Lambda vectors and their replication

• 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 c

• 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

λ 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

cos

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 λ− 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

λ gt10 and λ ZAP

3 Cosmids- are hybrid vectors of λ

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

-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

•

replication enzymes Without replication origin, DNA cannot be replicated in the cell

ture6

• 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

• Many cloning vectors contain a

multiple cloning site (DNA segment

with several unique sites for restriction nucleases located next to each other)

• Gene to be cloned can be introduced into the cloning

vector at one of the restriction sites present in the cloning site.

http://www.gmu.edu/departments/biology/385-Ch04c-rDNA/

Brock Biology of Microorganisms, 9 th Edition

(2000) Prentice Hall, Madigan, Martinko, Parker

steps in cloning with λ :

– 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

check recombinant phage for

the presence of desired foreign

DNA sequence by observing

its genetic properties.

Molecular Biology of the Cell, 3 rd Edition, Garland Publishing, Inc 1983

micro.msb.le.ac.uk/224/Phages.html#Lambda

http://www-•

-PL ( promoter) for transcription for the

left side of λ 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.

•

Life cycle of lambda

1 Virus enters cell

2 PL and PR gets activated

3 PL transcribes to make N protein

4 PR transcribes to make cro protein

5 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)

6 There are also termination sites next to Q

protein Q protein will allow transcription past this site

7 If Cro protein blocks production of cI (goes lytic)

8 If cII and cIII activates transcription to make cI

(goes lysogenic)

9 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

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.

• Ori λ –Origin of phage λ (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 ori λ

O.P.dnaB complex to liberate dnaB

• dnaB initiates unwinding of duplex.

• Primase starts chain initations and polII starts elongation.

DNA Replication, W.H Freeman and Co (1992)

Kornberg,A.

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.

• Brock Biology of Microorganisms, 9 th 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

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