Estrogenic antagonists bearing dicarba-closo-dodecaborane as a hydrophobic pharmacophore

Dicarba-closo-dodecaboranes (carboranes), which have spherical geometry and hydrophobicity, are applicable as a hydrophobic pharmacophore of biologically active molecules. We have designed and synthesized estrogenic antagonists based on the structure of the potent agonist 1-hydroxymethyl-12-(4-hydroxyphenyl)-1,12-dicarba-closo-dodecaborane, which we have developed. The compounds showed potent antagonistic activity in luciferase reporter gene assay using COS-1 cells transfected with rat ER-expression plasmid and an appropriate reporter plasmid.     

PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca

ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - NOS nopaline synthase - CCC covalently closed circular DNA - L linear DNA - PEG polyethylene glycol - HS herring sperm DNA - P protoplasts - PCM protoplast culture medium - MES morpholinoethane-sulfonic acid - Cm chloramphenicol - Ac acetylated - MUG 4-methyl umbelliferyl ß-D-glucuronide - TLC thin layer chromatography     

Control of gene expression in plant cells using a 434:VP16 chimeric protein

The herpes simplex virus type 1 VP16 polypeptide is a potent trans-activator of viral gene expression. We have tested the ability of the VP16 activation domain to activate gene expression in plant cells. A plasmid encoding a translational fusion between the full-length 434 repressor and the C-terminal 80 amino acids of VP16, was constructed. When expressed in Escherichia coli, the chimeric protein binds efficiently to 434-binding motifs (operators). For expression in plant cells, the chimeric activator gene was placed between the cauliflower mosaic virus (CaMV) 35S promoter and nos terminator sequences in a pUC-based plasmid. The 434 operators were placed upstream of a minimal CaMV 35S promoter linked to the E. coli gus reporter gene. This reporter-expression cassette was then incorporated into the same plasmid as the 434 cI/VP16 activator-expression cassette. Two control plasmids were also constructed, one encoding the 434 protein with no activator domain and the second a chimeric activator with no DNA-binding domain. The chimeric activator was tested for its ability to activate gene expression in a tobacco protoplast transient assay system. Results are presented to show that we can obtain in plant cells significant activation of gene expression that is dependent on both DNA-binding and the presence of the activator domain.     

Comparison of the immunogenicities of HIV-1 mutants based on structural modification of env

Eleven env mutants were designed and generated by site-directed mutagenesis of the regions around NAb epitopes and deletions of variable regions in env. The immunogenicities of the generated mutants were evaluated using single-cycle infection neutralization assays with two pseudoviruses and IFN-γ ELISPOT. Overall, five mutants (dWt, M2, M5-2, M5-1 and dM7) induced higher neutralization activities for both pseudoviruses than plasmid Wt, while only two of the mutants (dWt and M5-2) showed significant differences (P<0.05). Two mutants (M2 and dM2) induced more Env-specific T cells than plasmid Wt. Statistically however, significance was only reached for mutant M2. Thus, properly modified HIV-1 Env may have the potential to induce potent cellular and humoral immune responses.      

检测大肠杆菌内c-di-GMP水平的双荧光素报告质粒的构建及应用

细菌通过调控第二信使环二鸟苷酸(cyclic diguanylate, c-di-GMP)而促进其适应环境、存活及致病。【目的】本研究旨在建立有效的c-di-GMP水平检测方法,为大肠杆菌内c-di-GMP水平检测提供便利条件。【方法】根据c-di-GMP核糖开关受体的调控方式、荧光报告基因等设计引物,通过重叠聚合酶链反应(overlap polymerase chain reaction, overlap PCR)和同源重组酶构成基于核糖开关的双荧光素报告质粒pAmCherry-Vc2EGFP(pACVcE),然后构建c-di-GMP代谢基因过表达菌株和缺失菌株,利用pACVcE检测大肠杆菌内c-di-GMP水平。【结果】OverlapPCR扩增产物与目的靶序列一致,测序结果证明pACVcE序列正确。表达c-di-GMP合成酶DgcZ的大肠杆菌胞内c-di-GMP水平显著升高,而表达c-di-GMP降解酶PdeK的大肠杆菌胞内c-di-GMP水平显著降低。禽致病性大肠杆菌的胞内c-di-GMP水平检测发现c-di-GMP降解酶基因pdeK缺失后胞内的c-di-GMP水平显著升高。【结...     

Transfer of maternally administered fusogenic liposome-DNA complexes into monkey fetuses in a pregnancy model

Background

Materno‐fetal transfer of intravenously administered liposome‐plasmid DNA complexes has been demonstrated only in mice. Studies on its materno‐fetal transfer in the pregnant monkey model is needed because of critical differences in placental structure between primates including humans and rodents.

Methods

The reporter plasmid pEGFP‐C1 was formulated in cationic lipid containing polybrene and vesicular stomatitis virus G protein. The fusogenic liposome‐plasmid DNA complexes were intradermally injected into pregnant common marmosets (N=2), a New World monkey, near term. DNA extracted from fetal tissues was subjected to PCR for detection of the egfp gene. Confocal microscopy and immunostaining were performed to determine the sites of transgene expression in the fetal organs.

Results

The egfp gene was detected in fetal blood and major organs (heart, liver, lung). The encoded protein was mainly produced in the endothelial cells of blood vessels in the fetal lungs.

Conclusions

This is the first report on materno‐fetal transfer of intradermally administered fusogenic liposome‐plasmid DNA complexes and fetal expression of a transgene in primates. Copyright © 2002 John Wiley & Sons, Ltd.

     

Gene Transfer to Lentil Protoplasts by Lipofection and Electroporation

Abstract

Cationic liposomes made of dipalmitoylphosphatidylcholine and stearylamine (9:1) were prepared by reverse-phase evaporation and were able to interact spontaneously with plasmid DNA. The loaded vesicles delivered a β-glucuronidase (GUS)-carrying plasmid to lentil Lens culinaris) protoplasts, leading to transient expression of the GUS reporter gene. The transfection efficiency achieved by using stearylamine-containing liposomes (lipofection) was comparable to the one obtained by electroporating the protoplasts at 500 μF and different field strengths. Furthermore, the combination of electroporation and lipofection, though reducing cell survival, increased the activity of the reporter enzyme detected in the cell lysates, yielding transient expression levels higher than those recorded after lipofection or electroporation alone.     

Mono- and bis-cobalt complexes with carbonyl, nitrosyl, and monocarbollide ligands

Reaction of [Co(CO)₃(NO)] with [2-NMe₃-closo-2-CB₁₀H₁₀] in refluxing CH₂Cl₂ affords the mono- and di-cobalt complexes [1-NMe₃-2-CO-2-NO-closo-2,1-CoCB₁₀H₁₀] (3) and [2,7-{Co(CO)(NO)}-7-(μ-H)-1-NMe₃-2-CO-2-NO-closo-2,1-CoCB₁₀H₉] (4), respectively, of which 4 contains formally both Co(I) and Co(-I) centers. Compound 4 reacts with CO to give 3, or with donor ligands L in the presence of Me₃NO to afford simple substituted species, [1-NMe₃-2-L-2-NO-closo-2,1-CoCB₁₀H₁₀] (compounds 5; L = PEt₃, PPh₃, CNBu^t).     

Normalization of muscle plasmid uptake by Southern blot: application to SERCA1 promoter analysis

Directinjection of plasmid DNA into muscle allows the study of promoters in aphysiological environment. Because of the variability of reporter geneactivity, attempts have been made to normalize activity to muscleplasmid uptake by coinjection of a second "control" plasmid whose reporter gene is constitutively expressed by a viral promoter. The purpose of this study was to evaluate the use of acontrol plasmid vs. Southern blot to normalize for differences inuptake of plasmids containing promoter fragments of the skeletal muscle-specific sarco(endo)plasmic reticulumCa²⁺-ATPase (SERCA1) gene. Resultsshowed that the correlation of luciferase activity from control vs.SERCA1 plasmids is poor and that normalization by a virally drivencontrol plasmid increased variability of SERCA1 luciferase activity. Inseveral cases, the presence of a control plasmid inhibited SERCA1reporter expression. When Southern blot analysis was used to normalizefor differences in plasmid uptake there was less variability than withcoinjection, and correlations between plasmid uptake and SERCA1luciferase activity were better. Moreover, there were no inhibitoryeffects of a control plasmid allowing for optimization of injectionconditions of the SERCA1 deletion constructs. The use of Southernanalysis is suggested to determine whether plasmid uptake isdifferentially affected by physiological stimuli, muscle types, orplasmid sizes under study.

     

食品级乳酸乳球菌pNZ8149-luc质粒的构建及表达

目的:构建能够稳定表达萤火虫荧光素酶报告基因(luc)的乳酸乳球菌(Lactococcus lactis, L.lactis)食品级表达系统,以便后续研究对目的基因进行示踪。方法:从pGL4.10质粒中PCR扩增萤火虫荧光素酶报告基因,测序,克隆至载体pNZ8149,构建pNZ8149-luc表达质粒;电击转化宿主乳酸乳球菌NZ3900,采用乳糖筛选法获得重组的乳酸乳球菌,Nisin诱导,采用微孔板发光检测仪检测荧光素酶的存在,Western Blot检测目标蛋白luc的表达。结果:PCR扩增的荧光素酶报告基因成功克隆至pNZ8149质粒,并电击转化宿主乳酸乳球菌NZ3900,得到乳酸乳球菌表达系统NZ3900/pNZ8149-luc。Nisin诱导后,检测到荧光素酶随诱导时间的延长活性逐渐增强,时间超过24 h之后荧光素酶活性逐渐下降。Western Blot检测到目标蛋白luc在胞内表达。结论:成功构建了p NZ8149-luc表达载体,并能够在乳酸乳球菌体内稳定表达。     

Construction of a bioluminescence reporter plasmid for Francisella tularensis

A Francisella tularensis shuttle vector that constitutively expresses the Photorhabdus luminescens lux operon in type A and type B strains of F. tularensis was constructed. The bioluminescence reporter plasmid was introduced into the live vaccine strain of F. tularensis and used to follow F. tularensis growth in a murine intranasal challenge model in real-time by bioluminescence imaging. The results show that the new bioluminescence reporter plasmid represents a useful tool for tularemia research that is suitable for following F. tularensis growth in both in vitro and in vivo model systems.     

Construction of a Tightly Regulated Plasmid Vector for Streptococcus pneumoniae: Controlled Expression of the Green Fluorescent Protein

We have constructed a regulated plasmid vector for Streptococcus pneumoniae, based on the streptococcal broad-host-range replicon pLS1. As a reporter gene, we subcloned the gfp gene from Aequorea victoria, encoding the green fluorescent protein. This gene was placed under the control of the inducible P_M promoter of the S. pneumoniae malMP operon which, in turn, is regulated by the product of the pneumococcal malR gene. Binding of MalR protein to the P_M promoter is inactivated by growing the cells in maltose-containing media. Highly regulated gene expression was achieved by cloning in the same plasmid the P_M-gfp cassette and the malR gene, thus providing the MalR regulator in cis. Pneumococcal cells harboring this vector gave a linear response of GFP synthesis in a maltose-dependent mode without detectable background levels in the absence of the inducer.     

Role of a FMRFamide‐like family of neuropeptides in the pharyngeal nervous system of Caenorhabditis elegans

The nervous system of C. elegans has a remarkable abundance of flp genes encoding FMRFamide‐like (FLP) neuropeptides. To provide insight into the physiological relevance of this neuropeptide diversity, we have tested more than 30 FLPs (encoded by 23 flps) for bioactivity on C. elegans pharynx. Eleven flp genes encode peptides that inhibit pharyngeal activity, while eight flp genes encode peptides that are excitatory. Three potent peptides (inhibitory, FLP‐13A, APEASPFIRFamide; excitatory, FLP‐17A, KSAFVRFamide; excitatory, FLP‐17B, KSQYIRFamide) are encoded by flp genes, which, according to reporter gene constructs, are expressed in pharyngeal motoneurons. Thus, they may act through receptors localized on the pharyngeal muscle. The two other potent peptides, FLP‐8 (excitatory AF1, KNEFIRFamide,) and FLP‐11A (inhibitory, AMRNALVRFamide), appear to be expressed in extrapharyngeal neurons and are therefore likely to act either indirectly or as neurohormones. Intriguingly, a single neuron can express peptides that have potent but opposing biological activity in the pharynx. Only five flp genes encode neuropeptides that have no observable effect on the pharynx, but none of these have shown reporter gene expression in the pharyngeal nervous system. To examine the roles of multiple peptides produced from single precursors, a comparison was made between the bioactivity of different neuropeptides for five flp genes (flp‐3, flp‐13, flp‐14, flp‐17, and flp‐18). For all but one gene (flp‐14), the effects of peptides encoded by the same gene were similar. Overall, this study demonstrates the impressive neurochemical complexity of the simple circuit that regulates feeding in the nematode, C. elegans. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Stable expression of the GUS reporter gene in chrysanthemum depends on binary plasmid T-DNA

Three chrysanthemum (Dendranthema grandiflora) cultivars were cocultivated with 2 Agrobacterium tumefaciens strains in combination with 4 pBIN19 derived binary plasmids, all carrying the Nosnptll selection gene and 35Sgus(intron) reporter gene. All binary plasmids transferred DNA to chrysanthemum explants but only pMOG410 gave good stable expression of GUS. This plasmid differs from the other plasmids in 2 aspects: 1) It carries a restored nptll gene and 2) the selection gene is positioned at the left border side of the reporter gene. Cocultivation with AGLO(pMOG410) yielded up to 13 GUS positive shoots per 100 explants. The presence of the gus and nptll gene in recovered shoots was confirmed by PCR and Southern blot analysis.     

pTn5cat:A Tn5-Derived Genetic Element to Facilitate Insertion Mutagenesis, Promoter Probing, Physical Mapping, Cloning, and Marker Exchange in Phytopathogenic and Other Gram-Negative Bacteria

A Tn5-derived mobile element has been constructed to identify genes and promoters related to pathogenesis and virulence inPseudomonas syringaepv.phaseolicola.To enhance the rate of mutation this Tn5derivative was constructed carrying a mutant transposase which was placed incisto the transposable element, but just outside the inverted repeats, therefore eliminating secondary transposition and increasing the stability of the insertion. The new element also contains a promoterlesscat(chloramphenicol acetyltransferase) gene as reporter to allow for positive selection of promoters being expressed under specific conditions. To facilitate cloning and manipulations inEscherichia coli,a ColE1 origin of replication has been included within the transposable element as well as the Mob region from the broad-host-range plasmid RP4, which allows this element to be efficiently mobilized by a triparental mating or by using anE. colistrain such as S17-1 to provide thetrafunctions. Sites for the rare cuttersPacI andPmeI have also been included to facilitate locating the insertions on aPacI and/orPmeI physical map. This construction combines the properties of both a mobilizable plasmid and a transposon and therefore has been termed pTn5cat.It is almost the same size as the wild-type Tn5, 5877 bp, and has successfully been tested inP.s. phaseolicolaandXanthomonas campestrispv.campestris.     

A synthetic xylanase as a novel reporter in plants

Transient gene expression assays are often used to screen promoters before stable transformation. Current transient quantification methods have several problems, including a lack of reporter gene stability and expense. Here we report a synthetic, codon-optimised xylanase gene (sXynA) as a reporter gene for quantitative transient analyses in plants. Azurine-crosslinked xylan (AZCL-xylan) was used as a substrate for assaying xylanase activity. The enzymatic nature of the protein allows for sensitive assays at the low levels of transgene protein found in transiently transformed tissue extracts. The xylanase (XYN) protein is stable, activity slopes are linear over long time periods and assays are cost-effective. Coupled with the GUSPlus reporter gene, the XYN reporter allows sensitive and accurate quantification of gene control sequences in transient expression systems.Abbreviations Act1 Rice actin promoter - AZCL-xylan Azurine cross-linked xylan - AU absorbance units - Blt4.9 Barley lipid transfer protein promoter - GEB GUS extraction buffer - GFP Green fluorescent protein - GluB-1 Rice glutelin B-1 promoter - GUS -Glucuronidase - LUC Luciferase - sXynA Synthetic xylanase A gene - Ubi-1 Maize ubiquitin promoter - XAB Xylanase assay buffer - XYN Xylanase Communicated by P. Lakshmanan     

Effects of antibiotics on the elimination of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> from loblolly pine (<Emphasis Type="Italic">Pinus taeda</Emphasis>) zygotic embryo explants and on transgenic plant regeneration

Three antibiotics were evaluated for their effects on the elimination of Agrobacterium tumefaciens during the genetic transformation of loblolly pine ( Pinus taeda L.) using mature zygotic embryos as targets. Agrobacterium tumefaciens strains, EHA105, GV3101, and LBA 4404, all harbouring the plasmid pCAMBIA1301, which carries the selectable marker gene, hygromycin phosphotransferase ( hpt) controlled by the cauliflower mosaic virus 35S promoter and terminator, and the uidA reporter gene (GUS) driven by the cauliflower mosaic virus 35S promoter and the terminator of nopaline synthase gene, were used in this study. Exposure to 350 mg l^-1 carbenicillin, claforan, and timentin respectively for up to 6 weeks did not eliminate the Agrobacterium, while antibiotics at 500 mg l^-1 eradicated them from the co-cultivated zygotic embryos. All three antibiotics increased callus growth and shoot regeneration at 350 and 500 mg l^-1 each, but reduced callus growth and shoot regeneration at 650 mg l^-1 when compared with controls. Putative transgenic calli were selected for continued proliferation and differentiation on 4.5 mg l^-1 hygromycin-containing medium. Transformed calli and transgenic plants produced on a selection medium containing 4.5 mg l^-1 hygromycin were confirmed by GUS histochemical assays, by polymerase chain reaction (PCR), and by Southern blot analysis. These results are useful for future studies on optimizing genetic transformation procedures in loblolly pine.     

A Clostridium perfringens Vector for the Selection of Promoters

A promoter selection vector for Clostridium perfringens genes was constructed from a C. perfringens-Escherichia coli shuttle vector, pJIR418. The plasmid carries a promoterless chloramphenicol acetyltransferase gene (catP), derived from pIP401, downstream of the multiple cloning sites of pUC18. When a promoter region of the phospholipase C gene was inserted into one of the cloning sites, derivatives of C. perfringens strain 13 carrying the resultant plasmid acquired resistance to chloramphenicol. This plasmid should be a useful reporter system for C. perfringens genes.