Choosing a Cloning Vector

Vectors must be relatively small molecules for convenience of manipulation. They must be capable of prolific replication in a living cell, thereby enabling the amplification of the inserted donor fragment. Another important requirement is that there be 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 that there be a mechanism for easy identification and recovery of the recombinant molecule. There are numerous cloning vectors in current use, and the choice between them often depends on the size of the DNA segment that needs to be cloned. We will consider several commonly used types.

Plasmids

As described earlier, bacterial plasmids are small circular DNA molecules that are distinct from, as well as additional to, the main bacterial chromosome. 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 9, 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 12. 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 10-6 on the following page. 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 drug-resistance 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 then will no longer confer tetracycline resistance, and the cell will be sensitive to that drug. Therefore, the cloning procedure would be to mix the samples of cut plasmid and donor DNA, transform bacteria, and select first for ampicillin-resistant 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.

Figure 10-6

 

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 occurs in which the partial proteins coded 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.

Plasmids that contain large inserts of foreign DNA tend to spontaneously lose the insert; therefore, plasmids are not useful for cloning DNA fragments larger than 20 kb.

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-digested 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 10-7). 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.

Figure 10-7

 

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 10-8.

Figure 10-8

 

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 specific gene 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 Chapter 12) 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 10-9). Because it is made from mRNA, cDNA is devoid of both upstream and downstream regulatory sequences and of introns. This means that cDNA from eukaryotes can be translated into functional protein in bacteria—an important feature when expressing eukaryotic genes in bacterial hosts.

Figure 10-9

 

The choice between genomic DNA and cDNA depends on the situation. If a specific gene 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 libraries are bigger, they do have the benefit of containing genes in their native form, including introns and regulatory sequences. If the purpose of constructing the library is a prelude to cloning an entire genome, then a genomic library is necessary at some stage.

In some cases, it is possible to narrow down the genomic fraction used in library construction, to more easily detect the desired gene. This approach is possible if the experimenter already knows which chromosome contains the gene. One technique used in mammalian molecular genetics is to sort the chromosomes with an instrument called a flow cytometer. A suspension of chromosomes is passed through the apparatus, which sorts the chromosomes according to size (this procedure is discussed in more detail in Chapter 12). The appropriate chromosomal fraction is then used to make the library.

Another technique possible in organisms with small chromosomes is to fractionate whole chromosomes by using pulsed field gel electrophoresis (PFGE). Electrophoresis is a general technique that fractionates nucleic acids or proteins according to size on gels under the influence of a strong electric field. This type of procedure separates shorter DNA fragments. PFGE is a specialized type of electrophoresis useful for very long DNA molecules. It uses several oscillating electric fields oriented in several different directions. This enables large DNA molecules such as whole chromosomes to snake through the gel to different positions according to their size. The appropriate chromosome can be identified on the gel by probing with a chromosome-specific probe (see the next subsection). Then the desired chromosome can be cut out, eluted from the gel, and used to make a chromosome-specific library.

How can an experimenter determine whether a library is large enough to contain any one unique sequence of interest with a reasonable degree of certainty? There are formulas for calculating the minimum number of clones needed, but a rough idea of the general order of magnitude of the library can be obtained simply by taking the total genome size and dividing by the average size of the inserts carried by the vector being used. Generally, this number will be at least doubled, but it does provide a rough estimate of the magnitude of the job of library construction.

Finding Specific Clones by Using Probes

The library, which might contain as many as hundreds of thousands of cloned fragments, must be screened to find the recombinant DNA molecule containing the gene of interest. Such screening is accomplished by using a specific probe that will find and mark the clone for the researcher to identify. Broadly speaking, there are two types of probes: those that recognize DNA and those that recognize protein.

Probes for finding DNA

These probes depend on the natural tendency of a single strand of nucleic acid to find and hyb ridize to another single strand with a complementary base sequence. A probe that is itself DNA, when denatured (made single-stranded by unwinding the two halves of the double helix), will therefore find and bind to other similar denatured DNAs in the library.

Identification of a specific clone in a library is a two-step procedure (Figure 10-10). First, colonies or plaques of the library on a petri plate are transferred to an absorbent membrane (often nitrocellulose) by simply laying the membrane on the surface of the medium. The membrane is peeled off, and colonies or plaques clinging to the surface are lysed in situ and the DNA denatured. The next step is to bathe the membrane with a solution of a probe that is specific for the DNA being sought. The probe must be labeled either with radioactivity or a fluorescent dye. Generally, the probe is itself a cloned piece of DNA that has a sequence homologous to the desired gene. The probe DNA must be denatured; it will then bind only to the DNA of the clone being sought. The position of a positive clone will become clear from the position of the concentrated label, often as a spot on an autoradiogram.

Figure 10-10

Facing page: (a) A genomic library can be made by cloning genes in λ bacteriophages.

Where does the DNA to make a probe come from?

The DNA can be from one of several sources. One source is cDNA from tissue that expresses the gene of interest. The idea is that, because the mRNA of a gene is abundant, many of the cDNAs made from this tissue and inserted individually into vectors will very likely be for the desired gene. For example, in mammalian reticulocytes, 90 percent of the mRNA is known to be transcribed from the β-globin gene, so reticulocytes would be a good source of mRNA for making a cDNA probe to find a genomic globin gene. In this case, a genomic library would be probed. The need for this kind of analysis depends on which questions are to be asked about the gene. If only the transcribed sequence is of interest, then the cDNA clone itself could provide that information just as well. However, if introns and control regions are needed, the genomic clone must be obtained.

Another source of DNA for a probe might be a homologous gene from a related organism. For example, if a certain gene has been cloned in the ascomycete fungus Neurospora, then it is very likely that this gene can be used as a probe to find the homologous gene in the related fungus Podospora. This method depends on the evolutionary conservation of DNA sequences through time. Even though the probe DNA and the DNA of the desired clone might not be identical, they are often similar enough to promote hybridization. The method is jokingly called “clone by phone” because, if you can phone a colleague who has a clone of your gene of interest but from a related organism, then your job of cloning is made relatively easy.

 Probe DNA can be synthesized if the protein product of the gene of interest is known and an amino acid sequence has been obtained. Synthetic DNA probes are designed on the basis of knowledge of the genetic code, so an amino acid sequence merely has to be translated backward to obtain the DNA sequence that encoded it. However, because of the redundancy of the code—in other words, the fact that most amino acids are coded by more than one codon—several possible DNA sequences could have encoded the protein in question. To get around this problem, a short stretch of amino acids with minimal redundancy is selected. The nucleotide sequence is calculated by using the codon dictionary. The chemical DNA synthesizing reaction is a step-by-step process, so wherever in the sequence there are alternative nucleotides, a mixture of those alternative nucleotides is fed into the reaction and all possible DNA strands are synthesized. Figure 10-11 shows an example in which there are five positions of redundancy, showing 2, 3, 2, 2, and 2 alternatives, respectively. The reaction would make 2 × 3 × 2 × 2 × 2 = 48 oligonucleotide strands at the same time. This “cocktail” of oligonucleotides would be used as a probe. The correct strand within this cocktail would find the gene of interest. Twenty nucleotides embody enough specificity to find one unique DNA sequence in the library. 

Figure 10-11

A short sequence of a protein is used to design a set of redundant oligonucleotides for use as a probe to recover the gene that encoded the protein.

Additionally, free RNA can be radioactively labeled and used as a probe. This is possible only when a relatively pure population of identical molecules of RNA can be isolated, such as rRNA or fractionated tRNAs.

Probes for finding proteins

If the protein product of a gene is known and isolated in pure form, then this protein can be used to detect the clone of the corresponding gene in a library. The process is described in Figure 10-12. An antibody to the protein is prepared, and this antibody is used to screen an expression library. These libraries are made by using expression vectors designed to express high levels of a specific bacterial protein. To make the library, cDNA is inserted into the vector in frame with the bacterial protein, and the cells will make a fusion protein. A membrane is laid over the surface of the medium and removed with an imprint of colonies. It is dried and bathed in a solution of the antibody. Positive clones are revealed by making an antibody to the first antibody; the second antibody is labeled by a radioactive isotope or a chemical that will fluoresce or become a colored dye. By detecting the correct protein, the antibody effectively identifies the clone containing the gene that must have synthesized that protein.

Figure 10-12

Finding the clone of interest by using antibody. An expression library made with phage derivative λgt11 is screened with a protein-specific antibody.

At the beginning of the chapter, we asked how it might be possible to find the gene for human albinism. It was in fact cloned by using an antibody to the enzyme that is known to be defective in this condition, the enzyme tyrosinase. This enzyme, like any protein, can be purified by standard biochemical procedures, and subsequently an antibody to the enzyme was prepared in rabbits. From tyrosinase-producing cells, mRNA was isolated and used to make cDNA. This cDNA was used to make an expression vector library. The library was probed with the antibody to tyrosinase, and several positive clones were detected. The cDNA in the positive clones was sequenced and found to contain a gene whose exons total 1590 nucleotide pairs. The cDNA was used to probe a library of human genomic DNA, and, in this process, the intact tyrosinase gene was found. It proved to have five exons and four introns.

MESSAGE

A cloned gene can be selected from a library by using probes for the gene’s DNA sequence or for the gene’s protein product.

Finding Specific Clones by Functional Complementation

Specific clones in a bacterial or phage library can be detected through their ability to confer a missing function on a mutant line of the donor organism, which acts as the transformation recipient. This procedure is called functional complementation. Here the protocol is:

This method depends on the ability to transform the donor organism, often a eukaryote. We have already considered transformation in prokaryotes (Chapters 2 and 9), but eukaryotes can be transformed, too. The procedure differs among eukaryotes, but generally some special treatment of recipient cells is required. For example, to transform fungi, generally the cell walls must be removed enzymatically. Let’s assume that we have isolated a mutant that is relevant to some biological process that interests us. For the present purpose, we will assume that it is an auxotrophic mutation in a fungus. We shall use DNA from the library to transform the auxotrophic mutant strain and then plate these recipient fungal cells on minimal medium. Fungal cells that contain the wild-type allele (from the wild-type culture used to make the library) will transform the auxotroph to prototrophy and allow growth on minimal medium.

The reason that this transformation method works is that the transforming fragment functionally complements the deficiency caused by the mutant allele in the recipient. The production of a wild-type phenotype from the union of two mutant genomes. However, the transforming vector contributes something that the recipient genome lacks (the wild-type allele being sought), and the recipient genome contributes something that the vector lacks (the entire remainder of the genome), so a type of complementation is involved.

If the transformation recipient is an organism in which plasmid vectors replicate autonomously (mainly bacteria and yeasts), then the transforming insert can be recovered simply by isolating the plasmid. However, as we shall see, in most eukaryotic organisms the bacterial or phage vector cannot replicate and must insert into the genome to achieve stable transformation. In these cases, the transforming fragment is relatively inaccessible and must be retrieved from the successful clone in the library. This method uses a library in which the clones are laid out as a collection of numbered bacterial cultures in tubes or microtiter dishes. DNA is isolated in bulk from all the strains in specific subsets of the library, and transformation is attempted. By a process of narrowing down the library subsets that successfully transform, the clone with the wild-type allele can be identified The process is illustrated in Figure 10-13, using as an example the Neurospora trp3 gene discussed at the beginning of the chapter. In this case, a cosmid library was used. The cosmid must also carry a marker gene that can be used to select for successful transformants of the fungus. A gene for hygromycin resistance is commonly used in fungi, which are normally sensitive to this drug. Subsets of the cosmid library made from wild-type Neurospora DNA were used to transform trp3 mutant cells, and trp3⁺ clones were selected by plating transformed cells on medium containing hygromycin but lacking tryptophan. Colonies that grow are likely to contain the trp3⁺ allele and are isolated from the plate.

Figure 10-13

Finding a cloned gene by using progressively smaller pooled DNA samples in transformation. In this example, the quest is for the trp3 gene of Neurospora

In most cases, transformants are found to contain the vector carrying the wild-type allele inserted into one of the recipient’s chromosomes at a location that is different from the mutant locus in the recipient. This is called ectopic insertion (Figure 10-14). Less commonly, the transforming wild-type allele replaces the resident auxotrophic mutation by a double-crossover-like process.

Figure 10-14

The possible fates of transforming DNA. A donor wild-type allele A⁺ (cloned in a bacterial vector) transforms an A⁻ recipient by one of three.

If a eukaryotic gene is cloned on a prokaryotic vector but a specific eukaryotic sequence is known that can act as an origin of replication, this sequence can be added to the vector. Then the vector will be able to replicate in both bacterial and eukaryotic cells, and insertion into the chromosome is not essential. These types of vectors are called shuttle vectors because they can be moved back and forth between different hosts. Without an origin of replication, the donor DNA must integrate into the eukaryotic chromosome to effect stable transformation.

Positional Cloning

Information about a gene’s position in the genome can be used to circumvent the hard work of assaying an entire library to find the clone of interest. Positional cloning is a term that can be applied to any method that makes use of such information. Often both probing and complementation are part of positional cloning. A common starting point is the availability of another cloned gene or other marker known to be closely linked to the gene being sought. The linked marker acts as the departure point in a process, called chromosome walking, that will terminate at the target gene. Figure 10-15 summarizes the procedure of chromosome walking. End fragments of a clone of the linked marker are used as probes to select other clones from the library. These probes will detect clones of DNA regions that overlap with the initial clone. Restriction maps (pages 327–329) are made of the DNA of this second set of clones, and, again, outward fragments are used for a new round of selection of overlapping clones from the library. Hence the walking process moves outward in two directions from the start site. Each clone can be sequenced or otherwise tested, depending on the intent of the exploration.

Figure 10-15

Chromosome walking. One recombinant phage obtained from a phage library made by the partial EcoRI digest of a eukaryotic genome can be used to isolate another recombinant phage.

Sometimes a large insert that is known to contain the linked marker will also luckily contain the sought gene, and subcloning and transformation will narrow down the appropriate region of the cosmid. The availability of a large number of neutral DNA markers (restriction fragment length polymorphisms) dispersed throughout most genomes has provided many useful start points. Positional cloning has been particularly useful for cloning human genes, many of which have no known biochemical function and cannot be easily selected by functional complementation. The human gene for cystic fibrosis, mentioned at the beginning of the chapter, was cloned by chromosome walking, and we shall examine its cloning in more detail in Chapter 12. For any case of chromosome walking, there must be some type of criterion to assess each step of the walk for the gene of interest, and these criteria depend on the individual gene concerned.

Cloning a Gene by Tagging

Tagging is a cloning method that zeros in on the desired gene directly by inducing a mutation in that gene by using a specific piece of DNA as an insertional mutagen. The specific sequence is then used as a tag to recover the gene. The approach is summarized in Figure 10-16. One type of tag is transforming DNA. When exogenous DNA is added by transformation or by other methods such as injection, it can integrate into the genome and become part of the chromosome. Ectopic integration is random throughout the genome, and apparently no segment of chromosomal DNA is immune to integration. When integration takes place within or near a gene, the integrating fragment acts as a mutagen, disrupting the function of the gene. This property can be used to good advantage. Suppose that we use a specific cloned gene x⁺ and transform x⁻ cells of the donor organism into x⁺. Many of the x⁺ transformants will be mutant for the genes into which the transforming DNA has inserted ectopically. A subset of such x⁺ cells will be mutant for the target gene a⁺, the gene of interest, and will be of phenotype a⁻. Hence among the x⁺ transformants, a⁻ phenotypes are identified. The next step is to cross the transformants to determine if the a⁻ phenotype segregates with x⁺. If it does, the mutation is likely to have been caused by the integration of the fragment containing x⁺. The DNA of this mutant line is used to construct a library, and gene x⁺ can be used as a probe to recover the clone of the disrupted a gene. To recover the intact wild-type a gene, a fragment of the disrupted a gene sequence is used in another round of probing, this time with a wild-type library.

Figure 10-16

Using DNA insertion as a tag for marking and recovering a gene from the genome. The tag DNA can be transforming DNA or an endogenous transposon (movable element).

A similar approach uses transposons as tags. Transposons are naturally mobile DNA fragments found in many organisms. When they move, they can insert anywhere in the genome. If they insert into or near a gene, they can create a null mutation. (Transposons are described in more detail in Chapter 13). In a line containing an active transposon, mutants for the desired gene are selected. Many of these mutants will be caused by the insertion of the transposon. This mutant line is used to make a library. A cloned part of the transposon DNA can then be used as a tag to recover the gene, in a manner similar to that shown in Figure 10-16.