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  • 1.

    【2hr】Stress-Induced Modulators of Repeat Instability and Genome Evolution

    包量

    机译 重复诱导不稳定性和基因组进化的应激诱导调节剂
    摘要：Evolution hinges on the ability of organisms to adapt to their environment. A key regulator of adaptability is mutation rate, which must be balanced to maintain genome fidelity while permitting sufficient plasticity to cope with environmental changes. Multiple mechanisms govern an organism's mutation rate. Constitutive mechanisms include mutator alleles that drive global, permanent increases in mutation rates, but these changes are confined to the subpopulation that carries the mutator allele. Other mechanisms focus mutagenesis in time and space to improve the chances that adaptive mutations can spread through the population. For example, environmental stress can induce mechanisms that transiently relax the fidelity of DNA repair to bring about a temporary increase in mutation rates during times when an organism experiences a reduced fitness for its surroundings, as has been demonstrated for double-strand break repair in Escherichia coli. Still, other mechanisms control the spatial distribution of mutations by directing changes to especially mutable sequences in the genome. In eukaryotic cells, for example, the stress-sensitive chaperone Hsp90 can regulate the length of trinucleotide repeats to fine-tune gene function and can regulate the mobility of transposable elements to enable larger functional changes. Here, we review the regulation of mutation rate, with special emphasis on the roles of tandem repeats and environmental stress in genome evolution.
  • 2.

    【2hr】What Limits the Efficiency of Double-Strand Break-Dependent Stress-Induced Mutation in Escherichia coli?

    包量

    机译 是什么限制了大肠杆菌中双链断裂依赖性应激诱导突变的效率？
    摘要：Stress-induced mutation is a collection of molecular mechanisms in bacterial, yeast and human cells that promote mutagenesis specifically when cells are maladapted to their environment, i.e. when they are stressed. Here, we review one molecular mechanism: double-strand break (DSB)-dependent stress-induced mutagenesis described in starving Escherichia coli. In it, the otherwise high-fidelity process of DSB repair by homologous recombination is switched to an error-prone mode under the control of the RpoS general stress response, which licenses the use of error-prone DNA polymerase, DinB, in DSB repair. This mechanism requires DSB repair proteins, RpoS, the SOS response and DinB. This pathway underlies half of spontaneous chromosomal frameshift and base substitution mutations in starving E. coli [Proc Natl Acad Sci USA 2011;108:13659–13664], yet appeared less efficient in chromosomal than F′ plasmid-borne genes. Here, we demonstrate and quantify DSB-dependent stress-induced reversion of a chromosomal lac allele with DSBs supplied by I-SceI double-strand endonuclease. I-SceI-induced reversion of this allele was previously studied in an F′. We compare the efficiencies of mutagenesis in the two locations. When we account for contributions of an F′-borne extra dinB gene, strain background differences, and bypass considerations of rates of spontaneous DNA breakage by providing I-SceI cuts, the chromosome is still ∼100 times less active than F. We suggest that availability of a homologous partner molecule for recombinational break repair may be limiting. That partner could be a duplicated chromosomal segment or sister chromosome.
  • 3.

    【2hr】The smallest active carbamoyl phosphate synthetase was identified in the human gut archaeon Methanobtrevibacter smithii

    包量

    机译 在人类肠道古细菌Methanobtrevibacter smithii中鉴定出最小的活性氨基甲酰磷酸合成酶
    摘要：The genome of the major intestinal archaeon, Methanobrevibacter smithii, contains a complex gene system coding for carbamoyl phosphate synthetase (CPSase) composed of both full-length and reduced-size synthetase subunits. These ammonia-metabolizing enzymes could play a key role in controlling ammonia assimilation in M. smithii, affecting the metabolism of gut bacterial microbiota, with an impact on host obesity. In this study, we isolated and characterized the small (41 kDa) CPSase homolog from M. smithii. The gene was cloned, overexpressed in E. coli, and the recombinant enzyme was purified in one step. Chemical cross-linking and size exclusion chromatography indicated a homodimeric/tetrameric structure, in accordance with a dimer-based CPSase activity and reaction mechanism. This small enzyme, MS-s, synthesized carbamoyl phosphate from ATP, bicarbonate, and ammonia, and catalyzed the same ATP-dependent partial reactions observed for full-lenght CPSases. Steady state kinetics revealed a high apparent affinity for ATP and ammonia. Sequence comparisons, molecular modeling and kinetic studies suggest that this enzyme corresponds to one of the two synthetase domains of the full-length CPSase that catalyze the ATP-dependent phosphorylations involved in the three step synthesis of carbamoyl phosphate. This protein represents the smallest naturally occurring active CPSase characterized thus far. The small M. smithii CPSase appears to be specialized for carbamoyl phosphate metabolism in methanogens.
  • 4.

    【2hr】Identification of two DNA helicases UvrD and DinG as suppressors for lethality caused by mutant cspA mRNAs

    包量

    机译 鉴定两个DNA解旋酶UvrD和DinG作为突变cspA mRNA致死性的抑制剂
    摘要：CspA is a major cold-shock inducible protein (70 aa), and its major role in the cold-shock response was shown to be as an RNA chaperone destabilizing secondary structure of mRNAs at low temperature. Previously, we showed that the overexpression of mutant cspA containing premature nonsense codons at various positions led to stalled ribosomes on mutant cspA transcripts, ultimately leading to cell death. This lethality is primarily due to the highly translatable cspA 5′-UTR that recruits most of the ribosomes from other mRNAs, which are then stalled at the abnormal stop codon. This was called the ‘LACE’ effect. We show here that nonsense mutation even at 67th position as well as substitutions of aromatic amino acid residues present on the RNA-binding surface of CspA protein to alanine caused the LACE effect by trapping a substantial amount of ribosomes on cspA mRNAs.In an attempt to identify a suppressor(s), which may help the cells to recover from the inhibitory LACE effect, genetic screening of an E. coli genomic library was performed. We isolated suppressors that contained the genomic fragments encoding uvrD and dinG, respectively, whose gene products are ATP-dependent DNA helicases. The nucleic acid-binding and ATPase activities of these two helicases were found to be essential for their suppression activity. This genomic screening offers an approach to shed light on the mechanistic of 5′-UTR of cspA mRNA and novel roles of E. coli helicases that function in DNA repair.
  • 5.

    【2hr】Phylogenetic Characterization of Transport Protein Superfamilies: Superiority of SuperfamilyTree Programs over Those Based on Multiple Alignments

    包量

    机译 系统发育特征的运输蛋白超家族：SuperfamilyTree程序优于那些基于多个比对的程序。
    摘要：Transport proteins function in the translocation of ions, solutes and macromolecules across cellular and organellar membranes. These integral membrane proteins fall into >600 families as tabulated in the Transporter Classification Database (). Recent studies, some of which are reported here, define distant phylogenetic relationships between families with the creation of superfamilies. Several of these are analyzed using a novel set of programs designed to allow reliable prediction of phylogenetic trees when sequence divergence is too great to allow the use of multiple alignments. These new programs, called SuperfamilyTree1 and 2 (SFT1 and 2), allow display of protein and family relationships, respectively, based on thousands of comparative BLAST scores rather than multiple alignments. Superfamilies analyzed include: (1) Aerolysins, (2) RTX Toxins, (3) Defensins, (4) Ion Transporters, (5) Bile/Arsenite/Riboflavin Transporters, (6) Cation: Proton Antiporters, and (7) the Glucose/Fructose/Lactose superfamily within the prokaryotic phosphoenol pyruvate-dependent Phosphotransferase System. In addition to defining the phylogenetic relationships of the proteins and families within these seven superfamilies, evidence is provided showing that the SFT programs outperform programs that are based on multiple alignments whenever sequence divergence of superfamily members is extensive. The SFT programs should be applicable to virtually any superfamily of proteins or nucleic acids.
  • 6.

    【2hr】Specific and Nonspecific Host Adaptation during Arboviral Experimental Evolution

    包量

    机译 树木病毒实验进化过程中的特异性和非特异性宿主适应
    摘要：During the past decade or so, there has been a substantial body of work to dissect arboviral evolution and to develop models of adaptation during host switching. Regardless of what species serve as host or vectors, and of the geographic distribution and the mechanisms of replication, arboviruses tend to have slow evolutionary rates in nature. The hypothesis that this is the result of replication in the disparate environments provided by host and vector did not receive solid experimental support in any of the many viral species tested. Instead, it seems that from the virus's point of view, either the two environments are sufficiently similar or one of the environments so dominates viral evolution that there is tolerance for suboptimal adaptation to the other environment. Replication in alternating environments has an unexpected cost in that there is decreased genetic variance that translates into a compromised adaptability for bypassed environments. Arboviruses under strong and continuous positive selection may have unusual patterns of genomic changes, with few or no mutations accumulated in the consensus sequence or with dN/dS values typically consistent with random drift in DNA-based organisms.
  • 7.

    【2hr】Transposon-Mediated Adaptive and Directed Mutations and Their Potential Evolutionary Benefits

    包量

    机译 转座子介导的自适应和定向突变及其潜在的进化利益。
    摘要：Transposons, mobile genetic elements that can hop from one chromosomal location to another, are known to be both beneficial and deleterious to the cell that bears them. Their value in accelerating evolutionary adaptation is well recognized. We herein summarize published research dealing with these elements and then move on to review our own research efforts which focus on a small transposon that can induce mutations under the control of host factors in a process that phenotypically and mechanistically conforms to the definition of ‘directed mutation’. Directed mutations occur at higher frequencies when they are beneficial, being induced by the stress condition that they relieve. Here, we review evidence for transposon-mediated directed mutation in Escherichia coli. Deletion mutants in the crp gene can not grow on glycerol (Glp−); however, these cells mutate specifically to efficient glycerol utilization (Glp+) at rates that are greatly enhanced by the presence of glycerol or the loss of the glycerol repressor (GlpR). These rates are greatly depressed by glucose or by glpR overexpression. Of the four tandem GlpR-binding sites (O1–O4) in the control region of the glpFK operon, O4 (downstream) specifically controls glpFK expression while O1 (upstream) controls mutation rate. Mutation is due to insertion of the small transposon IS5 into a specific site just upstream of the glpFK promoter. Mutational control by the glycerol regulon repressor GlpR is independent of the selection and assay procedures, and IS5 insertion into other gene activation sites is unaffected by the presence of glycerol or the loss of GlpR. The results establish the principle of transposon-mediated directed mutation, identify a protein responsible for its regulation, and define essential aspects of the mechanism.
  • 8.

    【2hr】Transcriptional De-Repression and Mfd Are Mutagenic in Stressed Bacillus subtilis Cells

    包量

    机译 转录的抑制和Mfd是致病的枯草芽孢杆菌细胞中的诱变。
    摘要：In recent years, it has been proposed that conflicts between transcription and active chromosomal replication engender genome instability events. Furthermore, transcription elongation factors have been reported to prevent conflicts between transcription and replication and avoid genome instability. Here, we examined transcriptional de-repression as a genetic diversity-producing agent and showed, through the use of physiological and genetic means, that transcriptional de-represssion of a leuC defective allele leads to accumulation of Leu+ mutations. We also showed, by using riboswitches that activate transcription in conditions of tyrosine or methionine starvation, that the effect of transcriptional de-repression of the leuC construct on the accumulation of Leu+ mutations was independent of selection. We examined the role of Mfd, a transcription elongation factor involved in DNA repair, in this process and showed that proficiency of this factor promotes mutagenic events. These results are in stark contrast to previous reports in Escherichia coli, which showed that Mfd prevents replication fork collapses. Because our assays place cells under non-growing conditions, by starving them for two amino acids, we surmised that the Mfd mutagenic process associated with transcriptional de-repression does not result from conflicts with chromosomal replication. These results raise the interesting concept that transcription elongation factors may serve two functions in cells. In growing conditions, these factors prevent the generation of mutations, while in stress or non-growing conditions they mediate the production of genetic diversity.
  • 9.

    【2hr】Functional Analysis of the p40 and p75 Proteins from Lactobacillus casei BL23

    包量

    机译 干酪乳杆菌BL23 p40和p75蛋白的功能分析
    摘要：The genomes of Lactobacillus casei/paracasei and Lactobacillus rhamnosus strains carry two genes encoding homologues of p40 and p75 from L. rhamnosus GG, two secreted proteins which display anti-apoptotic and cell protective effects on human intestinal epithelial cells. p40 and p75 carry cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and NLPC/P60 domains, respectively, which are characteristic of proteins with cell-wall hydrolase activity. In L. casei BL23 both proteins were secreted to the growth medium and were also located at the bacterial cell surface. The genes coding for both proteins were inactivated in this strain. Inactivation of LCABL_00230 (encoding p40) did not result in a significant difference in phenotype, whereas a mutation in LCABL_02770 (encoding p75) produced cells that formed very long chains. Purified glutathione-S-transferase (GST)-p40 and -p75 fusion proteins were able to hydrolyze the muropeptides from L. casei cell walls. Both fusions bound to mucin, collagen and to intestinal epithelial cells and, similar to L. rhamnosus GG p40, stimulated epidermal growth factor receptor phosphorylation in mouse intestine ex vivo. These results indicate that extracellular proteins belonging to the machinery of cell-wall metabolism in the closely related L. casei/paracasei-L. rhamnosus group are most likely involved in the probiotic effects described for these bacteria
  • 10.

    【2hr】An Escherichia coli-Based Cell-Free System for Large-Scale Production of Functional Mammalian Membrane Proteins Suitable for X-Ray Crystallography

    包量

    机译 基于大肠杆菌的无细胞系统，用于大规模生产适用于X射线晶体学的功能性哺乳动物膜蛋白
    摘要：A cell-free expression system using an Escherichia coli extract was adapted for large-scale expression and purification of mammalian membrane proteins. The system was tested with a set of human membrane proteins of different sizes, numbers of transmembrane domains, oligomeric arrangements, and native membrane locations. Tens of milligrams of protein were readily expressed and purified from an overnight cell-free reaction. Both reaction ‘mode A’ (proteins were expressed as precipitant) and ‘mode B’ (proteins were expressed in the presence of mild detergents to keep them soluble) were investigated. The combination of ‘mode B’ and the right detergents, used in the subsequent extraction and purification steps, is critical for obtaining properly folded proteins (CX32 and VDAC1) that can be crystallized and diffracted (VDAC1). The E. coli cell-free system is capable of efficient expression of many mammalian membrane proteins. However, fine-tuning of the system, especially to facilitate proper protein folding, will be required for each specific target.
  • 11.

    【2hr】High Efficiency of a Sequential Recombinase-Mediated Cassette Exchange Reaction in Escherichia coli

    包量

    机译 大肠杆菌中顺序重组酶介导的盒式交换反应的高效
    摘要：A comparison between the efficiency of recombinase-mediated cassette exchange (RMCE) reactions catalyzed in Escherichia coli by the site-specific recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both recombinases is very inefficient. However, the same reaction catalyzed by each recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the recombinase. The results suggest that the RMCE reaction is sequential.
  • 12.

    【2hr】The Autoinducer-2 Exporter Superfamily

    包量

    机译 Autoinducer-2出口商超家族
    摘要：The TqsA (YdgG) protein of Escherichia coli has been shown to export the autoinducer-2 (AI-2) molecule, a furanosyl borate diester that bears little resemblance to previously characterized biological molecules. TqsA belongs to a large superfamily, the AI-2 exporter (AI-2E) superfamily, of putative transporters with no other functionally characterized members. These proteins derive exclusively from bacteria. Many different bacterial kingdoms contain them, although several kingdoms do not. These proteins exhibit a uniform topology with 8 putative transmembrane segments (TMSs) which we show probably arose from a 4-TMS precursor in a process that involved at least one and possibly two intragenic duplication event(s). The first halves of these proteins are more diverse in sequence than the second halves, suggesting that the first halves may serve substrate-specific functions while the second halves serve family-specific functions. Conserved residues and motifs in these proteins are identified. Some homologues include extra catalytic domains including those involved in purine nucleotide biosynthesis, ATP and GTP binding, and molecular signaling. The results presented provide guides for future functional studies on members of this superfamily of bacterial transporters.
  • 13.

    【2hr】Bioinformatic Analyses of Transmembrane Transport: Novel Software for Deducing Protein Phylogeny, Topology, and Evolution

    包量

    机译 跨膜转运的生物信息学分析：推论蛋白质系统发生，拓扑结构和进化的新型软件。
    摘要：During the past decade, we have experienced a revolution in the biological sciences resulting from the flux of information generated by genome-sequencing efforts. Our understanding of living organisms, the metabolic processes they catalyze, the genetic systems encoding cellular protein and stable RNA constituents, and the pathological conditions caused by some of these organisms has greatly benefited from the availability of complete genomic sequences and the establishment of comprehensive databases. Many research institutes around the world are now devoting their efforts largely to genome sequencing, data collection and data analysis. In this review, we summarize tools that are in routine use in our laboratory for characterizing transmembrane transport systems. Applications of these tools to specific transporter families are presented. Many of the computational approaches described should be applicable to virtually all classes of proteins and RNA molecules.
  • 14.

    【2hr】The Vibrio cholerae Mrp System: Cation/Proton Antiport Properties and Enhancement of Bile Salt Resistance in a Heterologous Host

    包量

    机译 霍乱弧菌Mrp系统：异质宿主中的阳离子/质子反转运性质和胆汁盐抗性的增强
    摘要：The mrp operon from Vibrio cholerae encoding a putative multisubunit Na+/H+ antiporter was cloned and functionally expressed in the antiporter-deficient strain of Escherichia coli EP432. Cells of EP432 expressing Vc-Mrp exhibited resistance to Na+ and Li+ as well as to natural bile salts such as sodium cholate and taurocholate. When assayed in everted membrane vesicles of the E. coli EP432 host, Vc-Mrp had sufficiently high antiport activity to facilitate the first extensive analysis of Mrp system from a Gram-negative bacterium encoded by a group 2 mrp operon. Vc-Mrp was found to exchange protons for Li+, Na+, and K+ ions in pH-dependent manner with maximal activity at pH 9.0–9.5. Exchange was electrogenic (more than one H+ translocated per cation moved in opposite direction). The apparent Km at pH 9.0 was 1.08, 1.30, and 68.5 mM for Li+, Na+, and K+, respectively. Kinetic analyses suggested that Vc-Mrp operates in a binding exchange mode with all cations and protons competing for binding to the antiporter. The robust ion antiport activity of Vc-Mrp in sub-bacterial vesicles and its effect on bile resistance of the heterologous host make Vc-Mrp an attractive experimental model for the further studies of biochemistry and physiology of Mrp systems.
  • 15.

    【2hr】A specialized version of the HD hydrolase domain implicated in signal transduction

    包量

    机译 HD水解酶结构域的专门版本涉及信号转导
    摘要：
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