How to Transfer DNA

Question 1

complementation

Answer 1

• 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

Question 2

Griffith Discovered transformation while working with

Answer 2

Streptococcus pneumoniae

Question 3

Transformation experiment

Answer 3

• 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

Question 4

naked DNA

Answer 4

not within cell or phage

Question 5

What is needed for transformation?

Answer 5

DNA

Question 6

Experiment that proved DNA as transformation

Answer 6

• 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

Question 7

Competence

Answer 7

• cells that have the ability to take up naked DNA

Question 8

Transformation in S. pneumoniae - gram positives

Answer 8

• 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

Question 9

Transformation in H. influenzae - gram negatives

Answer 9

• 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.

Question 10

Forced transformation

Answer 10

• destabilize the cell membrane

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

Question 11

two ways to make cells competent

Answer 11

• chemical

- electrical

Question 12

chemical transformation

Answer 12

• 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.

Question 13

electrical transformation

Answer 13

• 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.

Question 14

Transformation with DNA fragments

Answer 14

• 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.

Question 15

Transformation with a plasmid

Answer 15

• uptake of plasmid and stable transformation

- replicate if they have an origin of replication.

Question 16

conjugation

Answer 16

• direct cell to cell DNA transfer

- DNA transferred from donor to recipient through a pilus.

Question 17

The F factor

Answer 17

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

Question 18

Rolling circle replication

Answer 18

• 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.

Question 19

High frequency recombinants

Answer 19

• if an F plasmid integrated into the chromosome

Question 20

Hfr and chromosome mapping

Answer 20

• 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.

Question 21

Transduction

Answer 21

• 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.

Question 22

lytic cycle

Answer 22

• 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.

Question 23

lysogenic cycle

Answer 23

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

Question 24

generalized transduction

Answer 24

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

Question 25

specialized transduction

Answer 25

- the transfer of only a few specific genes when phage incorrectly excised from chromosome. - lysogenic process

Question 26

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

Answer 26

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

Question 27

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

Answer 27

recombination.

Question 28

linear DNA recombination

Answer 28

- requires large stretches of DNA | - homologous DNA is recombined with chromosomal DNA

Question 29

The Rec system

Answer 29

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

Question 30

RecBCD

Answer 30

- 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

Question 31

RecA

Answer 31

- 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.

Question 32

RuvAB proteins

Answer 32

- mediate branch migration - displaces more of recipient with donor strand - formation of Holliday junctions

Question 33

RuvC

Answer 33

- 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.

Question 34

Holliday resolution

Answer 34

- repair all mismatches - now new version of the gene.

Question 35

DNA exchange with plasmids

Answer 35

- 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.

Question 36

plasmid maintenance strategies

Answer 36

- high copy number - partitioning genes - resistance markers - addiction modules

Question 37

relaxed plasmids

Answer 37

- 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 -

Question 38

stringent plasmids

Answer 38

- only replicate when chromosome does. | - low copy number

Question 39

partitioning genes

Answer 39

- 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.

Question 40

resistance markers

Answer 40

- resistance genes give cell advantage | - in antibiotics must keep plasmid to encode resistance

Question 41

addiction module

Answer 41

- 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.