How to Transfer DNA Flashcards
complementation
- giving a mutant strain back its function.
- if piece of DNA is large and contains many genes, cut up the piece and figure out which gene or genes control the function
Griffith Discovered transformation while working with
Streptococcus pneumoniae
Transformation experiment
- smooth - capsule + pathogenic
- rough - no capsule + nonpathogenic
- smooth - dead mice
- rough - live mice
- heat killed smooth - live mice
- heat killed smooth + rough - dead mice
- all rough cells came out smooth - transformed
naked DNA
not within cell or phage
What is needed for transformation?
DNA
Experiment that proved DNA as transformation
- Avergy, MacLeod, and McCarthy
- fractionated S cells
- tested carb, protein, lipid, RNA, and DNA
- genes are made out of DNA
- only cells exposed to S cell DNA were transformed
Competence
- cells that have the ability to take up naked DNA
Transformation in S. pneumoniae - gram positives
- competence is cell cycle dependent only, and is induced by a competence stimulating peptide
- cells induced by CSP produce at least a dozen protein on their cell membrane which bind DNA.
- once DNA bound, it is transported via a translocasome which engulfs it, and the DNA is recombined into the chromosome
Transformation in H. influenzae - gram negatives
- competence in mediated by starvation: under nutrient depletion, cAMP levels rise, inducing 6 competence genes comA-F
- Cells then change the outer cell membrane, exhibiting elevated levels of LPS.
- Vesicles, called transformasomes, bud from the surface and specifically bind to the DNA which is recognized by the conserved sequence.
Forced transformation
- destabilize the cell membrane
- E. Coli isn’t normally competent, so it’s made to take up DNA in the lab
two ways to make cells competent
- chemical
- electrical
chemical transformation
- CaCl2 binds to peptidoglycan in cell wall and destabilize
- membrane opens up briefly and some DNA gets inside
- immediately add fresh media and let them recover.
electrical transformation
- wash in water to remove ions
- suspend in 10% glycerol solution
- DNA added to cells and put into cuvette. 2500V electricity put through
- DNA taken up.
Transformation with DNA fragments
- most bacteria don’t like linear DNA fragments so they get degraded in the cytoplasm
- if not, they undergo integration by nonreciprocal recombination for a stable transformation.
Transformation with a plasmid
- uptake of plasmid and stable transformation
- replicate if they have an origin of replication.
conjugation
- direct cell to cell DNA transfer
- DNA transferred from donor to recipient through a pilus.
The F factor
- has everything required to transfer itself
- pilus genes
- origin of replication (orin)
- ssDNA polymerase
- fertility factor
- codes for the pilus
Rolling circle replication
- Rep protein nicks one strand of the plasmid in the phosphate backbone
- DNA pol translates 5’-3’ displacing parent strand
- displaced strand send through pilus to recipient bacterium where the complementary strand is made.
- the complementary strand is then polymerized for the displaced strand
- one strand of DNA is made at a time.
High frequency recombinants
- if an F plasmid integrated into the chromosome
Hfr and chromosome mapping
- they can be isolated with F in many locations
- if you stop the mating at different times, you can tell how far the gene you are mapping is from the integration site.
Transduction
- bacteriophage mediated DNA exchange
- phage will sometimes pack DNA from its host into its head, instead of the viral genome. when the phage infects the next cell, it injects the host DNA instead.
lytic cycle
- if conditions are not so good
- multiply immediately
- takes over cell machinery to make progeny phage
- when cell is full of phage it bursts and progeny released
- kills host cell. progeny move to new cells.
lysogenic cycle
- phage DNA insert itself into bacterial genome - prophage
- inactive in cell chromosome but replicates each time bacteria replicates
- more efficient replication in good conditions.
generalized transduction
- host DNA packaged into phage particle instead of phage genome
- lytic process
specialized transduction
- the transfer of only a few specific genes when phage incorrectly excised from chromosome.
- lysogenic process
fates of DNA once it is passed from one bacteria to the next
- circular, can replicate autonomously
- lienar DNA is incorporated into the chromosome - not replicated.
- DNA not able to replicate or incorporated is degraded.
the integration of donor DNA into a bacterial genome is mediated by
recombination.
linear DNA recombination
- requires large stretches of DNA
- homologous DNA is recombined with chromosomal DNA
The Rec system
- strand breakage
- strand pairing
- strand assimilation
- crossover formation
- breakage and reunion
- mismatch repair
RecBCD
- binds to the end of linear DNA
- exhibits 5’-3’ and 3’-5’ exonuclease and helices activity
- unwinds and cleaves the duplex DNA
- just before coming to a CHI site, the 3’-5’ exonuclease activity stops, yielding single stranded DNA, which is bound by recA
RecA
- coats the ssDNA, and binds to homologous dsDNA forming a triple stranded intermediate
- ATP dependent
- the donor strand then progressively displaces the recipient strand through branch migration and replaces with donor strand.
RuvAB proteins
- mediate branch migration
- displaces more of recipient with donor strand
- formation of Holliday junctions
RuvC
- bind to Holliday junctions as a dimer and cleaves the DNA asymmetrically to leave ligatable products
- cross resolves, leaving a hybrid of recipient and donor DNA
- hybrid will contain mismatches.
Holliday resolution
- repair all mismatches - now new version of the gene.
DNA exchange with plasmids
- plasmids are extra-chromosomal DNA elements
- they are circular so will not be chewed up by exonuclease - no need for recombination
- in order to be maintained they must have an origin of replication or it will be lost by dilution.
plasmid maintenance strategies
- high copy number
- partitioning genes
- resistance markers
- addiction modules
relaxed plasmids
- origin of replication at any time - continuously replicated
- hundreds of copies in a single cell
- ## when cell divides, both daughter cells will have plasmid and be maintained
stringent plasmids
- only replicate when chromosome does.
- low copy number
partitioning genes
- encode proteins that will bind to certain elements in plasmid and carry one copy to end each of dividing cell to make sure each daughter cell gets a copy.
resistance markers
- resistance genes give cell advantage
- in antibiotics must keep plasmid to encode resistance
addiction module
- poison (long lasting) and antidote (short lived) in order to survive poison the antidote must be continuously made
- need to make antidote from plasmid.
- if you don’t have plasmid the cell dies.
