P1, PAC & MAC Vectors

Promila SheoranPh.D. BiotechnologyGJU S&T Hisar

P1 Vector

• P1 is a bacteriopahge that infects Escherichia coli and some other bacteria. When undergoing a lysogenic cycle the phage genome exists as a plasmid in the bacteriumunlike other phages (e.g. the lambda phage) that integrate into the host DNA.

•The P1 phage has gained research interest because it can be used to transfer DNA from one bacterial cell to another in a process known as transduction.

•As it replicates during its lytic cycle it captures fragments of the host chromosome. If the resulting viral particles are used to infect a different host the captured DNA fragments can be integrated into the new host's genome.

•This method of in vivo genetic engineering was widely used for many years and is still used today, though to a lesser extent. P1 can also be used to create the P1-derived artificial chromosome cloning vector which can carry relatively large fragments of DNA.

P1 Vector

•P1 encodes a site-specific recombinase, Cre, that is widely used to carry out cell-specific or time-specific DNA recombination by flanking the target DNA with loxP sites.

•To overcome some of the shortcomings of the cosmid and yeast cloning systems and to provide an alternative system to clone large molecular weight DNAs, a bacteriophage P1 cloning system has been developed.

•The P1 system permits the cloning, isolation, and recovery of DNA inserts as large as 100 kbp with an efficiency that is intermediate between that of cosmids and YACs.

Construction of P1 Vector The construction and structure of the two P1 cloning vectors pNS358 and pNS582 are described in Fig.

The starting plasmid is pBS69. It contains two directly repeated P1 loxP recombination sites flanking the pBR322 bla (amp') gene and multicopyreplicon on one side, and the S. cerevisiae LEU2 gene on the other.

To construct the P1 cloning vectors the P1 packaging site was first inserted as a 450-bp XhoI/Sal I fragment at the unique Xho I site in the ampr domain of the vector.

The aminoglycoside 3'-phosphotransferase (kanr) gene from Tn903 was then inserted as a 1.3-kbp Acc I fragment into the unique Cla I site in the LEU2 gene.

The P1 plasmid maintenance region containing plasmid replicon and partition systems was then inserted as a 7.2-kbp Kpn I fragment at the unique Kpn I site.

To complete the construction of pNS358, a polylinker containing unique BamHI, Xba I, NotI, Sal I, and SnaBI sites was inserted at the unique BamHI site.

pNS358 was converted to pNS582 by inserting the P1 lytic replicon as a 2-kbp Hpa I fragment into the unique SnaBI site of pNS358. The activity of that replicon is regulated by the lacoperon promoter.

A final vector used, pNS582tetl4, is one in which the polylinker in pNS582 is replaced by the tetracycline resistance (tet) gene from pBR322. pNS582tetl4 contains unique BamHIand Sal I restriction sites located in tet.

P1 Cloning Vectors pNS358 and pNS582Each of these vectors consists of two

domains that are flanked by P1 loxPrecombination sites that are oriented in the same direction.

Recombination between these sites mediated by the P1 Cre recombinaseseparates the two domains of the vector into discrete circles.

One of the domains of the vector has the pBR322 plasmid ampr gene, the pBR322 multicopy replicon, and the P1 pac site. The latter was oriented so as to direct packaging counterclockwise on the vector.

The second vector domain contains a P1 plasmid replicon, a kanr gene from Tn903, and a polylinker cloning site containing unique BamHI, SnaBI, Sal I, Not I, and Xba I sites.

pNS582 differs from pNS358 in that it contains a lac promoter-regulated P1 lytic replicon in the kanr domain of the vector.

An in Vitro P1 Packaging System

•Bacteriophage P1 packages DNA by a headful mechanism. The process is initiated when P1-encoded pacase proteins recognize and cleave pac in the phage DNA.

• The DNA on one side of that cleavage is then packaged into an empty phage prohead.

•Once the head has been filled, a second DNA cleavage event (the headful cut) occurs that separates the DNA inside of the head from that outside of the head.

• The headful cleavage is not sequence specific.

•Phage tails are then added to the filled heads to complete particle formation. Note that in this process pac is cleaved only during the initiation of packaging and not at its termination.

• Thus, the two ends of the packaged DNA are different.

In Vitro Packaging of Vector DNA

•To assess the efficiency of the in vitro packaging reaction, pNS582 vector DNA wascleaved at one of the restriction sites in the polylinker, ligated to produce concatemersconsisting of 3-10 vector units, and that DNA was then packaged in vitro.

•The phage produced generate 2 x 106 kanr transformants per /microgram ofvector used when the infected strain (NS2974) contains the Cre recombinase.

• If the infected strain does not contain Cre (JM109) then the yield of kan' transformantsis reduced -50-fold.

•Presumably, Cre-mediated recombination between loxP sites is necessary to cyclize the infecting linear DNA so that it can be faithfully maintained in the transformed cells.

Products generated by the packaging of various ligated products

A. Singly digested vector DNA was ligated to form a long concatemer. Packaging of that DNA into large P1 heads followed by the infection of a Cre' strain cyclizes the DNA between IoxP sites and produces compatible kanr and ampr domain plasmids.

B. Vector DNA was ligated to small insert DNA to generate a mixed concatemer. Headful packaging of that DNA from a pac site produces an ampr domainplasmid and three kanr plasmids. Two of those contain different, small inserts and the third does not. The kanrplasmids are incompatible and, therefore, segregate away from each other into separate cells.

(C) A single large insert was ligated between two vector molecules. Headful packaging of that DNA generates only a kanr plasmid with the large insert.

Conclusion

•The size of the DNA that is cloned in the P1 vector is determined by the headfulsize of P1 (110-115 kbp of DNA) and by the amount of DNA between any two directly repeated loxP sites in the ligated concatemer.

• If the inserted DNA fragment is >100 kbp, then the distance between loxP sites in the kanr domain of the plasmid will be greater than a P1 headful, and the packaged DNA will not contain two loxP sites.

• When that DNA is injected into a Cre-containing host, it will not be cyclized and, therefore, will not be recoverable as a kanr transformant.

• If the inserted DNA fragment is smaller than 100 kb it can be packaged between loxP sites and will be cyclized and recovered after injection.

PAC vectors

•Phage artificial chromosome or P1-derived artificial chromosome (PAC) is a form of chromosome derived through biological manipulation and it originates from a ‘phage’ instead of a ‘plasmid’, as seen in the construction of many other artificial chromosomes.

•Genomic libraries constructed in the original Bacterial Artificial Chromosome (BAC) and P1 Artificial Chromosome (PAC) cloning systems were very useful for completion of the Human Genome Project.

•Libraries constructed in these vector systems were used to generate physical maps of all twenty three-chromosome pairs and served as the templates for DNA sequencing.

•PACs can accommodate larger inserts of DNA than a plasmid or many other types of vectors. Sometimes, the number of inserts can be as high as 300 kilobase paires.

Construction of PACs through electroporation:

•During the construction of PACs, P1 phage containing cells will undergo a process known as ‘electroporation’, which will increase the permeability of the cell membrane and allow DNA material to enter the cell and couple with the existing DNA.

•This process will give rise to PACs and from there onwards, the PACs can replicate within the cell through ‘lysogeny’, without destructing the cell or incorporating into rest of the chromosomes.

Construction of PAC vector pJCPAC-Mam2 The PAC vector pJCPAC-Mam2 is a 23 kilobase (kb) shuttle vector that is highly versatile and useful for functional studies in human tissue culture cell lines.

pJCPAC-Mam2 contains the P1 single-copy replicon for low copy expression and a multi-copy lytic replicon under the control of the lac repressor for high copy expression in Escherichia coli,

wild type and mutant loxP sites for generation of bidirectional nested deletions in any clone of interest and

the Epstein Barr Virus (EBV) latent replication origin oriP, the Epstein Barr Nuclear Antigen 1 (EBNA1) gene, and a puromycin-resistance gene for propagation in mammalian cells.

Uses of PACs:

•PACs are in high demand when it comes to cloning important biomedical sequences, which are essential for many scientific functions.

•One of its main uses is the genome analysis and map based cloning of complex plants and animals, which requires isolation of large pieces of DNA rather than smaller segments.

•Furthermore, PAC based cloning is useful in the study of ‘phage therapy’ and in scientific studies focusing on how antibiotics act on a particular bacteria.

•Although there are other forms of artificial chromosomes which can accommodate more base pairs than PACs, relative user friendliness of these vectors makes it a popular choice among many biomedical researchers.

Mammalian Artificial Chromosomes (MAC) Vector

•Chromosomes in eukaryotes have evolved as vehicles for nuclear genes and have developed specialized nucleoprotein structures for this purpose, some of them are centromeres, telomeres and origin of replication.

•Alphoid arrays are found at all human centromeres and consist of a 171 bpmonomer organized in higher order repeats encompassing 0.5-5 Mb.

•They have been considered the best candidate for the specific DNA requirement for centromere function.

•MAC formation was observed following transfection of a 100 kb yeast artificial chromosome (YAC) containing alphoid sequence from chromosome 21 with uniform higher order repeats and frequent CENP-B boxes, a conserved motif binding the CENP-B protein.

•The YAC had been retrofitted with terminal human telomere sequence, but no other human DNA was included.

•This YAC construct generated cell lines containing MACs at frequencies ranging from 10 to 100% cells.

•A human artificial chromosome (HAC) is a mini-chromosome that is constructed artificially in human cells. Using its own self-replicating and segregating systems, a HAC can behave as a stable chromosome that is independent from the chromosomes of host cells.The essential elements for chromosome maintenance and transmission are the following three regions:(1) the “replication origin,” from which the duplication of DNA begins,(2) the “centromere,” which functions in proper chromosome segregation during cell division, and(3) the “telomere,” which protects the ends of linear chromosomes.

•The particular DNA sequences specifying the replication origin and centromerewere unresolved in higher eukaryotes such as mammals.

•The research group of Tsuneko Okazaki, Ph.D., the founder of Chromo Research, investigated these sequences and succeeded in building a HAC with type I alphoidDNA from human chromosome 21.

• HAC, which is maintained as an extra chromosome, duplicates synchronously with host cell chromosomes at each host cell division and is transmitted stably to daughter cells.

Production of HAC by bottom-up construction strategies

Tsuneko Okazaki and her collaborators discovered that HACs were frequently generated de novo in the human fibroblast cell line HT1080 upon introduction of precursor DNA constructs in YACs or BACs.

These YACs or BACs contained up to 70 kilobases of human α-satellite (alphoid) DNA derived from human chromosome 21, as well as marker genes and, in the case of YAC constructs, telomere sequences at both DNA ends.

•A HAC can be detected by FISH analysis as a “mini-chromosome” generated by multimerization of the introduced DNA. A HAC made from a YAC is linear with telomericstructures, whereas a HAC generated from a BAC is circular and lacks telomeres. •Dr. Okazaki’s findings revealed an important principal: “type I alphoid DNA with frequent CENP-B boxes is necessary for de novo centromere/kinetochore formation.”

•On the basis of these results, Chromo Research scientists were the first to reproducibly manufacture HACs.

•The “bottom-up construction” strategy of Chromo Research involves the de novo construction of HACs by introducing necessary DNA elements for the maintenance of chromosome function into cells.

• On the other hand, “top-down construction” refers to the truncation of natural chromosomes into smaller sizes by using targeting vectors containing telomericsequences.

Various useful applications

•In bottom-up construction, multimerization of transfected DNA occurs during HAC formation. Thus, HACs that contain transgenes are generated de novo from precursor BAC or YAC vectors that contain the transgene and an alphoid array in separate vectors.

•A HAC can carry a site-directed insertion system. Because a transgene (cDNA or genomic DNA) can be inserted at a certain position in this HAC, the transgene in the HAC can be expressed in mammalian cells in a promoter-dependent manner under the desired stable control.

•Microcell-mediated chromosome transfer (MMCT) enables HACs to be transferred into various cell types and maintained stably. HACs can be transferred into mouse embryonic stem cells by MMCT, so a transgenic mouse containing exogenous genes can be readily created. The establishment of a reliable method to create a transgenic animal will enable HAC vector utilization for gene therapy and regenerative medicine.

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