10 Генетическая инженерия (1160079), страница 3
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Some of the important features of a cloning vector are illustrated by the E. coli plasmidpBR322 (Fig. 28-5): (1) the origin of replication is required to propagate the plasmid and helps maintain it at a level of 10 to 20 copies percell; (2) two genes that confer resistance to different antibiotics allowthe selection of cells that contain the plasmid or a recombinant versionof it (Fig.
28-6); (3) several unique recognition sequences for differentrestriction endonucleases provide sites where the plasmid can be cutand foreign DNA inserted; and (4) an overall small size facilitates theplasmid's entry into cells. The efficiency of bacterial transformationdecreases as plasmid size increases, and it is difficult to clone DNAsegments longer than about 15,000 base pairs when plasmids are usedas the vector.Chapter 28 Recombinant DNA TechnologyEcoRl991BamHlPBR322plasmidsPstloAmpicillinresistance(ampR)SailForeign DNAOrigin ofreplication(ori)Figure 28-5 The constructed plasmid pBR322,showing the location of some important restrictionsites, antibiotic-resistance genes, and the replication origin (ori).
This plasmid, constructed by Herbert Boyer and coworkers in 1977, was one of theearly plasmids designed expressly for cloning inE. coli.selection oftransformed cellsFigure 28-6 Cloning foreign DNA in E. coli withpBR322. If foreign DNA is inserted at the Pstl restriction site, the ampicillin-resistance element isdisrupted and inactivated.
After ligation of theDNA and transformation of E. coli cells, the cellsare grown on agar plates containing tetracycline toselect for those that have taken up a plasmid. Bymeans of sterile toothpicks, individual colonies fromthese agar plates are transferred to the same position within a grid on two additional plates; oneplate contains tetracycline (a control) and the othercontains both tetracycline and ampicillin. Thosecells that grow in the presence of tetracycline, butdo not form colonies on the plate containing tetracycline plus ampicillin, contain recombinant plasmids (the ampicillin-resistance element is nonfunctional). Cells that contain pBR322 that was ligatedwithout the insertion of a foreign DNA fragmentretain ampicillin resistance and grow on bothplates.
Note that in this and other experimentsthat involve the use of two or more plate replicasan orienting mark is put on the back of each plateso that the colonies on different plates can be readily aligned.All colonieshave plasmidsColonies withrecombinantplasmidsAgar containingtetracycline (control)Agar containingtetracyclinecolonies transferredfor testingAgar containingampicillin + tetracyclinePart IV Information Pathways992restrictionendonucleaseFiller DNA (not neededfor packaging)Recombinant DNAsLack essential DNAneeded and/or are toosmall to be packagedin vitropackagingX bacteriophagecontaining foreign DNASomewhat larger DNA segments can be cloned using bacteriophage A as a vector.
Bacteriophage A has a very efficient mechanismfor delivering its 48,502 base pairs of DNA into a bacterium. The general procedure for cloning DNA in bacteriophage A (Fig. 28-7) is basedon two key features of the A genome: (1) about one-third of the genomeis nonessential and can be replaced with foreign DNA, and (2) DNAwill be packaged into infectious phage particles only if it is between40,000 and 50,000 base pairs long. Bacteriophage A vectors have beendeveloped that can be readily cleaved into three pieces, two of whichcontain essential genes but which together are only about 30,000 basepairs long.
Additional DNA must therefore be inserted between themto produce viable phage particles. Bacteriophage A vectors permit thecloning of DNA fragments up to 23,000 base pairs long, and their design ensures that all viable phage particles will contain a foreign DNAfragment.Once the bacteriophage A fragments are ligated to foreign DNAfragments of suitable size, the resulting recombinant DNAs can bepackaged into phage particles by adding them to crude bacterial cellextracts containing all the proteins needed to assemble a completephage. This is called in vitro packaging (Fig. 28-7).
The bacteriophage vector is now ready for insertion of the recombinant DNA intoE. coli cells.Cosmids are recombinant plasmids that combine useful featuresof both plasmids and bacteriophage A. They are designed to permit thecloning of even larger DNA fragments (up to 45,000 base pairs). Cosmids (Fig. 28-8) are small (typically 5,000 to 7,000 base pairs), circularDNA molecules that contain (1) a plasmid origin of replication, (2) oneor more selectable markers, (3) a number of unique restriction sitesTable 28-3 Types of cloning vectors used in E.
coliFigure 28-7 Bacteriophage A cloning vectors. Recombinant DNA methods have been used to removenonessential genes and certain restriction sitesfrom the bacteriophage A genome. The remaininggenes (essential for bacteriophage production) areclustered in two large fragments at either end ofthe linear chromosome. Bacteriophage A vectorsgenerally have a piece of "filler" DNA in place ofthe eliminated genes to make the vector DNA largeenough for packaging into phage particles. Thisfiller can be replaced with foreign DNA in cloningexperiments. Recombinants are packaged into viable phage particles only if they are of the appropriate size (i.e., they include an appropriately sizedforeign DNA fragment) and include both of the essential A DNA end fragments.
The recombinantDNA molecules are packaged into phage particlesin vitro.Type of vectorPlasmids;modified byrecombinantDNA techniquesMethod ofintroduction intoE. coliTransformation;cells madecompetent to takeup recombinantvector, thentransformed cellsselected usingselectable markerBacteriophage APhage infection,following invitro packagingof recombinantvector into phageparticlesCosmids,Either of aboveconstructedmethods,from plasmiddepending onand A DNA genessize of DNAfragment inserted;larger fragmentsrequire in vitro ApackagingMethod ofpropagationSize of DNAfragment thatcan be clonedPlasmidreplicationUp to 15,000 bpPhagereplicationUp to 23,000 bpPlasmid-typereplicationUp to 45,000 bpChapter 28 Recombinant DNA TechnologyFigure 28—8 Cloning with cosmids.
A cosmid contains a replication origin (ori) for propagation as aplasmid, and a cos site required for the packagingof DNA into A phage particles. Unique restrictionsites and antibiotic-resistance elements aid cloningand selection. Inserting a large foreign DNA fragment into the cosmid precludes bacterial transformation but, if the recombinant DNA molecule is asuitable size, it can be packaged into phage particles in vitro. The phage A particles are then used tointroduce the cosmid into bacterial cells, where thecosmid is propagated as a plasmid.993CosmidKrolU restrictu(MidonudeaseLarge foreign DNAfragment with EcoRlsticky endswhere foreign DNA can be inserted, and (4) a cos site (a DNA sequencein bacteriophage A that is required for packaging).Cosmids contain no other bacteriophage A genes and can be propagated in E. coli like plasmids. When a large foreign DNA fragment iscloned into them, transformation of E.
coli with these recombinantDNA constructs becomes difficult. If the cosmid contains a largeenough insert of foreign DNA to be packaged into a phage particle, invitro A packaging systems permit the bacteriophage A particle to be thevehicle for introducing the cosmid DNA efficiently into the bacterialcell. Once in the cell, the cosmid is again propagated as a plasmid, as itlacks the A genes needed to make phage particles in the cell.The three types of cloning vectors are summarized in Table 28-3.Recombinantcosmidin vitro ApackagingIsolating a Gene from a Cellular ChromosomeRecombinant cosmidpacked in phage particleBecause a single gene is only a very small part of a chromosome, isolating a DNA fragment containing a particular gene often requires twoprocedures.
First, a DNA library is constructed that contains manythousands of DNA fragments derived from a cellular chromosome. Second, the DNA fragment containing the gene of interest is identified bytaking advantage of the one property that distinguishes it from theother DNA fragments—its sequence.infection ofE. coliCloning a Gene Often Requires a DNA LibraryA DNA library is a collection of DNA fragments derived from thegenome of a particular organism, with each fragment attached to acloning vector. In short, genomic DNA is cleaved into thousands offragments, and all of them are cloned.
Thus, the total information content of an organism is represented by all the fragments in the library inmuch the same way as human knowledge is represented by all thevolumes in a book library. A vector is prepared for cloning by cleavingpurified vector DNA at an appropriate position with a restriction endonuclease. Genomic DNA to be cloned is reduced to appropriately sizedfragments by partial digestion with a restriction endonuclease (usuallythe same one used to prepare the vector), followed by a procedure suchas sucrose density gradient (isopycnic) centrifugation to remove fragments that are too large or too small to be cloned into the chosen vector.
Fragments are then mixed with the cleaved vector DNA and ligated. The mixture is used to transform bacterial cells or is packagedselection oftetracyclineresistant cellsAgar containingtetracyclineColonies withrecombinant cosmidsPart IV Information Pathways994mRNA5'AAAAAAAAimRNA template isannealed to syntheticoligonucleotide (oligo dT) primer5'AAAAAAAA3'mRNA-DNA hybrid5'irri JTI rpTT\rryTT\try nriReverse transcriptase anddNTPs yield a complementaryDNA strandAAAAAAAArp rri rr\ rv\ rr\ ryi rri3'FT\mRNA is degradedwith alkali.rji rry rp rri rrt ryi rri rp3'Duplex DNA5'3'iDNA polymerase I and dNTPsyield double-stranded DNAAAAAAAAAf|\r 1 1 ft\f|1fII "i /^ii r i ^ r*|iFigure 28-9 Constructing a cDNA library frommRNA. In practice, the mRNA from a cell will include transcripts from thousands of genes, and thecDNAs generated will be correspondingly heterogeneous.
The duplex DNA produced by this method isinserted into an appropriate cloning vector.into bacteriophage particles as described above. The final result is alarge population of bacteria or bacteriophages each harboring a different recombinant DNA molecule. Ideally, nearly all of the DNA in thegenome will be represented in the library, and libraries constructed inthis way are referred to as genomic libraries. Each transformed bacterium grows into a colony or "clone" of cells, all of which have thesame recombinant plasmid. In the case of bacteriophages, each type ofrecombinant phage creates a plaque—a clear region of lysed cellswithin a lawn of bacteria distributed evenly on an agar plate; all of therecombinant bacteriophages within a plaque are identical.
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