Flashcards in How to Transfer DNA Deck (41):
- 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
- 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
not within cell or phage
What is needed for transformation?
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
- 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.
- 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
- 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.
- 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.
- 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.
- 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.
- 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.
- phage DNA insert itself into bacterial genome - prophage
- inactive in cell chromosome but replicates each time bacteria replicates
- more efficient replication in good conditions.
- host DNA packaged into phage particle instead of phage genome
- lytic process
- 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
linear DNA recombination
- requires large stretches of DNA
- homologous DNA is recombined with chromosomal DNA
The Rec system
1. strand breakage
2. strand pairing
3. strand assimilation
4. crossover formation
5. breakage and reunion
6. mismatch repair
- 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
- 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.
- mediate branch migration
- displaces more of recipient with donor strand
- formation of Holliday junctions
- 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.
- 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
- 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
- only replicate when chromosome does.
- low copy number
- 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 genes give cell advantage
- in antibiotics must keep plasmid to encode resistance