A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells.

A composite mammalian cell-E. coli shuttle vector was developed based on the human papova virus BK and pSV-neo. The vector contains a dioxin-responsive enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the inducible expression of inserted genes. In human cells the vector replicates episomally, presumably utilizing the BKV rather than the SV40 origin, and expresses the BK T/t antigens. A deletion in the late BK region precludes the expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the infectious lytic cycle. HeLa cells which were transfected with this vector and selected for resistance to the antibiotic G418 maintained the construct primarily in episomal form during more than one year of continuous culture, with little or no integration into the host genome. Transformed cells cultured in higher concentrations of G418 contained higher copy numbers of the vector. This permits one to vary the dosage of an inserted gene easily and reversibly without the need of conventional amplification techniques and clonal analysis. Using a chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the MMTV promoter, we found that CAT expression was greater in clones with higher vector copy number. CAT expression was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but inducibility was found to be inversely proportional to the copy number. Transformation of bacteria with plasmid molecules retrieved from the mammalian host was efficient, making this vector well adapted for the screening of cDNA libraries for the ability to express a phenotype in mammalian cells. Moreover, DNA sequences were stable during long-term passage in mammalian cells; vector passaged continuously for more than one year retained fully functional bacterial genes for resistance to chloramphenicol and ampicillin.     

Chilli leaf curl virus-based vector for phloem-specific silencing of endogenous genes and overexpression of foreign genes

Geminiviruses are the largest and most devastating group of plant viruses which contain ssDNA as a genetic material. Geminivirus-derived virus-induced gene silencing (VIGS) vectors have emerged as an efficient and simple tool to study functional genomics in various plants. However, previously developed VIGS vectors have certain limitations, owing to their inability to be used in tissue-specific functional study. In the present study, we developed a Chilli leaf curl virus (ChiLCV)-based VIGS vector for its tissue-specific utilization by replacing the coat protein gene (open reading frame (ORF) AV1) with the gene of interest for phytoene desaturase (PDS) of Nicotiana benthamiana. Functional validation of ChiLCV-based VIGS in N. benthamiana resulted in systemic silencing of PDS exclusively in the phloem region of inoculated plants. Furthermore, expression of enhanced green fluorescence protein (EGFP) using the same ChiLCV vector was verified in the phloem region of the inoculated plants. Our results also suggested that, during the early phase of infection, ChiLCV was associated with the phloem region, but at later stage of pathogenesis, it can spread into the adjoining non-vascular tissues. Taken together, the newly developed ChiLCV-based vector provides an efficient and versatile tool, which can be exploited to unveil the unknown functions of several phloem-specific genes.

     

A novel 2A-peptide-containing plasmid to generate stable Perkinsus marinus cells expressing organelle-targeted genes

Genetic manipulation techniques for marine protists are not well-established, despite immense efforts. However, Perkinsus marinus is an exception and can be developed as a genetically tractable model organism for related protists. Here, we designed a new plasmid for P. marinus that allows two proteins from a single mRNA to be differently localized using a self-cleaving 2A peptide. This enabled us to establish a stable transfectant expressing a mitochondrially targeted fluorescent protein. The system can be applied to any protein in theory and would make a powerful tool for investigating unique organelles in P. marinus and related dinoflagellates.     

Mulcos: a vector for amplification and simultaneous expression of two foreign genes in mammalian cells

A method was developed for amplification and expression of foreign genes in mammalian cells. This procedure exploits the fact that an SfiI cleavage site, GGCCGCCT/CGGCC (the recognition sequences are underlined), is present at the SV40 replication origin and the cleaved ends, CCT-3' and AGG-3', are not rotationally equivalent. Thus DNA fragments flanked by the SfiI sites can be ligated in head-to-tail tandem arrays and cloned in cosmids; the resulting construct is called a mulcos. The cosmid vector we have used, pCHD2L, contains the single SfiI site as well as HmBR and dhfr genes, selectable markers in mammalian cells. Cassette plasmid pmoRH contains two expression units, each of which consists of SV40 early promoter, EcoRI or HindIII cloning site, small T splicing region, and poly(A) signal, and the two units as a whole are flanked by the SfiI sites. A set of alpha- and beta-chain cDNAs of a human major histocompatibility class-II antigen were inserted into the EcoRI and HindIII sites, respectively. The purified SfiI fragment, containing both expression units, was then ligated with SfiI-linearized cosmid vector pCHD2L at a molar ratio of 20:1. A mulcos containing eight pairs of the alpha- and beta-chain expression units was isolated by in vitro packaging in phage lambda heads and subsequent transfection into Escherichia coli. Drug-selected cells transfected with the mulcos contained significantly higher copy numbers of the expression units and higher expression levels than those obtained using conventional plasmids. More than 85% of these cells expressed class-II antigen on their cell surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)     

Versatility of a vector for expressing foreign polypeptides at the surface of gram-negative bacteria

A wide variety of peptides in terms of length and sequence can be expressed at the surface of the bacterium Escherichia coli by genetic insertion into a 'permissive' site of the outer membrane protein LamB, used as a carrier. The resulting hybrid proteins essentially keep their biological activities with inserts of up to about 60 amino acid residues, and of a large range of predicted structures or hydrophobicities. This reflects a remarkable flexibility in the organization of the protein, but also in the export machinery. The method used to select such a permissive site is quite general and its potential to generate applications, including a versatile type of live bacterial vaccine, are discussed.     

Synthesis of a novel nucleic acid mimic: P-boranomethylphosphonate

A new type of non-ionic nucleotide analogue with a doubly modified internucleotide linkage, P-boranomethylphosphonate, has been successfully synthesized and characterized. Dithymidine boranomethylphosphonate 5 is the first example of a P-boranomethylphosphonate compound; it is a highly lipophilic phosphodiester analog, which is almost totally resistant to both snake venom phosphodiesterase (SVPDE) and bovine spleen phosphodiesterase (BSPDE). P-boranomethylphosphonates are expected to be promising candidates for mechanistic, diagnostic and therapeutic applications.     

小鼠T-bet逆转录病毒载体的构建与功能验证

为在小鼠细胞中表达并研究T-bet功能,首先构建了含有报告基因Thy1.1的小鼠T-bet逆转录病毒载体,并将构建的载体质粒转染病毒包装细胞系包装成重组病毒,再利用重组病毒分别感染NIH-3T3和D9细胞系检测其感染能力。之后,使用该重组病毒感染T-bet敲除小鼠的CD4+T淋巴细胞,流式细胞术检测T-bet及其下游靶基因Ifng的表达情况。经验证,重组的逆转录病毒感染T-bet敲除小鼠T淋巴细胞后可以在细胞中表达T-bet,并进一步引起下游靶基因Ifng的表达上调,证明本研究中外源表达的转录因子T-bet具有正常功能。综上所述,本实验成功构建了含有报告基因的小鼠T-bet重组逆转录病毒载体,为进一步在小鼠细胞中研究T-bet功能奠定了基础。     

Construction of a new vector for the expression of foreign genes inZymomonas mobilis

Summary Broad host range plasmids have previously been shown to be suitable as vectors to introduce antibiotic resistance genes intoZ. mobilis. However, attempts to use these vectors to carry other genes with enteric promoters and controlling elements have resulted in limited success due to poor expression. Thus we have constructed a promoter cloning vector in a modified pBR327 and used this vector to isolated 12 promoters fromZ. mobilis which express various levels of -galactosidase inEscherichia coli. Four of these were then subcloned into pCVD 305 for introduction intoZ. mobilis. All expressed -galactosidase inZ. mobilis with activities of 100 to 1800 Miller units. One of these retained aBamHl site into which new genes can be readily inserted immediately downstream from theZ. mobilis promoter. Genetic traits carried by pCVD 305 were initially unstable but spontaneous variants were produced during sub-culture in which the plasmid was resistant to curing at elevated temperature. One of these variants was examined in some detail. The increased stability of this variant appears to result from an alteration in the plasmid rather than a chromosomal mutation or from chromosomal integration.     

Development of a novel pretargeting system with bifunctional nucleic acid molecules

This study was aimed at exploring a novel pretargeting system based upon bifunctional nucleic acid molecules that are comprised of a nucleic acid aptamer and a nucleic acid tail. The properties of bifunctional molecules were investigated by both theoretical prediction and experimental determination. Different from the algorithm-based structure prediction, the experimental data showed that some nucleic acid tails could significantly decrease the binding capability of the aptamer. It was also found that the effectiveness of bifunctional molecules in labeling cells was dependent on the hybridization length. Based on these understandings, one bifunctional molecule was selected to study pretargeting. The results demonstrated that the bifunctional molecule could not only bind to target cells, but also hybridize with its complementary oligonucleotide on the cell surface. Thus, bifunctional nucleic acid molecules hold great potential for pretargeting applications.     

Interactions of molecules with nucleic acids. I. An algorithm to generate nucleic acid structures with an application to the B-DNA structure and a counterclockwise helix.

An algorithm is developed that enables the routine determination of backbone conformations of nucleic acids. All atomic positions including hydrogen are specified in accord with experimental bond lengths and angles but with theoretically determined conformational angles. For two Watson-Crick base pairs at a separation of 3.38 Å, and perpendicular to a common helical axis, minimum energy configurations are found for all 10 combinations at helical angles of α ～ 36°–38°, corresponding to the B-DNA structure with C(2′)-endo sugar puckers. Backbone configurations exist only within the range 35.5° ? α ? 42°, which suggests the origin of the 10-fold helix. Calculated stacking energies for the B-DNA structure increases for each of the clustered groups of base pairs: G·C with G·C, G·C with A·T, and A·T with A·T, and they are in approximate agreement with experimental observations. The counter-clockwise helix is examined, and physically meaningful structures are found only when the helical axes of successive base pairs are disjointed.     

Interactions of molecules with nucleic acids. XI. Generalization of techniques to generate nucleic acid structures with applications to intercalation sites and kinked structures

A generalized procedure to generate nucleic acid structures is presented. In this procedure, the bases of a base pair are oriented first for characterization of particular DNA receptor sites. The resultant sites are then used in the study of specific molecule–DNA interactions. For example, intercalation sites, kinked DNA, and twisted and tilted bases are envisioned. Alterations of structures via anti → syn orientations of bases, as well as crankshaft motion about collinear bonds, provide additional conformations without disrupting the overall backbone structure. These approaches to the generation of nucleic acid structures are envisioned as required in studies of the intercalation phenomenon, minor adjustments of DNA to accommodate denaturation, binding of carcinogens to DNA, complex formation of transition metals with DNA, and antitumor agents as ligands. For these base-pair and base orientations, backbone orientations are calculated by the AGNAS technique to yield physically meaningful conformations, namely, those conformations for which nonbonded contacts are favourable. A procedure is presented to generate dimer duplex units that are physically meaningful and to assemble these units into a polynucleotide duplex. Double helices that begin with B-DNA, undergo a transition to one of the above-mentioned receptor sites, and return to B-DNA can be assembled from a catalog of dimer duplexes. Stereographic projections of the various receptor sites already being used to model binding to DNA are presented.     

Introduction of foreign genes into Pharbitis nil calli using a vector derived from Agrobacterium pTi

Transgenic calli of Pharbitis nil which grow in the presence of kanamycin were obtained by introduction of plasmid pBI121, which carries kanamycin resistance and the gene for beta-glucuronidase. The calli were shown to have fragments of vector DNA in their genome and high levels of beta-glucuronidase activity. This is the first report of the introduction of T-DNA into P. nil and the T-DNA has been shown to be integrated without apparent rearrangement in its genome. The range of copy numbers was between 3 and 5. The beta-glucuronidase activities measured were about 10 times higher than those of transgenic tobacco by introduction of the same plasmid as previously described by Jefferson et al. (1987). Thus, the widely used CaMV 35S promoter also appears to be very active in P. nil.Abbreviations CaMV Cauliflower Mosaic Virus - NAA naphthylacetic acid - BA benzylaminopurine - NPT neomycinphosphotransferase - GUS beta-glucuronidase - CTX Cefotaxime - MS Murashige and Skoog - 4-MU 4-methylumbelliferone - NOS nopaline synthase - CTAB hexadecyltrimethylammonium bromide     

The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists

The ability to design agonists that target peptide signaling is a strategy to delineate underlying mechanisms and influence biology. A sequence that uniquely characterizes a peptide provides a distinct site to generate novel agonists. Drosophila melanogaster sulfakinin encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH₂) and nsDSK II (GGDDQFDDYGHMRF-NH₂). Drosulfakinin is typical of sulfakinin precursors, which are conserved throughout invertebrates. Non-sulfated DSK II is structurally related to DSK I, however, it contains a unique 5-residue N-terminal extension; drosulfakinins signal through G-protein coupled receptors, DSK-R1 and DSK-R2. Drosulfakinin II distinctly influences adult and larval gut motility and larval locomotion; yet, its structure-activity relationship was unreported. We hypothesized substitution of an N-terminal extension residue may alter nsDSK II activity. By targeting the extension we identified, not unexpectedly, analogs mimicking nsDSK II, yet, surprisingly, we also discovered novel agonists with increased (super) and opposite (protean) effects. We determined [A3] nsDSK II increased larval gut contractility rather than, like nsDSK II, decrease it. [N4] nsDSK II impacted larval locomotion, although nsDSK II was inactive. In adult gut, [A1] nsDSK II, [A2] nsDSKII, and [A3] nsDSK II mimicked nsDSK II, and [A4] nsDSK II and [A5] nsDSK II were more potent; [N3] nsDSK II and [N4] nsDSK II mimicked nsDSK II. This study reports nsDSK II signals through DSK-R2 to influence gut motility and locomotion, identifying a novel role for the N-terminal extension in sulfakinin biology and receptor activation; it also led to the discovery of nsDSK II structural analogs that act as super and protean agonists.     

Plasticity of axial identity among somites: cranial somites can generate vertebrae without expressing Hox genes appropriate to the trunk

Classic studies have shown that the presomitic mesoderm is already committed to a specific morphological fate, for example, the ability to generate a rib. Hox gene expression in the paraxial mesoderm has also been shown to be fixed early and not susceptible to modulation by an ectopic environment. This is in contrast to the plasticity of Hox expression in neuroectodermal derivatives. We reexamine here the potential of somites for morphological plasticity by transplanting the cranial (occipital) somites 1-4, that normally produce small contributions to the skull, to the trunk of avian embryos. Surprisingly, the transposed cranial somites are able to form reasonably normal vertebral anlage. In addition, the cranial somitic mesoderm produces intervertebral disks, structures not normally found in the skull. These somites are however unable to generate some elements of the vertebrae, such as the costal process. In contrast to the morphogenetic plasticity of the occipital somites, their characteristic inability to support survival of dorsal root ganglia was not significantly modified by posterior transplantation. Dorsal root ganglia initially developed and then degenerated with the same morphological stages as normally observed. In striking contrast to the plasticity of morphology, we found that all four members of the of the fourth paralogous group of Hox genes that are expressed endogenously at the level of the graft are not upregulated in the caudad-transposed cranial mesoderm. It therefore appears that genes other than those of the Hox family normally expressed at this axial level control the position-specific morphogenesis of ectopic vertebrae formed from cranial somites. In evolutionary terms, the present results imply that occipital somites that were incorporated into the "New Head" retain the ability to develop according to their original morphogenetic fate, into vertebrae.     

卵周隙内注射慢病毒生产转基因绵羊的方法

目的提高并稳定转基因绵羊生产效率。方法采用卵周隙内注射慢病毒的方法生产转基因绵羊。比较了不同发育阶段卵母细胞注射慢病毒对其后期发育及基因表达的影响,对照了注射不同慢病毒剂量以及不同熟练程度人员操作,对胚胎发育及基因表达率的影响。结果 (1)受精前或受精后注射慢病毒对胚胎后期发育和阳性率没有影响(P>0.05);(2)在受精后的单细胞注射或者2～4细胞期注射慢病毒对胚胎后期发育和阳性率也都没有影响(P>0.05);(3)慢病毒注射剂量在50～100 pL之间对转基因效率没有影响(P>0.05);(4)操作人员的熟练程度不影响胚胎成活率和转基因效率(P>0.05)。结论建立了卵周隙内注射慢病毒生产转基因绵羊的方法,并使转基因胚胎阳性率稳定在70%以上。     

Oncolytic effects of a novel influenza A virus expressing interleukin-15 from the NS reading frame

Oncolytic influenza A viruses with deleted NS1 gene (delNS1) replicate selectively in tumour cells with defective interferon response and/or activated Ras/Raf/MEK/ERK signalling pathway. To develop a delNS1 virus with specific immunostimulatory properties, we used an optimised technology to insert the interleukin-15 (IL-15) coding sequence into the viral NS gene segment (delNS1-IL-15). DelNS1 and delNS1-IL-15 exerted similar oncolytic effects. Both viruses replicated and caused caspase-dependent apoptosis in interferon-defective melanoma cells. Virus replication was required for their oncolytic activity. Cisplatin enhanced the oncolytic activity of delNS1 viruses. The cytotoxic drug increased delNS1 replication and delNS1-induced caspase-dependent apoptosis. Interference with MEK/ERK signalling by RNAi-mediated depletion or the MEK inhibitor U0126 did not affect the oncolytic effects of the delNS1 viruses. In oncolysis sensitive melanoma cells, delNS1-IL-15 (but not delNS1) infection resulted in the production of IL-15 levels ranging from 70 to 1140 pg/mL in the cell culture supernatants. The supernatants of delNS1-IL-15-infected (but not of delNS1-infected) melanoma cells induced primary human natural killer cell-mediated lysis of non-infected tumour cells. In conclusion, we constructed a novel oncolytic influenza virus that combines the oncolytic activity of delNS1 viruses with immunostimulatory properties through production of functional IL-15. Moreover, we showed that the oncolytic activity of delNS1 viruses can be enhanced in combination with cytotoxic anti-cancer drugs.