Frequent,independent transfers of a catabolic gene from bacteria to contrasted filamentous eukaryotes

Even genetically distant prokaryotes can exchange genes between them, and these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain eukaryotes have acquired genes of bacterial origin. However, gene acquisitions in eukaryotes are thought to be much less important in magnitude than in prokaryotes. Here, we describe the complex evolutionary history of a bacterial catabolic gene that has been transferred repeatedly from different bacterial phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to multiple acquisitions of the gene in these filamentous eukaryotes—as many as 15 different events for 65 microeukaryotes. Furthermore, once transferred, this gene acquired introns and was found expressed in mRNA databases for most recipients. Our results show that effective inter-domain transfers and subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen at an unprecedented magnitude.     

小鼠的胚胎移植方法

全面而详细地描述了小鼠胚胎移植中所采用的输卵管移植及子宫移植方法,包括作者的改进。介绍了寄母小鼠的选择、配种及孕鼠的编号方法等,具有较强的实用意义。     

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology, agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer (MGCMGT)or female germ cell mediated gene transfer(FGCMGT)technique.Sperm-mediated gene transfer (SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been extensively used for producing transgenic animals.The newly developed approach of FGCMGT,ovary-mediated gene transfer(OMGT) is also a novel and useful tool for efficient transgenesis.This review highlights an overview of the recent progress in germ cell mediated gene transfer techniques,methods developed and mechanisms of nucleic acid uptake by germ cells.     

Exploring the mechanism of DNA polymerases by analyzing the effect of mutations of active site acidic groups in Polymerase β

Elucidating the catalytic mechanism of DNA polymerase is crucial for a progress in the understanding of the control of replication fidelity. This work tries to advance the mechanistic understanding by analyzing the observed effect of mutations of the acidic groups in the active site of Polymerase β as well as the pH effect on the rate constant. The analysis involves both empirical valence bond (EVB) free energy calculations and considerations of the observed pH dependence of the reaction. The combined analysis indicates that the proton transfer (PT) from the nucleophilic O3′ has two possible pathways, one to D256 and the second to the bulk. We concluded based on calculations and the experimental pH profile that the most likely path for the wild‐type (WT) and the D256E and D256A mutants is a PT to the bulk, although the WT may also use a PT to Asp 256. Our analysis highlights the need for very extensive sampling in the calculations of the activation barrier and also clearly shows that ab initio QM/MM calculations that do not involve extensive sampling are unlikely to give a clear quantitative picture of the reaction mechanism. Proteins 2016; 84:1644–1657. © 2016 Wiley Periodicals, Inc.     

Crystallization and preliminary X-ray analysis of electron transfer flavoproteins from human and Paracoccus denitrificans.

Mammalian electron transfer flavoprotein (ETF) is a soluble, heterodimeric flavoprotein responsible for the oxidation of at least nine primary matrix flavoprotein dehydrogenases. Crystals have been obtained for the recombinant human electron transfer flavoprotein (ETFhum) by the sitting-drop vapor diffusion technique using polyethylene glycol (PEG) 1500 at pH 7.0 as the precipitating agent. ETFhum crystallizes in the monoclinic space group P2(1), with unit cell parameters a = 47.46 angstrum, b = 104.10 angstrum, c = 63.79 angstrum, and beta = 110.02 degrees. Based on the assumption of one alpha beta dimer per asymmetric unit, the Vm value is 2.69 angstrum 3/Da. A native data set has been collected to 2.1 angstrum resolution. One heavy-atom derivative has also been obtained by soaking a preformed crystal of ETFhum in 2 mM thimerosal solution for 2h at 19 degrees C. Patterson analysis indicates one major site. The analogous electron transfer flavoprotein from Paracoccus denitrificans (ETFpar) has also been crystallized using PEG 8000 at pH 5.5 as the precipitating agent. ETFpar crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit cell parameters a = 79.98 angstrum, b = 182.90 angstrum, and c = 70.07 angstrum. The Vm value of 2.33 angstrum 3/Da is consistent with two alpha beta dimers per asymmetric unit. A native data set has been collected to 2.5 angstrum resolution.     

细菌胞际电子转移及其生态生理学意义研究进展

胞际电子转移是指细胞内电子以间接或直接的方式传递到细胞外,最终到达细胞周围电子受体的过程.胞际电子转移普遍存在于自然界,尤其存在于电子受体相对匮乏的环境中.胞际电子转移可分为间接和直接胞际电子转移.间接胞际电子转移(胞际基质转移)是主要借助氢、甲酸以及其他代谢产物的电子传递;而直接胞际电子转移则由胞内电子转移偶联胞外电子传递实现.胞际电子转移促进了细胞的基质代谢活性,拓展了细胞的作用空间,具有重要的生理意义.胞际电子转移产生了电流,实现了菌间能源共享,驱动了胞外物质(如重金属、腐殖质)转化,具体重大的生态意义.本文总结相关文献,对细菌胞际电子转移的过程、特点、机理及其生态生理学意义作了系统的分析和探讨.     

Progress in gene transfer by germ cells in mammals

Use of germ cells as vectors for transgenesis in mammals has been well developed and offers exciting prospects for experimental and applied biology,agricultural and medical sciences.Such approach is referred to as either male germ cell mediated gene transfer(MGCMGT) or female germ cell mediated gene transfer(FGCMGT) technique.Sperm-mediated gene transfer(SMGT),including its alternative method,testis-mediated gene transfer(TMGT),becomes an established and reliable method for transgenesis.They have been exten...     

Production of identical twins by separating two-cell rat embryos

Rat identical twins were produced from two-cell embryos. In the presence of cytochalasin B, rat two-cell embryos could be separated efficiently into two blastomeres by micromanipulation. Isolated blastomeres, embedded in agar cylinders and cultivated in ligated rat oviducts for 3 days, developed to the morula or blastocyst stage. After removing the agar, pairs of developed one-half embryos were transferred into Day 1 oviducts or Day 4 uteri of pseudopregnant rats. The percentage of embryos, separated either in the presence or absence of cytochalasin B, that developed into live fetuses was higher in cases of uterine transfer than in cases of oviduct transfer (38% vs. 18%, 31% vs. 15%, respectively). Throughout the present experiment, nine pairs of identical twins were successfully produced. This is the first report of the production of identical rat twins by separating two-cell embryos.     

Design principles of photo‐bioreactors for cultivation of microalgae

The present hype in microalgae biotechnology has shown that the topic of photo‐bioreactors has to be revisited with respect to availability in really large scale measured in hectars footprint area, minimization of cost, auxiliary energy demand as well as maintenance and life span. This review gives an overview about present designs and the basic limiting factors which include light distribution to avoid saturation kinetics, mixing along the light gradient to make use of light/dark cycles, aeration and mass transfer along the vertical or horizontal main axis for carbon dioxide supply and oxygen removal and last but not least the energy demand necessary to fulfil these tasks. To make comparison of the performance of different designs easier, a commented list of performance parameters is given. Based on these critical points recent developments in the areas of membranes for gas transfer and optical structures for light transfer are discussed. The fundamental starting point for the optimization of photo‐bioprocesses is a detailed understanding of the interaction between the bioreactor in terms of mass and light transfer as well as the microalgae physiology in terms of light and carbon uptake kinetics and dynamics.     

Attempts to observe through-chain energy transfer and electron transfer on α-helical polypeptide chain

Singlet singlet energy transfer between the two terminal chromophores attached to an α-helical polypeptide chain has been studied. The transfer efficiency was satisfactorily explained by Förster's theory when the interchromophore distance was calculated from the α-helical structure. Therefore, it was concluded that no particular effect from the possible energy band structure of the α-helical conformation was detected in the end-to-end energy transfer. Similarly, end-to-end electron transfer was attempted between the electron donor acceptor pair attached to the ends of α-helcial polypeptide chain. However, no intramolecular interaction was found between the donor acceptor pair, indicating that the exciton structure of the α-helical polypeptides is not effective enough to realize through-chain electron transfer.     

植物转基因方法概述

综述了植物基因转化的不同方法。植物基因转化方法主要包括直接基因转移和农杆菌介导的转化,直接基因转移又包括PEG介导、电激介导和基因枪方法。相比之下,农杆菌转化方法具有明显的优势;本文对其方法和机制作了较为全面的介绍。     

Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase

The c-type nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans is an integral membrane protein that catalyzes NO reduction; 2NO + 2e⁻ + 2H⁺ → N₂O + H₂O. It is also capable of catalyzing the reduction of oxygen to water, albeit more slowly than NO reduction. cNORs are divergent members of the heme-copper oxidase superfamily (HCuOs) which reduce NO, do not pump protons, and the reaction they catalyse is non-electrogenic. All known cNORs have been shown to have five conserved glutamates (E) in the catalytic subunit, by P. denitrificans numbering, the E122, E125, E198, E202 and E267. The E122 and E125 are presumed to face the periplasm and the E198, E202 and E267 are located in the interior of the membrane, close to the catalytic site. We recently showed that the E122 and E125 define the entry point of the proton pathway leading from the periplasm into the active site [U. Flock, F.H. Thorndycroft, A.D. Matorin, D.J. Richardson, N.J. Watmough, P. Ädelroth, J. Biol. Chem. 283 (2008) 3839-3845]. Here we present results from the reaction between fully reduced NOR and oxygen on the alanine variants of the E198, E202 and E267. The initial binding of O₂ to the active site was unaffected by these mutations. In contrast, proton uptake to the bound O₂ was significantly inhibited in both the E198A and E267A variants, whilst the E202A NOR behaved essentially as wildtype. We propose that the E198 and E267 are involved in terminating the proton pathway in the region close to the active site in NOR.     

Identification of proton transfer pathways in the X-ray crystal structure of the bacterial reaction center from Rhodobacter sphaeroides

Structural features that have important implications for the fundamental process of transmembrane proton transfer are examined in the recently published high resolution atomic structures of the reaction center (RC) from Rhodobacter sphaeroides in the dark adapted state (DQ_AQ_B) and the charged separated state (D⁺Q_AQ_B ^–); the latter is the active state for proton transfer to the semiquinone. The structures have been determined at 2.2 Å and 2.6 Å resolution, respectively, as reported by Stowell et al. (1997) [Science 276: 812–816]. Three possible proton transfer pathways (P1, P2, P3) consisting of water molecules and/or protonatable residues were identified which connect the Q_B binding region with the cytoplasmic exposed surface at Asp H224 & Asp M240 (P1), Tyr M3 (P2) and Asp M17 (P3). All three represent possible pathways for proton transfer into the RC. P1 contains an uninterrupted chain of water molecules. This path could, in addition, facilitate the exchange of quinone for quinol during the photocycle by allowing water to move into and out of the binding pocket. Located near these pathways is a cluster of electrostatically interacting acid residues (Asp-L213, Glu-H173, Asp-M17, Asp H124, Asp-L210 and Asp H170) each being within 4.5 Å of a neighboring carboxylic acid or a bridging water molecule. This cluster could serve as an internal proton reservoir facilitating fast protonation of Q_B ^– that could occur at a rate greater than that attainable by proton uptake from solution.     

Characterization of second site mutations show that fast proton transfer to QB − is restored in bacterial reaction centers of Rhodobacter sphaeroides containing the Asp-L213 → Asn lesion

The structural basis for proton coupled electron transfer to Q_B in bacterial reaction centers (RCs) was studied by investigating RCs containing second site suppressor mutations (Asn M44 Asp, Arg M233 Cys, Arg H177 His) that complement the effects of the deleterious Asp L213 Asn mutation [DN(L213)]. The suppressor RCs all showed an increased proton coupled electron transfer rate k _AB ⁽²⁾(Q_A ^–Q_B ^– + H⁺ Q_AQ_BH^–) by at least 10³ (pH 7.5) and a recombination rate k _BD (D⁺Q_AQ_B ^– DQ_AQ_B) 15–40 times larger than the value found in DN(L213) RCs. Proton transfer was studied by measuring the dependence of k _AB ⁽²⁾ on the free energy for electron transfer (G_et). k _AB ⁽²⁾ was independent of G_et in DN(L213) RCs, but dependent on G_et in native and all suppressor RCs. This shows that proton transfer limits the k _AB ⁽²⁾ reaction with a rate of 0.1s^–1 in DN(L213) RCs but is not rate limiting and at least 10⁸-fold faster in native and 10⁵-fold faster in the suppressor RCs. The increased rate of proton transfer by the suppressor mutations are proposed to be due to: (i) a reduction in the barrier to proton transfer by providing a more negative electrostatic potential near Q_B ^–; and/or (ii) structural changes that permit fast proton transfer through the network of protonatable residues and water molecules near Q_B.     

Phylogeny rather than ecology or lifestyle biases the construction of Escherichia coli–Shigella genetic exchange communities

Genetic material can be transmitted not only vertically from parent to offspring, but also laterally (horizontally) from one bacterial lineage to another. Lateral genetic transfer is non-uniform; biases in its nature or frequency construct communities of genetic exchange. These biases have been proposed to arise from phylogenetic relatedness, shared ecology and/or common lifestyle. Here, we test these hypotheses using a graph-based abstraction of inferred genetic-exchange relationships among 27 Escherichia coli and Shigella genomes. We show that although barriers to inter-phylogenetic group lateral transfer are low, E. coli and Shigella are more likely to have exchanged genetic material with close relatives. We find little evidence of bias arising from shared environment or lifestyle. More than one-third of donor–recipient pairs in our analysis show some level of fragmentary gene transfer. Thus, within the E. coli–Shigella clade, intact genes and gene fragments have been disseminated non-uniformly and at appreciable frequency, constructing communities that transgress environmental and lifestyle boundaries.     

Hydrodynamics-based transfection of the liver: entrance into hepatocytes of DNA that causes expression takes place very early after injection

BACKGROUND: The mechanism of gene transfer into hepatocytes by the hydrodynamics-based transfection procedure is not clearly understood. It has been shown that, after a hydrodynamic injection, a large proportion of plasmid DNA remains intact in the liver where it is bound to plasma membrane and suggested that this DNA could be responsible for the efficiency of the transfection. METHODS: We have investigated the problem by giving mice a hydrodynamic injection of isotonic NaCl, followed at different time intervals by a conventional injection of DNA, cold or labelled with (35)S, with cDNA of luciferase as a reporter gene. Then, we determined the consequences of that dual injection on luciferase expression and on DNA uptake by the liver and its intracellular fate. By such experiments, it is possible to establish the time dependency of the induction of liver changes caused by a hydrodynamic injection on the one hand and the expression and DNA uptake and fate on the other. Moreover, some experiments have been performed on primary cultures of hepatocytes isolated after a hydrodynamic injection of DNA. RESULTS: When DNA is given to mice by a conventional injection a few seconds after an hydrodynamic injection of isotonic NaCl, luciferase expression in the liver is considerably lower than that observed after a single hydrodynamic injection of the plasmid. On the other hand, as assessed by the rate of DNA degradation and by centrifugation results obtained after injection of (35)S-DNA, the uptake and the intracellular fate of the bulk of DNA are similar whether DNA is administered by a single hydrodynamic injection or by a conventional injection given up to at least 2 h after a hydrodynamic injection of isotonic NaCl. Hepatocytes isolated a few minutes after a hydrodynamic injection exhibit a maximal expression that does not depend on the large amount of DNA that remains bound to the plasma membrane for a relatively long time. CONCLUSIONS: Our results show that the efficiency of hydrodynamics-based transfection depends on a process that takes place very quickly after injection and is not linked to a delay of DNA degradation and the persistence of a large proportion of DNA bound to hepatocytes of the plasma membrane, strongly suggesting that expression after a hydrodynamic injection is caused by a small proportion of DNA molecules that rapidly enter the cytosol probably by plasma membrane pores generated by the hydrodynamic pressure.