p1 derived artificial chromosome (pac) vector

1. Pronabjyoti Mahatta.
M.Sc. 4th semester
Microbiology
Roll No. 10
P1 derived artificial
chromosome (PAC)
vector

2. INTRODUCTION
 A vector is a DNA molecule that has the
ability to replicate autonomously in an
appropriate host cell into which the DNA
fragment to be cloned (DNA insert) is
integrated for cloning.
 The vectors used for propagation of DNA
inserts in a suitable host are called cloning
vectors.
 There are many vectors which are
developed time to time for different
purposes.

3.  Plasmid vectors
 pSC101, pBR322, pUC18, pUC19, Pgem3z
 Bacteriophage vectors
 Lambda phage vectors, phage M13 vectors
 Cosmid vectors
 pJB8
 Phagemid vectors
 pBluescriptSK
 Phasmid vectors
 Lamba ZAP
 Artificial chromosome vectors
 PAC, BAC, YAC.

4. PAC VECTOR OR PHAGE P1 VECTOR
 Phage P1 is a temperate bacteriophage which has
been extensively used for genetic analysis of
Escherichia coli because it can mediate generalized
transduction.
 Sternberg and co-workers have developed a P1
vector system which has a capacity for DNA
fragments as large as 85 - 100 kb.
(sternberg 1990, Pierce et al.1992)

5.  The capacity is about twice that of cosmid clones
but less than that of yeast artificial chromosome
(YAC) clones.
 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 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 .

6.  Clones are maintained in E. coli as low-copy-number
plasmids by selection for a vector kanamycin-
resistance marker.
 A high copy number can be induced by exploitation
of the P1 lytic replicon.
 This P1 system has been used to construct genomic
libraries of mouse, human, fission yeast, and
Drosophila.

8. DESCRIPTION OF THE DIAGRAM :
 The phage P1 vector system. The P1 vector Ad10
(Sternberg 1990) is digested to generate short and
long vector arms. These are dephosphorylated to
prevent self-ligation. Size-selected insert DNA (85–
100 kb) is ligated with vector arms, ready for a two-
stage processing by packaging extracts.
 First, the recombinant DNA is cleaved at the pac
site by pacase in the packaging extract.

9.  Then the pacase works in concert with head/tail
extract to insert DNA into phage heads, pac site first,
cleaving off a headful of DNA at 115 kb. Heads and
tails then unite. The resulting phage particle can inject
recombinant DNA into host E. coli. The host is cre+.
The cre recombinase acts on loxP sites to produce a
circular plasmid.
 The plasmid is maintained at low copy number, but can
be amplified by inducing the P1 lytic operon.