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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).
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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
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.
-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
steps in cloning with l:
-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://www-micro.msb.le.ac.uk/224/Phages.html#Lambda
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.DNA Replication, W.H. Freeman and Co. (1992) Kornberg,A.
-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.DNA Replication, W.H. Freeman and Co. (1992) Kornberg,A.