文档库 最新最全的文档下载
当前位置:文档库 › Core proteome of the minimal cell comparative proteomics of three mollicute species

Core proteome of the minimal cell comparative proteomics of three mollicute species

Core proteome of the minimal cell comparative proteomics of three mollicute species
Core proteome of the minimal cell comparative proteomics of three mollicute species

Core Proteome of the Minimal Cell:Comparative Proteomics of Three Mollicute Species

Gleb Y.Fisunov1*,Dmitry G.Alexeev1,3,Nicolay A.Bazaleev1,Valentina https://www.wendangku.net/doc/734007817.html,dygina1,Maria A. Galyamina1,Ilya G.Kondratov1,Nadezhda A.Zhukova2,Marina V.Serebryakova1,Irina A.Demina1, Vadim https://www.wendangku.net/doc/734007817.html,orun1,2,3

1Scientific Research Institute of Physical-Chemical Medicine,Federal Bio-Medical Agency of Russia,Moscow,Russia,2Russian Research Centre Kurchatov Institute, Moscow,Russia,3Moscow Institute of Physics and Technology,Moscow,Russia

Abstract

Mollicutes(mycoplasmas)have been recognized as highly evolved prokaryotes with an extremely small genome size and very limited coding capacity.Thus,they may serve as a model of a‘minimal cell’:a cell with the lowest possible number of genes yet capable of autonomous self-replication.We present the results of a comparative analysis of proteomes of three mycoplasma species:https://www.wendangku.net/doc/734007817.html,idlawii,M.gallisepticum,and M.mobile.The core proteome components found in the three mycoplasma species are involved in fundamental cellular processes which are necessary for the free living of cells.They include replication,transcription,translation,and minimal metabolism.The members of the proteome core seem to be tightly interconnected with a number of interactions forming core interactome whether or not additional species-specific proteins are located on the periphery.We also obtained a genome core of the respective organisms and compared it with the proteome core.It was found that the genome core encodes73more proteins than the proteome core.Apart of proteins which may not be identified due to technical limitations,there are24proteins that seem to not be expressed under the optimal conditions.

Citation:Fisunov GY,Alexeev DG,Bazaleev NA,Ladygina VG,Galyamina MA,et al.(2011)Core Proteome of the Minimal Cell:Comparative Proteomics of Three Mollicute Species.PLoS ONE6(7):e21964.doi:10.1371/journal.pone.0021964

Editor:Vladimir Brusic,Dana-Farber Cancer Institute,United States of America

Received January26,2011;Accepted June14,2011;Published July19,2011

Copyright:?2011Fisunov et al.This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.

Funding:The study was supported by a grant from the Ministry of Education and Science of the Russian Federation02.512.12.2042.The funders had no role in study design,data collection and analysis,decision to publish,or preparation of the manuscript.

Competing Interests:The authors have declared that no competing interests exist.

*E-mail:herr.romanoff@https://www.wendangku.net/doc/734007817.html,

Introduction

A bacterial genome’s length varies from180Kb in obligate intracellular symbiont Carsonella rudii to13Mb in soil bacterium Sorangium cellulosum.The distribution of genome sizes between bacterial species with sequenced genomes has a bimodal structure with two peaks at2Mb and5Mb,dividing bacterial genomes into ‘small’and‘large’[1].Mycoplasmas belong to the group of bacteria with small genomes;their genomes rarely exceed1Mb. Mycoplasma genitalium,with a580Kb chromosome,is considered to be an organism with the smallest genome among autonomously-replicating bacteria.The sequencing of a M.genitalium genome led to the emergence of the minimal cell and minimal genome concepts[2].

Since the publication of the first sequenced genomes[3],a number of approaches to the identification of minimal gene sets from autonomously-replicating organisms have been developed. These include comparative genomics,comparative proteomics, experimental identification of essential genes,and theoretical modeling.

The comparative genomics approach was developed first and initially resulted in the identification of256genes[3].However,as the number of sequenced genomes increased,the set of genes conserved in all known genomes dropped to about50[4].It is now clear that,on the one hand,the comparison of distantly related organisms leads to the underestimation of the minimal gene set because of the non-orthologous displacement of essential genes[5]. On the other hand,the identification of the genes conserved between closely-related species results in the overestimation of the minimal gene set because,apart from essential genes,some non-essential genes(but important under the particular conditions)are included in the conserved core.

The next approach to discover the minimal gene set was the identification of essential genes by global transposon mutagenesis or single gene deletions.The first attempt to find essential genes of M.genitalium showed that from265to350out of517genes are essential[6].Enhancing the method gave382essential genes[7]. To date,there are a number of publications on the allocation of essential genes;their results are summarized in the Database of Essential Genes(DEG)[8].For example,a set of essential genes for Bacillus subtilis comprises271genes[9].Nonetheless,the estimation of the minimal gene set through gene deletion may face some obstacles.First,some identifications may be false-positive because of mutated genes and their altered products may affect a number of cellular processes like the metabolism and expression of downstream genes.Second,the effect of knocking out all non-essential genes at one time may not be equivalent to the step by step deletion in terms of cell survival.At the same time,some non-essential genes may be conserved among a number of species. According to Fang et al,these genes are involved in cellular homeostasis maintenance and adaptation to stress[10].The deletion of such genes does not have any effect under laboratory

conditions,but they may be crucial for cell survival in long-term periods in the natural environment.

The theoretical modeling of the minimal function set required for cell replication results in the allocation of151genes which are necessary and sufficient to sustain a living cell under the most favorable conditions[11].However,cellular organization on the whole is far from being fully understood.In particular,trans-criptome and interactome complexity issues are left out of con-sideration in this approach.

It is now clear that a minimal gene set greatly depends on selected approaches and organisms and may constitute from151 [11]to several hundreds[7].The number of genes in a minimal set tends to rise as the complexity of the selected organism increases.For example,the gene inactivation approach reveals 712[12]and614[13]essential genes found in large bacteria, E.coli and B.subtilis,and only382[7]essential genes are found in M.genitalium.Moreover,taking into account the recent data on a large number of untranslated RNAs[14],it is reasonable to consider that the use of only the genomic or proteomic approach is insufficient to discover a minimal gene set.Thus, a proteogenomic approach should be applied in this case. Comparative proteogenomics,proposed by Gupta et al[15], imply the use of genomic techniques to characterize genome and also imply proteomic techniques to increase reliability,correct genome annotation,and identify expressed open reading frames (ORFs).

Callister et al[16]applied the proteogenomic approach to identify a conserved core of6bacterial species.However,the selected species lived in different environments,were grown under different conditions and were phylogenetically distant from each other.This resulted in some genes,previously thought to be indispensable,not being included in the core genome.

In this study,we propose to explore the core proteome shared by three mycoplasma species using the proteogenomic approach. These species occupy different ecological niches,but can grow under the same conditions and are phylogenetically close to each other.Thus,this gives us a chance to avoid the non-orthologous displacement of essential genes,and,at the same time,it may help to exclude genes responsible for adaptation to the niche specific conditions.

Methods

Strains and Growth Conditions

Acholeplasma laidlawii PG-8A and Mycoplasma gallisepticum S6were grown in a modified Edward’s medium(Tryptose20g/L,NaCl 5g/L,NaOAc5g/L,KCl1.3g/L,Tris3g/L,yeast dialysate 5%,horse serum6%,glucose0.5%,pH7.6)at37u C for18and 24hours,respectively.The cells were cultured in500mL flasks containing300mL medium under aerobic conditions.The mycoplasma mobile was grown in an Aluotto medium(Heart infusion broth25g/L,yeast extract5%,horse serum20%, pH7.6)[17].The Acholeplasma laidlawii PG-8A strain was provided by Prof.H.Wroblewsky,Universite′de Rennes.The Mycoplasma gallisepticum S6strain was provided by Prof.S.N.Borkhsenius, Institute of Cytology,Russian Academy of Science.

RNA isolation and real-time PCR

The total RNA was extracted from a cell culture in the mid-logarithm growth phase with the aid of a Trizol LS reagent (Invitrogen).Then,RNA samples were treated with DNAse I (Fermentas)and used for cDNA synthesis with Mu-MLV reverse transcriptase(Fermentas).Real-time PCR using SYBR Green PCR Master Mix(ABI)and an ABI Prism SDS7000(ABI)instrument was then performed.Amplicons were designed to cover the middle of each annotated ORF.Primers were designed with PerlPrimer software(Supplementary tables S13and S14).

SDS-PAGE

Proteins were solubilized by boiling them in a sample buffer and were then separated by SDS/PAGE gels consisting of7.5%T or 16.5%T and2.6%C(%T,gel acrylamide concentration;%C, degree of crosslinking within the polyacrylamide gel),according to the Laemmli method[18].The gels were fixed and stained with Coomassie G-250.

Two-dimensional PAGE

Before carrying out,the2D PAGE cells were treated with a nuclease mix(Amersham Bioscience).The cells were centrifuged and the cell pellet was dissolved in a buffer(10m l):8M urea,2M thiourea,4%CHAPS,2%(w/v)NP-40,1%Triton X-100,2% Ampholytes,pH range3to10,80mM DTT.Protein concentra-tion was determined using Quick start Bradford dye reagent(Bio-Rad,USA).Isoelectrofocusing was performed using tube gels (20cm61.5mm)containing carrier ampholytes and applying a voltage gradient in an IEF-chamber Protean II XL cell(Bio-Rad). After IEF,the ejected tube gels were incubated in an equilibration buffer(125mM TrisHCl,40%(w/v)glycerol,3%(w/v)SDS, 65mM DTT,pH6.8)for30min.The tube gels were placed onto polyacrylamide gels(9–16%)of 1.5-mm thickness,20618cm (Protean II Multi-Cell,Bio-Rad,USA),and fixed using0.9%(w/v) agarose containing0.01%(w/v)bromphenol blue.The electro-phoresis was carried out for12–14hours.

Gel Staining and Detection of Proteins

The gels were fixed and silver stained as described by Shevchenko et al.[19].An image analysis was performed using PDQest software(Bio-Rad,USA).All spots were extracted for MALDI-MS analysis.

Trypsin Digestion and Mass Spectrometry

The protein bands/spots after1D or2D-PAGE were subjected to trypsin in-gel hydrolysis,mainly as described in[20].1mm3gel pieces were excised and washed twice with100m L of0.1M NH4HCO3(pH7.5)and40%acetonitrile mixture for30min at 37u C,dehydrated with100m L of acetonitrile,and air-dried. Then,they were treated by3m L of12mg/mL solution of trypsin (Promega)in50mM ammonium bicarbonate for12h at37u C. Peptides were extracted with6m L of0.5%trifluoroacetic acid water solution for30min.

MALDI analysis

Aliquots(1m L)from the sample were mixed on a steel target with0.3m L of2,5-dihydroxybenzoic acid(Aldrich)solution (10mg mL–1in30%acetonitrile/0.5%trifluoroacetic acid),and the droplet was left to dry at room temperature.Mass spectra were recorded on the Ultraflex II MALDI-ToF-ToF mass spectrometer (Bruker Daltonik,Germany)equipped with an Nd laser.The [MH]+molecular ions were measured in reflector mode,the accuracy of the mass peak measurement was0.007%. Fragment ion spectra were generated by laser-induced dissoci-ation,slightly accelerated by low-energy collision-induced disso-ciation,using helium as a collision gas.The accuracy of the fragment ions mass peak measurement was1Da.Correspondence of the found MS/MS fragments to the proteins was performed with the help of Biotools software(Bruker Daltonik,Germany)and a Mascot MS/MS ion search.

Protein identification was carried out by a peptide fingerprint search with the use of Mascot software(Matrix Science Inc.,USA) through a NCBI https://www.wendangku.net/doc/734007817.html,idlawii protein database.One missed cleavage,Met oxidation and Cys-propionamide settings were permitted for peptide search.Protein scores greater than44were considered to be significant(p,0.05).Two dimensional PAGE and subsequent MALDI analysis was performed five times for each species with three replicates per sample.

LC-ESI-MS analysis

LC-ESI-MS analyses(of tryptic peptides after1D-SDS-PAGE separation of proteins)were performed on a Agilent1100series HPLC-ESI/MSD Trap(Agilent Technologies,USA)equipped with a Zorbax300-SB C18column and nano-ESI source.The elution conditions consisted of a0.3m l/min20-min ablution by 5%solvent B(80%acetonitrile,20%water,0.1%formic acid),a 50-min gradient5–60%,and then a20-min gradient60–90% from solvent B into A(0.1%formic acid water solution).The [MH]1+-3+ions were detected in the200–2200m/z range optimized to800.MS/MS spectra were obtained automatically for all perceptible MS signals.The accuracy of the mass peak measurement was0.5Da.Protein identification was carried out by a MS/MS ion search using Mascot software,as mentioned before. Protein scores greater than31were considered significant. Additional validation was performed on ad-hoc software modules taking into account gel bands,physical properties of proteins,and unspecific trypsin digestion for high abundant proteins.The same software allowed us to scan the acquired spectra for incorrectly annotated N-terminal sequences or misannotated proteins.There were three biological replicates and one technical replicate per each biological replicate of each species subjected to LC-MS analysis.

Hydrophobic proteins extraction

Cells were resuspended in water and exposed to ultrasound (220Hz)on ice four times for15sec.Then,the intact cells were centrifuged on25000g for5min at4u C.Supernatant was transferred to new tubes and centrifuged on85000g for1h at 4u C.The pellet was resuspended in water and centrifuged a second time under the same conditions.The pellet was then resuspended in a buffer containing mM NaCl,10mM Tris,and 1%Triton X-114(pH7.5),and incubated on ice for30min. Then,the solution was centrifuged on16000g for30min, transferred to a new tube,and left for5min to allow the phases to separate.The lower phase was used to perform PAGE. Comprehensive proteome analysis

Comprehensive proteome analysis workflow implies a sequen-tial set of protein identification runs.After several runs it reaches a maximum of identified proteins when further runs don’t give more proteins.The identified proteins are considered,taking into account technical limitations,to sustain a comprehensive proteo-mic content of a cell[21].The overall view on comprehensive proteome analysis workflow is depicted in figure1.The main issue in comprehensive proteome analysis is a completeness of an acquired proteome.We considered ESI mass spectrometry to be a limiting stage in the way of protein’s identification.If there are multiple peptides in a single chromatography peak analyzed by ESI at a time,only major ones will be detected.

To ensure the comprehensiveness of obtained data we used1D gels zooming.First,the gel gradient was adjusted to a particular proteins mass range.Next,we obtained a full size1D gel for a selected mass range which gave a significantly higher resolution. We came to the conclusion that gels with100–120bands allow to identify all possible proteins for a particular species on our equipment.Further zooming didn’t result in the increase in the identified proteins number.

The abundance of high-copied molecular complexes(ribosome, GroEL,pyruvate dehydrogenase,and RNA-polymerase)was estimated by Kuhner et al[22]and was about several hundreds of copies per mycoplasma cell.This means that the dynamic range of protein abundance per mycoplasma cell is about102.

The sensitivity of2D electrophoresis coupled with MALDI is about102.LC-MS allows for the identification of a substantial number of additional proteins,like DNA-polymerase III subunits or components of DNA repair machinery(uvrA,uvrB,uvrC).The sensitivity of LC-MS is100fmol and the dynamic range of peptide identification reaches105.Thus,the dynamic range of our technique sufficiently covers the dynamic range of protein abundance in mycoplasma cells and allows for the identification of several copies of protein per cell.

Mycoplasma mobile proteomic data

The results of the proteomic study for M.mobile were taken from work of Jaffe et al[17].

Results

Identification of core proteomes

The studied species have small genomes(633genes in M.mobile, 763genes in M.gallisepticum,and1380genes in https://www.wendangku.net/doc/734007817.html,idlawii)and,in spite of living in different environments,the species can be cultivated in the same medium.This may allow one to identify the minimal protein set required to sustain a free living cell and to cut off proteins involved in niche adaptation and stress response, which in turn may shed light on the understanding of a vital function set.

Recently,we sequenced the Acholeplasma laidlawii genome (Refseq ID:NC_010163)and carried out its proteogenomic annotation.It resulted in the identification of1380ORFs.We also resequenced Mycoplasma gallisepticum S6strain genome.There are 762ORFs(Whole Genome Shotgun ID:AFFR00000000)and there are633ORFs in the Mycoplasma mobile genome(Refseq ID: NC_006908)according to NCBI.

The proteomic core and genomic core of the minimal free living cell was identified by a comprehensive proteome analysis and genome analysis,respectively,for three mycoplasma species.A comprehensive proteomic analysis of https://www.wendangku.net/doc/734007817.html,idlawii showed an expression of803ORFs(58%of annotated ORFs,Supplementary Table S8).The respective proteomic analysis of M.gallisepticum allowed us to identify481expressed proteins(66%of annotated ORFs,Supplementary Table S9)[23].The proteome of M.mobile, identified by Jaffe et al,consisted of557proteins(88%of annotated ORFs).

Clusters of orthologous genes(COGs)assigned to the annotated ORFs were used to compare the genomes and proteomes(Fig.2). The https://www.wendangku.net/doc/734007817.html,idlawii genome encodes783COGs and560unique ORFs(without COG assignment);the M.gallisepticum genome encodes409COGs and304unique ORFs;the M.mobile genome encodes404COGs and216unique ORFs.The respective proteomes consist of567COGs and88unique proteins in A. laidlawii,321COGs and109unique proteins in M.gallisepticum, and374COGs and127unique proteins in M.mobile.

The distribution of COGs in genomes and proteomes common for three species and for the pairs of species,for unique COGs, and for unique proteins without a COG were investigated. Genomic and proteomic cores shared by the three species are composed of284and212COGs,respectively.The proteomic

core further underwent a functional analysis in order to study the completeness of essential cellular machineries (replication,transcription,translation,and protein folding)and metabolic pathways.

Comparison of the proteome core with the genome core showed 72additional ORFs.These may be absent from the proteome core for two reasons:first,they may have extreme physical and chemical properties,like low mass and high hydrophobicity,which makes them hard to be detected by proteomics techniques;second,those proteins may be dispensable under the optimal growth conditions and expressed only in stress.Some of the COGs absent from the proteome core are membrane proteins and small ribosomal proteins and they were likely not detected in our study.There are 37proteins of this type.Another 35proteins were predicted not to have extremal properties and should be detected by our approach if presented in a few copies per cell.Their absence in identified proteins means that they are found in less than every cell,even if they are expressed on some level in

the

Figure 1.Saturating proteomics workflow chart.

doi:10.1371/journal.pone.0021964.g001

Figure https://www.wendangku.net/doc/734007817.html,parison of genomes and proteomes of three mycoplasma species.Venn diagrams shows the number of common COG’s,numbers outside from green circle represent genes and respective proteins without COG.A-diagram for genomes,B-diagram for proteomes.doi:10.1371/journal.pone.0021964.g002

whole population.It can be concluded that other proteins are apparently not required for cells to grow under the optimal conditions(Supplementary,Table S1).

The genome and proteome cores were compared with other minimal gene sets obtained by other methods.In particular,the comparison of the proteomic core with the list of essential genes may help to estimate the viability of an organism with such proteomic content.Taking into account the list of essential genes of M.genitalium determined by Venter el al[7],only26proteins from the proteome core are dispensable(Supplementary Table S10).There are also proteins essential for M.genitalium,but absent from the proteome core(111COGs,Supplementary Table S11). Most of them are small or membrane proteins and may not be identified,or they are absent in one or more of the three mycoplasmas.Thus,the proteomic core meets a good agreement with the list of M.genitalium essential genes.

However,it would not be correct to conclude that close relations cause dispensable proteins to be considered as essential ones.This may be true for organisms with larger genomes’and redundant proteins’functionality.In the case of mycoplasmas, which underwent significant genome deterioration and then diverged into different niches,it is not correct.Rather,it indicates the impossibility of building a minimal proteome core based on evolutionary distant species because it leads to the loss of truly essential genes.

Potential antisense RNAs in core proteome Remarkably,the proteome core has very few regulation potentials,compared to real cells,even when reduced as mycoplasmas.The role of possible antisense RNA in the regulation of the proteome core was evaluated using data on antisense RNA found in M.pneumonia[24].The intersection of M. pneumonia antisense RNAs with the sequences of core proteome genes shows that35of its members have antisense transcripts in M. pneumonia(Supplementary Table S5).

Functional characteristics of core proteome

The obtained proteome core contains all or a nearly complete list of the molecular machinery known to be necessary for a free living cell(Supplementary Table S10).It has all the components of basic a DNA replication apparatus except for DNA polymerase I which is absent in genomes of some mycoplasmas.DNA repair systems of the proteome core are represented by a very limited number of proteins.The most complete one is the nucleotide excision repair(NER)system with a total number of5proteins, including uvrA,uvrB,uvrC,and two uvrD-like helicases.

The core contains all subunits of RNA polymerase and three transcription factors,which is the maximum number of transcrip-tional apparatus components in some mycoplasmas.It also has all ribosomal parts that are known to be necessary to its functioning including ribosomal proteins and translation factors,except for IF-1and RF-2.IF-1is rather small and was likely to be missed in our proteomics study,while RF-2is possibly not essential in mycoplasmas as it recognizes stop codon which is replaced by tryptophan codon in mycoplasmas(but not in Acholeplasma).In addition,we identified a set of translation coupled proteins like protein chain release factor A,the ribosome recycling factor,and several other proteins.

Moreover,the core proteome contains several molecular chaperones including the DnaK-DnaJ system.The core compo-nents found also form a limited number of metabolic pathways, including glycolysis,the non-oxidative part of pentose phosphate pathway,and glycerophospholipid biosynthesis from fatty acids and glycerol.The synthesis of nucleotide triphosphates seems to be a main metabolic capacity of the core.

The most surprising thing in the core proteome is low abundance of cell division proteins compared to the amount of proteins from other essential cellular machineries.There are only two such proteins in the core:FtsH and a Smc-like protein.Although some two of three species have FtsK and FtsZ expressed in the proteome, M.mobile doesn’t even have corresponding genes in its genome.This may indicate that the cell division mechanism shows greater plasticity compared to other essential systems of the cell. Comparative genomics versus comparative proteomics To test comparative proteomics versus comparative genomics we built a genome core for16mollicute species and compared the results with the proteome core to estimate the strong and weak points of the two approaches(Supplementary Table S12).The more genomes are taken into comparison the fewer COGs are found in the core(Fig3).Nevertheless,the curve eventually comes to saturation,i.e.the minimal gene set for the particular phylogenetic group is reached.The common dispensable genes are likely to already be present in this set.

The intersection of the16mollicute genomes gives a core of 189COGs.The proteome core has an amount similar to COGs: 212.However,there are only156COGs common for the two cores.The detailed view on the16genome cores shows that15 protein products are supposed to have extreme physico-chemical properties and should be excluded from consideration.

The proteome core,in its turn,has56COGs more than the genomic core for16mollicutes.More than a half of them are involved in nutrients transport and metabolism.This may indicate that the metabolism is the most variable component of the cell. Metabolic COGs may be treated as adaptive ones because energy sources and available nutrients depend on the particular environment.On the assumption of the proteome core function-ality,the main function of the metabolism is to provide the cell with nucleotide triphosphates.

However,the proteome core lacks14COGs involved in the efficiency and fidelity of translation and DNA repair.On the one hand,these functions are adaptive,as different environmental con-ditions require different systems of fidelity maintenance.On the other hand,they may have a complex effect on fidelity and cell survival. These COGs may be essential if all or most of them are deleted at once,being,at the same time,dispensable if deleted individually. The comparison of the genomic core and our proteome core for the16mollicute species shows that there are some conserved metabolic enzymes that do not line up in a complete pathway.For example,there are not any annotated phosphofructokinase and fructose-bisphosphate aldolase enzymes in M.arthritidis,nor are there any glyceraldehyde-3-phosphate dehydrogenase enzymes in the Ureaplasma species.According to Commichau et al[25],some glycolytic enzymes form complexes with proteins from other cellular systems,like transcription and translation machines. Metabolic enzymes,besides their primary functions,seem to have other activities which they may carry out even in the absence of their metabolic pathways[25].

Thus,comparative genomics help to allocate a core that consists of the basic mechanisms of life but is viable on its own. Comparative proteomics allow us to identify an extended core that approximates the composition of real living cells. Transcriptional analysis of ORFs from https://www.wendangku.net/doc/734007817.html,idlawii and M. gallisepticum

We studied the transcriptional activity of ORFs with unknown functions which were not found in proteomic studies of https://www.wendangku.net/doc/734007817.html,idlawii

and M.gallisepticum by real-time PCR.The respective protein products were supposed not to have extreme physico-chemical functions and should be detected by proteomic techniques.It was found that 151from 165studied ORFs of https://www.wendangku.net/doc/734007817.html,idlawii and 101from 162studied ORFs of M.gallisepticum are expressed (Supplementary Tables S6and S7).mRNAs of 101ORFs of https://www.wendangku.net/doc/734007817.html,idlawii and 88ORFs of M.gallisepticum were found at amounts not less than the amount of a b -subunit of RNA polymerase mRNA indicating that a significant number of annotated ORFs in mycoplasmas produce transcripts which do not undergo translation.Rasmussen et al.[14]and Toledo-Arana et al.[26]obtained similar results for Lysteria monocytogenes and B.subtilis .These studies showed that 98%and 77%of annotated ORFs are expressed in the respective organisms.

The core interactome

We estimated a possible interaction that may be found in the proteome core based on Mycoplasma pneumonia interactome data [22].According to M.pneumonia data,most of the COGs in the proteome core (140COGs)are associated in complexes (Fig 4,Supplementary Table S2).Moreover,54COGs participate in more than one complex.Most of the COGs that do not form complexes are ribosomal proteins that are absent in M.pneumonia ,are transport COGs,or are unknown proteins (Supplementary Table S3).

Among the proteins found only in the genome core,35proteins take part in complex formation.Most of them probably were not identified during proteomic studies due to their extreme physico-chemical properties of having low molecular weight,hydropho-bicity,and other preventing factors for reliable MS identification.Only 20proteins which are not likely to be expressed in the proteome core but are found in the genome core actually make complexes (Supplementary Table S4).Most of them are the parts of the defense and DNA repair systems.

Based on M.pneumonia and B.subtilis protein complexes data,it is possible to estimate the composition of protein complexes in the proteome core.The interactions among selected members of the proteome core are displayed in Figure 5.Proteins of the proteome core form multifunctional complexes.For example,phosphoglyc-erate kinase,enolase,and two ribosomal proteins (S2and L5),according to Commichau et al [25],form interactional bridges between glycolysis,a translation apparatus;chaperones hold together glycolysis,translation,transcription,and DNA-binding proteins.

Core proteins,in addition,are bound to membrane proteins (Fig.5),and respective membrane proteins are specific to M.

pneumonia .This may indicate that the proteome core is anchored to the membrane through interactions between protein complexes.Thus the main part of the proteome core is bound together into a single complex,starting from DNA and ending on the membrane.Such an arrangement of cellular content may allow for the faster procedure of biological processes,as though it takes place on a conveyor belt.

Discussion

Undoubtedly,the amount of information provided by compar-ative proteomics is not less than that of comparative genomics or transcription profiling.The complexity of building a comparison model,which implicates the careful selection of organisms and media,is rewarded by more precise results.

It is now clear that universally conserved genes are too few to sustain a free living cell [4].Thus life is not invariant to the number of genes in different organisms.More or less,the universal core may be established only for a particular phylogenetic group of organisms.Our core is,more or less,close to the invariant of the mycoplasma clade and may not fit other bacteria.

In a similar study,the core proteome common for 17bacterial species was found to be composed of 105proteins,which make up about 74%of the total number of orthologs [16].In comparison with the mycoplasma proteome core identified by the current study,almost the same proportion of genes (70%)conserved in the genome was found in the proteome core.These two proteome cores,bacterial and mycoplasma,do not match each other completely.This may be explained by the significant genome reduction of mycoplasmas when a number of proteins common in larger bacteria is absent in the mycoplasmas and in the proteome core.At the same time,our core contains 100more proteins,possibly because we analyzed more close species in contrast with Callister et al [16].

Researchers are paying increased attention to the spatial structure of the cell.For example,Kuhner et al [22],besides a huge amount of interactions covering most cellular proteins,found complexes formed by functionally distant proteins like those involved in translation and metabolism.This led to the conclusion that there are a number of interactions besides classical protein interactions.However,the role of these interactions is poorly understood.The recent data for B.subtilis [25]leads to the suggestion that the persistence of glycolytic enzymes genes may have an explanation other than having a metabolic role.They may represent a scaffold for complex formation and modulate the activity of their partners in such

complexes.

Figure 3.Dependence of genome core size on the number of compared genomes.The plot reaches saturation.doi:10.1371/journal.pone.0021964.g003

The possibility of an alternative role of known proteins easily explains the existence of disrupted metabolic pathways,like in Mycoplasma hominis [27].This bacterium has an incomplete glycolytic pathway,the existence of which is even more questionable taking into account that it may fully cover its energy costs through the utilization of arginine.It is possible that those glycolytic proteins are complex-formers rather than metabolic enzymes.

An increasing number of publications show that the cellular organization of bacteria is not less complex than the eukaryotic one [28].Like eukaryotes,bacteria have a network of intracellular fila-ments for ordering cellular space,keeping DNA in a particular orien-tation,and helping it to distribute to daughter cells after replication.Most,if not all,cellular proteins are participants in multiple complexes,and their partners are also members of other complexes.Thus,cellular proteins make a spatial and functional network,which probably holds all cellular content together with the membrane.Moreover,the periphery of the complex network seems to bind species-specific membrane proteins (Fig.5)which may be required for fine tuning of complexes.Different species possess different membrane proteins which are bound to different complex components.The changes in membrane protein

expression may also affect the spatial organization of the bound complexes in the inner cellular space.This may be a way for cellular reaction on different stimuli in the absence of other regulators,which is common in mycoplasmas.

The composition and structure of protein complexes may maintain genome stability or direct the evolution,as the changes in internal components with a large number of interactions is less likely than changes in periphery which may not affect basic life machinery.Thus,the more a given protein is integrated into the complexes,the less dispensable it is.This is demonstrated by mycoplasmas preservation of the main parts of the proteome core and the changed or lost peripheral parts.

According to this concept,a minimal genome is treated not as a set of essential functions but as a set of essential structures.On the one hand,this structure exposes integrity,consistency,and compactness,and,on the other hand,it has potential to interact with the periphery and to change its configuration and composition.It is important that the proteome core matches the list of essential genes.However,the comparative proteomic approach may remove issues of non-orthologous gene displacement and false-positive essential genes because it shows the real

proteome

Figure https://www.wendangku.net/doc/734007817.html,plexes in the proteome core based on M.pneumonia data.Proteins that do not form complexes (35proteins)are shown on the left.Proteins that are found in complexes (140proteins)are shown on the right.Color indicates COG functional category (1sector per protein).Only the most representative complexes are shown.The key for COG functional categories letter code:C Energy production and conversion.D Cell cycle control,cell division,chromosome partitioning.F Nucleotide transport and metabolism.G Carbohydrate transport and metabolism.H Coenzyme transport and metabolism.J Translation,ribosomal structure and biogenesis.K Transcription.L Replication,recombination and repair.O Posttranslational modification,protein turnover,chaperones.P Inorganic ion transport and metabolism.R General function prediction only.U Intracellular trafficking,secretion,and vesicular transport.doi:10.1371/journal.pone.0021964.g004

content of the cell.The issue of importance of persistent genes –genes that are conserved but not essential –also gets a solution.Those that keep expression and participate in complex formation under the optimal conditions are important.Those which are not expressed are dispensable.

According to Jain et al [29],with the increase in the number of interacting partners it becomes harder to remove or alter a single protein in the network.Hence,all main proteins of the most important complexes should tend to conserve their sequence,even under a high level of mutagenesis,and keep their expression level under the different conditions.

Returning to the mycoplasmas,it is reasonable to pay attention to the high divergence of several genes [30]and overall high intraspecie proximity as a result of a limited MMR system [31].This results in the rapid adaptation of mycoplasmas to the particular environmental conditions by the use of periphery proteins alterations while keeping genes of the core proteins intact.Taking this into account,the successful transplantation of the

Mycoplasma mycoides genome into Mycoplasma capricolum [32]is not surprising because these species have essentially the same protein complex core.

The role of a large amount of non-translated RNAs,described in recent publications,is still unclear.It was found that B.subtilis expresses 84non-coding and 127antisense RNAs [14].Similar phenomena were found in small bacteria like M.pneumonia;with a genome size six times smaller than the B.subtilis genome,it produces 117non-coding transcripts which comprise 15%of the total number of transcripts [24].Taking into account a very limited regulatory potential of mycoplasmas (1–2transcriptional factors per genome),it is reasonable to conclude that non-coding RNAs may play a significant role in gene expression regulation.It is also known that gene regulation does not just control the organism’s responses to stimuli but may also be treated as a second genetic code,a key factor which drives genetic differences in more complex organisms [33].Non-coding RNAs may connect metabolism and gene expression through the riboswitch

mecha-Figure 5.Core proteins interaction chart based on M.pneumoniae and B.subtilis data.Proteins are divided to groups:DNA handling,transcription,translation,chaperones,glycolysis.Yellow indicates membrane-bound proteins of M pneumonia that interact with core proteins.ENO,PGK,S2and L5are found in a large number of bacterial species.doi:10.1371/journal.pone.0021964.g005

nism[34].Some core proteome genes also seem to have non-coding antisense transcripts.This may indicate an active usage of antisense RNAs in gene regulation in reduced cells.Hence,the studies of the minimal cell are not possible without taking into account non-coding RNAs.

The obtained systemic comparison of proteomes is in accordance with the latest publications on the composition of the minimal cell and is consistent with the concept of the organization of inner cellular machinery into a number of complexes modulating the member proteins.The common proteome core of the three mycoplasmas seems to support all processes required for a minimal cell to survive in a rich medium and is consistent with the data on essential genes.Moreover,the comparative proteomics approach allows for the separation of proteins required to sustain main cellular functions and proteins involved in adaptation to a particular lifestyle.These facts shows that mycoplasmas are the most suitable object for studying minimal cell content,and the used approach has to include all levels,from genomic to proteomic.

It becomes obvious that the additional layer of comparison on the protein expression level does not just allow us to obtain information that is unavailable on the genomic level,but to introduce a new dimension in the information flow:the spatial organization of the main cellular functions.Thus,studies on the spatial interactions between DNA,RNA,and proteins are inseparable from the studies on minimal cells.

Supporting Information

Table S1Proteins with non-extreme properties found in genome core but absent from proteome core.

(DOC)

Table S2Proteins from core proteome found in M pneumonia complexes.

(DOC)

Table S3Proteins of the core proteome that are not found in complexes.

(DOC)

Table S4Proteins found in complexes of M pneumoniae and found in genome core but not included in proteome core. (DOC)

Table S5Antisense transcripts of Mycoplasma pneumonia overlap-ping with genes coding proteins of the proteome core. (DOC)Table S6ORFs of Acholeplasma laidlawii which are transcribed but not translated.

(DOC)

Table S7ORFs of Mycoplasma gallisepticum which are transcribed but not translated.

(DOC)

Table S8Peptide coverage of predicted Acholeplasma laidlawii ORFs.

(DOC)

Table S9Peptide coverage of predicted Mycoplasma gallisepti-cum ORFs.

(DOC)

Table S10COG’s found in the core genome(the whole table) and the core proteome(highlighted in the‘‘Core Proteome’’column).

(DOC)

Table S11COG’s essential in Mycoplasma genitalium but not found in genome core.

(DOC)

Table S12COG’s conserved in genomes of16Mycoplasma species.

(XLS)

Table S13Primers for Acholeplasma laidlawii ORFs.

(DOC)

Table S14Primers for Mycoplasma gallisepticum ORFs. (DOC)

Acknowledgments

The mass-spectrometry data obtained in this study has been submitted to the PRIDE database and may be accessed with following details: Accession number:15476-15477

Password:review30396

Login:Zs3‘RB9h

Author Contributions

Conceived and designed the experiments:GYF DGA VMG.Performed the experiments:DGA NAB VGL MAG IGK NAZ MVS IAD.Analyzed the data:GYF DGA.Contributed reagents/materials/analysis tools:DGA NAB VGL MAG IGK NAZ MVS IAD.Wrote the paper:GYF DGA VMG.

References

1.Koonin EV,Wolf YI(2008)Genomics of bacteria and archaea:the emerging

dynamic view of the prokaryotic world.Nucleic acids research36:6688–719.

2.Fraser CM,Gocayne JD,White O,Adams MD,Clayton RA,et al.(1995)The

minimal gene complement of Mycoplasma genitalium.Science270:397–403.

3.Mushegian AR,Koonin EV(1996)A minimal gene set for cellular life derived

by comparison of complete bacterial genomes.Proc Natl Acad Sci U S A93: 10268–10273.

4.Harris JK,Kelley ST,Spiegelman GB,Pace NR(2003)The Genetic Core of the

Universal Ancestor.Genome Research13:407–412.

5.Koonin EV,Mushegian AR,Bork P(1996)Non-orthologous gene displacement.

Trends Genet12:334–6.

6.Hutchison III CA(1999)Global Transposon Mutagenesis and a Minimal

Mycoplasma Genome.Science286:2165–2169.

7.Glass JI,Assad-garcia N,Alperovich N,Yooseph S,Lewis MR,et al.(2006)

Essential genes of a minimal bacterium.Proc Natl Acad Sci U S A103: 425–430.

8.Zhang R,Lin Y(2009)DEG 5.0,a database of essential genes in both

prokaryotes and eukaryotes.Nucleic acids research37:D455–8.

9.Kobayashi K,Ehrlich SD,Albertini A,Amati G,Andersen KK,et al.(2003)

Essential Bacillus subtilis genes.Proceedings of the National Academy of Sciences of the United States of America100:4678–83.10.Fang G,Rocha E,Danchin A(2005)How essential are nonessential genes?

Molecular biology and evolution22:2147–56.

11.Forster AC,Church GM(2006)Towards synthesis of a minimal cell.Molecular

Systems Biology.

12.Baba T,Ara T,Hasegawa M,Takai Y,Okumura Y,et al.(2006)Construction

of Escherichia coli K-12in-frame,single-gene knockout mutants:the Keio collection.Molecular systems biology2:2006.0008.

13.Sassetti CM,Boyd DH,Rubin EJ(2003)Genes required for mycobacterial

growth defined by high density mutagenesis.Molecular microbiology48: 77–84.

14.Rasmussen S,Nielsen HB,Jarmer H(2009)The transcriptionally active regions

in the genome of Bacillus subtilis.Molecular microbiology73:1043–57.

15.Gupta N,Benhamida J,Bhargava V,Goodman D,Kain E,et al.(2008)

Comparative proteogenomics:combining mass spectrometry and comparative genomics to analyze multiple genomes.Genome research18:1133–42.

16.Callister SJ,McCue LA,Turse JE,Monroe ME,Auberry KJ,et al.(2008)

Comparative Bacterial Proteomics:Analysis of the Core Genome Concept.

PLoS One3:e1542.

17.Jaffe JD,Stange-Thomann N,Smith C,DeCaprio D,Fisher S,et al.(2004)The

complete genome and proteome of Mycoplasma mobile.Genome research14: 1447–61.

https://www.wendangku.net/doc/734007817.html,emmli UK(1970)Cleavage of structural proteins during the assembly of the

head of bacteriophage T4.Nature227:680–685.

19.Shevchenko A,Wilm M,Vorm O,Mann M(1996)Mass spectrometric

sequencing of proteins silver-stained polyacrylamide gels.Anal Chem68:850–8.

20.Jensen ON,Wilm M,Shevchenko A,Mann M(1999)Sample preparation

methods for mass spectrometric peptide mapping directly from2-DE gels.\ Methods Mol.Biol112:513–530.

21.Beck M,Claassen M,Aebersold R(2010)Comprehensive proteomics.Current

opinion in biotechnology1-6.

22.Ku¨hner S,Noort V van,Betts MJ,Leo-Macias A,Batisse C,et al.(2009)

Proteome organization in a genome-reduced bacterium.Science(New York, N.Y.)326:1235–40.

23.Demina IA,Serebryakova MV,Ladygina VG,Rogova MA,Zgoda VG,et al.

(2009)Proteome of the bacterium Mycoplasma gallisepticum.Biochemistry (Moscow)74:165–174.

24.Gu¨ell M,Noort V van,Yus E,Chen W-H,Leigh-Bell J,et al.(2009)

Transcriptome complexity in a genome-reduced bacterium.Science(New York, N.Y.)326:1268–71.

https://www.wendangku.net/doc/734007817.html,michau FM,Rothe FM,Herzberg C,Wagner E,Hellwig D,et al.(2009)

Novel activities of glycolytic enzymes in Bacillus subtilis:interactions with essential proteins involved in mRNA processing.Molecular&cellular proteomics:MCP8:1350–60.26.Toledo-Arana A,Dussurget O,Nikitas G,Sesto N,Guet-Revillet H,et al.(2009)

The Listeria transcriptional landscape from saprophytism to virulence.Nature 459:950–6.

27.Pereyre S,Sirand-Pugnet P,Beven L,Charron A,Renaudin H,et al.(2009)Life

on arginine for Mycoplasma hominis:clues from its minimal genome and comparison with other human urogenital mycoplasmas.PLoS genetics5: e1000677.

28.Gitai Z(2005)The new bacterial cell biology:moving parts and subcellular

architecture.Cell120:577–86.

29.Jain R,Rivera MC,Lake JA(1999)Horizontal gene transfer among genomes:

the complexity hypothesis.Proceedings of the National Academy of Sciences of the United States of America96:3801–6.

30.Peterson SN,Fraser CM(2001)The Complexity of Simplicity.Genome Biol2.

31.Eisen JA(1998)A phylogenomic study of the MutS family of proteins.Nucleic

acids research26:4291–300.

https://www.wendangku.net/doc/734007817.html,rtigue C,Vashee S,Algire MA,Chuang R-Y,Benders GA,et al.(2009)

Creating bacterial strains from genomes that have been cloned and engineered in yeast.Science(New York,N.Y.)325:1693–6.

33.Pennisi E(2004)Searching for the Genome’s Second Code.Science306:

632–635.

34.Grundy FJ,Henkin TM(2006)From ribosome to riboswitch:control of gene

expression in bacteria by RNA structural rearrangements.Crit Rev Biochem Mol Biol41:329–38.

The way常见用法

The way 的用法 Ⅰ常见用法: 1)the way+ that 2)the way + in which(最为正式的用法) 3)the way + 省略(最为自然的用法) 举例:I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法: 在当代美国英语中,the way用作为副词的对格,“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2)The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3)The way =how/ how much No one can imagine the way he missed her. 4)The way =because

Barratt冲动量表(巴瑞特冲动性人格问卷)(BIS-11)

巴瑞特衝動性人格問卷 通常情況下,人們思考問題的方式不同,採取的行動也不同。這部分的目的是瞭解您在一些情況下思考問題與行動的方式。下邊有30個問題,請您從“不是”、“極少”、“有時’,、 “經常”、“總是”五個答案中選擇一個最適合您情況的答案。答案不存在對與錯,不要花太多時間思考每個問題。如果您不太清楚如何回答,請儘量估計。

[量表介紹] 本研究採用的Barratt Impulsiveness Seale(BIS一11)中文版問卷由北京心理危機研究與干預中心翻譯修訂。自Barratt1959年制定以來,該量表被修訂過11版,現被廣泛使用的是JimH.Patl995年修訂的BIS一11,該自評量表用來評估個體的衝動性人格特徵的情況中心首先對BIS一11英文版進行翻譯,通過歷時5次的修改評定,在符合各分量表原意的前提下,對部分條目做了修改,以適合中國本土文化對衝動性的理解。隨後又進行了兩次預實驗,根據預實驗的結果對問卷部分條目進行修改。修訂後的中文版Barratt衝動性人格量表由30個條目組成,包括三個維度:運動衝動性(Motor Impulsiveness),包括條目2、5、8、11、14、17、20、23、26和29;認知衝動性(cognitive Impulsiveness),包括條3、6、9、12、15、18、21、24、27和30;以及無計畫衝動性(No Planning Impulsiveness),包括條目1、4、7、10、13、16、19、22、25和28。採用Likerts級評分(不是、極少、有時、經常、總是),每一組題目從1一5分記分,總分在30一150分之間變化,高分分別代表多動、注意力不集中和缺少計畫。各個分量表因數分等於所含條目得分之和,最低分為0分,最高分為50分;為了將分量表得分轉換成0一100分,分量表得分等於“[(所含條目得分之和一10)/40]*100”;總分為“各個分量表得分之和/3”.得分越高,衝動性越強。 [衝動性人格與自身意念的關係:] 國內學者通過艾森克人格問捲進行調查發現:人格是自殺意念的基礎性因素,神經質和精神質主要通過中間變數(應對、防禦、支持等)等對自殺意念間接起作用[23]。即使一些小事也能給神經質傾向者造成巨大的心理壓力,使他們產生強烈的情緒反應和對生活的無望感,而且他們對事物常常採用過激的應對方式因此容易出現自殺意念。 高衝動性人格的人帶有顯著的情緒色彩,多數表現為情緒不穩定,常因一點小事而使心情變壞,是情緒的焦慮反應[31]、神經質與自殺意念呈正相關[28]。國內胡冬梅等人應用心理解剖的方法研究自殺死亡者的危險因素,結果顯示神經質是殺的最主要的危險因素[42】。 國外對大五人格與自殺意念的研究表明:神經質和開放性與自殺意念呈正相關,得分越高,越容易產生自殺意念[43];責任感【44】、外傾性[45l、宜人性【46】與自殺意念呈負相關。翟書濤認為人格障礙是自殺意念的一個重要因素團】,張道祥的研究表明幾乎所有有自殺想法和行為的人都有人格不良傾向[48]。國外Hull一Blank等人的研究表明衝動性人格特徵容易產生自殺意念[5l]。 Jelena等對青年人的研究表明,衝動性人格特徵影響自殺意念,但是性別對其影響大小產生了一定的調節作用,與衝動性的男性相比,女性出現較強自殺意念的可能性比較 大[56] (參考來源:安靜(2008)衝動性人格特徵與自殺意念關係的研究. 中國地質大學碩士學位論文。指導老師:郭蘭心、費立鵬)

The way的用法及其含义(二)

The way的用法及其含义(二) 二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语: 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势,忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中

Barratt冲动量表(BIS-11)―巴瑞特冲动性人格问卷

Barratt 沖動量表/巴瑞特衝動性人格問卷/ BIS-11 通常情況下,人們思考問題的方式不同,採取的行動也不同。這部分的目的是瞭解您在一些情況下思考問題與行動的方式。下邊有30 個問題,請您從“不是”、“極少”、“有時'、“經常”、“總是”五個答案中選擇一個最適合您情況的答案。答案不存在對與錯,不要花太多時間思考每個問題。如果您不太清楚如何回答,請儘量估計。 1. 我認真安排每件事。 2. 我做事不加思考。 3. 遇到問題時我能想出好辦法。 4. 我對未來有計劃。 5. 我不能很好地控制自己的行為。 6. 必要時我能夠長時間考慮一個問題。 7. 我有規律地存錢或攢錢。 8. 我難以控制自己的脾氣。 9. 我能從不同的角度考慮問題。 10. 我對工作和獲得收入有計劃。 11. 我說話不加思考。 12. 遇到問題時我喜歡慢慢考慮。 13. 我做事比較理智。 14. 我激動時難以控制自己的行為。 15. 遇到難題時我能耐心思考解決問題的辦法。

16. 我有規律地安排飲食起居。 17. 我容易衝動行事 18. 做決定前,我喜歡仔細考慮得失。 19. 我離開家之前把事情都安排好。 20. 我不考慮後果而立即行動。 21. 我冷靜地思考問題。 22. 我做事時能按計劃完成。 23. 我容易衝動性購物。 24. 遇到難題時我不會輕易下結論。 25. 我花錢有計劃性。 26. 我做事十分莽撞。 27. 我思考問題時能集中注意力。 28. 我很看重對未來的安排。 29. 我想到什麼就馬上去做。 30. 我容易想出新的辦法來解決遇到的困難。不是 1 極少 2 有時 3 經常 4 總是5[量表介紹] 本研究採用的BarrattlmpulsivenessSeale(BIW 11)中文版問卷由北京心理危機研究與干預中心翻譯修訂。自Barratt1959 年制定以來,該量表被修訂過11 版,現被廣泛使用的是JimH.Patl995年修訂的BIS一11,該自評量表用來評估個體的衝動性人格特徵的情況中心首先對BIS 一11英文版進行翻譯,通過歷時 5 次的修改評定,在符合各分量表原意的前提下,對部分條目做了修改,以適合

(完整版)the的用法

定冠词the的用法: 定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸 或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...

Dickman冲动性量表在大学生中应用的信效度分析

Advances in Psychology 心理学进展, 2016, 6(12), 1267-1272 Published Online December 2016 in Hans. https://www.wendangku.net/doc/734007817.html,/journal/ap https://www.wendangku.net/doc/734007817.html,/10.12677/ap.2016.612160 文章引用: 郑丽君, 张婷(2016). Dickman 冲动性量表在大学生中应用的信效度分析. 心理学进展, 6(12), 1267-1272. Reliability and Validity of Dickman’s Impulsivity Inventory in Chinese College Students Lijun Zheng, Ting Zhang * Faculty of Psychology, Southwest University, Chongqing Received: Nov. 25th , 2016; accepted: Dec. 12th , 2016; published: Dec. 15th , 2016 Copyright ? 2016 by authors and Hans Publishers Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). https://www.wendangku.net/doc/734007817.html,/licenses/by/4.0/ Abstract Objective: To introduce and revise Dickman impulse scale (Dickman’s Impulsivity Inventory, DII) and test its reliability and validity among college students. Methods: The DII was tested in 500 Chinese college students, and retested after four weeks. Results: The DII internal consistency coef-ficient is 0.649; subscales of internal consistency coefficients are above 0.7; retest reliability is 0.748; it has a significant correlation with the Buss and Perry attack questionnaire; confirmatory factor analysis showed that the structure fitting is good. Conclusion: The DII is a good tool for a better assessment of impulsive behavior. Keywords Impulsive Scale, Functional Impulsivity, Dysfunctional Impulsivity, Reliability, Validity Dickman 冲动性量表在大学生中 应用的信效度分析 郑丽君,张 婷* 西南大学心理学部,重庆 Open Access * 通讯作者。

“the way+从句”结构的意义及用法

“theway+从句”结构的意义及用法 首先让我们来看下面这个句子: Read the followingpassageand talkabout it wi th your classmates.Try totell whatyou think of Tom and ofthe way the childrentreated him. 在这个句子中,the way是先行词,后面是省略了关系副词that或in which的定语从句。 下面我们将叙述“the way+从句”结构的用法。 1.the way之后,引导定语从句的关系词是that而不是how,因此,<<现代英语惯用法词典>>中所给出的下面两个句子是错误的:This is thewayhowithappened. This is the way how he always treats me. 2.在正式语体中,that可被in which所代替;在非正式语体中,that则往往省略。由此我们得到theway后接定语从句时的三种模式:1) the way+that-从句2)the way +in which-从句3) the way +从句 例如:The way(in which ,that) thesecomrade slookatproblems is wrong.这些同志看问题的方法

不对。 Theway(that ,in which)you’re doingit is comple tely crazy.你这么个干法,简直发疯。 Weadmired him for theway inwhich he facesdifficulties. Wallace and Darwingreed on the way inwhi ch different forms of life had begun.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 This is the way(that) hedid it. I likedthe way(that) sheorganized the meeting. 3.theway(that)有时可以与how(作“如何”解)通用。例如: That’s the way(that) shespoke. = That’s how shespoke.

barratt冲动量表(巴瑞特冲动性人格问卷)(bis-)

巴瑞特冲动性人格问卷 通常情况下,人们思考问题的方式不同,采取的行动也不同。这部分的目的是了解您在 一些情况下思考问题与行动的方式。下边有30个问题,请您从“不是”、“极少”、“有时’,、 “经常”、“总是”五个答案中选择一个最适合您情况的答案。答案不存在对与错,不要 花太多时间思考每个问题。如果您不太清楚如何回答,请尽量估计。

[量表介绍] 本研究采用的Barratt Impulsiveness Seale(BIS一11)中文版问卷由北京心

理危机研究与干预中心翻译修订。自Barratt1959年制定以来,该量表被修订过11版,现被广泛使用的是JimH.Patl995年修订的BIS一11,该自评量表用来评估个体的冲动性人格特徵的情况中心首先对BIS一11英文版进行翻译,通过历时5次的修改评定,在符合各分量表原意的前提下,对部分条目做了修改,以适合中国本土文化对冲动性的理解。随後又进行了两次预实验,根据预实验的结果对问卷部分条目进行修改。修订後的中文版Barratt冲动性人格量表由30个条目组成,包括三个维度:运动冲动性(Motor Impulsiveness),包括条目2、5、8、11、14、17、20、23、26和29;认知冲动性(cognitive Impulsiveness),包括条3、6、9、12、15、18、21、24、27和30;以及无计画冲动性(No Planning Impulsiveness),包括条目1、4、7、10、13、16、19、22、25和28。采用Likerts级评分(不是、极少、有时、经常、总是),每一组题目从1一5分记分,总分在30一150分之间变化,高分分别代表多动、注意力不集中和缺少计画。各个分量表因数分等於所含条目得分之和,最低分为0分,最高分为50分;为了将分量表得分转换成0一100分,分量表得分等於“[(所含条目得分之和一10)/40]*100”;总分为“各个分量表得分之和/3”.得分越高,冲动性越强。 [冲动性人格与自身意念的关系:] 国内学者通过艾森克人格问卷进行调查发现:人格是自杀意念的基础性因素,神经质和精神质主要通过中间变数(应对、防御、支持等)等对自杀意念间接起作用[23]。即使一些小事也能给神经质倾向者造成巨大的心理压力,使他们产生强烈的情绪反应和对生活的无望感,而且他们对事物常常采用过激的应对方式因此容易出现自杀意念。 高冲动性人格的人带有显着的情绪色彩,多数表现为情绪不稳定,常因一点

way 用法

表示“方式”、“方法”,注意以下用法: 1.表示用某种方法或按某种方式,通常用介词in(此介词有时可省略)。如: Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法,其后可接不定式或of doing sth。 如: It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词),也可跟由that 或in which 引导的定语从句,但是其后的从句不能由how 来引导。如: 我不喜欢他说话的态度。 正:I don’t like the way he spoke. 正:I don’t like the way that he spoke. 正:I don’t like the way in which he spoke. 误:I don’t like the way how he spoke. 4.注意以下各句the way 的用法: That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看,你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题: ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A,而不选其他几项,则要弄清选项中含way的四个短语的不同意义和用法,下面我们就对此作一归纳和小结。 一、in a way的用法 表示:在一定程度上,从某方面说。如: In a way he was right.在某种程度上他是对的。注:in a way也可说成in one way。 二、on the way的用法 1、表示:即将来(去),就要来(去)。如: Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示:在路上,在行进中。如: He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示:(婴儿)尚未出生。如: She has two children with another one on the way.她有两个孩子,现在还怀着一个。 She's got five children,and another one is on the way.她已经有5个孩子了,另一个又快生了。 三、by the way的用法

The way的用法及其含义(一)

The way的用法及其含义(一) 有这样一个句子:In 1770 the room was completed the way she wanted. 1770年,这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么?其意义如何?在阅读时,学生经常会碰到一些含有the way 的句子,如:No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中,the way之后的部分都是定语从句。第一句的意思是,“没人知道他是怎样发明这台机器的。”the way的意思相当于how;第二句的意思是,“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中,the way也是as的含义。随着现代英语的发展,the way的用法已越来越普遍了。下面,我们从the way的语法作用和意义等方面做一考查和分析: 一、the way作先行词,后接定语从句 以下3种表达都是正确的。例如:“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如:“火灾如何发生的,有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.

Barratt冲动量表(BIS-11)—巴瑞特冲动性人格问卷

Barratt沖動量表/ 巴瑞特衝動性人格問卷/ BIS-11 通常情況下,人們思考問題的方式不同,採取的行動也不同。這部分的目的是瞭解您在一些情況下思考問題與行動的方式。下邊有30個問題,請您從“不是”、“極少”、“有時’、“經常”、“總是”五個答案中選擇一個最適合您情況的答案。答案不存在對與錯,不要花太多時間思考每個問題。如果您不太清楚如何回答,請儘量估計。

[量表介紹] 本研究採用的Barratt Impulsiveness Seale(BIS一11)中文版問卷由北京心理危機研究與干預中心翻譯修訂。自Barratt1959年制定以來,該量表被修訂過11版,現被廣泛使用的是JimH.Patl995年修訂的BIS一11,該自評量表用來評估個體的衝動性人格特徵的情況中心首先對BIS一11英文版進行翻譯,通過歷時5次的修改評定,在符合各分量表原意的前提下,對部分條目做了修改,以適合中國本土文化對衝動性的理解。隨後又進行了兩次預實驗,根據預實驗的結果對問卷部分條目進行修改。 修訂後的中文版Barratt衝動性人格量表由30個條目組成,包括三個維度:運動衝動性(Motor Impulsiveness),包括條目2、5、8、11、14、17、20、23、26和29;認知衝動性(cognitive Impulsiveness),包括條3、6、9、12、15、18、21、24、27和30;以及無計畫衝動性(No Planning Impulsiveness),包括條目1、4、7、10、13、16、19、22、25和28。採用Likerts級評分(不是、極少、有時、經常、總是),每一組題目從1一5分記分,總分在30一150分之間變化,高分分別代表多動、注意力不集中和缺少計畫。各個分量表因數分等於所含條目得分之和,最低分為0分,最高分為50分;為了將分量表得分轉換成0一100分,分量表得分等於“[(所含條目得分之和一10)/40]*100”;總分為“各個分量表得分之和/3”.得分越高,衝動性越強。 [衝動性人格與自身意念的關係:] 國內學者通過艾森克人格問捲進行調查發現:人格是自殺意念的基礎性因素,神經質和精神質主要通過中間變數(應對、防禦、支持等)等對自殺意念間接起作用[23]。即使一些小事也能給神經質傾向者造成巨大的心理壓力,使他們產生強烈的情緒反應和對生活的無望感,而且他們對事物常常採用過激的應對方式因此容易出現自殺意念。 高衝動性人格的人帶有顯著的情緒色彩,多數表現為情緒不穩定,常因一點小事而使心情變壞,是情緒的焦慮反應[31]、神經質與自殺意念呈正相關[28]。國內胡冬梅等人應用心理解剖的方法研究自殺死亡者的危險因素,結果顯示神經質是殺的最主要的危險因素[42】。 國外對大五人格與自殺意念的研究表明:神經質和開放性與自殺意念呈正相關,得分越高,越容易產生自殺意念[43];責任感【44】、外傾性[45l、宜人性【46】與自殺意念呈負相關。翟書濤認為人格障礙是自殺意念的一個重要因素團】,張道祥的研究表明幾乎所有有自殺想法和行為的人都有人格不良傾向[48]。國外Hull一Blank等人的研究表明衝動性人格特徵容易產生自殺意念[5l]。 Jelena等對青年人的研究表明,衝動性人格特徵影響自殺意念,但是性別對其影響大小產生了一定的調節作用,與衝動性的男性相比,女性出現較強自殺意念的可能性比較 大[56] (參考來源:安靜(2008)衝動性人格特徵與自殺意念關係的研究. 中國地質大學碩士學位論文。指導老師:郭蘭心、費立鵬)

way 的用法

way 的用法 【语境展示】 1. Now I’ll show you how to do the experiment in a different way. 下面我来演示如何用一种不同的方法做这个实验。 2. The teacher had a strange way to make his classes lively and interesting. 这位老师有种奇怪的办法让他的课生动有趣。 3. Can you tell me the best way of working out this problem? 你能告诉我算出这道题的最好方法吗? 4. I don’t know the way (that / in which) he helped her out. 我不知道他用什么方法帮助她摆脱困境的。 5. The way (that / which) he talked about to solve the problem was difficult to understand. 他所谈到的解决这个问题的方法难以理解。 6. I don’t like the way that / which is being widely used for saving water. 我不喜欢这种正在被广泛使用的节水方法。 7. They did not do it the way we do now. 他们以前的做法和我们现在不一样。 【归纳总结】 ●way作“方法,方式”讲时,如表示“以……方式”,前面常加介词in。如例1; ●way作“方法,方式”讲时,其后可接不定式to do sth.,也可接of doing sth. 作定语,表示做某事的方法。如例2,例3;

情绪效价线索对大学生香烟依赖者行为抑制功能的影响香烟依赖者; 行为抑制; 冲动性; 情绪

香烟依赖俗称烟瘾,属于成瘾行为,是长期吸烟行为所导致的一种慢性高复发性疾病,其本质是尼古丁依赖。近年来,有关成瘾行为的研究颇多,主要集中在香烟成瘾、吸毒成瘾、酒精成瘾、网络成瘾等领域。大量研究表明,个体的冲动性[1]和行为抑制能力[2]是造成成瘾的重要因素。在大量物质依赖的研究中发现,长期物质滥用行为将导致个体冲动性行为的显著增加以及行为抑制能力的显著降低[3]。其中,个体行为抑制能力是成瘾的关键因素,能否有效降低复吸率实现成功戒断与其息息相关。香烟依赖是物质依赖的一种形式,个体的行为抑制功能对烟瘾的戒断是否同样关键,有待于更进一步的研究证实。除此,有研究表明,情绪效价线索对正常个体行为抑制能力有一定影响,具体表现为: 负性情绪线索减弱个体的行为抑制能力,正性情绪线索促进个体行为抑制能力[4]。对于香烟依赖者行为抑制功能而言,情绪效价线索的影响是否与正常人一致,积极情绪能否提高香烟依赖者的抑制能力,从而有效的实现戒断,目前尚未清楚。为此,本文采用双选择情绪oddball 范式考察情绪效价线索对香烟依赖者行为抑制能力的影响。该范式包含两类刺激,标准刺激与偏差刺激。通过操纵偏差刺激的效价,记录被试的行为反应时,便可考察情绪对个体行为抑制的影响。1 实验方法1. 1 被试本研究包括实验组和对照组两组被试。通过尼古丁依赖检验量表[5]筛选出35 名香烟依赖者作为实验组,平均烟龄22. 46( 0. 85) ; 另外,随机选取正常组被试35 名,无吸烟史,平均年龄21. 43( 1. 27) ,独立样本t 检验表明两组被试年龄无显著差异。所有被试均为男性大学生,身心健康,右利手,视力或矫正视力正常,均签署了实验知情同意书。1. 2 量表实验开始前所有被试需完成尼古丁依赖检验量表与巴瑞特冲动人格问卷[6]。尼古丁依赖检验量表共有6 道题目,总分在0 ~10 之间,得分越高表示吸烟程度越严重( ≥6 分提示有尼古丁依赖) 。巴瑞特冲动人格问卷中文修订版[6]共30 个题目,采用5 点评分,包括认知冲动性、运动冲动性、无计划冲动性三个维度( 各10 个题目) ,三个维度的克隆巴赫α 系数依次为0. 73、0. 84、0. 88,总量表的α 系数为0. 89。量表得分越高代表冲动性越高。1. 3 刺激材料与程序本研究采用情绪双选择oddball 任务。实验刺激分为标准刺激和偏差刺激( 70% vs 30% ) 。其中标准刺激为一张凳子的自然景物图片,偏差刺激为正性、中性和负性图片各一张,各效价偏差刺激图片呈现概率为10% ,所有图片均来自于国际情绪图片库( IAPS) 。实验程序通过E -prime 编程,包括200 个试次,其中大概率刺激呈现140 次,各效价偏差刺激均呈现20 次,呈现顺序完全随机。实验开始,首先在电脑屏幕中央呈现“+ ”字,持续300ms,然后空屏500 ms,随后随机呈现图片刺激800 ms,最后空屏800 ms。被试的任务是标准刺激按F 键,偏差刺激按J 键。正式实验开始之前,所有被试均进行练习,练习正确率达100% 方可进行正式实验。2 实验结果2. 1 实验组对照组被试冲动量表得分实验组与对照组巴瑞特冲动人格问卷得分如表1 所示,两组被试在运动冲动性、认知冲动性、无计划冲动性各维度上得分均有显著性差异( P <0. 001) ; 除此,两组被试在冲动性总分上差异同样显著( P <0. 001) ,具体表现为实验组被试冲动性得分显著高于对照组。A 组与B 组香烟依赖者在冲动性得分上存在显著性差异( P <0. 05) ,具体表现为B 组香烟依赖者冲动性得分显著性高于A 组; 另外,无论效价,两组被试在行为抑制反应时方面差异没有统计学意义。详见表2。2. 3 实验组与对照组被试任务反应时无论效价,实验组与对照组标准刺激与偏差刺激下任务反应时如表3 所示。重复测量方差分析表明,刺激类型主效应显著( F( 1,68) = 486. 62,P <0. 001) ,偏差刺激反应时显著长于标准刺激反应时; 组别主效应显著( F ( 1,68 ) = 14. 00,P <0. 001) ,实验组任务反应时显著长于对照组任务反应时; 交互作用显著( F( 1,68) = 9. 81,P <0. 01) 。进一步的简单效应分析表明,标准刺激任务下,实验组与对照组任务反应时差异不显著; 偏差刺激任务下,两组被试任务反应时差异显著( P <0. 001) ,具体表现为实验组被试的偏差刺激反应时显著长于对照组偏差刺激反应时。重复测量方差分析表明,刺激

Barratt冲动量表巴瑞特冲动性人格问卷BIS

B a r r a t t冲动量表巴瑞特 冲动性人格问卷B I S The latest revision on November 22, 2020

巴瑞特冲动性人格问卷 通常情况下,人们思考问题的方式不同,采取的行动也不同。这部分的目的是了解您在 一些情况下思考问题与行动的方式。下边有30个问题,请您从“不是”、“极少”、“有时’,、 “经常”、“总是”五个答案中选择一个最适合您情况的答案。答案不存在对与错,不要

本研究采用的Barratt Impulsiveness Seale(BIS一11)中文版问卷由北京心理危机研究与干预中心翻译修订。自Barratt1959年制定以来,该量表被修订过11版,现被广泛使用的是年修订的BIS一11,该自评量表用来评估个体的冲动性人格特徵的情况中心首先对BIS一11英文版进行翻译,通过历时5次的修改评定,在符合各分量表原意的前提下,对部分条目做了修改,以适合中国本土文化对冲动性的理解。随後又进行了两次预实验,根据预实验的结果对问卷部分条目进行修改。修订後的中文版Barratt冲动性人格量表由30个条目组成,包括三个维度:运动冲动性(Motor Impulsiveness),包括条目2、5、8、11、14、17、20、23、26和29;认知冲动性(cognitive Impulsiveness),包括条3、6、9、12、15、18、21、24、27和30;以及无计画冲动性(No Planning Impulsiveness),包括条目1、4、7、10、13、16、19、22、25和28。采用Likerts级评分(不是、极少、有时、经常、总是),每一组题目从1一5分记分,总分在30一150分之间变化,高分分别代表多动、注意力不集中和缺少计画。各个分量表因数分等於所含条目得分之和,最低分为0分,最高分为50分;为了将分量表得分转换成0一100分,分量表得分等於“[(所含条目得分之和一10)/40]*100”;总分为“各个分量表得分之和/3”.得分越高,冲动性越强。[冲动性人格与自身意念的关系:] 国内学者通过艾森克人格问卷进行调查发现:人格是自杀意念的基础性因素,神经质和精神质主要通过中间变数(应对、防御、支持等)等对自杀意念间接起作用[23]。即使一些小事也能给神经质倾向者造成巨大的心理压力,使他们产生强烈的情绪反应和对生活的无望感,而且他们对事物常常采用过激的应对方式因此容易出现自杀意念。 高冲动性人格的人带有显着的情绪色彩,多数表现为情绪不稳定,常因一点小事而使心情变坏,是情绪的焦虑反应[31]、神经质与自杀意念呈正相关[28]。国内胡冬梅等人应用心理解剖的方法研究自杀死亡者的危险因素,结果显示神经质是杀的最主要的危险因素[42】。 国外对大五人格与自杀意念的研究表明:神经质和开放性与自杀意念呈正相关,得分越 高,越容易产生自杀意念[43];责任感【44】、外倾性[45l、宜人性【46】与自杀意念呈负相关。翟书涛认为人格障碍是自杀意念的一个重要因素团】,张道祥的研究表明几乎所有有自杀想法和行为的人都有人格不良倾向[48]。国外Hull一Blank等人的研究表明冲动性人格特徵容易产生自杀意念[5l]。 Jelena等对青年人的研究表明,冲动性人格特徵影响自杀意念,但是性别对其影响大小产生了一定的调节作用,与冲动性的男性相比,女性出现较强自杀意念的可能性比较 大[56] (参考来源:安静(2008)冲动性人格特徵与自杀意念关系的研究. 中国地质大学硕士学位论文。指导老师:郭兰心、费立鹏)

the-way-的用法讲解学习

t h e-w a y-的用法

The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.

way的用法总结大全

way的用法总结大全 way的用法你知道多少,今天给大家带来way的用法,希望能够帮助到大家,下面就和大家分享,来欣赏一下吧。 way的用法总结大全 way的意思 n. 道路,方法,方向,某方面 adv. 远远地,大大地 way用法 way可以用作名词 way的基本意思是“路,道,街,径”,一般用来指具体的“路,道路”,也可指通向某地的“方向”“路线”或做某事所采用的手段,即“方式,方法”。way还可指“习俗,作风”“距离”“附近,周围”“某方面”等。 way作“方法,方式,手段”解时,前面常加介词in。如果way前有this, that等限定词,介词可省略,但如果放在句首,介词则不可省略。

way作“方式,方法”解时,其后可接of v -ing或to- v 作定语,也可接定语从句,引导从句的关系代词或关系副词常可省略。 way用作名词的用法例句 I am on my way to the grocery store.我正在去杂货店的路上。 We lost the way in the dark.我们在黑夜中迷路了。 He asked me the way to London.他问我去伦敦的路。 way可以用作副词 way用作副词时意思是“远远地,大大地”,通常指在程度或距离上有一定的差距。 way back表示“很久以前”。 way用作副词的用法例句 It seems like Im always way too busy with work.我工作总是太忙了。 His ideas were way ahead of his time.他的思想远远超越了他那个时代。 She finished the race way ahead of the other runners.她第一个跑到终点,远远领先于其他选手。 way用法例句

相关文档
相关文档 最新文档