盐酸胍浓度对变性溶菌酶复性的影响

研究了复性液中盐酸胍浓度对变性溶菌酶复性的影响。变性酶的复性收率与复性液中盐酸胍度浓度紧密相关,获得高复性收率所需的盐酸胍浓度随酶浓度提高而增大。当酶浓度较低时（0．06－0．21g/L）,0．7mol/L的盐酸胍即可溶菌酶完全复性;当酶浓度较高时（0．6－1．05g/L）,提高盐酸胍浓度至1．0－1．5mol/L才可使复性收率达到95％以上。另外,酶的复性速率随盐酸胍浓度增大而下降。因此,根据酶浓度选择最佳盐酸胍浓度是提高蛋白质复性收率的关键。     

Denaturation and Renaturation of Viral Ribonucleic Acid I. Annealing R17 Ribonucleic Acid with Denatured Replicative Form or with Denatured Replicative Intermediate

Purified replicative form (RF) and replicative intermediate (RI) prepared from Escherichia coli infected with R17 were denatured in 0.15 m NaCl, 0.015 m sodium citrate containing 65% dimethylsulfoxide. Denaturation of RF or RI was demonstrated spectrophotometrically, chromatographically, and by sedimentation analysis. Denatured RF or RI was annealed by carefully decreasing the temperature from 62 to 20 C. Annealing was accompanied by a decreased absorbance at 260 mmu. The decrease in absorbance during annealing appeared to be dependent upon the rate of cooling and the concentration of ribonucleic acid (RNA). Denatured RF or RI was annealed with R17 RNA which was labeled with (3)H-uridine. The annealed product was 73 to 82% resistant to 0.1 mug/ml of ribonuclease. Annealing R17 RNA with either denatured RF or RI resulted in the formation of a ribonuclease-resistant product with a sedimentation profile resembling that of native RI. Melting the annealed products in 85.7% dimethyl sulfoxide produced 27S single-stranded R17 RNA and a heterogeneous population of more slowly sedimenting RNA.     

Renaturation of denatured DNA in NaI gradients

We show that denatured DNA from Tetrahymena mitochondria or phage T₇, partially renatures during centrifugation in NaI equilibrium density gradients. This makes these gradients unsuitable for the analysis of single-stranded complementary nucleic acids, especially if their complexity and mole percent G+C are low.     

Renaturation and Hybridization Studies of Mitochondrial DNA

The products of the renaturation reaction of mitochondrial DNA from oocytes of Xenopus laevis have been studied by electron microscopy and CsCl equilibrium density gradient centrifugation. The reaction leads to the formation of intermediates containing single-stranded and double-stranded regions. Further reactions of these intermediates result in large complexes of interlinking double-stranded filaments. The formation of circular molecules of the same length as native circles of mitochondrial DNA was also observed. The formation of common high molecular weight complexes during joint reannealing of two DNA's with complementary sequences was used as a method to detect sequence homology in different DNA samples. Although this method does not produce quantitative data it offers several advantages in the present study. No homologies could be detected between the nuclear DNA and the mitochondrial DNA of X. laevis or of Rana pipiens. In interspecies comparisons homologies were found between the nuclear DNA's of X. laevis and the mouse and between the mitochondrial DNA's of X. laevis and the chick, but none between the mitochondrial DNA's of X. laevis and yeast. These results are interpreted as indicating the continuity of mitochondrial DNA during evolution.     

A Mesoscale Model of DNA and Its Renaturation

A mesoscale model of DNA is presented (3SPN.1), extending the scheme previously developed by our group. Each nucleotide is mapped onto three interaction sites. Solvent is accounted for implicitly through a medium-effective dielectric constant and electrostatic interactions are treated at the level of Debye-Hückel theory. The force field includes a weak, solvent-induced attraction, which helps mediate the renaturation of DNA. Model parameterization is accomplished through replica exchange molecular dynamics simulations of short oligonucleotide sequences over a range of composition and chain length. The model describes the melting temperature of DNA as a function of composition as well as ionic strength, and is consistent with heat capacity profiles from experiments. The dependence of persistence length on ionic strength is also captured by the force field. The proposed model is used to examine the renaturation of DNA. It is found that a typical renaturation event occurs through a nucleation step, whereby an interplay between repulsive electrostatic interactions and colloidal-like attractions allows the system to undergo a series of rearrangements before complete molecular reassociation occurs.     

Renaturation of denatured, covalently closed circular DNA

The rate of renaturation of denatured, covalently closed, circular DNA (form Id DNA) of the phi X174 replicative form has been investigated as a function of pH, temperature, and ionic strength. The rate at a constant temperature is a sharply peaked function of pH in the range of pH 9 to 12. The position on the pH scale of the maximum rate decreases as the temperature is increased and as the ionic strength is increased. The kinetic course of renaturation is pseudo-first order: it is independent of DNA concentration, but falls off in rate from a first order relationship as the reaction proceeds. The rate of renaturation depends critically on the temperature at which the denaturation is carried out. Form Id, prepared at an alkaline pH at 0 degrees C, renatures from 5 to more than 100 times more rapidly than that similarly prepared at 50 degrees C. Both the heterogeneity in rate and the effect of the temperature of denaturation depend, in part, on the degree of supercoiling of the form I DNA from which the form Id is prepared. However, it is concluded that a much larger contribution to both arises from a configurational heterogeneity introduced in the denaturation reaction. The renaturation rate was determined by neutralization of the alkaline reaction and analytical ultracentrifugal analysis of the amounts of forms I and Id. The nature of the proximate renatured species at the temperature and alkaline pH of renaturation was investigated by spectrophotometric titration and analytical ultracentrifugation. It is concluded that the proximate species are the same as the intermediate species defined by an alkaline sedimentation titration of the kind first done by Vinograd et al. ((1965) Proc. Natl. Acad. Sci. U. S. A. 53, 1104-1111). Observations are included on the buoyant density of form Id and on depurination of DNA at alkaline pH values and high temperatures.     

Denaturation and Renaturation of Viral Ribonucleic Acid II. Characterization of the Products Resulting from Annealing R17 Ribonucleic Acid with Denatured Replicative Form or with Denatured Replicative Intermediate

The ribonucleic acid (RNA) product resulting from annealing R17 RNA with denatured replicative form or replicative intermediate could be divided into two distinct types of RNA by precipitation in 1.5 m NaCl. The RNA found in the salt supernatant fluid was resistant to digestion by ribonuclease, had a sedimentation coefficient of 15S, and displayed a sharp thermal transition. The RNA in the salt supernatant fluid appeared to be identical to replicative form. The RNA found in the salt precipitate was resistant to digestion by ribonuclease, but possessed both single- and double-stranded characteristics. The RNA sedimented as a broad band in a sucrose gradient, with a sedimentation coefficient of 15S, and displayed a melting transition characteristic of a mixture of single- and double-stranded RNA. Mild ribonuclease digestion of the salt-precipitable RNA produced a ribonuclease-resistant material with sedimentation properties identical to the RNA found in the salt supernatant fluid.     

变性双链法实现葡萄糖异构酶基因敲除的研究

对７号淀粉酶菌Ｍ１０３３的遗传背景进行分析,建立了其原生质体制备、转化的优化条件。构建了葡萄糖异构酶（ＧＩ）结构基因内插千方百计以链丝菌肽基因（ｔｓｒ）的置换型同源重组质粒,利用其变性双链片段实现与Ｍ１０３３菌株染色体上基因的同源重组,获得置换型葡萄异构酶缺陷型蓖Ｍ１０３３ＬＪ。为在染色体上引入突变位点实现染色体上分子定点改造造尊定了基础。     

Renaturation of DNA in the presence of ethidium bromide

The rate of renaturation of T2 DNA has been studied as a fuction of ethidium bound per nucleotide of denatured DNA. The Binding constants and number of binding sites for ethidium have been determined by spectral titration for denatured DNA at 55, 65, and 75°C and for native DNA at 65°C in 0.4M Na⁺. The rate of renaturation of T2 DNA was found to be independentof ethidium binding up to 0.03 moles per mole of nucleotide. Above 0.03 moles, the rate drops off precipitously approaching zero at 0.08 and 0.06 moles bound ethidium per nucleotide at 65°C respectively. A study was also made of the use of bound ethidium fluorescence as a probe for monitoring DNA renaturation reactions.     

Renaturation of DNA: a novel reaction of histones.

Histones isolated from several sources, either singly or in combination promote the renaturation of complementary single strands of DNA, as measured by the acquisition of resistance to S1 nuclease. The reaction is rapid (T1/2 less than 1 min), and is stoichiometric rather than catalytic. Renaturation is stimulated by Mg2+, Mn2+, and Ca2+, but is strongly inhibited by Zn2+. Crude extracts of early embryos of Drosophila melanogaster possess renaturation activity which is protease sensitive, heat-stable, and acid-soluble, suggesting that most or all of it can be attributed to histones. This observation thus provides a functional assay for histones that should prove useful in studies of chromatin and histone-DNA interactions, as well as for the identification and isolation of histones and histone-like proteins in crude extracts.     

Renaturation of copper solutions of DNA. I. Phenomenological analysis

A study of the kinetics of renaturation of cupric DNA solutions has been carried out by two physical properties methods. The use of a purely phenomenological analysis leads nevertheless to interesting conclusions concerning the transformation mechanism. We have found that: in the presence of Cu⁺⁺ ions, different. DNA samples exhibit an equivalent kinetic behavior; the system can be decomposed into two parts: DNA and copper ions, and the rate of formation of DNA should be faster than that of copper ions; the mechanism of transformation may be bimolecular, but further investigation is necessary to confirm this.     

蛋白质复性研究进展

许多蛋白在大肠杆菌中高效表达时,其产物常以无活性的包含体形式存在,包含体蛋白的复性往往是制备这些蛋白的关键步骤之一,蛋白复性包括肽链折叠和分子内二硫键的氧化这两个互相影响的过程,本文综述了蛋白折叠过程的研究进展,及促进蛋白折叠和二硫键氧化的方法。     

Renaturation of complementary DNA strands by herpes simplex virus type 1 ICP8.

ICP8, the major single-stranded DNA-binding protein of herpes simplex virus type 1, promotes renaturation of complementary single strands of DNA. This reaction is ATP independent but requires Mg2+. The activity is maximal at pH 7.6 and 80 mM NaCl. The major product of the reaction is double-stranded DNA, and no evidence of large DNA networks is seen. The reaction occurs at subsaturating concentrations of ICP8 but reaches maximal levels with saturating concentrations of ICP8. Finally, the renaturation reaction is second order with respect to DNA concentration. The ability of ICP8 to promote the renaturation of complementary single strands suggests a role for ICP8 in the high level of recombination seen in cells infected with herpes simplex virus type 1.