PAC

P1 derived Artificial Chromosomes (PAC) is a genome derived from Phage P1. P1 phage utilises PAC sites for efficient breaking and packaging of the genome and its efficient delivery in transfection stage.

1. P1 derived ARTIFICIAL CHROMOSOME PAC Presented by Nagendra P 16PBT2004 1

2. Cloning Vectors Key features of an efficient vector. Origin of replication Multiple cloning site(MCS) Selectable marker Easy isolation from host cell 2

3. Cloning vectors Plasmids Lambda phages Cosmids Phasmids and Phagemids BACs PACs YACs Plant transformation vectors 3

4. Choice of vectors Vector Host Insert size Lambda phage E.coli 5-25kb Lambda cosmids E.coli 35-45kb P1 phage E.coli 70-100kb PACs E.coli 100-300kb BACs E.coli ≤ 300kb YACs S.cerevisiae 200-2000kb 4

5. The major advantage of the cosmid system for cloning is that the DNA insert can be packaged efficiently into phage lambda particles. However , the maximum amount of DNA that can be cloned into any one cosmid vector , 45-48 kilobase pairs ( kbp ), is limited by the size of the lambda head. In contrast, YAC cloning vectors can accept and propagate DNA that is as large as 200-800 kbp . Limitations of the YAC system are ( i ) the low efficiency of transformation with vector DNA containing large inserts ( ii) the decreasing transformation efficiency with increasing insert size ( iii) the need to process transformants individually prior to screening and ( iv) the difficulty in obtaining large amounts of insert DNA from transformed cells . 5

6. PAC Bacteriophage P1 was isolated in 1951 by Giuseppe Bertani from the Escherichia coli strain. Phage P1 is a temperate bacteriophage which has been extensively used for genetic analysis of Escherichia coli. I t can mediate generalized transduction. Sternberg and co-workers have developed a P1 vector system. H as a capacity for DNA fragments as large as 100 kb. Thus the capacity is about twice that of cosmid clones but less than that of yeast artificial chromosome (YAC) clones. 6

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8. Structure and Genome : P1 has an icosahedral “head” containing the phage DNA attached to a 220nm long tube, contractile tail or sheath , and base plate with six tail fibers . The P1 capsid is composed of 15 head proteins, 9 tail proteins, and 4 proteins of undetermined location In a same virion population, 80% of virions have a head diameter of 85nm, while the other 20% have a diameter of 65nm. 8

9. Genome The genome of P1 Phage is linear double stranded DNA , about 94 Kbp .  The genome is longer, about 120Kbs, than the actual length when in viral particle as it is created by cutting an appropriately sized fragment from a  concatemeric  DNA chain having multiple copies of the genome.  Due to this, the ends of the DNA molecule are identical and are referred to as being  “terminally redundant” . It has large, about 15 kbp terminal redundancy.  I n the viral particle it is in the form of a linear double stranded DNA molecule, once inserted into the host it circularizes because of the its large terminal redundancy and replicates as a plasmid. 9

10. Two origins of replication, oriR which replicates it during the lysogenic cycle and oriL which replicates it during the lytic stage. The genome has117 genes that are organized in 45 operons. 4 are responsible for the choice between lysis and lysogeny and another 4 are responsible for plasmid maintenance. The remaining 37 are invovled in lytic development . Maintains one copy per host cell. 10

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12. The Life Cycle of P1 Phage 1. Adsorption , Injection and protection of the genome P1 adsorbs to the receptors on the host cells – terminal glucose on the lipopolysaccharide present on the outer surface of the outer membrane of the host cell . A lytic transglycosylase faclitates the puncture of the bacterial cell wall. P1 contracts its tail and inject its DNA into the cell. Once inside the cell, P1 DNA circularizes by homologous recombination because these DNA when packed it is packed by  Head full Mechanism . This can be done either by host or phage recombination system. 12

13. 2. P1 DNA Replication and Phage Assembly. (Lytic Cycle) The P1 plasmid has a separate origin of replication ( oriL ) that is activated during the lytic cycle. Early P1 replication takes place by the theta mode of replication. Later in infection, P1 switch to rolling circle replication. Rolling circle replication produces concatemers for packaging into phage heads. At approx 45 minutes after the infection, the cells are filled with concatemers of phage DNA, assembled phage heads, and assembled phage tails. Now complete phage must take place. A protein made from phage genome cre recombinase recognizes a site on the concatemers of phage DNA called the  pac  site. The protein cuts the DNA, making a double-stranded end. This end is inserted into a phage head. The DNA continues to be pushed inside the head until the head is full, a process called head-full packaging . After the head is full of DNA, a double stranded cut is made and a tail is attached. Once the complete virions are assembled, the host cell is lysed, releasing the viral particles. 13

14. 3.The Location of the P1 Prophage in a lysogen (Lysogenic Cycle ) Prophages can be physically located in one or two places in a lysogen unlike in case of Lambda, the phage genome is recombined into the bacterial chromosome. P1 contains an origin for DNA replication and once the phage genome is converted to circular, double-stranded DNA, it is maintained in the cytoplasm as a stably inherited extra-chromosomal piece of DNA or plasmid. Coordinated DNA replication occurs, in order to maintain its population in all daughter cells. If not, when multiplied by binary fission only one daughter cell will posses the phage DNA and another daughter cell does not. 14

15. 4. P1 Transducing Particles F ormation of transducing particles or phage particles that contain chromosomal DNA instead of phage DNA.  E-Coli chromosomes contains many pseudopac sites or sites that can be used to initiate packaging of host chromosomal DNA into maturing phage. These pseudopac sites are used much less frequently than the phage pac sites but they are used. So t he resulting phage carry random pieces of the chromosome in place of phage genomes. The ability to package any piece of chromosomal DNA instead of phage DNA makes P1 a generalized transducing phage .   15

16. CONSTRUCTION OF PAC P1 vector contains a packaging site ( pac ) which is necessary for in vitro packaging of recombinant molecules into phage particles. The vectors contain two loxP sites. These are the sites recognized by the phage cre recombinase , the product of the phage cre gene, and which lead to circularization of the packaged DNA after it has been injected into an E. coli host expressing the recombinase . Cre ( C auses Re combination) and the recombination site was named loxP (locus of crossing (x) over, P1 ) 16

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18. In strains containing a lacI repressor, the P1 plasmid replicon maintains the DNA at about one copy per host chromosome . If IPTG is added to the medium the P1 lytic replicon in the vector is induced and the copy number of the plasmid increases up to 25 folds in 6 generations. 18

19. USES PACs are in high demand when it comes to cloning important biomedical sequences , which are essential for many scientific functions. This P1 system has been used to construct genomic libraries of mouse, human, fission yeast, and Drosophila 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. 19

20. REFERENCES Sternberg N. Bacteriophage P1 cloning system for the isolation, amplification, and recovery of DNA fragments as large as 100 kilobase pairs. Proceedings of the National Academy of Sciences. 1990 Jan 1;87(1):103-7 Lehnherr H, Yarmolinsky MB, Blattner FR. Genome of bacteriophage P1. JOURNAL OF BACTERIOLOGY. 2004 Nov:7032-68 . Abremski K, Hoess R. Bacteriophage P1 site-specific recombination. Purification and properties of the Cre recombinase protein. Journal of Biological Chemistry. 1984 Feb 10;259(3):1509-14 . Nagy A. Cre recombinase : the universal reagent for genome tailoring. genesis. 2000 Feb 1;26(2):99-109. https://biotechkhan.wordpress.com/2014/07/08/bacteriophage-p1-structure-and-life-cycle/ http://bio.classes.ucsc.edu/bio105l/EXERCISES/P1/introduction.pdf 20

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