Optical Feedback Loop Involving Dinoflagellate Symbiont and Scleractinian Host Drives Colorful Coral Bleaching

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Rapid production of a chimeric antibody-antigen fusion protein based on 2A-peptide cleavage and green fluorescent protein expression in CHO cells

To enable large-scale antibody production, the creation of a stable, high producer cell line is essential. This process often takes longer than 6 months using standard limited dilution techniques and is very labor intensive. The use of a tri-cistronic vector expressing green fluorescent protein (GFP) and both antibody chains, separated by a GT2A peptide sequence, allows expression of all proteins under a single promotor in equimolar ratios. By combining the advantages of 2A peptide cleavage and single cell sorting, a chimeric antibody-antigen fusion protein that contained the variable domains of mouse IgG with a porcine IgA constant domain fused to the FedF antigen could be produced in CHO-K1 cells. After transfection, a strong correlation was found between antibody production and GFP expression (r = 0.69) using image analysis of formed monolayer patches. This enables the rapid selection of GFP-positive clones using automated image analysis for the selection of high producer clones. This vector design allowed the rapid selection of high producer clones within a time-frame of 4 weeks after transfection. The highest producing clone had a specific antibody productivity of 2.32 pg/cell/day. Concentrations of 34 mg/L were obtained using shake-flask batch culture. The produced recombinant antibody showed stable expression, binding and minimal degradation. In the future, this antibody will be assessed for its effectiveness as an oral vaccine antigen.     

Comparison of label-free and GFP multiphoton imaging of hair follicle-associated pluripotent (HAP) stem cells in mouse whiskers

Hair-follicle-associated pluripotent (HAP) stem cells can differentiate into many cell types, including neurons and heart muscle cells, and have been shown to repair peripheral nerves and the spinal cord in mice. HAP stem cells can be obtained from each individual patient for regenerative medicine which overcomes problems with immune rejection. Previously, we have demonstrated that genetically-encoded protein markers such as GFP in transgenic mice can be used to visualize HAP stem cells in vivo by multiphoton tomography. Detection and visualization of stem cells in vivo without exogenous labels such as GFP would be important for human application. In the present report, we demonstrate label-free visualization of hair follicle stem cells in mouse whiskers by multiphoton tomography due to the intrinsic fluorophores such as NAD(P)H/flavins. We compared multiphoton tomography of GFP-labeled HAP stem cells and unlabeled stem cells in isolated mouse whiskers. We show that observation of HAP stem cells by label-free multiphoton tomography is comparable to detection using GFP-labeled stem cells. The results described here have important implications for detection and isolation of human HAP stem cells for regenerative medicine.     

A mini-Tn5-derived transposon with reportable and selectable markers enables rapid generation and screening of insertional mutants in Gram-negative bacteria

We re-engineered a classic tool for mutagenesis and gene expression studies in Gram-negative bacteria. Our modified Tn5-based transposon contains multiple features that allow rapid selection for mutants, direct quantification of gene expression and straightforward cloning of the inactivated gene. The promoter-less gfp-km cassette provides selection and reporter assay depending on the activity of the promoter upstream of the transposon insertion site. The cat gene facilitates positive antibiotic selection for mutants, while the narrow R6Kγ replication origin forces transposition in recipient strains lacking the pir gene and enables cloning of the transposon flanked with the disrupted gene from the chromosome. The suicide vector pCKD100, a plasmid that could be delivered into recipient cells through biparental mating or electroporation, harbours the modified transposon. We used the transposon to mutagenize Pectobacterium versatile KD100, Pseudumonas coronafaciens PC27R and Escherichia coli 35150N. The fluorescence intensities of mutants expressing high GFP could be quantified and detected qualitatively. Transformation efficiency from conjugation ranged from 1600 to 1900 CFU per ml. We sequenced the upstream flanking regions, identified the putative truncated genes and demonstrated the restoration of the GFP phenotype through marker exchange. The mini-Tn5 transposon was also utilized to construct mutant a library of P. versatile for forward genetic screens.     

Cis-regulatory characterization of sequence conservation surrounding the Hox4 genes

Hox genes are key regulators of anterior-posterior axis patterning and have a major role in hindbrain development. The zebrafish Hox4 paralogs have strong overlapping activities in hindbrain rhombomeres 7 and 8, in the spinal cord and in the pharyngeal arches. With the aim to predict enhancers that act on the hoxa4a, hoxb4a, hoxc4a and hoxd4a genes, we used sequence conservation around the Hox4 genes to analyze all fish:human conserved non-coding sequences by reporter assays in stable zebrafish transgenesis. Thirty-four elements were functionally tested in GFP reporter gene constructs and more than 100 F1 lines were analyzed to establish a correlation between sequence conservation and cis-regulatory function, constituting a catalog of Hox4 CNEs. Sixteen tissue-specific enhancers could be identified. Multiple alignments of the CNEs revealed paralogous cis-regulatory sequences, however, the CNE sequence similarities were found not to correlate with tissue specificity. To identify ancestral enhancers that direct Hox4 gene activity, genome sequence alignments of mammals, teleosts, horn shark and the cephalochordate amphioxus, which is the most basal extant chordate possessing a single prototypical Hox cluster, were performed. Three elements were identified and two of them exhibited regulatory activity in transgenic zebrafish, however revealing no specificity. Our data show that the approach to identify cis-regulatory sequences by genome sequence alignments and subsequent testing in zebrafish transgenesis can be used to define enhancers within the Hox clusters and that these have significantly diverged in their function during evolution.     

Spatial distribution and antifungal interactions of a Bacillus biological control agent on wheat surfaces

The proximity of a biological control agent and its associated anti-microbial metabolites to pathogens on plant surfaces can determine the outcome of disease control. In this study we investigated whether deficiencies in inoculum deposition and localization could explain the inability of the biological control agent Bacillus amyloliquefaciens strain TrigoCor to consistently control Fusarium head blight in the field, despite producing effective and consistent disease control in greenhouse settings. Using epifluorescent stereomicroscopy and confocal laser scanning microscopy, we visualized the coverage of wheat spike surfaces by Bacillus post-application in greenhouse and field environments, and determined that there are large unprotected areas on wheat spikes sprayed with commercial-scale field equipment, as compared to typical greenhouse applications. Additionally, we found that in conditions of low relative humidity, antifungal compounds produced by Bacillus were not able to diffuse across wheat surfaces in biologically relevant amounts, further suggesting that the inadequate coverage of wheat surfaces by Bacillus could be directly limiting disease control. Bacillus cells were easily rinsed off wheat surfaces within 8 h of application, indicating that rainfastness might be an additional limitation of biological control in field settings. Finally, we observed the inhibition of Fusarium graminearum spore germination by TrigoCor inoculum on wheat surfaces, confirming this as a mode of action for TrigoCor biocontrol. Future optimization efforts for biological control agents applied to above-ground plant parts should focus on enhancing the rainfastness, quantity, and spatial coverage of the inoculum on plant surfaces.     

Biochemical and structural insights into intramembrane metalloprotease mechanisms

Intramembrane metalloproteases are nearly ubiquitous in living organisms and they function in diverse processes ranging from cholesterol homeostasis and the unfolded protein response in humans to sporulation, stress responses, and virulence of bacteria. Understanding how these enzymes function in membranes is a challenge of fundamental interest with potential applications if modulators can be devised. Progress is described toward a mechanistic understanding, based primarily on molecular genetic and biochemical studies of human S2P and bacterial SpoIVFB and RseP, and on the structure of the membrane domain of an archaeal enzyme. Conserved features of the enzymes appear to include transmembrane helices and loops around the active site zinc ion, which may be near the membrane surface. Extramembrane domains such as PDZ (PSD-95, DLG, ZO-1) or CBS (cystathionine-β-synthase) domains govern substrate access to the active site, but several different mechanisms of access and cleavage site selection can be envisioned, which might differ depending on the substrate and the enzyme. More work is needed to distinguish between these mechanisms, both for enzymes that have been relatively well-studied, and for enzymes lacking PDZ and CBS domains, which have not been studied. This article is part of a Special Issue entitled: Intramembrane Proteases.     

Expression of pH-sensitive green fluorescent protein in Arabidopsis thaliana

This is the first report on using green fluorescent protein (GFP) as a pH reporter in plants. Proton fluxes and pH regulation play important roles in plant cellular activity and therefore, it would be extremely helpful to have a plant gene reporter system for rapid, non‐invasive visualization of intracellular pH changes. In order to develop such a system, we constructed three vectors for transient and stable transformation of plant cells with a pH‐sensitive derivative of green fluorescent protein. Using these vectors, transgenic Arabidopsis thaliana and tobacco plants were produced. Here the application of pH‐sensitive GFP technology in plants is described and, for the first time, the visualization of pH gradients between different developmental compartments in intact whole‐root tissues of A. thaliana is reported. The utility of pH‐sensitive GFP in revealing rapid, environmentally induced changes in cytoplasmic pH in roots is also demonstrated.     

DNA错配修复蛋白MutS和MutL的相互作用研究

MutL 和 MutS 是DNA错配修复系统中起关键作用的修复蛋白. 利用基因融合技术高效表达了MutL 和 MutS融合蛋白,并利用它们发展了一种研究二者相互作用的简便方法. 融合蛋白MutL-GFP (Trx-His6-GFP-(Ser-Gly)6-MutL),MutL-Strep tagⅡ (Trx-His6-(Ser-Gly)6-Strep tagⅡ-(Ser-Gly)6-MutL) 和 MutS (Trx-His6-(Ser-Gly)6-MutS) 被构建并在大肠杆菌中高效表达. 收集菌体细胞、超声波破碎后离心取上清进行SDS-聚丙烯酰胺凝胶电泳 (SDS-PAGE) 分析,结果表明有与预期分子质量相应的诱导表达条带出现,其表达量约占全细胞蛋白的30％且以可溶形式存在. 利用固定化金属离子配体亲和层析柱分别纯化融合蛋白,其纯度达到90％. 通过将MutS蛋白固定的方法研究两种MutL融合蛋白分别与MutS之间的相互作用. 结果表明：只有MutS蛋白与含有错配碱基DNA分子结合后才与MutL蛋白发生相互作用. 通过检测MutL融合蛋白标记的绿色荧光信号或酶学显色信号来鉴定相互作用的发生. 建立的融合分子系统方法也为研究其他的蛋白质或生物大分子之间的相互作用提供了一个技术平台.