Screening of oilseed rape and other brassicaceous genotypes for susceptibility to Ceutorhynchus pallidactylus (Mrsh.)

Production of oilseed rape, Brassica napus L., is affected by various insect pests. The cabbage stem weevil, Ceutorhynchus pallidactylus (Mrsh.) (Col.: Curculionidae), is one of the most damaging pests in Northern and Central Europe that requires regular control measures. Host plant resistance is a key factor in integrated pest management systems. To evaluate a large number of genotypes for their susceptibility to infestation by C. pallidactylus, new screening techniques were developed for testing both, the amount of feeding and the number of eggs deposited by adult C. pallidactylus on accessions of Brassicaceae under controlled conditions. In no‐choice screening tests, the leaf area consumed by adult cabbage stem weevil was quantified on a wide spectrum of 107 brassicaceous genotypes (B. napus, Brassica rapa L. and Brassica oleracea L. cultivars, breeding lines, resynthesized rapeseed lines and wild Brassicaceae). In comparison to feeding on the standard cultivar ‘Express’, the average leaf area consumed by C. pallidactylus on nine oilseed rape cultivars, four resynthesized rapeseed lines and five other accessions [B. oleracea, Camelina alyssum (Mill.) and Lunaria annua L.] was significantly reduced by 44–90%. In dual‐choice screening tests for the evaluation of oviposition preferences on 42 genotypes, female C. pallidactylus laid significantly fewer eggs into plants of two oilseed rape cultivars, five resynthesized rapeseeds and three accessions of B. oleracea and Brassica fruticulosa Cyrillo, respectively, than into plants of the standard cv ‘Express’. Results of both laboratory screening tests were confirmed by results of additional field testing.     

蜕皮激素诱导下家蚕CYP3基因家族的表达变化

为了研究家蚕Bombyx mori CYP3家族基因经蜕皮激素诱导后的表达变化, 用蜕皮激素溶液（2×10^-3 μg/μL）浸泡的桑叶喂食家蚕B. mori 5龄幼虫, 以不用蜕皮激素处理的桑叶喂食家蚕为对照, 采用双跟踪标定定量PCR（dual-spike-in qPCR）方法, 检测在蜕皮激素诱导下家蚕中肠和脂肪体内CYP3基因家族的转录水平。结果表明: 与对照相比, 在蜕皮激素诱导下家蚕幼虫体内脂肪体中CYP302, CYP306和CYP339的转录水平分别上升了191.4, 7.4和421倍, 在中肠中变化不显著; 其余基因转录水平变化不明显或检测不到转录活性。结果说明CYP339基因有可能参与家蚕蜕皮激素的代谢, 这为进一步研究P450基因与内源物质的关系提供理论基础。     

Unnatural amino acids as probes of protein structure and function

Nonsense suppression methodology, for incorporating unnatural amino acids into proteins, has enabled a wide range of studies into protein structure and function using both in vitro and in vivo translation systems. Although methodological challenges remain, scores of unnatural amino acids have been employed that include both subtle and dramatic variants of the natural set. A number of insights that would not have been possible using conventional site-directed mutagenesis have been gained.     

A simple strategy for generation of gene knockdown constructs with convergent H1 and U6 promoters

RNA interference (RNAi) is a powerful tool for functional genetic studies in model organisms and mammalian cells. To facilitate rapid construction of gene knockdown constructs and RNAi libraries for known genes of mammalian cells, a new and simple strategy to produce small interfering RNA (siRNA) expression vectors with two opposing polymerase III promoters was developed. The design involved a one-step PCR amplification and single cloning procedure to construct a dual promoter siRNA expression vector. The forward primer is identical for all PCR reactions, only a single reverse primer that contains the siRNA targeting sequence has to be synthesized in the construction of each individual vector. This single primer design is cost-effective and it reduces the risk of sequence errors during synthesis of long oligos. Sense and antisense strands of siRNA duplexes were transcribed from the same template and this eliminated the need to synthesize long hairpin-forming oligonucleotides. Our study demonstrated that this vector design could mediate potent inhibition of expression of both exogenous and endogenous genes in mammalian cells.     

改造地高辛标记DNA和检测试剂盒用于凝胶阻滞实验的新方法

凝胶阻滞实验（gel retardation）,又称为电泳迁移率变动实验（Electrophoretic mobility shift assay,EMSA）,是研究蛋白和DNA相互作用的一种技术。传统的³²P标记探针的凝胶阻滞实验,具有很高的灵敏度,然而也有接触危险性的放射性同位素且不容易定量分析的缺点。最近利用非放射性标记的凝胶阻滞实验,已有很多成功的报导,该方法快捷,安全,灵活,但非放射性标记探针的凝胶阻滞试剂盒的费用却很高。在论文中,我们提供了一种改造地高辛标记DNA和检测试剂盒用于凝胶阻滞实验的新方法。首先将双链DNA探针末端引入EcoRI 粘性末端以便进行3′末端标记,然后利用价格比较便宜的地高辛标记DNA和检测试剂盒(DIG High Prime DNA Labeling and Detection Starter Kit II, Rohe) 进行探针标记和凝胶阻滞信号检测。经过多次实验参数的摸索,最终得到了成功的结果,为利用地高辛标记DNA和检测试剂盒进行凝胶阻滞实验提供了成功的例子和方法。      

Crystal structures of beryllium fluoride-free and beryllium fluoride-bound CheY in complex with the conserved C-terminal peptide of CheZ reveal dual binding modes specific to CheY conformation

Chemotaxis, the environment-specific swimming behavior of a bacterial cell is controlled by flagellar rotation. The steady-state level of the phosphorylated or activated form of the response regulator CheY dictates the direction of flagellar rotation. CheY phosphorylation is regulated by a fine equilibrium of three phosphotransfer activities: phosphorylation by the kinase CheA, its auto-dephosphorylation and dephosphorylation by its phosphatase CheZ. Efficient dephosphorylation of CheY by CheZ requires two spatially distinct protein-protein contacts: tethering of the two proteins to each other and formation of an active site for dephosphorylation. The former involves interaction of phosphorylated CheY with the small highly conserved C-terminal helix of CheZ (CheZ(C)), an indispensable structural component of the functional CheZ protein. To understand how the CheZ(C) helix, representing less than 10% of the full-length protein, ascertains molecular specificity of binding to CheY, we have determined crystal structures of CheY in complex with a synthetic peptide corresponding to 15 C-terminal residues of CheZ (CheZ(200-214)) at resolutions ranging from 2.0 A to 2.3A. These structures provide a detailed view of the CheZ(C) peptide interaction both in the presence and absence of the phosphoryl analog, BeF3-. Our studies reveal that two different modes of binding the CheZ(200-214) peptide are dictated by the conformational state of CheY in the complex. Our structures suggest that the CheZ(C) helix binds to a "meta-active" conformation of inactive CheY and it does so in an orientation that is distinct from the one in which it binds activated CheY. Our dual binding mode hypothesis provides implications for reverse information flow in CheY and extends previous observations on inherent resilience in CheY-like signaling domains.     

Declaration of Taking Twice: The Fazendeville Community of the Lower Ninth Ward

The Fazendeville Village was a residential community founded during the Reconstruction era in 1867 on the land where the Battle of New Orleans (1815) was fought during the War of 1812 in what is now Chalmette, LA. The entire community was displaced and their homes were razed in 1964 to provide more land for the National Historical Park to commemorate the battle. Most of the residents moved to the Lower Ninth Ward in New Orleans and now, 42 years later, they are displaced again and their homes (those that are still standing) will be razed because of the devastation of Hurricanes Katrina and Rita and the floods. Previous research has proven that vernacular networks affect the exodus, return, recovery, and rebuilding of certain communities after traumatic situations. In this study, I suggest that in the Fazendeville community during past trauma, the maintenance of cultural livelihood was caused by communality, spirituality, and traditionality. In addition, I propose that these are also the vernacular networks that are catalyst for community renewal and empowerment—after hurricanes, floods, and historic displacement by the federal government. My ultimate goal is to transform ethnographies into a praxis capable of making the community present and not marginalized or excluded from history and the strategic plan for rebuilding New Orleans.     

Direct observation of the distribution of fluorescent probes in phosphatidylcholine/cholesterol vesicles using flow microfluorometry

The technique of flow microfluorometry has been extended to the study of small lipid complexes to assess either the lipid (hydrophobic) or aqueous (hydrophilic) compartments of selected natural or model membrane systems. sn-1-Palmitoyl-sn-2-oleoyl-phosphatidylcholine/cholesterol unilamellar vesicles, averaging 268 nm in diameter and containing varying concentrations of the synthetic lipophile probe, sn-1-palmitoyl-sn-2-12-[N-4-nitrobenzo-2-oxa-1,3-diazole]-aminocaproyl-phosphatidylcholine (NBD-PC), were analyzed using an Ortho Series 50-H Cytofluorograf and an Ortho 2150 computer system. NBD-labeled vesicles were analyzed for green fluorescence and the intensity of scattered light, the later being analyzed both at low angle (2–5°) and at 90° to the incident beam. At the high amplification required for vesicle detection, background signals from the sheath buffer, nonspecific laser light, and electronic noise were observed. However, this background noise signal was removed by appropriately setting a discriminator window. Profiles of signals falling within this region were then constructed. For the settings selected, more than 98% of data recorded could be attributed to observations on vesicles. Size information from the intensity of scattered light was obtained by comparison of the sample with fluorescent microspheres after correcting for the particle-scattering function difference between hollow and solid spheres and for refractive index differences. Additionally, cytograms and profiles were constructed for vesicles containing 5 m 6-carboxyfluorescein, 3′,6′-dihydroxy-3-oxospiro(isobenzofuran-1 (3H),9′-(9H)xanthen)-6-carboxylic acid, trapped in the aqueous core. Thus, the utility of flow microfluorometry has been extended to much smaller particle populations than studied previously by this technique. It has significant potential for studying several important properties of selected populations of vesicles and lipoproteins including (i) the size and fluorescence distribution of particles, (ii) the equilibrium distribution of probes among different size populations and among different domains within populations, (iii) the time dependence of probe transfer from a specific labeled population to a specific unlabeled population, (iv) the time dependence of vesicle fusion (combining aqueous compartments), and (v) sorting particles which are labeled differently.     

A method for recovering strand-specific probes from nick-translated DNA fragments

A method of preparing strand-specific probes for DNA X DNA or DNA X RNA hybridizations is described. Double-stranded DNA fragments are first isolated from any recombinant DNA clone containing the desired sequence, and then labeled in vitro by nick-translation (T. Maniatis, A. Jeffrey, and D. G. Kleid (1975) Proc. Natl. Acad. Sci. USA 72, 1184-1188; P. W. J. Rigby, M. Dieckmann, C. Rhodes, and P. Berg (1977) J. Mol. Biol. 113, 237-251). Sequences homologous to the desired strand are captured by annealing the denatured nick-translate to viral strands of an appropriate M13 clone, and recovered by elution of the resulting hybrids from a column of agarose A50M (Bio-Rad). By this method, separate probes with specificity to either strand, as well as the double-stranded probe, may conveniently be prepared from a single nick-translation reaction. Probes may be obtained which are homologous either to the full length of the cloned region or to selected portions thereof by selecting appropriate M13 clones for annealing. The probe is recovered as a population of fragments several hundred bases or less in length, which have been found ideal for saturating liquid hybridizations, and should be similarly well suited for in situ hybridizations to cytological preparations.