Overexpression limits of fission yeast cell‐cycle regulators in vivo and in silico

Cellular systems are generally robust against fluctuations of intracellular parameters such as gene expression level. However, little is known about expression limits of genes required to halt cellular systems. In this study, using the fission yeast Schizosaccharomyces pombe, we developed a genetic ‘tug‐of‐war’ (gTOW) method to assess the overexpression limit of certain genes. Using gTOW, we determined copy number limits for 31 cell‐cycle regulators; the limits varied from 1 to >100. Comparison with orthologs of the budding yeast Saccharomyces cerevisiae suggested the presence of a conserved fragile core in the eukaryotic cell cycle. Robustness profiles of networks regulating cytokinesis in both yeasts (septation‐initiation network (SIN) and mitotic exit network (MEN)) were quite different, probably reflecting differences in their physiologic functions. Fragility in the regulation of GTPase spg1 was due to dosage imbalance against GTPase‐activating protein (GAP) byr4. Using the gTOW data, we modified a mathematical model and successfully reproduced the robustness of the S. pombe cell cycle with the model.     

Improvement in Oil Production by Increasing Malonyl-CoA and Glycerol-3-Phosphate Pools in Scenedesmus quadricauda

In recent years, microalgae have attracted considerable interest as a biofuel resource owing to their rapid growth, tolerance to harsh conditions, and ability to accumulate a large amount of triacylglycerols (TAGs). However, the economic effectiveness of algal biofuel is still low. In this study, we attempted to increase oil production of the microalga Scenedesmus quadricauda by elevating intracellular malonyl-CoA and glycerol-3-phosphate (G3P) pools. To increase intracellular oil content, yeast-derived genes encoding acetyl-CoA carboxylase (ACC1), glycerol kinase (GPD1), and glycerol-3-phosphate dehydrogenase (GUT1) were overexpressed under the control of CaMV 35S and NOS promoters with SV40 large T antigen components. Fatty acid profiling, G3P content, and the number of cells with high oil content were analyzed by gas chromatography-mass spectrometry, G3P assay kit, and flow cytometry, respectively. Overexpression of ACC1 increased the total fatty acid content by 1.6-fold. Overexpression of GPD1 and GUT1 increased intracellular G3P content by 1.6- and 1.9-fold, respectively. Multi-gene expression of ACC1, GPD1, and GUT1 increased the number of cells with high oil content by 1.45-fold compared with that observed with the wild-type. This study is the first to report increased oil production by overexpression of the key genes (ACC1, GPD1, and GUT1) for TAG biosynthesis in microalgae.

Electronic supplementary material

The online version of this article (doi:10.1007/s12088-015-0546-4) contains supplementary material, which is available to authorized users.     

The role of thionins in rice defence against root pathogens

Thionins are antimicrobial peptides that are involved in plant defence. Here, we present an in‐depth analysis of the role of rice thionin genes in defence responses against two root pathogens: the root‐knot nematode Meloidogyne graminicola and the oomycete Pythium graminicola. The expression of rice thionin genes was observed to be differentially regulated by defence‐related hormones, whereas all analysed genes were consistently down‐regulated in M. graminicola‐induced galls, at least until 7 days post‐inoculation (dpi). Transgenic lines of Oryza sativa cv. Nipponbare overproducing OsTHI7 revealed decreased susceptibility to M. graminicola infection and P. graminicola colonization. Taken together, these results demonstrate the role of rice thionin genes in defence against two of the most damaging root pathogens attacking rice.     

拟南芥GH3.9基因的过表达及其表型分析

为研究GH3.9基因在植物生长发育过程中的作用,利用RT-PCR成功克隆到GH3.9基因,该基因全长为1 750bp。通过构建pEGAD-GH3.9过表达载体转化拟南芥,获得过表达GH3.9基因纯系转基因株系GH3.9ox-3和GH3.9ox-7。对拟南芥野生型(WT)和转基因株系(GH3.9ox-3和GH3.9ox-7)幼苗用不同光强和光质进行处理,结果显示:在蓝光、红光、远红光等不同光照强度下培养,过表达株系幼苗下胚轴的生长均明显受到抑制,且较野生型明显;采用不同光周期处理拟南芥幼苗,过表达幼苗下胚轴的伸长明显低于野生型;对成年植株表型进行观察,发现过表达株系植株矮小、雄蕊变短、果荚短小。研究表明:GH3.9基因参与了拟南芥生长发育调控,过表达GH3.9基因对拟南芥生长有抑制作用。     

Protein kinase Cδ promotes proliferation and induces malignant transformation in skeletal muscle

In this paper, we investigated the isoform‐specific roles of certain protein kinase C (PKC) isoforms in the regulation of skeletal muscle growth. Here, we provide the first intriguing functional evidence that nPKCδ (originally described as an inhibitor of proliferation in various cells types) is a key player in promoting both in vitro and in vivo skeletal muscle growth. Recombinant overexpression of a constitutively active nPKCδ in C2C12 myoblast increased proliferation and inhibited differentiation. Conversely, overexpression of kinase‐negative mutant of nPKCδ (DN‐nPKCδ) markedly inhibited cell growth. Moreover, overexpression of nPKCδ also stimulated in vivo tumour growth and induced malignant transformation in immunodeficient (SCID) mice whereas that of DN‐nPKCδ suppressed tumour formation. The role of nPKCδ in the formation of rhabdomyosarcoma was also investigated where recombinant overexpression of nPKCδ in human rhabdomyosarcoma RD cells also increased cell proliferation and enhanced tumour formation in mouse xenografts. The other isoforms investigated (PKCα, β, ε) exerted only minor (mostly growth‐inhibitory) effects in skeletal muscle cells. Collectively, our data introduce nPKCδ as a novel growth‐promoting molecule in skeletal muscles and invite further trials to exploit its therapeutic potential in the treatment of skeletal muscle malignancies.     

Cloning of the thermostable α-amylase gene from Pyrococcus woesei in Escherichia coli

Pyrococcus woesei (DSM 3773) α-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)α-amyl and pYTB2α-amyl vectors obtained were used for expression of thermostable α-amylase or fusion of α-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of α-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation—they exhibit only 35% of total cell activity—and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable α-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75°C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95°C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120°C. Maltose was the main end product of starch hydrolysis catalyzed by this α-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

人类重组PIF1蛋白的表达纯化和解螺旋酶活性的分析

Pif1解螺旋酶家族在酵母到人的进化中非常保守, 在生物体内具有很多重要的生理作用。为了从生物化学水平研究人类PIF1的功能, 从HeLa细胞的cDNA文库中克隆得到人类PIF1全长基因, 通过共转化一个携带稀有遗传密码tRNA1的质粒和一个编码分子伴侣的质粒, 增加了PIF1蛋白在大肠杆菌中的表达, 最后通过快速液相色谱纯化系统, 采用亲和层析和凝胶过滤, 纯化了人类重组PIF1蛋白。生物化学活性检测证明了纯化的人类PIF1蛋白具有ATP酶及解螺旋酶活性。人类PIF1蛋白的纯化为我们从分子水平理解PIF1在体内的功能奠定了基础。     

CYP9A17v2组成型过量表达参与棉铃虫对拟除虫菊酯的抗性

微粒体细胞色素P450氧化酶介导的解毒代谢增强是棉铃虫Helicoverpa armigera对拟除虫菊酯类杀虫剂产生抗性的主要原因。作者前期的研究表明, CYP9A12和CYP9A14组成型过量表达与棉铃虫YGF品系对拟除虫菊酯的高水平抗性相关, CYP9A12和CYP9A14的功能表达研究结果为其参与对拟除虫菊酯抗性提供了直接证据。本研究通过对棉铃虫CYP9A17v2的克隆、mRNA表达水平和功能表达的研究, 以期明确该基因是否参与棉铃虫对拟除虫菊酯的抗性。结果表明: CYP9A17v2与CYP9A12的氨基酸序列具有很高的相似性（94%）。与棉铃虫对照品系（YG）相比, CYP9A17v2在YGF抗性品系末龄幼虫脂肪体中具有10.9倍的组成型过量表达, 而在中肠中未发现过量表达。用酿酒酵母Saccharomyces cerevisiae异源表达的CYP9A17v2能够代谢多种拟除虫菊酯（顺式氰戊菊酯、溴氰菊酯和氟氯氰菊酯）。据此认为CYP9A17v2组成型过量表达参与了棉铃虫对拟除虫菊酯的抗性。至此, CYP9A亚家族中已有3个P450基因（CYP9A12, CYP9A14 和 CYP9A17v2）被证实参与了棉铃虫对拟除虫菊酯的氧化解毒代谢。     

Gain of function of Tbx1 affects pharyngeal and heart development in the mouse

Mammalian development is highly sensitive to Tbx1 gene dosage reduction. Gene function insights can also be learned from increased or ectopic expression. The authors generated a novel mouse transgenic line, named COET, which expresses Tbx1 upon Cre‐mediated recombination. The authors crossed this transgenic line with Tbx1^Cre animals to activate expression in the Tbx1‐expression domain. Compound mutant COET;Tbx1^Cre/+ animals died after birth and showed heart enlargement. At E18.5, compound mutants showed ventricular septal defects and thymic abnormalities. The authors crossed compound mutants into a Tbx1 null background to understand whether this phenotype is caused by gene overdosage. Results showed that gene dosage reduction at the endogenous locus could not rescue heart and thymic defects, although the transgene rescued the loss of function phenotype. Thus, the transgenic phenotype appears to be due to gain of function. Resultant data demonstrate that Tbx1 expression must be tightly regulated to be compatible with normal embryonic development. genesis 47:188–195, 2009. © 2009 Wiley‐Liss, Inc.     

Nitrogen use efficiency. 2. Amino acid metabolism

In a previous article, we highlighted the latest developments in the isolation and characterisation of genes involved in the uptake of nitrogen from the soil, which might be used to improve the nitrogen use efficiency (NUE) of crop plants. In this article, we have concentrated on the genes controlling the enzymes of amino acid metabolism that may be involved in transferring nitrogen to the protein in the grain. Evidence is now accumulating from the use of knockout mutants, of the role of individual isoenzymes involved in amino acid metabolism, which are encoded by specific genes that are often members of a multigene family. In addition, a significant number of overexpressing plant lines have been obtained, which have increased activities of cytosol located, glutamine synthetase, asparagine synthetase and alanine aminotransferase that appear to have improved NUE.