物定路段路面使用性能的预测模型

为了最大限度地利用信息量,提高路面管理系统中预测模型的精度,提出了综合平均模型、路段数据和专家经验等信息的特定路段模型,分析了特定路段模型的三种建模方法,并给出了特定路段模型的应用实例。     

Site-specific phosphorylation induces disassembly of vimentin filaments in vitro

Intermediate filaments are a major component of the cytoskeleton of eukaryotic cells. Although there appear to be at least five distinct classes of these filaments, cells of mesenchymal origin and most cells in culture contain the intermediate filament composed of the subunit protein vimentin. Vimentin exists in a nonphosphorylated as well as in a phosphorylated form, and there is increased phosphorylation of this protein when the filament undergoes marked redistribution in various cells. The role of phosphorylation on assembly-disassembly and organization of the vimentin filament has remained obscure. We report here a stable and purified system allowing biochemical examination of vimentin filament assembly and disassembly. Using this in vitro system, we carried out stoichiometrical phosphorylations, using purified protein kinases. We obtained evidence for site-specific, phosphorylation-dependent disassembly of the vimentin filament.     

DNA replication and recombination

Knowledge of the structure of DNA enabled scientists to undertake the difficult task of deciphering the detailed molecular mechanisms of two dynamic processes that are central to life: the copying of the genetic information by DNA replication, and its reassortment and repair by DNA recombination. Despite dramatic advances towards this goal over the past five decades, many challenges remain for the next generation of molecular biologists.     

DNA mutagenesis and recombination

The polymerase chain reaction is used for site-specific mutagenesis and for DNA recombination without any enzymatic reaction in vitro, apart from DNA amplification.     

重组质粒βhCG—pPIC9K的构建及嗜甲醇酵？…

目的：为用基因重组技术表达ｈＣＧ避孕疫苗抗原,构建在酵母细胞中表达的重组质粒βｈＣＧ－ｐＰＩＣ９Ｋ,转化嗜甲醇酵母,方法：根据βｈＣＧ的ｃＤＮＡ序列设计两条引物,使上游带ＥｃｏＲⅠ酶切位点,下游带ＮｏｔⅠ酶切位点,以质粒βｈＣＧ－ＰＢＳＫＳ为模板,进行ＰＣＲ扩增反应;将所得的ＤＮＡ片段经ＥｃｏＲⅠ和ＮｏｔⅠ双酶切后用Ｔ４连接酶与ｐＰＩＣ９Ｋ质粒进行连接,然后导入大肠杆菌ＤＨ５α,用ＰＣＲ筛选阳性克     

Site-specific uv crosslinking of minihelix DNA and TrpRS fromBacillus subtilis

In order to investigate the recognition mechanism and the relationship between structure and function of minihelix DNA with Tryptophanyl-tRNA Synthetase (TrpRS), TrpRS from Bacillus Subtilis was purified. Four minihelix DNAs were chemically synthesized and the photoreactive reagent s⁴T was incorporated into three of them at the positions of G73, T72 and T55 corresponding to tRNA^Trp. The apparatus for uv crosslinking was devised and the parameters for uv crosslinking were optimized. The results indicated that the G73 and T72 base of minihelix DNA interacted with TrpRS directly. The uv crosslinking reaction was improved by the dose of uv irradiation and the concentration of both TrpRS and minihelix DNA.     

Site-specific DICER and DROSHA RNA products control the DNA-damage response

Non-coding RNAs (ncRNAs) are involved in an increasingly recognized number of cellular events. Some ncRNAs are processed by DICER and DROSHA RNases to give rise to small double-stranded RNAs involved in RNA interference (RNAi). The DNA-damage response (DDR) is a signalling pathway that originates from a DNA lesion and arrests cell proliferation3. So far, DICER and DROSHA RNA products have not been reported to control DDR activation. Here we show, in human, mouse and zebrafish, that DICER and DROSHA, but not downstream elements of the RNAi pathway, are necessary to activate the DDR upon exogenous DNA damage and oncogene-induced genotoxic stress, as studied by DDR foci formation and by checkpoint assays. DDR foci are sensitive to RNase A treatment, and DICER- and DROSHA-dependent RNA products are required to restore DDR foci in RNase-A-treated cells. Through RNA deep sequencing and the study of DDR activation at a single inducible DNA double-strand break, we demonstrate that DDR foci formation requires site-specific DICER- and DROSHA-dependent small RNAs, named DDRNAs, which act in a MRE11–RAD50–NBS1-complex-dependent manner (MRE11 also known as MRE11A; NBS1 also known as NBN). DDRNAs, either chemically synthesized or in vitro generated by DICER cleavage, are sufficient to restore the DDR in RNase-A-treated cells, also in the absence of other cellular RNAs. Our results describe an unanticipated direct role of a novel class of ncRNAs in the control of DDR activation at sites of DNA damage.     

粗糙脉孢菌BenR基因在哈茨木霉中的定点转化

以β2-微管蛋白基因为定点整合位点,通过原生质体法将多菌灵抗性基因转化到哈茨木霉中,获得了具有多菌灵抗性的生物防治工程菌株．多菌灵抑制抗性转化子菌丝生长的ECso值达471．26μg／mL,比哈茨木霉原菌株提高1200倍以上;转化子对多菌灵的抗性具有遗传稳定性,且在无选择压力下菌丝生长速度及菌落形态与原菌株无显著差别;抑菌活性检测结果表明,3个转化子对立枯丝核菌均具有较强的抑菌活性,对菌丝生长的抑制率分别为87．5％、86．3％和85％．     

Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

For the temporally and spatially regulated expression of the barnase gene in plant, two kinds of plasmids with cre gene and its directly repeat recognition sitesiox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure. The transgenic plants were conformed by PCR analysis. The blocking fragment between the twolox directly repeat sites was excised by Cre protein in the transgenic plant genome. Cloning and sequencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre/lox site-specific recombination.     

Co-transformation to tobacco of Cre/lox site-specific recombination system and its precise recombination

For the temporally and spatially regulated expression of the barnase gene in plant,two kinds of plasmids with cre gene and its directly repeat recognition sites lox from bacteriophage P1 were constructed and co-transformed into tobacco by agrobacterium mediated procedure.The transgenic plants were conformed by PCR analysis.The blocking fragment between the two lox directly repeat sites was excised by Cre protein in the transgenic plant genome.Cloning and sequencing the DNA fragment from the co-transformed plant DNA showed that the precise DNA excision occurred in transgenic tobacco genome directed by Cre/lox site-specific recombination.