DNA elements are asymmetrically joined during the site-specific recombination of kappa immunoglobulin genes

Immunoglobulin K genes are constructed during lymphocyte differentiation by the joining of two DNA elements, VK and JK, to form both a VKJK coding unit and a reciprocal recombination product. The two products formed in single VK-to-JK joining events can be directly isolated through the use of a retrovirally introduced recombination substrate. The structural analysis of a number of recombinants and the derivation of secondary recombination products define some of the basic features of the mechanism of immunoglobulin gene assembly.     

Cre／loxp位点特异性重组系统在转基因植物中的应用

位点特异性重组系统能够在植物遗传转化中更准确、可靠的操纵外源DNA的引入或删除,已成为植物遗传操作中的重要工具。本文简要介绍目前应用最为广泛的Cre/loxp位点特异性重组系统的重组机制,并着重阐述Cre/loxp系统在删除转基因植物中标记基因及定点整合等方面的应用。     

利用体外特异位点重组反应构建黄瓜灰霉菌cDNA文库

体外特异位点重组技术应用于cDNA文库的构建,克服了传统文库构建方法中的常见问题。本研究利用这一技术,以能够分离得到植物激活蛋白的黄瓜灰霉菌(B.cinerea)菌株为材料,用TRIZOL试剂提取总RNA,从中分离纯化出mRNA,用预先接有attB2位点的Oligo(dT)引物和SuperScriptTMⅡ反转录酶合成cDNA,双链cDNA与attB1接头连接,经重组、转化后构建了其cDNA文库。文库滴度为2.3×106pfu/mL,文库总容量达2.53×107。随机挑取24个克隆,经酶切检测,平均插入片断为1466bp,重组率达100%。     

DNA干扰现象及其作用机理研究进展

与体内某一基因相同的DNA序列可特异性抑制细胞内靶标基因的表达,这种现象称之为DNA干扰(DNAi)。DNAi是随着转录后基因沉默现象而在烟草属植物上被发现,之后在一些动植物及其细胞上也被发现。在原核生物中也存在DNAi现象,且原核生物的Ago在体外也能实现DNAi。原核生物DNAi的机理主要是Ago以DNA为向导切割DNA或RNA,而真核生物可能是基因转录后抑制、基因组甲基化和启动子结合的转录抑制等。本文还对DNAi的进一步研究和应用进行了讨论和展望。     

Association of crossover points with topoisomerase I cleavage sites: a model for nonhomologous recombination

Nonhomologous DNA recombination is frequently observed in somatic cells upon the introduction of DNA into cells or in chromosomal events involving sequences already stably carried by the genome. In this report, the DNA sequences at the crossover points for excision of SV40 from chromosomes were shown to be associated with eukaryotic topoisomerase I cleavage sites in vitro. The precise location of the cleavage sites relative to the crossover points has suggested a general model for nonhomologous recombination mediated by topoisomerase I.     

DNA: a model compound for solution studies of macromolecules

Well-defined, monodisperse, homologous series of oligonucleotides and DNA restriction fragments may now be produced and used as models of rigid and semirigid rodlike molecules in solution. Information from optical experiments on these model systems aids in the formulation and testing of theories of macromolecular dynamics in both dilute and concentrated solution.     

表达Cre重组酶的真核细胞系的建立及在重组伪狂犬病病毒研究中的应用

根据Cre基因序列设计并合成1对引物,PCR扩增出Cre基因编码区,克隆于pIREShyg载体,得到重组质粒pIREShyg-Cre,转染HEK-293A细胞后经400 μg·mL-1 Hygromycin B的筛选,将其中1个阳性克隆命名为293A-Cre.利用伪狂犬病病毒(Pseudorabies virus,PRV)S03109株(带有gfp报告基因表达盒及loxP位点)感染293A-Cre细胞,荧光显微镜观察、PCR及Western blot检测gfp基因的表达.结果表明,S03109感染293A-Cre二代后loxP位点间的gfp表达盒被删除,获得重组病毒S0419.将S0419感染已转染pBlulox的293A-Cre细胞,在RK13细胞上筛选得到表达LacZ的重组病毒S06293.S06293在293A-Cre细胞上传代2次,X-Gal原位染色表明LacZ的表达消失.测序结果证实,在Cre重组酶作用下,2个同向loxP序列之间的外源基因序列被正确除去.以上结果表明,本研究构建的稳定表达Cre重组酶的293A-Cre细胞系可以用于在包含有loxP位点的PRV基因组中外源基因的快速删除或整合.     

Synthesis of a site-specific DNA-binding peptide

The Hin recombinase binds to specific sites on DNA and mediates a recombination event that results in DNA inversion. In order to define the DNA-binding domain of the Hin protein two peptides 31 and 52 amino acids long were synthesized. Even though the 31mer encompassed the sequence encoding the putative helix-coil-helix-binding domain, it was not sufficient for binding to the 26-base pair DNA crossover site. However, the 52mer specifically interacted with the site and also effectively inhibited the Hin-mediated recombination reaction. The 52mer bound effectively to both the 26-base pair complete site and to a 14-base pair "half site." Nuclease and chemical protection studies with the 52mer helped to define the DNA base pairs that contributed to the specificity of binding. The synthetic peptide provides opportunities for new approaches to the study of the nature of protein-DNA interaction.     

A novel mechanism of somatic rearrangement predicted by a human T-cell antigen receptor beta-chain complementary DNA

The T-cell antigen receptor is a cell surface molecule vital in mediating the cellular immune response. The arrangement and rearrangement of the gene segments encoding the beta-chain polypeptide of the receptor are similar to those of immunoglobulin gene segments. The two constant region genes of the human T-cell antigen receptor are 8 kilobases apart with a cluster of joining segments located 5' of each constant region gene. Although most beta-chain gene rearrangements involve the variable, diversity, and joining segments, analysis of a beta-chain complementary DNA clone suggests the occasional occurrence of another type of rearrangement.     

Establishment of Ecotilling for Discovery of DNA Polymorphisms in Brassica rapa Natural Population

Ecotilling is a new approach based on enzyme-mediated heteroduplex cleavage to discover DNA polymorphisms in natural population. We used mung bean nuclease（MBN） instead of routinely used CELI to cleave single base pair mismatches in heteroduplex DNA templates. Nested set of primers were designed to amplify targeted region to avoid the influence of the variation in quality and quantity of the genomic DNA. To reduce the costs in fluorescently labeled primers, we added M13 adapter to 5＇end of gene specific primers to make IRD dye labeled M13 forward and reverse primers possibly universal for different genes. A Brassica rapa ZIP gene homologue was subjected to the analysis to practise the feasibility of the method in polymorphisms detection. Our experiment showed this method is efficient in discovering DNA polymorphisms in Brassica rapa natural population.     

High-efficiency ligation and recombination of DNA fragments by vertebrate cells

DNA-mediated gene transfer (transfection) is used to introduce specific genes into vertebrate cells. Events soon after transfection were quantitatively analyzed by determining the infectivity of the DNA from an avian retrovirus and of mixtures of subgenomic fragments of this DNA. The limiting step of transfection with two DNA molecules is the uptake by a single cell of both DNA's in a biologically active state. Transfected cells mediate ligation and recombination of physically unlinked DNA's at nearly 100 percent efficiency.     

Excel在油菜菌核病灰色预测模型构建中的应用

灰色系统理论，为病虫害预测预报提供了新的途径与方法。灰色预测模型的计算，其中关键是计算微分方程^AX（1）（K 1)=(x^(0)(1)-u/a)e^-ak u/a的解。利用Excel计算求解，具有操作方法简便易行，结果准确可靠、实用性强的优点。本文根据洞庭湖区1980～1992年油菜菌核病发病资料，利用Microsoft Excel 200进行计算，建立灰色GM(1，1)长期预测模型，经检验该模型精确度高，预测结果可靠。     

Scissors-grip model for DNA recognition by a family of leucine zipper proteins

C/EBP is a sequence-specific DNA binding protein that regulates gene expression in certain mammalian cells. The region of the C/EBP polypeptide required for specific recognition of DNA is related in amino acid sequence to other regulatory proteins, including the Fos and Jun transforming proteins. It has been proposed that these proteins bind DNA via a bipartite structural motif, consisting of a dimerization interface termed the "leucine zipper" and a DNA contact surface termed the "basic region." An evaluation of the properties of conserved amino acids within the basic region of 11 deduced protein sequences, coupled with the observation that they are located at an invariant distance from the leucine zipper, has led to the formulation of a "scissors-grip" model for DNA binding. The architectural features of this model are well suited for interaction with directly abutted, dyadsymmetric DNA sequences. Data supportive of the model were obtained with chemical probes of protein: DNA complexes.     

Delineating site-specific management zones on pasture soil using a probabilistic and objective model and geostatistical techniques

Precision Agriculture - In recent years, different algorithms have been utilised to delineate management zones, areas with similar properties, within agricultural fields. However, there are few...     

Multiplicity reactivation as a test for recombination function

Multiplicity reactivation of bacteriophage inactivated by ultraviolet light is dependent on the recombination function of either the host bacterial cell or the infecting bacteriophage. Absence of both recombination systems leads to a loss of multiplicity reactivation.     

Regional model for nitrogen fertilization of site-specific rainfed corn in haplustolls of the central Pampas,Argentina

In semi-arid regions, soil water and nitrogen (N) are generally limiting factors for corn (Zea mays L.) production; hence, implementation of appropriate N fertilization strategies is needed. The use of precision agriculture practices based on specific site and crop properties may contribute to a better allocation of fertilizer among management zones (MZ). The aim of this study was to develop a model for diagnosis of N availability and recommendation of N fertilizer rates adjusted to MZ for dryland corn crops growing in Haplustolls. The model considered variability between MZ by including site-specific variables [soil available water content at sowing (SAW) and Available Nitrogen (soil available N-NO₃ at planting + applied N, Nd)] using spatial statistical analysis. The study was conducted in Córdoba, Argentina in Haplustolls and consisted in four field trials of N fertilizer (range 0–161 kg N ha⁻¹) in each MZ. The MZ were selected based on elevation maps analysis. Grain yields varied between MZ and increased with larger SAW and Nd at sowing. Grain responses to Nd and SAW in any MZ were not different between sites, allowing to fit a regional model whose parameters (Nd, Nd², SAW, SAW²) contributed significantly (p < 0.001) to yield prediction. Agronomical and economically optimum N rates varied among MZs. However, the spatial variability of optimum N rates among MZs within sites was not enough to recommend variable N fertilizer rates instead of a uniform rate. Variable N fertilizer rates should be recommended only if variability in SAW and soil N among MZ is greater than that found in this work.     

几种微生物杀线虫剂对烟草根结线虫病的防效试验

为了确定3种微生物杀线虫剂IPC、ZK7、412对烟草根结线虫害的防治效果，通过田间小区试验，从烤烟的生物学性状、发病情况、经济学性状等方面进行了全面考察。结果表明，3种微生物杀线虫剂的防效均优于生物农药，其防治效果分别为61.7%、64.7%、70.5%,对烤烟生物学性状、经济学性状的改善效果也明显。因此，微生物杀线虫剂可在烤烟生产上推广应用。     

Chemical conversion of a DNA-binding protein into a site-specific nuclease

The tryptophan gene (trp) repressor of Escherichia coli has been converted into a site-specific nuclease by covalently attaching it to the 1,10-phenanthroline-copper complex. In its cuprous form, the coordination complex with hydrogen peroxide as a coreactant cleaves DNA by oxidatively attacking the deoxyribose moiety. The chemistry for the attachment of 1,10-phenanthroline to the trp repressor involves modification of lysyl residues with iminothiolane followed by alkylation of the resulting sulfhydryl groups with 5-iodoacetamido-1,10-phenanthroline. The modified trp repressor cleaves the operators of aroH and trpEDCBA upon the addition of cupric ion and thiol in a reaction dependent on the corepressor L-tryptophan. Scission was restricted to the binding site for the repressor, defined by deoxyribonuclease I footprinting. Since DNA-binding proteins have recognition sequences approximately 20 base pairs long, the nucleolytic activities derived from them could be used to isolate long DNA fragments for sequencing or chromosomal mapping.     

Widescale testing of the Crop-meter for site-specific farming

A sensor for measuring crop biomass density has been designed and developed to meet the demands for practical use in site-specific farming. The mechanical sensor named ‘Crop-meter’ is based on the pendulum principle. The suitability and measuring stability of the Crop-meter has been confirmed under field conditions in different regions of Germany. Significant correlations were obtained between Crop-meter signals and soil electrical conductivity (R ²=0.16−0.66) and grain yield (R ²=0.42−0.57). To test the suitability of the Crop-meter for site-specific management, it was used to control variable application rates for nitrogen fertiliser, growth regulators and fungicides in real time. A small increase in yield (3.1%) as well as reduced application rates for agrochemicals (14.6% nitrogen fertilisers; 23.1% fungicides and growth regulators) were proved in large-scale trials.