Segregation into Chiral Enantiomeric Conformations of an Achiral Molecule by Concomitant Polymorphism

An analogue of the green fluorescent protein (GFP) luminophore crystallizes from a methanol solution impregnated with dichloromethane, into a pair of chiral crystals. Thermal analysis, fluorescence emission studies, and crystal packing analysis show that the two crystals are different materials. The two polymorphs arise from the rotation of a monosubstituted benzene ring about a C-N bond which results in the formation of two strong bifurcated C-H center dot center dot center dot O intermolecular bonds to oxygen O(6). The color difference has been ascribed to a difference in the packing of the two crystal forms. Theoretical studies supported by low temperature NMR show low kinetic energy barriers (similar to 10 kJ mol(-1)) separating the asymmetric units of the two crystal structures, suggesting that the driving force for the polymorphism could be the result of packing of two different asymmetric units.

G-Quadruplex Structures Formed at the HOX11 Breakpoint Region Contribute to Its Fragility during t(10;14) Translocation in T-Cell Leukemia

The t(10;14) translocation involving the HOX11 gene is found in several T-cell leukemia patients. Previous efforts to determine the causes of HOX11 fragility were not successful. The role of non-B DNA structures is increasingly becoming an important cause of genomic instability. In the present study, bioinformatics analysis revealed two G-quadruplex-forming motifs at the HOX11 breakpoint cluster. Gel shift assays showed formation of both intra- and intermolecular G-quadruplexes, the latter being more predominant. The structure formation was dependent on four stretches of guanines, as revealed by mutagenesis. Circular dichroism analysis identified parallel conformations for both quadruplexes. The non-B DNA structure could block polymerization during replication on a plasmid, resulting in consistent K K+-dependent pause sites, which were abolished upon mutation of G-motifs, thereby demonstrating the role of the stretches of guanines even on double-stranded DNA. Extrachromosomal assays showed that the G-quadruplex motifs could block transcription, leading to reduced expression of green fluorescent protein (GFP) within cells. More importantly, sodium bisulfite modification assay showed the single-stranded character at regions I and II of HOX11 in the genome. Thus, our findings suggest the occurrence of G-quadruplex structures at the HOX11 breakpoint region, which could explain its fragility during the t(10;14) translocation.

Spatial filtering nearly eliminates the side-lobes in single- and multi-photon 4pi-type-C super-resolution fluorescence microscopy

Super-resolution microscopy has tremendously progressed our understanding of cellular biophysics and biochemistry. Specifically, 4pi fluorescence microscopy technique stands out because of its axial super-resolution capability. All types of 4pi-microscopy techniques work well in conjugation with deconvolution techniques to get rid of artifacts due to side-lobes. In this regard, we propose a technique based on spatial filter in a 4pi-type-C confocal setup to get rid of these artifacts. Using a special spatial filter, we have reduced the depth-of-focus. Interference of two similar depth-of-focus beams in a 4 pi geometry result in substantial reduction of side-lobes. Studies show a reduction of side-lobes by 46% and 76% for single and two photon variant compared to 4pi - type - C confocal system. This is incredible considering the resolving capability of the existing 4pi - type - C confocal microscopy. Moreover, the main lobe is found to be 150 nm for the proposed spatial filtering technique as compared to 690 nm of the state-of-art confocal system. Reconstruction of experimentally obtained 2PE - 4pi data of green fluorescent protein (GFP)-tagged mitocondrial network shows near elimination of artifacts arising out of side-lobes. Proposed technique may find interesting application in fluorescence microscopy, nano-lithography, and cell biology. (C) 2013 AIP Publishing LLC.

Genetically engineered sensors of cell signaling

Measuring electrical activity in large numbers of cells with high spatial and temporal resolution is a fundamental problem for the study of neural development and information processing. To address this problem, we have constructed FlaSh: a novel, genetically-encoded probe that can be used to measure trans-membrane voltage in single cells. We fused a modified green fluorescent protein (GFP) into a voltage-sensitive potassium channel so that voltage dependent rearrangements in the potassium channel induce changes in the fluorescence of GFP. A voltage sensor encoded into DNA has the advantage that it may be introduced into an organism non-invasively and targeted to specific developmental stages, brain regions, cell types, and sub-cellular compartments.

We also describe modifications to FlaSh that shift its color, kinetics, and dynamic range. We used multiple green fluorescent proteins to produce variants of the FlaSh sensor that generate ratiometric signal output via fluorescence resonance energy transfer (FRET). Finally, we describe initial work toward FlaSh variants that are sensitive to G-protein coupled receptor (GPCR) activation. These sensors can be used to design functional assays for receptor activation in living cells.

花粉管通道遗传转化方法的新证据

将pBIN35S-mGFP4质粒转入受体菌DH5α中，扩繁后提取纯化质粒DNA，待棉花盛花期时，用花粉管通道转化方法将其注射到授粉24小时左右的子房中。在被检测的950个发育10～20天左右的幼胚中，发现七个幼胚在蓝光(460～490nm)激发下发出绿色荧光，而对照发出程度不同的红色荧光。将收获的“转化种子”浸泡萌发得到生长5～6天的黄化无菌苗，在200粒种子来源的无菌苗中，检出两棵转化植株。在紫外光照射灯下转化植株发出绿色荧光，其叶片和下胚轴横切面在蓝光激发下也与对照明显不同。PCR及Southern blotting结果均证实转化植株的真实性，从而为花粉管通道转化方法的可行性提供了直接可靠的细胞及分子生物学证据。 将GFPmut1质粒的gfp通过一端平接一端粘接后重组到pBI121的BamH1和Sal1限制性酶切位点从而代替GUS基因，然后将新的重组质粒用三亲交配法转入到LBA4404菌株中得到双元载体，用于棉花下胚轴切段的转化。结果表明，gfp象gus一样可作为报告基因用于农杆菌介导的棉花转化。在对筛选培养基上生长的“抗性愈伤”进一步进行报告基因检测时，只需手持紫外灯就可以检出GFP阳性愈伤，大大减少了工作量和试验费用。 另外，在进行农杆菌转化前的棉花卡那霉素敏感性实验中发现，卡那霉素对棉花下胚轴的愈伤组织形成和增殖均有明显的抑制作用，随其浓度的增加，愈伤组织形成的频率降低，增殖的倍数减小;当浓度增加到100mg/L时，愈伤组织严重褐化，其正常生长受到完全抑制;下胚轴形态学上端切段较下端更易受卡那霉素的影响。

Upstream regulatory region of zebrafish lunatic fringe: Isolation and promoter analysis

Lunatic fringe (Lfng), one modulator of Notch signaling, plays an essential part in demarcation of tissues boundaries during animal early development, especially somitogenesis. To characterize the promoter of zebrafish 1fng and generate somite-specific transgenic zebrafish, we isolated the upstream regulatory region of zebrafish 1fng by blast search at the Ensembl genome database (http://www. ensembl.org) and analyzed the promoter activity using green fluorescent protein (GFP) as a reporter. Promoter activity assay in zebrafish shows that the 0.2-kb fragment containing GC-box, CAAT-box, and TATA-box can direct tissue-specific GFP expression, while the 0.4-kb and 1.2-kb fragments with further upstream sequence included drive GFP expression more efficiently. We produced 1fngEGFP-transgenic founders showing somite-specific expression of GFP and consequently generated a hemizygous 1fngEGFP-transgenic line. The eggs from 1fngEGFP-transgenic female zebrafish show strong GFP expression, which is consistent to the reverse-transcription polymerase chain reaction PCR (RT-PCR) detection of 1fng transcripts in the fertilized eggs. This reveals that zebrafish 1fng is a maternal factor existing in matured eggs, suggesting that fish somitogcnesis may be influenced by maternal factors.

Integration of double-fluorescence expression vectors into zebrafish genome for the selection of site-directed knockout/knockin

Production of zebrafish by modifying endogenous growth hormone (GH) gene through homologous recombination is described here. We first constructed the targeting vectors pGHT1.7k and pGHT2.8k, which were used for the knockout/knockin of the endogenous GH gene of zebrafish, and injected these two vectors into the embryos of zebrafish. Overall, the rate of targeted integration with the characteristic of germ line transmission in zebrafish was 1.7x10(-6). In one experimental patch, the integrating efficiency of pGHT2.8k was higher than that of pGHT1.7k, but the lethal effect of pGHT2.8k was stronger than that of pGHT1.7k. The clones with the correct integration of target genes were identified by a simple screening procedure based on green fluorescent protein (GFP) and RFP dual selection, which corresponded to homologous recombination and random insertion, respectively. The potential homologous recombination zebrafish was further bred to produce a heterozygous F-1 generation, selected based on the presence of GFP. The potential targeted integration of exogenous GH genes into a zebrafish genome at the P-0 generation was further verified by polymerase chain reaction and Southern blot analysis. Approximately 2.5% of potential founder knockout and knockin zebrafish had the characteristic of germ line transmission. In this study, we developed an efficient method for producing the targeted gene modification in zebrafish for future studies on genetic modifications and gene functions using this model organism.

Functional analysis of FP25K of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus

The fp25k gene of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) was studied. HearNPV fp25k gene transcription was found starting from about 18 h post-infection, and protein could be detected from the same time with antiserum against FP25K. To study the function of HearNPV fp25k, a recombinant HearNPV (HaBacWD11) with an enhanced green fluorescent protein (GFP) gene replacing the fp25k was constructed using HaBacHZ8, a bacmid of HearNPV that lacks the polyhedrin gene. Growth curve analysis showed that HaBacWD11 produced higher titres of budded viruses (BVs) than its wild-type counterpart HaBacHZ8-GFP. Electron microscopic analysis indicated that at the late stage of infection, the number of intranuclear enveloped nucleocapsids in HaBacWD11-infected cells was much less than that of HaBacHZ8-GFP. A rescue recombinant virus HaBacWD14 was constructed by reintroducing fp25k gene into HaBacWD11. The growth curve and electron microscopic analysis of the rescued recombinant confirmed that the increase of BV yield and the decrease of the virion production in infected cells were the result of fp25k deletion. The expression of membrane fusion protein (Ha133) and ODV-E66 were studied using the FP25K mutants HaBacWD11 and HaBacHZ8-GFP. Unlike FP25K mutants in Autographa californica multicapsid NPV (AcMNPV), which caused an increase in the expression of membrane fusion protein GP64 and a decrease of ODV-E66, no obvious changes at the expression level of Ha133 and ODV-E66 were observed in HearNPV FP25K mutant.

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic leach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

基于错配修复系统的基因突变检测生物芯片研究

许多人类疾病和微生物抗药性的产生都是由基因组中单个碱基的替换、插入或缺失等基因突变引起的。因此，迫切需要发展快速、高通量基因突变检测方法来实现对基因疾病和细菌抗药性的早期诊断。本研究针对匕述需求发展了纂于DN八错配修复系统的墓因突变检测生物芯片方法。根据DNA错配修复MtltS蛋白结构与功能上的高度保守性，通过PCR从E.coli K-12基因组中扩一增出DNA错配修复基因，甩石（2.56kb）。通过基因水平的分子操纵，构建了Trx-His6-MutS（THM）、Trx-His6-Linker peptide-Muts（THLM）、Trx-His6-GFP-Linker peptide-MutS（THGLM）和Trx-His6-Linker peptide-Strep-tagll-Linker peptide-MutS（THLSLM）的融合基因并在大肠杆菌中进行了IPTG诱导表达。SDS-PAGE分析表明均有一与预期分子量相应的诱导表达条带出现，其表达量占菌体蛋白的30％左右，且以可溶形式存在。融合蛋白中Trx和His6亲和肤能增加表达蛋白的可溶性及便于蛋白的纯化。连接肤的加入增大了融合蛋白各个成分之间的距离，减少空间位阻，使各个蛋白能够较大程度地保持其原有的生物活性。MLltS融合蛋白的生物活性鉴定结果表明：它们既能识别、结合含有错配碱基的DNA双链，又保留了其它融合成分的生物活性。利用融合蛋白THLSLM中的Strep-tagII与Streptavidin相互作用的天然特性，使融合蛋白THLSLM在StrePtavidin修饰过的芯片基质上自动布阵沉积，制作成蛋白质芯片来识别、结合样品中含有错配或未配刘碱基的DNA双链。THGLM、THLM-Cy3和THLSLM能够使MutS蛋白显示不同的标记信号，通过它们识别并结合固定在DNA芯片基质上的基因片段来发展基因突变检测DNA芯片方法。利用基于MutS的蛋白质芯片和DNA芯片方法对含有不同错配类型、不同长度的DNA片段和错配序列背景对错配结合的影响做了深入研究，证明了MutS介导的基因突变检测生物芯片方法的可行性。基于MutS蛋白的鳌因突变检测生物芯片方法借用了生物系统本身的DNA错配修复（Mismatch Repair，MMR）机制。DNA错配修复过程是许多修复蛋白之间的相互作用共同完成的，其中蛋白MutS、MutL和MutH在肠道细菌例如大肠杆菌的甲基定向错配修复中起决定作用。这些修复蛋白的相关研究也引起了越来越多学者的关注，但对于MutL蛋白的体外生物功能一直存在争议，从而限制了该蛋白的应用研究。本研究利用基因的体外拼接技术构建了融合蛋白Trx-Hi56-Linker peptide-MutL（THLL）、Trx-His6-GFP-Linker peptide-MutL（THGLL）和Trx-His6-Linker peptide-Strep-tagII-Linker peptide-MutL（THLSLL）。非变性凝胶电泳鉴定MutL融合蛋白体外生物功能结果表明：THLL、THGLL和THLsLL都能增加融合蛋白Trx-His6-Linker peptide-MutS（THLM）与含有错配碱基DNA双链的结合，但受ATP浓度变化的影响很大。通过融合蛋白THGLL中绿色荧光蛋白（Green Fluorescent Protein，GFP）的荧光信号或THLSLL中Strep-tagII的特性并利用酶学反应来指示该蛋白的存在，发展了体外研究DNA错配修复蛋白MtuS和MutL之间相互作用的简便方法。本研究以构建的MutS融合蛋白为分子识别元件发展了基因突变检测生物芯片并利用构建的MutL融合蛋白发展了体外研究DNA错配修复蛋白MLuS和MutL之间相互作用的简便方法。建立的融合分子系统方法也为研究其它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。此外，本研究构建的融合蛋白THGLL及其 DNA错配修复蛋白与GFP的融合构想还可用来进行DNA错配修复基因产物的表达与基因突变频率和人类肿瘤恶性程度的相关性研究。

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase1. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Investigation of lantibiotic regulation, immunity and synergy

Due to the increasing incidence of antibiotic resistant strains, the use of novel antimicrobials, such as bacteriocins, has become an ever more likely prospect. Lacticin 3147 (of which there are two components, Ltnα and Ltnβ) and nisin belong to the subgroup of bacteriocins called the lantibiotics, which has attracted much attention in recent years. The lantibiotics are antimicrobial peptides that contain unusual amino acids resulting from a series of enzyme-mediated post translational modifications. Given that there have been relatively few examples of lantibiotic-specific resistance; these antimicrobials appear to represent valid alternatives to classical antibiotics. However, the fact that lantibiotics are naturally only produced in small amounts often hinders their commercialisation. In order to overcome this bottleneck, several approaches can be employed. For example, we can create a situation that reduces the quantity of a lantibiotic required to inhibit a target by combining it with other antimicrobials. Here, following an initial screen involving lacticin 3147 and several classical antibiotics, it was observed between lacticin 3147 and the commercial antibiotics polymyxin B/E function synergistically. This reduced the amounts of the individual antimicrobials required for kill and broadened the spectrum of inhibition of both agents. Upon combination with polymyxins, lacticin 3147, which has been associated with Gram positive targets only, actively targeted Gram negative species such as Escherichia coli and Cronobacter sp. An alternative means of addressing problems associated with lantibiotic yield is to better understand how production is regulated, and ultimately use this information to enhance peptide levels. With this in mind the regulation of lacticin 3147 production from the promoter Pbac was investigated using a green fluorescent protein (GFP) expression reporter system. This revealed that elements within both of the divergent operons of the lacticin 3147 gene cluster are involved in Pbac regulation. That is, LtnR, already established as a negative regulator of itself and the lacticin 3147 associated immunity genes, also acts as an activator of Pbac transcription. In contrast, an enhanced level of expression is observed in the absence of the lacticin 3147 structural genes, ltnA1 and ltnA2, indicating that these genes/gene products are involved in Pbac repression. In fact, through complementation of the ltnA2 gene, it was revealed that this regulation is more likely to be dependent on the presence of the gene transcript rather that the corresponding prepropeptide or modified Ltnβ. It may be that if lacticin 3147 production is successfully enhanced, the ability of the producing cell to protect itself may become an issue. To prepare for such a possibility a bioengineered derivative of the lacticin 3147 immunity protein LtnI (LtnI I81V) which provides enhanced protection was discovered through an in depth investigation involving the site and saturation mutagenesis of this protein. In addition, the creation of truncated forms of LtnI allowed the identification of important and essential regions of this immunity protein. Finally, as mentioned, self-immunity is essential to prevent self-killing. However the discovery of nisin U immunity and regulatory gene homologues (spiFEGRR’K) within the pathogenic strain S. infantarius subsp. infantarius is a cause for concern as it represents an example of immune mimicry, a form of lantibiotic-specific resistance. The ability of spiFEG to confer protection was apparent when they successfully provided protection to nisin A, F, Z, Q and U when expressed heterologously in the nisin sensitive L. lactis HP host. As a consequence of the studies presented in this thesis, it is likely that strategies will emerge that will facilitate the production of greater levels of lacticin 3147 production and lead to enhanced immunity in lactococcal backgrounds. Alternatively the need for enhanced production could be avoided through the use of antimicrobial combinations. In addition, providing awareness of the threats of the emergence of resistance through immune mimicry can allow researchers to develop strategies to prevent this phenomenon from leading to the dissemination of lantibiotic resistance.

Functional comparison of the endothelial nitric oxide synthase Glu298Asp polymorphic variants in human endothelial cells

The G894T endothelial nitric oxide synthase (eNOS) polymorphism results in a Glu to Asp substitution at position 298. This position is located externally on the protein and as the regulation of eNOS is dependent on its subcellular localization and interaction with modulatory proteins, we aimed to address whether the substitution of Asp at 298 had any effect on these mechanisms. Initially, we developed a novel method to accurately determine molar quantities of each variant by expressing them as green fluorescent protein (GFP) fusion proteins and using recombinant adenoviruses to facilitate transient infection of human microvascular endothelial cells. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting of eNOS298Asp revealed a 135-kDa proteolytic fragment which was not present with eNOS298Glu. This proteolysis was prevented by using LDS buffer confirming that this differential cleavage is an artefact of sample preparation and unlikely to occur intracellularly. Nitric oxide was measured following stimulation with calcium ionophore or oestrogen in the presence of varying sepiapterin concentrations. GFP fluorescence was used to quantify the amount of fusion protein and calculate intracellular specific activity. There was no significant difference in intracellular specific activity between Glu298 and Asp298 eNOS in response to calcium ionophore or oestrogen. Tetrahydrobiopterin supplementation increased eNOS activity of both variants in an identical manner. The presence of the GFP also facilitated the visualization of the variants by confocal microscopy and demonstrated that both localized to the plasma membrane and the Golgi. These findings demonstrate that the Asp substitution at 298 does not have a major effect in modulating eNOS activity in vivo.

The tissue plasminogen activator gene promoter: a novel tool for radiogenic gene therapy of the prostate?

Radiation therapy is a treatment modality routinely used in cancer management so it is not unexpected that radiation-inducible promoters have emerged as an attractive tool for controlled gene therapy. The human tissue plasminogen activator gene promoter (t-PA) has been proposed as a candidate for radiogenic gene therapy, but has not been exploited to date. The purpose of this study was to evaluate the potential of this promoter to drive the expression of a reporter gene, the green fluorescent protein (GFP), in response to radiation exposure. METHODS: To investigate whether the promoter could be used for prostate cancer gene therapy, we initially transfected normal and malignant prostate cells. We then transfected HMEC-1 endothelial cells and ex vivo rat tail artery and monitored GFP levels using Western blotting following the delivery of single doses of ionizing radiation (2, 4, 6 Gy) to test whether the promoter could be used for vascular targeted gene therapy. RESULTS: The t-PA promoter induced GFP expression up to 6-fold in all cell types tested in response to radiation doses within the clinical range. CONCLUSIONS: These results suggest that the t-PA promoter may be incorporated into gene therapy strategies driving therapeutic transgenes in conjunction with radiation therapy.