噬菌体434单链阻遏蛋白的高亲和力DNA结合序列的设计和筛选

一． 设计和筛选单链阻遏蛋白的高亲和力DNA结合序列 　　单链阻遏蛋白RRTRES是噬菌体434阻遏蛋白的衍生物，它是噬菌体434阻 遏蛋白的N端DBD(1-69位氨基酸)组成的共价二聚体。这个单链分子有两个DBD，一个是野生型噬菌体434的DBD-R，另一个是突变了的DBD - RTRES，二者用重组接头以头接尾的方式连接起来。在RTRES的α3-螺旋中．1、1、2、5位氨基酸与DNA识别紧密相关，它们分别为T、R、E、S。为了筛选出突变的RTRES的DNA结合位点，设计了核心序列为CATACAAGAAAGNNNNNNTTTATG随机DNA库，通过RRTRES与随机DNA库的体外结合和循环筛选。将筛选到的群体克隆并测序。通过与单链阻遏蛋白RRTRES的亲和力测定，对每一个筛选到的序列进行特性分析。结果表明，当结合位点（上述划线部分）为TTAC或TTCC时为最适操纵区序列。它们与单链阻遏蛋白RRTRES的亲和力很高，Kd值在1-10pM的范围。其中随机部分为TTTACG的操纵区与RRTRES的亲和力最高，Kd值约为lpM;当结合位点为TTAC时，平均Kd值为3pM：当结合位点为 TTCC时，Kd值在5-lOpM之间。天然噬菌体434阻遏蛋白与其操纵区的亲和力的Kd值在nM数量级，与之相比，所筛选操纵区的亲和力明显提高。此外，亲和力大小还受到结合位点两侧的碱基的影响，特别是5'位碱基的影响。 　　表达纯化同源双突变的单链阻遏蛋白RTRESRTRE'根据RTRES的以上识别特一点，设计了一系列新的操纵区序列，它们的共有序列为GTAAGAAARNTTACN，或GGAAGAAARNTTCCN，并测定它们与RTRES RTRE之间的结合特异性。结果表明，它们可被RTRES RTRES特异识别，且亲和力也很高，Kd值在5-40pM之间。其中GTAAGAAAGTTTACG与RTRES RTRES之间结合的Kd值约为5pM。同样，表达了异源双突变的单链阻遏蛋白R*RTRES，然而它与 设计的相关操纵区的亲和力并不很高，Kd值约为lOOpM。利用本工作中的随机筛选和合理设计的原则，得到了新的具有特异性识别和高亲和力的蛋白一DNA相互作用。这个方法可望用于其他DBP的新的结合特异性的筛选。 二． 非同位素的方法筛选单链阻遏蛋白的最佳DNA结合序列初探 　　克隆和表达了带半胱氨酸尾的单链阻遏蛋白，利用已包被了马来酰胺的活性板可以与自由巯基结合的特性，将蛋白固定在活性板表面。体外筛选RTRES RTRES的最佳DNA结合序列，得到了一些与RTRES RTRES结合的序列，但Kd值nM数量级。此方法需进一步优化。

The News, 31 October 1813 (no. 434) - Newspaper

Volumes of interest were published between 1812 and 1815 with articles about the War of 1812.

HG 434.40-434.55

Brown sediment with clasts ranging from small to medium in size. The clast shape ranges from sub-angular to sub-rounded. Grain crushing is abundant throughout the sample, and mainly involves medium sized clasts. Lineations can also be seen. A finer, and darker brown domain is also present within the sample.

The 434(G > C) polymorphism in the eosinophil cationic protein gene and its association with tissue eosinophilia in oral squamous cell carcinomas

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Resolución 434(XIX): Proyecto de plan de mediano plazo del sistema de la CEPAL para el período 1984-1989 = Resolution 434(XIX): Draft medium-term plan of the CEPAL system for the period 1984-1989 = Résolution 434(XIX): Project de Plan à moyen terme du système de la CEPAL pour la période 1984-1989

Publicado separadamete en cada idioma

Na 50 Nachlass Arthur Schopenhauer - 'Schopenhauer-Archiv', 434 - Abschrift von Gottlob Ernst Schulzes Rezension zu "Über die vierfache Wurzel des Satzes vom zureichenden Grunde" im Göttingischen Gelehrten Anzeiger

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Ms.Ff.F.Stoltze 3. 434 - Brief von Friedrich Stoltze an Hermann Stoltze

Krankheit Friedrich Stoltzes II, Stoltzes Reise nach Zürich

(Table 1) Composition of paraffin hydrocarbons at DSDP Site 56-434

Geochemical investigation of 18 samples of sediments from Site 434 involved determining the content of organic carbon, of bitumoid A (The chloroform A-chl and alcohol-benzene A-alb extracts) and its various fractions, and of individual hydrocarbons as well as the structural group composition of resins. We identified certain samples that differed sharply from the rest by their increased bitumen content and relatively low molecular hydrocarbons and by the fact that their resinous components were more neutral and aliphatic in composition. The distribution of bitumoid and its components seems to reflect migration processes in operation during the early stages of the transformation of organic matter.

(Table 1, page 434-435) Bulk chemical composition of S.W. Pacific island arc Mn-oxide crusts

Hydrothermal Mn-oxide crusts have been removed from the Tonga-Kermadec Ridge, the first such hydrothermal deposits to be reported in the S.W. Pacific island arc. In several respects the deposits are similar to hydrothermal Mn-crusts from oceanic spreading centre settings. They are limited in areal extent, comprise well-crystalline birnessite and generally display extreme fractionation of Mn from Fe. They are strongly depleted in many elements compared to hydrogenous Mn deposits but are comparatively enriched in Li, Zn, Mo and Cd. The Group IA and Group IIA metals show strong intercorrelations and the behaviour of Mg in the purest samples may indicate the extent to which normal seawater has influenced the composition of the deposits. Certain aspects of the deposits are not typical of hydrothermal Mn deposits. In particular at least some of the crusts have developed on a sediment or unconsolidated talus substrate. Some crusts, or specific layers within some crusts, display a chemical composition which suggests a significant input from normal seawater.