嗜盐古菌染色体DNA片段在大肠杆菌中的启动子功能

以启动子探针质粒pKK232-8为载体, 用微量量热法研究了源自盐生盐杆菌R1染色体的RM13 DNA片段在大肠杆菌HB101中的真细菌启动子功能, 该启动子RM13 DNA片段的大小为1000bp(碱基对), 它能启动探针质粒pKK232-8上的氯霉素乙酰水解酶基因(即:氯霉素抗性基因Cmr), 氯霉素抗性水平可达到150 mg•L－1, 抗性水平较高, 启动活性较大.研究结果表明, 在盐生盐杆菌染色体上可能存在具有双功能或多功能的启动子DNA片段, 这对系统发育、微生物遗传学和生物热化学等均有重要的意义.     

Microcalorimetric Studies on the Promoter Function in E. Coli TG1 from P. Maltophilia AT18 Chromosome DNA

In this paper, a promoter-probe plasmid pKK232-8 was used as a vector, which functioned in Escherichia coli TG₁ host. The plasmid DNA fragments from Pseudomonas maltophilia AT18 chromosome DNA active as promoter inEscherichia coli TG₁, the promoter function was studied by means of microcalorimetry, the promoter is about 800 bp DNA, it can promote the chloramphenicol (Cm) gene in plasmid pKK232-8, the Cm resistance level is about 80 μg mL^–1, the promoter activity is high. It implicates that there are probably many promoters in Pseudomonas maltophilia AT18 chromosome. All these information is readily obtained by an LKB 2277-204 heat conduction microcalorimeter. Microcalorimetry is a quantitative, inexpensive, and versatile method for microbiological genetic research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Microcalorimetric Studies of the Toxic Action of La^3＋ on Halobacterium Halobium R1 Growth

A microcalorimetric technique was used to evaluate the influence of La^3 on Halobacterium halobium R1 growth.By means of LKB-2277 bioactivity monitor,ampoule methos at 37℃,the thermogenic curves of Halobacterium halobium R1 growth were obtained.In order to analyze the results,the maximum power Pm and the growth rate constants k were determined,showing that values of Pm and k are linked to the concentration of La^3 .Addition of low concentration of La^3 can cause a decrease of the maximum heat production and growth rate constant.However,high concentration of La^3 may promote growth of Halobacterium halobium R1,but at much higher concentration of La^3 ,the growth of Halobacterium halobium R1 is inhibited again.For comparison,the shapes of Halobacterium halobium R1 cell were observed by means of transmission electron microscope.According to the thermogenic curves and TEM photos of Halobacterium halobium R1 under different conditions,it is clear that metabolic mechanism of Halobacterium halobium R1 growth is changed with the addition of La^3 .     

极端嗜盐古生菌启动子序列缺失突变的微量热研究

用微量热方法和DNA缺失突变技术研究了来源于极端嗜盐古生菌R1上的一个推测的启动子片段(RM10)在大肠杆菌中的启动子功能. 启动子片段融合到质粒pKK232-8上无启动子的氯霉素乙酰转移酶(CAT)基因前来检测它驱动基因表达的能力, 缺失分析RM10启动子片段定位具有启动活性的重要功能区. 实验结果从热动力学角度揭示, 这个启动子片段上含有－35区和－10区特征的1382～1517 bp(碱基对)区段是它在大肠杆菌中具有启动子功能的关键部分; 在1～1382 bp区段或1571～1848 bp区段上还存在它的负调控区. 该研究为基因启动子功能研究提供了一种新的、更加灵敏便捷的、化学与生物学相结合的方法.     

Notiz über mikrobiologische Umsetzungen mit Halobacterium halobium: Reduktion von 3-Oxobutansäure-ethylester und Hydrolyse von 3-Hydroxybutansäure-ethylester. Cooperative Effekte von Reduktase und Hydrolase

Note on Biotransformations with Halobacterium halobium: Reduction of Ethyl 3-Oxobutanoate and Hydrolysis of Ethyl 3-Hydroxybutanoate. Cooperative Effects of Reductase and Hydrolase The archaebacterium Halobacterium halobium, growing in saturated NaCl solution, is tested for its ability to achieve biotransformations. We found that this microorganism does accept only a small variety of compounds as substrates. Ethyl acetoacetate ( 1 ) is reduced to ethyl (S)-3-hydroxybutanoate ( 2 ) of optical purity of 40–76%, but in low chemical yields. The reduction is accompanied by hydrolysis of the hydroxy-ester to 3-hydroxybutanoic acid ( 3 ). Hydrolysis of rac-ester 2 by Halobacterium halobium gives (R)- 2 of optical purity of up to 88%, depending upon reaction time, together with the almost racemic hydroxy-acid 3 , both in low chemical yields. Hopes that the ‘extremist’ Halobacterium halobium would be able to effect unique conversions were not fulfilled.     

ULTRAVIOLET QUANTUM YIELDS OF PHOTOPHOSPHORYLATION IN Halobacterium halobium*

Quantum yields of photophosphorylation in Halobacterium halobium were determined for ultraviolet spectral bands between 276 and 365 nm, and at 565 nm wavelength, based on integral spectral cell absorptance, bacteriorhodopsin-specific cell absorptance and the corresponding quantum dose rates. In the ultraviolet, there is an almost linear decline of the quantum yields of photophosphorylation from 365 to 276 nm wavelength, despite the peak absorption of bacteriorhodopsin at 280 nm. The cycling quantum yield for 276 nm excitation of bacteriorhodopsin was determined as 4.5 ± 1.8%, which is about one fourth of the value of 19% for solubilized bacteriorhodopsin. Threshold energy fluence rates of 20 W m^?2 for UV-B radiation typify the photophosphorylation as three orders less sensitive than the sensory UV-B avoidance response that needs 0.02 W m^?2 as the threshold. Thus, UV-B avoidance appears as the dominating strategy for survival of the archaic bacterium H. halobium, rather than possible photoenergetic use of UV-B radiation and photorepair of UV-damage.     

BisPNA‐assisted Detection of Double‐stranded DNA via Electrochemical Impedance Spectroscopy

A highly effective strategy for quantification of plasmid which was a special dsDNA based on bisPNA by electrochemical impedance spectroscopy was presented in this work. Firstly, through Au?S bond, thiol‐terminated bisPNA probes were immobilized onto the gold electrode surface. Then bisPNA probes directly hybridized with target plasmid DNA pBR322 based on the PNA.DNA‐PNA invasion triplex without denaturation. In the presence of redox electroactive ions [Fe(CN)₆]^3?/4? as hybridization indicator, the charge transfer resistance (R_ct) was produced, and R_ct was measured via electrochemical impedance spectroscopy. Under optimal conditions, this strategy showed a good linear relationship between the ΔR_ct which was the difference of R_ct obtained before and after bisPNA hybridized with plasmid pBR322, and logarithm of the concentration of plasmid pBR322 within the range from 1 nM to 100 nM (R²=0.993), with a limit of detection (LOD) of 0.1 nM. Furthermore, this bisPNA‐assisted biosensor showed good stability and satisfactory analytical reliability. In addition, this novel bisPNA‐assisted biosensor also exhibited excellent analytical results in human serum.     

Construction of a Novel Expression System in <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> and its Application for 1,3-Propanediol Production

A novel expression system of Klebsiella pneumoniae was developed in order to improve 1,3-propanediol (1,3-PD) production using a K. pneumoniae–Escherichia coli shuttle vector pET28a consisting of the kanamycin-resistance gene promoter Pkan. The recombinant plasmid pETPkan-cat carrying the chloramphenicol acetyltransferase gene cat as selectable marker was constructed to test the availability of the promoter Pkan in K. pneumoniae. The results showed that the chloramphenicol acetyltransferase was apparently expressed in K. pneumoniae, and the recombinant strain had a high-level resistance to chloramphenicol, suggesting that the promoter Pkan was efficient in K. pneumoniae. Then, the expression system was applied to the expression of 1,3-PD oxidoreductase in K. pneumoniae. The enzyme was over-expressed, and the recombinant K. pneumoniae showed a nearly 3.0-fold decrease in peak level of the intermediary metabolite 3-hydroxypropionaldehyde and an increase of 16.5% in yield of 1,3-PD with respect to the wild-type strain. From these results, the first reported expression system has paved the way for improvement of 1,3-PD production and will be available and efficient for other heterologous gene expression in K. pneumoniae.     

STEADY HYPSOCHROMIC SHIFT OF PEAK RESPONSIVITY TO ATTRACTANT LIGHT FROM 590 nm TO 560 nm IN Halobacterium halobium*

Abstract— Peak responsivity of photoattraction in Halobacterium halobium cells shows steady hypsochromic shift from 590 nm wavelength under low irradiance conditions to 560 nm under high irradiance conditions. Inversion of the photoattractant response, as dependent on blue vs red background light, is compatible with the known properties of photochromic sensory rhodopsin-I (SR-I) with ground state maximum absorption at 587 nm. Relaxation of the photoattractant response in H. halobium, as a function of wavelength and irradiance, gives a hint at an antagonistic pigment or intermediate state, different from ground state SR-I, with peak sensitivity at 620 nm or even above. The less sensitive photoattractant response at 560 nm persists without photorelaxation and represents the peak responsivity under high irradiance conditions.     

Improvement of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> Endo-β-1,4-Xylanase Expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis> by Codon Optimization and Analysis of the Enzymic Characterization

The gene xynB from Aspergillus sulphureus encoding the endo-β-1,4-xylanase was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA and wild-type DNA were placed under the control of a glyceraldehyde-3-phosphate dehydrogenase gene promoter (GAP) in the constitutive expression vector plasmid pGAPzαA and electrotransformed into the P. pastoris X-33 strain, respectively. The transformants screened by Zeocin were able to constitutively secrete the xylanase in YPD liquid medium. The maximum yield of the recombinant xylanase produced by the synthetic DNA was 105 U ml⁻¹, which was about 5-fold higher than that by wild-type DNA under the flask culture at 28 °C for 3 days. The enzyme showed optimal activity at 50 °C and pH 5.0. The residual activity remained above 90% after the recombinant xylanase was pretreated in Na₂HPO₄–citric acid buffer (pH 2.4) for 2 h. The xylanase activity was significantly improved by Zn²⁺. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.     

Structure‐Based Design of Platinum(II) Complexes as c‐myc Oncogene Down‐Regulators and Luminescent Probes for G‐Quadruplex DNA

A series of platinum(II) complexes with tridentate ligands was synthesized and their interactions with G‐quadruplex DNA within the c‐myc gene promoter were evaluated. Complex 1 , which has a flat planar 2,6‐bis(benzimidazol‐2‐yl)pyridine (bzimpy) scaffold, was found to stabilize the c‐myc G‐quadruplex structure in a cell‐free system. An in silico G‐quadruplex DNA model has been constructed for structure‐based virtual screening to develop new Pt^II‐based complexes with superior inhibitory activities. By using complex 1 as the initial structure for hit‐to‐lead optimization, bzimpy and related 2,6‐bis(pyrazol‐3‐yl)pyridine (dPzPy) scaffolds containing amine side‐chains emerge as the top candidates. Six of the top‐scoring complexes were synthesized and their interactions with c‐myc G‐quadruplex DNA have been investigated. The results revealed that all of the complexes have the ability to stabilize the c‐myc G‐quadruplex. Complex 3 a ([Pt^II L2R ] ⁺ ; L2 =2,6‐bis[1‐(3‐piperidinepropyl)‐1H‐enzo[d]imidazol‐2‐yl]pyridine, R =Cl) displayed the strongest inhibition in a cell‐free system (IC₅₀=2.2 μM ) and was 3.3‐fold more potent than that of 1 . Complexes 3 a and 4 a ([Pt^II L3R ]⁺; L3 =2,6‐bis[1‐(3‐morpholinopropyl)‐1H‐pyrazol‐3‐yl]pyridine, R =Cl) were found to effectively inhibit c‐myc gene expression in human hepatocarcinoma cells with IC₅₀ values of ≈17 μM , whereas initial hit 1 displayed no significant effect on gene expression at concentrations up to 50 μM . Complexes 3 a and 4 a have a strong preference for G‐quadruplex DNA over duplex DNA, as revealed by competition dialysis experiments and absorption titration; 3 a and 4 a bind G‐quadruplex DNA with binding constants (K) of approximately 10⁶–10⁷ dm³ mol^?1, which are at least an order of magnitude higher than the K values for duplex DNA. NMR spectroscopic titration experiments and molecular modeling showed that 4 a binds c‐myc G‐quadruplex DNA through an external end‐stacking mode at the 3′‐terminal face of the G‐quadruplex. Intriguingly, binding of c‐myc G‐quadruplex DNA by 3 b is accompanied by an increase of up to 38‐fold in photoluminescence intensity at λ_max=622 nm.     

Direct Detection of DNA and DNA‐Ligand Interaction by Impedance Spectroscopy

In this work we present an impedimetric detection system for DNA‐ligand interactions. The sensor system consists of thiol‐modified single‐stranded DNA chemisorbed to gold. Impedance measurements in the presence of the redox system ferri‐/ferrocyanide show an increase in charge transfer resistance (R_ct) after hybridisation of a complementary target. Different amounts of capture strands, used for gold electrode modification, result in surface coverages between 3 and 15 pmol/cm² ssDNA. The relative change in R_ct upon hybridisation increases with increasing amount of capture probe on the electrode from 1.5‐ to 4.5‐fold. Impedimetric detection of binding events of a metal‐intercalator ([Ru(phen)₃]²⁺) and a groove binder (spermine) to double‐stranded DNA is demonstrated. Binding of [Ru(phen)₃]²⁺ and spermine exhibits a decrease in charge transfer resistance. Here, the ligand’s interaction leads to electrostatic shielding of the negatively charged DNA backbone. The impedance changes have been evaluated in dependence on the concentration of both DNA binders. Furthermore, the association of a single‐stranded binding protein (SSBP) is found to cause an increase in charge transfer resistance only when incubated with single‐stranded DNA. The specific binding of an anti‐dsDNA antibody to the dsDNA‐modified electrode surface decreases in contrast the interfacial impedance.     

DNA Interactions of the Functionalized (Mixed Polypyridine)ruthenium(II) Complex Bis(2,2′‐bipyridine‐κN1,κN1′)(methyl dipyrido[3,2‐a : 2′,3′‐c]phenazine‐11‐carboxylate‐κN4,κN5)ruthenium(2+) ([Ru(bpy)2(dppz‐11‐CO2Me)]2+)

A novel polypyridine ligand, dipyrido[3,2‐a:2′,3′‐c]phenazine‐11‐carboxylic acid methyl ester (=dppz‐11‐CO₂Me), and its ruthenium(II) complex, [Ru(bpy)₂(dppz‐11‐CO₂Me)]²⁺ ( 1 ), were synthesized and characterized. The binding properties of this complex to calf‐thymus DNA (CT‐DNA) were investigated by different spectrophotometric methods and viscosity measurements. The results suggest that the complex binds to DNA in an intercalative mode and serves as a molecular ‘light switch’ for DNA. When irradiated at 365 nm, the complex 1 promoted the photocleavage of plasmid pBR‐322 DNA.     

Synthesis,magnetic properties,DNA binding and cleavage activity of a new oxalato bridged copper(II) complex

A novel oxalato‐bridged copper(II) complex has been prepared and structurally characterized: [Cu(bpa)(μ‐C₂O₄)].H₂O (bpa = bis(2‐pyridylmethyl) amine). In the complex, the copper ion is linked in an unusual μ_1,2,3‐C₂O₄^2? bridging mode, generating one‐dimensional zigzag chain disposition. Variable‐temperature magnetic susceptibility studies (2–300 K) reveal a weak ferromagnetic coupling, J = 0.63 cm^?1, between the copper ions. The interaction of the complex with CT‐DNA has been studied using UV–visible absorption and emission spectral methods, and the binding constant of the complex with CT‐DNA is K_app = 9 × 10⁴ m ^?1, which indicates that the interaction of the complex with DNA is a moderate intercalative mode. Furthermore, the complex cleaves supercoiled plasmid DNA efficiently in the presence of hydrogen peroxide. The mechanistic investigations suggest that the hydroxyl radical and singlet oxygen are involved in the DNA degradation. Copyright © 2012 John Wiley & Sons, Ltd.     

Preparation,characterizations and biological evaluations of new copper(II) complexes containing ONO pincer type ligands

A new heterocyclic Schiff bases, 6‐methyl/8methyl‐2‐oxo‐1,2‐dihydroquinoline‐3‐carboxaldehyde semicarbazones (H₂‐6MOQsc‐H) ( H ₂ L ¹ ) and (H₂‐8MOQsc‐H) ( H ₂ L ² ) and their corresponding copper(II) complexes [CuCl₂(H₂‐6MOQsc‐H)].3H₂O ( 1 ), [CuCl₂(H₂‐8MOQsc‐H)].3H₂O ( 2 ), [CuNO₃(H₂‐6MOQsc‐H)(H₂O)].NO₃ ( 3 ) and [CuNO₃(H₂‐8MOQsc‐H)(H₂O)].NO₃ ( 4 ) have been synthesized and characterized by various physicochemical techniques. The single crystal X‐ray diffraction and spectral data revealed that all of the complexes ( 1‐4 ), the ligands coordinated to the Cu(II) ion in a neutral manner via ONO donor atoms and all the complexes exhibited distorted squarepyramidal geometry. The consequence of electronegativity and ring size of nitrogen heterocyclic moiety of ONO donor type of copper(II) chelates on nucleic acid interaction and albumin binding was investigated by in vitro experiments. The interaction of compounds with calf‐thymus DNA (CT‐DNA) has been explored by absorption and emission titration, which exposed those ligands/complexes, could bind with CT‐DNA through electrostatic interaction. The results of gel electrophoresis proved the ability of complexes ( 1‐4 ) to cleave the pBR322 plasmid DNA. The interaction of serum albumin (BSA) was investigated by UV‐Vis, fluorescence, synchronous and three dimensional fluorescence spectra. In addition, radical scavenging activity, antifungal activity and cytotoxicity of the newly synthesized compounds were also evaluated. From the results of in vitro studies, it is seen that complex 3 has more potential as compared with other complexes and ligands.     

Stabilization of G‐Quadruplex DNA with Platinum(II) Schiff Base Complexes: Luminescent Probe and Down‐Regulation of c‐myc Oncogene Expression

The interactions of a series of platinum(II) Schiff base complexes with c‐myc G‐quadruplex DNA were studied. Complex [PtL ^1a ] ( 1 a ; H₂L ^1a =N,N′‐bis(salicylidene)‐4,5‐methoxy‐1,2‐phenylenediamine) can moderately inhibit c‐myc gene promoter activity in a cell‐free system through stabilizing the G‐quadruplex structure and can inhibit c‐myc oncogene expression in cultured cells. The interaction between 1 a and G‐quadruplex DNA has been examined by ¹H NMR spectroscopy. By using computer‐aided structure‐based drug design for hit‐to‐lead optimization, an in silico G‐quadruplex DNA model has been constructed for docking‐based virtual screening to develop new platinum(II) Schiff base complexes with improved inhibitory activities. Complex [PtL ³ ] ( 3 ; H₂L ³ = N,N′‐bis{4‐[1‐(2‐propylpiperidine)oxy]salicylidene}‐4,5‐methoxy‐1,2‐phenylenediamine) has been identified with a top score in the virtual screening. This complex was subsequently prepared and experimentally tested in vitro for its ability to stabilize or induce the formation of the c‐myc G‐quadruplex. The inhibitory activity of 3 (IC₅₀=4.4 μM ) is tenfold more than that of 1 a . The interaction between 1 a or 3 with c‐myc G‐quadruplex DNA has been examined by absorption titration, emission titration, molecular modeling, and NMR titration experiments, thus revealing that both 1 a and 3 bind c‐myc G‐quadruplex DNA through an external end‐stacking mode at the 3’ terminal face of the G‐quadruplex. Such binding of G‐quadruplex DNA with 3 is accompanied by up to an eightfold increase in the intensity of photoluminescence at λ_max=652 nm. Complex 3 also effectively down‐regulated the expression of c‐myc in human hepatocarcinoma cells.     

多吡啶锌配合物键合与切割DNA研究

The complex of Zn[(phen)(dione)Cl]ClO_4·H_2O(where phen is 1,10-phenanthroline and dione is 1,10-phenan-throline-5,6-dione)has been synthesized and characterized.The interaction of the complex with DNA was investi-gated using UV absorption,fluorescence spectroscopy and electrophoresis measurements.The results show that thecomplex mainly binds to the double helix of DNA with intercalation mode and the binding constant K is 2.4×10~4mol~(-1)·L.Moreover,the complex can efficiently cleave plasmid DNA at physiological pH and temperature.Thecleavage occurs via a hydrolysis mechanism,which is showed by adding radical scavengers,rigorously anaerobicexperiments,analysis for malondialdehyde-like products,and the hydrolysis experiment of BDNPP with a rate con-stant k_(obs)of 5.3×10~(-6)s~(-1).     

Synthesis, Characterization, DNA-binding Properties and DNA Cleavage of a New Ternary Copper(Ⅱ) Complex with Mixed-ligands of Tridentate Schiff Base and 1,10-Phenanthroline

A new copper(II) complex, [Cu(naph‐leu)phen]CH₃OH·0.5H₂O, in which naph‐leu is the tridentate Schiff base ligand derived from the condensation of 2‐hydroxy‐1‐naphthaldehyde and L‐leucine, phen is phenanthroline, has been synthesized and characterized by elemental analyses, IR spectra and single crystal X‐ray diffraction. The DNA‐binding properties of this complex have been investigated by absorption spectra, fluorescence spectra and circular dichroism (CD) spectra, as well as viscosity measurement. Results show that this copper(II) complex binds to calf thymus DNA (CT‐DNA) in an intercalative mode and its intrinsic binding constant K_b is 4.87×10³ L·mol^?1. Furthermore, the DNA cleavage activity of this copper(II) complex has also been investigated by submarine gel electrophoresis. Interestingly, it was found that this complex can cleave the supercoiled plasmid pBR322 DNA to both nicked and linear forms.     

A Microcalorimetric Method for Studying Halobacterium halobium

The application of a microcalorimetric method to the study of extremophiles is described briefly. Using the LKB 2277 Bioactivity Monitor, the growth thermogenic curves of three strains of Halobacterium halobium were determined at 37°C, and compared with the spectrophotometric curves. Then the suitable growth thermokinetic equation was established based on the characteristics of growth thermogenic curves. By using cycle-flow method, all of the growth thermogenic curves of H. halobium strains displayed a brief lag phase before the onset of exponential growth when they were cultured in Halo-2 medium. This revised version was published online in August 2006 with corrections to the Cover Date.