Prokaryotic Gene and Sex Exchange

Question 1

Bacteria have 3 main ways of acquiring DNA

Answer 1

transformation, transduction and conjugation

Question 2

Describe transformation

Answer 2

• common amongst gram positive bacteria
• acquisition of DNA directly from the environment
• very powerful for genetic exchange
• acquiring new functions such as antibiotic resistance

Question 3

During transformation, DNA acquired must be

Answer 3

recombined into the host, except if it is a self-replicating plasmid

Question 4

Transformation occurs by

Answer 4

• nicking of one DNA strand
• a single stranded binding protein attaches
• allows cross strand exchange between the DNA chi

Question 5

What nicks the DNA strand during transformation?

Answer 5

an endonuclease

Question 6

What is a splice in transformation?

Answer 6

When the nick is on the external strand of the chi

Question 7

What is a patch in transformation?

Answer 7

when the cut is on the internal strand of the chi

Question 8

Transformation can be monitored in the lab using

Answer 8

a marker that tracks the path of recombination

Question 9

Trp- means a bacteria

Answer 9

can’t grow on Trp

Question 10

Plating cells with no Trp and seeing when they begin to grow proves that

Answer 10

they have taken up some DNA

Question 11

What does measuring DNA uptake require

Answer 11

an auxotrophic marker, or antibiotic resistance system

Question 12

Described induction of transformation in gram negative bacteria

Answer 12

• bacterial cells bind the DNA at the surface and strip it with nuclease
• it enters the cell single stranded
• recombination allows the cell to replicate
• makes the cell very leaky

Question 13

Inducing transformation in gram negative bacteria involves

Answer 13

• electroporation
• surface shielding of the extracellular polysachharide and S protein layers stops external molecules such as toxins, and antibiotics from invading,
• stops immune attack.

Question 14

Describe electroporation

Answer 14

an electric pulse knocks holes in the membrane

Question 15

Give an example of a naturally transformable clade of bacteria

Answer 15

the Streptococci

Question 16

Generalised transduction involves

Answer 16

aquiring DNA from a phage

Question 17

Describe generalised transduction

Answer 17

• a lytic phage enters the cell and replicates
• some phage DNA combines with any part of the chromosomal or plasmid DNA
• creates a transducing particle
• transfers genes from one strain to another, from host to recipient cells

Question 18

Describe the basics of specialised transduction

Answer 18

• requires a transducing phage
• activated by processes such as the LexA SOS cycle

Question 19

Give an example of a transducing phage

Answer 19

lambda phage

Question 20

Describe transducing phages

Answer 20

composed of a phage head and a linear molecule

Question 21

Describe the specifics of specialised transduction

Answer 21

• the transducing phage cyclises in the host, forming a sphericule with adhesive ends
• site-specific endonucleases cut at ATBs
• allows DNA insertion and integration between the galactose and biotin genes, forming a prophage
• prophage can then excise and replicate if the lytic cycle is induced

Question 22

ATBs

Answer 22

• attachment sites in bacterial chromosomes
• complementary to ATPs in the phage

Question 23

Describe rolling circle replication

Answer 23

• a way of making multiple copies of a genome
• used by the lambda phage genome
• catalysis occurs from 5’ to 3’

Question 24

Describe rolling circle replication in lambda phage genomes

Answer 24

can make hundreds of lambda phage genomes as it continuously replicates

Question 25

What is a ‘normal event’?

Answer 25

- induction by the lytic phage - excision - rolling circle replication - transcription and translation of the capsid protein - cell lysis - phage release

Question 26

What is the function of the capsid protein?

Answer 26

encapsulates the DNA

Question 27

Describe a ‘rare’ event

Answer 27

- the excising DNA takes another gene because the excision cut in the wrong place - creates a hybrid phage containing chromosome - phage loses some of its own DNA - only the genes very close to the phage are at risk of becoming hybridised

Question 28

Give an example of phage DNA in loss in a rare event

Answer 28

excision at the junction between ATB and ATP.

Question 29

Hybrid phages are

Answer 29

often defective

Question 30

‘Rare’ phages will therefore have been

Answer 30

mixed in with lots of wild phages

Question 31

What do wild phages provide?

Answer 31

the necessary proteins in transduction

Question 32

How are rare events isolated in the lab?

Answer 32

removal of the wildtype

Question 33

Describe the major constraint acting on transduction

Answer 33

- capsid proteins have a 45-50kb packaging limit - if the package is either too small or too large, the phage head won’t fit

Question 34

Describe conjugation in bacteria

Answer 34

- what causes the ‘sex change’ - transfer of the F plasmid across the conjugation bridge

Question 35

Describe the F plasmid

Answer 35

- contains the genes for the sex pillus - often recombines into the chromosome through insertion sequences - can exist as both plasmid and chromosomal DNA

Question 36

What is the OriT?

Answer 36

the transfer origin of the plasmid

Question 37

Give an example of an insertion sequence for the F plasmid

Answer 37

IS3 (insertion sequence 3)

Question 38

Integration direction of the F plasmid depends upon

Answer 38

orientation of the insertion sequence

Question 39

F episomes

Answer 39

transfer chromosomal DNA

Question 40

Describe wholesale transfer of E. coli

Answer 40

- almost all of its chromosome can be transferred from one strain to another in 100 minutes - between Hfr strains - can be used to show the order of genes in the chromosome by interrupting the experiment at various intervals

Question 41

Describe classical genetic mapping experiments

Answer 41

- landmark - use wholesale transfer

Question 42

Describe archaeal gene transfer

Answer 42

Archaea display a TraG-like homologue for stability - mobile elements have inverted repeats at either end, and composite transposons with 2 sets of insertion sequences at eirther end (L and R)

Question 43

Name an archaeal mobile element

Answer 43

insertion sequence 2

Question 44

Name an example of the composite transposons contained within mobile elements

Answer 44

IS50R and IS50L

Question 45

IS

Answer 45

insertion sequence

Question 46

Conservative transposition involves

Answer 46

- the transposon being excised from the donor and transferring to a new location - the donor has break in it

Question 47

replicative transposition involves

Answer 47

transposon duplication in its original position; it is not lost from donor strain

Question 48

Describe conjugation in E. coli

Answer 48

- E. Coli can only be conjugated by a narrow host range of plasmids

Question 49

Describe broad host range plasmids

Answer 49

can be conjugated by a large host range of plasmids

Question 50

What is necessary for conjugation?

Answer 50

replication origin and machinery recognised

Question 51

Describe some bacteria with a broad range of host plasmids

Answer 51

- Pseudomonas - Rhizobium

Question 52

How can DNA integrity be protected from foreign DNA?

Answer 52

using various genetic elements such as restriction endonucleases which express methyltransferase, or spacers

Question 53

Give an example of a restriction endonuclease used to protect DNA integrity against foreign DNA

Answer 53

- EchoK - EchoR1 - K9 E. Coli's

Question 54

Describe the action of methyltransferase in the protection of DNA integrity against foreign DNA

Answer 54

- methylates the recognition sequence of the DNA, to protect it - non-methylated DNA will be cut, but methylated DNA will not.

Question 55

Describe spacers, that protect DNA integrity from foreign DNA damage

Answer 55

- past viral DNA that has been integrated - adaptive immunity - e.g. CRISPR arrays