应用细菌内同源重组机制快速克隆腺病毒基因组的研究

以犬2型腺病毒为载体进行疫苗和基因治疗的研究,是近年的研究热点.目前的焦点问题是新型载体的构建,经典的方法是体外连接,费时、费力.     

应用细菌内同源重组机制快速克隆腺病毒基因组的研究

A novel procedure was used for cloning large adenovirus genome fragment by the homologous recombination in E.coli strain BJ5183. The 11.2Kb downstream fragment of the CAV-2 strain YCA18 genome was cloned by homologous recombination, the 1029bp left end and the 970bp fight end of this fragment were separately amplified by PCR. They were then cloned into plasmid pPoly2 with direction from left fragment to fight fragment, obtaining a “rescue” plasmid pT615. The pT615 was liberalized by Hind Ⅲ and PstⅠ digestion and was cotransformed with the purified CAV-2 genome which was cut by BstBI into competent E.coli strain BJ5183. Recombinant plasmids harboring the 11.2Kb downstream fragment of CAV-2 genome were obtained after bacterial intermolecular homologous recombination. The recombinant efficiency of all E.coli strains tested was 78.3%. One of the recombinant plasmids, pT618, was further identified by enzyme digestion analysis and PCR amplification. The results showed the plasmids contained the 11.2kb fragment downstream the genome of CAV-2.     

Concatamerization of Adeno-Associated Virus Circular Genomes Occurs through Intermolecular Recombination

Long-term recombinant AAV (rAAV) transgene expression in muscle has been associated with the molecular conversion of single-stranded rAAV genomes to high-molecular-weight head-to-tail circular concatamers. However, the mechanisms by which these large multimeric concatamers form remain to be defined. To this end, we tested whether concatamerization of rAAV circular intermediates occurs through intra- or intermolecular mechanisms of amplification. Coinfection of the tibialis muscle of mice with rAAV alkaline phosphatase (Alkphos)- and green fluorescent protein (GFP)-encoding vectors was used to evaluate the frequency of circular concatamer formation by intermolecular recombination of independent viral genomes. The GFP shuttle vector also encoded ampicillin resistance and contained a bacterial origin of replication to allow for bacterial rescue of circular intermediates from Hirt DNA of infected muscle samples. The results demonstrated a time-dependent increase in the abundance of rescued plasmids encoding both GFP and Alkphos, which reached 33% of the total circular intermediates by 120 days postinfection. Furthermore, these large circular concatamers were capable of expressing both GFP- and Alkphos-encoding transgenes following transient transfection in cell lines. These findings demonstrate that concatamerization of AAV genomes in vivo occurs through intermolecular recombination of independent monomer circular viral genomes and suggest new viable strategies for delivering multiple DNA segments at a single locus. Such developments will expand the utility of rAAV for splicing large gene inserts or large promoter-gene combinations carried by two or more independent rAAV vectors.     

A Primary Linkage Map of the Porcine Genome Reveals a Low Rate of Genetic Recombination

A comprehensive genetic linkage map of the porcine genome has been developed by typing 128 genetic markers in a cross between the European Wild Boar and a domestic breed (Large White). The marker set includes 68 polymerase chain reaction-formatted microsatellites, 60 anchored reference markers informative for comparative mapping and 47 markers which have been physically assigned by in situ hybridization. Novel multipoint assignments are provided for 54 of the markers. The map covers about 1800 cM, and the average spacing between markers is 11 cM. We used the map data to estimate the genome size in pigs, thereby addressing the total recombination distance in a third mammalian species. A sex-average genome length of 1873 +/- 139 cM was obtained by comparing the recombinational and physical distances in defined regions of the genome. This is strikingly different from the length of the human genome (3800-4000 cM) and is more similar to the mouse estimate (1600 cM). The recombination rate in females was significantly higher than in males.     

Intermolecular Transposition of Is10 Causes Coupled Homologous Recombination at the Transposition Site

Interplasmid and chromosome to plasmid transposition of IS10 were studied by assaying inactivation of the phage 434 cI gene, carried on a low copy number plasmid. This was detected by the activity of the tet gene expressed from the phage 434 P(R) promoter. Each interplasmid transposition resulted in the fusion of the donor and acceptor plasmids into cointegrate structure, with a 9-bp duplication of the target DNA at the insertion site. Cointegrate formation was abolished in δrecA strains, although simple insertions of IS10 were observed. This suggests a two-stage mechanism involving IS10 conservative transposition, followed by homologous recombination between the donor and the acceptor. Two plasmids carrying inactive IS10 sequences were fused to cointegrates at a 100-fold lower frequency, suggesting that homologous recombination is coupled to and stimulated by the transposition event. Each IS10 transposition from the chromosome to the acceptor plasmid involved replicon fusion, providing a mechanism for IS10-mediated integration of extrachromosomal elements into the chromosome. This was accompanied by the formation of an additional copy of IS10 in the chromosome. Thus, like replicative transposition, conservative transposition of IS10 is accompanied by cointegrate formation and results in duplication of the IS10.     

Homologous Recombination: A ring for a warhead

The active ‘warhead’ RuvB of the Escherichia coli protein that catalyzes the branch-migration step of homologous recombination is a helicase that binds DNA as a double-ring oligomer.     

Targeted Recombination Demonstrates that the Spike Gene of Transmissible Gastroenteritis Coronavirus Is a Determinant of Its Enteric Tropism and Virulence

Targeted recombination within the S (spike) gene of transmissible gastroenteritis coronavirus (TGEV) was promoted by passage of helper respiratory virus isolates in cells transfected with a TGEV-derived defective minigenome carrying the S gene from an enteric isolate. The minigenome was efficiently replicated in trans and packaged by the helper virus, leading to the formation of true recombinant and pseudorecombinant viruses containing the S proteins of both enteric and respiratory TGEV strains in their envelopes. The recombinants acquired an enteric tropism, and their analysis showed that they were generated by homologous recombination that implied a double crossover in the S gene resulting in replacement of most of the respiratory, attenuated strain S gene (nucleotides 96 to 3700) by the S gene of the enteric, virulent isolate. The recombinant virus was virulent and rapidly evolved in swine testis cells by the introduction of point mutations and in-phase codon deletions in a domain of the S gene (nucleotides 217 to 665) previously implicated in the tropism of TGEV. The helper virus, with an original respiratory tropism, was also found in the enteric tract, probably because pseudorecombinant viruses carrying the spike proteins from the respiratory strain and the enteric virus in their envelopes were formed. These results demonstrated that a change in the tropism and virulence of TGEV can be engineered by sequence changes in the S gene.     

A Single-Sampling Hair Trap for Mesocarnivores

Abstract Although techniques to analyze and quantify DNA-based data have progressed, methods to noninvasively collect samples lag behind. Samples are generally collected from devices that permit coincident sampling of multiple individuals. Because of cross-contamination, substantive genotyping errors can arise. We developed a cost-effective (US$4.60/trap) single-capture hair trap for American martens (Martes americana). In the field, traps effectively targeted martens; >75% of all hair samples were identified as marten. Eighty percent of marten hair (n = 180) contained sufficient quality for DNA-based analyses. This effective and affordable trap can be used by managers to monitor mesocarnivore populations noninvasively.     

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.Download video file.^{(62M, mov)}     

A Temperature-Jump Optical Trap for Single-Molecule Manipulation

To our knowledge, we have developed a novel temperature-jump optical tweezers setup that changes the temperature locally and rapidly. It uses a heating laser with a wavelength that is highly absorbed by water so it can cover a broad range of temperatures. This instrument can record several force-distance curves for one individual molecule at various temperatures with good thermal and mechanical stability. Our design has features to reduce convection and baseline shifts, which have troubled previous heating-laser instruments. As proof of accuracy, we used the instrument to carry out DNA unzipping experiments in which we derived the average basepair free energy, entropy, and enthalpy of formation of the DNA duplex in a range of temperatures between 5°C and 50°C. We also used the instrument to characterize the temperature-dependent elasticity of single-stranded DNA (ssDNA), where we find a significant condensation plateau at low force and low temperature. Oddly, the persistence length of ssDNA measured at high force seems to increase with temperature, contrary to simple entropic models.     

黄病毒检测工程细胞系的构建及功能鉴定

目的:构建黄病毒检测工程细胞系并对其功能进行鉴定。方法:以含有登革4型病毒814669株全长感染性克隆的质粒P4质粒为基础,缺失pr M-E基因1 945bp,构建含有红色荧光蛋白m Cherry报告基因的登革病毒复制子载体P4-△pr ME-m Cherry。以P4-△pr ME-m Cherry为基础,通过融合PCR方法,以真核表达载体p CDNA3.1+为骨架,构建用于黄病毒检测细胞筛选的缺陷型真核表达质粒p CDNA3.1-P4-m Cherry。脂质体转染法将质粒p CDNA3.1-P4-m Cherry转染BHK-21细胞,G418进行筛选,获得的阳性细胞克隆经96孔板系列稀释筛选、纯化克隆细胞BHKFlavivirus。结果:BHK-Flavivirus细胞感染登革病毒60h后,能够在荧光显微镜下检测到红色荧光蛋白m Cherry的表达,说明BHK-Flavivirus能够用于外源黄病毒的检测。BHK-Flavivirus细胞分别感染黄病毒属的乙脑病毒(P3)、4型登革病毒(P4),可以检测到红色荧光;而辛德毕斯病毒(XJ-160)、和基孔肯雅病毒(SD08Pan)、盖塔病毒(HB0234)等正链RNA病毒感染后则不出现红色荧光。结论:以上结果表明BHK-Alphavirus细胞可用于未知蚊媒黄病毒检测,该方法通过报告基因表达与否,能够高效、特异的甄别主要蚊媒甲病毒和黄病毒,同时该方法有利于稀缺病毒的分离、保存,操作方法简单、直观,有望应用于临床检测及病毒性生物战剂的早期甄别。     

A Two-Hybrid-Receptor Assay Demonstrates Heteromer Formation as Switch-On for Plant Immune Receptors

Receptor kinases sense extracellular signals and trigger intracellular signaling and physiological responses. However, how does signal binding to the extracellular domain activate the cytoplasmic kinase domain? Activation of the plant immunoreceptor Flagellin sensing2 (FLS2) by its bacterial ligand flagellin or the peptide-epitope flg22 coincides with rapid complex formation with a second receptor kinase termed brassinosteroid receptor1 associated kinase1 (BAK1). Here, we show that the receptor pair of FLS2 and BAK1 is also functional when the roles of the complex partners are reversed by swapping their cytosolic domains. This reciprocal constellation prevents interference by redundant partners that can partially substitute for BAK1 and demonstrates that formation of the heteromeric complex is the molecular switch for transmembrane signaling. A similar approach with swaps between the Elongation factor-Tu receptor and BAK1 also resulted in a functional receptor/coreceptor pair, suggesting that a “two-hybrid-receptor assay” is of more general use for studying heteromeric receptor complexes.Cell surface receptors are chemical sensors, often with an exquisite specificity and sensitivity, which detect extracellular signals and initiate corresponding intracellular response programs. Many of these receptors are transmembrane proteins with an extracellular ligand-binding domain and an intracellular protein kinase domain. Higher plants, such as Arabidopsis (Arabidopsis thaliana), have several hundred genes encoding receptor like kinases (Shiu and Bleecker, 2001; Shiu and Li, 2004). How are these receptor like kinases activated by their ligands, and how do they initiate a subsequent intracellular signaling cascade? In our work, we used the leucine-rich repeat receptor kinase (LRR-RK) Flagellin sensing2 (FLS2), which specifically detects bacterial flagellin or its peptide epitope flg22 at subnanomolar concentrations (Gomez-Gomez and Boller, 2000; Gómez-Gómez et al., 2001; Chinchilla et al., 2006). FLS2 undergoes heteromeric complex formation with brassinosteroid receptor1 associated kinase1 (BAK1) within seconds after application of the flagellin-derived peptide ligand flg22 (Chinchilla et al., 2007; Heese et al., 2007; Schulze et al., 2010). Thus, BAK1 might act as a coreceptor of FLS2. However, as previously observed (Chinchilla et al., 2007; Roux et al., 2011), FLS2 is still functional in the absence of BAK1, although with a reduced efficiency. This raises the question whether the ligand-induced heteromeric complex has merely an enhancing effect or whether association with BAK1 or a functional substitute acts as the essential switch-on for transmembrane signaling of FLS2. BAK1 is one of the five members that form the somatic embryogenesis receptor kinase (SERK) family (Albrecht et al., 2008), and other members of this family might partially substitute for BAK1 (Roux et al., 2011). However, a rigorous genetic approach to delineate the role of these potential substitutes is not feasible because triple mutants (serk1 serk3 serk4) and quadruple mutants (serk1 serk2 serk3 serk4) exhibit severe general phenotypes of dwarfing or even lethality at the early embryo stage (He et al., 2007; Gou et al., 2012) that might be due to the important role of SERKs in plant developmental processes (Li et al., 2002; Nam and Li, 2002).To address the role of the heteromeric complex with BAK1 in the absence of other interfering SERKs, we took a two-hybrid-receptor approach based on the premise that the apoplastic and cytoplasmic domains of FLS2 and BAK1 function in a modular manner. A heteromeric complex might thus also form and function when the roles of FLS2 and BAK1 are reversed by reciprocal swapping of their cytoplasmic protein kinase domains (Fig. 1A, schematic view).Open in a separate windowFigure 1.Heteromeric complex formation of FLS with BAK1 switches on flagellin-dependent transmembrane signaling. A, Model for the flg22-dependent heteromeric receptor complex. Schematic representation of FLS2 (blue), BAK1 (red), and the chimeric receptor constructs Ftm-B and Btm-F. Ftm-B comprises the extracellular and the transmembrane domains of FLS2 (blue) and the cytoplasmic domain of BAK1 (red). The receptor chimera Btm-F represents the reciprocal construct with the cytoplasmic part of BAK1 replaced by that of FLS2. The cytoplasmic domain of FLS2 was C-terminally tagged with a GFP. B and C, Functional comparison of native FLS2 and BAK1 (B) with the two hybrid receptors Ftm-B and Btm-F (C). The experiments show luciferase activity in Arabidopsis fls2 bak1-4 double mutant protoplasts cotransformed with pFRK1::Luciferase as a reporter and the receptor constructs indicated. At 0 h (dashed line) protoplasts were treated with 100 nm of flg22 (black diamonds) or 100 nm of the inactive analog flg22^Atum (white circles). Light emission of the protoplasts was measured with a luminometer. Values represent averages and sds of three replicates. Data shown are representative for at least three independent repetitions of the experiments with all constructs. D and E, Dose-response relationship for flg22-dependent induction of pFRK1::Luciferase in protoplasts expressing the receptor constructs indicated. Values represent increase in luciferase activity after 5 h of treatment as percentage of the increase observed with FLS2 plus BAK1 treated with saturating doses of greater than or equal to 10 nm flg22. Comparison of (half-maximal stimulation values in D and E shows that cells coexpressing Ftm-B plus Btm-F responded at least as sensitive to flg22 as cells coexpressing FLS2 plus BAK1. A combination of Ftm-B with the kinase dead version Btm-F_KD was not functional, even when treated with 1,000 nm flg22. LU, Light units.     

Genetic Change of Recombination Value in DROSOPHILA MELANOGASTER. I. Artificial Selection for High and Low Recombination and Some Properties of Recombination-Modifying Genes

A high and low selection line were formed by individual selection of females on the basis of their recombination. In the high line, recombination between Gl and Sb was increased from 14.8 percent to about 30 percent in twelve generations, when a plateau was apparently reached. Realized heritability was 0.12. The absence of a response to selection for low recombination is attributed mainly to genetic random drift, and partially to directional dominance and directional gene frequencies. Natural selection was found to act against increases of recombination above a level of about twenty percent in the measured interval. High recombination tended to be recessive to low recombination. In both selected lines and unselected stocks, intervals proximal to the centromere tended to have a higher recombination variance than distal intervals.     

Leaf Epidermal Cells： A Trap for Lipophilic Xenobiotics

Plant surfaces are covered by a layer of cuticle, which functions as a natural barrier to protect plants from mechanical damage, desiccation, and microbial invasion. Results presented in this report show that the epicuticular wax and the cuticle of plant leaves also play an important role in resisting xenobiotic invasion. Although the epicuticular wax is impermeable to hydrophilic xenobiotics, the cuticle not only restricts the penetration of hydrophilic compounds into leaf cells, but also traps lipophilic ones. The role of the epidermal cells of plant leaves in resisting xenobiotic invasion has been neglected until now. The present study shows, for the first time, that the epidermal cells may reduce or retard the transport of lipophilic xenobiotics into the internal tissues through vacuolar sequestration. Although the guard cells appear to be an easy point of entry for xenobiotics, only a very small proportion of xenobiotics present on the leaf surface actually moves into leaf tissues via the guard cells .     

A Repurposing Strategy for Hsp90 Inhibitors Demonstrates Their Potency against Filarial Nematodes

Novel drugs are required for the elimination of infections caused by filarial worms, as most commonly used drugs largely target the microfilariae or first stage larvae of these infections. Previous studies, conducted in vitro, have shown that inhibition of Hsp90 kills adult Brugia pahangi. As numerous small molecule inhibitors of Hsp90 have been developed for use in cancer chemotherapy, we tested the activity of several novel Hsp90 inhibitors in a fluorescence polarization assay and against microfilariae and adult worms of Brugia in vitro. The results from all three assays correlated reasonably well and one particular compound, NVP-AUY922, was shown to be particularly active, inhibiting Mf output from female worms at concentrations as low as 5.0 nanomolar after 6 days exposure to drug. NVP-AUY922 was also active on adult worms after a short 24 h exposure to drug. Based on these in vitro data, NVP-AUY922 was tested in vivo in a mouse model and was shown to significantly reduce the recovery of both adult worms and microfilariae. These studies provide proof of principle that the repurposing of currently available Hsp90 inhibitors may have potential for the development of novel agents with macrofilaricidal properties.     

Pairing for Recombination in Lgv of Caenorhabditis Elegans: A Model Based on Recombination in Deficiency Heterozygotes

The effect of deficiencies on recombination was studied in Caenorhabditis elegans. Heterozygous deficiencies in the left half of linkage group V [LGV(left)] were shown to inhibit recombination to their right. Fourteen deficiencies, all to the left of unc-46, were analyzed for their effect on recombination along LGV. The deficiencies fell into two groups: 10 "major inhibitors" which reduce recombination to less than 11% of the expected rate between themselves and unc-46; and four "minor inhibitors" which reduce recombination, but to a much lesser extent. All four minor inhibitors delete the left-most known gene on the chromosome, while six of the ten major inhibitors do not (i.e., these are "internal" deficiencies). Where recombination could be measured on both sides of a deficiency, recombination was inhibited to the right but not to the left. In order to explain these results we have erected a model for the manner in which pairing for recombination takes place. In doing so, we identify a new region of LGV, near the left terminus, that is important for the pairing process.