A gene involved in the metabolic control of ppGpp synthesis

Summary A genetic locus has been identified which controls the basal synthesis of ppGpp in growing E. coli. Cells carrying a recessive allele of the relX gene have a very low concentration of ppGpp during balanced growth, and fail to accumulate ppGpp in response to carbon/energy source downshift. Moreover, the recessive relX allele renders the cells unable to grow at 42° C and, when coupled with relA, makes the cells sensitive to the presence of leucine in minimal medium. RelX is cotransduced with fuc and relA and located at approximately 59.4 min on the E. coli genetic map.     

黑河上游灌丛建群种中国沙棘自由授粉子代父本分析和花粉流

利用SSR分子标记对中国沙棘（Hippophae rhamnoides ssp. sinensis Rousi）自由授粉的种子进行父本分析,研究其子代的父本来源和花粉散布模式。结果显示：在80%的置信水平上,193个子代中有104个个体可以确定花粉来源;在20个确定的父本中,16个为中国沙棘,4个为肋果沙棘（H.neurocarpa S.W.Liu et T.N.He）。传粉格局分析结果显示,中国沙棘有效花粉散布范围为3~71 m,平均距离为20.4 m,2株母本分别有87.23%和78.95%的有效花粉来自距其30 m的范围之内,研究结果表明中国沙棘自然种群以近距离传粉为主。此外,在黑河上游沙棘杂交带中,中国沙棘子代中的肋果沙棘花粉平均贡献率达到14.84%,表明中国沙棘与肋果沙棘存在较高的种间当代花粉流。     

植物蛋白质S-亚硝基化修饰的检测与分析

S-亚硝基化是一种重要的蛋白质翻译后修饰方式, 是指一氧化氮(NO)基团共价连接至靶蛋白特定半胱氨酸残基的自由巯基, 从而形成S-亚硝基硫醇(SNO)的过程。S-亚硝基化修饰广泛存在于各有机体中, 通过改变蛋白质生化活性、稳定性、亚细胞定位以及蛋白质-蛋白质相互作用等机制而调控不同的生物学过程或信号通路。在蛋白质S-亚硝基化检测分析方法中, 最为广泛使用的是生物素转化法(biotin switch assay), 其基本原理是首先封闭未被修饰的自由巯基, 进而将被修饰的SNO基团特异地还原为自由巯基并使用生物素将其特异标记。被生物素标记的半胱氨酸残基(即被修饰位点)可进一步通过蛋白质免疫印迹和/或质谱等方法进行检测分析。该文详细描述了植物蛋白质样品的体内和体外生物素转化法的实验流程, 并对实验过程中的注意事项进行了讨论。     

Partial purification of alpha-hemolysin inEscherichia coli

The present paper describes isolation and purification ofa-hemolysin ofEscherichia coli. The optimum production medium was found to be the Todd—Hewitt broth. Out of thirteen fractions obtained after separation on Sephadex G-200, two fractions possessed the highest relative specific activity.     

根癌农杆菌对单子叶植物竹子的细胞转化

本实验证明:具有重要经济价值的禾本科植物——竹子(bamboo)可被根癌农杆菌转化。在经有毒性的根癌农杆菌C_(580、A_(348)感染的竹子材料中,分别测到了专性的胭脂碱和章鱼碱,而对照处理及无毒性的根癌农杆菌VirA~-_(211)mx、NTI感染的竹子材料中,测不到冠瘿碱。     

建立生态经济型橡胶园橡胶咖啡间作模式

一、云南植胶区的自然地理条件云南橡胶种植区位于云南高原南部,北纬20°09′—24°59′,东经93°31′—105°08′,海拔100—1000m之间的热带季风地区。包括文山、红河、思茅、西双版纳、临沧和德宏地区     

显微高速摄影技术及其在血液微流变学研究中的应用

本文选用金黄地鼠的微血管,利用显微高速摄影技术,放大微观流场和血细胞,连续地“冻结”短瞬间的变化状态,把物理图象呈现在胶片上,经图像分析和数据处理,从定性及定量方面研究血液微流变学,为生命科学和医学研究提供了又一种新的方法。     

A spin label study of immobilized enzyme spectral subpopulations

Electron spin resonance (ESR) spin label studies have been carried out to examine the active site conformation of alpha-chymotrypsin before and after immobilization on two types of organic polymer supports: Amberlite XAD-8 and XAD-2. alpha-Chymotryspin was first chemically modified by reaction with methyl-4-phenylbutyrimidate and then inhibited by the active site spin label 4-(2,2,6,6-tetramethyl-piperdine-1-oxyl)-m-flurosulfonylbenzamide. In general, the ESR spectra of the active site lable revealed no significant changes in conformation for most of the enzyme before or after derivatization. On the other hand, two spectral subpopulations (A and B) of spin-labeled enzyme were characterized on the basis of their ESR spectra after immobilization on Amberlite XAD-8. Spectral subpopulation A (distinguished by a highly restrained spectrum) appeared to retain its active site structure and conformation and represented a large majority of the labeled chymotrypsin on the beads. Its presence correlated with the high activity and stability of phenylbutyramidinated chymotryspin on the Amberlite XAD-8 beads. Spectral subpopulation B (distinguished by a very weakly constrained spectrum) appeared to reflect loosely bound or denatured enzyme which was removable upon washing with 40% (v/v) ethylene glycol. Two methods for examining solvent accessibility to the active site lable of the kinetics of ascorbate reduction suggested that both spectral subpopulations had identical accessibilities to the bulk solvent. Paramagnetic broadening of the signal by K(3)Fe(CN)(6) revealed differences in the spin-spin broadening of the A and B components but is deemed and inappropriate indicator of solvent accessibility.     

Key residues in the allosteric transition of Bacillus stearothermophilus pyruvate kinase identified by site-directed mutagenesis.

The structural gene for pyruvate kinase from Bacillus stearothermophilus has been cloned in Escherichia coli and sequenced. The open reading frame from the ATG start codon to the TAG stop codon is 1482 base-pairs and encodes a peptide of relative molecular mass 52,967. In the expression vector pKK223-3, containing the synthetic tac promoter, the gene is overexpressed in E. coli cells to an estimated level of 30% total soluble cell protein. A purification procedure for the overexpressed protein has been established. The construction and characterization of a pair of mutant proteins has given insight into the structural basis of allosteric regulation in the tetrameric enzyme. Substituting tryptophan for tyrosine at position 466 (mutant Trp466-->Tyr) resulted in an activated form of the enzyme, having a reduced K1/2 for the substrate phosphoenolpyruvate. We propose that the characteristics of this mutant might be the result of bulk removal releasing steric inhibition to the formation of an interdomain salt bridge between Asp356 and Arg444. The regulatory behaviour of the double mutant produced by making the additional substitution aspartate for glutamate at position 356 (Trp466-->Tyr/Asp356-->Glu) corroborates this. The position of the salt bridge is such that it might be pivotal to the conformation of a pocket that is proposed to open up when the active R-conformation is adopted. We suggest that the mechanism of activation of B. stearothermophilus pyruvate kinase by ribose-5-phosphate might hinge on an interaction with, or indirectly through, residue Trp466, removing it from the vicinity of the potential salt bridge between Asp356 and Arg444 and thus effecting a closing together of the protein structure concomitant with an opening up of the pocket region.