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Impact of a Disruption of a Pathway Delivering Copper to Mitochondria on Podospora anserina

E UKARYOTIC C ELL,Feb.2004,p.200–211Vol.3,No.1 1535-9778/04/$08.00?0DOI:10.1128/EC.3.1.200–211.2004

Impact of a Disruption of a Pathway Delivering Copper to Mitochondria on Podospora anserina Metabolism and Life Span?

Stefan W.Stumpferl,Oliver Stephan,?and Heinz D.Osiewacz*

Johann Wolfgang Goethe Universita¨t,Botanisches Institut,60439Frankfurt,Germany

Received17July2003/Accepted27October2003

A global depletion of cellular copper as the result of a de?ciency in high-af?nity copper uptake was previously

shown to affect the phenotype and life span of the?lamentous fungus Podospora anserina.We report here the

construction of a strain in which the delivery of copper to complex IV of the mitochondrial respiratory chain

is affected.This strain,PaCox17::ble,is a PaCox17-null mutant that does not synthesize the molecular

chaperone targeting copper to cytochrome c oxidase subunit II.PaCox17::ble is characterized by a decreased

growth rate,a reduction in aerial hyphae formation,reduced female fertility,and a dramatic increase in life

span.The mutant respires via a cyanide-resistant alternative pathway,displays superoxide dismutase(SOD)

activity pro?les signi?cantly differing from those of the wild-type strain and is characterized by a stabilization

of the mitochondrial DNA.Collectively,the presented data de?ne individual components of a molecular

network effective in life span modulation and copper as an element with a dual effect.As a cofactor of complex

IV of the respiratory chain,it is indirectly involved in the generation of reactive oxygen species(ROS)and

thereby plays a life span-limiting role.In contrast,Cu/Zn SOD as a ROS-scavenging enzyme lowers molecular

damage and thus positively affects life span.Such considerations explain the reported differences in life span

of independent mutants and spread more light on the delicate tuning of the molecular network in?uencing

biological ageing.

1The ascomycete Podospora anserina is a?lamentous fungus characterized by a limited life span(54).After a strain-speci?c growth period,the growth of an individual culture ceases and ?nally the mycelium dies at the hyphal tips.Life span and ageing depend on environmental factors and genetic traits(for detailed reviews,see references8,22,44,and48).A variety of mutants characterized by different degrees of life span increase are available.Some of them are characterized by enormous differences in life span,even though the same molecular path-ways are affected.Examples are mutants in which the respira-tion is switched from a cytochrome c oxidase(COX)-depen-dent to an alternative pathway(14,19,25,37,39,46,57).The observed differences make it clear that a variety of different factors contribute to the complex molecular network modulat-ing life span.The details of the contribution of individual factors and their interactions are not known in any system. Previously,we described the characteristics of the nuclear long-lived mutant grisea,a strain with a loss-of-function mu-tation in a gene coding for the copper-regulated transcription factor GRISEA.As a consequence,the target genes,including a gene encoding the high-af?nity copper transporter PaCTR3, are not transcribed,and cellular copper levels are consequently low.Since copper is a cofactor of a variety of different enzymes (e.g.,COX,tyrosinase,and laccase),different pathways are affected(9–13,46).The contribution of every single pathway to the longevity phenotype is unclear.

In the present study,our aim was to address more carefully the effect of de?ned copper-dependent functions with respect to life span control.Speci?cally,we constructed a strain in which copper delivery to the COX was disrupted.Experimen-tally,this was possible via the generation of a transgenic strain in which a gene coding for the putative copper chaperone PaCOX17was replaced by a selectable marker gene.Here we present the data demonstrating an important dual effect of copper on longevity.

MATERIALS AND METHODS

Strains and growth conditions.The P.anserina wild-type strain s(21),the mitochondrial mutant strain ex1(57),the nuclear mutant strains grisea(49)and PaCox17::ble(both derived from strain s;the present study)were used through-out the present study(Table1).P.anserina strains were grown on standard cornmeal agar(BMM)at27°C in the light(21).For the construction of the cDNA library,strains were grown with additional33?M BCS(bathocu-proinedisulfonic acid)and1mM ascorbic acid to limit copper and to maximize the expression of genes that are repressed by this metal.For the preparation of RNA,native proteins,or mitochondria,cultures were subsequently grown in liquid complete medium(CM)for3or7days(14).To increase the copper concentration in the medium,?lter-sterilized copper sulfate was added to a?nal concentration of10?M or250?M Cu2?.BMM containing60mM ammonium acetate was used as germination medium for ascospores.

The Saccharomyces cerevisiae strains(W303,W303?COX17,W303?COX17-pAD4,and W303?COX17-PaCox17)used in the present study(Table1)were grown on yeast extract-peptone-dextrose(YPD)or YPG(YPD in which the dextrose is replaced by3%[vol/vol]glycerol)medium at30°C(59).The ability to grow on a nonfermentable carbon source was tested on YPG.All yeast mutants are in the genetic background of the strain W303?COX17(28),which was kindly provided by A.Tzagoloff(New York,N.Y.).

Preparation of a cDNA library and transformation of yeast.P.anserina wild-type strain s was grown on BMM and subsequently in liquid CM containing33?M BCS(a Cu?chelator)and1mM ascorbic acid(reduces Cu2?to Cu?).RNA was isolated as described previously(14).Synthesis of cDNA was performed by

*Corresponding author.Mailing address:Molekulare Entwicklungs-biologie und Biotechnologie,Botanisches Institut,Johann Wolfgang Goethe Universita¨t,Marie-Curie-Str.9,60439Frankfurt,Germany. Phone:49-69-798-29264.Fax:49-69-798-29363.E-mail:Osiewacz@em

.uni-frankfurt.de.

?Dedicated to K.Esser on the occasion of his80th birthday.

?Present address:Paul-Ehrlich-Institute,Paul-Ehrlich-Str.51-59, 63225Langen,Germany.

200 at Penn State Univ on February 12, 2008 https://www.sodocs.net/doc/7310282312.html, Downloaded from

using the Superscript II kit(Gibco).The cDNA fragments were ligated to Hin dIII and Xho I adapters and cloned in the yeast expression vector pAD4cut with Hin dIII and Sal I,whereby Sal I and Xho I generate compatible ends(pAD4 was kindly provided by S.M.Jazwinski,New Orleans,La.).The cDNA library was transformed into Escherichia coli MRF?cells(Stratagene)and grown on standard Luria-Bertani(LB)plates supplemented with50mg of ampicillin/liter (LBA).Colonies were washed from the surface of agar plates in a minimal volume of LBA.For the isolation of plasmids,2ml of suspension was used to in-oculate500ml of LBA liquid medium,and the culture was grown overnight at37°C. Yeast transformation was performed by using the lithium acetate method(56). To identify plasmids carrying a cDNA complementing the mutant strain W303?COX17,colonies were washed from the sorbitol-dextrose(Leu?)plates with sterile water and plated onto YPG plates.

Isolation and sequencing of PaCox17.The cDNA on plasmid pAD4-PaCox17 selected from a yeast clone complemented to respiration competence was se-quenced on both strands,starting from the ADH-promoter and from the ADH-terminator,by using oligonucleotides AD4(5?-TAA TCT TTT GTT TCC TCG-3?)and AD4-ter(5?-TTA GAA GTG TCA ACA ACG-3),respectively (accession number AJ578462).Further sequencing was performed by primer walking.The PaCox17-cDNA clone was used to screen a genomic cosmid library of wild-type strain s.An8-kbp subfragment(cut with Hin dIII and Xho I)con-taining the open reading frame(ORF)of PaCox17and?anking regions was cloned into pBluescript SK II(?)(Stratagene),resulting in plasmid pPaCox17g-1 (accession number AJ578463;Fig.3).

The sequence of both strands of all constructs was determined by automated sequencing.BLASTN and BLASTX were used to search the databases(2). The Tfsitescan program of the Institute for Transcriptional Informatics(IFTI [https://www.sodocs.net/doc/7310282312.html,/]),which has access to the object-oriented transcription fac-tor database ooTFD,was used to analyze putative transcription factor binding sites of the PaCox175?region(26).Sequence information provided by(3,18,27) was utilized to analyze the DNA sequence at the PaCox17locus.

Plasmid construction.Plasmid pPaCox17g-1containing a genomic fragment with the PaCox17ORF and?anking regions was utilized for the construction of the PaCox17gene replacement plasmid pBHP-9(Fig.1).First,the two?anking regions upstream of position?138and downstream of position?429of the PaCox17locus were ampli?ed with two sets of primers.In each PCR,one primer was either the universal(5?-GTA AAA CGA CGG CCA GT-3?)or the reverse (5?-GGA AAC AGC TAT GAC CAT G-3?)primer of pBluescript SK II(?),and the second primer was complementary to the sequence of the PaCox17locus upstream(CB[5?-TAT GGA TCC GAT ATG GGA TTG-3?)or downstream (CN[5?-CCC GCT AGC ATT GCC GCT GAG CCG-3?)of the PaCox17ORF (Fig.1).The latter primers were designed to result in the introduction of speci?c restriction sites.After digestion with the appropriate enzymes,these two frag-ments,the Kpn I/Nco I-digested pBSSK and a short Bam HI/Nhe I linker,were ligated.The resulting hybrid plasmid,pPa?Cox17,contains the?anking regions of the PaCox17ORF upstream of position?138and downstream of position ?429.In a series of subsequent subcloning experiments the bleocin resistance cassette(BRC)and the hygromycin B resistance gene were introduced into pPa?Cox17.In addition,the BRC cassette located on a Hin dIII/Bgl II fragment of pUT703(15)was used to replace the Bam HI/Nhe I linker fragment in pPa?Cox17.This cloning step led to plasmid pPaCox17::ble-hph(not shown). Subsequently,the hygromycin B resistance gene on a Hin dIII/Bgl II fragment of plasmid pAN7-1(51)was introduced,resulting in plasmid pBHP-9(Fig.1and

4A).In this plasmid the BRC is?anked by a continuous2.5-kbp5?and a

continuous1.6-kbp3?region of the genomic PaCox17locus.

Plasmid pSP17containing two copies of the?rst intron(pl-intron)of the

mitochondrial gene PaCoxI was used for the isolation of the plDNA probe(63).

The probe was labeled by using the digoxigenin(DIG)system. Production,regeneration,and transformation of spheroplasts.P.anserina strain s was grown3days on?ve BMM plates and subsequently for2days in a

total volume of1liter of CM.Then,30g of mycelium was washed with TPS(5

mM Na2HPO4?2H2O,45mM KH2PO4,0.8M sucrose),chopped in a Waring blender(twice for5s each time[slowly],twice for5s each time[quickly]),and

incubated at35°C for2h with20mg of?lter-sterilized Glucanex(Novo Nordisk

Ferment AG)/ml.Subsequently,the suspension was?rst?ltered through gauze

and then three to four times through glass wool.Spheroplasts were pelleted by

centrifugation for10min at4,000rpm.Subsequently,the pellet was washed with

TPS three times.

In order to regenerate the spheroplasts,the pellet was recovered in TPS and

different dilutions were plated onto plates containing regeneration agar(70mM

NH4Cl,2g of tryptone/liter,1g of Casamino Acids/liter,1g of yeast extract/liter,

50mM glucose,1M sucrose,550?M KH2PO4,335?M KCl,and110?M

MgSO4,and MnSO4,FeSO4,CuSO4,and ZnSO4[each at50?g/liter])with10?g of bleocin/ml.After7days of growth,mycelia of developing cultures were transferred to BMM.DNA transformation of spheroplasts of P.anserina strain s(mat?)was performed as previously described(47).

Northern analysis.RNA of strains s(wild type),grisea,and PaCox17::ble-37312was prepared,blotted,and washed as described previously(14).To con-?rm equal loading of RNA samples,blots were reprobed with the ribosomal DNA(rDNA)probe(plasmid pMY60[67],containing a Hin dIII fragment of the rDNA unit of Saccharomyces carlsbergensis).

Western blot analysis.Mitochondrial proteins(strains s,grisea,ex1,and PaCox17::ble-37312)were isolated as described previously(14).From each sam-ple,60?g of protein was loaded onto a sodium dodecyl sulfate-polyacrylamide gel and subsequently blotted onto nitrocellulose?lters.The alternative oxidase of P.anserina(PaAOX)was detected by using a monoclonal mouse AOX antibody,generated against the alternative oxidase of Sauromatum guttatum(20) and the alkaline-phosphatase labeled goat anti-mouse secondary antibody.To con?rm the equal loading of the mitochondrial protein probes,the blots were reprobed with an anti-?ATPase V rabbit monoclonal antibody(42).

Oxygen consumption measurements.Respiration of P.anserina strains s(wild type),grisea,and PaCox17::ble-37312was determined with a Clark-type elec-trode(Rank Bros.)as described previously(14).To distinguish between COX-and AOX-dependent respiration,potassium cyanide(KCN;2.2mM)and sali-cylhydroxamic acid(SHAM;4mM)were used as inhibitors.

SOD activity assay.Native proteins from wild-type strain s,mutants grisea, ex1,and PaCox17::ble-37312were isolated as described previously(14).To detect SOD activity,60?g of each protein sample were separated on a8.5% nondenaturing polyacrylamide gel.Puri?ed SOD1was used as a positive control. Gels were stained once in2g of nitroblue tetrazolium solution/liter and once in 2mg of ribo?avin solution/liter containing0.1%N,N,N?,N?-tetramethylethyl-enediamine(TEMED)for20min in the dark(24).Gels were then developed on a light box(neon light)and scanned with a Hewlett-Packard Scanjet4470c.

TABLE1.Strains of P.anserina and S.cerevisiae used in this study

Strain Genotype Source or reference

s21

grisea grisea49

ex1ex134

T28heterokaryotic contining wild-type and PaCox17::ble nuclei This study

PaCox17::ble-12PaCox17::ble This study a

PaCox17::ble-37312PaCox17::ble This study b

PaCox17::ble-47690PaCox17::ble This study b

PaCox17::ble-47692PaCox17::ble This study b

W303ade2-1his3-1,15leu2-3,112trp1-1ura3-1–c

W303?COX17ade2-1his3-1,15leu2-3,112trp1-1ura3-1?cox17::TRP128

W303?COX17-pAD4ade2-1his3-1,15leu2-3,112trp1-1ura3-1?cox17::TRP1pAD4This study

W303?COX17-PaCox17ade2-1his3-1,15leu2-3,112trp1-1ura3-1?Cox17::TRP1PaCox17This study

a Derived from a homokaryotic spheroplast of strain T28,which bears the PaCox17::ble mutation.

b Derived from the progeny(monokaryoti

c ascospore)of a cross between PaCox17::ble-12(mat?)an

d wild-typ

e strain s(mat?).

c Rodney Rothstein,Department of Human Genetics,Columbia University.

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Mitochondrial DNA (mtDNA)analysis.Total DNA of wild-type strain s and

PaCox17::ble-47690and -47692strain mycelia of different ages was isolated as described elsewhere (35).Digestion with restriction enzymes,gel electrophore-sis,blotting onto a nitrocellulose membrane,and hybridization with a DIG-labeled pl-intron probe (plasmid pSP17)were performed according to standard protocols (10,55,63).Life span measurements.Mononucleate ascospores were isolated from inde-

pendent crosses between wild-type strain s and the long-lived mutant PaCox17::ble (isolate 37312).After germination for 3days,the central piece of the culture was transferred to a race-tube containing BMM agar.Tubes were incubated at 27°C in the light.Growth was measured every day until the cultures reached a point where they stopped growing.The time elapsed to this point was taken as

the life span of an individual culture.Accession numbers.The sequences of the cDNA and of the genomic copy of PaCox17were submitted to the EMBL data bank.The accession numbers are AJ578462and AJ578463,respectively.RESULTS Cloning of PaCox17,a gene coding for an ortholog of the COX17copper chaperone of yeast.Previously,we reported that lowering the cellular copper levels affects the life span and ageing of P.anserina cultures signi ?cantly.This conclusion was derived from the characterization of the long-lived grisea mu-tant.The mutant is defective in high-af ?nity copper uptake.As a result,all cellular compartments are depleted of copper and various activities that depend on this metal are impaired (9,10,12,14,46).In order to de ?ne the functions that affect life span more precisely,we set out to construct a transgenic strain in

which copper depletion is restricted to mitochondria.More speci ?cally,our goal was to disrupt the pathway delivering copper to complex IV in the mitochondrial respiratory chain.To do this,we cloned PaCox17,a gene coding for a molecular chaperone targeting copper to subunit II of the COX.PaCox17was isolated by heterologous complementation of the Cox17-de ?cient yeast strain W303?COX17(28)with a yeast expres-sion cDNA library constructed from RNA of the P.anserina wild-type strain s grown on copper-depleted medium.Under

these conditions maximal expression of PaCox17was expected to occur.The cDNA library was used to transform the yeast strain W303?COX17,a strain unable to respire and thus not

growing on medium containing a nonfermentable carbon

source such as glycerol (YPG)as sole carbon source.Success-

ful complementation by a transforming plasmid led to yeast

cells able to grow on YPG.From such transformants plasmids were isolated.One plasmid,later termed pAD4-PaCox17,con-taining a 0.4-kbp insert was further veri ?ed.Serial dilutions of the recipient strain transformed with this plasmid were plated onto YPD and in parallel onto YPG (Fig.2).As controls,dilutions of the respiratory competent wild-type strain,the Cox17de ?ciency recipient strain,and the same strain trans-formed with the yeast expression vector pAD4containing no insert DNA were inoculated onto the same media.

Clearly,FIG.

Construction of knockout plasmid pBHP-9.In the ?rst step,parts of the plasmid upstream and downstream of the PaCox17ORF were

ampli ?ed by PCR and ligated into pBSSK in the presence of a short Bam HI/Nhe I linker,resulting in plasmid pPa ?Cox17.The genomic sequence

located upstream of the PaCox17ORF which turned out to be discontinuous in pPa ?Cox17was replaced by a continuous genomic fragment.The Bam HI/Nhe I linker was replaced by a ble resistance cassette,and then a hygromycin B resistance cassette (hph )was integrated in the bacterial backbone.The resulting plasmid is pBHP-9.202STUMPFERL ET AL.E UKARYOT .C ELL at Penn State Univ on February 12, 2008

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plasmid pAD4-PaCox17was found to reproducibly comple-ment the de ?ciency of strain W303?COX17to grow on YPG.Subsequently,the insert fragment of pAD4-PaCox17was sequenced and analyzed.This fragment contains an ORF (Fig.3A)able to code for a protein of 80amino acids with a signif-icant homology to the COX17copper chaperone from differ-ent organisms.Importantly,the four cysteine residues that are essential for the function of COX17are conserved in the dif-ferent proteins (Fig.3B)(32).In the genomic sequence of PaCox17,which was isolated from a cosmid library of the P.anserina wild-type strain s by using the cDNA as a probe,two short introns were found to interrupt the PaCox17ORF.In the 5?upstream region a pu-tative CCAAT (?113)and CAAT box (?145)(66),and puta-tive binding sites for different regulatory proteins such as GRISEA (?98),NRF-2(?51and ?182),MIG1(?214),and BRL A (?258,?404,?950,and ?1001)(1,16,38,68)were localized.Downstream of the PaCox17stop codon a putative polyadenylation site AATAGA (consensus,AATAAA)was identi ?ed in a region with an increased AT content (3).These data suggest that both regions are involved in controlling the expression of the PaCox17ORF.Finally,hybridization experiments revealed that PaCox17is a single-copy gene (data not shown)corresponding to the sit-uation in yeast and mice (28,65).In contrast,two Cox17copies are found in other organisms,such as Arabidopsis thaliana ,and in humans (52,69).Construction of a PaCox17deletion strain.In order to gen-erate a PaCox17-de ?cient strain,plasmid pBHP-9(Fig.1and 4A)was constructed in which the sequence from ?138to ?429(containing the PaCox17ORF)is replaced by the bleocin re-sistance cassette (BRC)of pUT703(15).A 2.5-kbp region upstream and a 1.6-kbp region downstream of the PaCox17ORF are retained in pBHP-9.These two regions allow cross over to occur on both sides of the BRC,a prerequisite for the exchange of the chromosomal PaCox17copy by the selectable BRC of pBHP-9(Fig.4A).In order to discriminate between transformants arising from a double cross over and from the integration of the complete transforming plasmid via a single crossing over,pBHP-9carries a second resistance marker (hy-gromycin B).Transformants of interest in which the PaCox17gene is exchanged by the BRC should be resistant to bleocin but sensitive to hygromycin B.In order to select such trans-formants,we transformed spheroplasts of P.anserina wild-type strain s with pBHP-9and isolated 649independent bleocin-resistant primary transformants.Only 93of them were sensi-tive to hygromycin B.Unfortunately,a Southern blot analysis revealed that none of the transformants was homokaryotic,containing exclusively nuclei with the deleted PaCox17gene.However,among the selected transformants one transformant (T28)was identi ?ed as displaying a hybridization pattern char-acteristic for a heterokaryon containing predominantly nuclei with the PaCox17wild-type gene but also a few nuclei with the PaCox17::ble mutation (Fig.4B).This strain was chosen to select a genetically homogenous PaCox17::ble strain.In a ?rst series of experiments,T28was crossed with wild-type strain s and 22monokaryotic ascospores were isolated and germinat-ed.Unfortunately,no homokaryotic PaCox17-de ?cient trans-genic strain could be selected.Therefore,as an alternative approach,mycelium of strain T28was treated with the cell wall-degrading enzyme mixture Glucanex,and spheroplasts were subsequently selected on bleocin-containing media.Twelve transformants were veri ?ed by Southern blot analysis.Al-though the two Pvu II fragments of 1.0and 2.6kbp containing the PaCox17wild-type gene copy do not appear in all of these transformants (two are shown in Fig.4B),a novel 2.8-kbp fragment hybridizes that results from the replacement of the PaCox17ORF through the BRC of pBHP-9.These hybridiza-tion data clearly show that the selected strains are homokary-otic for the PaCox17::ble locus.In order to further purify the PaCox17::ble strain,one of the selected primary transformants (PaCox17::ble-12)was crossed with wild-type strain s (mat ?),and 42monokaryotic ascospores were isolated.Three of these secondary trans-formants,PaCox17::ble-37312,-47690,and -47692,were used in subsequent experiments.PaCox17::ble-37312was crossed with strain s,and 52tetrads were isolated and analyzed.The germination rate of isolated ascospores from these tetrads

that FIG.

https://www.sodocs.net/doc/7310282312.html,plementation of the yeast mutant strain W303?COX17by pAD4carrying the P.anserina cDNA of PaCox17.The yeast strains are as follows (both plates):?rst line,wild-type W303;second line,respiratory-de ?cient Cox17mutant strain W303?COX17;third line,mutant W303?COX17transformed with empty pAD4;fourth line,mutant W303?COX17transformed with PaCox17cDNA cloned in yeast expression plasmid pAD4.Strains were grown to an optical density at 600nm of 1.0.Serial 10-fold dilutions were prepared,and 5?l was spotted onto YPD or YPG plates.These strains were grown for 2days (30°C)in YPG (right plate)or in YPD (left plate)medium.V OL .3,2004COPPER DELIVERY TO MITOCHONDRIA OF PODOSPORA ANSERINA 203 at Penn State Univ on February 12, 2008

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FIG.

3.(A)Sequence of the PaCox17locus and of the derived amino acid sequence.The coding sequence is shown in capital letters.Two introns (58and 71bp,respectively)disrupt the ORF.The 5?splice sites GTATGT and GTCCGT resemble the consensus (GTANGT)for

?lamentous

fungi,and the

two 3?splice sites ATAG match with the consensus (A/C)(C/T)AG.A putative polyadenylation site AATAGA (consensus,AATAAA)is found at position 495(4).Protein-binding sites are described in the text.PaCox17codes for a copper chaperone of 80

amino acids.The sequence of the cDNA is underlined.

Putative

promoter

binding

sites are shown in boldface,with the name of the binding protein

below.(B)Amino acid comparison between PaCOX17and COX17from other species (Neurospora crassa ;S.cerevisiae ,accession number

NP ?013092;Mus musculus ,accession number BAB32486;Chlamydomonas reinhardtii ,accession number AAF82382;and Arabidopsis thaliana ,

accession numbers AAK73496and AAK73497).Identical amino acids are shown by inverted colors.In the ?rst lane,positions which were recently shown to be critical for restoring the respiratory competence of a Cox17-null mutant are indicated by “#”(53).The four essential cysteinyl residues are marked by asterisks.204STUMPFERL ET AL.E UKARYOT .C ELL at Penn State Univ on February 12, 2008

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were derived from several crosses was between 90and 93%.In these experiments,it turned out that ascospores giving rise to colonies with the mutant phenotype germinated only very poorly on germination medium (BMM containing 60mM am-monium acetate),and the few developing hyphae stopped growing after about 1to 5mm of growth.However,it was possible to rescue these cultures by the transfer of hyphae to standard medium (BMM)without ammonium acetate.On this medium,the corresponding colonies are characterized by a strong reduction in the formation of aerial hyphae,a retarded pigmentation,strongly retarded fertility,and a growth rate (3.9?0.2mm/day)signi ?cantly lower than that of the wild-type strain s (6.5?0.5mm/day)(Fig.4C).Perithecia of the mutant are formed after about 3to 5weeks in comparison to about

2FIG.4.(A)Plasmid pBHP-9containing

the PaCox17locus in which the PaCox17ORF is replaced by a bleocin resistance cassette (ble R ).Further,the backbone of pBHP-9contains a hygromycin resistance cassette (hph R )as a second marker.Crossing over on both sides of the genomic PaCox17ORF

lead to its

replacement by the bleocin resistance cassette of plasmid pBHP-9,resulting in the formation of transgenic strain

PaCox17::ble.Distances in the schematic drawings are not drawn to scale.(B)Southern analysis of wild-type strain s and mutant strains PaCox17::

ble-12(PaCox17::ble,lane 1)and PaCox17::ble-37312(PaCox17::ble,lane 2).Isolated genomic DNA

was

cut with Pvu

II (positions

shown in panel

A)and

hybridized to the DIG-labeled PaCox17S1*S2probe,which encompasses the second half of the PaCox17-ORF and a 122-bp 3?region (black bars in panel A).The ?rst two lanes contain DNA of knockout plasmid pBHP-9and DNA from wild-type strain s ?(monokaryotic)as

controls;the

third lane

contains

DNA of the

heterokaryotic primary transformant T28.The fourth and ?fth lanes contain DNA from the two

examples of progeny (mutants PaCox17::ble)derived from monokaryotic ascospores of the primary transformant and wild-type strain s,respec-

tively.DIG-labeled DNA of phage lambda was cut with Hin dIII and used as length standard (sixth lane at the right).(C)Phenotype of wild-type

and mutant PaCox17::ble.Wild-type strain s is shown on the left;the mutant PaCox17::ble-37312strain is shown on the right side on one agar plate.In contrast

to the wild type,the mutant shows slower growth and mycelia lacking aerial hyphae.(D)Northern analysis of wild-type strain s (lane

1),mutant PaCox17::ble-37312(lane 2),and mutant grisea (lane 3).An rDNA probe (plasmid pMY60[67]containing a Hin dIII fragment of the

rDNA unit of S.carlsbergensis )and an ethidium bromide-stained gel were used as loading controls.V OL .3,2004COPPER DELIVERY TO MITOCHONDRIA OF PODOSPORA ANSERINA 205 at Penn State Univ on February 12, 2008

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weeks in wild-type strains.In addition,they are only about two-thirds of the size of the wild-type strain and contain only few asci.A tetrad analysis revealed that the mutant phenotype (slow-growth,reduced aerial hyphae)was strictly linked to bleocin resistance.In addition,both phenotypes show a post-reduction frequency(second strand division)of79%.This is good initial evidence for a location of the bleocin gene at the chromosomal position at which PaCox17is located in the wild-type genome.

The expression levels of PaCox17in wild-type strain s,mu-tant PaCox17::ble,and mutant grisea were subsequently ana-lyzed in Northern blot experiments.Although the grisea mu-tant displays lower PaCox17transcript levels than the wild type,no transcript of this gene was detectable in mutant PaCox17::ble(Fig.4D).

Finally,in order to experimentally demonstrate that the phenotype of PaCox17::ble-12is the result of the deletion of the PaCox17ORF and not due to other affected genes,trans-genic strain PaCox17::ble-12was transformed with plasmid pPaCox17g-2.This plasmid consists of pUC18and a genomic fragment containing the wild-type copy PaCox17,including 2.5kbp upstream and0.3kbp downstream of the ORF. Hygromycin B-resistant transformants were selected after cotransformation of PaCox17::ble-12with pAN7-1(51)and pPaCox17g-2.Southern blot analysis revealed that of34hy-

gromycin B-resistant primary transformants,11had integrated pPaCox17g-2at ectopic positions in the genome.These strains all displayed the wild-type phenotype(data not shown). Collectively,the data from the molecular and the genetic analysis are all consistent and demonstrate the successful ex-change of the PaCox17gene by the selectable bleocin marker gene,resulting in a PaCox17-null mutant strain.The mutant phenotype of this strain is the result of the deletion of the PaCox17ORF.

Induction of PaAOX in the PaCox17::ble transgenic strain. Since PaCox17is not expressed in the PaCox17::ble transgenic strain,delivery of copper to the COX subunit II and conse-quently the assembly of complex IV of the respiratory chain were expected to be affected(6,28,33,53,60).Previous in-vestigation revealed that in COX de?ciency strains an alter-native cyanide-resistant pathway is induced(12,14,19,57). Consequently,we addressed this possibility.A Western blot analysis revealed high levels of PaAOX in the transgenic PaCox17::ble strain.PaAOX levels are higher than in the copper uptake mutant grisea and reach almost those levels found in the immortal ex1mutant.Moreover,increasing copper in the growth medium lowers PaAOX levels(Fig.5). This is in agreement with previous results demonstrating a copper-dependent reduction of transcription of PaAox(14). Consistent results were obtained in oxygen consumption ex-periments.In these experiments mycelial pellets from liquid cultures were incubated in a buffer saturated with oxygen and subsequently the consumption of oxygen was determined.The addition of KCN or SHAM allowed a discrimination between a PaCOX and a PaAOX-dependent respiration.The PaCox17:: ble strain was clearly found to respire via a SHAM-sensitive and KCN-resistant alternative pathway.In contrast to the cop-per uptake mutant grisea,the molecular phenotype cannot be reverted by the addition of10?M CuSO4(Fig.6).Reversion was also not observed when cultures were grown in medium containing250?M CuSO4(data not shown).Finally,it is re-markable that oxygen uptake by the PaCox17::ble strain is signi?cantly higher than in the wild-type strain or in the grisea mutant.It may be speculated that,for yet unknown reasons, the uptake of oxygen by the mycelium of the Cox17de?ciency strain is enhanced.Thus,at the physiological level,mutants grisea and PaCox17::ble can clearly be differentiated corre-sponding to the fact that in both mutants different steps of the copper metabolism are impaired.

Cu/Zn SOD activities document the availability of copper in the cytoplasm of the PaCox17::ble strains.The aim of the present study was to construct a P.anserina strain in which copper depletion is restricted to the mitochondrial respiratory chain and not,as in mutant grisea,global in all cellular com-partments.In the PaCox17::ble strain depletion of copper at the mitochondrial respiratory chain is suggested by the ob-served COX de?ciency and the induction of the alternative respiratory pathway.Cytoplasmic copper levels were subse-quently investigated indirectly via the determination of super-oxide dismutase(SOD)activity.The activity of the copper depending SOD1located in the cytoplasm and the mitochon-drial intermembrane space depends on the availability of cop-per as a cofactor.Previously,we demonstrated that,due to cellular copper depletion,the copper uptake mutant grisea lacks a functional PaSOD1.This de?cit can be overcome via the addition of extracellular copper(14).SOD activity analysis revealed that,in clear contrast to the wild-type strain s, PaSOD1activities in PaCox17::ble are already high in juvenile cultures and remain at this level also in older age.In contrast to mutant grisea,in which the SOD1activity can be induced by the addition of extracellular copper,such an induction is not or only very poorly observed in strain PaCox17::ble.Finally,in contrast to the juvenile wild-type strain,no PaSOD2activity is detectable in juvenile cultures of PaCox17::ble strain(Fig.

7A). FIG.5.Western blot analysis of AOX proteins in PaCox17::ble strains grown in rich medium or rich medium with250?M copper sulfate.The juvenile wild-type(WT)strains,mutant ex1,mutant grisea on rich medium,or on mutant grisea on medium containing250?M additional copper serve as controls.Mitochondria were isolated,sep-arated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and blotted onto nitrocellulose.PaAOX was detected with monoclonal mouse antibodies against the AOX of Sauromatum guttatum strain Schott(20).An antibody against mitochondrial ATPase subunit?was used as loading control.

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These data indicate that cytoplasmic copper levels are rather high in cultures of the new transgenic strain from juvenile to older age.This possibility was experimentally addressed by Northern blot analysis.As shown in Fig.7B,transcripts of PaCtr3encoding the high-af ?nity copper transporter of P.an-serina are detected in RNA preparations of the wild-type strain s but not in mutant grisea and the transgenic strain PaCox17::ble.In mutant grisea the failure to transcribe PaCtr3is the result of a null mutation in the gene encoding transcription factor GRISEA (10).In contrast,in transgenic strain PaCox17::ble increases in cytoplasmic copper levels seem to repress the transactivation activity of GRISEA and consequently the tran-scription of PaCtr3.The mtDNA is stabilized in the PaCox17::ble strain.A hall-mark of ageing of wild-type cultures of P.anserina is the ex-tensive reorganization of the mtDNA.The most prominent age-related rearrangements occur in the region of the ?rst PaCoxI intron,giving rise to the age-dependent ampli ?

cation FIG.

6.Oxygen uptake in the wild-type strain s,mutant grisea,and the transgenic PaCox17::ble strain.The type of respiration was discriminated by the addition of respiratory inhibitors (either 1mM KCN or 4mM SHAM).PaCox17::ble strains were compared to wild-type strain s and mutant grisea.Strains were grown in rich medium or in rich medium with an additional 10?M copper sulfate.These experiments were each carried out at least ?ve times with two or more independent

isolates.FIG.

7.Activity of SODs and expression level of PaMt1.(A)Native proteins were isolated from PaCox17::ble strains that were juvenile (7days)and middle aged (m.a.;36days),grown with or without 250?M copper sulfate;wild-type strain s (juvenile [7days]or senescent [36days]);and

mutant ex1(ex)

and mutant grisea (with or without 250?M copper sulfate)as indicated.Subsequently,the proteins were separated by native

polyacrylamide gel electrophoresis (see Materials and Methods).(B)Northern

analysis

of mutant

grisea,wild-type strain s,and mutant PaCox17::ble-37312.PaCtr3was used as probe;therefore,plasmid pAD4-PaCtr3(12)was radioactively labeled.An rDNA probe (plasmid pMY60[67]containing a Hin dIII fragment of the rDNA unit of S.carlsbergensis )was used as a loading control.V OL .3,2004COPPER DELIVERY TO MITOCHONDRIA OF PODOSPORA ANSERINA 207 at Penn State Univ on February 12, 2008

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of the so-called plDNA (17,45,61,62).This circular plasmid becomes ampli ?ed during ageing of the wild-type strain s on both standard and copper-supplemented growth media (Fig.8B).In the long-lived copper-uptake mutant grisea grown on standard medium the mtDNA is stabilized.On medium sup-plemented with 250?M CuSO 4copper grisea displays the wild-type speci ?c instabilities (10).In sharp contrast,in the PaCox17::ble mutant the wild-type speci ?c age-related mtDNA reorganizations are neither observed in cultures grown in stan-dard growth medium nor in medium supplemented with 250?M CuSO 4.Under both conditions the mtDNA is stabilized.PaCox17::ble displays a long-life phenotype.Finally,we ad-dressed the question whether the life span is affected in the newly generated PaCox17::ble strain.Initial measurements with four of the primary transformants indeed suggested a signi ?cant life span increase.These strains are now still grow-ing after more than 1year.In order to raise statistical relevant life span data,we gene-rated a larger number of independent cultures of mutant PaCox17::ble.These are derived from monokaryotic ascospores of a cross of the secondary transformant PaCox17::ble (strain 37312)mat ?with wild-type strain s (strain 690766)mat ?.Sixty cul-tures (mat ?[n ?32];mat ?[n ?28])with the mutant pheno-type were selected,and life span measurements were per-formed in race tubes.Until now,after more than 300days of cultivation on BMM,only 20cultures expressed a senescent phe-notype and stopped growing (Fig.9).The remaining 40cultures are still growing and do not display any symptoms of senescence.DISCUSSION

In the ?lamentous ascomycete P.anserina a strong link be-tween the type of respiration and life span has been demon-strated repeatedly.A switch from the standard COX-depen-dent respiration to an alternative respiration is accompanied by an increase in life span (14,19,37,57).However,different COX-impaired mutants are characterized by extreme life span differences (e.g.,60%in the grisea mutant to immortality in the ex1mutant),indicating that a variety of factors,in addition to the type of respiration,contribute to life span control.The systematic construction of speci ?c transgenic strains and the comparative characterization of these strains in a more holistic sense is an important approach to de ?ne the relevant traits and their interactions.

Using this strategy,we focused on the relevance of cellular copper,which is essential for a COX-dependent respiration since copper is a cofactor of COX.The starting point for this analysis were previous data derived from the characterization of the long-lived grisea mutant (12,13,46).In comparison to other COX de ?ciency mutants,the grisea mutant is character-ized by a rather moderate life span increase of ?60%.In the present study,our speci ?c interest was to de ?ne the molecular traits responsible for these differences and to investigate more thoroughly the impact of copper on ageing processes.Toward this goal,we cloned and characterized PaCox17and demon-strated that it encodes an ortholog of the COX17copper chap-erone of yeast.COX17recruits copper probably in the

cyto-FIG.8.Southern

blot

analysis

the mitochondrial

PaCoxI region in two wild-type strains (B)and in two PaCox17::ble strains (C).(A)Bgl II

restriction map of the PaCoxI and PaCyt b region of the mtDNA.The position of the hybridizing pl-intron and Bgl II fragments 17and 5are

indicated.(B and C)Strains were grown on complex medium (left half of panels B and C)or with an additional 250?M CuSO 4(right half of panels

and C).

DNA samples

were

digested

with

Bgl

II,separated

an 1%

agarose

gel,blotted,and hybridized to a plDNA speci ?c probe.Signals corresponding to the mtDNA fragments 17(1.9kbp)and 5(4.5kbp)and the ampli ?ed plDNA are indicated by arrows.(B)DNA of two wild-type strains (7,14,21,

and 28days and senescent [ca.36days])was used.(C)DNA of two independent PaCox17-null mutants was used (aged 7,21,96,157,and 300days).208STUMPFERL ET AL.E UKARYOT .C ELL at Penn State Univ on February 12, 2008

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plasm or directly from a copper importer by binding copper in a oligomeric polycopper complex.It is thought that COX17delivers copper to SCO1,which is anchored in the inner mi-tochondrial membrane and contains a copper-binding site pro-truding in the mitochondrial intermembrane space.Subse-quently,SCO1passes copper to subunit II of COX located in the inner mitochondrial membrane (7,29,33,43,53,58).Cloning of PaCox17via complementation of a yeast Cox17-null mutant was possible because the amino acid sequence of the Podospora protein shares identical amino acids at positions that,in a recent mutagenesis analysis,were demonstrated to be important to restore wild-type respiration in a yeast Cox17-null mutant (53).In this study 11mutations leading to a single amino acid exchange each at eight different positions were found to result in partial or complete failure to complement the Cox17-null mutant,demonstrating that these amino acids are of critical functional importance.Three amino acids were already known from an independent analysis to be of func-tional signi ?cance (32).Interestingly,in the P.anserina protein at all eight positions the same amino acid sequence was found as in the yeast protein,although only 28of the 80PaCOX17amino acid residues were conserved in the protein of the two fungi.In P.anserina ,as in yeast,the deletion of Cox17was found to be nonlethal.However,yeast and P.anserina strains survive for different reasons.Yeast Cox17mutants are able to grow as “petite ”colonies without the generation of ATP via respira-tion.In contrast,in the P.anserina Cox17-null mutant,as in the grisea mutant,an alternative respiratory pathway relying on the activity of the copper-independent AOX is induced.Where-as respiration and all other mutant characteristics of the grisea mutant can be reverted by growing this copper uptake mutant in medium containing increased amounts of copper,such a reversion is not possible in the PaCox17::ble strain.The two strains affected in copper metabolism are thus clearly distin-guishable from each other.The Cox17-null phenotype of yeast mutant strains can be reverted by growth in medium containing increased amounts of copper.Reversion is dependent on ScoI ,a gene encoding a mitochondrial membrane protein accepting copper from COX17and delivering it to COXII (28,29).Although P.an-serina also contains a nuclear gene coding for a putative SCO1homolog (unpublished data),reversion of the PaCox17-null phenotype is not possible by simply growing the mutant in medium with increased amounts of copper.The reason for this is yet unknown.In contrast to the two fungal systems,in mice the deletion of Cox17is lethal.During development of the homozygous Cox17knockout strains died early during embry-ogenesis around the time of gastrulation (64).The lethality of Cox17deletion in mice appears to be a consequence of the fact that mammals cannot meet their ATP demands by glycolysis and are not able to express an alternative respiratory chain.They rely on the generation of ATP via a COX-dependent respiration.These data demonstrate clearly that the molecular machinery involved in copper homeostasis,although conserved among species in many aspects,differs in details between spe-cies.Impairments of these machineries,depending on different characteristics of the individual system (e.g.,ability to induce an alternative respiratory pathway or not),consequently lead to different phenotypes.One of the most striking results obtained in the presented study is a clear correlation of the PaAOX levels in the different analyzed strains and the degree of life span increase.Impor-tantly,in the PaCox17::ble strain,signi ?cant higher amounts of AOX are found than in the grisea mutant,reaching almost the level of the immortal ex1mutant.However,it should be stressed at this point that the induction of the AOX in these strains is the result of impairments in the

COX-dependent FIG.9.Life span determination of transgenic strain PaCox17::ble.A total of 60cultures derived from monokaryotic ascospores were grown on BMM agar in race tubes at 27°C in the light.After 320days,40cultures were still alive,displaying no symptoms of aging.V OL .3,2004COPPER DELIVERY TO MITOCHONDRIA OF PODOSPORA ANSERINA 209 at Penn State Univ on February 12, 2008

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pathway and appears to be well regulated via yet-unknown mechanisms.The forced transgenic overexpression of the AOX in the wild-type background was found to have no impact on phenotypic characteristics.Even more,in the long-lived cox5::ble mutant background which,like the long-lived mutants investigated in the present study,respires via an alternative pathway,an increase of AOX levels above those normally found in this mutant leads to a reversion of the mutant phe-notype(37).These data show that life span control depends on a delicate?ne-tuning of the different types of respiration,as it naturally occurs in P.anserina.Modifying just one single com-ponent of this molecular network without adjusting other com-ponents may not lead to the desirable long-life phenotype. From plants and P.anserina it is known that the respiration via the alternative pathway leads to a lower generation of reactive oxygen species(ROS)than in a COX-dependent res-piration(19,40).The increase in life span observed in the various COX de?ciency mutants is in good agreement with the “mitochondrial free radical theory of ageing,”which postulates that ageing of biological systems is the result of a time-depen-dent accumulation of molecular damage,resulting from ROS mainly produced at the respiratory chain(reviewed in refer-ences5,23,30,31,and41).In this context the level of ROS is of great signi?cance.Lowering ROS levels can be achieved by interfering with the processes leading to their production(e.g., a switch from a COX-dependent pathway to an alternative pathway)or into the cellular ROS scavenging system.SODs are part of the latter system.In P.anserina we previously investigated two SODs,the Cu/Zn SOD(PaSOD1)and the MnSOD(PaSOD2)(12–14).In the wild-type strain s,PaSod1 was found to be constitutively expressed,but the activity of the encoded enzyme,which depends on the availability of copper, was found to increase during senescence.In accordance with other data,we concluded that during ageing the apoprotein is activated due to an increase of cytoplasmic copper level(12, 36,50).In the long-lived copper uptake mutant grisea,no PaSOD1activity was measured during the whole life time since copper is depleted in all cellular compartments.In addition,in this mutant the PaSod2gene encoding the mitochondrial Mn-SOD is not transcribed due to the absence of the GRISEA transcription factor.In contrast,in the immortal mutant ex1 PaSOD2activity and a very strong PaSOD1activity were de-tected.In the present study,we report that the copper delivery mutant PaCox17::ble resembles the SOD activity pro?les of the ex1mutant(Fig.7A).PaSOD1activity was demonstrated to be rather high,although lower than in ex1,throughout the whole life span.These data and the results from the transcrip-tion analysis failing to detect transcripts of PaCtr3clearly show that copper levels are increased in the cytoplasm of strain PaCox17::ble and that this mutant is well protected against cytoplasmic ROS and ROS generated in the mitochondrial intermembrane space.

Finally,a stabilization of the mtDNA was observed in the PaCox17::ble strain even after a very long period of growth.In contrast to mutant grisea,however,this holds true in strains grown on medium supplemented with copper salts.If in the transgenic strain the delivery of copper to the respiratory chain is speci?cally affected,this result clearly points to an indirect effect of copper on the mtDNA metabolism since in this case copper levels in the mitochondrial matrix should be normal.In contrast to the wild-type strain in which the mtDNA is ef?-ciently rearranged,the mtDNA of mutant PaCox17::ble re-mains stable.This allows the expression of mtDNA encoded genes also in older cultures of the mutant and the remodeling of damaged mitochondrial proteins(e.g.,proteins of the respi-ratory chain)and contributes to maintenance of functional mitochondria and to increased life span.

Collectively,the results of the present study demonstrate that very speci?c genetic modi?cations,as generated in the copper delivery mutant PaCox17::ble,may lead to multiple consequences.In the present study,the dual role of copper on life span control became obvious.On the one hand,copper indirectly leads to the age-related increase of mitochondrial oxidative stress and thus is active in limiting life span.On the other hand,as a cofactor of SOD1,it is part of a system lowering oxidative stress and as such life span increasing.This demonstrates that successful interference with the ageing pro-cess needs to be well designed.Transgenic strain PaCox17::ble is an example of an engineered strain with superior character-istics.However,still this strain has undesired attributes.Most importantly,due to the reduced ATP generation via the alter-native pathway,PaCox17::ble is growing slower than the wild type and is affected in fertility.It will be interesting to see whether the observed impact on growth and life span can be dissected by speci?c manipulations.

ACKNOWLEDGMENTS

We thank A.Tzagoloff(New York,N.Y.)for yeast strains,T.Elthon (Lincoln,Nebr.)for monoclonal AOX antibody,B.Ludwig(Frankfurt, Germany)for the?ATPase V antibody(42),and S.M.Jazwinski(New Orleans,La.)for plasmid pAD4.The excellent technical assistance of A.Werner(Frankfurt)is gratefully acknowledged.

This study was supported by a grant of the Deutsche Forschungs-gemeinschaft(Bonn,Germany)to H.D.O.

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最全商业地产专业术语及解释

商业地产是一个综合的领域，涉及商业、经济、建筑、设计等多方面的知识。刚入门的小白常常会被一些专业术语弄得一头雾水。小编在这里整理了一份商业地产相关术语及解释，助你快速掌握该领域，给你最全面的指引。商业地产概念商业地产: 作为商业用途的地产，用于各种零售、批发、餐饮、娱乐、健身、休闲等经营用途的房地产形式，国外用的比较多的词汇是零售地产的概念。 1.商圈：是指商业中心以其所在地点为圆心，沿着一定的方向和距离扩展，吸引顾客的辐射范围。 2. 所有权：是所有人依法对自己财产所享有的占有,使用,收益和处分的权利。 3.产权：产权是经济所有制关系的法律表现形式。它包括财产的所有权、占有权、支配权、使用权、收益权和处置权。 4. 经营权：经营者对其经营财产的一种占有、使用和收益的权利。 5.零售业：是一种交易形式,可定义为将商品或劳务直接出售给最终消费者的销售活动,也是向消费者提供销售商品的一种商业活动环境，直接关系到居民的生活质量和生活方式，商业行为的最后一步。 6. 开发商：开发项目，做为项目的第一承担人、所有人。 7.商业用地：商业用地是指规划部门根据城市规划所规定该宗地块的用地性质是用于建设商业用房屋，出让后用地的使用年限为40年。

8. 国内一线城市：指对本国经济、政治具有重要作用的大都市。在城市规模、基建、财政收入、消费、对人才吸引力等各层面，领先于其他城市。如：上海、北京、广州、深圳、天津 9. 国内二线城市：指除一线城市以外，省会、旅游城市，京广、京沪等主要干线旁边的城市等，如：成都、重庆、武汉、南京、苏州、无锡、济南、长沙、郑州、西安、哈尔滨、杭州、沈阳、青岛、大连、宁波、厦门等 10. 国内三线城市：昆山、江阴、常熟、张家港、义乌、洛阳、柳州、株洲、长春、温州、佛山、东莞等其它较发达城市。 11. 五证一书：“五证一书”指的是商品房屋预售许可证、建设工程规划许可证、建设用地许可证、国有土地使用规划许可证、建筑工程施工许可证及项目选址意见书。商业建筑 1.占地面积：占地面积是指建筑物所占有或使用的土地水平投影面积，计算一般按底层建筑面积。 2. 建筑面积：建筑展开面积，它是指建筑外墙外围线测定的各层平面面积之和。它是表示一个建筑物建筑规模大小的经济指标。 3. 经营面积（实用面积、柜台面积）：它是“建筑面积”扣除公共分摊面积后的余额。

超级一键网克使用方法

超级一键网克使用方法进行网克需要的条件：一键网克软件：超级一键网克，当然其他的网克软件也可以。一台已经装好系统的机器（操作系统98以上即可，推荐XP）用作服务器，一台已经装好全部软件并且配置好的机器作为母盘，当然，如果你只对系统盘进行网克的话，这台机器也可以兼做服务器，但是如果你想全盘网克的话，那就需要另外一台机器做服务器了，道理嘛，因为做全盘镜像的时候镜像文件当然不可能放在源盘上，而必须放在另外一块硬盘上，就好象你左手永远不可能摸到左手的手背一样，呵呵。其它客户机，数量255台以下，当然多于255台也可以，只是下面的IP地址和子网掩码要重新配置，大家参看有关资料。不过我想大家一次网克也不可能会有那么多台机器的，呵呵。注意事项：首先，这些机器间物理网络要保持畅通（废话！）。另外，如果你的网吧是杂牌军的话，那就不要试了，因为每台机器的配置如果不一样的话，所装的驱动也不一样，否则网克后可能有太多的不可预知的后果，经常蓝屏肯定是家常便饭了，当然也有可能根本就启动不了。以上说的配置主要是显卡和主板（如果你的声卡是集成的话）。硬盘无所谓，但要注意，目标盘的容量一定要大于母盘。当然了，最好是配置完全一样，这样基本上不会出什么问题。

当然，如果你的机器配置不一样的话，也可以进行网克，大家可以把雨林木风ghost版里面的ghost文件提取出来作为镜像，这样也比一台一台装系统要快得多了。不过那应该是分区网克，具体方法你学会了全盘网克那自然不在话下。下面开工。第一步，制作镜像，如果只制作系统盘的镜像，那么在单机操作就行了。具体操作我想大家都会。下面讲的是制作全盘镜像，当然你不必拆开机器挂两个硬盘了，网克嘛！ 1、配置tftpd32 解压超级一键网克到服务器的任意盘，比如E盘，双击TFTPD32.EXE，关闭防火墙，或者在应用规则里设为允许访问网络。最好关掉，否则下面你要点若干次允许。点DHCP server标签，这里有几个地方要填一下，当前目录和server不要填，程序自动判断。IP地址池随便填，但要注意不要跟服务器的IP地址冲突，当然要符

实时频谱仪—工作原理

实时频谱分析仪（RTSA），这是基于快速傅利叶（FFT）的仪表，可以实时捕获各种瞬态信号，同时在时域、频域及调制域对信号进行全面分析，满足现代测试的需求。一、实时频谱分析仪的工作原理在存在被测信号的有限时间内提取信号的全部频谱信息进行分析并显示其结果的仪器主要用于分析持续时间很短的非重复性平稳随机过程和暂态过程，也能分析40兆赫以下的低频和极低频连续信号，能显示幅度和相位。傅里叶分析仪是实时式频谱分析仪，其基本工作原理是把被分析的模拟信号经模数变换电路变换成数字信号后，加到数字滤波器进行傅里叶分析；由中央处理器控制的正交型数字本地振荡器产生按正弦律变化和按余弦律变化的数字本振信号，也加到数字滤波器与被测信号作傅里叶分析。正交型数字式本振是扫频振荡器，当其频率与被测信号中的频率相同时就有输出，经积分处理后得出分析结果供示波管显示频谱图形。正交型本振用正弦和余弦信号得到的分析结果是复数，可以换算成幅度和相位。分析结果也可送到打印绘图仪或通过标准接口与计算机相连。二、实时频谱分析仪中的数字信号处理技术 1. IF 数字转换器一般会数字化以中间频率(IF)为中心的一个频段。这个频段或跨度是可以进行实时分析的最宽的频率范围。在高IF 上进行数字转换、而不是在DC 或基带上进行数字转换，具有多种信号处理优势(杂散性能、DC抑制、动态范围等)，但如果直接处理，可能要求额外的计算进行滤波和分析。 2. 采样内奎斯特定理指出，对基带信号，只需以等于感兴趣的最高频率两倍的速率取样 3. 具有数字采集的系统中触发能够以数字方式表示和处理信号，并配以大的内存容量，可以捕获触发前及触发后发生的事件。数字采集系统采用模数转换器(ADC)，在深内存中填充接收的信号时戳。从概念上说，新样点连续输送到内存中，最老的样点将离开内存。

商场营销专业术语

商场商业术语营销术语大全 1、返券：即在促销活动期间内，购物满一定金额赠送不同或相同金额的抵用券，如满500元返20元券。 2、折扣：折扣是一种最常见、也最容易操作的促销方式。即全场商品或部分商品进行不同程度的折扣销售。 3、特价：即限定某个时间段内某些商品执行特价销售，超出时间自动恢复，一般建议在客流比较少的时段做，这样可以拉平卖场人气。 4、限定条件优惠：如，限会员购买优惠或会员达到一定消费积分时优惠，还有买购多少额度优惠或返利。 5、限总量优惠：即在优惠的商品出售够一定数量时，特价终止，恢复原价销售。 6、限客单量优惠：即限定每客最多购买特价商品的数量，若超过限量则超过的部分按原价销售计算。 7、购买超过一定量优惠：即某个单品一次购买超过几个，就按设定的优惠价计算销售。 8、商品碰头分组促销：此项促销事先设定好分组的商品（必须是两个单品），定好优惠价格，只有消费者在购买时，在规定的时间同时找到两个对应的单品时则按优惠价销售；此活动一般适应与卖场布局死角多的地方，将两个分组商品放置在不同的区域，让消费者去寻找，以提升卖场动线和消费者购物的乐趣。 9、捆绑销售：即将一些关联性强的商品放在一起，并打包给予一定的优惠进行销售。 10、消费购买一定额度，可以购买超低价商品，如：购物满50元可以1元钱买一斤色拉油等。 11、买赠促销：即买指定的东西送赠品，还有买够多少钱送不同的赠品；赠品可以是商品也可以是企业的广告礼品（如：茶杯、围君、广告衫、广告伞、遮阳帽等）。 12、有奖销售：即购物满一定条件可以参加摸奖或抽奖活动，主要是奖品一定要丰富，并且有公众吸引力。 13、商家联盟促销：即购物满一定条件或消费达到某种条件，可以提供消费者到其他商家消费的打折权利，比如：在超市买够500元送餐饮或娱乐项目的赠票或折扣券，或者在某餐饮或娱乐项目消费单位满多少元可以送超市的优惠卡或折扣券。 14、购物送服务：购物满一定条件可以免费送货、报销车费、代大扫除、免费般移大件物品、免费维修家具或电器和通讯工具等。

常用的商业术语(ok)

常用的商业术语口部（部口）：地下商城的专用名词，即出入口。坪（平）效:指单位面积的销售额或租金额。动线: 指商场的布局，使顾客自然行走、购物的轨迹。良好的动线规划可诱导顾客在店内顺畅地选购商品，避免卖场产生死角，提高卖场坪效。动线设计对超市尤其重要。主动线：也称主通道，顾客在一走进商场就可以看到的货架间的通道，主要指入口到末端也指顾客经过的主要通道，也是商品陈列的最佳地段。主通道的宽度应在3米以上。副动线：也称副通道，指专柜之间的通道。副通道宽度应在2米以上。水平动线：指在一个层面中,人们行走的最佳路线。垂直动线：是指不同标高空间或楼层的垂直联系如楼梯、电梯和自动扶梯。引导动线：指通过一定的手段（比如刀旗、横幅等）将顾客引导到指定目的地的路线安排。开间：又称面宽，即商铺对外可见的宽度距离。进深：指商铺纵深的长度，与面宽线段到商铺墙面垂直的距离。建筑面积：建筑面积亦称建筑展开面积，包含主要设备间室、动线、出入口等。使用面积：指商铺中直接供用户经营使用的净平面面积之和。套内建筑面积：成套商铺的套内建筑面积，成套商铺的套内建筑面积由套内商铺使用面积，套内墙体面积，套内公共面积三部分组成。

层高：层高是指顶板到地面的垂直距离。内净高：指装修落成后的地面与形成最终铺设的棚顶间的间距。门楣：门楣，就是商铺正门上方的横梁，用于悬挂或粘贴牌匾的地点。一装：一般指商场公共区间的原始装修，如墙面、地面、柱体、卫生间等，一装将确立商场的主题定位思想，也将引导商户店面装饰的方向。二装：即商户进场后对其商铺的装修、装饰。均价：某一个个案的总销金额除以总建面得出的单价。基价：某一个个案的最低价。订金：商铺订购金，又称协议金、诚意金等等，可退还给客户，或抵用购买、租赁商铺的费用。定金：商铺定购金，又称大定金等。如购买者超出定购期未付全款，该定金归出售方所有，不可退还给认购人；如签订全款合同可抵用购买商铺的费用。经销商：是产品的经销环节，直接面向销售终端。供应商：为零售案场提供商品，销售终端的货品供给商。代理商：是货品生产厂家的合作者。生产厂家在某一区域内设立代理商，代理商负责该区域的品牌宣传、推广、分销等活动，拥有该商品品牌的独家经销权，及该区域内市场拓扑分布的规划、统筹权。渠道商：渠道商是指连接制造商和消费者之间的众多中间商家,包括:批发商,零售商,代理商和佣金商等。码单：指供货商提供给经销商货品的尺码单据。

商场商业术语营销术语大全

商场商业术语营销术语大全（不断更新）商场商业术语营销术语大全 1、返券：即在促销活动期间内，购物满一定金额赠送不同或相同金额的抵用券，如满500元返20元券。 2、折扣：折扣是一种最常见、也最容易操作的促销方式。即全场商品或部分商品进行不同程度的折扣销售。 3、特价：即限定某个时间段内某些商品执行特价销售，超出时间自动恢复，一般建议在客流比较少的时段做，这样可以拉平卖场人气。 4、限定条件优惠：如，限会员购买优惠或会员达到一定消费积分时优惠，还有买购多少额度优惠或返利。 5、限总量优惠：即在优惠的商品出售够一定数量时，特价终止，恢复原价销售。 6、限客单量优惠：即限定每客最多购买特价商品的数量，若超过限量则超过的部分按原价销售计算。 7、购买超过一定量优惠：即某个单品一次购买超过几个，就按设定的优惠价计算销售。 8、商品碰头分组促销：此项促销事先设定好分组的商品（必须是两个单品），定好优惠价格，只有消费者在购买时，在规定的时间同时找到两个对应的单品时则按优惠价销售；此活动一般适应与卖场布局死角多的地方，将两个分组商品放置在不同的区域，让消费者去寻找，以提升卖场动线和消费者购物的乐趣。

9、捆绑销售：即将一些关联性强的商品放在一起，并打包给予一定的优惠进行销售。 10、消费购买一定额度，可以购买超低价商品，如：购物满50元可以1元钱买一斤色拉油等。 11、买赠促销：即买指定的东西送赠品，还有买够多少钱送不同的赠品；赠品可以是商品也可以是企业的广告礼品（如：茶杯、围君、广告衫、广告伞、遮阳帽等）。 12、有奖销售：即购物满一定条件可以参加摸奖或抽奖活动，主要是奖品一定要丰富，并且有公众吸引力。 13、商家联盟促销：即购物满一定条件或消费达到某种条件，可以提供消费者到其他商家消费的打折权利，比如：在超市买够500元送餐饮或娱乐项目的赠票或折扣券，或者在某餐饮或娱乐项目消费单位满多少元可以送超市的优惠卡或折扣券。 14、购物送服务：购物满一定条件可以免费送货、报销车费、代大扫除、免费般移大件物品、免费维修家具或电器和通讯工具等。 15、POP：POP是英文point of purchase的缩写，意为“卖点广告”,其主要商业用途是刺激引导消费和活跃卖场气氛。她的形式有户外招牌，展板，橱窗海报，店内台牌，价目表，吊旗，甚至是立体卡通模型等等。常用的POP为短期的促销使用，其表现形式夸张幽默，色彩强烈，能有效地吸引顾客的视点唤起购买欲，她作为一种低价高效的广告方式已被广泛应用。

安装系统的几种方法

一、光盘安装 1、安装盘安装这是最常规的方法，也是最基本的方法，特点是安全、稳定，缺点是速度慢，需要另外安装驱动程序和软件。简单介绍一下步骤，开机进入BIOS，设置从光驱启动（CD-ROM），放入安装盘，重启电脑，选择相应的菜单，就会进入windows安装程序，选择你想安装的分区，然后根据需要对其进行重分区或格式化操作，然后回车，开始安装。期间会碰到输入密钥、时区、键盘类型等信息，一一输入即可，如果用的是无人职守安装盘，等着就行了，重启几次后，就安好了，记得还要安装驱动。 2、ghost盘安装 GHOST意为幽灵，是赛门铁克公司推出的硬盘分区备份恢复软件，具有简单易行、速度快的特点，一般在ghost光盘启动后会有一键恢复到C盘的选项，也可以进入WINPE，在PE环境下运行GHOST，进行分区恢复。打开GHOST后，先点一下OK，然后依次选择local→partition →from image,找到合适的映像文件，然后选择需要恢复的分区，一般是C盘，确定后等上最多七八分钟就可以了，重启后会进入一个安装程序界面，这是在给你的电脑安装驱动、常用软件的过程，再次重启，系统就装好了。二、U优盘安装这个方法适用于没有光驱的电脑。要求电脑支持USB启动，当然，只要不是太旧的电脑，一般都支持的。其中制作相应的启动盘是关键。安装前记得进入BIOS，设置相应的启动方式。 1、优盘启动盘安装

可以是DOS启动盘，或者是PE启动盘，另外需要有GHOST 映像或者光盘映像。开机设置启动方式，这个根据启动盘的模式，可以是USB-HDD、USB-ZIP、USB-FDD等，DOS环境下，打开GHOST，然后还原C盘分区，在PE环境下，也可以打开GHOST 还原分区，这两种方法和用ghost盘安装方法是一样的，只是实现途径不一样。在PE下还可以用虚拟光驱加载光盘映像，然后运行相应的安装程序，开始系统安装，这和安装盘安装方法类似。 2、量产U盘为CD-ROM 准备好U盘、光盘镜像和量产工具，一步一步制作好启动盘，然后，进入BIOS，设置为USB-CDROM启动，随后方法就和光盘安装方法一样了，不过速度要比光盘安装快，这个和优盘读写速度快有关。三、硬盘安装这个安装方法几乎不需要什么别的硬件，速度也快。需要可以安装到硬盘的pe，光盘镜像。先在硬盘上安装PE，然后重启，进入PE环境，运行虚拟光驱，加载光盘镜像。还是那样，根据光盘镜像的类型，是安装盘就运行相应的安装程序，是ghost盘就运行ghost恢复C盘分区。还有就是如果要安装双系统，那么用硬盘安装就方便多了，不用刻盘，也不用制作启动优盘，只要先进入已有的系统，加载光盘镜像，然后运行相应的安装程序就可以了。这里如果是安装盘镜像，启动菜单就已经修改好了，如果是ghost盘，那么，还要使用相应的软件修改启动菜单。

商业零售106个基本专业术语

卖场106个基本专业术语——好东东呀营运的基本术语 1、货架：商场上用来存放商品、展示商品的金属架。通常有几种类型，有承重式的高达几米的，有较矮的，与人的身高差不多。每一种货架都有其专用的配件。 2、端架：货架两断的位置，也是顾客在卖场回游经过频率最高的地方。 3、堆头：即“促销区”，通常用栈板、铁筐或周转箱堆积而成。 4、收银台端架：收银台前面用来陈列货物的货架。 5、专柜：指精品区、烟酒区用来陈列贵重商品的玻璃柜。 6、冷藏柜：用来陈列需要冷藏食品的冷柜，温度在0℃～5℃。 7、冷冻柜：用来陈列需要冷冻食品的冷柜，温度在—18℃以下。 8、冷藏库：用来储存需要冷藏食品的冷库，温度在0℃～5℃。 9、冷冻库：用来储存需要冷冻食品的冷库，温度在—18℃以下。 10、购物车/篮：顾客购物时用的推车和篮子。 11、促销车：专门用来在超市中做商品展示，度吃等活动的车子。 12、冰台：超市中专门用来展示、陈列商品的金属台，台上覆盖冰碎以保持温度。 13、铝梯：超市重用来到高处取放货物时用的铝质梯子，带自锁安装。 14、叉车：超市中用来运输货物的车辆，有手动和电动两种。 15、卡板：木制或胶制的用于放货运货的栈板。 16、货架配件：货架上的配件，主要有层架、支架、挂钩、篮筐、挂篮、挡篮等。 17、主通道：商场布局中的主要通道，一般比较宽，是客人大量通过的地方。 18、电脑中心：商场里的电脑信息中心。 19、销售区域：商场中销售商品的区域，也是客人可以自由购物的区域。 20、精品区：不适合用开架方式进行销售的商品封闭区域，一般采取单独付款方式。 21、员工通道：超市内部员工上下班进出的通道。 22、安全通道：是超市建筑物在设计时留出来的防火通道，以应付紧急情况疏散。 23、紧急出口：当发生紧急情况如火灾时，可以逃离商场的出口。平时不使用。 24、用具间：超市中用来存放与商品陈列有关的用具、道具的房间。 25、洗手池：生鲜部专门用来洗手的地方。 26、更衣室：员工用来更换工装的地方。 27、更衣柜：员工用来存放工装或私人物品的柜子，一般设在商场外部。 28、电子称：对以重量进行销售的商品进行称重的设备。 29、压纸机：对商场内的空纸箱进行压制处理的机器。一般设在收货部。 30、收银机：又称POS（POINT OF SA LES）机、销售信息管理系统、主要执行收银的功能。 31、防盗门：超市中设置的电子系统防盗门，具有防盗报警功能。 32、防盗标签：用来防止盗窃的磁性标签或磁扣。一般对贵重商品、服饰、鞋等，多用防盗标签。 33、取订器：用来取防盗磁扣的设备。 34、收银小票：批顾客购物结账后给顾客的以商品的电脑小单，可作为客人付款的赁证。 35、药箱：超市中配备的药箱，以应付员工的普通外伤的初级处理。 36、对讲机：超市范围内的主要通讯工具。一般有两个频道。 37、垃圾桶：超市中专门用来装垃圾的桶。有销售区域用和生鲜区专用之分。 38、促销员：厂商为了更好销售、宣传其商品而派驻商场的员工。 39、优秀员工：超市中每月评比出的表现优秀的员工。 40、无星级收银员：收银部门最出色的收银员。 41、培训教练：负责培训本部门员工的资深职员。一般由经验丰富、熟悉工作、表现优秀的人员

软盘、光盘、硬盘克隆

软盘、光盘、硬盘克隆一个也不能少经常玩电脑朋友，肯定常常复制软盘，那么如何才能使复制的软盘与源盘文件完全一样，引导信息、文件分配表（FAT）都完全一样呢？甚至将源盘作成IMG映像保存到硬盘上，在需要时再做成软盘呢？如何将光盘做成一个文件，以便于存放到硬盘上，或再刻录出与源光盘一模一样的光盘呢？如何将硬盘的启动分区做成一个文件，在操作系统出现问题时，能够整盘恢复回去，使操作系统恢复正常，而省去了重新安装操作系统和各种驱动程序的烦恼呢？下面分别进行介绍，不过这需要三款工具的帮忙，它们分别是HD-Copy、WinIso、Ghost，一、软盘的克隆使用过DOS的朋友大多都使用过HD-COPY，它是德国人Oliver Fromme编写的一个磁盘对拷程序。功能非常强大，可以用作格式化软盘、软盘扩容、修复坏软盘、保存软盘信息、读取坏软盘、清洗软驱磁头等用处。最大的好处是它能将源盘“克隆”成一个映像（IMG格式)，在需要做成软盘时再恢复就可以了。所以从92年问世至今一直都深受人们的喜爱。 HD-COPY的使用非常简单，在DOS下运行HD-Copy，其主界面如图1，菜单中的英文依次为READ（读取）、WRITE(写入）、VERIFY DESTINATION（校验目标盘）、VERIFY SOURCE（校验源盘）、FORMAT DESTINATION（格式化磁盘）、PUT TO FILE（存入映象文件）、GET FROM FILE（读取映象文件）、SPECIAL MENU（特殊菜单）、ABOUT（关于）、Escapr(exit) （退出），菜单栏的右侧是一些开关。图1

克隆软盘的操作方法是：先启动HD-COPY再插入源盘，选择READ（或按R键）读取源盘信息到缓冲区（缓冲区是程序在内存中划出的一块用作临时数据交换的区域）。结束后再插入目标盘，选择Write（或直接按W）。如果目标盘中以前存有数据，HD-COPY将会问你是否确定覆盖（Y/N），当然是Y，然后程序将执行对拷动作。如果你不想拷贝目标盘，只想将源盘留一个备份（例如借来的软盘）可以在第一步完成后选择PUT TO FILE（按P键），输入你想要存放的路径和文件名。注意的是文件必须是DOS下的8.3格式，就是说文件名不能超过8个英文字母或4个中文字。将来需要的时候可以把你保存的映象文件找来GET FROM FILE（快捷键G）再执行第二步就可以了。二、光盘的克隆随着大容量硬盘的普遍采用，人们已经习惯将光盘拷贝成光盘映像文件使用，普遍采用的便是大名鼎鼎的ISO 9660国际标准格式，因此光盘映像文件也简称ISO文件。因为ISO文件保留了光盘中的全部数据信息（包括光盘启动信息），你可以方便地采用常用光盘刻录软件（如Nero Burning-ROM）通过CD-R/RW烧录成光碟，也可以通过虚拟光驱软件(如Daemon-Tools)直接使用。 WinISO 就是一个CD-ROM 映像文件格式转换工具，并且可以直接编辑光盘映像文件！通过WinISO，你可以在映像文件内部添加/删除/重命名/提取文件。你可以将其他格式的映像文件转换为标准的ISO 格式，同时你也可以从你的CD-ROM 中创建ISO 映像文件。将光盘“克隆”成ISO镜像文件的方法如下： 1.启动WinISO之后将欲克隆的光盘放入光驱中，点击菜单“操作”→“从CDROM制作ISO”项，弹出如图2所示的窗口，在其中的“来源CD-ROM”栏选择光盘所在的盘符，在“输出文件”栏设定好存放光盘镜像文件的路径和文件名，同时要在下部选中“ASPI”来制作ISO文件。图2

商场购物时常用英文用语

商场购物时常用英文用语我们去(商店)逛逛吧! Let's go window-shopping. I'm flat broke. (我身无分文。) Let's go window-shopping anyway. (不管怎么说，我们去逛逛吧。) Why don't we go window-shopping? Shall we go window-shopping? (去逛商店吗?) 人多得要命! What a crowd! It's so crowded! What a big crowd! 商店几点开门? When does the store open? When does the store open? (商店几点开门?) It's closed today. (今天休息。) When do you open? What time do you open? How soon does it open? What time does the store open? When do the doors open?

商店几点关门? When does the store close? When does the store close? (商店几点关门?) We close at seven. (7点。) What time does the store close? When is closing time? (几点打烊?) 我们11点才开门呢。 We won't open until eleven. We won't open until eleven. (11点才开门。) That late? (那么晚?) It opens at eleven. (11点开门。) From eleven. (11点开始。) Our business hours begin at eleven. (我们11点开始上班。) 卖鞋的专柜在哪儿呀? Where's the shoe department? Where's the shoe department? (卖鞋的专柜在哪儿呀?) I'm lost, too. (我也糊涂了。) *department store “商场”。 Where can I find the shoe department? Where is the shoe department located? 在3楼。 It's on the third floor. *on the...floor是“(楼的)……层”。美国的1楼是the first floor，2层是the second floor。英国的1楼是

超级一键使用方法

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商场商业术语集锦

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市场化经营商场化管理：指采取"整体规划、招租经营、统一管理、自收自付"，各柜相对自主经营，并以规范化的商场管理形成专业特色的经营管理模式。精细化管理：是以"精确、细致、深入、规范"为特点的全面化的管理模式。全面化是指精细化管理的思想和作风贯彻到整个企业的所有管理活动中。精细化管理包括：精细化的操作、精细化的控制、精细化的核算、精细化的分析、精细化的规划。购物中心交通规划：购物中心交通规划分为两部分，一部分是周围道路把交通引入购物中心，另一部分是购物中心自身用地范围内的交通组织和规划，以及建筑内部的人流组织。购物中心的步行人流购物中心的步行人流分为两种：一种是从停车场到购物中心的运动；另一种是从购物中心到购物中心的运动。购物中心的汽车交通车流包括购物车流、货运车流和公共交通车流。交通组织的原则

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学校机房计算机硬件系统的一致性，进行克隆系统就能保证稳定与统一。一、母盘的制作 1、视具体使用情况，将一台学生计算机硬盘分为若干个区，可设置第一个分区为NTFS文件系统的主分区，然后安装操作系统及相应设备驱动程序。 2、应用软件安装根据教学需求及机房应用方向，选择安装所需的应用软件。同时一定要确保每一个软件都能正常运行，否则重新制作母盘将导致后面的工作前功尽弃。 3、DeefFreeze的安装（1）在教师机或服务器上安装DeefFreeze。（2）用DeefFreeze配置管理器生成客户端安装程序和客户端种子文件。（3）在作为母盘的学生机上安装种子文件。在利用克隆的方法将这台计算机的系统安装到所有学生计算机上后，再利用用DeefFreeze控制台程序进行客户端安装程序的运行，可以大大提高效率。 4、系统的全面清理

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卖场专业术语大全

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(超级一键网克)PXE网克最详细图文教程

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