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Download Cloning Vectors A cloning vector is a DNA molecule that can carry
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Cloning Vectors A cloning vector is a DNA molecule that can carry inserted DNA and be perpetuated in a host system. It is also called cloning vehicle. There are many such systems: – Plasmids – Cosmids – Phages – Phasmids – Artificial vehicles: YAC, BAC etc. Features of a ideal vector 1. Small size which is necessary for the efficiency of transfer of foreign DNA 2. Unique (single) restriction endonuclease recognition site into which the insert DNA can be cloned 3. One or more selectable genetic markers for identifying recepient cells that carry the cloning vector – insert DNA construct Plasmids It is an autonomous, double stranded, self-replicating, circular, extra chromosomal DNA molecule. Virtually all bacterial genera have plasmids. Some carry information for their own transfer from one cell to another – F plasmids. Some encode resistance to antibiotics – R plasmid. Some carry specific set of genes for the utilization of unusual metabolites – Degradative plasmids. And others have no apparent function at all – Cryptic plasmids Plasmids can range in size from less than 1 to more than 500 kb. Each plasmid has a sequence that functions as the origin of replication of DNA – without this site it cannot replicate in the host. A plasmid is considered to be a suitable cloning vector if it possesses the following features: 1. Easily isolatable 2. Possessing single restriction site for one or more restriction enzymes 3. Insertion of linear molecule at on of theses sites should not alter its replication properties 4. Reintroducible into host – but carrying identifiable marker – enabling easy selection 5. Do not occur free in nature but are found in other bacterial cells Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes. Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. 1 Plasmid Natural Occurrence Size (Kb) S. Marker pACYC177 E. coli 3.7 Ampr, Kanr pBR322 E. coli 4.0 Ampr, Tetr pBR324 E. coli 8.3 Ampr ,Tetr pMB9 R. coli 5.8 Tetr pRK646 E. coli 3.4 Ampr pC194 Staphylococcus aureus 3.6 Eryr p SA 0501 S. aureus 4.2 Strr p BS 161-1 Bacillus subtillus 3.65 Tetr p WWO Pseudomonas putida 117 Kanr Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest. Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics. However, a plasmid can only contain inserts of about 1–10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used. One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain socalled tra-genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium. Non-conjugative plasmids are incapable of initiating conjugation, hence they can only be transferred with the assistance of conjugative 2 plasmids, by 'accident'. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can 'parasitize' a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases. It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. But related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups. Another way to classify plasmids is by function. There are five main classes: • Fertility-F-plasmids, which contain tra-genes. They are capable of conjugation (transfer of genetic material between bacteria which are touching). • Resistance-(R)plasmids, which contain genes that can build a resistance against antibiotics or poisons and help bacteria producepili. Historically known as R-factors, before the nature of plasmids was understood. • Col-plasmids, which contain genes that code for (determine the production of) bacteriocins, proteins that can kill other bacteria. • Degradative plasmids, which enable the digestion of unusual substances, e.g., toluene or salicylic acid. • Virulence plasmids, which turn the bacterium into a pathogen (one that causes disease). Plasmids can belong to more than one of these functional groups. Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems which attempt to actively distribute a copy to both daughter cells. Some plasmids or microbial hosts include an addiction system or "post segregational killing system (PSK)", such as the hok/sok (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli. This variant produces both a long-lived poison and a short-lived antidote. Several types of plasmid addiction systems (toxin/ antitoxin, metabolism-based, ORT systems) were described in the literature and used in biotechnical (fermentation) or biomedical (vaccine therapy) applications. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell. Finally, the overall productivity could be enhanced. pBR322 pBR322 is a plasmid and for a time was one of the most commonly used E. coli cloning vectors. Created in 1977, it was named eponymously after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez. pBR322 is 4361 base pairs in length and contains a replicon region (source plasmid pMB1), the ampR gene, encoding the ampicillin resistance protein (source plasmid RSF2124) and the tetR gene, encoding the tetracycline resistance protein (source plasmid pSC101). The plasmid has unique restriction sites for more than forty restriction enzymes. 11 of these 40 sites lie within the tetR gene. There are 2 sites for restriction enzymes HindIII and ClaI within the 3 promoter of the tetR gene. There are 6 key restriction sites inside the ampR gene. The origin of replication or ori site in this plasmid is pMB1 (a close relative of ColE1). The ori encodes two RNAs (RNAI and RNAII) and one protein (called Rom or Rop). The circular sequence is numbered such that 0 is the middle of the unique EcoRI site and the count increases through the tet genes. The ampicillin resistance gene is a penicillin betalactamase. Promoters P1 and P3 are for the beta-lactamase gene. P3 is the natural promoter, and P1 is artificially created by the ligation of two different DNA fragments to create pBR322. P2 is in the same region as P1, but it is on the opposite strand and initiates transcription in the direction of the tetracycline resistance gene pACYC177 • • • • • Type Cloning Origin of Replicationp 15A Copy #low, 15 copies per cell Markers ampicillin kanamycin Link to Sequence Compatible with ColE1 Cloning vectors – Phages The bacteriophage lambda is important in molecular biology because it is used in constructing vectors for gene cloning. Plasmids do this as well, but bacteriophages have an advantage over plasmids. Plasmids are circular, indpendently replicating, doublestranded DNA, most often found in bacteria. They replicate quickly and are easily manipulated in the laboratory. Plasmids are typically 2-10 thousand base pairs in size (Corbley, 1999). While this small size allows plasmids the two aforementioned attributes, it also means that plasmids are limited in the DNA fragments they can clone. They are typically limited to fragments around 5 thousand base pairs (King, 2002). While plasmids are great vector vehicles for many molecular endeavors, if the fragment to be cloned is larger than the available insertion space of the plasmid, the plasmid vector will probably not transfer the fragment of DNA successfully. Plasmid based cloning vectors can generally carry only a 10 kb insert of DNA. For formation of a library it is helpful to have larger fragments of DNA. Therefore a different vector is required. A bacteriophage is considered a much more effective insertion vector for the formation of libraries. The most commonly used phage vectors are derived form lambda (λ) phages that infect E. coli A bacteriophage is any one of a number of viruses that infect bacteria. Bacteriophages are among the most common biological entities on Earth. Typically, bacteriophages consist of an outer protein capsid enclosing genetic material. The genetic material can be ssRNA,dsRNA, ssDNA, or dsDNA along with either circular or linear arrangement. Bacteriophages are much smaller than the bacteria they destroy. In its life cycle the λ phage, infects E. coli, after injection of the viral DNA two possibilities exist. It can enter a lytic cycle, which after 20 min results in the lysis of the host cell and the release of about 100 phage particles. Alternatively the DNA can be integrated in the host 4 genome as a prophage. The prophage can be maintained more or less indefinitely as a benign guest [Lysogeny]. Under conditions of nutritional and environmental stress the integrated prophage can be excised and enter the lytic cycle. The λ phage DNA is 50 kb in length, of which approximately 20 kb are essential for the excision – integration events. For forming genomic libraries this 20 kb is replaced by 20 kb of cloned DNA. The resultant recombinant phage would go through compulsory lysogenic cycles Lytic Cycle – Important Molecular events The lytic cycle is typically considered the main method of viral replication, since it results in the destruction of the infected cell. An infective phage has a head packed with 50 kb DNA. The production and assembly of the heads and tails and the packing of DNA is considered to a highly coordinated sequence of events. The 50 kb in the head is a linear molecule with a 12 base single strand extension at the 5prime end of each strand. These extensions are called cohesive or cos ends. They contain sequences complementary to each other. After the injection into the host the cos ends pair to form a circular DNA molecule. During the early phase of the lytic cycle, replication from the circular molecule generates a linear DNA strand that is a continuous length of many 50 kb molecules. Each new head is filled with one 50 kb unit before the tail is attached The volume of the head in an infective phage particle is 50 kb, if 38 kb is packed in the head a non infective particle is produced. In contrast more than 52 kb can not fit into the head capsule. The location of the cos sequence, which is 50 kb apart, ensures that each head receives the correct amount of DNA. At the opening of the head is located a enzyme that identifies the ds cos sequence and cuts the DNA. In experiments the phage DNA was cut with BamHI, it produced 3 fragments • The left arm region (L region) containing the genetic information for the production of heads and tails • The right arm region (R region) containing genes for replication and cell lysis • A middle fragment (I/E) that has the genes for the integration – excision process By this method it was determined that the middle fragment could be replaced by cloned DNA. The length of the middle piece is around 20 kb 5 6 Lysogenic cycle The phage genome intergrates at an attachment (att) site with a partially homologous on the E. coli genome. Two events are considered to be obligatory to establish lysogenic cycle: – (i) the synthesis of all late proteins must be stopped, and – (ii) the lambda genome must integrate into the bacterial chromosome. To prevent the synthesis of late proteins, the product of the cI gene must be synthesized. The cI gene product is the lambda repressor protein. The latter, if synthesized, represses the synthesis of all other lambda genome-encoded proteins. The cI gene occurs between PL and PR (promoter left and promoter right) which are oriented in such a way that neither transcribe the cl gene. The cI genes represses all the genes responsible for the lytic pathways. No phage structural proteins are synthesized.The insertion of the proteins of the cI genes (phage) and cro genes (E. coli) decides the events of the required pathways either lytic or lysogenic. Only 50 % of the phage DNA is required for growth and plaque formation Advantages over plasmids • • • • • • DNA can be packed in vitro into phage heads and transduced into E. coli with high efficiency Foreign DNA up to 25 kb can be inserted into phage vector Screening and storage of the rDNA is easier Before using the phage as a vector it is necessary to remove the stuffer fragment using restriction enzymes Restriction sites can be obtained by inducung mutations or deletions Two types of phage cloning vectors have been constructed • Insertion vectors • Replacement vectors Insertion vector • They have unique cleavage site into which relatively small pieces of DNA (35 – 53 kb) are inserted • The maximum size of foreign DNA is about 18 kb Replacement Vector • They have cleavage sites present on either side of a length of non essential DNA of phage • As a result of cleavage left and right arms are formed each having a terminal cos site • The middle replaceable DNA is called stuffer DNA / stuffer region / stuffer fragment • The maximum size of insertable DNA depends on how much is non essential • The substituted vectors are gt, WES, λ • The non essential part can be separated from the arms by electrophoresis or velocity gradient ultracentrifugation (size differences) 7 • • • • Formation of multiple inserts can be used by using alkaline phosphatase before ligation with insert fragment r DNA formed by multiple inserts has too large a genome to be packed into the head Optimum distance between cos sites governs efficiency of packing Bacteriophage libraries can be screened using DNA probes or immunological assays COSMIDS A cosmid, first described by Collins and Hohn in 1978, is a type of hybrid plasmid (often used as a cloning vector) that contains cos sequences, DNA sequences originally from the Lambda phage. Cosmids can be used to build genomic libraries. Cosmids are able to contain 37 to 52 kb of DNA, while normal plasmids are able to carry only 1–20 kb. They can replicate as plasmids if they have a suitable origin of replication: for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNA replication or f1 ori for single-stranded DNA replication in prokaryotes. They frequently also contain a gene for selection such as antibiotic resistance, so that the transfected cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would be unable to grow. Unlike plasmids, they can also be packaged in phage capsids, which allow the foreign genes to be transferred into or between cells by transduction. Plasmids become unstable after a certain amount of DNA has been inserted into them, because their increased size is more conducive to recombination. To circumvent this, phage transduction is used instead. This is made possible by the cohesive ends, also known as cos sites. In this way, they are similar to using the lambda phage as a vector, but only that all the lambda genes have been deleted with the exception of the cos sequence. Cos sequences are ~200 base pairs long and essential for packaging. They contain a cosN site where DNA is nicked at each strand, 12bp apart, by terminase. This causes linearization of the circular cosmid with two "cohesive" or "sticky ends" of 12bp. (The DNA must be linear to fit into a phage head.) The cosB site holds the terminase while it is nicking and separating the strands. The cosQ site of next cosmid (as rolling circle replication often results in linear concatemers) is held by the terminase after the previous cosmid has been packaged, to prevent degradation by cellular DNases Cosmids are predominantly plasmids with a bacterial oriV, an antibiotic selection marker and a cloning site, but they carry one, or more recently two cos sites derived from bacteriophage lambda. Depending on the particular aim of the experiment broad host range cosmids, shuttle cosmids or 'mammalian' cosmids (linked to SV40 oriV and mammalian selection markers) are available. The loading capacity of cosmids varies depending on the size of the vector itself but usually lies around 40–45 kb. The cloning procedure involves the generation of two vector arms which are then joined to the foreign DNA. Selection against wildtype cosmid DNA is simply done via size exclusion. Cosmids, however, always form colonies and not plaques. Also the clone density is much lower with around 105 - 106 CFU per µg of ligated DNA. 8 After the construction of recombinant lambda or cosmid libraries the total DNA is transferred into an appropriate E.coli host via a technique called in vitro packaging. The necessary packaging extracts are derived from E.coli cI857 lysogens (red- gam- Sam and Dam (head assembly) and Eam (tail assembly) respectively). These extracts will recognize and package the recombinant molecules in vitro, generating either mature phage particles (lambda-based vectors) or recombinant plasmids contained in phage shells (cosmids). These differences are reflected in the different infection frequencies seen in favour of lambda-replacement vectors. This compensates for their slightly lower loading capacity. Phage library are also stored and screened easier than cosmid (colonies!) libraries. Target DNA: the genomic DNA to be cloned has to be cut into the appropriate size range of restriction fragments. This is usually done by partial restriction followed by either size fractionation or dephosphorylation (using calf-intestine phosphatase) to avoid chromosome scrambling, i.e. the ligation of physically unlinked fragments. Based on the properties of DNA and Col E1 plasmids a group of Japanese scientists (Fukumaki et al., 1976) showed that the presence of a small segment of λ phage DNA containing cohesive end on the plasmid molecule is a sufficient prerequisite for in vitro packaging of this DNA into infectious particles. A new breed of hybrid vectors was thereby derived from the fusion of plasmids and bacteriophages. The first type were called cosmids. Cosmids were developed by Collins and Hohn in 1978. They contain the cos site of the bacteriophage DNA in association with the plasmid DNA. Cosmids lack genes encoding for viral proteins – therefore neither viral particles are formed in the host cell nor does lysis occur. Certain special features are observed in the cosmids • The presence of the origin of replication • A marker gene encoding for antibiotic resistance • A special cleavage site for the insertion of foreign DNA • Small size • Presence of a cos site (12 bases) The cos site helps to ligate and circularize the whole genome. The cosmids have a length of 5 kb, the upper size limit of the foreign DNA fragment is approximately 45 kb. According to the size of the cos sites and the upper size limit in the head of the phage, the foreign DNA can be packed in vitro. Cosmids have been used as gene cloning vectors in conjugation with the in vitro packaging system. The cosmid vector can be packed and the resultant particle can be infected into a suitable host. The injected recombinant cosmid DNA circularizes like phage DNA but replicated like a normal plasmid without the expression of any phage functions. Transformer cells are selected on the basis of the presence of a vector drug resistance marker. Cosmids provide an excellent means of cloning large pieces of DNA. Because of their capacity for large fragments they are particularly attractive vectors for the construction of libraries of eukaryotic genome fragments. Partial digestion with restriction endonuclease provides suitably large fragments. However there is a potential problem in the use of partial digests produced this way. There is a possibility of two or more DNA fragments joining together in the ligation reaction, this may create a clone containing fragments that were not initially adjacent to each other in the genome. This would give an incorrect picture of their genomic location. The problem can be over come by size fractionation of the partial digest. Even with sized DNA, min practice cosmid clones may be produced that contain 9 non-contiguous DNA fragments ligated from a single insert. This is solved by dephosphorlyating the foreign DNA fragments so as to prevent their ligation together. The above mentioned method is sensitive to the exact ratio of target to vector DNAs because vector to vector ligation can occur. Furthermore recombinants with a duplicate vector are unstable and breakdown in the host by recombination, resulting in the propagation of non recombinant cosmid vectors. Such difficulties have been over come a few examples are listed • 1981 – Ish – Horowicz and Burke – pJB8 : • purified left hand and right hand vector ends – incapable of self ligation • Can accept dephosphorylated foreign DNA • Eliminated the need to size the fragments and prevents the formation of clones containing short foreign DNA or multiple vector sequences • 1983 – Bates and Swift – c2XB : • carries a BamH1 insertion site and two cos sites seperated by a blunt end restriction site • they ligate ineffectively under conditions used this too prevents self ligation • Modern cosmids of the pWE and sCos series contain the following features • Multiple cloning sites for simple cloning using non size selected DNA • Phage promoters flanking the cloning site • Unique NotI, SacII or SfiI sites (rare cutters) flanking the cloning site to permit the removal of the insert form the vector as a single fragment Mammalian expression modules encoding dominant selectable markers may also be present for gene transfer to mammalian cells if required. There are additional cosmid vectors based on the λ phage as well as other phages that infect E. coli. The genome of P1 bacteriophage is 115 kb long and the cos mid can therefore carry an 85 kb insert. The advantages of using a cosmid are twofold • First the capacity of a cosmid is more than a plasmid,gene clusters and larger genes are easier to clone • Second, for creating a library, a large insert in the cloning vector means that fewer clones have to be screened pLFR-5 The commonly used cosmid pLFR-5 (approximately 6 kb) has – two cos sites from the λ phage seperated by ScaI site – A multiple cloning sequence with six unique sites (HindIII, PstI, SalI, BamH1, SmaI and EcoRI) – An origin of DNA replication (ori) – Tetracycline resistant gene (Tet r) This cosmid can carry around 40 kb DNA, that are purified by sucrose density gradient centrifugation from a partial digestion of source DNA with BamHI. The pLFR-5 DNA is initially cleaved by ScaI and the with BamHI. The two DNA samples are mixed and ligated . Some of the products will have a 40 kb DNA piece inserted between the two fragments that are derived from the digestion of the pLFR-5 DNA. These molecules will be about 50 kb in length, with cos sequences 50 kb apart. These DNA constructs can be successfully packed into a λ phage head in vitro. Since the packaging of the head accepts 10 only 50 kb DNA reconstituted pLFR-5 without inserts will not be packed. After assembly the phage particles the DNA is delivered by infection into E. coli. Fosmids Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host (usually E. coli) can only contain one fosmid molecule. Fosmids are 40 kb of random genomic DNA. Fosmid library is prepared from a genome of the target organism and cloned into a fosmid vector. Low copy number offers higher stability than comparable high copy number cosmids. Fosmid system may be useful for constructing stable libraries from complex genomes. Fosmid clones were used to help assess the accuracy of the Public Human Genome Sequence. Fosmids are plasmids that use the F-plasmid origin of replication and partitioning mechanisms to allow cloning of large DNA fragments. A library that provides 20–70-fold redundant coverage of the genome can easily be prepared Bacterial artificial chromosome A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. The bacterial artificial chromosome's usual insert size is 150-350 kbp, but can be greater than 700 kbp. A similar cloning vector called a PAC has also been produced from the bacterial P1plasmid. BACs are often used to sequence the genome of organisms in genome projects, for example the Human Genome Project. A short piece of the organism's DNA is amplified as an insert in BACs, and then sequenced. Finally, the sequenced parts are rearranged in silico, resulting in the genomic sequence of the organism. Common gene components • oriS, repE - F – for plasmid replication and regulation of copy number. • parA and parB – for partitioning F plasmid DNA to daughter cells during division and ensures stable maintenance of the BAC. • A selectable marker – for antibiotic resistance; some BACs also have lac Z at the cloning site for blue/white selection. • T7 & Sp6 – phage promoters for transcription of inserted genes. Contribution to models of disease Inherited disease BACs are now being utilized to a greater extent in modelling genetic diseases, often alongside transgenic mice. BACs have been useful in this field as complex genes may have several regulatory sequences upstream of the encoding sequence, including various promoter sequences that will govern a gene's expression level. BACs have been 11 used to some degree of success with mice when studying neurological diseases such as Alzheimer's disease or as in the case of aneuploidy associated with Down syndrome. There have also been instances when they have been used to study specific oncogenes associated with cancers. They are transferred over to these genetic disease models by electroporation/transformation, transfection with a suitable virus or microinjection. BACs can also be utilised to detect genes or large sequences of interest and then used to map them onto the human chromosome using BAC arrays. BACs are preferred for these kind of genetic studies because they accommodate much larger sequences without the risk of rearrangement, and are therefore more stable than other types of cloning vectors. Infectious disease The genomes of several large DNA viruses and RNA viruses have been cloned as BACs. These constructs are referred to as "infectious clones", as transfection of the BAC construct into host cells is sufficient to initiate viral infection. The infectious property of these BACs has made the study of many viruses such as the herpesviruses, poxviruses and corona viruses more accessible. Molecular studies of these viruses can now be achieved using genetic approaches to mutate the BAC while it resides in bacteria. PAC The P1-derived artificial chromosome are DNA constructs that are derived from the DNA of P1 bacteriophage. They can carry large amounts (about 100-300 kilobases) of other sequences for a variety of bioengineering purposes. It is a type of vector used to clone DNA fragments (100- to 300-kb insert size; average, 150 kb) in Escherichia coli cells. YAC A yeast artificial chromosome (YAC) is a vector used to clone DNA fragments larger than 100 kb and up to 3000 kb. YACs are useful for the physical mapping of complex genomes and for the cloning of large genes. First described in 1983 by Murray and Szostak, a YAC is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. A YAC is built using an initial circular plasmid, which is typically broken into two linear molecules using restriction enzymes; DNA ligase is then used to ligate a sequence or gene of interest between the two linear molecules, forming a single large linear piece of DNA. Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids), have an advantage over bacterial artificial chromosomes (BACs) in that they can be used to express eukaryotic proteins that require posttranslational modification. However, YACs have been found to be less stable than BACs, producing chimeric effects. 12 Human artificial chromosome A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6-10 megabases in size, and able to carry new genes introduced by human researchers. Yeast artificial chromosomes and bacterial artificial chromosomes were created before human artificial chromosomes, which first appeared in 1997. They are useful in expression studies as gene transfer vectors and are a tool for elucidating human chromosome function. Grown in HT1080 cells, they are mitotically and cytogenetically stable for up to six months. John J. Harrington, Gil Van Bokkelen, Robert W. Mays, Karen Gustashaw & Huntington F. Willard of Case Western Reserve University School of Medicine published the first report of human artificial chromosomes in 1997. They were first synthesized by combining portions of alpha satellite DNA with telomeric DNA and genomic DNA into linear microchromosomes 13