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    9 views7 pages

    Arti 2 Protein

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    AMS.COL
    proteina AmyA

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    TENN 705 Biochemie Sclones Vol 6Nos Wy 001 Techniques: Recombinogenic engineering - new options for cloning and manipulating DNA Joep PP Muyrers, Youming Zhang and A. Francis Stewart Driven by the needs of functional genomics, DNA engineering by homologous recombination in Escherichia colihas emerged as a major addition to existing technologies. Two altemative approaches, Recfvdependent engineering and ET recombination, allow a wide variety of DNA modifications, including some Which are virtually impossible by conventional methods. These approaches do not rely onthe presence of suitable restriction sites and can be used to insert, delete or substitute DNA sequences at any desired position ona target ‘molecule. Furthermore, ET recombination can be used for direct subcloning and cloning of DNA sequences trom complex mixtures, including bacterial artificial chromosomes and genomic DNA preparations. The strategies reviewed in this article are applicable to modification of DNA molecules of any ‘ze, Including very large ones, and present powerfull new avenues for DNA ‘manipulation in general The completion of large DNA-sequencing projects, including the Human Genome Project, has generated an extraordinary amount of primary sequence data. The noxt major challenge isto investigate the ‘components that make up a genome, and is often called functional genomies. Escherichia coli vectors ‘that can contain large inserts, such as bacteria artificial chromosomes (BACs)', PI vectors# and PL artificial chromosomes (PACs)’, offer several advantages for functional genomics. They can carry sufficient DNA to encompass most eukaryotic genos, ‘including all cis-acting regulatory elements, as well as many eukaryotic gene clusters, prokaryotic rogulons and many complete viral genomes, ina single molecule*. However, conventional cloning. methods rely on the use of restriction enzymes and in vitro purification steps, which preclude engineering of large molecules. Consequently, the usefulness of such molecules has been limited until recently. Novel DNA engineering strategies that rely on homologous recombination in vivo in E.coli alleviate this imitation and allow a wide range of ‘modifications of DNA molecules at any chosen position. These strategies are the scope of this review. Program emis. genearages com Homologous recombination and DNA engineering: recombinogenic engineering Homologous recombination allows the exchange of genetic infarmation between two DNA moleculesin a precise, specific and faithful manner. These qualities are optimal or engineering a DNA molecule regardless of its size. Homologous recombination cecurs through ‘homology regions, which are stretches of DNA shared by Ue two molecules that recombine, Because the sequence ofthe homology regionscan be chosen reels, any position on atarget molecule ean be specifically altered. Because homologous recombination is a rare ‘event, some form of aelection is needed to identify the cells that carry the recombinant. Hence DNA. engineering by homologous recombination, or rrecombinogenic engineering, makes use ofa selection procedure that usually involv ‘genes, In certain cases, such as simply deleting a part ofa BAC, Pl-vector or PAC (Refs 5-8), or generating. mouse knockout constructs in one step*, the product ‘ofa single round of homologous recombination can be the dosired product (Fig. 1a).In many other eases, persistence ofthe selectable gene at the site of recombination in the product is undesirable. Consequently, recombinogenic engineering often involves two rounds of processing. In the first round, hhomelogous recombination is used to generate an initial product by integration ofthe selectable gene, ‘together with additional fmetional elements, at the intended site. In the second round, the extra antibiotic-resistant functional elements are used to remove the selectable gene, thereby generating the final product. By informed application of these principles, virtually any DNA iteration ean be accomplished. Variations of how additional functional elements can he used are shown in Fig. 1 and inelude the following: + Acounterselectable gene (Fig. 1b; for recent examples of the use of tho rpsl, ett and sacB counterselectable senes, see Refs 810-18). Here, the desired second: round product is identified by countersclection Dbocause homologous recombination can eliminate the counterselectable gene and flanking sequences, replacing it with the DNA region of interest. This approach can theret second: round product that earries na sca'from the first round of recombination, However, cunterselection is typically less ficient than positive selection, as the intended homologous recombination is only one of several solutions for counterselection pressure. Any other mutational event that alates expression of the counterselectable gene will also grow under counterselection pressure. Thus, candidates from second-round-countersclection stratogies need tobe serened find the intended recombinogenie engineering product. In practice, the ratio of anon rseed Pe sone-oon4oa8 4 a TES 0S oho Scenes VrTe ie Mny (2) One-stop positve selection _(b) Selection, countarsolaction {€) Selection ste-specife (€) Selection, restiction recombination digestion A B Aas 8 A= 8 At ate near ONA Linesr NA Linear DNA Linesr DNA, ne Target molecule Target molecule Target molecule Fist round Festmund Frstround Frstround rod rot prot Second + Delton Delon rom orem | through» rowan fcontioan Recomtnan\ ON finest conning ‘containing n 8 5 recombinant Escherichia colenromosome. targeting DNA hee stated as linear ONA i generoted BY PCRampliieaton toned wo eglons, Asnd 8, omologousoegions inthe age ralecule ‘Alo includedisselertablemarer gene (im). The simplest exerese( ivolvesinsetion. by between A ona (uncncanbe anywere beeen Dbp anda ast 0X8) ean Beuseatoaelee remveit Three waysteremove he selecinble gene re state npanets bead. panel | unter selectable gene fs iinsrted alongside the selecible gone nthe staund pode The sireesmeassete lcd na second oundet hmelogous recombination courteseleton ‘anges or nserion of BNAregion ofirerest compel ee of any perationalsequences sed for fengirering in ()sitespeciereeombinalion eget ses (S5RTs shown arevhesds} re sprcteracombinsian|s useatoremove the smeatsteoleavea single SSRT inthe pros! Tesvtion ates, Indcatedby an estes, re ncludedolnkineselectabie gene inte neat targeting motel immediatly adjacent a one or oth ofthe homlogy regions nat show). The poroaches ihusirated here are based on ET econbinogenicengineving bul te prices with intended-to-unwanted produete varies wildly leg. botwoen <1% (Ref. 14) and 15-85% (Refs 5,13) forreasons that are still undefined. + Flanking site-specific recombination target sites (SSRTe), such as the 34-bp recognition sites for (Big. Le; Refs 5,6,16,17). Here, the first products exposed tothe relevant site-specific recombinase, which deletes the selectable cassette to leave a single SSRT at the site of engineering, Thisis a high! cffcient way to eliminate the selectable cassette and is also useful method of placing an SSRT exactly hero itis required. However, the remaining 34 (or 86) bp scar (the SSRT) can be a problem ifleftin protein-coding, or regulatory, regions. + Flanking sites for restriction enzymo(s) (Fig Ld. Here, the first product is purified, cut with the relevant restriction enzyme and religated to give second-round product free ofthe first-round selectable gene, As with SSRTs, this is a useful way of placing a restriction enzyme site (or sites, including polylinkers) exactly where required. In this strategy, the second-round product includes the restriction site() and ean be used only for relatively small DNA molecules because it relies on restriction enzyme digestion Recombinogenic engineering in coll ‘Although £. coliis the premier host for conventional DNA engineering, Saccharomyces cerevisiae was, TENN 705 Biochemie Sclones Vol 6Nos Wy 001 ur until recently, the preferred host for recombinogenic engineering. This situation was due tothe remarkable proficieney of &. cerevisiae at homologous recombination, combined with certain complications inherent in the endogenous E.coli homologous recombination mechanism, Recombinogenie engineering in S. cerevisiae is straightforward because the linear DNA flanked by short homology rogions can be inserted into endogenous DNA targets. Linear DNA i particularly useful because DNA ends significantly promote recombination'®. Furthermore, ‘the homology regions can be as short as 42 nucleotides. Consequently oligonucleotide synthesis ‘ean be used to make the homology regions'®. Success in using yeast artificial chromosomes (YACs) and ‘yeast episomes in S. cerevisiae has produced several recombinogenie engincering breakthroughs, the most recent example being direct cloning by homologous -recombination?®2!. Although YACs are better than BACs, P1-veetors or PACs in one respect, that is they: ‘can carry larger inserts, recombinagenic engineering in S. cerevisioeis hampered by several difficulti as implicit genetic instability, low yields of purified DNA products and the care and labour-intensive skills, required to carry out the procedures. In B.coli, the endogenous homologous recombination mechanism initiated by cooperation between RecA (the most conserved strand invasion protein in evolution)#*#* and RecBCD (a large, functional enzyme composed of ReeB, RecC and ‘RecD subunits)##+" impedes the use of linear DNA fragments, probably because RecBCD is, among other functions, a very vigorous exonuclease. Therefore, RecBCD rapidly digesta any exogenously introduced linear DN Avariety of RecA-dependent approaches for ‘recombinogenic engineering have been published, For brevity only the approach that has found most, "use will be described here; however, the interested ‘reader might wish to consider other variations!™2, such molecules RecA dependent recombinogenic engineering Consistent with the difficulties of using linearized DNA in E.coli, early findings with RecA-dependent ‘recombinogenic engineering reported some successes using intermolecular recombination between two circular molecules", A significant advance in RecA-dependent strategies was the inclusion of a tomperature-sensitive (ts) plasmid origin. This permitted the construction of a targeting plasmid, grown at the permissive temperature, followed by a first round of homologous ‘recombination to integrate the plasmid into its target at a non-permissive temperature (Fig. 2). After identification of correct homologous ‘recombinants and further culturing in the presence of RecA, shifting back to the permissive temperature identifies those recombinants that have lost the ts origin (because its continued presence kills the ‘host). To improve the yield of intended product in the DNA of interest Target Homologous recombination through A Fret round DNA of imtorest Cointegrant LoS Resolution through A Resolution through B DNA of interest Ae alia ‘Second round 7 Fig. 2 RecA dpender-nedatearecombinagenc engheetng The most ley usedRecraepndenstegys shou Befoetecomansen, ‘ake mam). tempertre-ienaiv origina epson tr}, “recagene andtwonomology regions (a8 yea Te ntng herelogy em Aar or srl any recombination rowan Ceirnganisidetes by selection rhe Slate genet pear hat sperrsivefor hese Reson ca the reve second round, a counterselectable gene has been included near the ts origin. This strategy was developed for altering the B. coli chromosome", applied to recombinogenic engineering in BACs (Refs 11,15) and recently improved TSIEN FRENDS io ochonct Scones Vol eNO Moy 2001 An outline ofthis RecA-dependent strategy is shown in Fig. 2. Before the first round of recombinogenie engineering, a dedicated targeting plasmid containing several components must be built. ‘The components include: (1) ene( or selection and counterselection; 2) homology region(s), typically 1 xb or longer; (3) ats origin; and (4) the recA gene, Although the recA gene could be omitted ifa recA+ E.coli host is used, providing RecA in trans from the targeting plasmid permits the use ofthe common recA hosts and also limits the recombinogenie window. After integration ofthe intact targeting plasmid into the target to form a cointegrantin the first round (Fig. 2), activating the ts origin by temperature shift and exerting countersclection pressure drives resolution in the second round, Because resolution. climinates the backbone ofthe targeting plasmid, including the recA gene, the host carrying the second-round product reverts to rec; thus limiting the recombinogenie window and securing the genetic stability othe intended product, Resolution in the ‘second round can produce either the original target or the intended recombinant. Selected applications of RecA-dependent recombinogenie engineering are summarized in ‘Table 1. All RecA-dependent strategies appear to roquire relatively long homology region(s) and hence the construction of dedicated targeting plasmids, Furthermore, efficiencies are often low. [The term ‘efficiency’ refers throughout to both the total number of candidate recombination products achieved and the ratio of intended (romologous) to unwanted (non ‘homologous or rearranged) recombination products ‘among those candidates,] Recombinogenic engineering using ET recombination An alternative recombinogenic engineering strategy was developed whereby recombination is not dependent on RecA, but instead mediated by phage-derived protein pairs, either RecE/Rec from the Rac phage or Reda/Redf, from 2 phage". To coin simple term, this recombinogenie engineering strategy was initially termed ET recombination (or ET cloning)® and has also been called L-mediated recombination® and GET recombination". As established from both fundamental studios!®238 and applied studies with recombinogenic engineoring® "2199 or genotypic manipulation of E.coli hosts “8%, the RecB/RecT and Reda/RedB protein pairs are functionally and operationally equivalent, RecE and Reda are 5’-3" exonucleases, and RecT and Red are DNA annealing proteins, Alunetional interaction between Ree and Ree, or Redotand Redf, is also required for homologous recombination™ ‘As with recombinogenie engineering in S. cerevisiae, ET recombination encompasses the advantages inherent in the use of linear targeting DNAs and thus the increased efficiencies promoted by DNA ends. To secure this advantage, the endogenous RecBCD exonuclease activity must be absent or inactivated. I'the E.coli host in which BT recombination takes place is not recBCD, a recBCD phenocopy can be induced by the expression of Redy, the phage protein that inactivates ReeBCD (Ref. 88).A further similarity to S. cerevisiae is found in the fact that the RecE/ReeT and Reda/RedB protein pairs need only short homology regions for useful ET recombination efficioncies™#116173936, With BT recombination, as illustrated in Fig. 1a, a linear targeting DNA carrying short homology regions flanking a selectable gene (sm) is integrated into. circular target DNA. Although long homology rogions can bo used”, the length of homology required for efficient recombination is ;-60 nucleotides, and thus short enough tobe made by oligonucleotide synthesis. Ina very convenient application, the synthesized oligonucleotides also include polymerase chain reaction (PCR) primers for amplification ofthe selectable gene. Thus the linear targeting DNA can be a PCR product and no dedicated targeting plasmid needs to be constructed, For example, PCR products have been used to'shave’ target molecules, such as various BACs, by deletion of ‘Table 1. Summary of recombinogenic engineering applications+® Modification Target motecaTe E-collehromosome PIs/EACS/PACS RecA ET RecA, ET RecA, et Precise sequence deletion $2782) 4617.85-85) + (10-1215,1629) +8) 1252631) 159.58,36) Precise sequence insertion Small (6009, - +6. +110) +69 - “0 0g SSRThesriction sites Large, eg. rotein tags + QN82 46788-3855 101215.29 + 1.7) + (25.2628.30) +669) GFR.IRES. promoters, genes, ete Point mutations - « - sea = “6 modieatonsin varius target mokectes ere neate Accomp shnets of engineering exercises by RecA-deenden recombination RecA subaivised ini sll (20 bp sarge 40 bp} to cstinguish between those barea onthe convenience fered by algonurleoise TEIN 78055 ochericrScoces VraeNoS My 20 7° ‘unwanted DNA precisely in one step, POR products have also been used to generate targeting constructs for knockout experiments in mouse ES cellsin one stop?. Here, the selectable gene was chosen beeause it conveys resistance to antibiotics in both E. coli and cukaryoticcolls. Throe such genes are usoful, namely ‘those that convey resistance to kanamyein/neomycin, hygromycin and blasticidin. ET recombination was used to place one of these selectable genes exactly where desired for later sein knockout experiments. Strategies involving ET recombination and two rounds of recombinogenic engineering are illustrated in Fig. 1b-a. In particular, POR produets carrying selectable and counterselectable gene(s) (Fig. 1b) ‘have heen used to point mutate a plasmid? or BACs (Refs 13,14).As described above, Figs eand Id illustrate simple ways to delete the selectable gene ‘cassette and to introduce short sequences encoding SSRTS or restriction site(s) exactly where desired. In ‘these cases the SSRTs/restriction site(s) can also be ‘programmed into the synthesized oligonucleotides. Similarly other short ‘DNA-of interest’ sequences, such as a polyadenylation signal ora short protein ‘ag, can be programmed into a synthetic ‘oligonucleotide for convenient introduction. In eases in which the added DNA-of interest is beyond the limits of oligonucleotide synthesis, itean be introduced either in the second round of selection/eounterselection (Fig. 1b) orby use of plasmid template constructed tocarry the DNA-of- interest beside a selectable gene. (The construction of these plasmids is easily accomplished using one-step positive selection; Fig. 1.) note of caution ‘concerning the fidelity of PCR is appropriate here. In ‘many cases, the risk that a PCR amplification step introduces mutations in the DNA-of-nterest is “unacceptable, Fear of PCR mutagenesis does not apply, however, toamplification of selectable genes, ‘counterselectable genes, SSRTs, restriction sites or short DNA-of-interest regions because there are easy ways to test function or screen out the mutants, Ifwanted, the DNA-of.interest can be constructed in adedicated plasmid that is linearized by restriction enzyme digestion. These linear DNAs necessarily ‘have some non-homologous sequence at their very ends, However, erminal stretches of non-homology, ‘which are either the few nucleotides left by restriction enzyme cleavage, or up to several kilobases between, the end and the homology region, are eliminated by recombination through the homology regions toleave the intended product. Successful applications of ET recombination are ‘summarized in Table 1, In contrast to RecA strategies, the efficiencies achieved with ET recombination, using either RecE/RecT or RedaRed protein pairs, are consistently high, and typically more than 80% of ‘candidates are the correct recombinant, Hence, itis not necessary to analyse many clones to obtain the desired ‘recombinant (fora more detailed deseription of the involved experimental procedures, se Ref. 39). As evidence ofthe very high efficiencies achievable ‘with ET recombination, a recent publication has shown ‘that correet recombinants ean be identified without the need to employ antibiotically selectable gones®, Using oligonucleotides and short PCR products, absolute ET recombination efficiencies approaching 1 in 100 total colonies have been shawn. Hence, selective PCR screening methods on pools and individual colonies ean bboused to identify correct recombinants. Limiting the recombinogenie window [Recombinogenic engineering, either Rec dependent orby BT recombination, oocurs through homology regions. Any two regions homologous to each other ‘within # recombinogenic host can recombine. In practical terms, this means that unintended ‘additional homology rogions in the targeting DNA should he avoided, Probable unintended homologies include all or parts of plasmid origins, selectable markers or other commonly used DNA sequences such ‘as fragments carried over from common cloning vectors. Whereas unintended homologies in the targeting DNA can be avoided simply by good design, repeats occur randomly in cloned sections of cukaryotic DNA and cannot be avoided. Intramolecular recombination between such repeats is clearly undesirable. Conveniently, the homology regions that direct the intended ETT recombination product are stimulated by nearby DNA ends, whereas potential intramolecular recombination in the target DNA doos not have this advantage. Thus, intended EP eventa should be favoured over unintended background instabilities. Inour experience, thishas proven tobe the case, but moreexperienceusinga broader range of target molecules is needed to settle the matter, Regardless of the outcome, the issue of ‘genetic instability isan important concern for rrecombinogenie engincering and highlights the importance of strategies that limit the recombinogenic ‘window; that i, the time-span in which recombination cean take place, tothe smallest interval required for generation of the intended product. In addition to the RecA-dependent suicide strategy outlined in Fig. 2, ats RecA host has also been used as a way tolimit the recombinogenic ‘window to a period of expression aterculturo at the permissive temperature!®®®, For ET strategies, the rrecombinogenie window i limited by regulated ‘expression of at least one of the components ofan ET protein pair (RecE/RecT or Reda/Red) from an inducible promoter7IS7:S3, Most plasmids from. ‘which the ET'mediating proteins are expressed rely on the ParaB promoter and its properties of L-arabinose induction and glucose repression‘. Frequently, such ET plasmidsalso allow expression of Red so that ET recombination in any B. coli strain (including those ‘expressing RecBCD) can occur. Thus, these plasmids offer a simple way toimport ET recombination intonew hhosts, particularly ones that already contain a BAC of interest, and to limit the rocombinogenie window. me TREN 0S oho Scenes Vrae es My DNA of DNAof A interest a _ierest Purlied DNA preparation | Intact ereutar target as ‘a [ Recombinant } 5 DNA of interest a User tagetingmotcles ae generale polymersecaineation PER ocentan aepleaen Puried DNA proprton loin oan actu trget rola uber). The partied ONA ani a genomic DNA preprtton ands co-ecropraed th ne nes een mole aan molecule creategplsriarecombinanthatistertiiedy seectonr the selectable Recent developments Following the recombinogenic engineering initiatives pioneered in S. cerevisiae®2%, the potential of BT recombination for direct cloning and subcloning was explored. The principle ofthis strategy isillustrated in Fig. 3. In this variation, the linear targeting ‘molecule is a PCR-amplified plasmid backbone that contains a selectable gene and an origin of replication, ‘The oligonucleotides used for PCR also contain ‘homology regions that are chosen to define the exact boundaries ofthe DNA region ta he cloned or ‘subeloned. The chosen DNA region is copied into the plasmid backbone by homologous recombination. By a single-step process, chosen regions from plasmids and BACs, including a23-kb region of mouse genomic DNA, were subcloned at the high efficiencies that are typical of BT exercises, Similarly chosen regione from purified E.coli, east and mouse genomic DNA preparations were directly cloned'?, This presents a simple and efficient way to subelone that does not involve the tedium of purifying DNA, the required eogereering ne co's inomtegedrereby Irhuenmuyrerscoen, Supported by nts fom Feundsion orogrmmon prosence of convenient restriction sites or the ‘mutational risk of PCR. It also opena up a new way to clone without the need to make and serven libraries, ‘and to.amplify a chosen DNA region by a means other than PCR, Furthermore, unlike PCR, DNA molecules ofup to atleast 28 kb have been subcloned in a single step. These subcloned molecules (unlike those generated by PCR) are unlikely to contain any ‘unwanted mutations because the subeloned region is amplified in vivo by the endogenous E. coli replication ‘machinery and hence has been fully proaf reviewed. Concluding remarks and perspectives ‘Tho strategies presented outline how precise ‘modifications, including single-base changes, and variably sized insertions and deletions, can be made to DNA molecules in £ coli of any size Recombinogenic engineering strategies will undoubtedly accelerate progress in functional genomics, as they allow straightforward engineering oflarge DNA molecules, which are important for functional studies of genos in all aspects such as development, homeostasis and disease, Importantly, the strategies also provide alternatives to conventional, small-molecule engineering using restriction enzymes, POR and DNA ligase. Moreover, ET cloning and subcloning provide an alternative to PCR amplification, Because any recombination- based strategy is carried out in vivo, any recombinant molecule is generated and inspected by the endogenous F. coli replication and repair ayatems, Therefore, the recombinant product is virtually ensured tobe free of any undesired ‘mutations, Thus, with a little forethought, the ‘mutagenic ambiguities involved in the use of PCR or EtBr/UV exposure during DNA preparation can be avoided by use of a recombinogenic engineering alternative, Although recombinogenic engineering strategios were previously reliant on the use ofS. cerevisiae, the convenience of using B. coli as the host is now available, Consequently, commonly used cloning ‘molecules, which replicate in E coli,can be engincered directly: Moreover, whereas recombination potential is constitutively activein yeast, the E.coli-based recombinogenie engineoring systems have been developed to limit the recombinogenic window. The possibilities offered by homologous recombination prompt new ways of thinking about DNA engineering. Engineering strategies that were previously fat all, only applicable to small DNA. ‘molecules auch as plasmids, can now be applied to DNA molecules of any size and composition. Ata ‘minimum, the experimenter nevds tobe aware that, when conventional methods fail, are flawed, or are difficult to apply, alternative solutions exist. Adding recombinogenic engineering tothe repertoire of frequently used molecular biological techniques is likely to pay off, as it will provide effective solutions for many DNA engineering objectives. 1 Shinya, H eta. (1992) Cloningand stable ‘maintenance of 900-bane pale fragments human DNA in Baherchiacal wing an Pistorbased van, Pr, Natl, vad. Sel USA. 0, 974-8707 2 Sternberg, Ni (1982 Cloning igh molecular ‘ripht DNA Supmontab the biter P sytem, Trad Get, =18 $ Touro, PA. etal (1099 Anew bacteriophage Priderived eta forthe propagation a arge ‘human DNA fragmenta, Not Gone 6, 4-89 4 Brase, W eof (2000) Fareand wich BACS now toalaforhorpsvius genomic, Tends Genet 16, 254-258, 5 Thana, ¥ eta (1996) new ogiefor DNA gineringwsingrenmbination in Eosherichia col Net Gene, 20, 129-128 6 Miyrery PR.

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