Vectors-16.8.24-Sent.pdf - Cloning Vectors

Module 3 Vectors A K A Mandal 1  A DNA molecule needs to display several features to be able to act as a vector for gene cloning Most importantly: – It must be able to replicate within the host cell, so that numerous copies of the recombinant DNA molecule can be p...

Module 3 Vectors A K A Mandal 1  A DNA molecule needs to display several features to be able to act as a vector for gene cloning Most importantly: – It must be able to replicate within the host cell, so that numerous copies of the recombinant DNA molecule can be produced and passed to the daughter cells – A cloning vector also needs to be relatively small, ideally less than 10 kb in size, as large molecules tend to break down during purification, and are also more difficult to manipulate. Two kinds of DNA molecule that satisfy these criteria can be found in bacterial cells: – plasmids and – bacteriophage chromosomes 2  Plasmid Biology Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell Plasmids almost always carry one or more genes, and often these genes are responsible for a useful characteristic displayed by the host bacterium. For example, the ability to survive in normally toxic concentrations of antibiotics such as chloramphenicol or ampicillin is often due to the presence in the bacterium of a plasmid carrying antibiotic resistance genes In the laboratory, antibiotic resistance is often used as a selectable marker to ensure that bacteria in a culture contain a particular plasmid 3 Plasmids: independent genetic elements found in bacterial cells. 4  Most plasmids possess at least one DNA sequence that can act as an origin of replication, so they are able to multiply within the cell independently of the main bacterial chromosome The smaller plasmids make use of the host cell’s own DNA replicative enzymes in order to make copies of themselves, whereas some of the larger ones carry genes that code for special enzymes that are specific for plasmid replication A few types of plasmid are also able to replicate by inserting themselves into the bacterial chromosome. These integrative plasmids or episomes may be stably maintained in this form through numerous cell divisions, but always at some stage exist as independent elements 5 The use of antibiotic resistance as a selectable marker for a plasmid. RP4 (top) carries genes for resistance to ampicillin, tetracycline and kanamycin. Only those E. coli cells that contain RP4 (or a related plasmid) are able to survive and grow in a medium that contains toxic amounts of one or more of these antibiotics. 6 Replication strategies for (a) a non-integrative plasmid, and (b) an episome. 7 Some of the basic properties of plasmids Plasmids are replicons which are stably inherited in an extrachromosomal state Most plasmids exist as double-stranded circular DNA molecules If both strands of DNA are intact circles the molecules are described as covalently closed circles or CCC DNA If only one strand is intact, then the molecules are described as open circles or OC DNA When isolated from cells, covalently closed circles often have a deficiency of turns in the double helix, such that they have a supercoiled configuration 8  Because of their different structural configurations, supercoiled and OC DNA separate upon electrophoresis in agarose gels Addition of an intercalating agent, such as ethidium bromide, to supercoiled DNA causes the plasmid to unwind If excess ethidium bromide is added, the plasmid will rewind in the opposite direction Use of this fact is made in the isolation of plasmid DNA 9  The interconver sion of supercoiled, relaxed covalently closed circular DNA and open circular DNA. 10 11 12  The host range of plasmids is determined by the replication proteins that they encode Plasmids encode only a few of the proteins required for their own replication and in many cases encode only one of them. All the other proteins required for replication, e.g. DNA polymerases, DNA ligase, helicases, etc., are provided by the host cell Those replication proteins that are plasmid-encoded are located very close to the ori (origin of replication) sequences at which they act. Thus, only a small region surrounding the ori site is required for replication Other parts of the plasmid can be deleted and foreign sequences can be added to the plasmid and replication will still occur. This feature of plasmids has greatly simplified the construction of versatile cloning vectors 13  The host range of a plasmid is determined by its ori region Plasmids whose ori region is derived from plasmid Col E1 have a restricted host range: – they only replicate in enteric bacteria, such as E. coli, Salmonella, etc. Other promiscuous plasmids have a broad host range and these include – RP4 and RSF1010. Plasmids of the RP4 type will replicate in most Gram-negative bacteria, to which they are readily transmitted by conjugation. Such promiscuous plasmids offer the potential of readily transferring cloned DNA molecules into a wide range of genetic backgrounds 14  Plasmids like RSF1010 are not conjugative but can be transformed into a wide range of Gram-negative and Gram- positive bacteria, where they are stably maintained. Many of the plasmids isolated from Staphylococcus aureus also have a broad host range and can replicate in many other Gram- positive bacteria Plasmids with a broad host range encode most, if not all, of the proteins required for replication. They must also be able to express these genes and thus their promoters and ribosome binding sites must have evolved such that they can be recognized in a diversity of bacterial families 15  Size and copy number The size and copy number of a plasmid are particularly important as far as cloning is concerned. Plasmid size less than 10 kb is desirable for a cloning vector Plasmids range from about 1.0 kb for the smallest to over 250 kb for the largest plasmids, so only a few are useful for cloning purposes. However, larger plasmids can be adapted for cloning under some circumstances 16 Sizes of representative plasmids 17  The copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell The factors that control copy number are not well understood. Some plasmids, especially the larger ones, are stringent and have a low copy number of perhaps just one or two per cell; others, called relaxed plasmids, are present in multiple copies of 50 or more per cell Generally speaking, a useful cloning vector needs to be present in the cell in multiple copies so that large quantities of the recombinant DNA molecule can be obtained 18 Good plasmid cloning vehicles share a number of desirable features An ideal cloning vehicle would have the following three properties: – low molecular weight – ability to confer readily selectable phenotypic traits on host cells – single sites for a large number of restriction endonucleases (multiple cloning site – MCS), preferably in genes with a readily scorable phenotype 19 MCS 20  The advantages of a low molecular weight are several – First, the plasmid is much easier to handle, i.e. it is more resistant to damage by shearing, and is readily isolated from host cells – Secondly, low molecular weight plasmids are usually present as multiple copies, and this not only facilitates their isolation but leads to gene dosage effects for all cloned genes – Finally, with a low molecular weight there is less chance that the vector will have multiple substrate sites for any restriction endonuclease 21  Plasmid classification The most useful classification of naturally occurring plasmids is based on the main characteristic coded by the plasmid genes. The five major types of plasmid according to this classification are as follows: Fertility or F plasmids carry only tra genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids. A well-known example is the F plasmid of E. coli. 22  Resistance or R plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents, such as chloramphenicol, ampicillin, and mercury. R plasmids are very important in clinical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections. An example is RP4, which is commonly found in Pseudomonas, but also occurs in many other bacteria Col plasmids code for colicins, proteins that kill other bacteria. An example is ColE1 of E. coli Degradative plasmids allow the host bacterium to metabolize unusual molecules such as toluene and salicylic acid, an example being TOL of Pseudomonas putida Virulence plasmids confer pathogenicity on the host bacterium; these include the Ti plasmids of Agrobacterium tumefaciens, which induce crown gall disease on dicotyledonous plants 23 Plasmids in organisms other than bacteria Although plasmids are widespread in bacteria they are by no means as common in other organisms. The best characterized eukaryotic plasmid is the 2 Fm circle that occurs in many strains of the yeast Saccharomyces cerevisiae. The discovery of the 2 fm plasmid was very fortuitous as it allowed the construction of cloning vectors for this very important industrial organism. However, the search for plasmids in other eukaryotes (such as filamentous fungi, plants and animals) has proved disappointing, and it is suspected that many higher organisms simply do not harbor plasmids within their cells 24  Bacteriophages Bacteriophages, or phages as they are commonly known, are viruses that specifically infect bacteria. Like all viruses, phages are very simple in structure, consisting merely of a DNA (or RNA) molecule carrying a number of genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein molecules 25  The two main types of phage structure: (a) head-and tail (e.g. λ) (b) filamentous (e.g. M13) 26  The phage infection cycle The general pattern of infection, which is the same for all types of phage, is a three- step process: 1. The phage particle attaches to the outside of the bacterium and injects its DNA chromosome into the cell 2. The phage DNA molecule is replicated, usually by specific phage enzymes coded by genes in the phage chromosome 3. Other phage genes direct synthesis of the protein components of the capsid, and new phage particles are assembled and released from the bacterium 27  The phage infection cycle With some phage types the entire infection cycle is completed very quickly, possibly in less than 20 minutes. This type of rapid infection is called a lytic cycle, as release of the new phage particles is associated with lysis of the bacterial cell. The characteristic feature of a lytic infection cycle is that phage DNA replication is immediately followed by synthesis of capsid proteins, and the phage DNA molecule is never maintained in a stable condition in the host cell 28  In contrast to a lytic cycle, lysogenic infection is characterized by retention of the phage DNA molecule in the host bacterium, possibly for many thousands of cell divisions. With many lysogenic phages the phage DNA is inserted into the bacterial genome, in a manner similar to episomal insertion. The integrated form of the phage DNA (called the prophage) is quiescent, and a bacterium (referred to as a lysogen) that carries a prophage is usually physiologically indistinguishable from an uninfected cell. However, the prophage is eventually released from the host genome and the phage reverts to the lytic mode and lyses the cell. The infection cycle of lambda (λ), a typical lysogenic phage of this type. 29  The lysogenic infection cycle of bacteriophage λ. 30  A limited number of lysogenic phages follow a rather different infection cycle. When M13 or a related phage infects E. coli, new phage particles are continuously assembled and released from the cell. The M13 DNA is not integrated into the bacterial genome and does not become quiescent. With these phages, cell lysis never occurs, and the infected bacterium can continue to grow and divide, albeit at a slower rate than uninfected cells Although there are many different varieties of bacteriophage, only λ and M13 have found a major role as cloning vectors 31  The infection cycle of bacteriophage M13. 32  Gene organization in the λ DNA molecule λ is a typical example of a head-and-tail phage. The DNA is contained in the polyhedral head structure and the tail serves to attach the phage to the bacterial surface and to inject the DNA into the cell 33  The λ DNA molecule is 49 kb in size and has been intensively studied by the techniques of gene mapping and DNA sequencing. As a result, the positions and identities of all the genes in the λ DNA molecule are known. A feature of the λ genetic map is that genes related in terms of function are clustered together in the genome. For example, all of the genes coding for components of the capsid are grouped together in the left-hand third of the molecule, and genes controlling integration of the prophage into the host genome are clustered in the middle of the molecule. Clustering of related genes is profoundly important for controlling expression of the λ genome, as it allows genes to be switched on and off as a group rather than individually. Clustering is also important in the construction of λ -based cloning vectors. 34  The λ genetic map, showing the positions of the important genes and the functions of the gene clusters. 35  The linear and circular forms of λ DNA A second feature of λ that turns out to be of importance in the construction of cloning vectors is the conformation of the DNA molecule. The molecule showed in the figure is linear, with two free ends, and represents the DNA present in the phage head structure. This linear molecule consists of two complementary strands of DNA, base-paired according to the Watson–Crick rules (that is, double-stranded DNA). However, at either end of the molecule is a short 12- nucleotide stretch in which the DNA is single-stranded. The two single strands are complementary, and so can base pair with one another to form a circular, completely double- stranded molecule 36  Complementary single strands are often referred to as “sticky” ends or cohesive ends, because base pairing between them can “stick” together the two ends of a DNA molecule (or the ends of two different DNA molecules). The λ cohesive ends are called the cos sites and they play two distinct roles during the λ infection cycle. First, they allow the linear DNA molecule that is injected into the cell to be circularized, which is a necessary prerequisite for insertion into the bacterial genome. 37  The second role of the cos sites is rather different, and comes into play after the prophage has excised from the host genome. At this stage a large number of new λ DNA molecules are produced by the rolling circle mechanism of replication, in which a continuous DNA strand is “rolled off” the template molecule. The result is a catenane consisting of a series of linear λ genomes joined together at the cos sites. 38  The role of the cos sites is now to act as recognition sequences for an endonuclease that cleaves the catenane at the cos sites, producing individual λ genomes. This endonuclease, which is the product of gene A on the λ DNA molecule, creates the single stranded sticky ends, and also acts in conjunction with other proteins to package each λ genome into a phage head structure. The cleavage and packaging processes recognize just the cos sites and the DNA sequences to either side of them, so changing the structure of the internal regions of the λ genome, for example by inserting new genes, has no effect on these events so long as the overall length of the λ genome is not altered too greatly 39  The linear and circular forms of λ DNA. (a) The linear form, showing the left and right cohesive ends. (b) Base pairing between the cohesive ends results in the circular form of the molecule. (c) Rolling circle replication produces a catenane of new linear λ DNA molecules, which are individually packaged into phage heads as new λ particles are assembled. 40  M13—a filamentous phage M13 is an example of a filamentous phage and is completely different in structure from λ. Furthermore, the M13 DNA molecule is much smaller than the λ genome, being only 6407 nucleotides in length. It is circular and is unusual in that it consists entirely of single-stranded DNA. The smaller size of the M13 DNA molecule means that it has room for fewer genes than the λ genome. This is possible because the M13 capsid is constructed from multiple copies of just three proteins (requiring only three genes), whereas synthesis of the λ head-and-tail structure involves over 15 different proteins. In addition, M13 follows a simpler infection cycle than λ, and does not need genes for insertion into the host genome 41  Injection of an M13 DNA molecule into an E. coli cell occurs via the pilus, the structure that connects two cells during sexual conjugation Once inside the cell the single-stranded molecule acts as the template for synthesis of a complementary strand, resulting in normal double- stranded DNA This molecule is not inserted into the bacterial genome, but instead replicates until over 100 copies are present in the cell 42  When the bacterium divides, each daughter cell receives copies of the phage genome, which continues to replicate, thereby maintaining its overall numbers per cell New phage particles are continuously assembled and released, about 1000 new phages being produced during each generation of an infected cell 43 Several features of M13 make this phage attractive as a cloning vector: The genome is less than 10 kb in size, well within the range desirable for a potential vector In addition, the double-stranded replicative form (RF) of the M13 genome behaves very much like a plasmid, and can be treated as such for experimental purposes It is easily prepared from a culture of infected E. coli cells and can be reintroduced by transfection Most importantly, genes cloned with an M13-based vector can be obtained in the form of single-stranded DNA. Single- stranded versions of cloned genes are useful for several techniques, notably DNA sequencing and in vitro mutagenesis 44  Cloning in an M13 vector is an easy and reliable way of obtaining single-stranded DNA for this type of work. M13 vectors are also used in phage display, a technique for identifying pairs of genes whose protein products interact with one another 45 The M13 infection cycle, showing the different types of DNA replication that occur. (a) After infection the single-stranded M13 DNA molecule is converted into the double-stranded replicative form (RF). (b) The RF replicates to produce multiple copies of itself. (c) Single-stranded molecules are synthesized by rolling circle replication and used in the assembly of new M13 particles. 46  Viruses as cloning vectors for other organisms Most living organisms are infected by viruses and it is not surprising that there has been great interest in the possibility that viruses might be used as cloning vectors for higher organisms. This is especially important when it is remembered that plasmids are not commonly found in organisms other than bacteria and yeast. Several eukaryotic viruses have been employed as cloning vectors for specialized applications: for example, human adenoviruses are used in gene therapy , baculoviruses are used to synthesize important pharmaceutical proteins in insect cells, and caulimoviruses and geminiviruses have been used for cloning in plants 47  Cloning Vectors for E. coli The greatest variety of cloning vectors exists for use with E. coli as the host organism. This is not surprising in view of the central role that this bacterium has played in basic research over the past 50 years. The tremendous wealth of information that exists concerning the microbiology, biochemistry, and genetics of E. coli has meant that virtually all fundamental studies of gene structure and function were initially carried out with this bacterium as the experimental organism. Even when a eukaryote is being studied, E. coli is still used as the workhorse for preparation of cloned DNA for sequencing, and for construction of recombinant genes that will subsequently be placed back in the eukaryotic host in order to study their function and expression 48  Cloning Vectors for E. coli In recent years, gene cloning and molecular biological research have become mutually synergistic—breakthroughs in gene cloning have acted as a stimulus to research, and the needs of research have spurred on the development of new, more sophisticated cloning vectors 49  Cloning vectors based on E. coli plasmids The simplest cloning vectors, and the ones most widely used in gene cloning, are those based on small bacterial plasmids A large number of different plasmid vectors are available for use with E. coli, many obtainable from commercial suppliers They combine ease of purification with desirable properties such as – high transformation efficiency, – convenient selectable markers for transformants and recombinants – the ability to clone reasonably large (up to about 8 kb) pieces of DNA Most “routine” gene cloning experiments make use of one or other of these plasmid vectors 50  Cloning vectors based on E. coli plasmids One of the first vectors to be developed was pBR322 Although pBR322 lacks the more sophisticated features of the newest cloning vectors, and so is no longer used extensively in research, it still illustrates the important, fundamental properties of any plasmid cloning vector. We will therefore begin our study of E. coli vectors by looking more closely at pBR322. 51 The nomenclature of plasmid cloning vectors The name “pBR322” conforms with the standard rules for vector nomenclature: – “p” indicates that this is indeed a plasmid – “BR” identifies the laboratory in which the vector was originally constructed (BR stands for Bolivar and Rodriguez, the two researchers who developed pBR322). – “322” distinguishes this plasmid from others developed in the same laboratory (there are also plasmids called pBR325, pBR327, pBR328, etc.). 52  The useful properties of pBR322 The genetic and physical map of pBR322 gives an indication of why this plasmid was such a popular cloning vector The first useful feature of pBR322 is its size. pBR322 is 4363 bp, which means that not only can the vector itself be purified with ease, but so can recombinant DNA molecules constructed with it. Even with 6 kb of additional DNA, a recombinant pBR322 molecule is still a manageable size 53  The second feature of pBR322 is that it carries two sets of antibiotic resistance genes. Either ampicillin or tetracycline resistance can be used as a selectable marker for cells containing the plasmid, and each marker gene includes unique restriction sites that can be used in cloning experiments Insertion of new DNA into pBR322 that has been restricted with PstI, PvuI, or ScaI inactivates the ampR gene, and insertion using any one of eight restriction endonucleases (notably BamHI and HindIII) inactivates tetracycline resistance. This great variety of restriction sites that can be used for insertional inactivation means that pBR322 can be used to clone DNA fragments with any of several kinds of sticky end 54  A third advantage of pBR322 is that it has a reasonably high copy number. Generally there are about 15 molecules present in a transformed E. coli cell, but this number can be increased, up to 1000–3000, by plasmid amplification in the presence of a protein synthesis inhibitor such as chloramphenicol. An E. coli culture therefore provides a good yield of recombinant pBR322 molecules 55  A map of pBR322 showing the positions of the ampicillin resistance (ampR ) and tetracycline resistance (tetR ) genes, the origin of replication (ori) and some of the most important restriction sites. 56  pUC8—a Lac selection plasmid pUC8 is descended from pBR322, although only the replication origin and the ampR gene remain. The nucleotide sequence of the ampR gene has been changed so that it no longer contains the unique restriction sites: all these cloning sites are now clustered into a short segment of the lacZ′ gene carried by pUC8. pUC8 has three important advantages that have led to it becoming one of the most popular E. coli cloning vectors. The first of these is fortuitous: the manipulations involved in construction of pUC8 were accompanied by a chance mutation, within the origin of replication, which results in the plasmid having a copy number of 500–700 even before amplification. This has a significant effect on the yield of cloned DNA obtainable from E. coli cells transformed with recombinant pUC8 plasmids. 57  The second advantage is that identification of recombinant cells can be achieved by a single step process, by plating onto agar medium containing ampicillin plus X-gal. With both pBR322 and pBR327, selection of recombinants is a two-step procedure, requiring replica plating from one antibiotic medium to another. A cloning experiment with pUC8 can therefore be carried out in half the time needed with pBR322 or pBR327. 58 Blue-white selection- pUC8 vectors have a short segment of E. coli DNA, which contains the regulatory and coding sequences of Lac Z gene that codes for β- galactosidase enzyme. Isopropyl thiogalactoside (IPTG) is an inducer of Lac Z gene expression. β -galactosidase reacts with the chromogenic substrate 5-bromo-4-chloro- β-D-Galactoside (X-gal) and yields a blue colored product. A multiple cloning site (MCS) is engineered inside the coding region of the Lac Z gene. The MCS as such does not disrupt the reading frame and results only in insertion of a few amino acids in the amino terminal fragment of the β- galactosidase. Therefore, the colonies appear blue in color in the presence of IPTG and X-gal. However, when a insert is cloned in the MCS, that becomes a harmful insertion to the functional properties of β- galactosidase and it can no longer react with X-gal, and therefore, the colonies appear white in color. This is a simple visual color test that can be used to screen thousands of colonies to identify the presence of recombinant plasmids. 59 A schematic representation of a typical blue-white screening procedure 60 Blue/white colony 61  LIMITATIONS OF BLUE-WHITE SCREENING The blue-white technique is only a screening procedure; it is not a selection technique. The lacZ gene in the vector may sometimes be non- functional and may not produce β-galactosidase. The resulting colony will not be recombinant but will appear white. Even if a small sequence of foreign DNA may be inserted into MCS and change the reading frame of lacZ gene. This results in false positive white colonies. Small inserts within the reading frame of lacZ may produce ambiguous light blue colonies as β-galactosidase is only partially inactivated. 62  The third advantage of pUC8 lies with the clustering of the restriction sites, which allows a DNA fragment with two different sticky ends (say EcoRI at one end and BamHI at the other) to be cloned without resorting to additional manipulations such as linker attachment. Other pUC vectors carry different combinations of restriction sites and provide even greater flexibility in the types of DNA fragment that can be cloned. Furthermore, the restriction site clusters in these vectors are the same as the clusters in the equivalent M13mp series of vectors. DNA cloned into a member of the pUC series can therefore be transferred directly to its M13mp counterpart, enabling the cloned gene to be obtained as single-stranded DNA 63 The pUC plasmids. (a) The structure of pUC8. (b) The restriction site cluster in the lacZ′ gene of pUC8. (c) The restriction site cluster in pUC18. (d) Shuttling a DNA fragment from pUC8 to M13mp8. 64  pGEM3Z—in vitro transcription of cloned DNA pGEM3Z is very similar to a pUC vector: it carries the ampR and lacZ′ genes, the latter containing a cluster of restriction sites, and it is almost exactly the same size The distinction is that pGEM3Z has two additional short pieces of DNA, each of which acts as the recognition site for attachment of an RNA polymerase enzyme. These two promoter sequences lie on either side of the cluster of restriction sites used for introduction of new DNA into the pGEM3Z molecule. This means that if a recombinant pGEM3Z molecule is mixed with purified RNA polymerase in the test tube, transcription occurs and RNA copies of the cloned fragment are synthesized. The RNA that is produced could be used as a hybridization probe, or might be required for experiments aimed at studying RNA processing (e.g., the removal of introns) or protein synthesis. 65  pGEM3Z—in vitro transcription of cloned DNA The promoters carried by pGEM3Z and other vectors of this type are not the standard sequences recognized by the E. coli RNA polymerase. Instead, one of the promoters is specific for the RNA polymerase coded by T7 bacteriophage and the other for the RNA polymerase of SP6 phage. These RNA polymerases are synthesized during infection of E. coli with one or other of the phages and are responsible for transcribing the phage genes. They are chosen for use in in vitro transcription as they are very active enzymes – remember that the entire lytic infection cycle takes only 20 minutes, so the phage genes must be transcribed very quickly. These polymerases are able to synthesize 1–2 mg of RNA per minute, substantially more than can be produced by the standard E. coli enzyme. 66 pGEM3Z. (a) Map of the vector. (b) In vitro RNA synthesis. R = cluster of restriction sites for EcoRI, SacI, KpnI, AvaI, SmaI, BamHI, XbaI, SalI, AccI, HincII, PstI, SphI, and HindIII. 67  Cloning Vectors for Eukaryotes Most cloning experiments are carried out with E. coli as the host, and the widest variety of cloning vectors are available for this organism. E. coli is particularly popular when the aim of the cloning experiment is to study the basic features of molecular biology such as gene structure and function. However, under some circumstances it may be desirable to use a different host for a gene cloning experiment. This is especially true in biotechnology, where the aim may not be to study a gene, but to use cloning to obtain large amounts of an important pharmaceutical protein (e.g., a hormone such as insulin), or to change the properties of the organism (e.g., to introduce herbicide resistance into a crop plant). We must therefore consider cloning vectors for organisms other than E. coli 68  Artificial chromosomes can be used to clone long pieces of DNA in yeast Yeast artificial chromosome (YAC) presents a totally different approach to gene cloning. The development of YACs was a spin-off from fundamental research into the structure of eukaryotic chromosomes, work that has identified the key components of a chromosome as being : – The centromere, which is required for the chromosome to be distributed correctly to daughter cells during cell division; – Two telomeres, the structures at the ends of a chromosome, which are needed in order for the ends to be replicated correctly and which also prevent the chromosome from being nibbled away by exonucleases; – The origins of replication, which are the positions along the chromosome at which DNA replication initiates, similar 69 to the origin of replication of a plasmid.  Chromosome structure 70  Once chromosome structure had been defined in this way, the possibility arose that the individual components might be isolated by recombinant DNA techniques and then joined together again in the test tube, creating an artificial chromosome. As the DNA molecules present in natural yeast chromosomes are several hundred kilobases in length, it might be possible with an artificial chromosome to clone long pieces of DNA. 71 72 The structure and use of a YAC vector Several YAC vectors have been developed but each one is constructed along the same lines, with pYAC3 being a typical example At first glance, pYAC3 does not look much like an artificial chromosome, but on closer examination its unique features become apparent 73  The structure and use of a YAC vector pYAC3 is essentially a pBR322 plasmid into which a number of yeast genes have been inserted. Two of these genes, URA3 and TRP1, which are the selectable markers for YIp5 and YRp7, respectively. As in YRp7, the DNA fragment that carries TRP1 also contains an origin of replication, but in pYAC3 this fragment is extended even further to include the sequence called CEN4, which is the DNA from the centromere region of chromosome 4. The TRP1–origin–CEN4 fragment therefore contains two of the three components of the artificial chromosome. 74 A YAC vector and the way it is used to clone large pieces of DNA 75  The third component, the telomeres, is provided by the two sequences called TEL. These are not themselves complete telomere sequences, but once inside the yeast nucleus they act as seeding sequences onto which telomeres will be built. This just leaves one other part of pYAC3 that has not been mentioned: SUP4, which is the selectable marker into which new DNA is inserted during the cloning experiment. 76 The cloning strategy with pYAC3 is as follows: The vector is first restricted with a combination of BamHI and SnaBI, cutting the molecule into three fragments The fragment flanked by BamHI sites is discarded, leaving two arms, each bounded by one TEL sequence and one SnaBI site. The DNA to be cloned, which must have blunt ends (SnaBI is a blunt end cutter, recognizing the sequence TACGTA), is ligated between the two arms, producing the artificial chromosome. 77  Protoplast transformation is then used to introduce the artificial chromosome into S. cerevisiae The yeast strain that is used is a double auxotrophic mutant, trp1− ura3−, which is converted to trp1+ ura3+ by the two markers on the artificial chromosome Transformants are therefore selected by plating onto minimal medium, on which only cells containing a correctly constructed artificial chromosome are able to grow Any cell transformed with an incorrect artificial chromosome, containing two left or two right arms rather than one of each, is not able to grow on minimal medium as one of the markers is absent The presence of the insert DNA in the vector can be checked by testing for insertional inactivation of SUP4, which is carried out by a simple color test: white colonies are recombinants, red colonies are not 78  A YAC vector and the way it is used to clone large pieces of DNA. 79  Applications for YAC vectors The initial stimulus in designing artificial chromosomes came from yeast geneticists who wanted to use them to study various aspects of chromosome structure and behavior, for instance to examine the segregation of chromosomes during meiosis. These experiments established that artificial chromosomes are stable during propagation in yeast cells and raised the possibility that they might be used as vectors for genes that are too long to be cloned as a single fragment in an E. coli vector. Several important mammalian genes are greater than 100 kb in length (e.g., the human cystic fibrosis gene is 250 kb), beyond the capacity of all but the most sophisticated E. coli cloning systems, but well within the range of a YAC vector. 80  Applications for YAC vectors Yeast artificial chromosomes therefore opened the way to studies of the functions and modes of expression of genes that had previously been intractable to analysis by recombinant DNA techniques A new dimension to these experiments was provided by the discovery that under some circumstances YACs can be propagated in mammalian cells, enabling the functional analysis to be carried out in the organism in which the gene normally resides 81  Yeast artificial chromosomes are equally important in the production of gene libraries. With fragments of 300 kb, the maximum insert size for the highest capacity E. coli vector, some 30,000 clones are needed for a human gene library However, YAC vectors are routinely used to clone 600 kb fragments, and special types are able to handle DNA up to 1400 kb in length, the latter bringing the size of a human gene library down to just 6500 clones. Unfortunately these “mega- YACs” have run into problems with insert stability, the cloned DNA sometimes becoming rearranged by intramolecular recombination. Nevertheless, YACs have been of immense value in providing long pieces of cloned DNA for use in large- scale DNA sequencing projects 82  References Primrose SB and Twyman R M. Principles of Gene manipulation and Genomics, Seventh edition, Blackwell Scientific Publications, 2006. Terence A. Brown. Gene cloning and DNA analysis: an introduction, Wiley-Blackwell, 2006. 83