anintroductiontogeneticanalysis(遗传学英文教科书第十二章)

Chapter 12
Recombinant DNA Technology Key Concepts
Recombinant DNA is made by splicing a foreign DNA fragment into a small replicating molecule <such as a bacterial plasmid>, which will then amplify that fragment along with itself and result in a molecular clone of the inserted DNA.
Restriction enzymes cut DNA at specific target sites, resulting in defined fragments with sticky ends suitable for insertion into a vector that has been cut open by the same enzyme.
A collection of DNA clones that encompasses the entire genome of an organism is called a genomic library.
An individual DNA clone can be selected from a library by using a specific probe for the DNA or its protein product or by its ability to transform a null mutant.
DNA fragments of different sizes produced by restriction-enzyme digestion can be fractionated because they migrate to different positions on an electrophoretic gel.
RNA or restriction-enzyme-cut DNA molecules that have been fractionated by size in an electrophoretic gel can be probed to detect a specific molecule.
Restriction-enzyme target sites can be mapped, providing useful markers for DNA manipulation.
A gene can be found by testing overlapping clones radiating outward from a linked marker. After a gene has been cloned, its nucleotide sequence can be determined, and the sequence can be used to study gene function and evolution.
A pair of replication primers spanning a DNA sequence can be used to amplify that sequence for isolation.
Introduction
The goal of genetics is to study the structure and function of genes and genomes. Since Mendel's time, genes have been identified by observing standard phenotypic ratios in controlled crosses. Clues about gene function first came from correlating specific mutations with enzyme and other protein deficiencies. The correlation of mutant sites within a gene with amino acid substitutions in the appropriate protein led to a better understanding of gene structure and function. To these ideas were added discoveries about the nature of DNA and the genetic code, leading to a fairly comprehensive understanding of the basic nature of the gene. However, all were indirect inferences about genes; no gene had ever been isolated and its DNA sequence examined directly. Indeed, it seemed impossible to isolate an individual gene from the genome.
Although it is relatively easy to isolate DNA from living tissue, DNA in a test tube looks like a glob of mucus. How could it be possible to isolate a single gene from this tangled mass of DNA threads? Recombinant DNA technology provides the techniques for doing just that, and today individual genes and other parts of genomes are isolated routinely.
Why is gene isolation so important? First, isolation of a gene enables the determination of its nucleotide sequence. From this information, the internal landmarks of the gene can be determined—for example, intron number and position. A comparison of DNA sequences between genes also can lead to insights in gene evolution. Converting the DNA sequence of a gene into amino acid sequence by using the genetic code leads to comparisons with the protein products of known genes; and, from this knowledge, the function of the gene can often be inferred. Function can also be studied by direct modification of part of the gene's DNA sequence followed by the reintroduction of the gene into the genome. Furthermore, a gene can be moved from one organism to another. An organism containing a foreign gene is called transgenic.Transgenic organisms can be used either for basic research or for specialized commercial applications. One application has been to make valuable human gene products such as insulin in transgenic bacteria carrying the appropriate human gene. From this brief overview, we see that gene isolation has become an indispensable tool of modern genetic analysis.
What are some examples of interesting genes that could be isolated? The answer depends very much on which biological process is being studied. Let's look at a few cases. A fungal geneticist studying the cellular pathway for synthesizing tryptophan would be interested in the genes that, when mutated, confer an auxotrophic requirement for tryptophan, because each gene would represent a step in the synthetic pathway <see Chapter 10>. These genes can be identified through mutation, segregation, and mapping analysis. They would be named trp1, trp2, trp3,and so forth. This geneticist would be very interested in isolating and characterizing one or more of these genes. Likewise, human genes that have mutant alleles conferring some type of functional disorder are interesting for medical and biological reasons. We have seen that these genes are identified by pedigree analysis. Two examples covered in Chapters 2 and 9 are the recessive autosomal conditions albinism and alkaptonuria. In these cases, the general nature of the defect has been understood for some time <both are enzyme defects>, but it would be very useful to isolate the genes themselves. Other human genes are known from pedigree analysis, but no biochemical function is known for them. Isolating such genes would be particularly useful because the characterization of gene structure might lead to a determination of gene function and the nature of the disease. A good example is cystic fibrosis, a disease known from pedigree analysis to be caused by an autosomal recessive allele of a gene for which no function was known until the gene was isolated and sequenced. Cases such as these would be raised in all the organisms used in genetic research.
In our consideration of gene isolation, we shall first examine the nature of recombinant DNA and the principle whereby recombinant DNA technology can be used to isolate a gene. Next, we shall examine the methods for isolating specific genes such as those just discussed.
Making recombinant DNA
How does recombinantDNA technology work? The organism under study, which will be used
to donate DNA for the analysis, is called the donor organism.The basic procedure is to extract and cut up DNA from a donor genome into fragments containing from one to several genes and allow these fragments to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids. These small circular molecules act as carriers, or vectors, for the DNA fragments. The vector molecules with their inserts are called recombinant DNA because they consist of novel combinations of DNA from the donor genome <which can be from any organism> with vector DNA from a completely different source <generally a bacterial plasmid or a virus>. The recombinantDNA mixture is then used to transform bacterial cells, and it is common for single recombinant vector molecules to find their way into individual bacterial cells. Bacterial cells are plated and allowed to grow into colonies. An individual transformed cell with a single recombinant vector will divide into a colony with millions of cells, all carrying the same recombinant vector. Therefore an individual colony contains a very large population of identical DNA inserts, and this population is called a DNA clone. A great deal of the analysis of the cloned DNA fragment can be performed at the stage when it is in the bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA back into cells of the original donor organism to carry out specific manipulations of genome structure and function. Hence the protocol is often as follows:
MESSAGE
Cloning allows the amplification and recovery of a specific DNA segment from a
large, complex DNA sample such as a genome.
Inasmuch as the donor DNA was cut into many different fragments, most colonies will carry a different recombinantDNA <that is, a different cloned insert>. Therefore, the next step is to find a way to select the clone with the insert containing the specific gene in which we are interested. When this clone has been obtained, the DNA is isolated in bulk and the cloned gene of interest can be subjected to a variety of analyses, which we shall consider later in the chapter. Notice that the cloning method works because individual recombinantDNA molecules enter individual bacterial host cells, and then these cells do the job of amplifying the single molecules into large populations of molecules that can be treated as chemical reagents. Figure 12-1 gives a general outline of the approach.
The term recombinantDNA must be distinguished from the natural DNA recombinants that result from crossing-over between homologous chromosomes in both eukaryotes and
prokaryotes. RecombinantDNA in the sense being used in this chapter is an unnatural union of DNAs from nonhomologous sources, usually from different organisms. Some geneticists prefer the alternative name chimeric DNA, after the mythological Greek monster Chimera. Through the ages, the Chimera has stood as the symbol of an impossible biological union, a combination of parts of different animals. Likewise, recombinantDNA is a DNA chimera and would be impossible without the experimental manipulation that we call recombinantDNA technology.
Isolating DNA
The first step in makingrecombinantDNA is to isolate donor and vector DNA. General protocols for DNA isolation were available many decades before the advent of recombinantDNA technology. With the use of such methods, the bulk of DNA extracted from the donor will be nuclear genomic DNA in eukaryotes or the main genomic DNA in prokaryotes; these types are generally the ones required for analysis. The procedure used for obtaining vector DNA depends on the nature of the vector. Bacterial plasmids are commonly used vectors, and these plasmids must be purified away from the bacterial genomic DNA. A protocol for extracting plasmid DNA by ultracentrifugation is summarized in Figure 12-2. Plasmid DNA forms a distinct band after ultracentrifugation in a cesium chloride density gradient containing ethidium bromide. The plasmid band is collected by punching a hole in the plastic centrifuge tube. Another protocol relies on the observation that, at a specific alkaline pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization precipitates the genomic DNA, but plasmids stay in solution. Phages such as λalso can be used as vectors for cloning DNA in bacterial systems. Phage DNA is isolated from a pure suspension of phages recovered from a phage lysate.
Cutting DNA
The breakthrough that made recombinantDNA technology possible was the discovery and characterization of restriction enzymes. Restriction enzymes are produced by bacteria as a defense mechanism against phages. The enzymes act like scissors, cutting up the DNA of the phage and thereby inactivating it. Importantly, restriction enzymes do not cut randomly; rather, they cut at specific DNA target sequences, which is one of the key features that make them suitable for DNA manipulation. Any DNA molecule, from viral to human, contains restriction-enzyme target sites purely by chance and therefore may be cut into defined fragments of a size suitable for cloning. Restriction sites are not relevant to the function of the organism, and they would not be cut in vivo, because most organisms do not have restriction enzymes.
Let's look at an example: the restriction enzyme Eco RI <from E. coli> recognizes the following six-nucleotide-pair sequence in the DNA of any organism:
This type of segment is called a DNA palindrome, which means that both strands have the
same nucleotide sequence but in antiparallel orientation. Many different restriction enzymes recognize and cut specific palindromes. The enzyme Eco RI cuts within this sequence but in a pair of staggered cuts between the G and the A nucleotides.
This staggered cut leaves a pair of identical single-stranded "sticky ends." The ends are called sticky because they can hydrogen bond <stick> to a complementary sequence. Figure 12-3 shows Eco RI making a single cut in a circular DNA molecule such as a plasmid: the cut opens up the circle, and the linear molecule formed has two sticky ends. Production of these sticky ends is another feature of restriction enzymes that makes them suitable for recombinantDNA technology. The principle is simply that, if two different DNA molecules are cut with the same restriction enzyme, both will produce fragments with the same complementary sticky ends, making it possible for DNA chimeras to form. Hence, if both vector DNA and donor DNA are cut with Eco RI, the sticky ends of the vector can bond to the sticky ends of a donor fragment when the two are mixed.
MESSAGE
Restriction enzymes have two properties useful in recombinant DNA technology.
First, they cut DNA into fragments of a size suitable for cloning. Second, many
restriction enzymes make staggered cuts that create single-stranded sticky ends
conducive to the formation of recombinant DNA.
Dozens of restriction enzymes with different sequence specificities have now been identified, some of which are shown in Table 12-1. You will notice that all the target sequences are palindromes, but, like Eco RI, some enzymes make staggered cuts, whereas others make flush cuts. Even flush cuts, which lack sticky ends, can be used for makingrecombinantDNA.
DNA can also be cut by mechanical shearing. For example, agitating DNA in a blender will break up the long chromosome-sized molecules into flush-ended clonable segments.
Joining DNA
Most commonly, both donor DNA and vector DNA are digested with the use of a restriction enzyme that produces sticky ends and then mixed in a test tube to allow the sticky ends of vector and donor DNA to bind to each other and form recombinant molecules. Figure 12-4a shows a plasmid vector that carries a single Eco RI restriction site; so digestion with the restriction enzyme Eco RI converts the circular DNA into a linear molecule with sticky ends. Donor DNA from any other source <say, Drosophila> also is treated with the Eco RI enzyme to produce a population of fragments carrying the same sticky ends. When the two populations are mixed, DNA fragments from the two sources can unite, because double helices form between their sticky ends. There are many opened-up vector molecules in the solution, and many different Eco RI fragments of donor DNA. Therefore a diverse array of vectors carrying different donor inserts will be produced. At this stage, although sticky ends
have united to generate a population of chimeric molecules, the sugar-phosphate backbones are still not complete at two positions at each junction. However, the backbones can be sealed by the addition of the enzyme DNA ligase,which create phosphodiester bonds at the junctions <Figure 12-4b>. Certain ligases are even capable of joining DNA fragments with blunt-cut ends.
Amplifying recombinant DNA
The ligated recombinantDNA enters a bacterial cell by transformation. After it is in the host cell, the plasmid vector is able to replicate because plasmids normally have a replication origin. However, now that the donor DNA insert is part of the vector's length, the donor DNA is automatically replicated along with the vector. Each recombinant plasmid that enters a cell will form multiple copies of itself in that cell. Subsequently, many cycles of cell division will take place, and the recombinant vectors will undergo more rounds of replication. The resulting colony of bacteria will contain billions of copies of the single donor DNA insert. This set of amplified copies of the single donor DNA fragment is the DNA clone <Figure 12-5>.
Cloning a specific gene
The foregoing descriptions are generic approaches to creating recombinant DNA. However, a geneticist is interested in isolating and characterizing some particular gene of interest, so the procedures must be tailored to isolate a specific recombinant DNA clone that will contain that particular gene. The details of the process differ from organism to organism and from gene to gene. An important initial factor is the choice of an appropriate vector for the job at hand.
Choosing a cloning vector
The ideal vector is a small molecule, facilitating manipulation. It must be capable of prolific replication in a living cell, thereby enabling the amplification of the inserted donor fragment. Another important requirement is to have convenient restriction sites that can be used for insertion of the DNA to be cloned. Unique sites are most useful because then the insert can be targeted to one site in the vector. It is also important to have a method for easily identifying and recovering the recombinant molecule. Numerous cloning vectors are in current use, and the choice between them often depends on the size of the DNA segment that needs to be cloned and on the intended application for the cloned gene. We shall consider several commonly used types.
Plasmids.
As described earlier, bacterial plasmids are small circular DNA molecules that are not only distinct from the main bacterial chromosome, but also additional to it. They replicate their DNA independently of the bacterial chromosome. Many different types of plasmids have been found in bacteria. The distribution of any one plasmid within a species is generally sporadic; some cells have the plasmid, whereas others do not. In Chapter 7, we encountered the F plasmid, which confers certain types of conjugative behavior to cells of E. coli. The F plasmid can be used as a vector for carrying large donor DNA inserts, as we shall see in
Chapter 14. However, the plasmids that are routinely used as vectors are those that carry genes for drug resistance. The drug-resistance genes are useful because the drug-resistant phenotype can be used to select not only for cells transformed by plasmids, but also for vectors containing recombinant DNA. Plasmids are also an efficient means of amplifying cloned DNA because there are many copies per cell, as many as several hundred for some plasmids.
Two plasmid vectors that have been extensively used in genetics are shown in Figure 12-6. These vectors are derived from natural plasmids, but both have been genetically modified for convenient use as recombinant DNA vectors. Plasmid pBR322 is simpler in structure; it has two drugresistance genes, tet R and amp R. Both genes contain unique restriction target sites that are useful in cloning. For example, donor DNA could be inserted into the tet R gene. A successful insertion will split and inactivate the tet R gene, which will then no longer confer tetracycline resistance, and the cell will be sensitive to that drug. Therefore, the cloning procedure is to mix the samples of cut plasmid and donor DNA, transform bacteria, and select first for ampicillinresistant colonies, which must have been successfully transformed by a plasmid molecule. Of the Amp R colonies, only those that prove to be tetracycline sensitive have inserts; in other words, the Amp R Tet S colonies are the ones that contain recombinant DNA. Further experiments are needed to find the clones with the specific insert required. The pUC plasmid is a more advanced vector, whose structure allows direct visual selection of colonies containing vectors with donor DNA inserts. The key element is a small part of the E. coli β-galactosidase gene. Into this region has been inserted a piece of DNA called a polylinker or multiple cloning site, which contains many unique restriction target sites useful for inserting donor fragments. The polylinker is in frame translationally with the β-galactosidase fragment and does not interfere with its translation. The transformation protocol uses recipient cells that contain a β-galactosidase gene lacking the fragment present on the plasmid. An unusual type of complementation takes place in which the partial proteins encoded by the two fragments unite to form a functional β-galactosidase. A colorless substrate for β-galactosidase called X-Gal is added to the medium, and the functional enzyme converts this substrate into a blue dye, which colors the colony blue. If donor DNA is inserted into the polylinker, the enzyme fragment borne on the vector is disrupted, no complete β-galactosidase protein is formed, and the colony is white. Hence, selection for white Amp R colonies selects directly for vectors bearing inserts, and such colonies are isolated for further study.
Small plasmids that contain large inserts of foreign DNA tend to spontaneously lose the insert; therefore, these plasmids are not useful for cloning DNA fragments larger than 20 kb.
Viral vectors.
Viral vectors, in which the gene or genes of interest are incorporated into the genome of a virus, offer many advantages for cloning and the subsequent applications of cloned genes. Because viruses infect cells with high efficiency, the cloned gene can be introduced into cells at a significantly higher frequency than by simple transformation. Some viral vectors are specialized for producing high levels of proteins encoded by the cloned genes, as exemplified
by the use of insect baculovirus to express foreign proteins in a eukaryotic cell system, which is detailed in Chapter 13. Other viral vectors, such as the bacterial M13-based vectors, are designed to facilitate sequencing and the generation of mutations in cloned genes. Vectors derived from retroviruses can effect the stable integration into mammalian chromosomes of cloned DNA, allowing continued expression of the gene. Viral vectors are also the vehicles of choice for gene-therapy strategies. Some examples of viral vectors used in bacteria are described next.
Phage lambda.Phage λ is a convenient cloning vector for several reasons. First, λ phage heads will selectively package a chromosome about 50 kb in length, and, as will be seen, this property can be used to select for λ molecules with inserts of donor DNA. The central part of the phage genome is not required for replication or packaging of λ DNA molecules in E. coli, so the central part can be cut out by using restriction enzymes and discarded. The two "arms" are ligated to restriction-enzyme-cut donor DNA. The chimeric molecules can be either introduced into E. coli directly by transformation or packaged into phage heads in vitro. In the in vitro system, DNA and phage-head components are mixed together, and infective λ phages form spontaneously. In either method, recombinant molecules with 10- to 15-kb inserts are the ones that will be most effectively packaged into phage heads, because this size of insert substitutes for the deleted central part of the phage genome and brings the total molecule size to 50 kb. Therefore the presence of a phage plaque on the bacterial lawn automatically signals the presence of recombinant phage bearing an insert <Figure 12-7>. A second useful property of a phage vector is that recombinant molecules are automatically packaged into infective phage particles, which can be conveniently stored and handled experimentally.
Single-stranded phages.Some phages contain only single-stranded DNA molecules. On infection of bacteria, the single infecting strand is converted into a double-stranded replicative form, which can be isolated and used for cloning. The advantage of using these phages as cloning vectors is that single-stranded DNA is the very substrate required for the Sanger DNA-sequencing technique currently in widespread use <page 387>. Phage M13 is the one most widely used for this purpose.
Cosmids.
Cosmids are vectors that are hybrids of λ phages and plasmids, and their DNA can replicate in the cell like that of a plasmid or be packaged like that of a phage. However, cosmids can carry DNA inserts about three times as large as those carried by λ itself <as large as about 45 kb>. The key is that most of the λ phage structure has been deleted, but the signal sequences that promote phage-head stuffing <cos sites> remain. This modified structure enables phage heads to be stuffed with almost all donor DNA. Cosmid DNA can be packaged into phage particles by using the in vitro system. Cloning by cosmids is illustrated in Figure 12-8.
Expression vectors.
One way of detecting a specific cloned gene is by detecting its protein product expressed in the bacterial cell. Therefore, in these cases, it is necessary to be able to express the gene in bacteria; that is, to transcribe it and translate the mRNA into protein. Most cloning vectors do
not permit expression of cloned genes, but such expression is possible if special vectors are used. However, because bacteria cannot process introns, the cloned sequences must be stripped of introns. The cloned gene is inserted next to appropriate bacterial transcription and translation start signals. Some expression vectors have been designed with restriction sites located just next to a lac regulatory region. These restriction sites permit foreign DNA to be spliced into the vector for expression under the control of the lac regulatory system.
Making a DNA library
We have learned that the most important goal of recombinant DNA technology is to clone a particular gene or other genomic fragment of interest to the researcher. The approach used to clone a specificgene depends to a large degree on the gene in question and on what is known about it. Generally, the procedures start with a sample of DNA such as eukaryotic genomic DNA. The next step is to obtain a large collection of clones made from this original DNA sample. The collection of clones is called a DNA library. This step is sometimes referred to as "shotgun" cloning because the experimenter clones a large sample of fragments and hopes that one of the clones will contain a "hit"—the desired gene. The task then is to find that particular clone.
There are different types of libraries, categorized, first, according to which vector is used and, second, according to the source of DNA. Different cloning vectors carry different amounts of DNA, so the choice of vector for library construction depends on the size of the genome <or other DNA sample> being made into the library. Plasmid and phage vectors carry small amounts of DNA, so these vectors are suitable for cloning genes from organisms with small genomes. Cosmids carry larger amounts of DNA, and other vectors such as YACs and BACs <see Chapters 13 and 14> carry the largest amounts of all. Ease of manipulation is another important factor in choosing a vector. A phage library is a suspension of phages. A plasmid or a cosmid library is a suspension of bacteria or a set of defined bacterial cultures stored in culture tubes or microtiter dishes.
The second important decision is whether to make a genomic library or a cDNA library. cDNA,or complementary DNA, is synthetic DNA made from mRNA with the use of a special enzyme called reverse transcriptase originally isolated from retroviruses. With the use of an mRNA as a template, reverse transcriptase synthesizes a single-stranded DNA molecule that can then be used as a template for double-stranded DNA synthesis <Figure 12-9>. Because it is made from mRNA, cDNA is devoid of both upstream and downstream regulatory sequences and of introns. Therefore cDNA from eukaryotes can be translated into functional protein in bacteria—an important feature when expressing eukaryotic genes in bacterial hosts.
The choice between genomic DNA and cDNA depends on the situation. If a specificgene that is active in a specific type of tissue in a plant or animal is being sought, then it makes sense to use that tissue to prepare mRNA to be converted into cDNA and then make a cDNA library from that sample. This library should be enriched for the gene in question. A cDNA library is based on the regions of the genome transcribed, so it will inevitably be smaller than a complete genomic library, which should contain all of the genome. Although genomic。