Vectors for co-expression of an unrestricted number of proteins
线性代数的几何解释
• Author: Eero Simoncelli, Center for Neural Science, and Courant Institute of Mathematical Sciences. • Created: 20 January 1999. Last revised: 17 September 2001. • Send corrections or comments to eero.simoncelli@
v2
v v2 v1 v1
v1 v2
vN
2 The norm (or magnitude) of a vector is defined as: ||v || = n vn . Geometrically, this corresponds to the length of the vector. A vector containing all zero components has zero norm,
v
^) u ^ (v . u
^ u
3
Vector Spaces
Vectors live in vector spaces. Formally, a vector space is just a collection of vectors that is closed under linear combination. That is, if the two vectors {v, w} are in the space, then the vector av + bw (with a and b any scalars) is also in the space. All vector spaces include the zero vector (since multiplying any vector by the scalar zero gives you the zero vector). A subspace is a vector space lying within another vector space. This definition is somewhat abstract, as it implies that we construct vector spaces by starting with a few vectors and “filling out” the rest of the space through linear combination. But we have been assuming an implicit vector space all along: the space of all N -vectors (denoted R I N)
Vector space Review
V1 is itself a vector space!
Evangelos Milios (Dalhousie Univ.)
Dimensionality Reduction
February 11, 2007
6 / 43
Examples
In R3 A line through the origin is a subspace. A plane through the origin is a subspace. A line/plane not containing the origin is not subspace (why?) They are called affine subspaces
Dimensionality Reduction
Evangelos Milios
Dalhousie Univ., Faculty of Computer Science
February 11, 2007
Evangelos Milios (Dalhousie Univ.)
Dimensionality Reduction
Dimensionality Reduction
February 11, 2007
10 / 43
Projection onto subspace
V : vector space with inner product V1 : a subspace of V
Every vector v ∈ V can be decomposed into two orthogonal components: v1 that belongs to the subspace V1
February 11, 2007
里氏木霉外源表达载体的构建_英文_
Constructi on of Exogenous Expression Vector of Tri -choder ma reeseiZHANG X i ao-xuan 1,WANG Ao -xue2*1.Chengdong Co llege ,Nort heast Agricultural Univ ers ity ,Har b in 150030;2.Co llege of Lif e Sc iences ,Nort heas tAgricult ural Universit y ,Har -bin 150030Abstract [Objecti ve]The st udy w as t o construc ta ex ogenous expression v ector f or Tri chode r m a ree se i .[M et ho d]U sing CBH I pro moter and t er m inat or of T.ree se i strain 40359,we cons tructed an expr ession vec t or o f T.ree se i s train 40359f or expressing Hp t gene and got s ix strains capab l e o f grow ing on basic m ediu m c ontaining 175mg /L of hygro myc i n B ,f urt her c onduc t ed hygr omycin resis t ance tes.t [R es ults ]In co mpar-i son w ith t he ori g inal strain(w il d t ype),hygro myc in r esis t ance t he six engineer ed strains was increased by 75%;t he hygro myc i n resis t ance c oul d inherit st ab l y .[Co ncl usio n]Our results laid bas is f or bi o l o g i c al study on T.ree se i atm ol e cul a r and genetic a lly engineeri n g levels .Key wo r ds T ri cho de r m a ree se i ;Vec t or ;Genetic transf or mati o nReceived :February 18,2011 Accept ed :March 21,2011*C orresponding author .E -mai:l wangaoxue @yahoo .co mC BH I pro mot er fro m Tri choder m a reese i is a very strong pro m oter that is usually used for vector constructi o n f or gene-t i c m i provement of T.reese i ,i .e .,inserting t he gene of inter -est(GOI)int o t he space bet w een CBHI promot er and ter m-i nat or sequence .The reco mbined frag ment is transf or med into fil a ment ous fungi prot oplas,t and located and int egrat ed into chromoso m e by us i n g t he m ethod of t argeted gene replace -ment and int egrati o n .This makes the GO I dri v en by t he strong pro mot er CBH I and the required homologous or heter -ologous prot ein expressed secret ory under the induction of t he l e ader peptide of C BH I .This method has been successf ull y used f or construct engineered filament ous fungistai n s overex -pressing various t arget prot eins .For inst ance ,MU Jing -yu e t a l .[1]from Jilin Universit y succeeded i n overexpression of As-pe rg ill u s n i g er glucose ox i d ase gene under the control of CBH I pro m oter fro m T.reese i .I n vie w of t his ,we att empt ed to ex -press hygro mycin B phos photrans f erase gene i n T.reese i by us i n g CBH I promot er and ter m i n at or sequence ,and carri e d out hygromyc i n B resist ance t est on the y i e lded resistant strains and reco mbinants ,am i i n g at prov i d ing basis for bi o log -i c al study on T.reese i at molecular and genetically engineer -i n g l e vels .Mat eri a ls and MethodsExperi m entalmateri al sStrai ns and reagents Tri chode r m a reese i strain 40359was purchas ed from Chi n a Center of IndustrialCulture Collection ;plas m id pCAM1305.1harboring hygromyc i n B phos photrans -ferase gene was preserved by our research group ;vectors pMD18and pU C19were purchas ed fro m TaKaRa ;EF Taq DNA pol y merase ,plas m id DNA isolation k it and prm i ers were purchas ed from Beijing Langang B i o t ech Co .,Ltd .;T 4-DNA ligase ,DL2000Marker ,RT -PCR k i,t CTAB ,Tris #C,l B -mer -capt oet hano,l RNase and agarose were all purchas ed fro m TaKaRa ;all the restrictive endonulceas es used were pur -chased from MB;I agarose gel extrac tion k it was purchased fro m T iangen Biot ech (Beijing)Co .,Lt d .All other reagents were home made products at analyti c pure grade .M ed i a r eci pe(for 1L) Basalmediu m:20g glucose ,15gKH 2PO 4,5g(NH 4)2SO 4,0.6g CaC l 2#2H 2O,0.6g M g -SO 4#7H 2O,0.005g FeSO 4#7H 2O ,0.0016g M n SO 4#H 2O,0.0014g ZnSO 4#7H 2O ,0.002g Co C l 2#6H 2O,pH=5.5;aut o -cl a ving at 121e f or 30m in (addition of 2%of agar f or solid m ediu m and 1%agar for sem-i soli d medium ).Regenerati o n m edia :(1)l o wer layermediu m :additi o n of1mol/L of s orb-i t oland 2%of agar into basalm ediu m;(2)upper layermed-i u m :addition of 1mol/L of sorbit ol and 1%of agar into basal m ediu m.Experi m entalmethods Pri m er s and their sequences used for PCR a mplifi cati on Based on sequence inf or mation from GenBank ,soft ware Gene runnerwas e mployed to design prm i ers as f ollo w s :amplificati o n of CBH I promoter and signalpeptide(pCBHI ):P1:Sense :5c -GC GCATGC AATTCTGGAGACGGCTT -GTT -3c :S ph ÑP2:Ant -i s ens e :5c -GCGT CGACCTCCAAGT GTTGC -CAT CGTA -3c :S a lÑamplification of C BH I t er m inator(t CBHI ):T1:Sense :5c -GCGCGGATCCAGGTCACCTTCT CCAA -CATCA -3c :Bam H ÑT2:Ant -i sense :5c -GCGCGAATT CCACGAAGAGCG -GCGATTCTA -3c :Eco R Ñamplification of Hp t :H1:Sense :5c -GCGC GTCGAC ATGCCTGAACTCAC -CGCGAC -3c S a l ÑH2:Ant -i sens e :5c -GC GC GGAT CC CGGTCGGCATC -TACT CT ATT -3c Bam H Ñ.A mplifi cati on of target frag ment (1)PCR reacti o n volu me (50L l)f or amplifying t he upstrea m nucleoti d e fragment of T.reese i cbh 1gene was composed of 312.5L mol/L dNTPs ,2.5L mol/L of prm i er ,500ng of t emplate ,3.5-3.6mmol/L of MgC l 2,2.5U EF Taq DN A poly merase .These reacti o n co mponents were first react ed at 94e f or 7m in predenat ur -ati o n before t he addition of EF Taq DNA poly meras e ,then 30cyc l e s at 94e f or 1m in ,60e f or 1m in and 72e for 2.5m in ,finally at 72e f or 10m i n .(2)PCR reaction volume(50L l)f or a mplif y ing t he downstream nuc l e otide fragment of T.reese i cbh 1gene was composed of 200L mol/L of dNTPs ,1.5L mol/L of prm i er ,500ng of te mpl a t e ,1.5-2.5mmol/L ofAgri c ultural B i o technol o gyAgricult ural Sc i e nc e&Technology ,2011,12(2):308-312Copyright k 2011,I nf or mati o n Institute ofHAAS .A ll ri g ht s reserv ed .MgC l2,1.5U EF Taq DNA poly meras e.These reacti o n co mpo-nent s were first reac t ed at94e f or7m i n predenat urationbef ore the addition of EF Taq DNA poly meras e,t hen30cycl e s at94e f or1m in,55e f or1.5m in and72e f or1.5m in,fi n ally at72 e f or10m in.(3)PCR reac tion volu me(50L l)f or a mplifying the Hp t gene was co mposed of312.5L mol/L of dNTPs,1.0 L mol/L of prm i er,500ng of t emplate,2.5mmol/L ofMgC l2, 1.5U EF Taq DNA poly merase.These reaction co mponents were first react ed at94e f or7m i n predenaturati o n bef ore t he additi o n of EF Taq DNA poly merase,then30cycles at94e f or30s,60e f or1m in and72e f or2.5m in,finally at72e f or10m in.Constr uction of expr ession vector Geno m ic DNA of T.reese i[2-3]and P l a s m i d contai n ing Hygro m ycin B phospho-transferase gene was extracted as te mplat e f or PCR amplif-i cati o n using prm i ers P1and P2t o a mplif y T.reese iCBHI pro-mot er and CBHI signal pepti d e gene;the reacti o n products were ligat ed i n t o p MD18-T vect or;t he y i e lded vect orwas then double-digest ed by us i n g S phÑand S a lÑf or use.Likew ise,T. reese iCBH I promot erwas a mp lifi e d using prm i ers T1and T2 and ligated int o p MD18-T vector;the y i e lded vector was then double-digest ed by using Bam HÑand Eco RÑf or use.Esche-ri ch i a co l i hygromyc i n B(HygB)phosphotransferase gene was amplified using prm i ers H1and H2from pl a s m id DNA har-boring this gene,and li g at ed int o pMD18-T vec t or;t he yielded vec t or was t hen double-digest ed by us i n g S a lÑand Bam HÑf or use.The frag m ents yiel d ed above were li g at ed by usi n g T4 ligase at16e overni g h;t t he y i e lded productwas transfor med i n t o co mpet ent E.co li cells v i a heat shock method and coat ed on LB sel e cti o n pl a te cont aining Amp and HygB,the pl a tes were incubat ed at37e overni g h.t The recombi n antwas inoc-ulated i n t o LB li q ui d medium cont aini n g Amp and HygB and in-cubat ed at37e f or12-16h(200r/m in).The yiel d ed turbid bact erial solution was used to extract plas m i d DNA f or PCR and enzy mati c di g estion.The constructs consist s of CBH I promoter-CBH I signal peptide gene-Hyg gene-C BH I ter m i n at or-pUC19vector,desig-nat ed as Pcbh-HygB.Deter m i nation on the resi stance of T.reesei strain to HygB B.Se m-i solid basal medi a contai n ing25,50,75, 100,125,150,175and200mg/L of HygB were respectivel y prepared and m i x ed w ith appropriat e vol u me of T.reese i strain40359.Them ixtures were poured into the l o wer l a yerof plat e and cultured at28e for the observation of mycelial grow t h.Transfor mati on o f the pr o toplast of T.reesei strai n40359 Prot opl a st preparati o n and transfor mati o n was ref erred to the method of PenttiL¾M.et a l.[3].Appropriat e volu me of transfor mati o n reacti o n soluti o n was coated on the lower layer of regeneration mediu m and i n cubated at28e f or12-16h, then a l a yer of se m-i solid fil a ment ous f ungiMM mediu m cont a-i ning125mg/L of HygB was poured on the pl a t e used above and i n cubat ed at28e for3-4d.The Hyg B-resist ant transfor ma-nt s were sel e ct ed f or furt hermolecular identificati o n.PCR i dentifi cation of transgen i c str ai ns PCR a mplificati o n was perf or med by using genom ic DNA as t empl a t e and t he upstream and downstrea m prm i ers of Hp t as prm i ers,w ith ge-no m ic DNA of non-transf or med strain40359as negati v e con-trol and plas m id DNA as positive contro.lHpt-spec ific prm i ers:s ense:5c-ATGCCT GAACTCACCGCGAC-3c;ant-i sense:5c-CGGTCGGCATCTACT CTATT-3cFuncti onal i dentificati on o f transgenic strai ns Transgenic strains were inoculat ed on PDA media w it hout hygro mycin f or 6conti n uous generations.The yiel d ed spores were scraped int o ddH2O and coat ed on solid m edia containing diff erent concentrations of HygB;these medi a were incubat ed under dark f or12-24h,then appropriate vol u me of s em-i s olid me-dium cont aining s ame concentration of hygromycin was poured into thes e plates.Likew ise,non-transf or m ed strain 40359was used as negative control f or observing myceli a l grow t h.Results and AnalysisA mplifi cati on o f pCBH I,tCBHI and Hpt genesGenom ic DNA of T.reese i strai n YB40359was extract ed by using m i proved CTAB method(F i g.1).The y i e lded ge-nom ic DNAs were allw it h molecul a rweight hi g her t han20kb and hi g hly pure,whi c h could be used f or subsequent exper-i men.t PCR results of all the Hp t gene,t CBH I and pCBHI as-su med a singl e band,w ith Hp t gene of approxm i ately1000bp, t CBH I of approxm i ately600bp,pCBH I approxm i ately1600bp (F i g.2).M:Marker15000;lanes1-8:G enom i c DNA samples.Fi g.1E l e ctrophoresis patt ern of geno m ic DNA of T.reese i strain40359M:2000bp m arker;lanes1and2:Hyg;3-4:t C BHI;4-5:pC BH I.Fi g.2Elec tr ophores is patt ern of PCR a mp lific ati o n result sC l oning of pCBHI,t CBHI and Hpt genesThe a mplified results of pCBHI,t C BH I and Hp t genes were ligat ed into pMD18T-vect or and incubat ed at16e over-309ZHANG X i a o-xuan e t a l.Construc tion of Exogenous Ex pres s ion Vec t or o f Tri chode r m a reese inigh.t The li g at ed products were transfor med i n t o E.co li viaheat shock method and coated on LB pl a t e contai n ing Amp .Follow ing incubation for another 12-16h ,t wo white col o nies f or each gene were select ed f or PCR identificati o n .The re -sults showed that all six col o ni e s produced a band consistent w ith the size of t arget genes(Fig .3).The reco mbinants were identifi e d by using double diges -tion of Kpn I and S ph ,I the digestion produc t s were identifi e d w ith agarose gel electrophoresis(F i g .4).The double endonu -c l e ase di g esti o ns all produced t w o frag ments ,t he large one is the vector bond hi g her t han2.5kb ,the s mallone is the t arget genes :l a ne 1,about 600bp of t CBH I ;lane 2,about1000bp of Hp t ;lane 3,about 1600bp of pC BH I .This suggests that the t arget genes have been insert ed int o the c l o ning vec t or and successf ull y transf or med .The sequencing results (Shanghai Sangon Bioengineeri n g Co .,Lt d .)were blast ed and f ound an identity of 100%.M :2000bp marker ;1-2:Hyg ;3-4:t CB HI ;5-6:pC BH I .F i g .3 Bac t eriu m -based PCR a mplifi c ation on recombinantsM :2000bpmarker ;1:P l a s m id cont aini n g t CBH 1gene ;2:P l a s -m i d cont aini n g Hp t gene ;3:P l a sm i d cont ainin g pCBH 1gene .F i g .4 I dentifi c ation of reco mbined plas m id by double -di g esti o nI den tifi cati on expr essi on vector by usi ng PCR amp lifi ca -ti on and double endonucl ease digesti on As i n di c ated fro m Fig .5,PCR amplification results of all pCBHI ,t CBHI and Hp t genes accord w it h t hat us ed f or liga -tion ;l a ne 4is f or a mplificati o n of pC BH I p l u s Hp t gene ,w ith a size of approxm i atel y 2600bp ;lane 5is f or a mplificati o n of Hpt plus t CBH I gene ,w it h a size of approxm i at ely 1650bp .Endonucl e ase di g esti o n on recombinant s is using Eco R Ñpro -duced a s i z e of approxm i at ely 6000bp ,accordi n g w it h t he expect ed s iz e ;double endonulcease di g esti o n on recomb-inants is using S a l Ñand Bam H Ñto i d entif y Hp t gene ,whichproduced a larger band of approxm i at ely 4800bp(pU C19+pCBH I +t CBH I )and a s maller one of approxm i atel y 1000bp (Hp t gene);double endonul c ease digestion on reco mbinants is using S ph Ñand S a l Ñto identif y pCBHI ,which produced a lar -ger band of approxm i at ely 3300bp (pUC19+Hyg+t CBHI )and a s maller one of approxm i at ely 1600bp(pCBH I );double endonulcease di g esti o n on recombinant s is us i n g Bam H Ñand PstIto i d entif y t CBH I ,which produced a l a rger band of approx -m i at ely 4300bp(pUC19+Hyg+pCBHI )and a s maller one of approxm i at ely 600bp(t CBHI ).Not e :M:2000bp marker ;1:pC BHI ;2:t C BH I ;3:Hyg ;4:pC BH I and Hyg ;5:Hyg and t C BH I .Fi g.5 PCR a mplific ati o n on ex pres s ion vectorM1:15000bp mar ker ;M2:2000bp marker ;1:Sin g l e enzymedi g estion us i n g E co R Ñ;2:Dou b l e di g estion of S a l Ñand Bam H Ñt o i d entif y Hpt ;3:Doubl e di g estion of S ph Ñand S a l Ñto identif y pCB-H I ;4:Doubl e digesti o n of Bam H Ñand Pst Ñt o identif y t CBH I .Fi g.6 I dentifi c ation of ex press i o n by double -di g esti o nTransfor mati on of T.r eesei 40359and scr eeni ng of trans -for mantsRef erring t o PenttiL ¾M(1987),2-5L lof single endonu -clease di g est ed pUC19-Hyg was m ixed w it h the prot opl a st of T.reese i strain 40359(100s L l),using non -transfor med pro -t oplast as blank contro.l After coating on regenerationmedium containing HygB and incubat ed at 28e f or 3-4d ,six tran -f or m ants capabl e of grow ing on regeneration mediu m cont a-i ning 175mg/L of HygB .These six transf or m ants had st able and l a sting hygro mycin resi s tance aft er several conti n uous gen -erati o ns of culture .For the contro,l non -transf or med prot oplast of T.reesei strain 40359di d not grow onmediu m cont ai n ing 125mg/L of hygromycin ,indicati n g t hat there is no self resist ance m ut ation inw il d t ype strai n s .I dentificati on of tr ansgeni c strai ns PCR amp lifi cati on Geno m i c DNAs of hygro m ycin -resist ant strains all assumed a s i n gl e clear and orderly band(Fig .7).310Agricultural Sc i e nc e&Technology Vo.l 12,No .2,2011This indicat es that the DNA s amples have good qualit y and could be used for subsequent PCR amplifi c ati o n .Tab l e 1Gro w th sit uation of T.see se i strains on hy gro myc in Bp l a tesHy gro m y c in c oncentration M mg /LT.see se i Z40359T.see se i4035950++++75++++100+++125++-150++-175+-200--++repr es ent s we ll gro w th ;+r epres ents poor gro w th ;-repres ents no gro w th .F i g .7 E l e ctrophoresis patt ern of geno m ic DNA of genetic a llyengineered s tr a i nAs shown in Fig .8,PCR amplification on Hp t geneshowed t hat plas m id and six resist ant transf omants all produc -ed a target band of about 1000bp ,whil e non -transfor medstrain 40359di d no.t The results well demonstrat e that Hp t gene has transf or med and i n t egrated int o T.seese i strain 40359,designed as Z40359.M :2000bp marker ;1:Pos itive contro;l 2:Negati v e contro;l3-8:Transgenic s trains .Fi g.8 PCR det ection of pUC19-Hp t transgenic strainsResi stance and stab ility of tr ansgenic T.seesei strains tohygro myci n One of the T.seese i Z40359strains was cu-lt ured on solidm ediu m for s i x conti n uous generati o ns t o det ect its resistance t o hygro mycin(Fig .9).The results sho wed t hat T.seesei Z40359strains were res i s tant to175mg/L of hygro -m ycin ,and the resist ance l a st ed f or six generations .This confir m s that T.seeseiZ40359has a heredit ary stable resis-t ance t o hygromycin.1:Gene tically engineer ed T.ree se i s train Z40359on mediu m cont aining 100mg /L of hygro m yc in B ;2:P riginal strain 40359on med i u mcont aining 100mg/L hygro myc in B ;3:G eneti c all y engineered T.ree se i s train Z40359on mediu m cont aining 175mg/L of hygr omycin B ;4:O ri g i n a l s tr a i n 40359onm ediu m cont aining 175mg/L of hygro myc in B .Fi g.9 Toleranc e o fgeneticall y engineered T.reese i strain Z40359t o hygro myc i nD i s cussi o nThe re m arkable advantages of T.seese i inc lude its well capacit y of protein synt hesis and excretion and it s eukaryoti c excretory m echanis m ,as well as its protein modification per -for mance sm i ilar w ith ma mmal s yst e m ,such as hi g h -mannose type and N -gl y cosy l a tion .Thes e advant ages hugely pro mot e the geneti c modifi c ati o n of T.seese i strain ,and that construction of strong ext ernalgene expressi o n syst e m is t he precondition of itsmol e cul a r biol o gy st udy and genetic m i provement [4].The maj o r co mponent of T.reesei -excret ed extracellular prot ein i s cellul o se(CBH I ).So f ar ,t he cellulose cont ent of produced by T.reese i reached 40g/L ,several hundredstm i es over common bacteria [4].In the present st udy ,we e m -ployed PCR technique t o cl o ne the promot er and ter m i n at or of T.reese i strain YB40359,and f urt her est ablished strong ex -ternal gene expression geneti c transf or m ation syst em in T.reese i .Our results confir med the strong f unc tion of CBH I pro m oter and also lai d basis f or molecul a r st udy and geneti cm i provement of T.reese i .References[1]MU J Y(母敬郁),WANG Q(王峤),YAN G CZ (杨纯中),e ta l .Re -co mbinant Aspe r g ill u s n i ger gl u cose ox i d as e ex press ed in Tri -chode r m a reese i (瑞氏木霉表达黑曲霉葡萄糖氧化酶)[J].Chines eJ ournal of Biot echnol o gy (生物工程学报),2006,22(1):82-86.[2]JI A NG J(冮洁),DU LX (杜连祥),LU FP(路福平),e t a l .Themethod f or chro mos o me DNA pr eparation fro m reco mbinant Tri -chode r m a t ee se i (基因工程菌里氏木霉染色体DNA 的提取方法)[J].Bi o tec hn o l o gy(生物技术),2004,14(2):24-26.[3]PE NTT I L A M,NEVALAI NEN H ,R 'TT ;M ,e ta l .A v ersatile trans -f or mation syst e m f or t he cell u lol y tic fila ment ous fungus T ri chode r -rn a reese i [J].Gene ,1987,61(2):155-164.[4]WANG DH(汪大虹),WU ZH(吴志红).Construc ti o n of het er oge -n eous genes ex pression s yst e m of fila ment ous f ungus Tri chode r m ar e ese i (丝状真菌瑞氏木霉外源基因表达系统的构建)[J].Chines e J ournal of Bioc hem i s t ry and Molecul a r B i o logy (中国生物化学与分子生物学报),2003,19(6):736-742.Respons i bl e editor :DU AN Yong -boRespons i bl e proofreader :WU X i ao -yan311ZHANG X i a o -xuan e t a l .Construc tion of Exogenous Ex pres s ion Vec t or o f Tri chode r m a reese i里氏木霉外源表达载体的构建(摘要)张晓烜1,王傲雪2*(1.东北农业大学成栋学院,黑龙江哈尔滨150030;2.东北农业大学生命科学学院,黑龙江哈尔滨150030)[目的]构建里氏木霉外源表达载体。
C++出错提示英汉对照表
Ambiguous operators need parentheses -----------不明确的运算需要用括号括起Ambiguous symbol ''xxx'' ----------------不明确的符号Argument list syntax error ----------------参数表语法错误Array bounds missing ------------------丢失数组界限符Array size toolarge -----------------数组尺寸太大Bad character in paramenters ------------------参数中有不适当的字符Bad file name format in include directive --------------------包含命令中文件名格式不正确Bad ifdef directive synatax ------------------------------编译预处理ifdef有语法错Bad undef directive syntax ---------------------------编译预处理undef有语法错Bit field too large ----------------位字段太长Call of non-function -----------------调用未定义的函数Call to function with no prototype ---------------调用函数时没有函数的说明Cannot modify a const object ---------------不允许修改常量对象Case outside of switch ----------------漏掉了case 语句Case syntax error ------------------ Case 语法错误Code has no effect -----------------代码不可述不可能执行到Compound statement missing{ --------------------分程序漏掉"{"Conflicting type modifiers ------------------不明确的类型说明符Constant expression required ----------------要求常量表达式Constant out of range in comparison -----------------在比较中常量超出范围Conversion may lose significant digits -----------------转换时会丢失意义的数字Conversion of near pointer not allowed -----------------不允许转换近指针Could not find file ''xxx'' -----------------------找不到XXX 文件Declaration missing ; ----------------说明缺少"；" Declaration syntax error -----------------说明中出现语法错误Default outside of switch ------------------ Default 出现在switch语句之外Define directive needs an identifier ------------------定义编译预处理需要标识符Division by zero ------------------用零作除数Do statement must have while ------------------ Do-while语句中缺少while部分Enum syntax error ---------------------枚举类型语法错误Enumeration constant syntax error -----------------枚举常数语法错误Error directive :xxx ------------------------错误的编译预处理命令Error writing output file ---------------------写输出文件错误Expression syntax error -----------------------表达式语法错误Extra parameter in call ------------------------调用时出现多余错误File name too long ----------------文件名太长Function call missing -----------------函数调用缺少右括号Fuction definition out of place ------------------函数定义位置错误Fuction should return a value ------------------函数必需返回一个值Goto statement missing label ------------------ Goto语句没有标号Hexadecimal or octal constant too large ------------------16进制或8进制常数太大Illegal character ''x'' ------------------非法字符x Illegal initialization ------------------非法的初始化Illegal octal digit ------------------非法的8进制数字houjiumingIllegal pointer subtraction ------------------非法的指针相减Illegal structure operation ------------------非法的结构体操作Illegal use of floating point -----------------非法的浮点运算Illegal use of pointer --------------------指针使用非法Improper use of a typedefsymbol ----------------类型定义符号使用不恰当In-line assembly not allowed -----------------不允许使用行间汇编Incompatible storage class -----------------存储类别不相容Incompatible type conversion --------------------不相容的类型转换Incorrect number format -----------------------错误的数据格式Incorrect use of default --------------------- Default使用不当Invalid indirection ---------------------无效的间接运算Invalid pointer addition ------------------指针相加无效Irreducible expression tree -----------------------无法执行的表达式运算Lvalue required ---------------------------需要逻辑值0或非0值Macro argument syntax error -------------------宏参数语法错误Macro expansion too long ----------------------宏的扩展以后太长Mismatched number of parameters in definition---------------------定义中参数个数不匹配Misplaced break ---------------------此处不应出现break语句Misplaced continue ------------------------此处不应出现continue语句Misplaced decimal point --------------------此处不应出现小数点Misplaced elif directive --------------------不应编译预处理elifMisplaced else ----------------------此处不应出现else Misplaced else directive ------------------此处不应出现编译预处理elseMisplaced endif directive -------------------此处不应出现编译预处理endifMust be addressable ----------------------必须是可以编址的Must take address of memory location ------------------必须存储定位的地址No declaration for function ''xxx'' -------------------没有函数xxx的说明No stack ---------------缺少堆栈No type information ------------------没有类型信息Non-portable pointer assignment --------------------不可移动的指针（地址常数）赋值Non-portable pointer comparison --------------------不可移动的指针（地址常数）比较Non-portable pointer conversion ----------------------不可移动的指针（地址常数）转换Not a valid expression format type ---------------------不合法的表达式格式Not an allowed type ---------------------不允许使用的类型Numeric constant too large -------------------数值常太大Out of memory -------------------内存不够用Parameter ''xxx'' is never used ------------------能数xxx没有用到Pointer required on left side of -> -----------------------符号->的左边必须是指针Possible use of ''xxx'' before definition -------------------在定义之前就使用了xxx（警告）Possibly incorrect assignment ----------------赋值可能不正确Redeclaration of ''xxx'' -------------------重复定义了xxx Redefinition of ''xxx'' is not identical ------------------- xxx的两次定义不一致Register allocation failure ------------------寄存器定址失败Repeat count needs an lvalue ------------------重复计数需要逻辑值Size of structure or array not known ------------------结构体或数给大小不确定Statement missing ; ------------------语句后缺少"；" Structure or union syntax error --------------结构体或联合体语法错误Structure size too large ----------------结构体尺寸太大Sub scripting missing ] ----------------下标缺少右方括号Superfluous & with function or array ------------------函数或数组中有多余的"&"Suspicious pointer conversion ---------------------可疑的指针转换Symbol limit exceeded ---------------符号超限Too few parameters in call -----------------函数调用时的实参少于函数的参数不Too many default cases ------------------- Default太多(switch 语句中一个)Too many error or warning messages --------------------错误或警告信息太多Too many type in declaration -----------------说明中类型太多Too much auto memory in function -----------------函数用到的局部存储太多Too much global data defined in file ------------------文件中全局数据太多Two consecutive dots -----------------两个连续的句点Type mismatch in parameter xxx ----------------参数xxx类型不匹配Type mismatch in redeclaration of ''xxx'' ---------------- xxx 重定义的类型不匹配Unable to create output file ''xxx'' ----------------无法建立输出文件xxxUnable to open include file ''xxx'' ---------------无法打开被包含的文件xxxUnable to open input file ''xxx'' ----------------无法打开输入文件xxxUndefined label ''xxx'' -------------------没有定义的标号xxx Undefined structure ''xxx'' -----------------没有定义的结构xxxUndefined symbol ''xxx'' -----------------没有定义的符号xxx Unexpected end of file in comment started on line xxx----------从xxx行开始的注解尚未结束文件不能结束Unexpected end of file in conditional started on line xxx ----从xxx 开始的条件语句尚未结束文件不能结束Unknown assemble instruction ----------------未知的汇编结构Unknown option ---------------未知的操作Unknown preprocessor directive: ''xxx'' -----------------不认识的预处理命令xxxUnreachable code ------------------无路可达的代码Unterminated string or character constant -----------------字符串缺少引号User break ----------------用户强行中断了程序Void functions may not return a value ----------------- Void类型的函数不应有返回值Wrong number of arguments -----------------调用函数的参数数目错''xxx'' not an argument ----------------- xxx不是参数''xxx'' not part of structure -------------------- xxx不是结构体的一部分xxx statement missing ( -------------------- xxx语句缺少左括号xxx statement missing ) ------------------ xxx语句缺少右括号xxx statement missing ; -------------------- xxx缺少分号xxx'' declared but never used -------------------说明了xxx 但没有使用xxx'' is assigned a value which is never used----------------------给xxx赋了值但未用过Zero length structure ------------------结构体的长度为零。
线性代数》英文专业词汇
线性代数》英文专业词汇has a unique valueinvertible matrixadjoint matrixinverse matrixelementary matrix___column nrank of a matrixlinearly independentlinearly dependentspanbasis_______________diagonalizable matrix___ ___singular value nmatrix norm___positive definite matrixpositive semi-definite matrixnegative definite matrixnegative semi-definite matrix___ deals with the study of linear ___。
One of the key concepts in linear algebra is the determinant。
___ from a square matrix。
The ___.A matrix is a rectangular array of numbers that is used to ___。
The identity matrix is a special type of matrix that has ones on the___ matrix is equal to its transpose。
while a skew-symmetric matrix is equal to the negative of ___ that the order of matrix ___.The rank of a matrix is the maximum number of linearly independent rows or columns it contains。
psicheck2质粒说明书
Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·1.Description ..........................................................................................................12.Product Components and Storage Conditions ............................................23.General Considerations (3)A.siCHECK™ Vector Features...............................................................................3B.How the siCHECK™ Vectors Work..................................................................4C.Sample Experiments Using the siCHECK™ Vectors.. (6)4.siCHECK™ Vector Maps .................................................................................95.siCHECK™ Vector Restriction Enzyme Tables . (11)A.Restriction Enzyme Sites for the psiCHECK™-1 Vector..............................11B.Restriction Enzyme Sites for the psiCHECK™-2 Vector (13)6.siCHECK™ Vector Backbones and Components .....................................157.References .........................................................................................................168.Related Products ..............................................................................................181.DescriptionThe psiCHECK™-1 Vector (a–d)(Cat.# C8011) and psiCHECK™-2 Vector (a–f)(Cat.# C8021) are designed to provide a quantitative and rapid approach for optimization of RNA interference (RNAi). The vectors enable the monitoring of changes in expression of a target gene fused to the reporter gene. In both vectors, Renilla luciferase is used as a primary reporter gene, and the gene of interest can be cloned into the multiple cloning region located downstream of the Renilla luciferase translational stop codon. Initiation of the RNAi process toward a gene of interest results in cleavage and subsequent degradation of fusion mRNA. Measurement of decreased Renilla luciferase activity is a convenient indicator of RNAi effect (1).RNAi is a phenomenon by which double-stranded RNA complementary to a target mRNA can specifically inactivate gene function by stimulating the degradation of the target mRNA (2–4). Because of the ability to inactivate genes, RNAi has emerged as a powerful tool for analyzing gene function.siCHECK™ VectorsAll technical literature is available on the Internet at: /tbs Please visit the web site to verify that you are using the most current version of this Technical Bulletin. Please contact Promega Technical Services if you have questions on useof this system. E-mail: techserv@In mammalian systems, including cultured mammalian cells, chemicallysynthesized double-stranded short interfering RNA molecules (<30 nucleotides;siRNA) or endogenously expressed short hairpin RNA molecules (shRNA) result in dsRNA duplexes <30 base pairs in length that induce RNAi (5–10). RNAiduplexes >30bp induce the interferon response and nonspecific degradation ofmRNA and cannot be used as tools for specific gene silencing (11,12).Interestingly, a significant percentage of the siRNA or shRNA designed for aspecific gene are not effective (5,13–16). On average only 1 in 5 of thesiRNA/shRNAs selected for targeting a specific region show efficient genesilencing (16,17). Possible causes for the failure of a particular siRNA/shRNAmay be instability of an siRNA probe in vivo, inability to interact withcomponents of the RNAi machinery or the inaccessibility of the target mRNAdue to local secondary structural constraints. Analysis of nucleotide sequences,melting temperatures and secondary structures have not revealed any obviousdifference between effective and ineffective siRNA/shRNA (18).At present, one of the most serious limitations for the RNAi technology is thelack of a rapid, reliable, quantitative target-site screening method. Variousalgorithm programs exist that aid in the design of potential siRNA targets.However, an experimental method is needed to screen these siRNAs. Currentscreening technologies include such semi-quantitative, time-consuming methods as fluorescence change for GFP-target fusions, Western blot analysis, monitoringphenotypic changes or RT-PCR. In addition, the current screening technologiesare not easily modified for the rapid, simultaneous screening of multiplesiRNA/shRNA.2.Product Components and Storage ConditionsProduct Size Cat.# psiCHECK™-1 Vector20μg C8011 psiCHECK™-2 Vector20μg C8021 Storage Conditions:Store the psiCHECK™-1 and psiCHECK™-2 Vectors at–20°C.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·3.General Considerations3.A. siCHECK™ Vector FeaturesCurrent methods to monitor changes in gene expression as the result of RNAi are either semi-quantitative, time-consuming or not applicable to high-throughput screening. The siCHECK™ Vectors are easier to use than currently available methods, allow optimal quantitative target site selection and can be adapted for use in high-throughput methodologies.There are two siCHECK™ Vectors, the psiCHECK™-1 Vector and thepsiCHECK™-2 Vector. Both vectors contain as the primary reporter gene the synthetic version of Renilla luciferase, hRluc , which is used to monitor changes in expression as the result of RNAi induction. This synthetic gene is engineered for more efficient expression in mammalian cells and for reduced anomalous transcription.To aid in fusion of the target gene to the synthetic Renilla luciferase reporter gene, a region of restriction sites (i.e., the multiple cloning region) has been added 3´ to the Renilla translational stop. The restriction sites present in the multiple cloning region can be used to create genetic fusions between the gene of interest and the Renilla reporter gene. Because no fusion protein isexpressed, there is no need to be concerned about whether you have cloned into a proper translational reading frame.The multiple cloning region of the psiCHECK™-1 Vector contains unique restriction sites SgfI, XhoI, SmaI, EcoRI, PmeI and NotI. Due to the presence of the firefly expression cassette, the psiCHECK™-2 Vector contains fewer unique restriction sites. The restriction sites in the psiCHECK™-2 Vector multiple cloning region are SgfI, XhoI, PmeI and NotI.The promoter used for Renilla luciferase expression in the siCHECK™ Vectors is the SV40 promoter. Experimental results (data not shown) demonstrate that the SV40 promoter results in the best balance between Renilla luciferaseexpression and the detection of RNAi activity when used with siRNA or vectors expressing shRNA.The difference between the two siCHECK™ Vectors is that the psiCHECK™-2Vector possesses a secondary firefly reporter expression cassette. The firefly expression cassette consists of an HSV-TK promoter, a synthetic firefly luciferase gene and an SV40 late poly(A) signal. To reduce the potential for recombination events, the Renilla luciferase reporter gene in the psiCHECK™-2Vector uses a synthetic poly(A). This firefly reporter cassette has beenspecifically designed to be an intraplasmid transfection normalization reporter;thus when using the psiCHECK™-2 Vector, the Renilla luciferase signal can be normalized to the firefly luciferase signal.If no transfection normalization is required or one would prefer to have the transfection normalization reporter on a second plasmid, the psiCHECK™-1Vector is the vector of choice.3.A. siCHECK™ Vector Features (continued)The psiCHECK™-1 Vector is recommended for use in monitoring RNAieffects in live cells. The changes in Renilla luciferase activity are measuredwith EnduRen™ Live Cell Substrate (Cat.# E6481), which allows continuousmonitoring of intracellular Renilla luminescence (19; Figure 2). EnduRen™ LiveCell Substrate is for use only with Renilla luciferase.Promega offers several reagents that can be used in conjunction with thesiCHECK™ Vectors to monitor Renilla and/or firefly luciferase signals. For thepsiCHECK™-1 Vector, which only contains the Renilla luciferase reporter gene,the Renilla Luciferase Assay System (Cat.# E2810, E2820) can be used. ThepsiCHECK™-2 Vector, which contains Renilla and firefly luciferase reportergenes, requires the use of either the Dual-Luciferase®Reporter Assay System(Cat.# E1910) or the Dual-Glo™ Luciferase Assay System (Cat.# E2920) togenerate the firefly and Renilla luciferase signals.3.B. How the siCHECK™ Vectors WorkFigure 1 provides a basic description of how the siCHECK™ Vectors work.Using the unique restriction sites, the gene of interest is cloned into themultiple cloning region located 3´ to the synthetic Renilla luciferase gene andits translational stop codon. After cloning, the vector is transfected into themammalian cell line of choice, and a fusion of the Renilla luciferase gene andthe gene of interest is transcribed. Vectors expressing potential shRNA orsiRNA can be cotransfected simultaneously or sequentially, depending onyour experimental design. If a specific shRNA/siRNA binds to the targetmRNA and initiates the RNAi process, the fused Renilla luciferase:gene ofinterest mRNA will be cleaved and subsequently degraded, decreasing theRenilla luciferase signal.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·translation stopcleavage of mRNAlight mRNA5´3´5´3´4339M A 10_3A5´3´hRlucgene of interesthRluc Figure 1. Mechanism of action of the siCHECK™ Vectors.3.C. Sample Experiments Using the siCHECK™ VectorsTo demonstrate the utility of the siCHECK™ Vectors, two experiments are detailed in this Technical Bulletin. In the first experiment, human p53 cDNA was subcloned into the psiCHECK™-1 and the psiCHECK™-2 Vectors using the SgfI and NotI restriction sites located in the multiple cloning region of both vectors. Note the SgfI and NotI restriction sites are located 3´ to the Renilla luciferase translational stop codon. As shown in Figure 2, the psiCHECK™-1Vector containing the human p53 cDNA was cotransfected into HEK-293T cells with the psiLentGene™ Basic Vector expressing either a Renilla luciferase (hRluc ) or p53 shRNA. The negative control was the psiLentGene™ Basic Vector with a nonspecific 19bp insert. (A BLAST search using this 19bp sequence and a threshold >90% revealed no homology to any knownmammalian gene or to the synthetic Renilla luciferase gene.) This nonspecificsequence was used for all RNAi experiments in this Technical Bulletin.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·4398M A 11_3AR e n i l l a L u m i n e s c e n c e (R L U )Time Post-Transfection (hours)Negative Control Renilla p53Figure 2. Inhibition of Renilla luciferase expression by targeting either the Renilla luciferase or p53 gene.The human p53 cDNA was subcloned into the psiCHECK™-1Vector using the SgfI and NotI restriction sites located 3´ to the Renilla luciferase translational stop codon. To begin the transfection assay, HEK-293T cells were plated in a 96-well plate at 3,000 cells/well. After an overnight incubation, the cells were treated with a transfection mixture consisting of 35μl of serum-free medium,0.3μl of TransFast™ Transfection Reagent (Cat.# E2431), 0.02μg of psiCHECK™-1:p53vector and 0.08μg of psiLentGene™ Basic Vector per well. For this experiment, the psiLentGene™ Vector expressed shRNAs directed against human p53, Renilla luciferase or the nonspecific 19bp sequence, which serves as a negative control,(Section 3.C). After a one-hour incubation, 100μl of serum-containing medium was added to the wells. At 21 hours post-transfection, EnduRen™ Live Cell Substrate (Cat.# E6481) was added to a final concentration of 60μM, and Renilla luciferase activity was monitored. Renilla luciferase activities were normalized to the number of viable cells using the CellTiter-Glo ®Luminescent Cell Viability Assay (Cat.#G7573; 20).At 21 hours post-transfection, nonlytic EnduRen™ Live Cell Substrate wasadded to the wells; luminescence was monitored for the next 27 hours until48 hours post-transfection. The data in Figure 2 show that the psiLentGene™Basic Vector expressing either Renilla luciferase or p53 shRNA inhibits theexpression of the Renilla luciferase reporter gene from the psiCHECK™-1:p53vector. Interestingly, using either Renilla luciferase or p53 shRNA results invirtually identical inhibition of Renilla luciferase expression.In a second experiment, the human p53 cDNA used in Figure 2 was subclonedinto the psiCHECK™-2 Vector using the SgfI and NotI restriction sites. Fivepotential p53 shRNAs designed to bind to five different target sites were clonedinto the psiLentGene™ Basic Vector; the resulting vectors were named Site 1through Site 5. The control is a psiLentGene™ Vector containing the nonspecific19bp sequence. The psiCHECK™-2 Vector containing the p53 cDNA wascotransfected with the psiLentGene™ Vector expressing either a p53 shRNA(Figure 3, Sites 1–5) or the nonspecific shRNA into HEK-293T cells as describedin Figure 3. Forty-eight hours after transfection, the medium was removed andcells were lysed in Passive Lysis Buffer (Cat.# E1941). The firefly and Renillaluciferase signals were generated using the Dual-Luciferase®Reporter 1000Assay System (21).Figure 3, Panel A, displays the Renilla luciferase signal, while Figure 3, Panel B,shows the Renilla luciferase signal normalized (corrected for transfectionefficiency to the firefly luciferase signal). The data in Figure 3, Panel A, isdifficult to interpret due to transfection variations. The Renilla luciferasepositive control, which should demonstrate inhibition of reporter expression, isnot statistically different (i.e., overlapping error bars) from the negative control(no effect on reporter expression was detected). The inability to distinguishbetween the positive and negative controls renders any conclusion regardingthe effectiveness of potential shRNAs suspect.However, when the Renilla luciferase signals are normalized (see Figure 3,Panel B) to the internal firefly luciferase transfection control, the datainterpretation is different, as the Renilla luciferase positive control isstatistically different from the negative control. In addition, the normalizeddata allow the ability to distinguish the effectiveness of the various target siteshRNAs.Promega Corporation·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Figure 3. Target site selection using the psiCHECK™-2 Vector. HEK-293T cells were seeded into a 96-well plate at a density of 3,000 cells/well. Human p53 cDNA was subcloned into the psiCHECK™-2 Vector using the SgfI and NotI restriction sites. After an overnight incubation, the cells were treated with a transfection mixture consisting of 35μl of serum-free medium, 0.3μl of TransFast™ Transfection Reagent (Cat.# E2431), 0.02μg of psiCHECK™-2 Vector:p53 and 0.08μg of psiLentGene™Basic Vector per well. The psiLentGene™ Basic Vector expressed one of five different shRNAs directed against human p53, Renilla luciferase or a nonspecific 19bp sequence (Section 3.C) as a negative control. After a one-hour incubation, 100μl of serum-containing medium was added to the wells. Forty-eight hours post-transfection Renilla and firefly luciferase activities were measured using the Dual-Luciferase ®Reporter 1000 Assay System (Cat.# E1980; 21). Panel A displays the raw Renilla luciferase data, while in Panel B , the Renilla luciferase data has beennormalized to firefly luciferase data. The data represent the mean of 12 wells plus or minus the standard deviation. Note that in other experiments the ability of different shRNAs to inhibit gene expression might vary more dramatically.4399M A 11_3AA.B.S i t e1S i t e2S i t e3S i t e 4 S i t e5 R e ni l l a P o s it i ve C o n t r o l Ne g a t i v e C o n t r o l R e n i l l a L u m i n e s c e n c e (R L U )S i t e 1S i t e 2S i t e 3 S i t e 4 S i t e 5 R e n i l l a Po s i t i v e C o nt r o l N e g a t i v e C o n t r o lN o r m a l i z e d R e n i l l a L u m i n e s c e n c e (R L U )4.siCHECK™ Vector MapspsiCHECK™-1 Vector sequence reference points: SV40 early enhancer/promoter 7–425Chimeric intron489–621T7 RNA polymerase promoter666–684Synthetic Renilla luciferase gene (hRluc )694–1629Multiple cloning region 1636–1680Synthetic poly(A)1688–1736β-lactamase (Amp r ) coding region1874–2734Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USA·Fax 608-277-2516 ·4343M A 10_3ABglII 1Figure 4. psiCHECK™-1 Vector map. –^– denotes the intron.Synthetic poly(A) signal4342M A 10_3ACCCGGGAATTCGTTTAAACCTAGAGCGGCCGCTGGCCGC AATAAAATA . . . 3′5′ . . . GAGCAGTAA TTCTAGGCGATCGCTCGAG XhoISmaINotIEcoRIPmeISgfIhRlucFigure 5. psiCHECK™-1 Vector multiple cloning region.psiCHECK™-2 Vector sequence reference points:SV40 early enhancer/promoter 7–425Chimeric intron489–621T7 RNA polymerase promoter666–684Synthetic Renilla luciferase gene (hRluc )694–1629Multiple cloning region 1636–1680Synthetic poly(A)1688–1736HSV-TK promoter1744–2496Synthetic firefly luciferase gene (hluc +)2532–4184SV40 late poly(A)4219–4440β-lactamase (Amp r ) coding region4587–5447Figure 6. psiCHECK™-2 Vector map.–^– denotes the intron.4345M A 10_3ABglII 1Synthetic poly(A) signal4344M A 10_3ACCCGGGAATTCGTTTAAACCTAGAGCGGCCGCTGGCCGC AATAAAATA . . . 3′5′ . . . GAGCAGTAA TTCTAGGCGATCGCTCGAGXhoINotIPmeISgfIhRlucFigure 7. psiCHECK™-2 Vector multiple cloning region.5.siCHECK™ Vector Restriction Enzyme Tables5.A.Restriction Enzyme Sites for the psiCHECK™-1 VectorThe following restriction enzyme tables were constructed using DNASTAR ®sequence analysis software. Please note that we have not verified this information by restriction digestion with each enzyme listed. The location given specifies the 3´-end of the cut DNA (the base to the left of the cut site).For more information on the cut sites of these enzymes, or to report adiscrepancy, please contact your local Promega Branch or Distributor. In the U.S., contact Promega Technical Services at 800-356-9526. Vector sequences are available from the GenBank ®database (GenBank ®/EMBL accession number AY535006) and online at:/vectors/Enzyme # of Sites Location AatII 11391Acc65I 154AcyI 21388, 2121AflII 2452, 649Alw44I 21989, 3235AlwNI 13140AspHI 41091, 1993, 2078,3239AvaI 3715, 1643, 1649AvaII 22297, 2519AvrII 1404BamHI 11738BanI 354, 575, 2708BanII 3759, 899, 1650BbsI 1560BbuI 2152, 224BclI 2734, 1187BglI 3357, 694, 2543BglII 11BsaI 3514, 1234, 2595BsaOI51640, 1677, 2143,2292, 3215BsaBI 11453BsaHI 21388, 2121BspHI 21821, 2829BspMI 1476BssSI 21992, 3376Bst98I 2452, 649BstZI 11280Cfr10I 12576Enzyme # of Sites LocationDraI 41663, 2083, 2775,2794DraII 11539DraIII 1882DrdI 2441, 3447DsaI 415, 311, 692, 899EaeI 31674, 1681, 2268EagI 11674EarI 21193, 1862EclHKI 12661Eco52I 11674Eco81I 11280EcoRI 11654EcoRV 11179FspI 28, 2438HaeII 13309HgaI 41570, 2129, 2859,3437HindIII 1420Hsp92I 21388, 2121KpnI 158MspA1I 580, 1679, 2025,2966, 3211NciI 51650, 1651, 2125, 2476, 3172NcoI 315, 311, 692NheI 1684NotI 11674NruI 11355NsiI3154, 226, 913Table 1. Restriction Enzymes That Cut the psiCHECK™-1 Vector Between 1 and 5 Times.Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USAFax 608-277-2516 ·5.A.Restriction Enzyme Sites for the psiCHECK™-1 Vector (continued)Table 2. Restriction Enzymes That Do Not Cut the psiCHECK™-1 Vector.A ccB7I AccI AccIII AflIII AgeI ApaI AscI BalI BbeI BbrPI BlpI Bpu1102IBsaAI BsaMI BsmI Bsp120I BsrGI BssHII Bst1107I BstEII BstXI ClaI CspI Csp45IEco47III Eco72I EcoICRI EcoNI EheI FseI HincII HindII HpaI I-PpoI KasI MluINaeI NarI NdeI NgoMIV PacI PflMI PinAI PmlI PpuMI PshAI Psp5II RsrIISacI SacII SalI SgrAI SnaBI SpeI SplI SrfI Sse8387I SwaI XbaI XcmITable 3. Restriction Enzymes That Cut the psiCHECK™-1 Vector 6 or More Times. AciI AluI Alw26I BbvI BsaJI Bsp1286I BsrI BsrSI Bst71IBstOI BstUI CfoI DdeI DpnI DpnII Fnu4HI FokI HaeIIIHhaI HinfI HpaII HphI Hsp92II MaeI MaeII MaeIII MboIMboII MnlI MseI MspI NdeII NlaIII NlaIV PleI RsaISau3AI Sau96I ScrFI SfaNI TaqI Tru9I XhoIINote:The enzymes listed in boldface type are available from Promega.Enzyme # of Sites LocationNspI 2152, 224PaeR7I 11643PmeI 11663Ppu10I 3150, 222, 909PspAI 11649PstI 1462PvuI 21640, 2292PvuII 180ScaI 2662, 2180SfiI 1357SgfI 11640SinI 22297, 2519Enzyme # of Sites Location SmaI 11651SphI 2152, 224SspI 11856StuI 1403StyI 515, 311, 404, 692,701TfiI 2426, 805Tth111I 11390VspI 12486XhoI 11643XmaI 11649XmnI21228, 2061Table 1. Restriction Enzymes That Cut the psiCHECK™-1 Vector Between 1 and 5 Times (continued).5.B.Restriction Enzyme Sites for the psiCHECK™-2 VectorThe following restriction enzyme tables were constructed using DNASTAR ®sequence analysis software. Please note that we have not verified this information by restriction digestion with each enzyme listed. The location given specifies the 3´-end of the cut DNA (the base to the left of the cut site).For more information on the cut sites of these enzymes, or to report adiscrepancy, please contact your local Promega Branch or Distributor. In the U.S., contact Promega Technical Services at 800-356-9526. Vector sequences are available from the GenBank ®database (GenBank ®/EMBL accession number AY535007) and online at:/vectors/Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USAFax 608-277-2516 ·Enzyme # of Sites Location AatII 11391AccI 22079, 3132Acc65I 154AflII 4452, 649 , 1773,1897AflIII 12450Alw44I 24702, 5948AlwNI 22094, 5853ApaI 12562AvrII 2404, 2059BalI 31865, 3513, 4038BamHI 14451BanII 5759, 899, 1650,2050, 2562BbeI 42030, 2815, 3481,3613BbsI 2560, 1743BbuI 2152, 224BclI 5734, 1187, 3112,3853, 4147BglII 11BsaI 4514, 1234, 2123,5308BsaAI 22083, 3734BsaBI 41453, 2979, 4146,4450BsaMI 32504, 4270, 4363BsmI 32504, 4270, 4363Bsp120I 12558BspHI 33115, 4534, 5542BspMI2476, 3463Enzyme # of Sites Location BsrGI 13022BssHII 11978BssSI 33459, 4705, 6089Bst1107I 12080Bst98I 4452, 649, 1773, 1897BstXI 13650BstZI 31674, 4202, 4206Bsu36I 3 1280, 3145, 3745ClaI 14444Csp45I 12390DraI 51663, 4410, 4796,5488, 5507DraIII 1882DrdI 2441, 6160EagI 31674, 4202, 4206EarI 51193, 1874, 2616,2727, 4575EclHKI 15374Eco47III 13519Eco52I 31674, 4202, 4206Eco81I 31280, 3145, 3745EcoNI 32721, 3144, 4149EcoRI 21654, 2386EcoRV 11179EheI 42028, 2813, 3479,3611FseI 23943, 4208FspI 38, 3354, 5151HincII 14349HindII14349Table 4. Restriction Enzymes That Cut the psiCHECK™-2 Vector Between 1 and 5 Times.5.B.Restriction Enzyme Sites for the psiCHECK™-2 Vector (continued)Table 5. Restriction Enzymes That Do Not Cut the psiCHECK™-2 Vector.AccB7I AccIII AgeI AscI BbrPI BlpIBpu1102I BstEII CspI Eco72I EcoICRI I-PpoINdeI PacI PflMI PinAI PmlI PshAIRsrII SacI SalI SgrAI SnaBI SpeISplI SrfI Sse8387I SwaI XcmIEnzyme # of Sites Location HindIII 2420, 2497HpaI 14349KasI 42026, 2811, 3477,3609KpnI 158MluI 12450NaeI 33941, 3962, 4206NarI 42027, 2812, 3478,3610NcoI 515, 311, 692, 2067, 2530NgoMIV 33939, 3960, 4204NheI 1684NotI 11674NruI 11355NsiI 3154, 226, 913NspI 5152, 224, 2336, 3023, 3278PaeR7I 11643PmeI 11663Ppu10I3150, 222, 909Enzyme # of Sites LocationPpuMI 12056Psp5II 12056PspAI 21649, 2019PvuI 21640, 5005PvuII 380, 2268, 2606SacII 12036ScaI 3662, 2697 ,4893SfiI 1357SgfI 11640SmaI 21651, 2021SphI 2152, 224SspI 14569StuI 1403TfiI 2426, 805Tth111I 11390VspI 15199XbaI 14189XhoI 11643XmaI 21649, 2019XmnI 21228, 4774Table 4. Restriction Enzymes That Cut the psiCHECK™-2 Vector Between 1 and 5 Times (continued).6.siCHECK™ Vector Backbones and ComponentsThe vector backbones of the psiCHECK™-1 and psiCHECK™-2 Vectors are based on the phRL-SV40 Vector (Cat.# E6261). Both the psiCHECK™-1 Vector and psiCHECK™-2 Vector contain the synthetic Renilla luciferase reporter gene.The psiCHECK™-2 Vector also contains a synthetic firefly luciferase gene.These synthetic luciferase genes have been codon optimized for more efficient mammalian expression and have been designed with a greatly reduced number of consensus transcription factor binding sites for reduced risk of anomalous transcriptional behavior.SV40 Early Enhancer/PromoterThe psiCHECK™-1 Vector and psiCHECK™-2 Vector contain the SV40 early enhancer/promoter region, which provides strong, constitutive expression of Renilla luciferase in a variety of cell types.Chimeric IntronDownstream of the SV40 enhancer/promoter region is a chimeric introncomposed of the 5´-donor site from the first intron of the human β-globin and the branch and 3´-acceptor site from the intron that is between the leader and the body of an immunoglobin gene heavy chain variable region (22). The sequences of the donor and acceptor sites, along with the branch point site,have been changed to match the consensus sequence for splicing (23).Transfection studies have demonstrated that the presence of an intron flankingthe cDNA insert frequently increases the level of gene expression (24–27).Promega Corporation ·2800 Woods Hollow Road ·Madison, WI 53711-5399 USAFax 608-277-2516 ·Table 6. Restriction Enzymes That Cut the psiCHECK™-2 Vector 6 or More Times. AciI AcyI AluI Alw26I AspHI AvaI AvaII BanI BbvI BglI BsaOI BsaHI BsaJIBsp1286I BsrI Bsr SI Bst71I BstOI BstUI CfoI Cfr10I DdeI DpnI DpnII DraII DsaIEaeI Fnu4HI FokI HaeII HaeIII HgaI HhaI HinfI HpaII HphI Hsp92I Hsp92II MaeIMaeII MaeIII MboI MboII MnlI MseI MspI MspA1I NciI NdeII NlaIII NlaIV PleIPstI RsaI Sau3AI Sau96I ScrFI SfaNI SinI StyI TaqI Tru9I XhoIINote:The enzymes listed in boldface type are available from Promega.T7 PromoterA T7 RNA polymerase promoter is located downstream of the chimeric intron and immediately precedes the synthetic Renilla luciferase reporter gene. This promoter can be used to synthesize RNA transcripts in vitro using T7 RNA Polymerase (Cat.# P2075). Note that the T7 promoter has been verified by sequence only; there has been no functional testing of the T7 promoter. Polyadenylation Signals (SV40 Late and Synthetic)Polyadenylation signals are coupled to the termination of transcription by RNA polymerase II and signal the addition of approximately 200–250 adenosine residues to the 3´-end of the RNA transcript (28). Polyadenylation has been shown to enhance RNA stability and translation (29,30). The late SV40 polyadenylation signal is extremely efficient and has been shown to increase the steady-state level of RNA to approximately fivefold more than that of the early SV40 polyadenylation signal (31). The synthetic poly(A) was cloned from our pCI-neo Vector (Cat.# E1841). The synthetic poly(A) signal is based on the highly efficient polyadenylation signal of the rabbit β-globin gene (32).7.References1.Kumar, R., Conklin, D.S. and Mittal, V. (2003) High-throughput selection of effectiveRNAi probes for gene silencing. Genome Res.13, 2333–40.2.Bass, B.L. (2000) Double-stranded RNA as a template for gene silencing. Cell101, 235–8.3.Zamore, P.D. (2001) RNA interference: Listening to the sound of silence. Nature Struct.Biol.8, 746–50.4.Sharp, P.A. (2001) RNA interference—2001. Genes Dev.15, 485–90.5.Gil, J. and Esteban, M. (2000) Induction of apoptosis by the dsRNA-dependentprotein kinase (PKR): Mechanism of action. Apoptosis5, 107–14.6.Marcus, P.I. and Sekellick, M.J. (1985) Interferon induction by viruses. XIII. Detectionand assay of interferon induction-suppressing particles. Virology142, 411–5.7.Elbashir, S.M. et al.(2001) Duplexes of 21-nucleotide RNAs mediate RNA interferencein cultured mammalian cells. Nature411, 494–8.8.Brummelkamp, T.R., Bernards, R. and Agami, R. (2002) A system for stableexpression of short interfering RNAs in mammalian cell. Science296, 550–3.9.Elbashir, S.M. et al. (2002) Analysis of gene function in somatic mammalian cellsusing small interfering RNAs. Methods26, 199–213.10.Paddison, P.J. et al. (2002) Short hairpin RNAs (shRNAs) induce sequence-specificsilencing in mammalian cells. Genes Dev.16, 948–58.11.Paul, C.P. et al.(2002) Effective expression of small interfering RNA in human cells.Nature Biotechnol. 20, 505–8.。
Residue formulae for vector partitions and Euler-MacLaurin sums
f (x) =
j =1
e yj ,x Pj (x),
where y1 , . . . , ym ∈ CN , and the functions P1 , . . . , Pm are polynomials with complex coefficients. If the elements {yj , 1 ≤ j ≤ m} are such that there exists an integer M with M yj ∈ 2πiZN , then the function f is said to be periodic-polynomial. The restriction of such a function to any coset x + M ZN of ZN /M ZN is plainly polynomial.
ξ ∈ΠΦ (λ)∩ZN
f (ξ ).
Indeed, if f = 1, the function S [f, Φ] is just the function ιΦ . Such a sum S [f, Φ] will be called an Euler-MacLaurin sum. In this paper, we will search for “explicit” formulae for the function λ → S [f, Φ](λ) on Γ∗ . Let us recall some qualitative results about this function. We start with the following result of Ehrhart: for a rational polytope Π in Rr , consider the function k → #(kΠ ∩ Zr ), where #S stands for the cardinality of the set S . Ehrhart proved that this function is given by a periodic-polynomial formula for all k ≥ 0. More precisely (see [13] and references therein), if M is an integer such that all the vertices of the polytope M Π are in Zr , then there exist polynomial functions Pj , 0 ≤ j ≤ iπj M −1 2M k Pj (k). If f is a polynomial, then M − 1, such that #(kΠ ∩ Zr ) = j =0 e S [f, Φ](λ) consists of summing up the values of a polynomial over the integral points of the rational polytope ΠΦ (λ). If f is an exponential x → e y,x , then S [ey , Φ](λ) is the sum ξ ∈ΠΦ (λ)∩ZN e y,ξ ; such sums were evaluated “explicitly” by M. Brion [4] and by A.I. Barvinok [3] for generic exponentials. Assume first that Φ consists of n = dim V linearly independent vectors of Γ∗ . Denote by ρ the linear isomorphism from Rn to V ∗ defined by ρ(x) = n i=1 xi βi . The set ΠΦ (λ) is nonempty if and only if λ ∈ C (Φ) ∩ ZΦ. In this case, the set ΠΦ (λ) coincides with ρ−1 (λ), and our function λ → S [f, Φ](λ) on Γ∗ is just the function λ → f (ρ−1 (λ)) restricted to C (Φ) ∩ ZΦ. In general, the map ρ : RN → V ∗ defined by ρ(x) = N i=1 xi βi is a surjection, and the following qualitative statement holds: Theorem 0.1. For each conic chamber c of the cone C (Φ), there exists an exponentialpolynomial function P [c, f, Φ] on V ∗ such that for each λ ∈ c ∩ Γ∗ , we have S [f, Φ](λ) = P [c, f, Φ](λ). This theorem follows, for example, from [5], and there are many antecedents of this result in particular cases. The periodic-polynomial behavior of ιΦ (λ) on closures of conic chambers of the cone C (Φ) is proved in [21]. If f is a polynomial function, then the sum ξ ∈kΠ f (ξ ) is a polynomial function of k for k ≥ 0 if the vertices of Π have integral coordinates [13, 4, 9]. Let Π1 , Π2 , . . . , ΠN be rational polytopes in Rr . For a sequence [k1 , . . . , kN ] of nonnegative integers, denote by k1 Π1 + k2 Π2 + · · · + kN ΠN the weighted Minkowski sum of the polytopes Πi . Then, as proved in [18], there exists an periodic-polynomial function P on RN such that #((k1 Π1 + k2 Π2 + · · · + kN ΠN ) ∩ Zr ) = P (k1 , k2 , . . . , kN ). We explain in Section 3.2 how to pass from the setting of Minkowski sums to the setting of partition polytopes. Most of the investigations of the function S [f, Φ] ([16, 9, 5]), starting with the Euler-MacLaurin formula evaluating the sum B A f (k ) of the values of a function f at all integral points of an interval [A, B ], were dedicated to the fascinating relation of
机器学习 深度学习 笔记 (9)
minima or global minima of the cost function. Also note that the underlying
parameterization for hθ(x) is different from the case of linear regression, even though the form of the cost function is the same mean-squared loss.
θ := θ − α∇θJ (j)(θ)
(1.4)
Oftentimes computing the gradient of B examples simultaneously for the parameter θ can be faster than computing B gradients separately due to hardware parallelization. Therefore, a mini-batch version of SGD is most commonly used in deep learning, as shown in Algorithm 2. There are also other variants of the SGD or mini-batch SGD with slightly different sampling schemes.
3
Algorithm 2 Mini-batch Stochastic Gradient Descent
1: Hyperparameters: learning rate α, batch size B, # iterations niter. 2: Initialize θ randomly 3: for i = 1 to niter do 4: Sample B examples j1, . . . , jB (without replacement) uniformly from
A Tutorial on Support Vector Machines for Pattern Recognition
burges@
Bell Laboratories, Lucent Technologies Editor: Usama Fayyad Abstract. The tutorial starts with an overview of the concepts of VC dimension and structural risk minimization. We then describe linear Support Vector Machines (SVMs) for separable and non-separable data, working through a non-trivial example in detail. We describe a mechanical analogy, and discuss when SVM solutions are unique and when they are global. We describe how support vector training can be practically implemented, and discuss in detail the kernel mapping technique which is used to construct SVM solutions which are nonlinear in the data. We show how Support Vector machines can have very large (even infinite) VC dimension by computing the VC dimension for homogeneous polynomial and Gaussian radial basis function kernels. While very high VC dimension would normally bode ill for generalization performance, and while at present there exists no theory which shows that good generalization performance is guaranteed for SVMs, there are several arguments which support the observed high accuracy of SVMs, which we review. Results of some experiments which were inspired by these arguments are also presented. We give numerous examples and proofs of most of the key theorems. There is new material, and I hope that the reader will find that even old material is cast in a fresh light. Keywords: support vector machines, statistical learning theory, VC dimension, pattern recognition
3.1 Definitions of vector spaces
Definitions of vector spacesIntroductionThe operations of addition and scalar multiplication are used in many diverse contexts in mathematics. Regardless of the context, however, these operations usually obey the same set of algebraic rules.Thus, a general theory of mathematical systems involving addition and scalar multiplication will be applicable to many areas in mathematics.Mathematical systems of this form are called vector spaces or linear spaces.IntroductionVector space is not just a mathematical concept.The results about vector spaces have some applications.In this section we will give the definition of a vector space, show some examples and discuss some fundamental properties of vector spaces.Outline1.Definition of vector space2.Examples of vector spaces3.Fundamental properties of vector spacesDefinition of vector spaceLet V be a set on which the operations of addition and scalar multiplication are defined. By this we mean that, with each pair of elements x and y in V, we can associate a unique element x + y that is also in V, and with each element x in V and each scalar α, we can associate a unique element αx in V. Namely, linear combination αx + βy in V.The set V, together with the operations of addition and scalar multiplication, is said to form a vector space if the following axioms are satisfied:A1.x + y = y + x for any x and y in V.A2.(x + y) + z = x + (y + z) for any x, y, and z in V. A3.There exists an element 0 in V such that x + 0 = x for each x ∈V.A4. For each x ∈V, there exists an element −x in V such that x + (−x) = 0.A5. α(x + y) = αx + αy for each scalar αand any x and y in V.A6. (α+ β)x = αx + βx for any scalars αand βand any x ∈V.A7.(αβ)x = α(βx) for any scalars αand βand any x ∈V.A8.1 ·x = x for all x ∈V.Closure propertiesAn important component of the definition is the closure properties of the two operations. These properties can be summarized as follows: C1. If x ∈V and αis a scalar, then αx∈V.C2. If x, y ∈V, then x + y ∈V.Determine whether a set U is a vector space Given a set U on which the operations of addition and scalar multiplication have been defined and satisfy properties C1 and C2,then we must check to see if the eight axioms are valid in order to determine whether U is a vector space.Examples of vector spacesEuclidean vector spaces C[a , b ], the set of all real-valued functions that are defined and continuous on the closed interval [a , b ].R 1.n n ， the set of all polynomials of degree less than n .The vector space , the set of all m ×n matrices with real entries.P 0n n ，， m nRFundamental properties of vector spacesTh1 If V is a vector space and x is any element of V, then(i) 0x = 0.(ii) x + y = 0 implies that y = −x (i.e., the additive inverse of x is unique).(iii) (−1)x = −x.Fundamental properties of vector spaces(continued)(iv) 0 in a vector space is unique.(v)Let x, y, and z be vectors in V. If x + y = x + z, then y = z.(vi)β0 = 0 for each scalar β.(vii)if αx = 0, then either α = 0 or x = 0.Thank you!。
气候的影响英语作文
气候的影响英语作文Climate Change and Its Impact。
Climate change refers to the long-term alteration of temperature and typical weather patterns in a place. It is primarily caused by human activities, such as the burning of fossil fuels, deforestation, and industrial processes. The consequences of climate change are far-reaching and affect various aspects of our lives, including the environment, economy, and human health. In this essay, we will explore the different ways in which climate change impacts our world.One of the most significant impacts of climate change is the rise in global temperatures. As greenhouse gases, such as carbon dioxide, accumulate in the atmosphere, they trap heat and cause the Earth's temperature to increase. This rise in temperature leads to the melting of polar ice caps and glaciers, resulting in rising sea levels. Consequently, coastal areas are at risk of flooding, and low-lying islands face the threat of disappearing entirely. Additionally, higher temperatures also contribute to extreme weather events, such as hurricanes, droughts, and heatwaves, which can have devastating effects on communities and ecosystems.Another consequence of climate change is the disruption of ecosystems and biodiversity loss. Many species are highly sensitive to changes in temperature and precipitation patterns. As their habitats become inhospitable, they are forced to migrate or face extinction. This disruption in the natural balance of ecosystems can have cascading effects, impacting other species and ultimately destabilizing entire ecosystems. Furthermore, the loss of biodiversity also has implications for human well-being, as it reduces the availability of natural resources, such as clean water and food.The agricultural sector is heavily influenced by climate change. Changes in temperature and rainfall patterns can affect crop yields and livestock production. For instance, prolonged droughts can lead to crop failures, while increased rainfall can result in flooding and soil erosion. These extreme weather events not only threaten food security but also impact the livelihoods of farmers and rural communities. Moreover,climate change also affects the quality and availability of water resources, which are essential for irrigation and livestock rearing.In addition to environmental and economic impacts, climate change also poses risks to human health. Rising temperatures contribute to the spread of infectious diseases, as vectors like mosquitoes and ticks thrive in warmer climates. For example, the transmission of diseases such as malaria, dengue fever, and Lyme disease is closely linked to climate conditions. Furthermore, heatwaves can cause heatstroke and other heat-related illnesses, particularly among vulnerable populations such as the elderly and those with pre-existing health conditions. Additionally, the displacement of communities due to climate-related disasters can lead to mental health issues and social unrest.Addressing climate change requires global cooperation and immediate action. Governments, businesses, and individuals all have a role to play in reducing greenhouse gas emissions and transitioning to renewable energy sources. This includes investing in clean technologies, promoting energy efficiency, and adopting sustainable practices. Furthermore, efforts should be made to conserve and restore natural ecosystems, which can serve as carbon sinks and help mitigate the effects of climate change.In conclusion, climate change is a pressing issue that affects various aspects of our lives. From rising temperatures and sea levels to biodiversity loss and health risks, the consequences of climate change are far-reaching. It is crucial for us to recognize the urgency of the situation and take collective action to mitigate its impact. By working together, we can create a sustainable future for generations to come.。
英语技术写作试题及答案
英语技术写作试题及答案一、选择题（每题2分，共20分）1. The term "API" stands for:A. Application Programming InterfaceB. Artificially Programmed IntelligenceC. Advanced Programming InterfaceD. Automated Programming Interface答案：A2. Which of the following is not a common data type in programming?A. IntegerB. StringC. BooleanD. Vector答案：D3. In technical writing, what is the purpose of using the term "shall"?A. To indicate a requirement or obligationB. To suggest a recommendationC. To express a possibilityD. To denote a future action答案：A4. What does the acronym "GUI" refer to in the context of computing?A. Graphical User InterfaceB. Global User InterfaceC. Generalized User InterfaceD. Graphical Unified Interface答案：A5. Which of the following is a correct statement regarding version control in software development?A. It is used to track changes in software over time.B. It is a type of software testing.C. It is a method for encrypting code.D. It is a way to compile code.答案：A6. What is the primary function of a compiler in programming?A. To debug codeB. To execute codeC. To translate code from one language to anotherD. To optimize code for performance答案：C7. In technical documentation, what does "RTFM" commonly stand for?A. Read The Frequently Asked QuestionsB. Read The Full ManualC. Read The File ManuallyD. Read The Final Message答案：B8. Which of the following is a common method for organizing code in a modular fashion?A. LoopingB. RecursionC. EncapsulationD. Inheritance答案：C9. What is the purpose of a "pseudocode" in programming?A. To provide a detailed step-by-step guide for executing codeB. To serve as a preliminary version of code before actual codingC. To act as an encryption for the codeD. To be used as a substitute for actual code in production答案：B10. What does "DRY" stand for in software development?A. Don't Repeat YourselfB. Data Retrieval YieldC. Database Record YieldD. Dynamic Resource Yield答案：A二、填空题（每空2分，共20分）1. The process of converting a high-level code into machine code is known as _______.答案：compilation2. In programming, a _______ is a sequence of characters that is treated as a single unit.答案：string3. The _______ pattern in object-oriented programming is a way to allow a class to be used as a blueprint for creating objects.答案：prototype4. A _______ is a type of software development methodology that emphasizes iterative development.答案：agile5. The _______ is a set of rules that defines how data is formatted, transmitted, and received between software applications.答案：protocol6. In technical writing, the term "should" is used toindicate a _______.答案：recommendation7. The _______ is a type of software that is designed to prevent, detect, and remove malicious software.答案：antivirus8. A _______ is a variable that is declared outside the function and hence belongs to the global scope.答案：global variable9. The _______ is a programming construct that allows you to execute a block of code repeatedly.答案：loop10. In software development, the term "branch" in version control refers to a _______.答案：separate line of development三、简答题（每题10分，共40分）1. Explain the difference between a "bug" and a "feature" in software development.答案：A "bug" is an unintended behavior or error in a software program that causes it to behave incorrectly or crash. A "feature," on the other hand, is a planned and intentional part of the software that provides some functionality or capability to the user.2. What is the significance of documentation in technical writing?答案：Documentation in technical writing is significant as it serves to provide detailed information about a product or system, making it easier for users, developers, and other stakeholders to understand its workings, usage, and maintenance. It is crucial for training, troubleshooting, and future development.3. Describe the role of a software architect in a software development project。
气候变化对地球的影响英语作文高中
气候变化对地球的影响英语作文高中全文共3篇示例，供读者参考篇1Climate change, also known as global warming, is one of the most pressing environmental issues facing our planet today. It refers to the long-term changes in temperature and weather patterns that occur as a result of human activities such as burning fossil fuels and deforestation. These activities release greenhouse gases into the atmosphere, which trap heat and cause the Earth's temperature to rise.The effects of climate change on the Earth are far-reaching and can be seen in a variety of ways. One of the most obvious impacts is the increase in global temperatures. According to the Intergovernmental Panel on Climate Change (IPCC), the Earth's average temperature has risen by about 1.1 degrees Celsius since the pre-industrial era. This may not seem like a significant increase, but it has already led to more frequent and severe heatwaves, droughts, and wildfires in many parts of the world.Another consequence of climate change is the melting of polar ice caps and glaciers. This is causing sea levels to rise,which puts coastal communities at risk of flooding and displacement. In addition, the loss of ice in the Arctic and Antarctic regions is affecting wildlife habitats and accelerating the process of global warming.Climate change is also causing changes in weather patterns, leading to more extreme and unpredictable events such as hurricanes, typhoons, and heavy rainfall. These events can have devastating effects on communities, causing loss of life, property damage, and disruptions to food and water supplies.The impact of climate change is not limited to the environment – it also has serious implications for human health. Rising temperatures can lead to heat-related illnesses and exacerbate air pollution, which can worsen respiratory conditions such as asthma. Changes in weather patterns can also affect the spread of infectious diseases, as vectors such as mosquitoes and ticks thrive in warmer temperatures.In order to mitigate the effects of climate change, it is crucial for individuals, communities, and governments to take action. This can include reducing carbon emissions by using renewable energy sources, conserving water and energy, and planting trees to absorb greenhouse gases. It is also important to adapt to the changing climate by implementing measures such as buildingresilient infrastructure, protecting natural ecosystems, and preparing for extreme weather events.While the effects of climate change on the Earth are undeniable, there is still hope for the future. By working together to reduce our impact on the environment and adapt to a changing climate, we can help to protect our planet for future generations. It is up to all of us to take responsibility for our actions and make a positive difference in the fight against climate change.篇2Climate change has become one of the most pressing issues facing our planet today. The impact of climate change on the Earth is undeniable and far-reaching, affecting various aspects of our environment, economy, and society.One of the most significant effects of climate change on the Earth is the increase in global temperatures. The Earth's average temperature has been rising steadily over the past century, primarily due to human activities such as the burning of fossil fuels and deforestation. This increase in temperature has led to a number of consequences, including more frequent and severe heatwaves, droughts, and wildfires. These extreme weatherevents not only pose a threat to human health and safety but also have a damaging impact on ecosystems and wildlife.Another consequence of climate change is the melting of polar ice caps and glaciers. As global temperatures rise, ice caps and glaciers in the Arctic and Antarctic regions are melting at an alarming rate. This melting ice has contributed to the rise in sea levels, threatening coastal communities and low-lying islands with flooding and erosion. Furthermore, the loss of ice in these regions has a significant impact on global climate patterns, leading to changes in weather patterns and ocean currents.In addition to the physical effects of climate change, there are also economic and social impacts to consider. Climate change has the potential to disrupt food production and water resources, leading to food scarcity, water scarcity, and increased competition for resources. Communities that rely on agriculture and fishing for their livelihoods are particularly vulnerable to the effects of climate change, as changes in temperature and precipitation patterns can have a negative impact on crop yields and fish stocks.Furthermore, climate change can exacerbate social inequalities and contribute to conflicts over resources. Displacement due to sea-level rise and natural disasters can leadto mass migration and strain social services and infrastructure in host communities. In regions already facing social and political instability, climate change can further exacerbate existing challenges and contribute to conflict and unrest.In order to address the impact of climate change on the Earth, it is essential that we take action to reduce greenhouse gas emissions and mitigate the effects of global warming. This requires a collective effort from individuals, communities, businesses, and governments to transition to renewable energy sources, implement sustainable land-use practices, and promote conservation and adaptation strategies.By working together to address the root causes of climate change and its impacts, we can create a more sustainable future for our planet and ensure the well-being of current and future generations. Climate change is a global challenge that requires global solutions, and it is up to all of us to take action and protect the Earth for future generations.篇3Climate change is one of the most pressing issues facing our planet today, and its impacts are already being felt all around the world. From rising global temperatures to more frequentextreme weather events, the effects of climate change are varied and far-reaching. In this essay, we will explore the ways in which climate change is impacting the Earth and what can be done to mitigate its effects.One of the most obvious effects of climate change is the increased frequency and intensity of extreme weather events. Hurricanes, droughts, floods, and heatwaves are becoming more common, causing devastation to communities and ecosystems. For example, the recent wildfires in Australia and California have been some of the worst in history, with millions of acres of land destroyed and countless lives lost. These events are not only costly in terms of human lives and infrastructure, but also have long-lasting environmental consequences.Another significant impact of climate change is the melting of the polar ice caps and glaciers. As global temperatures rise, these ice masses are melting at an alarming rate, leading to rising sea levels and disruptions to ocean currents. This not only threatens coastal communities with flooding and erosion but also poses a serious risk to the stability of marine ecosystems. For example, the melting of Arctic sea ice is causing the habitat of polar bears and other animals to shrink, putting their survival at risk.In addition to extreme weather events and melting ice caps, climate change is also affecting biodiversity and ecosystems around the world. Many plant and animal species are struggling to adapt to the changing climate, leading to declines in populations and even extinction in some cases. Coral reefs, for example, are highly sensitive to changes in temperature and acidity levels in the ocean, and are already experiencing widespread bleaching and die-off as a result of climate change.The impacts of climate change are not limited to the environment – they also have significant social and economic consequences. For example, the increased frequency of extreme weather events is putting a strain on emergency response systems and healthcare infrastructure, leading to higher costs and reduced capacity to help those in need. Additionally, changes in agricultural production due to shifting climate patterns are impacting food security and livelihoods for millions of people around the world.So, what can be done to mitigate the effects of climate change and protect our planet for future generations? One of the key solutions is to reduce greenhouse gas emissions by transitioning to renewable energy sources such as wind, solar, and hydroelectric power. This will not only help to reduce thewarming of the planet but also create new jobs and economic opportunities in the clean energy sector. In addition, conservation efforts and sustainable land use practices can help to protect biodiversity and ecosystems from the impacts of climate change.Ultimately, addressing climate change requires a global effort to reduce emissions, adapt to the changing climate, and protect vulnerable communities and ecosystems. By working together to implement sustainable solutions, we can help to mitigate the effects of climate change and create a more resilient and sustainable future for all. The time to act is now – the future of our planet depends on it.。
4th Int'l Conf. On Principles Of DIstributed Systems. December 2000. DESCRIBING MOBILE COMP
4th Int’l Conf.On Principles Of DIstributed Systems.December2000. DESCRIBING MOBILE COMPUTATIONS WITH PATH VECTORSPHILIPPE QU´EINNEC,MAMOUN FILALI,PHILIPPE MAURAN,G´ERARD PADIOU Keywords:mobile agent,diffusing computation,termination detection,dis-tributed debugging1.I NTRODUCTIONRecently,mobile agents have been advocated as a useful mechanism to de-sign and implement Internet based services.They provide interesting proper-ties for handling dynamic network topologies,load balancing and disconnected operation.For instance,data mining requires to propagate a client request,first,toward some nodes,and then,recursively,from these nodes to further ones.This behavior can be viewed as a parallel and distributed diffusing com-putation.The concept of mobile agent provides an appropriate abstraction to describe such computations.An initial agent starts on the requesting node,12PHILIPPE QU´EINNEC,MAMOUN FILALI,PHILIPPE MAURAN,G´ERARD PADIOU and,after a local step,forks clones that migrate to further selected nodes.Each clone adopts the same behavior until no next node exists.Theflow of control generated by mobile agents from node to node can be abstracted as a tree.Figure1describes such a diffusing computation[DS80],and illustrates its interpretation as a set of paths from the root to theleaves.F IGURE1.Agent migration and its path interpretationDistributed paths describe theflow of control generated by the mobile agents and record a history of their mobility.These paths are represented by means of path vectors.Each agent is only required to piggyback a path vector during its migration.When an agent does not generate any further clones,it sends its current path vector to a collector process and terminates.The collector can take advantage of the following properties:path vectors allow to detect the termination of all agents;the knowledge of all leaf path vectors allows to reconstruct the compu-tation tree;the number of nodes and the identity of each visited node are not required to be known beforehand.An agent only has to record a list of couples (visited node,counter)during its migration.Wefirst describe our model of mobile agents.The next section defines distributed paths and path vectors.Section4presents an algorithm to detect distributed termination which does not need to know either the set of the visited nodes or the topology of the computation.The last section gives an algorithm to rebuild the computation tree from the collected vectors.2.D IFFUSING COMPUTATION AND MOBILE AGENTSMost mobile agent services provide two basic primitives:a fork-like method to duplicate the calling agent,and a migration statement to carry out the proac-tive mobility of agents.More precisely,we consider a service with the follow-ing API:DESCRIBING MOBILE COMPUTATIONS WITH PATH VECTORS3 Url fork(Set[Url]locations):the calling agent splits into as many clones as locations in the set.Moreover,the method returns a distinct Url from the set to each clone;go(Url location):this method performs the migration of the call-ing agent towards the specified location:the call begins on the caller’s node and returns on the destination node.With this API,a generic pattern of diffusing computation is:AgentModel()while(true)//Agent arrival at a node//Local step at the current nodeSet[Url]dest=url1,url2,;if(dest.isEmpty())break;//The agent ends//Possible multiple migrations to other nodesUrl s=fork(dest);go(s);//Performed by each cloneend();After a local step,the agent evaluates a list of further destinations.If this list is not empty,the agent splits into clones which migrate toward their desti-nation.If the list is empty,the agent terminates.This End event is interpreted as the end of a migration path.Each cloning and migration step generates new distributed paths.This step is called Split(n),where specifies the number of outgoing paths.In this model,the number of new migration paths is known by all the new clones.The agent mobility must be handled by an underlying migration service. We assume a migration server exists on each node.These servers have the following behavior:server MigrationHandling()while(true)Agent A=readAgent();//Wait for a new agentA.Context=LocalContext;//Give it a local contextA.resume();//Local return from go//Possible multiple migrations toward further nodes for each go-request(A’,d’)writeAgent(d’,A’);4PHILIPPE QU´EINNEC,MAMOUN FILALI,PHILIPPE MAURAN,G´ERARD PADIOU Figure2illustrates the underlying exchange of messages among visited servers.This computation pattern satisfies the assumptions of a diffusing com-putation.3.D ISTRIBUTED PATHSPath vectors provide a mechanism to count the number of path creations. Path vectors are vectors of non decreasing integer counters.Their handling is based on the following rules:A local counter at each node records the number of locally createdpaths.These counters are initialized to.Each migration message piggybacks a path vector.A vector of a given path is updated along the spreading of the path.This path vector records the number of new paths issued by the current path,and carries the causal history of these new paths.Actually,the“vector”is a mapping from node to natural.For the sake of simplicity,we will denote these mappings as vectors.An actual imple-mentation can use either vectors(if the set of nodes is known andfixed) or sparse association sets.3.1.Handling of path vectors.The diffusing computation spreads from a starting node.We describe the actions performed to handle path vectors when an agent arrives at site.Let be a received path vector.This vector is updated according to the following actions:End:the incoming agent ends(its associated path ends).The vectorcan be used as a stamp of the path ending with this leaf.Such a vector is called a leaf vector.This vector value can be sent to a collector process that handles this information according to specific goals(stable property detection,computation tree reconstruction).Split(p):the computation step ends by sending migration messages with:new paths are created and the incoming one follows its way.In this case,the local counter increases by the number of new paths.If messages are sent,paths are new.Therefore,the following increment is performed:Then,the-th component of is assigned the resulting value: .This updated vector is piggybacked in all sent messages.In this way,all new paths inherit the same causality from the incoming path.DESCRIBING MOBILE COMPUTATIONS WITH PATH VECTORS5S1S2S0F IGURE 2.Path vector handlingTherefore,every vector leaving a node exactly knows the number of paths created on this node.Follow :the incoming agent continues its way to one single node.The vector is left unchanged and is piggybacked on the agent.Note that a Follow could be considered as a Split (1).However,such an interpreta-tion leads to an superfluous assignment ().Initially,the first agent is created with an all-zero path vector.Figure 2is a timing diagram which illustrates the handling of path vec-tors for a diffusing computation with three sites.The initial site is .Local counters are written in bold and migration messages are labeled by their path vector.This computation has generated six Split (2)and seven End .Follow are completely transparent and are abstracted from the computation tree.There-fore,the corresponding underlying computation tree involves six internal nodes and seven leaves.The number of leaves is equal to the number of paths.Fig-ure 3describes this computation tree and its associated distributed paths.Each node is labeled by the path vector value of its input message.In this example,we obtain seven leaf path vectors:.3.2.Path vector properties.The next property is used to develop a new ter-mination detection algorithm (section 4),and the second property comes from the algorithm which reconstructs the computation tree based on the leaf path vectors (section 5).Prop.1.Let be the set of leaf path vectors of a diffusing computation andthe maximum vector defined by:Then,the number of paths is:6PHILIPPE QU´EINNEC,MAMOUN FILALI,PHILIPPE MAURAN,G´ERARD PADIOUF IGURE3.Path oriented computation treeThis property is an application of Euler’s theorem on planar graphs.In the following,is the set of leaf vertices of a tree,is the set of its internal vertices,is the number of outgoing edges of vertex.number of edges number of vertices(Euler)each vertex corresponds to a splitProp.2.The set of leaf path vectors of a diffusing computation provides enough knowledge to rebuild the whole computation tree.This reconstruction consists in computing all path vectors associated to in-ternal Split actions.For instance,from thefinal path vectors of the computa-tion tree in Figure3,we can deduce the path vectors,and associ-ated to the internal Split actions.We describe this algorithm in Section5.DESCRIBING MOBILE COMPUTATIONS WITH PATH VECTORS73.3.Path vector and time vector.Path vectors can be compared to time vec-tors[Mat89b,Fid88].Their main difference lies in the events they count.Time vectors record send,receive and internal events.Path vectors only record Split events,and ignore End and Follow events although such events generate mes-sages.Moreover,each node manages a time vector,whereas path vectors only require a scalar stly,the number of non-zero elements in a path vector is the number of distinct nodes which were visited by the agent along this path.Unlike a time vector,a path(its path vector)does not acquire any knowledge about what happened on other nodes when“crossing”another path (i.e.when reaching a node which was previously visited by another path). When using path vectors with mobile agents,this point also justifies the use of association sets instead of vectors,as the number of positive elements is at most the length of the path.4.T ERMINATION DETECTIONTo illustrate and motivate the use of path vectors,we develop a new termi-nation detection algorithm based on path vectors.4.1.Description.Property1entails the evaluation of,related to(the set of leaf path vectors)by the equation:.The number of leaves of a terminated computation is also the number of distributed paths.If each path ending is reported to a collector process,this collector can evaluate the current number of actualfinal leaves.Then,the termination and its detection can be defined as:termination:detection:[FMP00]presents a formal validation process,based upon refinement, which provides a formal proof of this algorithm.Algorithm of the ing the collected path vectors,the collector pro-gressively evaluates the number of created and ended paths.It handles a path vector and a counter.In the initial state,they are equal to zero.The collector gathers the path vectors until it can decide the termination. For each received vector,it performs the following algorithm:8PHILIPPE QU´EINNEC,MAMOUN FILALI,PHILIPPE MAURAN,G´ERARD PADIOUF IGURE4.A computation exampleprocessinteger/*maximal vector*/integer/*counter of terminated paths*/repeatintegerreceive();until/*Detection*/The timing diagram of Figure4illustrates a computation behavior.4.2.Distributed collection.The collecting process can be partially distributed by locally accumulating End messages.We add a weight to each vector.When an agent terminates and emits an End message,the weight of the vector is1. Two weighted messages are combined by:With this rule,we can merge End vectors in a distributed way.It can be applied to any site where two(or more)messages are available.Moreover,this rule can be used to reduce the number of messages sent to the collector.4.3.Related work.When considering algorithms for detecting distributed termination[Mat87,Ray92],three classes can be distinguished:algorithms which explicitly use structural properties of the computation;algorithms which build a logical structure of the nodes;and algorithms which are based on more abstract travel patterns.Thefirst category is illustrated by Chandy and Misra’s algorithm[CM90], which counts the number of incoming and outgoing messages of each channel of each site,or by Misra’s algorithm[Mis83]which uses a circuit including(atDESCRIBING MOBILE COMPUTATIONS WITH PATH VECTORS9 least once)all the sites and all the channels used by the distributed computa-tion,and a colored token circulating on this circuit.Thus,the configuration of the connections is supposed to befixed and known a priori.The colored-token algorithm by Dijkstra[DFG83],which relies on a virtual ring,is an example of the second category.Algorithms of this category require to build a logical structure,and to preserve it for the whole duration of the computation.Mattern’s four counters algorithm[Mat87]belongs to the third category. This algorithm uses successive waves visiting all the sites,to accumulate the local state of these sites.Each wave must visit all the sites which are used or which could be used by the diffusing computation:these sites must be known beforehand.Our contribution differs in several points:we need neither to memorize the logical structure of the diffusing com-putation,nor to build another dedicated structure;the graph containing the nodes of the computation can be unconnected,i.e.once a site has sent the computation to other sites(Split or Follow),the termination detection algorithm does not need this site to be reach-able,either by the collector,or by the root of the computation,or by the other sites;the visited nodes of the diffusing computation do not need to be known beforehand,as the algorithm for detecting termination does not visit these nodes to collect local data;one can accumulate the vectors of a subset of the sites in order to transmit only one message to the collector.Thefirst two points mean that the nodes which have been visited by the dif-fusing computation can be withdrawn or disconnected from the network after the visit of the computation.Moreover,they do not have to store information on the computation itself(except for the local counter,which is only used by a new visit of the same computation).Part of the graph of the diffusing com-putation can also be temporarily disconnected from the initiating site.We only need that the leaves eventually send their vectors to the collector.We know of only one other algorithm which has these same properties: Mattern’s credit algorithm[Mat89a].This algorithm is based on credit shares which are split when a site sends a message to another site(and thus activates it).When the computation at a site ends,its credit share is sent to the collector.10PHILIPPE QU´EINNEC,MAMOUN FILALI,PHILIPPE MAURAN,G´ERARD PADIOU When the collector has recovered the whole credit,it can conclude that the computation is terminated.Path vectors are more powerful,as they allow the whole computation tree to be reconstructed after termination has been detected.These properties are interesting when dealing with mobile agents,traveling on an indefinite network,whose components are unknown:an agent locally determines towards which sites the computation must continue,and the initia-tor of the computation does not know(and will never need to know)the visited sites.Some nodes can be mobile nodes or nodes with a temporary connection (such as portable devices)and some distant sub-networks can be temporarily isolated.The message sent to the collector at the end of a path can be seen as an electronic mail,which will be transmitted when connection becomes possible again.Lastly,the distributed collector can be used to accumulate the vectors re-sulting from an arbitrary subset of the visited sites(for example a distant sub-network)and to transmit only one message to the main collector.Thus,sending the message to the main collector can be delegated to one node,and makes it possible to consider the existence offirewalls which isolate the visited sites from the collecting site.5.C OMPUTATION TREE RECONSTRUCTIONIn this section,we show how the vectors collected at the end of each path enable to rebuild the computation tree.In the following,we only consider com-putations with no Follow operation(only End and Split operations).More-over,the destinations of a split cannot include the originating node,and all destinations must be distinct.We present a non-deterministic algorithm which computes an execution tree producing the collected vectors.This tree provides a global view of the execution.We can then analyze the agent paths in the network and the local history of each node.The computation can also be replayed,either totally or partially.The later case enables to restarta computation in any arbitrary state.5.1.Definitions.Def.1(Distance).The distance between two vectors is the number of different components in these vectors:Prop.3.The sequence of values taken by the local counter of node is the (sorted)projection of the-th components of the collected vectors.DESCRIBING MOBILE COMPUTATIONS WITH PATH VECTORS11 Thus,by examining the collected vectors,we can deduce the number of splits which occurred on each node,and the cardinality of each split(i.e.the number of paths issued from each split).For instance,in Figure3,the his-tory of node is,and so we know that three splits occurred on, that thefirst one generated two messages(the counter changed from0to1), etc.Consequently,the reconstruction does not have to deal with multiple oc-currences of identical vectors,and we can use sets of vectors instead of bags. When we build the tree by writing“add edges from to”,the number of new edges is determined by the cardinality of the split.Terminology.siblings:the vectors issued from a split.All siblings are identical.parent:the parent of a vector is the previous split in the path leading to that vector.Note that“uncles”and parent are indistinguishable.cousins:siblings of two brothers.Cousins have a common grand-parent.:vect vect bool:set of vect vect:set vect set add an element to a set:set vect set remove an element from a set5.2.Non-deterministic reconstruction.The algorithm works by recursively finding the“parent”of a vector(or of a bag of identical vectors).This is based on the following two propositions:Prop.4(Distance-1reduction).If one vector is the parent of another vector, the two vectors differ by only one component,and conversely:is the parent ofProp.5(Distance-2reduction).If two vectors have the same grand-parent, their distance is at most2.More precisely,if their parents are brothers,the distance is exactly2,and their parents are the minimum of the two vectors: are cousins their parents are These two reductions are illustrated in Figure5.In thefirst case,the collec-tor has received and,and,using the distance-1rule,one can deduce that the split on S2is the sibling of the split on S1.In the second case,by using, and(or just two of them),one can deduce the value of.For instance,in Figure3,the vector is the parent of and their distance is1;and are cousins,their distance is2and their parents are.Note that distance-2does not necessarily imply a12PHILIPPE QU´EINNEC,MAMOUN FILALI,PHILIPPE MAURAN,G´ERARD PADIOUF IGURE5.The two reductionsrelationship:the distance between and is also2,but they are not cousins.With distance-1,the two vectors differ by one component.This component is the node where the split occurred.The distance-2rule is true for a split of any cardinality:if there are brothers(identical vector),and they split, one needs to consider only two of the resulting messages tofind the vector of the parents.The algorithm.The algorithm consists in recursivelyfinding the parent of a vector and removing that vector,until all vectors have been reduced.To that purpose,we use the distance-1and distance-2properties.The algorithm is(initially is the set of collected vectors plus):trueWhen reducing with the distance-1rule,one adds a new instance of ver-tex and edges from to.When reducing with the distance-2rule,one adds a new vertex and edges from this vertex to. Convergence.This definition is sound.The distance-1rule removes a vector from,and so the size of is decreasing.The distance-2rule replaces one vector with a smaller one,and vectors are bounded by.Non-deterministic convergence towards true.There is(at least)one way to existentially choose the reductions so that is reduced to.DESCRIBING MOBILE COMPUTATIONS WITH PATH VECTORS13 Backtracking.From a programming point of view,this definition could be used to implement an algorithm by using backtracking:a spurious reduction eventually leads to a non-empty set with no possible reduction,or to a tree which does not correspond to a valid diffusing computation.We have shown that non-determinism can be progressively removed to get a more efficient de-terministic algorithm[QFMP00].Time complexity.In[QFMP00],we sketch a deterministic implementation whose time complexity is,where is the number of sites,and is the number of collected vectors(the number of splits in the tree is conse-quently).Note that this is a worst-case time complexity,and our implementation has shown in practice a complexity lower than.6.C ONCLUSIONPath vectors appear to befitted to the distributed and dynamic aspects of mobile computations:a distributed path can be associated to a mobile agent, and no interaction is required until this agent terminates.A distinctive feature of this coding,w.r.t.more classical ones such as time vectors,is that it is centered around the controlflow,whereas the coding of causality is usually based on the local history of sites.Consequently,this facil-itates the evaluation of global properties related to control,such as deadlock, controlflow topology,e.g.:width of the controlflow graph,existence of cycles (a path returns to the same site)The ability to rebuild the computation tree is also useful for debugging purposes.Currently,we are investigating otherfields of application such as global snapshots,and the evaluation of global predicates.An other ongoing work aims at providing a more efficient reconstruction algorithm.R EFERENCES[CM90]K.Mani Chandy and Jayadev Misra.Proofs of distributed algorithms:An exercice.In C.A.R.Hoare,editor,Developments in Concurrency and Communication,chap-ter11,pages305–332.Addison Wesley,1990.[DFG83] E.W.D.Dijkstra,W.H.J Feijen,and A.J.M.van Gasteren.Derivation of a termina-tion detection algorithm for distributed rmation Processing Letters,16:217–219,1983.[DS80] E.W.D.Dijkstra and C.S.Scholten.Termination detection for diffusing computations.Information Processing Letters,11(4):1–4,1980.[Fid88]Colin J.Fidge.Timestamps in message-passing systems that preserve the partial order-ing.In11th Australian Computer Science Conference,pages55–66,February1988.14PHILIPPE QU´EINNEC,MAMOUN FILALI,PHILIPPE MAURAN,G´ERARD PADIOU [FMP00]Mamoun Filali,Philippe Mauran,G´e rard Padiou,Philippe Qu´e innec,and Xavier Thirioux.Refinement based validation of an algorithm for detecting distributed ter-mination.In Jos´e Rolim et al.,editors,Int’l Workshop on Formal Methods for ParallelProgramming:Theory and Applications(FMPPTA2000),volume1800of LectureNotes in Computer Science.Springer-Verlag,May2000.[Mat87]Friedemann Mattern.Algorithms for distributed termination detection.Distributed Computing,2(3):161–175,1987.[Mat89a]Friedemann Mattern.Global quiescence detection based on credit distribution and rmation Processing Letters,30(4):195–200,February1989.[Mat89b]Friedemann Mattern.Virtual time and global state in distributed systems.In M.Cos-nard,Y.Robert,P.Quinton,and M.Raynal,editors,Int’l Workshop on Parallel andDistributed Algorithms,pages215–226.Elsevier Science Publishers,1989.[Mis83]Jayadev Misra.Detecting termination of distributed computation using markers.In 2nd Int’l Symposium on Principles of Distributed Computing,pages290–294,August1983.[QFMP00]Philippe Qu´e innec,Mamoun Filali,Philippe Mauran,and G´e rard Padiou.Distributed paths:Modelling and reversing mobile computations.Rapport de recherche,Institutde Recherche en Informatique de Toulouse,http://www.enseeiht.fr/sdl/mvr/publis/,April2000.21pages.[Ray92]Michel Raynal.Synchronisation et´Etat Global dans les Syst`e mes R´e partis,chapter D´e tection r´e partie de la terminaison,pages157–172.Edition Eyrolles,1992. Authors addresses:Philippe Mauran,G´e rard Padiou,Philippe Qu´e innec,Institut de Recherche en Informa-tique de Toulouse–ENSEEIHT,France,mauran,padiou,queinnec@enseeiht.fr Mamoun Filali,Institut de Recherche en Informatique de Toulouse,France,filali@irit.fr。
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英语专业八级考试TEM-8阅读理解练习册（1）(英语专业2012级)UNIT 1Text AEvery minute of every day, what ecologist生态学家James Carlton calls a global ―conveyor belt‖, redistributes ocean organisms生物.It’s planetwide biological disruption生物的破坏that scientists have barely begun to understand.Dr. Carlton —an oceanographer at Williams College in Williamstown,Mass.—explains that, at any given moment, ―There are several thousand marine species traveling… in the ballast water of ships.‖ These creatures move from coastal waters where they fit into the local web of life to places where some of them could tear that web apart. This is the larger dimension of the infamous无耻的，邪恶的invasion of fish-destroying, pipe-clogging zebra mussels有斑马纹的贻贝.Such voracious贪婪的invaders at least make their presence known. What concerns Carlton and his fellow marine ecologists is the lack of knowledge about the hundreds of alien invaders that quietly enter coastal waters around the world every day. Many of them probably just die out. Some benignly亲切地，仁慈地—or even beneficially — join the local scene. But some will make trouble.In one sense, this is an old story. Organisms have ridden ships for centuries. They have clung to hulls and come along with cargo. What’s new is the scale and speed of the migrations made possible by the massive volume of ship-ballast water压载水— taken in to provide ship stability—continuously moving around the world…Ships load up with ballast water and its inhabitants in coastal waters of one port and dump the ballast in another port that may be thousands of kilometers away. A single load can run to hundreds of gallons. Some larger ships take on as much as 40 million gallons. The creatures that come along tend to be in their larva free-floating stage. When discharged排出in alien waters they can mature into crabs, jellyfish水母, slugs鼻涕虫，蛞蝓, and many other forms.Since the problem involves coastal species, simply banning ballast dumps in coastal waters would, in theory, solve it. Coastal organisms in ballast water that is flushed into midocean would not survive. Such a ban has worked for North American Inland Waterway. But it would be hard to enforce it worldwide. Heating ballast water or straining it should also halt the species spread. But before any such worldwide regulations were imposed, scientists would need a clearer view of what is going on.The continuous shuffling洗牌of marine organisms has changed the biology of the sea on a global scale. It can have devastating effects as in the case of the American comb jellyfish that recently invaded the Black Sea. It has destroyed that sea’s anchovy鳀鱼fishery by eating anchovy eggs. It may soon spread to western and northern European waters.The maritime nations that created the biological ―conveyor belt‖ should support a coordinated international effort to find out what is going on and what should be done about it. (456 words)1.According to Dr. Carlton, ocean organism‟s are_______.A.being moved to new environmentsB.destroying the planetC.succumbing to the zebra musselD.developing alien characteristics2.Oceanographers海洋学家are concerned because_________.A.their knowledge of this phenomenon is limitedB.they believe the oceans are dyingC.they fear an invasion from outer-spaceD.they have identified thousands of alien webs3.According to marine ecologists, transplanted marinespecies____________.A.may upset the ecosystems of coastal watersB.are all compatible with one anotherC.can only survive in their home watersD.sometimes disrupt shipping lanes4.The identified cause of the problem is_______.A.the rapidity with which larvae matureB. a common practice of the shipping industryC. a centuries old speciesD.the world wide movement of ocean currents5.The article suggests that a solution to the problem__________.A.is unlikely to be identifiedB.must precede further researchC.is hypothetically假设地，假想地easyD.will limit global shippingText BNew …Endangered‟ List Targets Many US RiversIt is hard to think of a major natural resource or pollution issue in North America today that does not affect rivers.Farm chemical runoff残渣, industrial waste, urban storm sewers, sewage treatment, mining, logging, grazing放牧,military bases, residential and business development, hydropower水力发电,loss of wetlands. The list goes on.Legislation like the Clean Water Act and Wild and Scenic Rivers Act have provided some protection, but threats continue.The Environmental Protection Agency (EPA) reported yesterday that an assessment of 642,000 miles of rivers and streams showed 34 percent in less than good condition. In a major study of the Clean Water Act, the Natural Resources Defense Council last fall reported that poison runoff impairs损害more than 125,000 miles of rivers.More recently, the NRDC and Izaak Walton League warned that pollution and loss of wetlands—made worse by last year’s flooding—is degrading恶化the Mississippi River ecosystem.On Tuesday, the conservation group保护组织American Rivers issued its annual list of 10 ―endangered‖ and 20 ―threatened‖ rivers in 32 states, the District of Colombia, and Canada.At the top of the list is the Clarks Fork of the Yellowstone River, whereCanadian mining firms plan to build a 74-acre英亩reservoir水库，蓄水池as part of a gold mine less than three miles from Yellowstone National Park. The reservoir would hold the runoff from the sulfuric acid 硫酸used to extract gold from crushed rock.―In the event this tailings pond failed, the impact to th e greater Yellowstone ecosystem would be cataclysmic大变动的，灾难性的and the damage irreversible不可逆转的.‖ Sen. Max Baucus of Montana, chairman of the Environment and Public Works Committee, wrote to Noranda Minerals Inc., an owner of the ― New World Mine‖.Last fall, an EPA official expressed concern about the mine and its potential impact, especially the plastic-lined storage reservoir. ― I am unaware of any studies evaluating how a tailings pond尾矿池，残渣池could be maintained to ensure its structural integrity forev er,‖ said Stephen Hoffman, chief of the EPA’s Mining Waste Section. ―It is my opinion that underwater disposal of tailings at New World may present a potentially significant threat to human health and the environment.‖The results of an environmental-impact statement, now being drafted by the Forest Service and Montana Department of State Lands, could determine the mine’s future…In its recent proposal to reauthorize the Clean Water Act, the Clinton administration noted ―dramatically improved water quality since 1972,‖ when the act was passed. But it also reported that 30 percent of riverscontinue to be degraded, mainly by silt泥沙and nutrients from farm and urban runoff, combined sewer overflows, and municipal sewage城市污水. Bottom sediments沉积物are contaminated污染in more than 1,000 waterways, the administration reported in releasing its proposal in January. Between 60 and 80 percent of riparian corridors (riverbank lands) have been degraded.As with endangered species and their habitats in forests and deserts, the complexity of ecosystems is seen in rivers and the effects of development----beyond the obvious threats of industrial pollution, municipal waste, and in-stream diversions改道to slake消除the thirst of new communities in dry regions like the Southwes t…While there are many political hurdles障碍ahead, reauthorization of the Clean Water Act this year holds promise for US rivers. Rep. Norm Mineta of California, who chairs the House Committee overseeing the bill, calls it ―probably the most important env ironmental legislation this Congress will enact.‖ (553 words)6.According to the passage, the Clean Water Act______.A.has been ineffectiveB.will definitely be renewedC.has never been evaluatedD.was enacted some 30 years ago7.“Endangered” rivers are _________.A.catalogued annuallyB.less polluted than ―threatened rivers‖C.caused by floodingD.adjacent to large cities8.The “cataclysmic” event referred to in paragraph eight would be__________.A. fortuitous偶然的，意外的B. adventitious外加的，偶然的C. catastrophicD. precarious不稳定的，危险的9. The owners of the New World Mine appear to be______.A. ecologically aware of the impact of miningB. determined to construct a safe tailings pondC. indifferent to the concerns voiced by the EPAD. willing to relocate operations10. The passage conveys the impression that_______.A. Canadians are disinterested in natural resourcesB. private and public environmental groups aboundC. river banks are erodingD. the majority of US rivers are in poor conditionText CA classic series of experiments to determine the effects ofoverpopulation on communities of rats was reported in February of 1962 in an article in Scientific American. The experiments were conducted by a psychologist, John B. Calhoun and his associates. In each of these experiments, an equal number of male and female adult rats were placed in an enclosure and given an adequate supply of food, water, and other necessities. The rat populations were allowed to increase. Calhoun knew from experience approximately how many rats could live in the enclosures without experiencing stress due to overcrowding. He allowed the population to increase to approximately twice this number. Then he stabilized the population by removing offspring that were not dependent on their mothers. He and his associates then carefully observed and recorded behavior in these overpopulated communities. At the end of their experiments, Calhoun and his associates were able to conclude that overcrowding causes a breakdown in the normal social relationships among rats, a kind of social disease. The rats in the experiments did not follow the same patterns of behavior as rats would in a community without overcrowding.The females in the rat population were the most seriously affected by the high population density: They showed deviant异常的maternal behavior; they did not behave as mother rats normally do. In fact, many of the pups幼兽，幼崽, as rat babies are called, died as a result of poor maternal care. For example, mothers sometimes abandoned their pups,and, without their mothers' care, the pups died. Under normal conditions, a mother rat would not leave her pups alone to die. However, the experiments verified that in overpopulated communities, mother rats do not behave normally. Their behavior may be considered pathologically 病理上，病理学地diseased.The dominant males in the rat population were the least affected by overpopulation. Each of these strong males claimed an area of the enclosure as his own. Therefore, these individuals did not experience the overcrowding in the same way as the other rats did. The fact that the dominant males had adequate space in which to live may explain why they were not as seriously affected by overpopulation as the other rats. However, dominant males did behave pathologically at times. Their antisocial behavior consisted of attacks on weaker male,female, and immature rats. This deviant behavior showed that even though the dominant males had enough living space, they too were affected by the general overcrowding in the enclosure.Non-dominant males in the experimental rat communities also exhibited deviant social behavior. Some withdrew completely; they moved very little and ate and drank at times when the other rats were sleeping in order to avoid contact with them. Other non-dominant males were hyperactive; they were much more active than is normal, chasing other rats and fighting each other. This segment of the rat population, likeall the other parts, was affected by the overpopulation.The behavior of the non-dominant males and of the other components of the rat population has parallels in human behavior. People in densely populated areas exhibit deviant behavior similar to that of the rats in Calhoun's experiments. In large urban areas such as New York City, London, Mexican City, and Cairo, there are abandoned children. There are cruel, powerful individuals, both men and women. There are also people who withdraw and people who become hyperactive. The quantity of other forms of social pathology such as murder, rape, and robbery also frequently occur in densely populated human communities. Is the principal cause of these disorders overpopulation? Calhoun’s experiments suggest that it might be. In any case, social scientists and city planners have been influenced by the results of this series of experiments.11. Paragraph l is organized according to__________.A. reasonsB. descriptionC. examplesD. definition12．Calhoun stabilized the rat population_________.A. when it was double the number that could live in the enclosure without stressB. by removing young ratsC. at a constant number of adult rats in the enclosureD. all of the above are correct13.W hich of the following inferences CANNOT be made from theinformation inPara. 1?A. Calhoun's experiment is still considered important today.B. Overpopulation causes pathological behavior in rat populations.C. Stress does not occur in rat communities unless there is overcrowding.D. Calhoun had experimented with rats before.14. Which of the following behavior didn‟t happen in this experiment?A. All the male rats exhibited pathological behavior.B. Mother rats abandoned their pups.C. Female rats showed deviant maternal behavior.D. Mother rats left their rat babies alone.15. The main idea of the paragraph three is that __________.A. dominant males had adequate living spaceB. dominant males were not as seriously affected by overcrowding as the otherratsC. dominant males attacked weaker ratsD. the strongest males are always able to adapt to bad conditionsText DThe first mention of slavery in the statutes法令，法规of the English colonies of North America does not occur until after 1660—some forty years after the importation of the first Black people. Lest we think that existed in fact before it did in law, Oscar and Mary Handlin assure us, that the status of B lack people down to the 1660’s was that of servants. A critique批判of the Handlins’ interpretation of why legal slavery did not appear until the 1660’s suggests that assumptions about the relation between slavery and racial prejudice should be reexamined, and that explanation for the different treatment of Black slaves in North and South America should be expanded.The Handlins explain the appearance of legal slavery by arguing that, during the 1660’s, the position of white servants was improving relative to that of black servants. Thus, the Handlins contend, Black and White servants, heretofore treated alike, each attained a different status. There are, however, important objections to this argument. First, the Handlins cannot adequately demonstrate that t he White servant’s position was improving, during and after the 1660’s; several acts of the Maryland and Virginia legislatures indicate otherwise. Another flaw in the Handlins’ interpretation is their assumption that prior to the establishment of legal slavery there was no discrimination against Black people. It is true that before the 1660’s Black people were rarely called slaves. But this shouldnot overshadow evidence from the 1630’s on that points to racial discrimination without using the term slavery. Such discrimination sometimes stopped short of lifetime servitude or inherited status—the two attributes of true slavery—yet in other cases it included both. The Handlins’ argument excludes the real possibility that Black people in the English colonies were never treated as the equals of White people.The possibility has important ramifications后果，影响.If from the outset Black people were discriminated against, then legal slavery should be viewed as a reflection and an extension of racial prejudice rather than, as many historians including the Handlins have argued, the cause of prejudice. In addition, the existence of discrimination before the advent of legal slavery offers a further explanation for the harsher treatment of Black slaves in North than in South America. Freyre and Tannenbaum have rightly argued that the lack of certain traditions in North America—such as a Roman conception of slavery and a Roman Catholic emphasis on equality— explains why the treatment of Black slaves was more severe there than in the Spanish and Portuguese colonies of South America. But this cannot be the whole explanation since it is merely negative, based only on a lack of something. A more compelling令人信服的explanation is that the early and sometimes extreme racial discrimination in the English colonies helped determine the particular nature of the slavery that followed. (462 words)16. Which of the following is the most logical inference to be drawn from the passage about the effects of “several acts of the Maryland and Virginia legislatures” (Para.2) passed during and after the 1660‟s?A. The acts negatively affected the pre-1660’s position of Black as wellas of White servants.B. The acts had the effect of impairing rather than improving theposition of White servants relative to what it had been before the 1660’s.C. The acts had a different effect on the position of white servants thandid many of the acts passed during this time by the legislatures of other colonies.D. The acts, at the very least, caused the position of White servants toremain no better than it had been before the 1660’s.17. With which of the following statements regarding the status ofBlack people in the English colonies of North America before the 1660‟s would the author be LEAST likely to agree?A. Although black people were not legally considered to be slaves,they were often called slaves.B. Although subject to some discrimination, black people had a higherlegal status than they did after the 1660’s.C. Although sometimes subject to lifetime servitude, black peoplewere not legally considered to be slaves.D. Although often not treated the same as White people, black people,like many white people, possessed the legal status of servants.18. According to the passage, the Handlins have argued which of thefollowing about the relationship between racial prejudice and the institution of legal slavery in the English colonies of North America?A. Racial prejudice and the institution of slavery arose simultaneously.B. Racial prejudice most often the form of the imposition of inheritedstatus, one of the attributes of slavery.C. The source of racial prejudice was the institution of slavery.D. Because of the influence of the Roman Catholic Church, racialprejudice sometimes did not result in slavery.19. The passage suggests that the existence of a Roman conception ofslavery in Spanish and Portuguese colonies had the effect of _________.A. extending rather than causing racial prejudice in these coloniesB. hastening the legalization of slavery in these colonies.C. mitigating some of the conditions of slavery for black people in these coloniesD. delaying the introduction of slavery into the English colonies20. The author considers the explanation put forward by Freyre andTannenbaum for the treatment accorded B lack slaves in the English colonies of North America to be _____________.A. ambitious but misguidedB. valid有根据的but limitedC. popular but suspectD. anachronistic过时的，时代错误的and controversialUNIT 2Text AThe sea lay like an unbroken mirror all around the pine-girt, lonely shores of Orr’s Island. Tall, kingly spruce s wore their regal王室的crowns of cones high in air, sparkling with diamonds of clear exuded gum流出的树胶; vast old hemlocks铁杉of primeval原始的growth stood darkling in their forest shadows, their branches hung with long hoary moss久远的青苔;while feathery larches羽毛般的落叶松,turned to brilliant gold by autumn frosts, lighted up the darker shadows of the evergreens. It was one of those hazy朦胧的, calm, dissolving days of Indian summer, when everything is so quiet that the fainest kiss of the wave on the beach can be heard, and white clouds seem to faint into the blue of the sky, and soft swathing一长条bands of violet vapor make all earth look dreamy, and give to the sharp, clear-cut outlines of the northern landscape all those mysteries of light and shade which impart such tenderness to Italian scenery.The funeral was over,--- the tread鞋底的花纹/ 踏of many feet, bearing the heavy burden of two broken lives, had been to the lonely graveyard, and had come back again,--- each footstep lighter and more unconstrained不受拘束的as each one went his way from the great old tragedy of Death to the common cheerful of Life.The solemn black clock stood swaying with its eternal ―tick-tock, tick-tock,‖ in the kitchen of the brown house on Orr’s Island. There was there that sense of a stillness that can be felt,---such as settles down on a dwelling住处when any of its inmates have passed through its doors for the last time, to go whence they shall not return. The best room was shut up and darkened, with only so much light as could fall through a little heart-shaped hole in the window-shutter,---for except on solemn visits, or prayer-meetings or weddings, or funerals, that room formed no part of the daily family scenery.The kitchen was clean and ample, hearth灶台, and oven on one side, and rows of old-fashioned splint-bottomed chairs against the wall. A table scoured to snowy whiteness, and a little work-stand whereon lay the Bible, the Missionary Herald, and the Weekly Christian Mirror, before named, formed the principal furniture. One feature, however, must not be forgotten, ---a great sea-chest水手用的储物箱,which had been the companion of Zephaniah through all the countries of the earth. Old, and battered破旧的，磨损的, and unsightly难看的it looked, yet report said that there was good store within which men for the most part respect more than anything else; and, indeed it proved often when a deed of grace was to be done--- when a woman was suddenly made a widow in a coast gale大风，狂风, or a fishing-smack小渔船was run down in the fogs off the banks, leaving in some neighboring cottage a family of orphans,---in all such cases, the opening of this sea-chest was an event of good omen 预兆to the bereaved丧亲者;for Zephaniah had a large heart and a large hand, and was apt有…的倾向to take it out full of silver dollars when once it went in. So the ark of the covenant约柜could not have been looked on with more reverence崇敬than the neighbours usually showed to Captain Pennel’s sea-chest.1. The author describes Orr‟s Island in a(n)______way.A.emotionally appealing, imaginativeB.rational, logically preciseC.factually detailed, objectiveD.vague, uncertain2.According to the passage, the “best room”_____.A.has its many windows boarded upB.has had the furniture removedC.is used only on formal and ceremonious occasionsD.is the busiest room in the house3.From the description of the kitchen we can infer that thehouse belongs to people who_____.A.never have guestsB.like modern appliancesC.are probably religiousD.dislike housework4.The passage implies that_______.A.few people attended the funeralB.fishing is a secure vocationC.the island is densely populatedD.the house belonged to the deceased5.From the description of Zephaniah we can see thathe_________.A.was physically a very big manB.preferred the lonely life of a sailorC.always stayed at homeD.was frugal and saved a lotText BBasic to any understanding of Canada in the 20 years after the Second World War is the country' s impressive population growth. For every three Canadians in 1945, there were over five in 1966. In September 1966 Canada's population passed the 20 million mark. Most of this surging growth came from natural increase. The depression of the 1930s and the war had held back marriages, and the catching-up process began after 1945. The baby boom continued through the decade of the 1950s, producing a population increase of nearly fifteen percent in the five years from 1951 to 1956. This rate of increase had been exceeded only once before in Canada's history, in the decade before 1911 when the prairies were being settled. Undoubtedly, the good economic conditions of the 1950s supported a growth in the population, but the expansion also derived from a trend toward earlier marriages and an increase in the average size of families; In 1957 the Canadian birth rate stood at 28 per thousand, one of the highest in the world. After the peak year of 1957, thebirth rate in Canada began to decline. It continued falling until in 1966 it stood at the lowest level in 25 years. Partly this decline reflected the low level of births during the depression and the war, but it was also caused by changes in Canadian society. Young people were staying at school longer, more women were working; young married couples were buying automobiles or houses before starting families; rising living standards were cutting down the size of families. It appeared that Canada was once more falling in step with the trend toward smaller families that had occurred all through theWestern world since the time of the Industrial Revolution. Although the growth in Canada’s population had slowed down by 1966 (the cent), another increase in the first half of the 1960s was only nine percent), another large population wave was coming over the horizon. It would be composed of the children of the children who were born during the period of the high birth rate prior to 1957.6. What does the passage mainly discuss?A. Educational changes in Canadian society.B. Canada during the Second World War.C. Population trends in postwar Canada.D. Standards of living in Canada.7. According to the passage, when did Canada's baby boom begin?A. In the decade after 1911.B. After 1945.C. During the depression of the 1930s.D. In 1966.8. The author suggests that in Canada during the 1950s____________.A. the urban population decreased rapidlyB. fewer people marriedC. economic conditions were poorD. the birth rate was very high9. When was the birth rate in Canada at its lowest postwar level?A. 1966.B. 1957.C. 1956.D. 1951.10. The author mentions all of the following as causes of declines inpopulation growth after 1957 EXCEPT_________________.A. people being better educatedB. people getting married earlierC. better standards of livingD. couples buying houses11.I t can be inferred from the passage that before the IndustrialRevolution_______________.A. families were largerB. population statistics were unreliableC. the population grew steadilyD. economic conditions were badText CI was just a boy when my father brought me to Harlem for the first time, almost 50 years ago. We stayed at the hotel Theresa, a grand brick structure at 125th Street and Seventh avenue. Once, in the hotel restaurant, my father pointed out Joe Louis. He even got Mr. Brown, the hotel manager, to introduce me to him, a bit punchy强力的but still champ焦急as fast as I was concerned.Much has changed since then. Business and real estate are booming. Some say a new renaissance is under way. Others decry责难what they see as outside forces running roughshod肆意践踏over the old Harlem. New York meant Harlem to me, and as a young man I visited it whenever I could. But many of my old haunts are gone. The Theresa shut down in 1966. National chains that once ignored Harlem now anticipate yuppie money and want pieces of this prime Manhattan real estate. So here I am on a hot August afternoon, sitting in a Starbucks that two years ago opened a block away from the Theresa, snatching抓取，攫取at memories between sips of high-priced coffee. I am about to open up a piece of the old Harlem---the New York Amsterdam News---when a tourist。
二氧化碳对环境的危害和影响英语作文
二氧化碳对环境的危害和影响英语作文Carbon dioxide, commonly known as CO2, is a gas that plays a crucial role in the Earth's atmosphere and climate. While it is essential for the survival of many life forms, the excessive release of CO2into the environment has become a significant concern in recent years. The impact of CO2 on the environment can be far-reaching and detrimental, affecting various aspects of our planet's ecosystem.One of the primary dangers posed by excessive CO2 emissions is its contribution to the greenhouse effect. The greenhouse effect is a natural process that helps regulate the Earth's temperature by trapping heat from the sun's radiation. However, the increased concentration of CO2 and other greenhouse gases in the atmosphere has amplified this effect, leading to a phenomenon known as global warming. As the Earth's temperature rises, it can cause a wide range of environmental changes, including melting glaciers and ice caps, rising sea levels, and more frequent and severe weather events, such as hurricanes, droughts, and heatwaves.The impact of global warming on the environment can be devastating. As sea levels rise, coastal regions and islands are at risk of flooding, displacing millions of people and threatening theirhomes and livelihoods. Additionally, the melting of glaciers and ice caps can disrupt the delicate balance of water resources, leading to water scarcity and affecting the availability of drinking water and irrigation for agriculture.Moreover, the increased frequency and intensity of extreme weather events can have devastating consequences for both human and natural ecosystems. Droughts can lead to crop failures and food shortages, while floods and storms can cause widespread damage to infrastructure and disrupt essential services. These environmental changes can also contribute to the spread of diseases, as warmer temperatures and altered habitats can facilitate the proliferation of disease-carrying vectors, such as mosquitoes.The impact of CO2 on the environment is not limited to global warming and climate change. Excessive CO2 emissions can also have direct effects on the natural environment, particularly on the ocean's ecosystems. As the ocean absorbs a significant portion of the excess CO2 in the atmosphere, it undergoes a process known as ocean acidification. This process can disrupt the delicate balance of marine life, affecting the growth and survival of coral reefs, shellfish, and other organisms that rely on a specific pH level in the water.The consequences of ocean acidification can be far-reaching, as these marine ecosystems play a crucial role in the global food chainand the overall health of the planet. The loss of coral reefs, for example, can have a devastating impact on the diversity and abundance of marine life, as well as the livelihoods of communities that depend on them for fishing and tourism.In addition to the environmental impacts, the excessive release of CO2 can also have significant economic and social consequences. The costs associated with mitigating the effects of climate change, such as the development of renewable energy sources, the implementation of energy-efficient technologies, and the adaptation to changing weather patterns, can be substantial. Furthermore, the disruption of agricultural and fishing industries, as well as the displacement of populations due to environmental disasters, can lead to economic instability and social unrest.To address the dangers posed by excessive CO2 emissions, a comprehensive and coordinated global effort is required. This effort must involve a combination of policy changes, technological advancements, and individual actions. Governments, businesses, and individuals must work together to reduce CO2 emissions, promote the use of renewable energy sources, and implement sustainable practices that minimize the environmental impact of human activities.In conclusion, the dangers and impacts of excessive CO2 emissions on the environment are far-reaching and profound. Fromcontributing to global warming and climate change to directly affecting the health of marine ecosystems, the consequences of our actions can have lasting and devastating effects on the planet we call home. It is our collective responsibility to take immediate and decisive action to mitigate these threats and ensure a sustainable future for generations to come.。
infectious前缀
Infectious Diseases: An OverviewInfectious diseases have been a constant threat to humanity throughout history. From the devastating pandemics like the Black Death to the recent COVID-19 outbreak, these diseases have claimed countless lives and shaped the course of civilizations. In this article, we will explore the nature of infectious diseases, their causes, prevention, and the impact they have on societies.What are Infectious Diseases?Infectious diseases are illnesses caused by pathogenic microorganisms such as bacteria, viruses, fungi, and parasites. These microorganisms can enter the human body, multiply, and disrupt normal bodily functions, leading to various symptoms. Some examples of infectious diseases include influenza, tuberculosis, malaria, and HIV/AIDS.Modes of TransmissionInfectious diseases can spread in several ways. The most common modes of transmission are:1.Airborne Transmission: Pathogens can be carried in tiny dropletsthat are released into the air when an infected individual coughs or sneezes.Breathing in these droplets can lead to infection, as seen in the case of airborne diseases like tuberculosis and COVID-19.2.Direct Contact: Infections can be transmitted through direct physicalcontact with an infected person or their bodily fluids. Examples of diseasestransmitted through direct contact include sexually transmitted infections and skin infections like impetigo.3.Vector-Borne Transmission: Many diseases are transmitted throughvectors, such as mosquitoes, ticks, and fleas. These vectors act as carriers for the infectious agents, transferring them from one host to another. Malaria,dengue fever, and Lyme disease are examples of vector-borne diseases.4.Food and Waterborne Transmission: Consumption of contaminatedfood or water can introduce pathogens into the body. Contaminated watersources and improper food handling can lead to diseases like cholera,salmonellosis, and hepatitis A.Prevention and ControlPreventing and controlling the spread of infectious diseases is crucial to safeguarding public health. Several measures can be taken at individual and community levels:1.Vaccination: Vaccines have been instrumental in preventing thespread of various infectious diseases. Vaccination programs help developimmunity against pathogens by introducing weakened or inactivated forms of the disease-causing agents. This allows the body to mount a defense againstfuture infections.2.Hygiene Practices: Maintaining good hygiene can significantlyreduce the risk of infection. Regular handwashing with soap and water, using hand sanitizers, practicing safe food handling, and promoting personalcleanliness are essential to prevent the transmission of pathogens.3.Vector Control: Implementing effective strategies to control disease-carrying vectors is crucial to preventing vector-borne diseases. This mayinvolve measures like mosquito netting, insect repellents, and eliminatingbreeding sites for mosquitoes.4.Isolation and Quarantine: Isolating infected individuals andimplementing quarantine measures can help limit the spread of infectiousdiseases. This is particularly important during pandemics when the rapidtransmission of the disease poses a severe threat.Impact on SocietyInfectious diseases can have far-reaching consequences on societies, both in terms of public health and socio-economic aspects. These include:1.Healthcare Burden: Infectious diseases place a significant burden onhealthcare systems. Outbreaks often strain hospitals and medical resources,leading to a shortage of medical personnel, supplies, and adequate treatment facilities.2.Economic Consequences: Outbreaks of infectious diseases can havea substantial economic impact. The costs associated with healthcare,productivity losses, trade restrictions, and reduced tourism can cause severe damage to economies at local, national, and global levels.3.Disruption of Daily Life: Epidemics and pandemics often disrupt thenormal functioning of societies. School and workplace closures, travelrestrictions, and social distancing measures can lead to psychological distress, societal unrest, and economic instability.ConclusionInfectious diseases remain a significant global challenge, posing a threat to human health, societal well-being, and economic stability. Understanding the causes, modes of transmission, and effective prevention and control measures are vital in minimizing the impact of these diseases. By investing in research, healthcareinfrastructure, and public health initiatives, we can work towards a safer and healthier future for all.。
Lecture3 Cloning vector
Cloning vectors A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted, capable of infecting the host cell and existing steadily therein through replicating themselves.
Genetic Engineering
Dr. Zheng Xuesong Shanghai Institute of Technology
Lecture 3 CloningVectors for genetic engineering 3.1 Characteristics for vector
3.2 plasmid vector
3.1.1 Fundamental characteristics Some other possible features present in cloning vectors are: a. All plasmids must carry a functional origin of replication (Ori; Pro et al.)
• The lacI gene. This gene codes for the lac repressor protein.
• The lacZ' gene. This gene product is transcribed from the lac promoter and produces an amino-terminal fragment of the β-galactosidase protein.
Pointed and copointed Hopf algebras as cocycle deformations
0. Introduction Finite dimensional pointed Hopf algebras over an algebraically closed field of characteristic zero, particularly when the group of points is abelian, have been studied quite extensively with various methods in [AS, BDG, Gr1, Mu]. The most far reaching results as yet in this area have been obtained in [AS], where a large class of such Hopf algebras are classified. In the present paper we will show, among other things, that all Hopf algebras in this class can be obtained by cocycle deformations. We also consider the “dual” case, where the Jacobson radical is a Hopf ideal. In the non-pointed case, in particular when the coradical is not a Hopf algebra, very little is known. Few examples occur of the literature [Ra2, Be], but no general description or classification results are available. It is the aim of this paper to contribute to the construction and classification of such Hopf algebras, in particular the copointed kind. By a copointed Hopf algebra we mean a Hopf algebra H whose Jacobson radical Rad H is a Hopf ideal and H/Rad H is a group algebra. If H is a Hopf algebra with coradical a Hopf subalgebra then the graded coalgebra grc H associated with the coradical filtration is a graded Hopf algebra and its elements of positive degree form the radical. If the radical of H is a Hopf ideal then the graded algebra associated with the radical filtration is a graded Hopf algebra with Cor(gra H ) ∼ = H/ Rad H . In either case we have gr H ∼ = R#H0 , where
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Published online20February2007Nucleic Acids Research,2007,Vol.35,No.6e43doi:10.1093/nar/gkm067 Vectors for co-expression of an unrestricted number of proteinsChristoph Scheich1,Daniel Ku¨mmel2,3,Dimitri Soumailakakis1,Udo Heinemann2,3and Konrad Bu¨ssow1,*1Max Planck Institute for Molecular Genetics,Department of Vertebrate Genomics,Ihnestr.63-73,14195Berlin, Germany,2Max Delbru¨ck Center for Molecular Medicine,Robert-Ro¨ssle-Strasse10,13125Berlin,Germany and3Free University of Berlin,Institute of Chemistry and Biochemistry,Takustraße6,14195Berlin,Germany Received November10,2006;Revised January17,2007;Accepted January23,2007ABSTRACTA vector system is presented that allows generation of E.coli co-expression clones by a standardized, robust cloning procedure.The number of co-expressed proteins is not limited.Five‘pQLink’vectors for expression of His-tag and GST-tag fusion proteins as well as untagged proteins and for cloning by restriction enzymes or Gateway cloning were generated.The vectors allow proteins to be expressed individually;to achieve co-expression,two pQLink plasmids are combined by ligation-independent cloning.pQLink co-expression plasmids can accept an unrestricted number of genes.As an example,the co-expression of a heterotetrameric human transport protein particle(TRAPP)complex from a single plasmid, its isolation and analysis of its stoichiometry are shown.pQLink clones can be used directly for pull-down experiments if the proteins are expressed with different tags.We demonstrate pull-down experiments of human valosin-containing protein (VCP)with fragments of the autocrine motility factor receptor(AMFR).The cloning method avoids PCR or gel isolation of restriction fragments,and a single resistance marker and origin of replication are used, allowing over-expression of rare tRNAs from a second plasmid.It is expected that applications are not restricted to bacteria,but could include co-expression in other hosts such as Bacluovirus/ insect cells.INTRODUCTIONCo-expression of subunits is an important technique in the study of protein complexes.Proteins that form tight complexes with partners may not be soluble when over-expressed alone(1).Proteins that form stable homodimers may only form heterodimers upon co-expression,but not by mixing the purified complex partners(2,3).The Protein Structure Factory has estab-lished high throughput protein expression procedures for human proteins in E.coli(4,5).To achieve co-expression in a systematic way we have created a new vector system described here.Co-expression is often achieved with two or more plasmids,each carrying the gene of one subunit and a different selection marker(6).The plasmids should also have different compatible replicons,although co-expression with plasmids that have the same replicon has been demonstrated(7).Instead of multiple vectors, several genes can be inserted into the same vector.This requires a vector with more than one multiple cloning site and the use of different sets of restriction enzymes for each cloning step(8).Genes are transcribed either from individual promoters or from a single promoter,leading to a long polycistronic mRNA.Polycistronic expression has been reported to lead to lower expression of the more downstream encoded protein,which can be exploited to influence the stoichiometry of a protein complex(9). Several-fold higher expression with individual promoters compared to polycistronic transcription has been reported(10).A recent evaluation of co-expression experiments performed by the Structural Proteomics in Europe(SPINE)consortium compared examples of co-expression with various systems(11).The pETDuet system of Novagen allows construction of a co-expression plasmid for two genes in a single PCR step.Alternative approaches to co-expression from a single vector require different sets of restriction enzymes for multiple insertions.Berger et al.have developed a Baculovirus co-expression system that allows iterative addition of genes to a Baculovirus vector(12).Alexandrov et al.have developed a vector with a‘LINK’sequence that allows fusion of two plasmids into one(13).The method allows cloning of all cDNAs of interest into one standard expression vector*To whom correspondence should be addressed.Tel:þ493094062983;Fax:þ493094062925;Email:buessow@molgen.mpg.deß2007The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License(/licenses/ by-nc/2.0/uk/)which permits unrestricted non-commercial use,distribution,and reproduction in any medium,provided the original work is properly cited.by a standardized,high-throughput procedure.Then, co-expression plasmids are generated by fusion of LINK sequences with the ligation-independent cloning method (LIC)that avoids PCR,which may introduce mutations. Two alternative rare cutting restriction enzymes are used. Inserts to be cloned are unlikely to contain both sites. The method is limited to co-expression of two proteins. We have designed a modified version of the LINK sequence cloning technique.Vectors with two modified LINK sequences were created that allow the creation of co-expression plasmids by the established LINK cloning procedure but avoid the duplication of the vector back-bone.In principle,the vectors can accept an unlimited number of genes to be co-expressed.To demonstrate the usefulness of the method, co-expression and binding of VCP and the C-terminus of AMFR(14,15)is demonstrated.In addition,the co-expression of four subunits of the human transport protein particle(TRAPP)(16)from a single plasmid and an analysis of their stoichiometry is shown. MATERIALS AND METHODSVector constructionThe vector pQTEV2was constructed from pQE-2 (Qiagen)by replacing the multiple cloning site with synthetic oligonucleotides to introduce a TEV protease site and attB Gateway recombination sites.To obtain pQLinkH,LINK sequences were inserted by PCR amplification of bp2–379of pQTEV2with the primers 50-TTTCTCGAGC TTAATTAACA ACACCATTTG TCGAGAAATC ATAAAAAATT TATT-30and 50-TTTTGCTAGC CTTAATTAAC CACTCCATTT GTAACCACTC CATTTAAATG GTGTTGTTAC TTGGATTCTC ACCAATAAAA A-30and cloning of the product into pQTEV2using XhoI and NheI. To construct pQLinkG,a GST coding sequence was PCR amplified from pGEX-5X-1(GE Healthcare)with primers carrying EcoRI and PstI restriction sites. The fragment was cloned between the EcoRI and PstI sites of pQLinkH,thereby removing the His-tag. pQLinkHD and pQLinkGD were obtained from pQLinkH and pQLinkG—which contain attB sites—by Gateway BP reactions with pDONR221,which inserted the Gateway destination vector cassettes.pQLinkHD and pQLinkGD confer both ampicillin and chloramphenicol resistance and require the strain DB3Á1for propagation. The vector pQLinkN without a tag was generated from pQLinkH using QuikChange TM site-directed mutagenesis (17)with the double-stranded oligonucleotide 50-AAAGAGGAGA AATTAACTAT GGGATCCAGT CTTCGCATGC AT-30.The sequences of the new vectors were verified by sequencing and were submitted to GenBank(EF025686–EF025689,EF196676).Cloning of expression clonesThe E.coli strain SCS1with the helper plasmid pRARE for over-expression of rare tRNAs(18)was used as a host for cloning of expression and co-expression clones, unless indicated otherwise.The VCP coding sequence (GenBank NP_009057)was cloned into pQLinkH and AMFR C-terminal regions(GenBank NP_001135)and into pQLinkG(AMFR)with BamHI and NotI.BamHI/ NotI fragments of full-length cDNAs encoding four human TRAPP subunits were cloned into pQLinkH. The Bet3(GenBank AF041432)and Mum2(AAD44697) full-length cDNAs lack the native start and stop codons and were cloned with the BamHI site adjacent to the second codon and with a stop codon downstream of the NotI site,which encodes for three additional C-terminal alanine residues.Full-length Tpc6(CAD61947)and synbindin(AAH10866)cDNAs were cloned with the BamHI site adjacent to the native start codon and with a TGA stop codon replacing the native stop codon.The Bet3:Tpc6pETDuet-1clone was described previously(2). Cloning of co-expression clonesCo-expression plasmids were cloned according to Alexandrov et al.(13).To produce a co-expression plasmid from two pQLink plasmids,0.2–0.5m g of one plasmid was digested overnight with5units PacI in10m l at378C while the other was cleaved with5units SwaI at 258C.GST-plasmids were digested with PacI because the GST coding region contains a SwaI site.Enzymes were inactivated at658C for20min and5m l DNA was treated with1.3units LIC qualified T4DNA polymerase(Merck Biosciences)in20m l(50mM Tris-HCl,pH8.0,10mM MgCl2,5m g/ml BSA,5mM DTT,2.5mM dCTP for the PacI digest and2.5mM dGTP for the SwaI digest).Upon incubation for30min at258C and heat inactivation at 658C for20min,the two plasmids were mixed and heated to658C and cooled to room temperature for annealing. Two microlitres of25mM EDTA were added,followed by E.coli transformation.Transformants were tested for expected inserts by colony PCR with primers pQTEV3U, 50-TATAAAAATA GGCGTATCAC GAGG-30and pQTEV3L,50-CCAGTGATTT TTTTCTCCAT TTT-30 and598C annealing temperature.These primers are positioned upstream of the LINK1and downstream of the LINK2sequences,respectively.Co-expression of AMFR and VCP and GST pull downHis-VCP:GST-AMFR co-expression plasmids were expressed in2l with1mM IPTG for4h at168C.Cells were harvested,resuspended in20mM Tris-HCl,pH7.5, 150mM NaCl,2mM b-mercaptoethanol,1mM PMSF and1tablet/50ml lysate of Roche complete EDTA-free protease inhibitor cocktail and lysed via incubation with0.5mg/ml lysozyme for30min on ice,followed by addition of8units/ml benzonase and treatment with an Avestin Emulsiflex C5homogenizer.Insoluble material was removed by centrifugation(30min,75000g)and the lysate was loaded on a glutathione sepharose column.The column was washed with wash buffer(20mM Tris-HCl, pH7.5,150mM NaCl)and incubated overnight with 0.1mg/ml TEV protease in wash buffer at88C.Proteins mobilized by the protease were collected and analysed by SDS-PAGE.Co-expression and pull down with nickel chelating beads Protein expression and purification were performed as described(19).Briefly,proteins were expressed in1ml cultures with1mM IPTG at378C(TRAPP proteins)or 168C(AMFR and VCP).His-tag fusion proteins and their bound interaction partners were immobilized on Ni-NTA agarose(Qiagen),washed with20mM Tris-HCl,pH8.0, 300mM NaCl and20mM imidazole and then eluted with 70m l500mM imidazole in wash buffer.Six-microlitre aliquots of eluate were analysed by SDS-PAGE.BL21(DE3)E.coli clones with pETDuet-1Bet3:Tpc6 (2)and pQLinkH Bet3:pQLinkN Tpc6plasmids were cultured in50ml and protein expression was induced with 1mM IPTG at OD600$0.7for4h.Purification and characterization of TRAPP subcomplexes TRAPP co-expression plasmids were expressed in0.5l overnight express TM instant TB medium(Novagen).Cells were lysed using a French Press(SLM-AMINCO)and purified using Ni-NTA agarose(Qiagen),according to the manufacturer’s instructions.Gelfiltration was performed on a Superdex20026/60column(GE Healthcare) equilibrated with20mM Tris-HCl pH7.5,200mM NaCl and2mM DTT.RESULTSVector constructionWe have constructedfive vectors for protein expression in E.coli that allow preparation of co-expression constructs by a simple cloning procedure(Figure1).The vectors contain two LINK sequences(13)thatflank the expression cassette of promoter,multiple cloning site and a transcriptional terminator.LINK1contains a PacI restriction site and LINK2a SwaI and a PacI site (Figure1A).The LINK sequences allow insertion of a PacI fragment of one plasmid at the SwaI site of another plasmid by LIC(Figure1B).The vectors pQLinkH and pQLinkG have a His-tag and a GST-tag,respectively,followed by a TEV protease site and a multiple cloning site.The vector pQLinkN is used for expression of proteins without a tag.It contains a start codon adjacent to the BamHI site and can be used to express a protein complex with only one tagged subunit for isolation.In the vectors pQLinkHD and pQLinkGD, the TEV site and the multiple cloning site of pQLinkH and pQLinkG were replaced by a cassette for Gateway recombination cloning.The vectors pQLinkH and pQLinkG contain Gateway attB sites and inserts can be shuttled by a reverse Gateway reaction into a pDONR donor vector to obtain Entry clones.Vector sequences were submitted to GenBank(EF025686–EF025689, EF196676)and vectors will be available from Addgene. The cloning of co-expression plasmids by LIC was very robust in our hands and about90%of transformants contained the expected insert.Correct clones are easily recognized by the large product size of colony PCRs.Co-expression of VCP and AMFRThe GST coding sequence contains a SwaI site,which requires that pQLinkG clones are cut with PacI and not with SwaI when generating a co-expression clone.To circumvent this limitation,pQLinkG2was generated by introducing a silent mutation that removes this site in pQLinkG.To study the interaction of VCP and AMFR,a human VCP cDNA was cloned into pQLinkH and three C-terminal fragments of the643amino acid coding region of AMFR were cloned into pQLinkG.These fragments comprise the Cue domain(amino acids450–510),the downstream region up to the C-terminus(498–643)and a combination of both(455–643).Previously,the larger constructs were found to interact with VCP,but not with amino acids450–510(15).His-VCP:GST-AMFR co-expression clones were obtained by inserting a PacI fragment of the VCP plasmid into the LINK2sequence of the AMFR pQLinkG plasmids.Protein expression was induced and the expression products of two His-VCP:GST-AMFR co-expression clones were coupled to a glutathione agarose column.The column was treated with TEV protease to separate AMFR and GST and elute AMFR and any attached proteins.Co-expression was successful and,as shown in Figure2A,the longer AMFR fragment bound to His-VCP while the shorter one did not. Reverse pull-down experiments were also performed by coupling His-VCP to nickel chelating beads. Co-purification with this matrix was generally less efficient than with glutathione agarose.Co-purified bands were observed upon Coomassie staining and anti-GST western blotting for the AMFR constructs456–643and498–643 (Figure4B).Only a weak background of co-purified GST and of the small AMFR construct was observed. These results indicate that amino acids498–643of AMFR,the region downstream of the Cue domain,are responsible for the interaction with VCP,as it was found by Grelle et al.(15).Purification and characterization of TRAPP subcomplexes Upon construction of a co-expression plasmid,only the outer LINK sequences reconstitute(Figure1B). Therefore,it is possible to continuously increase the number of genes by combining plasmids that carry any number of expression cassettes.We have constructed a co-expression plasmid for four subunits of the human TRAPP complex.Bet3,Tpc6,Mum2and synbindin were cloned into pQLinkH,and the expression of individual proteins was tested by small-scale expression and metal chelate affinity chromatography(Figure3A).A Bet3 and Tpc6co-expression plasmid was constructed and a several-fold increase in the yield of purified Tpc6was achieved(Figure3A).This result is in agreement with earlier studies that showed that the Bet3:Tpc6heterodimer is likely to represent the physiologically relevant form of both proteins(2).Mum2was included in the plasmid, andfinally a plasmid with all four subunits,including synbindin,was constructed.The yield of purified Mum2 was also increased upon co-expression.All four subunits of the complex were successfully co-expressed and theRestriction digest,T4DNA polymerasetreatmentSwaIPacIAmpRQT1term.λ t 0H i n d I I N B a ma t t BgenespQLinkHpQLinkGpQLinkNABPlasmid 1,PacI digest and T4DNA pol.treatmentPlasmid 2,SwaI digest and T4DNA pol.treatmentAnnealing productLINK1LINK2LINK1LINK2LINK1C+Figure 1.Map of the pQLink vectors and construction of co-expression plasmids.(A )Map of vector pQLinkH and genetic elements of all pQLink vectors.The LINK sequences are shown with lines indicating overhangs generated by restriction digest and T4DNA polymerase treatment.MCS ¼multiple cloning site,TEV ¼TEV protease cleavage site,term.¼transcription terminator.(B )Construction of a co-expression plasmid from two pQLink plasmids with two different cDNA inserts,labelled 1and 2.The resulting plasmid can accept additional inserts,labelled 3and 4.S ¼SwaI,P ¼PacI.(C )The LINK sequences and their digestion and annealing.The lines indicate overhangs generated by restriction digest and T4DNA polymerase treatment.Two plasmids,identified by upper and lower case nucleotide codes,are digested and annealed,leading to a product with a LINK1and a LINK20sequence,slightly larger than the original LINK2sequence.yield of purified protein was equal to or better than that for the expression of the individual proteins.We compared the expression achieved by our system to the pETDuet co-expression system.The plasmids pETDuet-1Bet3:Tpc6and pQLinkH Bet3:pQLinkN Tpc6both encode for His-tagged Bet3and untagged Tpc6protein.The expression clones showed good expres-sion of both proteins,with comparable protein levels in both systems (Figure 3B).Bet3from pQLinkH was more strongly expressed and migrated at a higher molecular weight because of the TEV cleavage site not present in pETDuet-1.With expression constructs containing up to four subunits in hand,we used the pQLink system to study the assembly and stoichiometry of TRAPP binations of Bet3:Tpc6,Bet3:Tpc6:Mum2and Bet3:Tpc6:Mum2:synbindin were expressed and affinity purified using nickel chelating beads.Samples were then analysed by gel filtration to isolate the protein subcom-plexes (Figure 4A).As previously described,Bet3and Tpc6form a heterodimer,which binds Mum2(2).The isolated Bet3:Tpc6:Mum2complex exhibited amolecular weight of $90kDa.A heterotrimer would have a molecular weight of only 65kDa,which suggests that the observed complex contains a fourth TRAPP polypeptide.It might result from binding of a Mum2dimer to a Bet3:Tpc6heterodimer,since Mum2alone forms homodimers (unpublished observation).Upon including synbindin in the co-expression system,a complex of similar size was formed,corresponding to a 85-kDa tetramer containing one molecule each of Bet3,Tpc6,Mum2and synbindin.This tetrameric complex could be isolated and crystallized (not shown).DISCUSSIONThe vectors described here allow generation of expression clones for His-tag and GST-tag fusion proteins and untagged proteins by restriction enzyme or Gateway recombination-based methods.The vectors are capable of expressing single proteins and can be converted into co-expression plasmids by a simple,PCR-free LIC reaction that is suitable for high-throughput projects.Any combination of pQLink plasmids ispossible.VCPAMFR (aa 450-510)MTEV protease25015010075503725201510kDaVCP WP WP WP W P WPGST-AMFR (aa 450-510)GST-AMFR (aa 498-643)kDa V C PV C P +A M F R (a a 450-510)V C P +A M F R (a a 455-643)V C P +A M F R (a a 498-643)V C P +G S TA BV C P +A M F R (a a 455-643)V C P +A M F R (a a 45-510)25375075GST-AMFR (aa 498-643)a n t i -G S T W e s t e r n b l o tC o o m a s s i e s t a i n i n g10075503725Figure 2.Co-expression of His-tagged VCP with GST-tagged AMFR fragments.SDS-PAGE and Coomassie staining of whole cells solubilized in SDS-PAGE sample buffer (W)and proteins purified under non-denaturating conditions (P)(A )Proteins were eluted from a glutathione agarose column by TEV protease treatment and analysed by SDS-PAGE and Coomassie staining.Degradation products of the larger AMFR construct were observed.W ¼whole cellular protein extract,P ¼purified proteins,M ¼molecular weight marker.(B )Protein products of co-expression clones of His-VCP with GST-AMFR and GST alone were purified with nickel chelating beads and separated by SDS-PAGE and visualized by Coomassie staining (upper panel)or anti-GST western blot (lower panel,reagents:anti-GST HRP conjugate,GE-Healthcare,and Western Lightning Western Blot Chemiluminescence Reagent,Perkin Elmer).The use of different tags or the combination of tagged and untagged complex subunits facilitates the production of homogenous complex preparations.The presence of an internal SwaI or PacI site in the gene of interest would prevent the use of the system.However,SwaI and PacI are rarely cutting enzymes with eight base pair recognition sites,and one is free to choose either PacI or SwaI for a given gene.Therefore we expect that almost all genes of interest can be converted into co-expression clones.The comparison of yields showed that this expression system works equally well as the pETDuet system.The specific interaction of a C-terminal region of AMFR with VCP was confirmed.Co-expression of the four TRAPP subunits Bet3,Tpc6,Mum2and synbindin was demonstrated.Figure 3shows that co-expression generally does not decrease the yield of individual proteins.On the contrary,all four proteins were obtained with similar or increased yield upon co-expression and affinity purification.Gel filtration revealed that the four TRAPP subunits form a heterotetramer containing onemolecule of each of the four TRAPP subunits.Bet3,Tpc6and Mum2without synbindin form a complex of similar size,which suggests that this complex is also a tetramer that might include two Mum2molecules.These findings1001502002501001502002500.00.20.40.60.81.0A d s o r b t i o n [a .u .]0.00.20.40.60.81.00.00.20.40.60.81.0A d s o r b t i o n [m l ]A d s or b t i o n [a .u .]158kDa void440kDa 67kDa 43kDa 14kDa25 kDa Bet3:Tpc6:Mum2Bet3:Tpc6:Mum2:synbindinBAB e t 3:T pc 6B e t 3:T p c 6:M u m 2B e t 3:T p c 6:M u m 2:s y n b i nd iBet3:Tpc620 kDa***M a r k erVolume [ml]Volume [ml]100150200250Volume [ml]Figure 4.Analysis of TRAPP subcomplexes.(A )Proteins were co-expressed,purified with nickel chelating beads and loadedon a calibratedgel filtration column.(B )SDS-PAGE and Coomassie staining of isolated subcomplexes (marked with an asterisk in A).M W P W P W P W P W P W P W P+−−−+++−+−−+++−−+−−++−−−+−−+Bet3Tpc6Mum2Synbindin75503725201510kDa0I0Ip E T D u e t -1B e t 3:T p c 6p Q L i n k H B e t 3:p Q L i n k N T p c 6AB6745292112.5kDa Figure 3.Co-expression of four subunits of the human TRAPP complex.(A )Protein expression products and nickel affinity chromato-graphy products of seven clones expressing different subunits were analysed by SDS-PAGE and Coomassie staining.W ¼whole cellular protein extract,P ¼purified proteins,M ¼molecular weight marker.(B )Expression test with pETDuet-1Bet3:Tpc6and pQLinkH Bet3:pQLinkN Tpc6in BL21(DE3).Total cellular protein was analysed before (0)and after (I)4-h induction.agree with a recent study that identified TRAPP subcomplexes consisting of Bet3:Tpc6:Mum2and Mum2:synbindin,respectively(20).In addition to the co-expressions documented here, we generated co-expression constructs for20other pairs of human proteins using the Gateway pQLink vectors. Cloning was robust and expression levels of individual and co-expressed proteins were similar in virtually all cases(data not shown).This further demonstrates the robustness of our method.In principle,there is no limit to the number of proteins that can be co-expressed,even though one should expect that the cloning efficiency decreases as the size of the plasmid constructs increases.It seems likely that our method can be transferred to expression host systems other than E.coli.For Baculovirus vectors, a co-expression system that also relies on duplication of expression cassettes including promoters and transcription terminators has been described(12).This demonstrates that duplication of promoters and terminators in a Baculovirus vector is unproblematic as has been shown by Alexandrov et al.and in our work with E.coli vectors. 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