当前位置：文档库 › 2003 Understanding and improving transgene stability and expression

2003 Understanding and improving transgene stability and expression

Insect Biochemistry and Molecular Biology34(2004)121–130

https://www.wendangku.net/doc/8a10120897.html,/locate/ibmb Understanding and improving transgene stability and expression in insects for SIT and conditional lethal release programs

Alfred M.Handler?

Center for Medical,Agricultural,and Veterinary Entomology,Agricultural Research Service,U.S.Department of Agriculture,

1700S.W.23rd Drive,Gainesville,FL32608,USA

Received2December2002;received in revised form19March2003;accepted7August2003

Abstract

Genetically transformed insect pests provide signi?cant opportunities to create strains for improved sterile insect technique and new strategies based on conditional lethality.A major concern for programs that rely on the release of transgenic insects is the stability of the transgene,and maintenance of consistent expression of genes of interest within the transgene.Transgene instability would in?uence the integrity of the transformant strain upon which the e?ectiveness of the biological control program depends. Loss or intra-genomic transgene movement would result in strain attributes important to the program being lost or diminished, and the mass-release of such insects could signi?cantly exacerbate the insect pest problem.Instability resulting in intra-genomic movement may also be a prelude to inter-genomic transgene movement between species resulting in ecological risks.This is less of a concern for short-term releases,where transgenic insects are not expected to survive in the environment beyond two or three generations.Transgene movement may occur,however,into infectious agents during mass-rearing,and the potential for move-ment after release is a possibility for programs using many millions of insects.The primary methods of addressing potential trans-gene instability relate to an understanding of the vector system used for gene transfer,the potential for its mobilization by the same or a related vector system,and methods required to identify transformants and determine if unexpected transgene move-ment has occurred.Methods also exist for preventing transposon-mediated mobilization,by deleting or rearranging vector sequences required for transposition using recombination systems.Stability of transgene expression is also a critical concern, especially in terms of potential epigenetic interactions with host genomes resulting in gene silencing that have been observed in plants and fungi,and it must be determined if this or related phenomena can occur in insects.

Keywords:Transgenic insects;Sterile insect technique;Transgene stability;Transposable elements;Epigenetics

1.Introduction

The ability to genetically transform insect pests pre-sents a wide array of possibilities to create strains with speci?c attributes that can greatly improve existing bio-logical control methods,as well as the development of novel new strategies for control(see Handler,2002a). The e?ective use of such strains,however,relies on consistent and reliable expression of the integrated genes of interest,as well as maintenance of strain?t-ness and viability,especially under mass-rearing proto-cols.It is also essential that the transgene vector remain stably integrated,not only to maintain strain integrity,but also to prevent possible inter-species movement of the transgene into unintended hosts. Thus,the major areas of concern for the release of transgenic insects for sterile insect technique(SIT) and the release of conditional lethal(CLR)strains relate to risks associated with stability of the trans-gene vector and the expression of genes of interest within the vector.The potential for unintended trans-gene movement and its impact on program e?ective-ness and ecological risks must be considered in the context of several factors.These include the host insect,the vector system used for transformation,the methods and facilities for mass-rearing,and the anticipated persistence of the transformant strain in the?eld.The mechanisms and interactions that might result in strain instability will be a function of

?Tel.:+1-352-374-5793;fax:+1-352-374-5794.

E-mail address:handler@nersp.nerdc.u?.edu(A.M.Handler).

the number of generations the strain is maintained under mass-rearing,and the generations it is expec-ted to be in the?eld.For example,for programs such as SIT,the presence of the transgenic strain in the?eld is not expected to exceed the lifetime of the released insect(short-term single generation).While for CLR programs,such as release of insects with a dominant lethal(RIDL)(Thomas et al.,2000),auto-cidal biological control(ABC)(Fryxell and Miller, 1994),and similar systems(Heinrich and Scott,2000; Horn and Wimmer,2003)that rely on inherited leth-ality or sterility to suppress the released insect and its o?spring,several generations will be required for the transgenic insects to be eliminated(short-term multi-generational).

Transgene instability would?rst be evident by intra-genomic movement of the vector,that could in?uence the integrity of the transformant strain upon which the e?ectiveness of the biological control program depends. If the transgene is lost or transposes to another site within the genome,strain attributes important to the program may be lost or diminished,and the mass-release of such insects could signi?cantly worsen the targeted insect pest problem.Instability resulting in intra-genomic movement may also be a prelude to inter-genomic transgene movement between species resulting in ecological risks.While less of a concern for short-term releases,transgene movement into inter-mediary symbionts or infectious agents could occur during mass-rearing,allowing rapid movement into other hosts after release.For programs where many millions of insects are released,the potential for inter-species movement must be considered a statistical possibility.

The primary methods of addressing potential transgene instability relate to an understanding of the vector system used for gene transfer,the poten-tial for its mobilization by the same or a related vec-tor system,and methods required to identify transformants and determine if unexpected transgene movement has occurred.To prevent transposon-mediated mobilization,methods exist to delete or rearrange vector sequences critical for transposition post-integration using recombination systems such as FLP/FRT and Cre/loxP(see Rong and Golic,2000). Another potential source of instability in transgene expression may result from epigenetic interactions with the host genome that have been well-docu-mented as gene silencing systems in plants and fungi (see Martienssen and Colot,2001).It is currently unknown if,and to what extent,gene silencing resulting from RNA-mediated methylation occurs in insect systems,and further understanding of this phenomenon will be essential.2.Transposon-based vectors for gene transfer

An evaluation of risks associated with transgenic insect release and development of methods to abate these risks requires an understanding of the systems and processes used for gene transfer.The transform-ation systems available include those that result in the stable heritable integration of a transgene by germ-line transformation,while other systems include gene expression from transgenic symbionts(known as para-transgenesis),or by the extrachromosomal transient expression of a genetic system,usually mediated by a viral or bacterial system.For the applied use of gene expression systems in released insects,germ-line trans-formation typically mediated by a transposable element based system is currently the method of choice,and these are the systems we will focus on for this dis-cussion.

A critical assessment of risks associated with strain stability requires a thorough knowledge of the behavior and regulatory properties of the vector used for geno-mic integration.The vectors currently used for non-drosophilid germ-line transformation are Class II transposable elements that are short terminal inverted repeat(TIR)transposons that transpose via a DNA-mediated intermediate in a cut-and-paste fashion(see Handler,2001).These elements,generally,range in size from 1.3to 3.2kband have a transcriptional unit within the TIR sequences that encodes a transposase molecule that acts at or near the termini to catalyze excision and transposition of the complete element. The ability of the transposase to act in trans has allowed the development of binary vector-helper sys-tems where the mobile TIRs surround a marker gene and genes of interest within a vector plasmid,while the helper transposase,rendered immobile due to deletion of one or both of the terminal sequences,is provided on a separate plasmid(Rubin and Spradling,1982). After introduction into germ cells,the helper mediates transposition of the vector into the genome,but since the helper is unable to integrate,it is lost after sub-sequent cell divisions allowing the vector to remain sta-bly integrated.Thus,integrated vectors are typically defective non-autonomous transposons that require an exogenous source of transposase to be re-mobilized. Short-term concerns for transgene stability generally relate to the potential for mobilization by the same or a related transposon system.The current transposons used for insect germ-line transformation,that include piggyBac,Hermes,Minos,and mariner(see Handler, 2001;Atkinson et al.,2001),all have closely related elements that exist in divergent species.For some, inter-species movement has very likely occurred by horizontal transmission,and the mechanisms involved in this movement provide a major area of concern for potential transgene movement.

122 A.M.Handler/Insect Biochemistry and Molecular Biology34(2004)121–130

2.1.Hermes

Hermes was discovered in Musca domestica and is a member of the hobo,Activator,Tam3(hAT)family of transposons,and among functional transposons,is most closely related to hobo(Warren et al.,1994).Its existence was?rst postulated by the fact that hobo exci-sion from plasmids occurred in the absence of hobo transposase in Musca(Atkinson et al.,1993),and the same?nding was made for hobo in three tephritid fruit ?y species(Handler and Gomez,1996).Hermes has proven to be an e?ective transformation vector in Dro-sophila(O’Brochta et al.,1996),med?y(Michel et al., 2001),stable?y(O’Brochta et al.,2000)and several mosquito species.A unique aspect of Hermes transpo-sition is that unusual recombinant integrations that include the entire vector plasmid were observed in the culicine mosquitoes,Aedes aegypti(Jasinskiene et al., 1998)and Culex quinquefasciatus(Allen et al.,2001), while only precise integrations were reported for an Anopheles gambiae cell line(Zhao and Eggleston, 1998).It has been postulated that some of these are replicative recombination events caused by interactions with related genomic hAT elements(Jasinskiene et al., 2000).A more direct functional relationship between these elements has been shown by plasmid and germ-line excision assays where hobo transposase mobilized both Hermes and hobo terminal sequence(Sundarar-ajan et al.,1999).This is the?rst direct evidence for the cross-mobilization of related,yet distinct elements,and heightens the concern for hAT vector stability and transmission to non-target species.

2.2.Minos and mariner

Minos and mariner are members of the wide ranging mariner/Tc family.Minos is an e?ective vector system and has been tested in two non-drosophilids,the Medi-terranean fruit?y(Loukeris et al.,1995)and Anopheles stephensi(Catteruccia et al.,2000).It is most closely related to Tc elements originally discovered in nema-todes(Franz and Savakis,1991),although direct inter-action with these elements has not been reported. Embryonic and cell line transposition assays indicate that it has a wide range of function including insects and vertebrates(Klinakis et al.,2000;Zagoraiou et al., 2001).

Mariner was discovered in D.mauritiana(Medhora et al.,1988),and related elements have been found in a wide range of insects and other organisms(Robertson and MacLeod,1993),and horizontal transmission has been suggested for the Himar element which exists in two insect orders(Robertson and Lampe,1995).After transforming several Drosophila species(see Hartl et al., 1997),the mariner Mos1element was used to transform A.aegypti(Coates et al.,1998)and M.domestica (Yoshiyama et al.,2000)and,notably,it has also trans-formed chickens and zebra?sh(Sherman et al.,1998; Fadool et al.,1998).Given the demonstrated cross-mobilization between hAT transposons,and the wide ranging presence and function of mariner/Tc elements (Avancini et al.,1996;Robertson and MacLeod,1993), vector stability and the potential for horizontal trans-mission is a concern for these vectors.

2.3.piggyBac

The piggyBac transposon(originally IFP2)is part of a subclass of elements that insert exclusively in TTAA target sites and excise only in a precise fashion(Elick et al.,1995;Fraser et al.,1996),and it has been used to transform more than12insect species spanning four orders including the Diptera,Lepidopteran,Cole-optera,and most recently,the Hymenopotera(see Handler,2002b).Transformed species that may be controlled by SIT or CLR programs include med?y (Handler et al.,1998),carib?y(Handler and Harrell, 2001a),Oriental fruit?y(Handler and McCombs, 2000),pink bollworm(Peloquin et al.,2000),sheep blow?y(Heinrich et al.,2002),and Anopheles albima-nus(Perera et al.,in press),among several others.pig-gyBac was originally discovered as the causative agent of few polyhedra(FP)mutations in baculoviruses pas-sed through the Trichoplusia ni TN-368cell line(Fraser et al.,1983;Cary et al.,1989).Although it exists as a repetitive element in several T.ni cell lines(Fraser et al., 1996),it was not found in any other insect until recently when multiple complete,but non-functional piggyBac elements were discovered in the Oriental fruit ?y,Bactrocera dorsalis(Handler and McCombs,2000). piggyBac has since been discovered in eight sibling species of Bactrocera and three Spodoptera species (Handler,2002b;Zimowska and Handler,unpub-lished).Thus,the existence of piggyBac in phylogeneti-cally and geographically distinct species suggests that it has recently traversed insect orders by horizontal trans-mission,and probably exists in other species as well. The ability of piggyBac to transpose into an infectious baculovirus raises the possibility that its inter-species movement has been facilitated by a viral vector.

3.Identi?cation and characterization of germ-line transgenics

Risk assessment evaluation of transgenic insects will depend initially on their identi?cation and molecular characterization by methods that are e?cient,reliable, and consistent so that changes in transgene integrat-ion site or expression can be de?nitively assessed.It is also important to e?ciently distinguish transformant insects from non-transformed siblings so that potential

A.M.Handler/Insect Biochemistry and Molecular Biology34(2004)121–130123

intra-genomic movement of the transgene can be distinguished from inter-genomic movement within the same species.It should also be possible to deter-mine whether the transgene has been introduced into unrelated organisms,potentially transmitted by a non-vertical means of inheritance.If necessary,otherwise identical vectors may be marked with unique molecular tags(short DNA sequence)so their origin can be rap-idly de?ned.

Transformant selection and identi?cation in non-drosophilid insects originally used the eye color mutant-rescue selection routinely used in Drosophila (see Sarkar and Collins,2000).Since mutant hosts and cloned wild type alleles are not widely available for other insects,use of?uorescent protein markers,which are dominant-acting visible neomorphs,have found common use in non-drosophilids(see Horn et al., 2002).The primary marker used thus far has been enhanced GFP(EGFP)under polyubiquitin,3?P3,or actin promoter regulation,but other GFP variants and the DsRed?uorescent protein are?nding use as well (Matz et al.,1999;Handler and Harrell,2001b).These markers have the advantage of being easily and unam-biguously identi?ed,and they are less susceptible to position e?ects than white eye markers(Chal?e et al., 1994;Higgs and Lewis,2000;Handler and Harrell, 1999).False positives and mis-identi?cation are only problematic when indistinguishable auto?uoresence occurs,and this can be minimized by using promoters limiting expression to speci?c tissues.An important bene?t of using?uorescent protein markers is that they can be used as to detect released insects caught in traps (Handler,2002a),and when regulated by broadly active promoters,they may be used to determine inter-species movement of a transgene.

The number and general integrity of vector integra-tions requires Southern DNA hybridization using restriction digests and probes that independently ident-ify the50and30vector arms.Integration number should be consistent for both arms,with additional restriction digests used to clarify overlapping bands or incomplete digestions.Unusual insertions(e.g.recom-bination events)would typically result in anomalous hybridization patterns,though such patterns can also result from incomplete restriction enzyme digestion or from a mixed population of transformants having a varying number of unlinked transgene alleles.The use of Southern analysis as a test for transgene stability depends on unambiguous and consistent patterns of hybridization.

Insertion site sequencing allows the most de?nitive determination of transposon-mediated genomic inte-gration.This is achieved most simply by inverse PCR methods that isolate the junction sites independently, or together as part of a single PCR product(Ochman et al.,1993).The method used depends on restriction sites available and whether the genome contains a sin-gle or multiple integrations(determined by Southern analysis).Proximal genomic sequence then allows a de?nitive determination of transposition by performing PCR on transformed and non-transformed host species genomes.The vector should be detected in the trans-formed genome,with only the empty insertion site in the non-transformed genome.These primers then become essential to rapidly de?ning integration stab-ility in subsequent generations.

Chromosomal in situ hybridization also provides a de?nitive determination of a chromosomal integration and also allows a rapid determination of the number of genomic integrations and mapping.Hybridization to polytene chromosomes is most informative in species having a polytene map,but this is possible in only some dipteran species.An alternative is hybridization to mitotic chromosomes by?uorescent in situ hybridi-zation(FISH).

4.Potential for intra-and inter-genomic movement The primary concerns in evaluating transgenic insects for risk assessment and program e?ectiveness relate to transgene stability,which has implications for the integrity of the strain being released or studied, and potential risks if the transgene is transmitted to a non-host organism.For SIT and CLR programs,the primary risks for transgene instability relate to intra-genome movement,though the substantial number of organisms used in release programs raises the statistical possibility that inter-genomic movement may occur between species despite limited environmental exposure.For both transgene loss and transgene move-ment,a mobilizing system must exist within the trans-genic host that is the same or similar to the transposon vector.

Transgene loss would eliminate the bene?cial char-acteristics of the transgenic strain,and would be most devastating during mass-rearing if not rapidly detected before release.Intra-genome movement may result in di?ering position or dosage e?ects,or other genomic in?uences that can change the expressivity of the mar-ker and/or the gene of interest.This could a?ect de?ned characteristics of the strain that may be essen-tial to its e?ective use,and changes in marker gene expression may diminish the ability to detect transgenic insects for risk assessment and program analysis.Intra-genomic movement may also result in new integrations into lethal or semi-lethal sites that would diminish strain?tness or viability.

Inter-genome movement implies the horizontal trans-mission of a transgene between species,or di?erent strains of the same species.The existence of nearly identical transposons in insects from di?erent orders (e.g.Himar and piggyBac)has suggested that this is not

124 A.M.Handler/Insect Biochemistry and Molecular Biology34(2004)121–130

an uncommon natural phenomenon(Robertson and Lampe,1995;Handler and McCombs,2000).Such movement requires that the transposon is functionally autonomous,or that a cross-mobilizing system exists in the host genome that can de-stabilize the transgene, and the presence of a mechanism or vector that allows transmission of the transgene into a new genome.As discussed,the mobilizing system may be the same transposon as the gene transfer vector,or a related element.A primary question for risk assessment is how to detect and quantitate the activity of such a mobiliz-ing system,and how to determine if it resides in the host genome or a co-habiting or infectious endo-symbiont.

4.1.Evaluating the potential for transgene instability Transgene instability would most directly result from the existence of the same transposon as the vector in a host species,and these could be straightforwardly detected by hybridization or PCR analysis.Identi?-cation of related cross-mobilizing systems can be more di?cult since transposons,such as hobo and Hermes, may be functionally related but share insu?cient struc-tural similarity to make direct comparisons(Warren et al.,1994;Sundararajan et al.,1999).Thus,identi?-cation of such systems would rely on functional assays that can detect cross-mobilization.The most sensitive of these are embryonic or cell line excision assays per-formed in the absence of transposase,such as the hobo assays that implicated the existence of Hermes in M. domestica(Atkinson et al.,1993).Transposition assays could be similarly useful,but are more likely to detect only identical or nearly identical systems that can cata-lyze both excision and insertion(O’Brochta et al.,1994; Sarkar et al.,1997).A bene?t of transposition assays is that if a nearly identical transposon is detected in a host strain,they can allow a rapid determination of function.For example,the functional piggyBac element was discovered in a T.ni cell line(Fraser et al.,1983), yet,none of the piggyBac sequences isolated by PCR from T.ni larval tissue are identical to the original element(Zimowska and Handler,unpublished).Nega-tive data from transposition assays in T.ni embryos indicate that these in vivo elements are not functional (Lobo et al.,1999).

An important consideration for use of these assays is that the lack of a mobilizing system would be inferred by negative data;the absence of excision or transpo-sition.Therefore statistical tests are necessary to con-clude with con?dence that a mobilizing system does not exist,or that mobilization is limited to the extent that it is not a concern.Controls for these tests should include mobility assays in strains of the host species that have a genomic source of transposase,such as a jumpstarter strain.

If transgene mobilization within the host species is possible,then an area of concern is the potential for its integration into a symbiotic,infectious,or predatory system that could mediate transmission to another host.These systems include procaryotic bacteria or viruses,or eucaryotic predators such as mites or wasps, that could also harbor a mobilizing system.The possi-bility for an infectious virus transmitting a eucaryotic transposon became evident by the discovery of piggy-Bac when it transposed into an infecting AcNPV gen-ome(Fraser et al.,1983).Similar transpositions have been observed repeatedly from insect cell lines into sev-eral viral systems,as well as from a larval host(Jehle et al.,1998).While the piggyBac that transposed into AcNPV was an autonomous functional element,other TTAA elements discovered after transposition,such as tagalong,were defective elements that had to be cross-mobilized(Fraser et al.,1983).It is now important to determine whether transposon movement can occur from infectious agents or other potential vectors into a eucaryotic host genome,which would be required for horizontal transmission.For short-term release of transgenic strains,the highest likelihood would be for transposition into infectious agents during mass-rearing,that could have immediate consequences for inter-species movement into predatory organisms after release.Direct transposon movement from transgenic insects into predatory species or infectious agents after transgenic release would be less likely.Thus,a compre-hensive assessment of potential vector mobilization into symbiotic or infectious agents during mass-rearing over numerous generations should be a high priority. 5.Mechanisms for piggyBac vector immobilization 5.1.Vector stability and the need for immobilizable vectors

Determining the presence and function of a trans-posable element in a wide range of insects and organ-isms they associate with will provide important information for assessing vector stability and the pot-ential for breakdown of transgenic strains,as well as the potential for inter-genomic movement.E?ectively addressing this potential was a primary criticism of the Environmental Assessment for the?rst experimental release of a transgenic insect(the pink bollworm,Pecti-nophora gossypiella)when public comment was soli-cited by USDA-APHIS-PPQ.Since it is not feasible to determine if and to what extent all potential hosts and all environmental situations will support transposon mobilization,experimental studies can only determine the potential for inter-species movement,and the rela-tive risks to speci?c organisms if that were to occur. Eliminating the potential for transposon-mediated transposition may depend on the creation of vectors

A.M.Handler/Insect Biochemistry and Molecular Biology34(2004)121–130125

that are immobilized after their initial integration.It is not inconceivable that the?eld release of transgenic insects will depend on use of immobilized vectors, though further studies into transposon behavior may eventually?nd that the risks for re-mobilization of non-autonomous transgenes in the?eld are negligible. However,these conclusions will not be made in the near-term,and they will certainly be relative to the e?ect of the genes being vectored,the host organism, and the area of release.For example,there will be minimal ecological concern for the release of transgenic insects that are sterile for SIT,with slightly more con-cern for CLR insects that are expected to die along with all of their o?spring.For most transgenic releases in the foreseeable future,use of an immobilized vector, and data to support immobilization will present the best opportunity for these programs to go forward.

5.2.Vector immobilization

Transposon vector immobilization will be most sim-ply achieved by the deletion or rearrangement of vector DNA required for mobility.This typically includes the inverted terminal repeats and associated subterminal DNA.For the P element,the minimal DNA require-ments for mobility include138bp from the50terminus and216bp from the30terminus(Mullins et al.,1989). The minimal DNA requirements for mobility of most other transposon vectors are unclear,but at a mini-mum,the terminal TIRs are required in addition to subterminal inverted repeats if they exist.Typically adjacent DNA is required as well,but the actual sequences and length are speci?c to each transposon, and for most elements,the requisite terminal DNA for mobility must be empirically determined.A caveat is that these sequence requirements may di?er for plasmid and chromosomal transposition,and thus vector mod-i?cations should be tested in both contexts. Strategies to immobilize vectors include the internal deletion or rearrangement of subterminal sequences,or chromosomal inversions that displace one of the ter-mini.These manipulations can be achieved by use of recombinase systems such as the FRT/FLP recombi-nase system from the2l plasmid of yeast(Andrews et al.,1985),though other systems such as the bacterio-phage Cre-loxP system(Siegal and Hartl,1996)may be similarly used.A functional FRT site consists of two13 bp inverted repeats separated by an8bp spacer,which e?ciently and speci?cally recombines with other sites in the presence of FLP recombinase.Importantly, speci?city of FRT recombination depends on identical spacer sequences,so that FRT sites with di?erent spacer sequences can be used in the same genome to confer predicted rearrangements(Seneco?and Cox, 1986).Depending upon their location,FRT recombi-nation can occur within and between chromosomes causing translocations,inversions,insertions,duplica-tions,and deletions(Golic and Lindquist,1989;Golic et al.,1997;Golic and Golic,1996).FRT recombi-nation can also occur between plasmid DNA and chro-mosomes allowing insertion of the plasmid into a speci?c chromosomal target site.While there are advantages to using FRT target sites instead of trans-poson insertions for transformation,especially in terms of minimizing variable position e?ects,plasmid inte-gration by FRT recombination has yet to be demon-strated.

5.3.FRT vectors for immobilization

The deletion or rearrangement of sequences required for movement can be achieved by surrounding these sequences with FRT sites as direct or inverted repeats within the vector,respectively.Recombination between the FRT sites would be catalyzed by FLP recombinase introduced by either injection of an FLP helper plas-mid,FLP mRNA,or crosses to strains having a recombinase gene integration.Recombination between FRT direct repeats results only in deletion of sequences within the sites,with the rest of the vector and external marker DNA left intact.A secondary marker may be included within the FRT sites to visibly assess the deletion,though frequencies may be high enough so that PCR can be used to screen lines.Recombination between FRT inverted repeat sites results in inversions that‘‘displace’’or rearrange the critical sequences.This can be achieved within the vector,but can also occur between two independent vector integrations having the FRT s in opposite orientation resulting in a chromo-somal inversion.There are relative advantages and dis-advantages to these approaches in terms of practical implementation in non-drosophilid species.

5.3.1.Internal vector recombination

For internal vector deletions or rearrangements,one FRT site is required within the terminal sequence criti-cal for movement,with another site positioned intern-ally.Recombination can be monitored by having the sites surrounding a visible marker,such as white,that would be deleted by direct repeats,and this could be monitored as a somatic or germ-line event.For rear-rangements mediated by inverted FRT sites,the inter-nal FRT could be placed between the promoter and coding sequence,or within an intron,of a marker gene so that an internal inversion would disrupt its gene expression.A caveat for this strategy is the possibility that insertion of an FRT site into terminal sequences could negatively e?ect vector mobility,preventing the initial transformation event,and this may require test-ing various FRT insertion sites.

126 A.M.Handler/Insect Biochemistry and Molecular Biology34(2004)121–130

5.3.2.Chromosomal inversions

The second approach for vector immobilization relies on chromosomal inversions between two vector integrations.Single FRT sequences would be inserted into two vectors using the same transposon system,but in opposite orientation,or in two di?erent vector trans-posons(e.g.piggyBac and Hermes)in either orien-tation.Host insects would be transformed with the vector pairs and selected for linked integrations on the same chromosome having the FRT sites in an inverted or opposite https://www.wendangku.net/doc/8a10120897.html,e of the same transposon system would require the vectors having the same50–30 (head–tail)orientation resulting in the FRT sites being in opposite orientation.Recombination would result in a conversion of the50terminus of one vector for the30 terminus of the other,yielding head–head and tail–tail vectors,thus immobilizing both https://www.wendangku.net/doc/8a10120897.html,e of di?er-ent vector transposons would be simpler since seq-uential transformations will be more reliable(if same vector is used,the second transformation may de-stabilize the?rst integration).For di?erent transpo-sons,vector orientation would be irrelevant since conversion of either the30or50termini would immobi-lize both vectors,though FRT sites would still have to be in opposite orientation for a chromosomal inversion to occur.An added bene?t of this scheme is that the chromosomal inversion will inhibit normal recombi-nation,so if genes of interest are within the inversion, the strain will be further stabilized.

The advantage to this approach is that the FRT sites should not interfere with vector integration,and if properly positioned,the desired inversion and vector immobilization is highly feasible.The disadvantage to this approach is selecting transformants with linked integrations in the proper orientation.Increasing dis-tance between FRT sites also negatively a?ects recom-bination frequency,with distances greater than30Mb in Drosophila resulting in negligible recombination(a single chromosome arm in Drosophila is~30Mb)(see Rong and Golic,2000).Determining linkage by segre-gation of di?erently marked vectors should not be di?-cult,but determining orientation is more problematic for non-drosophilids.In Drosophila,and additional species that are being sequenced,a determination of vector orientation is possible by sequencing the inser-tion sites and determining chromosomal positions using the genome sequence database(that will also rea?rm linkage).Determining orientation in other insect species is presently not possible,and all lines having linked vectors would have to be tested with recombinase.Identifying successful inversions,how-ever,can be simpli?ed by having the FRT site,in at least one of the vectors,within a secondary marker as described for internal inversions.If placed in a non-translated region(e.g.transcript leader sequence or intron),the FRT inversion should eliminate the marker phenotype.

Another possibility is to use FRT s with a palin-dromic spacer,that would allow recombination in both vector orientations resulting in deletions,as well as inversions.In this strategy,both vector orientations could be used,but chromosomal deletions resulting from direct repeats would probably be lethal,allowing

a rapid selection for the desired inversions.

5.3.3.FLP recombinase

A reliable method of providing recombinase is by crossing the FRT strain to another strain having one or more hsp70-recombinase integrations which is done routinely in Drosophila(see Rong and Golic,2000). Having heat shock regulated recombinase allows some control over the level of recombination and,indeed, since FRT recombination can proceed in both direc-tions,the major caveat to this mechanism is the re-insertion of the excised product which will occur if recombinase activity is not down-regulated.Drosophila experiments have also used injection of FLP mRNA, produced in vitro from a T7promoter,which has the advantage of catalyzing high levels of germ-line recom-bination,without the need for additional inter-strain matings(Rong and Golic,2000).

For either vector deletions or inversions,the expec-ted recombination event would be veri?ed in transfor-mants by PCR to determine vector sequence.Vector immobilization then would be tested by crossing the lines to an appropriate jumpstarter strain having an integrated source of transposase and testing marker stability compared to controls.Testing vector stability as a function of marker phenotype can be assessed in somatic tissue or in the germ-line,though use of the white eye color marker is preferable for somatic events.

6.Epigenetic host genome–vector interactions Unanticipated mobilization of a transgene will cer-tainly a?ect strain stability,but another important con-sideration is epigenetic interactions between the transgene vector and the host genome that can in?u-ence transformant strain stability in terms of its?tness and expression of the desired phenotype.The?rst of these genomic interactions to be considered,that were discussed previously,relate to chromosomal position e?ects such as transcriptional interference and pro-moter suppression.These can be controlled or pre-vented to some extent by the use of transcription terminators and insulator elements,respectively,placed at appropriate sites within the vector(Bell et al.,2001). It is also likely that the e?ect of these interactions would be evident early on,with a?ected transgenic strains being selected against before further use in SIT or CLR programs.

A.M.Handler/Insect Biochemistry and Molecular Biology34(2004)121–130127

Other potential interactions involve transcriptional gene silencing(TGS)and post-transcriptional gene sup-pression(PTGS),and these are of concern since their a?ects may not be apparent in early generations,or may occur discontinuously in a transgenic population (see Fire,1999).These phenomena have been generally attributed to defense mechanisms against invasive nucleic acids in plants,typically from viruses,with double-stranded RNA(dsRNA)eliciting PTGS and TGS resulting from CpG methylation of promoter sequences(see Martienssen and Colot,2001).Transpo-sons are also major targets for methylation that can have a positive a?ect due to enhanced stability result-ing from repressed transposase expression.For defec-tive vectors,however,concern is focused on the silencing or diminished expression of genes within the vector.Gene silencing may also occur at particular insertion sites by a reverse enhancer e?ect resulting from proximal opposite-strand enhancers that promote dsRNA transcription.This could result in silencing of genes within the vector as well as homologous genes at chromosomal loci.Such epigenetic interactions could have a negative in?uence on marker gene expression that would impede identi?cation of transformant organisms,and if it occurred in generations subsequent to primary transformant selection,monitoring of the release program and risk assessment analysis would be seriously a?ected.In a worse case scenario,there could be unintended use or release of‘‘unmarked’’transgenic organisms,especially if marker suppression occurred discontinuously in the population.Similarly,repression of genes of interest in transgenic populations could result in fertile males being used for SIT or loss of con-ditional lethality for CLR,and if undetected,could ser-iously exacerbate the pest problem after release. Genomic interactions could also a?ect strain?tness and viability by mechanisms such as co-suppression or intercellular PTGS spreading,that result in repre-ssion of chromosomal genes vital to normal develop-ment,reproduction,or behavior(see Mlotshwa et al., 2002).Fitness can also be a?ected by transgene integra-tions that promote genomic rearrangements.Some hobo insertion sites are associated with chromosomal breakpoints in D.melanogaster(Lim,1981),and a mariner-related element in humans is associated with a recombination hotspot resulting in duplications and de?ciencies from unequal crossovers(Reiter et al., 1996).

The in?uence of CpG methylation on gene silencing in plants is well-documented,but it is very important to note that,presently,it is unknown if and to what extent this and associated mechanisms a?ect insects. Methylation occurs at very low levels in Drosophila, and it is generally associated with CpT and CpA dinu-cleotide sites during embryogenesis(see Lyko,2001). Any in?uence by methylation or dsRNA on transgene silencing in Drosophila has not been documented, though RNA-mediated repression of the LINE-related I element activity has been suggested(Jensen et al., 1999).It is not unlikely,however,that methylation is more pronounced in some insects,and that transpo-sons are di?erentially a?ected by PTGS,perhaps as a function of the genomic environment.

Su?ce it to say,a high priority for research is the assessment of potential epigenetic e?ects on speci?c vec-tors in speci?c host species.Gene silencing or variable expression would eventually manifest itself during mass-rearing,and until such concerns are deemed incon-sequential,careful monitoring of transgene presence and expression will be critical to program e?cacy and safety.Similar to the way that the?lter rearing system (Fisher and Caceres,2000)controls for low level recom-bination in mass-reared genetic-sexing strains in med?y, a?lter system may be used to maintain transgenic strains with consistent levels of gene expression.

7.Summary

Areas of concern for transgenic insect strains inten-ded for mass-release in SIT and CLR programs prim-arily relate to the stability of the transgene vector and expression of marker genes and genes of interest within the vector.Both these attributes are essential to the maintenance of transgenic strain integrity,so that the desired phenotype is reliably and consistently expres-sed.Vector instability and/or repression of transgene expression would seriously compromise program e?ec-tiveness,possibly exacerbating the targeted problem, and potentially creating environmental risks by the unintended behavior or release of the transgenic strain. Potential vector instability mediated by mobilization or cross-mobilization by related transposon systems may be anticipated by surveys of the host species and asso-ciated organisms for the presence and function of such systems.A more reliable method of managing the potential for transposase-mediated movement is by creating new vectors that can be immobilized post-integration.These would rely on recombination sys-tems that could delete or rearrange internal sequences necessary for transposition.A more daunting concern, however,relates to potential epigenetic interactions between transgene vectors and host genomes as observed in transgenic plants.While such interactions may have the positive bene?t of repressing transgene movement,they also have the potential for repressing or silencing transgene markers and genes of interest so that identi?cation and activity of the transgenic strain becomes https://www.wendangku.net/doc/8a10120897.html,e of insulator elements may pre-vent some types of repression,though continued research into transposon vector systems,potential host genomes,and their interactions will be essential for the e?ective use of transgenic insects for SIT and CLR.

128 A.M.Handler/Insect Biochemistry and Molecular Biology34(2004)121–130

Acknowledgements

Grateful appreciation is extended to Paul Eggleston and Grazyna Zimowska for comments on the manu-script,and support from the USDA-NRI Competitive Grants Program.

References

Allen,M.L.,O’Brochta,D.A.,Atkinson,P.W.,Levesque,C.S.,2001.

Stable,germ-line transformation of Culex quinquefasciatus(Dip-tera:Culicinae).J.Med.Entomol.38,701–710.

Andrews,B.J.,Proteau,G.A.,Beatty,L.G.,Sadowski,P.D.,1985.

The FLP recombinase of the2micron circle DNA of yeast:inter-action with its target sequences.Cell40,795–803.

Atkinson,P.W.,Pinkerton,A.C.,O’Brochta,D.A.,2001.Genetic trans-formation systems in insects.Annu.Rev.Entomol.46,317–346. Atkinson,P.W.,Warren,W.D.,O’Brochta,D.A.,1993.The hobo transposable element of Drosophila can be cross-mobilized in house?ies and excises like the Ac element of maize.Proc.Natl.

https://www.wendangku.net/doc/8a10120897.html,A90,9693–9697.

Avancini,R.M.,Walden,K.K.,Robertson,H.M.,1996.The gen-omes of most animals have multiple members of the Tcl family of transposable elements.Genetica98,131–140.

Bell,A.C.,West,A.G.,Felsenfeld,G.,2001.Insulators and bound-aries:versatile regulatory elements in the eukaryotic genome.

Science291,447–450.

Cary,L.C.,Goebel,M.,Corsaro,H.H.,Wang,H.H.,Rosen,E.,Fra-ser,M.J.,1989.Transposon mutagenesis of baculoviruses:analy-sis of Trichoplusia ni transposon IFP2insertions within the FP-Locus of nuclear polyhedrosis viruses.Virology161,8–17. Catteruccia,F.,Nolan,T.,Loukeris,T.G.,Blass,C.,Savakis,C., Kafatos,F.C.,Crisanti,A.,2000.Stable germline transformation of the malaria mosquito Anopheles stephensi.Nature405, 959–962.

Chal?e,M.,Tu,Y.,Euskirchen,G.,Ward,W.,Prasher,D.C.,1994.

Green?uorescent protein as a marker for gene expression.Science 263,802–805.

Coates,C.J.,Jasinskiene,N.,Miyashiro,L.,James,A.A.,1998.Mari-ner transposition and transformation of the yellow fever mosquito Aedes https://www.wendangku.net/doc/8a10120897.html,A95,3742–3751.

Elick,T.A.,Bauser,C.A.,Principe,N.M.,Fraser,M.J.,1995.PCR analysis of insertion site speci?city,transcription,and structural uniformity of the Lepidopteran transposable element IFP2in the TN-368cell genome.Genetica97,127–139.

Fadool,J.M.,Hartl,D.L.,Dowling,J.E.,1998.Transposition of the mariner element from Drosophila mauritiana in zebra?sh.Proc.

https://www.wendangku.net/doc/8a10120897.html,A95,5182–5186.

Fire, A.,1999.RNA-triggered gene silencing.Trends Genet.15, 358–363.

Fisher,K.,Caceres,C.,2000.A?lter rearing system for mass-reared med?y.In:Tan Penang,K.H.(Ed.),Area-wide Control of Fruit Flies and Other Insects Pests.In:Joint Proceedings of the Inter-national Conference on Area-wide Control of Insect Pests and the Fifth International Symposium on Fruit Flies of Economic Importance.Malaysia,pp.543–550.

Franz,G.,Savakis, C.,1991.Minos,a new transposable element from Drosophila hydei,is a member of the Tc-1-like family of transposons.Nucleic Acids Res.19,6646.

Fraser,M.J.,Smith,G.E.,Summers,M.D.,1983.Acquisition of host-cell DNA-sequences by baculoviruses—relationship between host DNA insertions and FP mutants of Autographa-californica and Galleria-mellonella nuclear polyhedrosis viruses.J.Virol.47, 287–300.Fraser,M.J.,Ciszczon,T.,Elick,T.,Bauser,C.,1996.Precise exci-sion of TTAA-speci?c lepidopteran transposons piggyBac(IFP2) and tagalong(TFP3)from the baculovirus genome in cell lines from two species of Lepidoptera.Insect Mol.Biol.5,141–151. Fryxell,K.J.,Miller,T.A.,1994.Autocidal biological control:a gen-eral strategy for insect control based on genetic transformation with a highly conserved gene.J.Econ.Entomol.85,1240–1245. Golic,K.G.,Lindquist,S.L.,1989.The FLP recombinase of yeast catalyzes site-speci?c recombination in the Drosophila genome.

Cell59,499–509.

Golic,M.M.,Rong,Y.S.,Petersen,R.B.,Lindquist,S.L.,Golic,K.G., 1997.FLP-mediated DNA mobilization to speci?c target sites in Drosophila chromosomes.Nucleic Acids Res.25,3665–3671. Golic,K.G.,Golic,M.M.,1996.Engineering the Drosophila genome: chromosome rearrangements by design.Genetics144,1693–1711. Handler,A.M.,2001.A current perspective on insect gene transfer.

Insect Biochem.Mol.Biol.31,111–128.

Handler,A.M.,2002a.Prospects for using genetic transformation for improved SIT and new biocontrol methods.Genetica116, 137–149.

Handler,A.M.,https://www.wendangku.net/doc/8a10120897.html,e of the piggyBac transposon for germ-line transformation of insects.Insect Biochem.Mol.Biol.32, 1211–1220.

Handler,A.M.,Gomez,S.P.,1996.The hobo transposable element excises and has related elements in tephritid species.Genetics143, 1339–1347.

Handler,A.M.,Harrell,R.A.,1999.Germline transformation of Dro-sophila melanogaster with the piggyBac transposon vector.Insect Mol.Biol.8,449–458.

Handler,A.M.,Harrell,R.A.,2001a.Transformation of the Car-ibbean fruit?y with a piggyBac transposon vector marked with polyubiquitin-regulated GFP.Insect Biochem.Mol.Biol.31, 199–205.

Handler,A.M.,Harrell,R.A.,2001b.Polyubiquitin-regulated DsRed marker for transgenic insects.Biotechniques31,820–828. Handler, A.M.,McCombs,S.D.,2000.The piggyBac transposon mediates germ-line transformation in the Oriental fruit?y and closely related elements exist in its genome.Insect Mol.Biol.9, 605–612.

Handler,A.M.,McCombs,S.D.,Fraser,M.J.,Saul,S.H.,1998.The lepidopteran transposon vector,piggyBac,mediates germline transformation in the Mediterranean fruit?y.Proc.Natl.Acad.

https://www.wendangku.net/doc/8a10120897.html,A95,7520–7525.

Hartl,D.L.,Lohe,A.R.,Lozovskaya,E.R.,1997.Modern thoughts on an ancyent marinere:function,evolution,regulation.Annu.

Rev.Genet.31,337–358.

Heinrich,J.C.,Scott,M.J.,2000.A repressible female-speci?c lethal genetic system for making transgenic insect strains suitable for a sterile-release https://www.wendangku.net/doc/8a10120897.html,A97, 8229–8232.

Heinrich,J.C.,Li,X.,Henry,R.A.,Haack,N.,Stringfellow,L., Heath, A.,Scott,M.J.,2002.Germ-line transformation of the Australian sheep blow?y,Lucilia cuprina.Insect Mol.Biol.11, 1–10.

Higgs,S.,Lewis,D.L.,2000.Green?uorescent protein(GFP)as a marker for transgenic insects.In:Handler,A.M.,James,A.A.

(Eds.),Insect Transgenesis:Methods and Applications.CRC Press,Boca Raton,Florida,pp.93–108.

Horn,C.,Schmid,B.G.M.,Pogoda,F.S.,Wimmer,E.A.,2002.Flu-orescent transformation markers for insect transgenesis.Insect Biochem.Mol.Biol.32,1221–1235.

Horn,C.,Wimmer,E.A.,2003.A transgene-based,embryo-speci?c lethality system for insect pest management.Nat.Biotechnol.21, 64–70.

Jasinskiene,N.,Coates,C.J.,Benedict,M.Q.,Cornel,A.J.,Ra?erty,

C.S.,James,A.A.,Collins,F.H.,1998.Stable,transposon medi-

ated transformation of the yellow fever mosquito,Aedes aegypti,

A.M.Handler/Insect Biochemistry and Molecular Biology34(2004)121–130129

using the Hermes element from the house?y.Proc.Natl.Acad.

https://www.wendangku.net/doc/8a10120897.html,A95,3743–3747.

Jasinskiene,N.,Coates,C.J.,James,A.A.,2000.Structure of Hermes integrations in the germline of the yellow fever mosquito,Aedes aegypti.Insect Mol.Biol.9,11–18.

Jehle,J.A.,Nickel,A.,Vlak,J.M.,Backhaus,H.,1998.Horizontal escape of the novel Tc1-like lepidopteran transposon TCp3.2into Cydia pomonella granulovirus.J.Mol.Evol.46,215–224. Jensen,S.,Gassarna,M.P.,Heidmann,T.,1999.Cosuppression of I transposon activity in Drosophila by I-containing sense and anti-sense transgenes.Genetics153,1767–1774.

Klinakis,A.G.,Loukeris,T.G.,Pavlopoulos,A.,Savakis,C.,2000.

Mobility assays con?rm the broad host-range activity of the Minos transposable element and validate new transformation tools.Insect Mol.Biol.9,269–275.

Lim,J.K.,1981.Site-speci?c intrachromosomal rearrangements in Drosophila melanogaster:cytogenetic evidence for transposable elements.Cold Spring Harbor Symp.Quant.Biol.45,553–560. Lobo,N.,Li,X.,Fraser,Jr.M.J.,1999.Transposition of the piggy-Bac element in embryos of Drosophila melanogaster,Aedes aegypti and Trichoplusia ni.Mol.Gen.Genet.261,803–810.

Loukeris,T.G.,Livadaras,I.,Arca, B.,Zabalou,S.,Savakis, C., 1995.Gene transfer into the Med?y,Ceratitis capitata,with a Drosophila hydei transposable element.Science270,2002–2005. Lyko,F.,2001.DNA methylation learns to?y.Trends Genet.17, 169–172.

Martienssen,R.A.,Colot,V.,2001.DNA methylation and epigenetic inheritance in plants and?lamentous fungi.Science293, 1070–1074.

Matz,M.V.,Fradkov,A.F.,Labas,Y.A.,Savitsky,A.P.,Zaraisky,

A.G.,Markelov,M.L.,Lukyanov,S.A.,1999.Fluorescent pro-

teins from nonbioluminescent Anthozoa species.Nat.Biotechnol.

17,969–973.

Medhora,M.M.,MacPeek,A.H.,Hartl,D.L.,1988.Excision of the Drosophila transposable element mariner:identi?cation and char-acterization of the Mos factor.EMBO J.7,2185–2189.

Michel,K.,Stamenova,A.,Pinkerton,A.C.,Franz,G.,Robinson,

A.S.,Gariou-Papalexiou, A.,Zacharopoulou, A.,O’Brochta,

D.A.,Atkinson,P.W.,2001.Hermes-mediated germ-line trans-

formation of the Mediterranean fruit?y Ceratitis capitata.Insect Mol.Biol.10,155–162.

Mlotshwa,S.,Voinnet,O.,Mette,M.F.,Matzke,M.,Vaucheret,H., Ding,S.W.,Pruss,G.,Vance,V.B.,2002.RNA silencing and the mobile silencing signal.Plant Cell14(Suppl.),S289–S301. Mullins,M.C.,Rio, D.C.,Rubin,G.M.,1989.Cis-acting DNA sequence requirements for P-element transposition.Cell3,729–738. O’Brochta,D.A.,Atkinson,P.W.,Lehane,M.J.,2000.Transform-ation of Stomoxys calcitrans with a Hermes gene vector.Insect Mol.Biol.9,531–538.

O’Brochta,D.A.,Warren,W.D.,Saville,K.J.,Atkinson,P.W.,1994.

Interplasmid transposition of Drosophila hobo elements in non-drosophilid insects.Mol.Gen.Genet.244,9–14.

O’Brochta,D.A.,Warren,W.D.,Saville,K.J.,Atkinson,P.W.,1996.

Hermes,a functional non-drosophilid insect gene vector.Genetics 142,907–914.

Ochman,H.,Ayala,F.J.,Hartl,D.L.,https://www.wendangku.net/doc/8a10120897.html,e of polymerase chain reaction to amplify segments outside boundaries of known sequences.Methods Enzymol.218,309–321.

Peloquin,J.J.,Thibault,S.T.,Staten,R.,Miller,T.A.,2000.Germ-line transformation of pink bollworm(Lepidoptera:Gelechiidae) mediated by the piggyBac transposable element.Insect Mol.Biol.

9,323–333.Perera,O.P.,Harrell,R.A.,Handler,A.M.,2002.Germ-line trans-formation of the South American malaria vector,Anopheles albi-manus,with a piggyBac/EGFP transposon vector is routine and highly e?cient.Insect Mol.Biol.11,291–297.

Reiter,L.T.,Murakami,T.,Koeuth,T.,Pentao,L.,Muzny,D.M., Gibbs,R.A.,Lupski,J.R.,1996.A recombination hotspot responsible for two inherited peripheral neuropathies is loca-ted near a mariner transposon-like element.Nat.Genet.12, 288–297.

Robertson,H.M.,Lampe,D.J.,1995.Recent horizontal transfer of a mariner transposable element among and between Diptera and Neuroptera.Mol.Biol.Evol.12,850–862.

Robertson,H.M.,MacLeod,E.G.,1993.Five major subfamilies of mariner transposable elements in insects,including the Mediterra-nean fruit?y,and related arthropods.Insect Mol.Biol.2, 125–139.

Rong,Y.,Golic,K.,2000.Site-speci?c recombination for the genetic manipulation of transgenic insects.In:Handler, A.M.,James,

A.A.(Eds.),Insect Transgenesis:Methods and Applications.

CRC Press,Boca Raton,Florida,pp.53–75.

Rubin,G.M.,Spradling, A.C.,1982.Genetic transformation of Drosophila with transposable element vectors.Science218, 348–353.

Sarkar,A.,Collins,F.H.,2000.Eye color genes for selection of trans-genic insects.In:Handler, A.M.,James, A.A.(Eds.),Insect Transgenesis:Methods and Applications.CRC Press,Boca Raton,Florida,pp.93–108.

Sarkar,A.,Coates,C.J.,Whyard,S.,Willhoeft,U.,Atkinson,P.W., O’Brochta,D.A.,1997.The Hermes element from Musca domes-tica can transpose in four families of cyclorrhaphan?ies.Genetica 99,15–29.

Seneco?,J.F.,Cox,M.M.,1986.Directionality in FLP protein-pro-moted site-speci?c recombination is mediated by DNA–DNA pairing.J.Biol.Chem.261,7380–7386.

Sherman,A.,Dawson,A.,Mather,C.,Gilhooley,H.,Li,Y.,Mitch-ell,R.,Finnegan,D.,Sang,H.,1998.Transposition of the Droso-phila element mariner into the chicken germ line.Nat.Biotechnol.

16,1050–1053.

Siegal,M.L.,Hartl, D.L.,1996.Transgene coplacement and high e?ciency site-speci?c recombination with the Cre/loxP system in Drosophila.Genetics144,715–726.

Sundararajan,P.,Atkinson,P.W.,O’Brochta,D.A.,1999.Transpos-able element interactions in insects:crossmobilization of hobo and Hermes.Insect Mol.Biol.8,359–368.

Thomas, D.T.,Donnelly, C.A.,Wood,R.J.,Alphey,L.S.,2000.

Insect population control using a dominant,repressible,lethal genetic system.Science287,2474–2476.

Warren,W.D.,Atkinson,P.W.,O’Brochta,D.A.,1994.The Hermes transposable element from the house?y,Musca domestica,is a short inverted repeat-type element of the hobo,Ac,and Tam3

(h AT)element family.Genet.Res.Camb.64,87–97. Yoshiyama,M.,Honda,H.,Kimura,K.,2000.Successful transform-ation of the house?y,Musca domestica(Diptera:Muscidae)with the transposable element,mariner.Appl.Entomol.Zool.35,321–325. Zagoraiou,L.,Drabek,D.,Alexaki,S.,Guy,J.A.,Klinakis,A.J., Langeveld,A.,Skavdis,G.,Mamalaki,C.,Grosveld,F.,Savakis,

C.,2001.In vivo transposition of Minos,a Drosophila mobile

element,in mammalian https://www.wendangku.net/doc/8a10120897.html,A98, 11474–11478.

Zhao,Y.G.,Eggleston,P.,1998.Transformation of an Anopheles gambiae cell line mediated by the Hermes mobile genetic element.

Insect Biochem.Mol.Biol.28,213–219.

130 A.M.Handler/Insect Biochemistry and Molecular Biology34(2004)121–130

2018全国计算机等级考试一级考试试题库

2018年全国计算机等级考试一级考试试题库 0401) 下列关于世界上第一台电子计算机ENIAC的叙述中，错误的是 A)它是1946年在美国诞生的 B)它主要采用电子管和继电器 C)它是首次采用存储程序控制使计算机自动工作 D)它主要用于弹道计算答案：C 0402) 一个字长为8位的无符号二进制整数能表示的十进制数值范围是 A)0-256 B)0-255 C)1-256 D)1-255 答案：B 0403) 二进制数1001001转换成十进制数是 A)72 B)71 C)75 D)73 答案：D 0404) 十进制数90转换成无符号二进制数是 A)1011010 B)1101010 C)1011110 D)1011100 答案：A 0405) 标准ASCII码用7位二进制位表示一个字符的编码，其不同的编码共有 A)127个 B)128个 C)256个 D)254个答案：B 0406) 根据国标GB2312-80的规定，总计有各类符号和一、二级汉字编码 A)7145个 B)7445个 C)3008个 D)3755个答案：B 0407) 运算器的主要功能是进行 A)算术运算 B)逻辑运算 C)加法运算 D)算术和逻辑运算答案：D 0408) 下列各存储器中，存取速度最快的是 A)CD-ROM

C)软盘 D)硬盘答案：B 0409) 假设某台式计算机的内存储器容量为256MB，硬盘容量为20GB。硬盘的容量是内存容量的 A)40倍 B)60倍 C)80倍 D)100倍答案：C 0410) 在外部设备中，扫描仪属于 A)输出设备 B)存储设备 C)输入设备 D)特殊设备答案：C 0411) 计算机能直接识别的语言是 A)高级程序语言 B)机器语言 C)汇编语言 D)C++语言答案：B 0412) 下列关于计算机病毒的叙述中，错误的是 A)计算机病毒具有潜伏性 B)计算机病毒具有传染性 C)感染过计算机病毒的计算机具有对该病毒的免疫性 D)计算机病毒是一个特殊的寄生程序答案：C 0413) Internet网中不同网络和不同计算机相互通讯的基础是 A)ATM B)TCP/IP C)Novell D)X.25 答案：B 0414) 已知一汉字的国标码是5E38，其内码应是 A)DEB8 B)DE38 C)5EB8 D)7E58 答案：A 0415) 已知三个字符为：a、X和5，按它们的ASCII码值升序排序，结果是 A)5,a,X B)a,5,X C)X,a,5 D)5,X,a 答案：D 0416) 度量计算机运算速度常用的单位是

项目管理软件project操作手册

项目管理软件p r o j e c t 操作手册 SANY标准化小组 #QS8QHH-HHGX8Q8-GNHHJ8-HHMHGN#

项目管理软件PROJECT 2010操作实例 Project工具一般用来管理一个项目，制定项目的执行计划。项目的三要素到底是时间,成本和范围。如何使用Project，必须明确如下几项： A，做什么事 B，这些事的时间有什么要求 C，要做的事之间有什么关系 D，做这些事的人员有谁 E，人员有特别的时间要求下面举一个具体例子，了解一下项目管理软件的操作过程。项目名称：电石炉主体安装工程项目的开始日期：2016年2月1日项目的结束日期：2016年6月11日日程排定方法：从项目的开始之日起项目日历：标准日历工作时间：每周工作7天，每天8小时项目目标：确保设备按期投产。可衡量结果：达到业主产量要求。一、任务清单列表：

大纲级别任务名称工期（工作日） 1（摘要）电石炉主体安装进度计划90 安装下料槽4 安装底部装置3 安装铜母线9 安装黄铜管3 安装电极壳3 安装电极柱2 安装下料仓2 安装下料管4 安装环形加料机10 安装炉顶加料皮带10 。。。。。。。。。。。电气安装57 安装低压配电室电气柜4 安装中控室电气柜、控制台5 安装3－4层电气柜3 安装电缆槽30 。。。。。。。。。。液压安装29 调试27 2配合投料 3投产保驾 4设备验收 5结束二、资源可使用情况：资源名称最大单位标准工资率每次使用成本成本累算方式钳工4￥工时按比例电工4￥工时按比例管工6￥工时按比例电焊工12￥工时按比例起重工2￥工作日按比例力工42￥工作日按比例电动葫芦卷扬机3￥自卸车1￥三、任务之间的相关性：放线→设备机、电、液安装→调试→投产保驾→竣工验收→结束四、具体操作步骤

Project2010中文版基础教程

Project 2010中文版基础教程了解Project 2010 Microsoft Project 2010 推出的新功能能够显著地改善您管理、计划和查看项目的方式。如果您已经熟悉早期版本的Microsoft Project，则单击此文档中的链接可获得Project 2010 中新功能和不同之处的相关信息。了解Project 2010 新增功能概括了解Project 2010 中的新功能，包括用户控制的计划、日程表视图、工作组计划程序以及功能区。用户控制的计划（即将推出）如手动计划任务、非活动任务、自顶向下计划一类的新功能使您更能随心撑控。了解功能区使用功能区可快速查找命令；查看针对项目管理使用此功能的方式。键盘快捷方式随着Project 2010 中增加了功能区，一些您最喜爱的键盘快捷方式可能已发生变化。项目规范（即将推出）弄清您可在项目计划中包括多少任务、资源、基线和其他元素。Microsoft Project 2010 的新增功能改进的界面 Project 2010 引入了多个能够显著改善查看和使用项目的功能。功能区简介当您首次启动Project 2010 时，您可能会对看到的内容感到惊讶。“功能区”取代了菜单和工具栏，它可帮助您快速找到完成任务所需的命令。这些命令按逻辑分组，并集中在各个选项卡下面。对于Project 2010，功能区上的所有选项卡和组都是可完全自定义的。如果您的组织具有一些业务上特有的功能，则可以将这些功能组织到独自的功能区选项卡上。欢迎使用Backstage 单击“文件”选项卡您将会转到Backstage，这是一个用于管理您的项目文件的一站式图形目的地。Backstage 包含可用来打开、保存和打印项目文件的基本命令，这些命令与

2011年3月全国计算机考试真题及答案

2011年3月全国计算机等级考试二级C++真题及答案解析一、选择题(每小题2分，共70分) 下列各题A、B、C、D四个选项中，只有一个选项是正确的，请将正确选项涂写在答题卡相应位置上，答在试卷上不得分。 1. 下列关于栈叙述正确的是 A. 栈顶元素最先能被删除 B. 栈顶元素最后才能被删除 C. 栈底元素永远不能被删除 D. 上述三种说法都不对答案：A 解析：在栈中，允许插入与删除的一端称为栈顶，而不允许插入与删除的另一端称为栈底。栈顶元素总是最后被插入的元素，从而也是最先能被删除的元素；栈底元素总是最先被插入的元素，从而也是最后才能被删除的元素。故本题选A。 2. 下列叙述中正确的是 A. 有一个以上根结点的数据结构不一定是非线性结构 B. 只有一个根结点的数据结构不一定是线性结构 C. 循环链表是非线性结构 D. 双向链表是非线性结构答案：B 解析：如果一个非空的数据结构满足以下两个条件：（1）有且只有一个根结点；（2）每个结点最多有一个前件，也最多有一个后件。则称该数据结构为线性结构。如果一个数据结构不是线性结构，则称之为非线性结构,故A项错误。有一个根结点的数据结构不一定是线性结构，如二叉树，B项说法正确。循环链表和双向链表都属于线性链表，故C、D项错误。 3. 某二叉树共有7个结点，其中叶子结点只有1个，则该二叉树的深度为（假设根结点在第1层）

A. 3 B. 4 C. 6 D. 7 答案：D 解析：根据二叉树的性质：在任意一棵二叉树中，度为0的结点（即叶子结点）总是比度为2的结点多一个。所以n2=0，由n=n0+n1+n2可得n1=6，即该二叉树有6个度为1的结点，可推出该二叉树的深度为7。 4. 在软件开发中，需求分析阶段产生的主要文档是 A. 软件集成测试计划 B. 软件详细设计说明书 C. 用户手册 D. 软件需求规格说明书答案：D 解析：软件需求规格说明书是需求分析阶段的最后成果，是软件开发中的重要文档之一。 5. 结构化程序所要求的基本结构不包括 A. 顺序结构 B. GOTO跳转 C. 选择（分支）结构 D. 重复（循环）结构答案：B 解析：结构化程序设计的三种基本控制结构为:顺序结构、选择结构和重复结构。 6. 下面描述中错误的是 A. 系统总体结构图支持软件系统的详细设计 B. 软件设计是将软件需求转换为软件表示的过程

2013年全国计算机等级考试二级C语言考试大纲及重点

2013年全国计算机等级考试二级C语言考试大纲 ◆基本要求 1.熟悉V isual C++ 6.0 集成开发环境。 2.掌握结构化程序设计的方法，具有良好的程序设计风格。 3.掌握程序设计中简单的数据结构和算法并能阅读简单的程序。 4.在V isual C++ 6.0 集成环境下，能够编写简单的C程序，并具有基本的纠错和调试程序的能力 ◆考试内容一、C语言程序的结构 1.程序的构成，main函数和其他函数。 2.头文件，数据说明，函数的开始和结束标志以及程序中的注释。 3.源程序的书写格式。 4.C语言的风格。二、数据类型及其运算 1.C的数据类型(基本类型，构造类型，指针类型，无值类型)及其定义方法。 2.C运算符的种类、运算优先级和结合性。 3.不同类型数据间的转换与运算。 4.C表达式类型(赋值表达式，算术表达式，关系表达式，逻辑表达式，条件表达式，逗号表达式)和求值规则。三、基本语句 1.表达式语句，空语句，复合语句。 2.输入输出函数的调用，正确输入数据并正确设计输出格式。四、选择结构程序设计 1.用if语句实现选择结构。 2.用switch语句实现多分支选择结构。 3.选择结构的嵌套。五、循环结构程序设计 1.for循环结构。 2.while和do-while循环结构。 3.continue语句和break语句。 4.循环的嵌套。六、数组的定义和引用 1.一维数组和二维数组的定义、初始化和数组元素的引用。 2.字符串与字符数组。七、函数 1.库函数的正确调用。 2.函数的定义方法。 3.函数的类型和返回值。 4.形式参数与实在参数，参数值传递。

2013年全国计算机等级二级C语言模拟试题及答案

2013年全国计算机等级二级C 语言模拟试题及答案

2013年3月份全国计算机等级二级C语言试题及答案一、填空题 1、C语言中基本的数据类型有：__________、__________ 、__________ 。 2、C语言中普通整型变量的类型说明符为__________，在内存中占__________字节，有符号普通整型的数据范围是__________。 3、整数-35在机内的补码表示为__________。 4、执行下列语句int a=8; a+=a-=a*a; 后，a的值是__________ 。 5、有如下语句：char A[ ]={”I am a student”}; 该字符串的长度是__________，A[3]=__________ 。 6、符号”a”和’a’的区别是__________。 7、所谓“指针”就是__________ 。 “&”运算符的作用是__________。 “*”运算符的作用是__________ 。 8、有如下输入语句：scanf(“a=%d,b=%d,c=%d”,&a,&b,&c);为使变量a的值为1，b的值为3，c的值为2，从键盘输入数据的正确形式应是__________。二、选择题 1、设整型变量a为5，使b不为2的表达式是（）。 A. b=a/2 B. b=6-(--a) C. b=a%2 D. b=a>3?2:1 2、为了避免嵌套的条件分支语句if-else的二义性，C语言规定：C程序中的else总是与（）组成配对关系。 A. 缩排位置相同的if B. 在其之前未配对的if C. 在其之前未配对的最近的if D.同一行上的if 3、以下程序的输出结果是( )。 int x=10,y=10; printf(“%d%d\n”,x--,--y); A. 10 10 B. 9 9 C. 9 10 D. 10 9 4、设A为存放（短）整型的一维数组，如果A的首地址为P，那么A中第i 个元素的地址为（）。A．P+i*2 B. P+(i-1)*2

全国计算机等级考试一级试题

一、选择题 1、以下名称是手机中的常用软件，属于系统软件的是（B ）。 A) 手机QQ B) android C) Skype D) 微信【解析】Andriod是手机操作系统，属于系统软件，直接排除A、C、D，答案选择B。 2、计算机操作系统通常具有的五大功能是（ C ）。 A) CPU管理、显示器管理、键盘管理、打印机管理和鼠标器管理 B) 硬盘管理、软盘驱动器管理、CPU的管理、显示器管理和键盘管理 C) 处理器(CPU)管理、存储管理、文件管理、设备管理和作业管理 D) 启动、打印、显示、文件存取和关机【解析】操作系统通常应包括下列五大功能模块：处理器管理、作业管理、存储器管理、设备管理、文件管理。 3、造成计算机中存储数据丢失的原因主要是( D ）。 A) 病毒侵蚀、人为窃取 B) 计算机电磁辐射 C) 计算机存储器硬件损坏 D) 以上全部【解析】造成计算机中存储数据丢失的原因主要是：病毒侵蚀、人为窃取、计算机电磁辐射、计算机存储器硬件损坏等等。因此答案选择D选项。 4、下列选项不属于"计算机安全设置"的是（ C ）。 A) 定期备份重要数据 B) 不下载来路不明的软件及程序 C) 停掉Guest 帐号

D) 安装杀（防）毒软件【解析】对于信息系统的使用者来说，维护信息安全的措施主要包括保障计算机及网络系统的安全，预防计算机病毒以及预防计算机犯罪等内容。在日常的信息活动中，我们应注意以下几个方面:①尊重知识产权，支持使用合法原版的软件，拒绝使用盗版软件；②平常将重要资料备份；③不要随意使用来路不明的文件或磁盘，若需要使用，要先用杀毒软件扫描；④随时注意特殊文件的长度和使用日期以及内存的使用情况；⑤准备好一些防毒、扫毒和杀毒的软件，并且定期使用。A、B、D选项都是属于安全设置的措施，C选项关于账号的停用不属于该范畴，因此选择C选项。 5、已知英文字母m的ASCII码值为6DH ，那么ASCII码值为71H的英文字母是（D ）。 A) M B) j C) p D) q 【解析】6DH为16进制（在进制运算中，B代表的是二进制数，D表示的是十进制数，O表示的是八进制数，H表示的是十六进制数）。m的ASCII码值为6DH，用十进制表示即为6×16+13=109（D在10进制中为13）。q的ASCII码值在m的后面4位，即是113 ，对应转换为16进制，即为71H，因此答案选择D。 6、一个汉字的内码长度为2个字节，其每个字节的最高二进制位的值依次分别是（D ）。 A) 0，0 B) 0，1 C) 1，0 D) 1，1 【解析】国标码是汉字信息交换的标准编码，但因其前后字节的最高位为0，与ASCII 码发生冲突，于是，汉字的机内码采用变形国标码，其变换方法为：将国标码的每个字节都加上128，即将两个字节的最高位由0改1，其余7位不变，因此机内码前后

2011年全国计算机等级二级C语言模拟试题及答案(1)

1、C语言中基本的数据类型有：__________、__________ 、__________ 。 2、C语言中普通整型变量的类型说明符为__________，在内存中占__________字节，有符号普通整型的数据范围是__________。 3、整数-35在机内的补码表示为__________。 4、执行下列语句int a=8; a+=a-=a*a; 后，a的值是__________ 。 5、有如下语句：char A[ ]={”I am a student”}; 该字符串的长度是__________，A[3]=__________ 。 6、符号”a”和’a’的区别是__________。 7、所谓“指针”就是__________ 。 “&”运算符的作用是__________。 “*”运算符的作用是__________ 。 8、有如下输入语句：scanf(“a=%d,b=%d,c=%d”,&a,&b,&c);为使变量a的值为1，b的值为3，c的值为2，从键盘输入数据的正确形式应是__________。二、选择题 1、设整型变量a为5，使b不为2的表达式是（）。 A. b=a/2 B. b=6-(--a) C. b=a%2 D. b=a>3?2:1 2、为了避免嵌套的条件分支语句if-else的二义性，C语言规定：C程序中的else总是与（）组成配对关系。 A. 缩排位置相同的if B. 在其之前未配对的if C. 在其之前未配对的最近的if D.同一行上的if 3、以下程序的输出结果是( )。 int x=10,y=10; printf(“%d%d\n”,x--,--y); A. 10 10 B. 9 9 C. 9 10 D. 10 9 4、设A为存放（短）整型的一维数组，如果A的首地址为P，那么A中第i 个元素的地址为（）。A．P+i*2 B. P+(i-1)*2 C. P+(i-1) D. P+i 5、选出下列标识符中不是合法的标识符的是（）。 A. hot_do B. cat1

Project操作手册打印版

使用Project编制进度计划 1.三套时间的概念与运用 1.1. 比较基准时间（）项目进度计划排定后，经审核通过，即可成为指导整个项目施工过程的基准。我们将这一计划中的时间明确记录下来，固定成为所谓的‘基准时间’。这一时间将成为在后续工作中进行对比分析偏差的‘原点’。 1.2. 实际时间在施工的实际过程中，生产人员根据现场当前实际开工的分项工程，和完成的分项工程，在进度计划中填写‘实际时间’，这一组时间是现实的客观记录。 1.3. 开始/完成时间（计划时间）这一计划在最初是和‘基准时间’完全相同的。过程中，随着每一次填写分项工程实际开始时间或结束时间，我们需要马上重新排定进度计划。比如，一个分项工作完成了，我们有了实际完成时间。根据原先确定的各分项工程间的逻辑关系和计划耗用时间，我们可以重新排定全部计划。完成时间=完成部分的实际用时+未完成部分的基准用时 2.进度计划的操作步骤 2.1 建立新项目； 2.2 修改日历； 2.3启用新日历； 2.4添加任务名称; 2.5 添加任务工期； 2.6 建立关系链接； 2.7设定任务间的关系； 2.8 建立比较基准； 2.9 增加比较基准时间和实际时间； 2.10更新任务； 2.1建立新项目 2.1.1 步骤：文件→新建→空白项目

2.1.2显示项目摘要任务信息

2.2修改日历 Project默认每周开始于周日，工作时间一般周六、周日为非工作时间。而建筑工程中每周开始于周一，编制进度计划是不考虑周六、周日的。所以要将Project中的默认工作时间进行更改，将周六、周日改为非默认工作时间。注：非工作时间在Project当中为灰色显示，工作时间为白色显示操作步骤:

全国计算机等级考试一级试题及答案

全国计算机等级考试一级试题及答案(25套) 一、选择题(每题1分，共20分) D (1)世界上第一台计算机诞生于哪一年 A) 1945年B)1956年C)1935年D)1946年 D( 2)第4代电子计算机使用的电子元件是 A)晶体管B)电子管C)中、小规模集成电路D)大规模和超大规模集成电路 D( 3)二进制数110000转换成十六进制数是 A) 77 B) D7 C) 7 D ) 30 A( 4)与十进制数4625等值的十六进制数为 A)1211 B) 1121 C) 1122 D) 1221 C( 5)二进制数110101对应的十进制数是 A)44 B) 65 C ) 53 D ) 74 C (6)在24X 24点阵字库中，每个汉字的字模信息存储在多少个字节中 A)24 B) 48 C ) 72 D ) 12 A (7)下列字符中，其ASCII码值最小的是 A) A B) a C ) k D ) M C (8)微型计算机中，普遍使用的字符编码是 A)补码B)原码C) ASCII码D)汉字编码 C( 9)网络操作系统除了具有通常操作系统的4大功能外，还具有的功能是 A)文件传输和远程键盘操作B)分时为多个用户服务C)网络通信和网络资源共享D)远程源程序开发 C (10 )为解决某一特定问题而设计的指令序列称为 A)文件B)语言C)程序D)软件 C (11)下列4条叙述中，正确的一条是 A)计算机系统是由主机、外设和系统软件组成的 B)计算机系统是由硬件系统和应用软件组成的

C)计算机系统是由硬件系统和软件系统组成的 D)计算机系统是由微处理器、外设和软件系统组成的 B（ 12）两个软件都属于系统软件的是 A）DOS口Excel B ）DOS口UNIX C）UNIX ffi WPS D Wore和Linux A （13）用数据传输速率的单位是 A）位/秒B）字长/秒C ）帧/秒D）米/秒 A（14）下列有关总线的描述，不正确的是 A）总线分为内部总线和外部总线B ）内部总线也称为片总线 C）总线的英文表示就是Bus D）总线体现在硬件上就是计算机主板 B （15）在Window环境中，最常用的输入设备是 A）键盘B）鼠标C）扫描仪D ）手写设备 D （16）下列叙述中，正确的是 A）计算机的体积越大，其功能越强 B）CD-RO M容量比硬盘的容量大 C）存储器具有记忆功能，故其中的信息任何时候都不会丢失 D）CPU!中央处理器的简称 B （17）已知双面高密软磁盘格式化后的容量为，每面有80个磁道，每个磁道有15个扇区，那么每个扇区的字节数是 A）256B B）512B C）1024B D）128B C （18）下列属于计算机病毒特征的是 A）模糊性B）高速性C）传染性D）危急性 A （19）下列4条叙述中，正确的一条是 A）二进制正数原码的补码就是原码本身 B）所有十进制小数都能准确地转换为有限位的二进制小数 C）存储器中存储的信息即使断电也不会丢失 D）汉字的机内码就是汉字的输入码 A（20）下列4条叙述中，错误的一条是 A）描述计算机执行速度的单位是MB B）计算机系统可靠性指标可用平均无故障运行时间来描述 C）计算机系统从故障发生到故障修复平均所需的时间称为平均修复时间

全国计算机等级考试的等级划分与内容分别是什么

全国计算机等级考试的等级划分与内容分别是什么,谢谢最佳答案全国的计算级等级考试有4个等级。一级：考核微型计算机基础知识和使用办公自动化软件及因特网（Internet）的基本技能。要求掌握字、表处理（Word）、电子表格（Excel）和演示文稿（PowerPoint）等办公自动化（Office）软件的使用及因特网（Internet）应用的基本技能，具备从事机关、企事业单位文秘和办公信息计算机化工作的能力。二级：考核计算机基础知识和使用一种高级计算机语言（包括JAVA、C、C++、ACCESS、Visual Basic、Visual FoxPro）编写程序以及上机调试的基本技能。要求能够使用计算机高级语言编写程序和调试程序，可以从事计算机程序的编制工作、初级计算机教学培训工作以及计算机企业的业务和营销工作。三级：分为“PC技术”、“信息管理技术”、“数据库技术”和“网络技术”四个类别。“PC 技术”考核PC机硬件组成和Windows操作系统的基础知识以及PC机使用、管理、维护和应用开发的基本技能。“信息管理技术”考核计算机信息管理应用基础知识及管理信息系统项目和办公自动化系统项目开发、维护的基本技能。“数据库技术”考核数据库系统基础知识及数据库应用系统项目开发和维护的基本技能。“网络技术”考核计算机网络基础知识及计算机网络应用系统开发和管理的基本技能。四级：考核计算机专业基本知识以及计算机应用项目的分析设计、组织实施的基本技能。四级证书表明持有人掌握计算机的基础理论知识和专业知识，熟悉软件工程、数据库和计算机网络的基本原理和技术，具备从事计算机信息系统和应用系统开发和维护的能力。。

2011年3月全国计算机等级考试二级Access笔试试题及参考答案

2011年3月全国计算机等级考试二级Access笔试试题及参考答案 2011年3月全国计算机等级考试二级Access笔试试题一、选择题 (1)下列关于栈叙述正确的是 A)栈顶元素最先能被删除 B)栈顶元素最后才能被删除 C)栈底元素永远不能被删除 D)以上三种说法都不对 (2)下列叙述中正确的是 A)有一个以上根结点的数据结构不一定是非线性结构 B)只有一个根结点的数据结构不一定是线性结构 C)循环链表是非线性结构 D)双向链表是非线性结构 (3)某二叉树共有7个结点，其中叶子结点只有1个，则该二叉树的深度为(假设根结点在第1层) A)3 B)4 C)6 D)7 (4)在软件开发中，需求分析阶段产生的主要文档是 A)软件集成测试计划 B)软件详细设计说明书 C)用户手册 D)软件需求规格说明书 (5)结构化程序所要求的基本结构不包括 A)顺序结构 B)GOTO跳转 C)选择(分支)结构 D)重复(循环)结构 (6)下面描述中错误的是 A)系统总体结构图支持软件系统的详细设计 B)软件设计是将软件需求转换为软件表示的过程 C)数据结构与数据库设计是软件设计的任务之一 D)PAD图是软件详细设计的表示工具 (7)负责数据库中查询操作的数据库语言是

A)数据定义语言 B)数据管理语言 C)数据操纵语言 D)数据控制语言 (8)一个教师可讲授多门课程，一门课程可由多个教师讲授。则实体教师和课程间的联系是 A)1:1联系 B)1:m联系 C)m:1联系 D)m:n联系 (9)有三个关系R、S和T如下：则由关系R和S得到关系T的操作是 A)自然连接 B)交 C)除 D)并 (10)定义无符号整数类为UInt,下面可以作为类UInt实例化值的是 A)-369 B)369 C)0.369 D)整数集合{1,2,3,4,5} (11)在学生表中要查找所有年龄大于30岁姓王的男同学，应该采用的关系运算是 A)选择 B)投影 C)联接 D)自然联接 (12)下列可以建立索引的数据类型是 A)文本

2011年9月全国计算机等级考试四级数据库工程师真题及答案

2011年9月全国计算机等级考试四级数据库工程师真题及答案百手整理起驾为您一、选择题（每小题1分，共40分）（1）下列不属于宽带城域网QoS技术的是 A）密集波分复用DWDM B）区分服务DiffServ C）资源预留RSVP D）多协议标记交换MPLS 答案：A （2）WLAN标准802.11a将传输速率提高到 A）5.5Mbps B）11Mbps C）54Mbps D）100Mbp 答案：C （3）ITU标准OC-12的传输速率为 A）51.84Mbps B）155.52Mbps C）622.08Mbps D）9.95328Gbps 答案：C （4）下列关于网络接入技术和方法的描述中，错误的是 A）“三网融合”中的三网是指计算机网络、电信通信网和广播电视网 B）宽带接入技术包括xDSL、HFC、SDH、无线接入等 C）无线接入技术主要有WLAN、WMAN等 D）Cable Modem的传输速率可以达到10～36Mbps 答案：C （5）当服务器组中一台主机出现故障，该主机上运行的程序将立即转移到组内其他主机。下列技术中能够实现上述需求的是 A）RAID B）Cluster C）RISC D）CISC 答案：B （6）下列不属于路由器性能指标的是 A）吞吐量B）丢失率 C）延时与延时抖动D）最大可堆叠数答案：D （7）若服务器系统年停机时间为55分钟，那么系统可用性至少达到 A）99％B）99.9％C）99.99％D）99.999％答案：C （8）IP地址块58.192.33.120/29的子网掩码可写为 A）255.255.255.192 B）255.255.255.224 C）255.255.255.240 D）255.255.255.248 答案：D

(完整word版)2019年全国计算机等级考试一级上机Word练习题汇总,推荐文档

2011年全国计算机等级考试一级上机Word练习题汇总第1题、 ****** 本套题共有2小题 ****** 1、在考生文件夹下打开文档WDT11.DOC，按照要求完成下列操作。 (1) 将文中所有错词“款待”替换为“宽带”;将标题段文字(“宽带发展面临路径选择”)设置为三号黑体、红色、加粗、居中并添加文字蓝色底纹，段后间距设置为16磅。 (2) 将正文各段文字(“近来，……设备商、运营商和提供商都难以获益。”) 设置为五号仿宋_GB2312，各段落左右各缩进2厘米，首行缩进0.8厘米，行距为2倍行距，段前间距9磅。 (3) 将正文第二段(“中国出现宽带接入热潮，……一个难得的历史机会。”) 分为等宽的两栏，栏宽为7厘米。并以原文件名保存文档。 2、在考生文件夹下打开文档WDT12.DOC，按照要求完成下列操作。 (1) 将文档中所提供的表格设置成文字对齐方式为垂直居中，段落对齐方式为水平居中。 (2) 在表格的最后增加一列，设置不变，列标题为“平均成绩”，计算各考生的平均成绩并插入相应单元格内，再将表格中的内容按“平均成绩”的递减次序进行排序。并以原文件名保存文档。第2题、 ****** 本套题共有2小题 ****** 1、在考生文件夹下打开文档WDT21.DOC，按照要求完成下列操作。 (1) 将文中所有“质量法”替换为“产品质量法”;将标题段文字(“产品质量法实施不力地方保护仍是重大障碍”)设置为三号、楷体_GB2312、蓝色、倾斜、居中并添加文字黄色底纹，并设置段后间距为18磅。 (2) 将正文各段落文字(“为规范……没有容身之地。”)设置为小四号宋体、加粗，各段落右缩进1厘米，悬挂缩进0.8厘米，行距为2倍行距。 (3) 将正文第一段(“为规范……重大障碍。”)和第二段合并,并将合并后的段落分为等宽的两栏，栏宽为7厘米。并以原文件名保存文档。 2、在考生文件夹下打开文档WDT22.DOC，按照要求完成下列操作。 (1) 将文档末尾所提供的5行文字转换成一个5行6列的表格，再将表格设置文字对齐方式为底端对齐，水平对齐方式为右对齐。 (2) 在表格的最后增加一行，设置不变，其行标题为“午休”，再将“午休”所在单元格设置成红色底纹填充,表格内实单线设置成0.75磅实线，外框实单线设置成1.5磅实线。并以原文件名保存文档。

全国计算机等级考试一级选择题以及答案

全国计算机等级考试一级选择题以及答案．．．．．．．．．．．．．．．．．． 1、世界上第一台电子计算机诞生于（ B ）年。 A）1939 B）1946 C）1952 D）1958 2、冯·诺依曼研制成功的存储程序计算机名叫（B ）。 A）EDV AC B）ENIAC C）EDSAC D）MARK-2 3、1949年，世界上第一台（ C ）计算机投入运行。 A）存储程序B）微型C）人工智能D）巨型 4、计算机的发展趋势是（ D ）、微型化、网络化和智能化。 A）大型化B）小型化C）精巧化D）巨型 5、新一代计算机是指（ B ）。 A）奔腾4系列B）人工智能计算机C）工作站D）多媒体计算机 6、计算机从其诞生至今已经经历了四个时代，这种对计算机划时代的原则是根据（A）。 A）计算机所采用的电子器件（即逻辑元件）B）计算机的运算速度 C）程序设计语言D）计算机的存储量 7、计算机采用的逻辑元件的发展顺序是（ B ）。 A）晶体管、电子管、集成电路、大规模集成电路 B）电子管、晶体管、集成电路、大规模集成电路 C）晶体管、电子管、集成电路、芯片 D）电子管、晶体管、集成电路、芯片 8、下列不属于第二代计算机特点的一项是（ A ）。 A）采用电子管作为逻辑元件 B）主存储器主要采用磁芯，辅助存储器主要采用磁盘和磁带 C）运算速度从每秒几万次提高到几十次，主存储器容量扩展到几十万字节D）出现操作系统，开始使用汇编语言和高级语言 9、在计算机时代的划分中，采用集成电路作为主要逻辑元件的计算机属于（ C ）。 A）第一代B）第二代C）第三代D）第四代 10、使用晶体管作为主要逻辑元件的计算机是（ B ）。 A）第一代B）第二代C）第三代D）第四代 11、用电子管作为电子器件制成的计算机属于（A ）。 A）第一代B）第二代C）第三代D）第四代 12、以大规模、超大规模集成电路为主要逻辑元件的计算机属于（D ）。A）第一代计算机B）第二代计算机C）第三代计算机D）第四代计算机 13、现代微机采用的主要元件是（ D ）。 A）电子管B）晶体管C）中小规模集成电路D）大规模、超大规模集成电路 14、计算机可分为数字计算机、模拟计算机和混合计算机，这是按（C ）进行分类。 A）功能和用途B）性能和规律C）工作原理D）控制器 15、专门为某种用途而设计的计算机，称为（A ）计算机。

Microsoft Project入门实例教程

软件项目管理（Microsoft Project）实验指导 1、实验内容本次实验是通过使用Microsoft Project完成项目管理的一些工作，目的是了解Microsoft Project工具的使用和项目管理的相关知识。实验内容和步骤如下： 1.1 建立项目管理文件在开始制定项目计划之前，要明确定义项目的一些基本属性信息，或者对项目有一个基本的定义，例如项目的名称、内容、开始时间、结束时间等。例如有一个《校园网站》项目，内容是通过网站介绍学校的基本情况，发布一些及时的信息，同时有论坛等讨论区域。然后开始在Project2003中创建项目，实验步骤如下： 1、新建项目选择[程序]-〉Microsoft Office ——〉Microsoft Office Project进入Project2003。选择[文件]-〉[新建]菜单命令，打开Project的“新建项目”向导，如图1，单击“空白项目”将出现“任务”导向，如图2。图1：新建项目

图2：文件向导 2、定义项目项目创建完成之后，需要定义项目。单击图2中的“定义项目”，将显示“定义项目”导向，利用向导可以很容易完成定义项目的操作。分三步： 1)输入项目的开始时间，如图3，将日期调整为项目的预计开始日期； 2)输入项目工作组选项，如图4，询问是否使用Project Server和Project Web Access ，本项目比较小，不需要Project Server的协调，选择“否”； 3)保存文件，将文件保存为SchoolWebside.mpp。图3：定义项目-a项

图4：定义项目-b项图5:文件保存 3、设置环境信息项目的环境信息包括工期、项目的日历等。选择[工具]-〉[选项]，弹出“选项”对话框，选择“选项”对话框中的“日历”选项卡可以输入、查看或者修改日期、时间等设置，如图6。选择“日程”选项卡可以输入、查看和修改排定任务日程的首选项。如图7。

2011年3月全国计算机等级考试二级C语言笔试真题与答案

2011年3月全国计算机等级考试二级C语言笔试真题一、选择题 (1)下列关于栈叙述正确的是 A)栈顶元素最先能被删除B)栈顶元素最后才能被删除 C)栈底元素永远不能被删除D)以上三种说法都不对 (2)下列叙述中正确的是 A)有一个以上根结点的数据结构不一定是非线性结构 B)只有一个根结点的数据结构不一定是线性结构 C)循环链表是非线性结构 D)双向链表是非线性结构 (3)某二叉树共有7个结点，其中叶子结点只有1个，则该二叉树的深度为(假设根结点在第1层) A)3 B)4 C)6 D)7 (4)在软件开发中，需求分析阶段产生的主要文档是 A)软件集成测试计划B)软件详细设计说明书 C)用户手册D)软件需求规格说明书 (5)结构化程序所要求的基本结构不包括 A)顺序结构B)GOTO跳转 C)选择(分支)结构D)重复(循环)结构 (6)下面描述中错误的是 A)系统总体结构图支持软件系统的详细设计 B)软件设计是将软件需求转换为软件表示的过程 C)数据结构与数据库设计是软件设计的任务之一 D)PAD图是软件详细设计的表示工具 (7)负责数据库中查询操作的数据库语言是 A)数据定义语言B)数据管理语言 C)数据操纵语言D)数据控制语言 (8)一个教师可讲授多门课程，一门课程可由多个教师讲授。则实体教师和课程间的联系是 A)1:1联系B)1:m联系C)m:1联系D)m:n联系 (9)有三个关系R、S和T如下：则由关系R和S得到关系T的操作是 A)自然连接B)交C)除D)并

(10)定义无符号整数类为UInt,下面可以作为类UInt实例化值的是 A)-369 B)369 C)0.369 D)整数集合{1,2,3,4,5} (11)计算机高级语言程序的运行方法有编译执行和解释执行两种，以下叙述中正确的是 A)C语言程序仅可以编译执行 B)C语言程序仅可以解释执行 C)C语言程序既可以编译执行又可以解释执行 D)以上说法都不对 (12)以下叙述中错误的是 A)C语言的可执行程序是由一系列机器指令构成的 B)用C语言编写的源程序不能直接在计算机上运行 C)通过编译得到的二进制目标程序需要连接才可以运行 D)在没有安装C语言集成开发环境的机器上不能运行C源程序生成的.exe文件 (13)以下选项中不能用作C程序合法常量的是 A)1,234 B)'\123'C)123 D)"\x7G" (14)以下选项中可用作C程序合法实数的是 A).1e0 B)3.0e0.2C)E9 D)9.12E (15)若有定义语句：int a=3,b=2,c=1;，以下选项中错误的赋值表达式是 A)a=(b=4)=3; B)a=b=c+1; C)a=(b=4)+c; D)a=1+(b=c=4); (16)有以下程序段 char name[20]; int num; scanf("name=%s num=%d",name;&num); 当执行上述程序段，并从键盘输入：name=Lili num=1001<回车>后，name的值为 A)Lili B)name=Lili C)Lili num= D)name=Lili num=1001 (17)if语句的基本形式是：if(表达式)语句，以下关于“表达式”值的叙述中正确的是 A)必须是逻辑值B)必须是整数值 C)必须是正数D)可以是任意合法的数值 (18)有以下程序 #include main() { int x=011; printf("%d\n",++x); }

Microsoft Project 2013操作手册

Project 2013操作手册

目录目录 1、启动阶段 (3) 1.1、前期准备 (3) 1.1.1、新建项目文件 (3) 1.1.2、设置项目信息 (3) 2、计划阶段 (5) 2.1、定义资源 (5) 2.2、建立任务 (6) 2.2.1、任务列表 (6) 2.2.2、插入周期性任务 (7) 2.2.3、任务日历 (8) 2.2.4、任务工期 (8) 2.2.5、建立限制条件 (8) 2.2.6、建立依赖关系 (9) 2.3、资源分配 (9) 3、跟踪阶段 (9) 3.1、比较基准 (9) 3.2、跟踪完成进度 (10) 4、报告与分析 (10) 4.1、盈余分析 (10) 5、多项目管理 (11) 5.1、创建共享资源 (11) 5.2、使用共享资源 (11) 6、其它 (11) 6.1、如何在“跟踪甘特图”中显示其它比较基准 (11) 6.2、基准保存技巧 (11) 6.4、如何进行计划的调整 (11) 6.5、查询可以调整的时间 (11) 6.6、关于加班 (12)

1、启动阶段 1.1、前期准备 1.1.1、新建项目文件选择 File—new 菜单，选择项目模版： 1.1.2、设置项目信息自定义日历选择“Project”下的“Change Working Time”菜单：

项目信息定义在“Project”中选择“Project Information”，打开项目信息窗口，在这个窗口中有几个重要信息要设置：开始日期、排序方法、项目日历：

2、计划阶段 2.1、定义资源在“Task”中选择将视图切换为“Resource Shee t”：资源分三类，分别为“work”、“Material”、“Cost”，三种类型均可以设置为费率；

全国计算机等级考试一级理论汇总(版)

基础知识 1. 下列存储器中，存取速度最快的是( )。B A. CD-ROM B. 存 C. 软盘 D. 硬盘 2. 在微机中，1MB准确等于( )。B A. 1024×1024个字 B. 1024×1024个字节 C. 1000×1000个字节 D. 1000×1000个字 3. 计算机存储器中，一个字节由( )位二进制位组成。B A. 4 B. 8 C. 16 D. 32 4. 下列各指标中，( )是数据通信系统的主要技术指标之一。D A. 重码率 B. 传输速率 C. 分辨率 D. 时钟主频 5. 十进制整数100化为二进制数是( )。D A. 1100100 B. 1101000 C. 1100010 D. 1110100 6. CPU主要由运算器和( )组成。A A. 控制器 B. 存储器 C. 寄存器 D. 编辑器 7. 计算机病毒是可以造成计算机故障的( )。B A. 一种微生物 B. 一种特殊的程序 C. 一块特殊芯片 D. 一个程序逻辑错误 8. 用高级程序设计语言编写的程序，要转换成等价的可执行程序，必须经过( )。D A. 汇编 B. 编辑 C. 解释 D. 编译和连接 9. 在微机的性能指标中，存储器容量指的是B A) ROM的容量B) RAM的容量C) ROM和RAM容量的总和D) CD-ROM的容量 10. 1GB等于（）D A) 1000×1000字节B) 1000×1000×1000字节C) 3×1024字节D) 1024×1024×1024字节 11. CPU中控制器的功能是（）C A) 进行逻辑运算B) 进行算术运算C) 分析指令并发出相应的控制信号D)只控制CPU的工作 12. 计算机软件分为（）B A) 程序与数据B) 系统软件与应用软件C) 操作系统与语言处理程序D) 程序、数据与文档 13. 与十进制数291等值的十六进制数为（）A A) 123 B) 213 C) 231 D) 132 14. 能将高级语言源程序转换成目标程序的是（）A A) 编译程序B) 解释程序C) 调试程序D) 编辑程序 15. 下列说法中错误的是 B A) 简单地来说，指令就是给计算机下达的一道命令。 B) 指令系统有一个统一的标准，所有的计算机指令系统相同 C) 指令是一组二进制代码，规定由计算机执行程序的操作 D) 为解决某一问题而设计的一系列指令就是程序