10 Генетическая инженерия (1160079), страница 2
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Over 100 different specific sequences arerecognized by one or more of these enzymes. These sequences are almost always short (four to six base pairs, occasionally more) and palindromic (see Fig. 12—20). A sampling of sequences recognized by sometype II restriction endonucleases is presented in Table 28-2. Note thatthe name of each enzyme consists of a three-letter abbreviation of thebacterial species from which it is derived (e.g., Bam for bacillussxnyloliquefaciens, Eco for Escherichia coli).In a few cases, the interaction between a restriction endonucleaseand its target sequence has been elucidated in exquisite molecular detail.
The complex comprising the type II restriction endonucleaseEcoRI and its target sequence is illustrated in Figure 28-2. DNA sequence recognition by Eco RI is mediated by 12 hydrogen bonds formedbetween the purines in the recognition site and six amino acid residuesin the dimeric endonuclease (one Glu and two Arg residues in eachsubunit). Some restriction endonucleases cleave both strands of DNAso as to leave no unpaired bases on either end; these ends are oftencalled blunt ends (Fig. 28-3a).
Others make staggered cuts on thetwo DNA strands, leaving two to four nucleotides of one strand unpaired at each resulting end. These are referred to as cohesive endsor sticky ends (Fig. 28-3a) because they can base-pair with eachother or with complementary sticky ends of other DNA fragments.987Table 28—2 Recognition sequences for sometype II restriction endonucleasesiBamHl(5')GGATCC(3')CCTAGGtClal(5') ATCGAT(3')TAGCTA* T1 *(5')GAATTC (3')CTTAAG* tEcoRlHaelllHindlll1*1*(5')GGCC(3')CCGG*T1(5')AAGCTT(3')TTCGAATNotlPstlPvullI(5')GCGGCCGC(3 ; )CGCCGGCGt*l(5')CTGCAG(3')GACGTC!*1(5)CAGCTG(3')GTCGACSmalt4(5)CCCGGG(3)GGGCCCTthllllt1(5') GACNNNGTC (3')CTGNNNCAGArrows indicate the phosphodiester bonds cleavedt by each restriction endonuclease.
Asterisks indicate bases that are methylated by the corresponding methylase (where known). N denotes any base. The Roman numerals included in the enzymenames (e.g., BamHl) distinguish different restriction endonucleases isolated from the same bacterial species rather thanthe type of restriction enzyme.Figure 28-2 The interaction of Eco RI endonuclease with its target sequence.
The dimeric enzyme(with its two subunits in gray and light blue) isshown bound to DNA. In the top view the DNAbinding site is facing the viewer. In the bottomview the bound DNA is facing away from theviewer and is not visible. In the bound DNA, thebases that make up the recognition site for EcoRlare shown in red.CleavagesiteChromosomal DNARecognition' sequencesCleavagesite- G G T G [ A A_T_T CAGCTTCGCATTAGCAGlCTGTAGCC C A C T T A A G T C G A A G C G T A A T C GTCiGAC ATCGtPniUrestrictionendonucleaserestrictionI'tidonucloascGGTIGCCACTTAAl[AATTCIAGCTTCGCATTAGCAGHTC-GAAGC GTAATC GTCSticky endsCTGTAGCGACATCGBlunt endsDXAPlasmidcloning vectorcleaved withEcoRl and PvullPstIBamHIHindlllSmalAATTGCTGCAGAAGCTTCCGGATCCCCGGGCGACGTCTTCGAAGGCCTAGGGGCCCTTAASynthetic polylinkerPlasmid cloning vectorcleaved with EcoRl(b)988(a)Figure 28-3 Cleavage of DNA molecules into reproducible fragments by restriction endonucleases.Restriction enzymes recognize and cleave only specific sequences, leaving either sticky ends (with protruding single strands) or blunt ends, (a) Cleavageof a DNA yields a characteristic set of fragments,and these fragments can be ligated to other DNAssuch as the cleaved cloning vector (a plasmid)shown here.
The ligation reaction is facilitated bythe annealing of complementary sticky ends. DNAfragments with blunt ends are ligated at a lowerefficiency than those with complementary stickyends, and DNA fragments with different (noncomplementary) sticky ends generally are not ligated.(b) A synthetic DNA fragment containing the recognition sequences for several restriction endonucleases can be inserted into a plasmid after it hasbeen cleaved by a restriction endonuclease; thiscreates a polylinker.The average size of the DNA fragments produced by cleavinggenomic DNA with a restriction endonuclease depends upon the frequency with which a particular restriction site occurs in a large DNAmolecule; this in turn depends largely on the size of the recognitionsequence.
In a DNA molecule with a random sequence in which all fournucleotides are equally abundant, a 6 base pair sequence recognized bya restriction endonuclease such as BamHI will occur on average onceevery 4 6 , or 4,096, base pairs. Enzymes that recognize a 4 base pairChapter 28 Recombinant DNA Technologysequence will produce smaller DNA fragments; a recognition sequenceof this size would be expected to occur on average every 256 base pairs.These sequences tend to occur less frequently than this because nucleotide sequences in DNA are not random and the four nucleotides arenot equally abundant. The average size of the fragments produced byrestriction endonuclease cleavage of a large DNA can be increased bysimply not allowing the reaction to go to completion. Such an incomplete reaction is often called a partial digest.Once a DNA molecule has been cleaved into fragments, a particular fragment that a researcher is interested in can be separated fromthe others by agarose gel electrophoresis (p.
347) or HPLC (p. 122).Because cleavage of a typical mammalian genome by a restriction endonuclease may yield several hundred thousand different fragments,isolation of a particular DNA fragment by electrophoresis or HPLC isoften impractical. In these cases an intermediate step in the cloning ofa specific gene or DNA segment of interest is the construction of a DNAlibrary, described later in the chapter.When the target DNA fragment is isolated, it is joined to a cloningvector using DNA ligase. The base-pairing of complementary stickyends greatly facilitates the ligation reaction (Fig.
28-3). Because different restriction endonucleases usually generate different stickyends, the efficiency of the ligation step is greatly affected by the endonucleases used to generate the DNA fragments. A fragment generatedby EcoRI generally will not be linked to a fragment generated byBamHl. Blunt ends can also be ligated, albeit less efficiently.Before ligating two DNA fragments, it is often useful to add recognition sequences for a restriction endonuclease (other than that used tocreate the fragments) at the junction to permit cleavage of the ligatedDNA at that location later on.
This is often done by inserting a synthetic DNA fragment containing the required recognition sequencebetween the two DNA fragments. Such a synthetic DNA fragment isgenerally called a linker. A synthetic fragment containing recognitionsequences for several restriction endonucleases is called a polylinker(Fig.
28-3b).The importance of sticky ends in efficiently joining two DNA fragments in a desired manner was apparent in the earliest recombinantDNA experiments. Before restriction endonucleases were widely available, some workers found that sticky ends could be generated by thecombined action of the bacteriophage A exonuclease and terminaltransferase (Table 28-1).
The fragments to be joined were given complementary homopolymeric tails (Fig. 28-4, p. 990). This method wasused by Peter Lobban and Dale Kaiser in 1971 in the first experimentsto join naturally occurring DNA fragments. Similar methods were usedsoon after in the laboratory of Paul Berg to join DNA segments fromsimian virus 40 (SV40) to DNA derived from bacteriophage A, therebycreating the first recombinant DNA molecule involving DNA segmentsfrom different species.Cloning Vectors Amplify Inserted DNA SegmentsThree types of cloning vectors—plasmids, bacteriophages, and cosmids—are commonly used in E. coli.
Plasmids (see Fig. 23-3) arecircular DNA molecules that replicate separately from the host chromosome. Naturally occurring bacterial plasmids range in size from5,000 to 400,000 base pairs.989Part IV Information Pathways9905'5'5'NNNNN3'3'3'NNNNNdATP,dTTP,f|*i5'Deoxynucleosidemonophosphatesrn fit fin nn np fit fitAAAAA3'•5'NNNNN3'NNannealingdTTP,5'rri rri tr\ r§t ro rii rr* rji3'AAAAA5'3'NNNNNTTTTTTTTNN(b)5'3'AAAAAAAA5'rri np nri rrt rri np np fin3'AAAAAAAAITIinn rrt rrifin jnrt rriFTI(a)Figure 28-4 Sticky ends generated by terminaltransferase can be used to join two DNA fragments,(a) Complementary homopolymeric tails are addedto the ends of the two fragments to be joined, forming sticky ends. After annealing, the gaps are filledand the nicks sealed by the action of DNA polymerase I and DNA ligase (Chapter 24).
(b) The optimalsubstrate for terminal transferase is the 3' OH atthe end of a single strand of DNA at least threenucleotides long. If the ends of the duplex DNAhave a 5' protruding single strand or are bluntends, the A exonuclease (which degrades DNAstrands in the 5'-»3' direction) can be used to create a good substrate for terminal transferase.N denotes any base.Plasmids can be introduced into bacterial cells by a process calledtransformation. To get the cells to take up the DNA, the cells andDNA are incubated together at 0 °C in a calcium chloride solution, thensubjected to heat shock by rapidly shifting the cells to temperatures of37 to 43 °C.
For reasons not entirely understood, cells so treated become "competent" to take up the DNA. Because only a few cells take upthe plasmid DNA, a method is needed for selecting those that do. Theusual strategy is to build into the plasmid a gene that the host cellrequires for growth under specific conditions. This makes a cell thatcontains the plasmid "selectable" if the cell is grown under those conditions. The gene, sometimes called a selectable marker, is often one thatconfers resistance to an antibiotic. Only those few cells that have beentransformed by the recombinant plasmid will be antibiotic resistantand thus able to grow in the presence of the antibiotic.Many different plasmid vectors suitable for cloning have been developed by modifying naturally occurring plasmids.
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