lambda – E. coli bacteriophage
48.5 kb genome – 12 bp single-stranded cohesive ends
– linear form (in phage particle)
– circular form (in cell)
– ligation of cohesive ends
temperate phage – two lifestyles
lysogeny – phage dormant
– inserted in host genome
lytic growth – progeny phage produced
– host cells lysed
infection – phage particle binds to LamB gene product
– (maltose transport protein)
– phage genome injected in cell
– lysis/lysogeny decision made – cII gene
– cII protein may be degraded by host Hfl protease
– if cII degraded, get lytic growth
– if cII not degraded, phage inserts in genome
(homologous recombination
– cI repressor keeps phage dormant
– cI inactivated (SOS system)
– phage reverts to lytic growth
– lytic growth
– phage genome replicated by rolling circle replication
– produces concatemers
– linear molecules with multiple phage genomes
– requires phage Gam protein
– stops host nuclease from digesting concatemers
– phage coat protein genes expressed
– head and tail self-assemble
– phage DNA packaged in phage heads
– requires 2 cos sites (cohesive packaging sites)
– must be 78%-105% of phage genome apart
– 38-52 kb apart
– cos sites cleaved to 12 bp single stranded
ends
– ends of phage genome
DNA sequence between cos sites – does not
matter
– any DNA molecule with cos sites at right
distance
– packaged in phage heads
– complete phage heads assemble – S protein lyses cell
– progeny phage released
lambda as cloning vector
– why use lambda?
– allows large fragments DNA to be cloned
– up to 24.6 kb – good for genomic libraries
– self-assembly of phage particles
– allows recombinant lambda to be packaged in vitro
– amplified by infecting E. coli
– large DNA fragments cloned efficiently
– screening for recombinants
– phage plaques easily screened for desired clone
– using nucleic acid probes – plaque lifts
– some vectors
– allow expression of cloned cDNAs
– can screen plaques for cloned protein
– using antibodies to protein