Flashcards
7 - Transduction
Flashcards do conjunto (9)
- 1Generalized TransductionP1 (E. coli) & P22 (S. typhimurium)
Phage infects bacteria
Host DNA hydrolyzes; Phage DNA & proteins synthesized
*Occasionally a bacterial DNA fragment is packaged into phage capsid*
Transducing phage infects another host cell --- recombination --> Recombinant bacteria
---*A from bact #1*--- - 2Specialized TransductionProphage integrates between genes
*Occasionally, prophage DNA exist incorrectly, taking adjoining bacterial DNA with it*
Transducing phage infects another host -- recombination --> recombinant bacteria
---!!!!!!!!!!!!*A from bact #1*!!!--- - 3Generalized Transduction: non-specific pac sitespac/cos sites: Where phage DNA is cut for packaging into T7 phage heads
Often found in concatemers of phage DNA produced during rolling circle replication
Enzyme recognizes pac site & makes regular cuts at unit lengths. Similar sites in bacterial chromosome: it will be packaged as well - 4Generalized Transduction: Headful MechanismOnce packaging starts, phage chromosome unit lengths are produced & packaged
- 5Uses of generalized transduction: MappingMapping: Two-factor Crosses -> the closer genes are, the greater probability of COTRANSDUCTION. Select for recombinants only by selecting for one marker and screening for the others
Co-transduction frequency = (cotransductants/total transductants) * 100 - 6Uses of Generalized Transduction: Strain ConstructionTo study gene function, compare ISOGENIC strains (differ only in the gene studied) using transduction/transformation to move mutations into a strain.
Can be done even if no way to select -> use closely linked marker. Select for marker, screen for presence of mutation - 7Specialized Transduction CharacteristicsDefective phage particles (lamba dgal lacks head & tail genes, so it only carries bacterial genes close to site of integration)
Defective excision is due to rare abnormal outlooping -> crossing over
Lambda p bio = {It can't integrate so it's not lysogenic}
Lambda d gal = {With no tail, it can't form a proper phage particle unless a wildtype copy can supply it}.
Requires HELPER PHAGE to produce more phage particles
Can create DI-LYSOGENS carrying specialized transducing phage + WT helper phage to permit isolation of HIGH FREQUENCY TRANSDUCTION (HGFT) LYSATES - 8Specialized/Restricted Transduction: Types of transductants (??) Slide 34Nonlysogenic gal+ cells: double crossover between λdgal+ and gal- yields stable gal+ transductants. Plated to get gal+ transductants
λdgal+ PROPHAGE: Single crossover between λdgal+ and gal- yields unstable transductants. Resuspended and plated gal+ transductant = gal + & gal - colonies - 9Lysogenic ConversionProphages carry genes for virulence factors/toxins called MORONS (more DNA)
-Expressed from own promotors, which are active inside hosts.
-Prophage induction would additionally elevate expression
Turning on maturation genes AND toxin genes {shiga toxins} (when cell gets stressed out).
Enterohemorrhagic E. Coli: bacterial host induces SOS response, inactivating Stx phage repressor -> elevated expression of stxAB genes -> Shiga toxin -> cell lyses
Vibrio cholerae: phage provides Toxin co-regulated pilus (TCP) used to attach to intestinal epithelium -> cholera toxin (CTX) -> Another phage uses TCP as receptor to infect cell and type Ii secretion system for CTX secretion to exit cell
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- FlashCards sobre 7 - Transduction, criado por blam2 em 14-10-2014.
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