isopropanol-producing strains were constructed by introduction of codon-optimized genes of acetoacetate decarboxylase (adc) and primary-secondary alcohol dehydrogenase (adh) from clostridia into glucose-utilizing and PHA-negative (ΔphaC1) strain of R. eutropha. Several genetic modifications showed that high expression of the isopropanol synthesis genes by using a strong synthetic promoter and deletion of NAD+-dependent (S)-3-hydroxybutyryl-CoA dehydrogenase genes (paaH1 and had) in addition to NADPH-dependent acetoacetyl-CoA reductase genes (phaB1 and phaB3) were effective for improving isopropanol production with low by-production of acetone. Isopropanol titer of 4.13 g/L was achieved by two-stage cultivation of the strain IP-007/pBj5c2-adh-adc, corresponding to overall yield of 0.6 mol mol-glucose−1. The fixation of sugar-derived CO2 during isopropanol synthesis was evaluated by 13C-labelling of the isopropanol produced from [1–13C]-glucose. The 13C-abundance in isopropanol synthesized by the engineered strain was significantly increased up to 4.8%, demonstrating actual reassimilation of CO2 emitted from glucose moiety by decarboxylation and potential contribution towards increase in the carbon yield of isopropanol on glucose.

Advances in genomics have expedited the improvement of several agriculturally important crops but similar efforts in wheat (Triticum spp.) have been more challenging. This is largely owing to the size and complexity of the wheat genome1, and the lack of genome-assembly data for multiple wheat lines2,3. Here we generated ten chromosome pseudomolecule and five scaffold assemblies of hexaploid wheat to explore the genomic diversity among wheat lines from global breeding programs. Comparative analysis revealed extensive structural rearrangements, introgressions from wild relatives and differences in gene content resulting from complex breeding histories aimed at improving adaptation to diverse environments, grain yield and quality, and resistance to stresses4,5. We provide examples outlining the utility of these genomes, including a detailed multi-genome-derived nucleotide-binding leucine-rich repeat protein repertoire involved in disease resistance and the characterization of Sm16, a gene associated with insect resistance. These genome assemblies will provide a basis for functional gene discovery and breeding to deliver the next generation of modern wheat cultivars., Comparison of multiple genome assemblies from wheat reveals extensive diversity that results from the complex breeding history of wheat and provides a basis for further potential improvements to this important food crop.

Summary Polyploidization is pervasive in plants, but little is known about the niche divergence of wild allopolyploids (species that harbor polyploid genomes originating from different diploid species) relative to their diploid progenitor species and the gene expression patterns that may underlie such ecological divergence. We conducted a fine‐scale empirical study on habitat and gene expression of an allopolyploid and its diploid progenitors.We quantified soil properties and light availability of habitats of an allotetraploid Cardamine flexuosa and its diploid progenitors Cardamine amara and Cardamine hirsuta in two seasons. We analyzed expression patterns of genes and homeologs (homeologous gene copies in allopolyploids) using RNA sequencing.We detected niche divergence between the allopolyploid and its diploid progenitors along water availability gradient at a fine scale: the diploids in opposite extremes and the allopolyploid in a broader range between diploids, with limited overlap with diploids at both ends. Most of the genes whose homeolog expression ratio changed among habitats in C. flexuosa varied spatially and temporally.These findings provide empirical evidence for niche divergence between an allopolyploid and its diploid progenitor species at a fine scale and suggest that divergent expression patterns of homeologs in an allopolyploid may underlie its persistence in diverse habitats.

Contemporary speciation provides a unique opportunity to directly observe the traits and environmental responses of a new species. Cardamine insueta is an allotriploid species that appeared within the past 150 years in a Swiss village, Urnerboden. In contrast to its two progenitor species, C. amara and C. rivularis that live in wet and open habitats, respectively, C. insueta is found in-between their habitats with temporal water level fluctuation. This triploid species propagates clonally and serves as a triploid bridge to form higher ploidy species. Although niche separation is observed in field studies, the mechanisms underlying the environmental robustness of C. insueta are not clear. To characterize responses to a fluctuating environment, we performed a time-course analysis of homeolog gene expression in C. insueta in response to submergence treatment. For this purpose, the two parental (C. amara and C. rivularis) genome sequences were assembled with a reference-guided approach, and homeolog-specific gene expression was quantified by using HomeoRoq software. We found that C. insueta and C. rivularis initiated vegetative propagation by forming ectopic meristems on leaves, while C. amara did not. We examined homeolog-specific gene expression of three species at nine time points during the treatment. The genome-wide expression ratio of homeolog pairs was 2:1 over the time-course, consistent with the ploidy number. By searching the genes with high coefficient of variation of expression over time-course transcriptome data, we found many known key transcriptional factors related to meristem development and formation upregulated in both C. rivularis and rivularis-homeolog of C. insueta, but not in C. amara. Moreover, some amara-homeologs of these genes were also upregulated in the triploid, suggesting trans-regulation. In turn, Gene Ontology analysis suggested that the expression pattern of submergence tolerant genes in the triploid was inherited from C. amara. These results suggest that the triploid C. insueta combined advantageous patterns of parental transcriptomes to contribute to its establishment in a new niche along a water-usage gradient.

To study the genetic structure of clonal plant populations, genotyping and genet detection using genetic markers are necessary to assign ramets to corresponding genets. Assignment is difficult as it involves setting a robust threshold of genetic distance for genet distinction as neighbouring genets in a plant population are often genetically related. Here, we used restriction site-associated DNA sequencing (RAD-seq) for a rhizomatous clonal herb, Cardamine leucantha [Brassicaceae] to accurately determine genet structure in a natural population. We determined a draft genome sequence of this species for the first time, which resulted in 66 617 scaffolds with N50 = 6086 bp and an estimated genome size of approximately 253 Mbp. Using genetic distances based on the RAD-seq analysis, we successfully distinguished ramets that belonged to distinct genets even from a half-sib family. We applied these methods to 372 samples of C. leucantha collected at 1-m interval grids within a 20 × 20 m plot in a natural population in Hokkaido, Japan. From these samples, we identified 61 genets with high inequality in terms of genet size and patchy distribution. Spatial autocorrelation analyses indicated significant aggregation within 7 and 4 m at ramet and genet levels, respectively. An analysis of parallel DNA microsatellite loci (simple sequence repeats) suggested that RAD-seq can provide data that allows robust genet assignment. It remains unclear whether the large genets identified here became dominant stochastically or deterministically. Precise identification of genets will assist further study and characterization of dominant genets., Clonal plants produce genetically identical offspring through vegetative growth. A ‘ramet’ refers to a single physiological individual produced by clonal propagation; a ‘genet’ refers to a group of ramets that originate from a single seed. Here we investigated how ramets that belong to different genets distribute in a natural population of Carrdamine leucantha using a genome-wide SNP markers. We applied the method to 372 samples collected at 1-m interval grids within a 20 × 20 m plot, and identified 61 genets with high inequality in terms of genet size and patchy distribution.

Background:Genetic variation in plants alters insect abundance and community structure in the field; however, little is known about the importance of a single gene among diverse plant genotypes. In this context,Arabidopsistrichomes provide an excellent system to discern the roles of natural variation and a key gene,GLABRA1, in shaping insect communities. In this study, we transplanted two independent glabrous mutants (gl1-1andgl1-2) and 17 natural accessions ofArabidopsis thalianato two localities in Switzerland and Japan.Results:Fifteen insect species inhabited plant accessions, with 10–30% broad-sense heritability of community indices being detected, such as species richness and diversity. The total abundance of leaf-chewing herbivores was negatively correlated with trichome density at both the field sites, while glucosinolates had variable effects on leaf chewers between the two sites. Interestingly, there was a parallel tendency for the abundance of leaf chewers to be higher ongl1-1andgl1-2than for their different parental accessions, Ler-1 and Col-0, respectively. Furthermore, the loss of function in theGLABRA1gene significantly decreased the resistance of plants to the two predominant chewers, flea beetles and turnip sawflies.Conclusions:Overall, our results indicate that insect community composition onA. thalianais heritable across two distant field sites, withGLABRA1playing a key role in altering the abundance of leaf-chewing herbivores. Given that such a trichome variation is widely observed in Brassicaceae plants, the present study exemplifies the community-wide impact of a single plant gene on crucifer-feeding insects in the field.

Mouse APOBEC3 (mA3) inhibits murine leukemia virus (MuLV) replication by a deamination-independent mechanism in which the reverse transcription is considered the main target process. However, other steps in virus replication that can be targeted by mA3 have not been examined. We have investigated the possible effect of mA3 on MuLV protease-mediated processes and found that mA3 binds both mature viral protease and Pr180gag-pol precursor polyprotein. Using replication-competent MuLVs, we also show that mA3 inhibits the processing of Pr65 Gag precursor. Furthermore, we demonstrate that the autoprocessing of Pr180gag-pol is impeded by mA3, resulting in reduced production of mature viral protease. This reduction appears to link with the above inefficient Pr65gag processing in the presence of mA3. Two major isoforms of mA3, exon 5-containing and -lacking ones, equally exhibit this antiviral activity. Importantly, physiologically expressed levels of mA3 impedes both Pr180gag-pol autocatalysis and Pr65gag processing. This blockade is independent of the deaminase activity and requires the C-terminal region of mA3. These results suggest that the above impairment of Pr180gag-pol autoprocessing may significantly contribute to the deaminase-independent antiretroviral activity exerted by mA3., Author summary Soon after the identification of the polynucleotide cytidine deaminase APOBEC3 as a host restriction factor against vif-deficient HIV, it was noticed that deamination-independent mechanisms are involved in the inhibition of viral replication in addition to the deaminase-dependent mechanism. We previously showed that mouse APOBEC3 (mA3) physiologically restricted mouse retrovirus replication in their natural hosts without causing significant G-to-A hypermutations. Inhibition of reverse transcription is reported to be the most plausible mechanism for the deamination-independent antiretroviral function. However, it remains unknown whether the inhibition of reverse transcription is the only way to explain the whole picture of deamination-independent antiviral activity exerted by APOBEC3. Here we show that mA3 targets the autoprocessing of Pr180gag-pol polyprotein. This activity does not require the deaminase catalytic center and mainly exerted by the C-terminal half of mA3. mA3 physically interacts with murine retroviral protease and its precursor Pr180gag-pol. mA3-induced disruption of the autocatalytic Pr180gag-pol cleavage leads to a significant reduction of mature viral protease, resulting in the inhibition of Pr65gag processing to mature Gag proteins. As the Pr180gag-pol autoprocessing is necessary for the maturation of other viral enzymes including the reverse transcriptase, its inhibition by host APOBEC3 may precede the previously described impairment of reverse transcription. Our discovery may lead to the development of novel antiretroviral drugs through the future identification of detailed molecular interfaces between retroviral Gag-Pol polyprotein and APOBEC3.

Although genome duplication is widespread in wild and crop plants, little is known about genome-wide selection due to the complexity of polyploid genomes. In allopolyploid species, the patterns of purifying selection and adaptive substitutions would be affected by masking owing to duplicated genes or ‘homeologs’ as well as by effective population size. We resequenced 25 distribution-wide accessions of the allotetraploid Arabidopsis kamchatica, which has a relatively small genome size (450 Mb) derived from the diploid species A. halleri and A. lyrata. The level of nucleotide polymorphism and linkage disequilibrium decay were comparable to A. thaliana, indicating the feasibility of association studies. A reduction in purifying selection compared with parental species was observed. Interestingly, the proportion of adaptive substitutions (α) was significantly positive in contrast to the majority of plant species. A recurrent pattern observed in both frequency and divergence-based neutrality tests is that the genome-wide distributions of both subgenomes were similar, but the correlation between homeologous pairs was low. This may increase the opportunity of different evolutionary trajectories such as in the HMA4 gene involved in heavy metal hyperaccumulation.

Finger millet (Eleusine coracana (L.) Gaertn) is an important crop for food security because of its tolerance to drought, which is expected to be exacerbated by global climate changes. Nevertheless, it is often classified as an orphan/underutilized crop because of the paucity of scientific attention. Among several small millets, finger millet is considered as an excellent source of essential nutrient elements, such as iron and zinc; hence, it has potential as an alternate coarse cereal. However, high-quality genome sequence data of finger millet are currently not available. One of the major problems encountered in the genome assembly of this species was its polyploidy, which hampers genome assembly compared with a diploid genome. To overcome this problem, we sequenced its genome using diverse technologies with sufficient coverage and assembled it via a novel multiple hybrid assembly workflow that combines next-generation with single-molecule sequencing, followed by whole-genome optical mapping using the Bionano Irys® system. The total number of scaffolds was 1,897 with an N50 length >2.6 Mb and detection of 96% of the universal single-copy orthologs. The majority of the homeologs were assembled separately. This indicates that the proposed workflow is applicable to the assembly of other allotetraploid genomes., DNA Research, 25 (1), ISSN:1340-2838, ISSN:1756-1663

Background and Aims The coexistence of hermaphrodites and female-sterile individuals, or androdioecy, has been documented in only a handful of plants and animals. This study reports its existence in the plant species Cardamine amara (Brassicaceae), in which female-sterile individuals have shorter pistils than seed-producing hermaphrodites. Methods Morphological analysis, in situ manual pollination, microsatellite genotyping and differential gene expression analysis using Arabidopsis microarrays were used to delimit variation between female-sterile individuals and hermaphrodites. Key Results Female sterility in C. amara appears to be caused by disrupted ovule development. It was associated with a 2.4- to 2.9-fold increase in clonal propagation. This made the pollen number of female-sterile genets more than double that of hermaphrodite genets, which fulfils a condition of co-existence predicted by simple androdioecy theories. When female-sterile individuals were observed in wild androdioecious populations, their ramet frequencies ranged from 5 to 54 %; however, their genet frequencies ranged from 11 to 29 %, which is consistent with the theoretically predicted upper limit of 50 %. Conclusions The results suggest that a combination of sexual reproduction and increased asexual proliferation by female-sterile individuals probably explains the invasion and maintenance of female sterility in otherwise hermaphroditic populations. To our knowledge, this is the first report of the coexistence of female sterility and hermaphrodites in the Brassicaceae.

Ralstonia eutropha is a facultative chemolithoautotrophic bacterium that uses the Calvin-Benson-Bassham (CBB) cycle for CO2 fixation. This study showed that R. eutropha strain H16G incorporated (13)CO2, emitted by the oxidative decarboxylation of [1-(13)C1]-glucose, into key metabolites of the CBB cycle and finally into poly(3-hydroxybutyrate) [P(3HB)] with up to 5.6% (13)C abundance. The carbon yield of P(3HB) produced from glucose by the strain H16G was 1.2 times higher than that by the CBB cycle-inactivated mutants, in agreement with the possible fixation of CO2 estimated from the balance of energy and reducing equivalents through sugar degradation integrated with the CBB cycle. The results proved that the 'gratuitously' functional CBB cycle in R. eutropha under aerobic heterotrophic conditions participated in the reutilization of CO2 emitted during sugar degradation, leading to an advantage expressed as increased carbon yield of the storage compound. This is a new insight into the role of the CBB cycle, and may be applicable for more efficient utilization of biomass resources.

Allopolyploidization combines parental genomes and often confers broader species distribution. However, little is known about parentally transmitted gene expression underlying quantitative traits following allopolyploidization because of the complexity of polyploid genomes. The allopolyploid species Arabidopsis kamchatica is a natural hybrid of the zinc hyperaccumulator Arabidopsis halleri and of the nonaccumulator Arabidopsis lyrata. We found that A. kamchatica retained the ability to hyperaccumulate zinc from A. halleri and grows in soils with both low and high metal content. Hyperaccumulation of zinc by A. kamchatica was reduced to about half of A. halleri, but is 10-fold greater than A. lyrata. Homeologs derived from A. halleri had significantly higher levels of expression of genes such as HEAVY METAL ATPASE4 (HMA4), METAL TRANSPORTER PROTEIN1 and other metal ion transporters than those derived from A. lyrata, which suggests cis-regulatory differences. A. kamchatica has on average about half the expression of these genes compared with A. halleri due to fixed heterozygosity inherent in allopolyploids. Zinc treatment significantly changed the ratios of expression of 1% of homeologous pairs, including genes putatively involved in metal homeostasis. Resequencing data showed a significant reduction in genetic diversity over a large genomic region (290 kb) surrounding the HMA4 locus derived from the A. halleri parent compared with the syntenic A. lyrata-derived region, which suggests different evolutionary histories. We also estimated that three A. halleri-derived HMA4 copies are present in A. kamchatica. Our findings support a transcriptomic model in which environment-related transcriptional patterns of both parents are conserved but attenuated in the allopolyploids., Molecular Biology and Evolution, 33 (11), ISSN:0737-4038, ISSN:1537-1719

Polyploidization, or genome duplication, has played a critical role in the diversification of animals, fungi and plants. Little is known about the population structure and multiple origins of polyploid species because of the difficulty in identifying multiple homeologous nuclear genes. The allotetraploid species Arabidopsis kamchatica is closely related to the model species Arabidopsis thaliana and is distributed in a broader climatic niche than its parental species. Here, we performed direct sequencing of homeologous pairs of the low-copy nuclear genes WER and CHS by designing homeolog-specific primers, and obtained also chloroplast and ribosomal internal transcribed spacer sequences. Phylogenetic analysis showed that 50 individuals covering the distribution range including North America are allopolyploids derived from Arabidopsis lyrata and Arabidopsis halleri. Three major clusters within A. kamchatica were detected using Bayesian clustering. One cluster has widespread distribution. The other two are restricted to the southern part of the distribution range including Japan, where the parent A. lyrata is not currently distributed. This suggests that the mountains in Central Honshu and surrounding areas in Japan served as refugia during glacial-interglacial cycles and retained this diversity. We also found that multiple haplotypes of nuclear and chloroplast sequences of A. kamchatica are identical to those of their parental species. This indicates that multiple diploid individuals contributed to the origin of A. kamchatica. The haplotypes of low-copy nuclear genes in Japan suggest independent polyploidization events rather than introgression. Our findings suggest that self-compatibility and gene silencing occurred independently in different origins.

Self-incompatibility (SI) systems in flowering plants distinguish self- and non-self pollen to prevent inbreeding. While other SI systems rely on the self-recognition between specific male- and female-determinants, the Solanaceae family has a non-self recognition system resulting in the detoxification of female-determinants of S-ribonucleases (S-RNases), expressed in pistils, by multiple male-determinants of S-locus F-box proteins (SLFs), expressed in pollen. It is not known how many SLF components of this non-self recognition system there are in Solanaceae species, or how they evolved. We identified 16?20 SLFs in each S-haplotype in SI Petunia, from a total of 168 SLF sequences using large-scale next-generation sequencing and genomic polymerase chain reaction (PCR) techniques. We predicted the target S-RNases of SLFs by assuming that a particular S-allele must not have a conserved SLF that recognizes its own S-RNase, and validated these predictions by transformation experiments. A simple mathematical model confirmed that 16?20 SLF sequences would be adequate to recognize the vast majority of target S-RNases. We found evidence of gene conversion events, which we suggest are essential to the constitution of a non-self recognition system and also contribute to self-compatible mutations., [プレスリリース]植物が自分以外の花粉を選ぶための遺伝子セットを特定！ ～生物の非自己認識システムの進化の謎解明へ～, [Author's version title]Gene duplication and genetic exchange drive the evolution of S-RNase based self-incompatibility

The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×10(6) to 2.2×10(8) cells ml(-1). We measured the activity of three common enzymes in the fluid: acid phosphatases, β-D-glucosidases, and β-D-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant.

Ever since Darwin's pioneering research, the evolution of self-fertilisation (selfing) has been regarded as one of the most prevalent evolutionary transitions in flowering plants. A major mechanism to prevent selfing is the self-incompatibility (SI) recognition system, which consists of male and female specificity genes at the S-locus and SI modifier genes. Under conditions that favour selfing, mutations disabling the male recognition component are predicted to enjoy a relative advantage over those disabling the female component, because male mutations would increase through both pollen and seeds whereas female mutations would increase only through seeds. Despite many studies on the genetic basis of loss of SI in the predominantly selfing plant Arabidopsis thaliana, it remains unknown whether selfing arose through mutations in the female specificity gene (S-receptor kinase, SRK), male specificity gene (S-locus cysteine-rich protein, SCR; also known as S-locus protein 11, SP11) or modifier genes, and whether any of them rose to high frequency across large geographic regions. Here we report that a disruptive 213-base-pair (bp) inversion in the SCR gene (or its derivative haplotypes with deletions encompassing the entire SCR-A and a large portion of SRK-A) is found in 95% of European accessions, which contrasts with the genome-wide pattern of polymorphism in European A. thaliana. Importantly, interspecific crossings using Arabidopsis halleri as a pollen donor reveal that some A. thaliana accessions, including Wei-1, retain the female SI reaction, suggesting that all female components including SRK are still functional. Moreover, when the 213-bp inversion in SCR was inverted and expressed in transgenic Wei-1 plants, the functional SCR restored the SI reaction. The inversion within SCR is the first mutation disrupting SI shown to be nearly fixed in geographically wide samples, and its prevalence is consistent with theoretical predictions regarding the evolutionary advantage of mutations in male components.

Background Ralstonia eutropha H16 is well known to produce polyhydroxyalkanoates (PHAs), which are potential bio-based biodegradable plastics, in an efficient manner as an energy storage material under unbalanced growth conditions. To obtain further knowledge of PHA biosynthesis, this study performed a quantitative transcriptome analysis based on deep sequencing of the complementary DNA generated from the RNA (RNA-seq) of R. eutropha H16. Results Total RNAs were extracted from R. eutropha cells in growth, PHA production, and stationary phases on fructose. rRNAs in the preparation were removed by repeated treatments with magnetic beads specific to bacterial rRNAs, and then the 36 bp sequences were determined using an Illumina high-throughput sequencer. The RNA-seq results indicated the induction of gene expression for transcription, translation, cell division, peptidoglycan biosynthesis, pilus and flagella assembly, energy conservation, and fatty acid biosynthesis in the growth phase; and the repression trends of genes involved in central metabolisms in the PHA production phase. Interestingly, the transcription of genes for Calvin-Benson-Bassham (CBB) cycle and several genes for β-oxidation were significantly induced in the PHA production phase even when the cells were grown on fructose. Moreover, incorporation of 13C was observed in poly(3-hydroxybutyrate) synthesized by R. eutropha H16 from fructose in the presence of NaH13CO3, and further gene deletion analyses revealed that both of the two ribulose 1,5-bisphosphate carboxylase (Rubiscos) in CBB cycle were actually functional in CO2 fixation under the heterotrophic condition. Conclusions The results revealed the phase-dependent transcriptomic changes and a CO2 fixation capability under heterotrophic conditions by PHA-producing R. eutropha.

Two forms of erythropoietin, EPO-bi and EPO-tetra, with different biological activities were isolated from the culture medium of a recombinant Chinese hamster ovary cell line, B8-300, into which the human erythropoietin gene had been introduced. EPO-bi, an unusual form, showed only one-seventh the in vivo activity and 3 times higher in vitro activity of the previously described recombinant human EPO (standard EPO). In contrast, EPO-tetra showed both in vivo and in vitro activities comparable to those of the standard EPO. EPO-bi, EPO-tetra, and the standard EPO had the same amino acid composition and immunoreactivity. However, structural analyses of their N-linked sugar chains revealed that EPO-bi contains the biantennary complex type as the major sugar chain, while EPO-tetra and the standard EPO contain the tetraantennary complex type as the major sugar chain. From examination of various preparations of recombinant human EPO, we found a positive correlation between the in vivo activity of EPO and the ratio of tetraantennary to biantennary oligosaccharides. These results suggest that higher branching of the N-linked sugar chains is essential for effective expression of in vivo biological activity of EPO.