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MB 414 > How to Transfer DNA > Flashcards

How to Transfer DNA Flashcards

1
Q

complementation

A
  • 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
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2
Q

Griffith Discovered transformation while working with

A

Streptococcus pneumoniae

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3
Q

Transformation experiment

A
  • 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
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4
Q

naked DNA

A

not within cell or phage

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5
Q

What is needed for transformation?

A

DNA

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6
Q

Experiment that proved DNA as transformation

A
  • 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
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7
Q

Competence

A
  • cells that have the ability to take up naked DNA
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8
Q

Transformation in S. pneumoniae - gram positives

A
  • 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
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9
Q

Transformation in H. influenzae - gram negatives

A
  • 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.
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10
Q

Forced transformation

A
  • destabilize the cell membrane

- E. Coli isn’t normally competent, so it’s made to take up DNA in the lab

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11
Q

two ways to make cells competent

A
  • chemical

- electrical

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12
Q

chemical transformation

A
  • 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.
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13
Q

electrical transformation

A
  • 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.
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14
Q

Transformation with DNA fragments

A
  • 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.
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15
Q

Transformation with a plasmid

A
  • uptake of plasmid and stable transformation

- replicate if they have an origin of replication.

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16
Q

conjugation

A
  • direct cell to cell DNA transfer

- DNA transferred from donor to recipient through a pilus.

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17
Q

The F factor

A
  • has everything required to transfer itself
  • pilus genes
  • origin of replication (orin)
  • ssDNA polymerase
  • fertility factor
  • codes for the pilus
18
Q

Rolling circle replication

A
  • 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.
19
Q

High frequency recombinants

A
  • if an F plasmid integrated into the chromosome
20
Q

Hfr and chromosome mapping

A
  • 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.

21
Q

Transduction

A
  • 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.
22
Q

lytic cycle

A
  • 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.
23
Q

lysogenic cycle

A
  • phage DNA insert itself into bacterial genome - prophage
  • inactive in cell chromosome but replicates each time bacteria replicates
  • more efficient replication in good conditions.
24
Q

generalized transduction

A
  • host DNA packaged into phage particle instead of phage genome
  • lytic process
25
Q

specialized transduction

A
  • the transfer of only a few specific genes when phage incorrectly excised from chromosome.
  • lysogenic process
26
Q

fates of DNA once it is passed from one bacteria to the next

A
  • circular, can replicate autonomously
  • lienar DNA is incorporated into the chromosome - not replicated.
  • DNA not able to replicate or incorporated is degraded.
27
Q

the integration of donor DNA into a bacterial genome is mediated by

A

recombination.

28
Q

linear DNA recombination

A
  • requires large stretches of DNA

- homologous DNA is recombined with chromosomal DNA

29
Q

The Rec system

A
  1. strand breakage
  2. strand pairing
  3. strand assimilation
  4. crossover formation
  5. breakage and reunion
  6. mismatch repair
30
Q

RecBCD

A
  • 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
31
Q

RecA

A
  • 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.
32
Q

RuvAB proteins

A
  • mediate branch migration
  • displaces more of recipient with donor strand
  • formation of Holliday junctions
33
Q

RuvC

A
  • 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.
34
Q

Holliday resolution

A
  • repair all mismatches - now new version of the gene.
35
Q

DNA exchange with plasmids

A
  • 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.
36
Q

plasmid maintenance strategies

A
  • high copy number
  • partitioning genes
  • resistance markers
  • addiction modules
37
Q

relaxed plasmids

A
  • 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
38
Q

stringent plasmids

A
  • only replicate when chromosome does.

- low copy number

39
Q

partitioning genes

A
  • 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.
40
Q

resistance markers

A
  • resistance genes give cell advantage

- in antibiotics must keep plasmid to encode resistance

41
Q

addiction module

A
  • 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.

MB 414 (26 decks)

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