Download Lambda Vectors and their replication

• Lambda vectors and their
replication
Sonita Gafary
• Biochem 72020
• Lambda was first discovered at the
Pasteur Institute by Andre Lwoff when he
observed strains of E. Coli.
• He showed that the cells of these bacterial
strains carried bacteriophage in a dormant
form (prophage).
• Phage can alternate between lysogenic
(non-productive) and lytic (productive)
growth cycles.
l Bacteriophage
• Double stranded DNA molecule
• 5' twelve-base-pair sticky ends (cos sites)
• It is used as a cloning vector, accommodating fragments of
DNA up to 15 kilobase pairs long. For larger pieces, the
cosmid or YAC’s are used.
• Will accept foreign DNA and still complete their life cycle.
• Distinguish cells that have foreign and non foreign DNA.
• Should replicate in host
• Gene of interest can be identified and grown in large
amounts.
• Non essential genes can be removed and replaced by foreign
gene.
Cont.
• Should carry one or more selectable markers
that identify the parent and recombinant vectors
• Should have restriction sites in non-essential
regions of DNA into which foreign DNA can be
inserted
• easy to make and maintain library
Enzymes needed:
1.Restriction enzymes: cuts the DNA at specific
sequences to generate a set of fragments
2. DNA ligase: inserts DNA restriction fragments into
replicating DNA molecules to produce recombinant
DNA
Lambda vectors
• 1) Insertion vectors
cos
cos
EcoRI
• 2) Replacement
vectors
cos
cos
20Kb
EcoRI
EcoRI
• http://www.sh.lsuhsc.edu/gradcore/IDSP117/16
limitations
• size of DNA to be introduced into the host cell
• Problem: when making genomic libarary of
large size (plants and mammals) only a portion
of those fragments will be represented. If gene
of interest is located in a large fragment, then
you won’t be able to isolate that gene from the
library.
• Solution :use a vector that can accept large
fragments of DNA
Vector types:
1. Plasmids- small circular DNA molecules
which can replicate their DNA
independently of their bacterial
chromosome. They are found naturally in
bacteria and replicate inside the bacterial
cell. They can insert pieces up to
10kb(kilobases) or 100 to 10,000 base
pairs. Examples: pBR322 and pUC18
2. Bacteriophage l- They are double
stranded linear DNA vector. They
replicate in E. Coli in the lytic or lysogenic
mode. They can insert fragments up to
15kb. Examples are lgt10 and lZAP
3. Cosmids- are hybrid vectors of l phage
and plasmids. They can replicate their
DNA in the cell with a plasmid and be
packaged like a phage. They can insert
up to 50kb.
4. Yeast artificial chromosomes (YAC)primarily used in genome sequencing
projects. They host large inserts up to
1000kb.
Vector
Insert size
(kb)
Plasmid
<10 kb
Bacteriophage
l
9-15 kb
Cosmids
33-50 kb
YACS
100-1000 kb
What determines choice of vector?
1.
2.
3.
4.
Insert size
Vector size
Restriction sites
Cloning efficiency
-Central 1/3 is the “stuffer” fragment.
-Segments of the lambda DNA are removed and a stuffer
fragment is put in, this keeps the vector at a correct size
•
• Origin of replication
is a DNA segment
recognized by the
cellular DNAreplication enzymes.
Without replication
origin, DNA cannot be
replicated in the cell
http://www.uic.edu/classes/phar/phar33
1/lecture6/
• Selective marker is
required for maintenance
of plasmid in the cell.
Because of the presence
of the selective marker
the plasmid becomes
useful for the cell. Under
the selective conditions,
only cells that contain
plasmids with selectable
marker can survive.
http://www.uic.edu/classes/phar/phar331/lec
ture6
• Many cloning vectors
contain a multiple
cloning site (DNA
segment with several
unique sites for
restriction nucleases
located next to each
other)
http://www.uic.edu/classes/phar/phar331/lecture6
• Gene to be cloned
can be introduced
into the cloning
vector at one of
the restriction
sites present in
the cloning site.
http://www.uic.edu/classes/phar/phar331/lecture6
http://www.gmu.edu/departments/biology/385-Ch04c-rDNA/
steps in cloning with l :
–
Isolate vector DNA and gene of
interest
–
Cut both with restriction
enzyme(EcoRI)
–
Connect two fragments of
foreign DNA with DNA ligase.
(recombinant DNA)
–
Package DNA by adding cell
extracts containing head and tail
proteins
–
Transfer recombinant molecules
into host cell (transform)
–
Grow/select transformants:
check recombinant phage for the
presence of desired foreign DNA
sequence by observing its
genetic properties.
Brock Biology of Microorganisms, 9th Edition
(2000)
Prentice Hall, Madigan, Martinko, Parker
Molecular Biology of the Cell, 3rd Edition, Garland Publishing, Inc. 1983
-PL ( promoter) for transcription for the left
side of l with N and cIII
-PR (promoter) for right, including cro, cII
and the genes encoding the structural
proteins.
-OL and OR is short non-coding region of
genome, they control the promoters.
-cI (repressor) protein of 236 a.a. which
binds to OR and OL, preventing
transcription of cro and N, but allowing
transcription of OL, and the other genes
in the left hand end.
-cII and cIII encode activator proteins which
bind to the genome.
-Cro (66 aa) protein which binds to OR and
OL, blocking binding of the repressor to
this site to prevent lysogeny.
-N codes an antiterminator protein and
allows transcription from PL and PR. It
also allows RNA polymerase to
transcribe a number of phage genes,
including those responsible for DNA
recombination and integration of the
prophage, as well as cII and cIII.
-Q is an antiterminator similar to N, but only
permits extended transcription from PR
-Two Termination sites- One between N
and CIII and other between cro and CII.
•
http://wwwmicro.msb.le.ac.uk/224/Phages.html#Lambda
•
http://www-micro.msb.le.ac.uk/224/Phages.html#Lambda
Life cycle of lambda
1.
2.
3.
4.
5.
6.
7.
8.
9.
Virus enters cell
PL and PR gets activated
PL transcribes to make N protein
PR transcribes to make cro protein
Termination sites stop transcription but when enough N
protein is made, transcription goes past these two stop
sites (you can now make cIII and cII, replication
proteins (O and P) and Q)
There are also termination sites next to Q protein. Q
protein will allow transcription past this site.
If Cro protein blocks production of cI (goes lytic)
If cII and cIII activates transcription to make cI (goes
lysogenic)
cI blocks PL and PR (stops transcription) by binding to
OL and OR.
How do cells leave lysogeny cycle
and go to lytic cycle?
• By stress
• ultraviolet irradiation of cells, this causes
induction of a host cell protein, RecA
whose normal function is to induce the
expression of cellular genes which permit
the cell to adapt to and survive in altered
environmental conditions. RecA cleaves
the cI repressor protein.
Which proteins determine the
cycle?
• Lysogenic cycle: cI
proteins predominate
• Lytic cycle: cro
proteins predominate
DNA lambda replication
• Initation of replication at the lambda origin
requires “activation” by transcription
starting from PR.
• DNA replication is between O and P gene
proteins.
• Oril –Origin of phage l (with 4 binding
sites adjacent to AT rich region)
•
•
•
•
•
•
•
•
DNA Replication, W.H. Freeman and Co. (1992)
Kornberg,A.
O protein binds to lambda origin
causing a structural change in the
origin.
P protein interacts with O protein
Lambda proteins O and P form a
complex with DnaB at the lambda
origin (complex is inactive) This
forms a spherical structure called an
“O-Some” (~100bp of DNA)
P protein (lambda’s) brings dnaB to
the origin making the duplex larger
(~160bp)
The AT rich region becomes
susceptible to nuclease attack
(recognizes unpaired DNA), melting
the DNA duplex.
Shock proteins (dnaK, dnaJ and
grpE gene) dissociate the oril
O.P.dnaB complex to liberate dnaB
dnaB initiates unwinding of duplex.
Primase starts chain initations and
polII starts elongation.
-l circles multiply by
DNA Replication, W.H. Freeman and Co. (1992)
Kornberg,A.
theta form(q) and
continues for 5-15
minutes after
infection.
-Rolling circles
predominates after
15 minutes and
produce linear
concatemers
(genomes linked end
to end).
-Packaging requires
THF (termination
host factor) provided
by the host cell.
References
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http://www.uic.edu/classes/phar/phar331/lecture6/
http://www.sh.lsuhsc.edu/gradcore/IDSP117/16
http://www.gmu.edu/departments/biology/385-Ch04c-rDNA
Brock Biology of Microorganisms, 9th Edition (2000)
Prentice Hall, Madigan, Martinko, Parker
Recombinant DNA: A short course, W.H. Freeman and Co.(1983) Watson, Tooze,
Kurtz
http://www-micro.msb.le.ac.uk/224/Phages.html#Lambda
DNA Replication, W.H. Freeman and Co. (1992) Kornberg,A.
Genes VII, Oxford Unine Press. (2000), Lewin Benjamin
http://www.biocan.com/pdf/FAQ%20TrueBlue%20Vectors.pdf
http://www.cc.ndsu.nodak.edu/instruct/mcclean/plsc731/cloning/cloning1.htm
http://www.biochem.arizona.edu/classes/bioc461/Chapter6Powerpoint.pdf