P1-Derived Artificial Chromosome or PAC

Keywords

P1 bacteriophages, bacterial artificial chromosome, bacteria, DNA cloning vectors, DNA fragments, Escherichia coli, yeast artificial chromosomes, complex genomes, gene cluster, restriction enzyme, ligation, restriction sites.

Introduction

P1-derived artificial chromosome, or PAC, is a DNA construct derived from the DNA of P1 bacteriophages and Bacterial artificial chromosome. It can carry large amounts of about 100-300 kilobases of other sequences for a variety of bioengineering purposes in bacteria.

It is one type of efficient cloning vector used to clone DNA fragments (100-300 kb insert size; average,150 kb) in Escherichia coli cells. DNA cloning vectors containing regions of the P1 phage genome and capable of accepting large DNA inserts (100 kilobases long). In this system, vector DNA and DNA fragments to be cloned are ligated and packaged in vitro into phage particles that can infect Escherichia coli.

PACs are useful for cloning large genes, chromosome walking, physical mapping, and shotgun sequencing of complex genomes. Compare with bacterial artificial chromosomes (BAC)s, yeast artificial chromosomes (YACs). PAC cloning system will be useful in the mapping and detailed analysis of complex genomes.

Even when using BAC and PAC vectors, the desired gene cluster may sometimes be split between two or more clones. It will often be necessary or desirable to join the two segments together in a single construct that can be used readily for heterologous expression. Achieving this by restriction enzyme digestion and ligation is usually not possible due to the lack of appropriate restriction sites.

History of PAC

The bacteriophage P1 was first isolated by Dr. Giuseppe Bertani. In his study, he noticed that the lysogen produced abnormal non-continuous phages, and later found phage P1 was produced from the Lisbonne lysogen strain, in addition to bacteriophages P2 and P3.

P1 can copy a bacteria's host genome and integrate that DNA information into other bacteria hosts, also known as generalized transduction. Later, P1 was developed as a cloning vector by Nat Sternberg and colleagues in the 1990s. It is capable of Cre-Lox recombination. The P1 vector system was first developed to carry relatively large DNA fragments in plasmids (95-100kb).

Construction

PAC has 2 loxP sites, which can be used by phage recombinases to form the product from its cre-gene recognition during Cre-Lox recombination. This process circularizes the DNA strand, forming a plasmid, which can then be inserted into bacteria such as Escherichia coli.

The transformation is usually done by electroporation, which uses electricity to allow the plasmids to permeate into the cells. If high expression levels are desired, the P1 lytic replicon can be used in constructs. Electroporation allows for lysogeny of PACs so that they can replicate within cells without disturbing other chromosomes.

Comparison with other Artificial Chromosomes

PAC is one of the artificial chromosome vectors. Some other artificial chromosomes include bacterial artificial chromosome, yeast artificial chromosome and the human artificial chromosome. Compared to other artificial chromosomes, it can carry relatively large DNA fragments, however less so than the yeast artificial chromosome (YAC).

Some advantages of PACs compared to YACs includes easier manipulation of bacteria system, easier separation from DNA hosts, higher transformation rate, more stable inserts, and they are non-chimeric which means they do not rearrange and ligate to form new DNA strand, allowing for a user-friendly vector choice.

Applications

PAC is commonly used as a large capacity vector which allows propagation of large DNA inserts in Escherichia coli. This feature has been commonly used for:

• Building genome libraries for human, mouse, etc., helps with projects such as Human Genome Project
• Libraries served as the template for gene sequencing (example: used as gene template in mouse gene function analysis)
• Genome analysis on specific functions of different genes for more complex organisms (plants, animals etc.)
• Facilitate gene expression.

Since PAC was derived from phages, PAC and its variants are also useful in the PAC-based phage therapy and antibiotic studies.

Conclusion

Bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) are large genomic clones stably maintained in bacteria and are very important in functional studies through transfection because of their large size and stability. Because most BAC or PAC vectors do not have a mammalian selection marker, transfecting mammalian cells with genes cloned in BACs or PACs requires the insertion into the BAC/PAC of a mammalian selectable marker.

Large-scale genomic sequencing projects have provided DNA sequence information for many genes, but the biological functions for most of them will only be known through functional studies. However, currently available procedures are not satisfactory in efficiency and fidelity.

BAC/PAC retrofitting vector is used on transformation into competent BAC or PAC strains, will catalyze the specific insertion of itself into BAC/PAC vectors through in vivo cre/loxP site-specific recombination.