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Phage replication strategies Flashcards

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Biology 101 flashcards
1
Q

what is bacteriophage

A

virus that infects bacteria and replicates inside it

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2
Q

why is bacteriophage studied

A

increase our understanding of fundamental biology

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3
Q

what do bacteriophages provide

A

a way to transfer genes between bacteria: useful in the laboratory and nature

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4
Q

what are bacteriophages exploited for uses

A
  • tools in molecular biology
  • biocontrol agents in agriculture e.g. to control infections
  • vehicles for vaccine delivery
  • detection and killing of pathogenic bacteria (using phages instead of antibiotics)
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5
Q

how are bacteriophages transferred

A

Can transfer between bacteria through conjugation

Can use phages to transfer bacteria

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6
Q

what are the bacteriophages that infect E.coli

A

ΦX174
M13 phage
Phage Lambda
Phage T4

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7
Q

what is ΦX174 (Phi X174)

A

Small and simple structure with no tail

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8
Q

what DNA does ΦX174 (Phi X174) have

A

Circular ssDNA

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9
Q

what is the structure of ΦX174 (Phi X174)

A

Head region

DNA inside of it

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10
Q

what helps ΦX174 (Phi X174) mature

A

proteins help it mature

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11
Q

what is a procapsid

A

immature phage

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12
Q

what happens when ΦX174 (Phi X174) mature

A

lose proteins

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13
Q

what is the structure os M13

A

Filamentous structure

Surrounded by coat proteins

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14
Q

what DNA does M13 phage have

A

circular ssDNA

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15
Q

what is phage lambda structure

A

Complex structure with a head and tail

long (injected into bacterium)

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16
Q

what is phage Lambda DNA

A

linear dsDNA

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17
Q

what is phage T4 structure

A

Complex structure with a head and tail
bigger fibres
has a tail

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18
Q

what is the structure of phage T4 DNA

A

linear dsDNA

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19
Q

what is bacteriophage lytic life cycle

A

Attach to bacteria surface, inject their DNA inside it (only part of the phage e.g. the tail inserts into the host)
Makes lots of copies of itself inside, replicate - copies made inside, cell eventually bursts and the phages are released from inside and these can go on to infect

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20
Q

what do bacteriophages contain

A

either RNA or DNA enclosed in a protein coat

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21
Q

how do bacteriophage infect bacteria

A

by attaching to a receptor on their surface and transmit their nucleic acid inside

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22
Q

what genes do bacteriophages have

A

have early, middle and late genes

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23
Q

what happens to the bacterium when inside the bacterial host cell

A

Once inside the bacterium the nucleic acid is replicated and new phage particles are assembled

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24
Q

what happens when host cell lyse

A

many new phage particles are released

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25
Q

do all phages enter bacteriophage lytic life cycle

A

some phages (such as lambda) may not always enter the lytic cycle and may instead establish lysogeny (stable relationship with the host cell), and M13 phage exits without lysis

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26
Q

what are the bacteriophages sets of genes for

A

allow their replication and subsequent assembly of new phage particles inside a bacterium

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27
Q

what are the bacteriophage replication of ssDNA

A

ΦX174 and M13

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28
Q

what are the bacteriophage replication of dsDNA

A

phage Lambda

phage T4

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29
Q

what does theta form replication require

A

circular DNA template RNA primer BUT IN BACTERIA DONT NEED RNA PRIMER
RecA

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30
Q

what is theta form replication like

A

Can be uni- or bi-directional

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31
Q

what does theta form replication make

A

2 circular DNA molecules

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32
Q

what happens in rolling circle replication

A
  1. concatamers represent linearly joined copies of the plasmid genes
  2. homologous sequences align and RecA promotes recombination and releases free plasmid molecules
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33
Q

in bacteria is rolling circle replication uni- or bi-directional

A

In bacteria some plasmids can use a different method of replication that is NOT bidirectional

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34
Q

how does DNA synthesis of rolling circle replication occur

A

occurs from ‘3 OH of DNA molecule

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35
Q

what is needed to finish replication

A

RecA protein

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36
Q

what replication is used for conjugation and bacteriophage

A

rolling circle replication

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37
Q

how is phage replication prepared

A

entering a bacterium phages need to prepare DNA or RNA for replication – it needs to be converted to the replicative form (RF)
ssDNA must become dsDNA
Linear DNA must become circular

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38
Q

how is ΦX174 and M13 replicated

A

infecting ss viral genome
theta form replciation copies RF dsDNA
replication (bi-directional ori)
gene II nicks +strand
rolling circle replication makes new ssDNA +strand genomes
gene II nicks completed and +strand circularisation of completed strand

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39
Q

what is ΦX174 and M13 replication similar to

A

RCR during plasmid conjugative transfer

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40
Q

what does ΦX174 and M13 genomes contain

A

small and circular ssDNA, and contain overlapping genes

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41
Q

what does the viral particle of ΦX174 and M13 contain

A

+strand

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42
Q

what happens after ΦX174 and M13 infection

A

host cell proteins perform complementary strand synthesis to make dsDNA (RF)

43
Q

what forms the RF copies in ΦX174 and M13 replication

A

Theta form replication is then used to make copies of RF

44
Q

what are the RF copies from ΦX174 and M13 used to make

A

more + strands by rolling circle replication (RCR)

45
Q

what happens to new +strands in ΦX174 replication

A

packaged into capsids (ΦX174) to make new phage particles

46
Q

what happens to new +strands in M13 replication

A

assembled with coat proteins at the membrane (M13) to make new phage particles

47
Q

what are M13 new ssDNA +strands stabilised by

A

binding proteins inside the host cell and transferred to the cell membrane

48
Q

what is the end of the M13 phage particle assembly

A

new viral DNA is extruded from cell and coat proteins are added during extrusion, finally releasing a complete new M13 phage particle

49
Q

what must first happen to Lambda and T4 phages before replication

A

Linear dsDNA must first become circular

50
Q

how is DNA packaged to new phage

A

DNA for packaging into new phage heads is synthesised by rolling circle replication (concatamers method)

51
Q

how are phage capsid forms

A

Phage capsids are pre-formed ready to package the DNA

52
Q

how is the complete phage for Lambda and T4 phage complete

A

phage tails and fibers are then connected to complete the new phage particle

53
Q

how is Lambda DNA circularised

A

Homologous 12 bp single stranded sticky ends to linear genome - Cohesive ends (cos)
entering cell sticky ends anneal to form circular dsDNA = Replicative Form (RF)

54
Q

what happens in the Lambda replication (lytic cycle)

A
  1. Linear dsDNA circularised by ligating cos sticky ends
  2. Theta form replication makes RF copies
  3. Rolling circle replication produces linear concatamers of dsDNA genome with identical ends (cos)
55
Q

how is Lambda phage packaged

A

Linear DNA packaged into empty capsid head until cos site enters
Enzyme localised to head structure recognises cos site and cuts DNA asymmetrically – leaving 12bp overhangs
amount of DNA packaged into head is whatever is between cos sites

56
Q

what is Int

A

integrase

57
Q

what are the two stages of lysogeny

A

integration

excision

58
Q

what happens in lysogeny integration stage

A

Integrase catalyses site-specific recombination between attP and attB 15 bp core sequences to integrate phage lambda into the bacterial chromosome
attP and attB are NOT identical, 2 different sequences are created

59
Q

what are the 2 different sequences that are created by integration

A

attL and attR =

60
Q

what is prophage

A

Integrated phage lambda

61
Q

what happens in excision

A

Int needs Xis to recognise attL and attR and recombine them again

62
Q

what does excision allow

A

induction into lytic cycle

63
Q

what happens if attB is deleted from bacterial chromosome

A

other sequences with similarity to attB may be used at lower frequencies
alternative sites for phage Lambda attachment to bacterial chromosome

64
Q

how does Lambda phage DNA effect cells in lysogeny

A

Phage DNA is maintained and replicated in the host cell without causing damage to the cell

65
Q

how does lambda phage integration occur in lysogeny

A

via semi-homologous recombination

66
Q

what is phage attachment in Lambda lysogeny

A

Phage attachment site is partially homologous to the chromosomal integration site (attP = phage sequence; attB = bacterial chromosome sequence)

67
Q

what does integrase do

A

catalyses the integration recombination

68
Q

what does integrase need

A

needs Xis to catalyse the excision recombination and allow induction of lytic cycle

69
Q

can lysogens form from Lambda mutants

A

can’t integrate may still form lysogens – maintained as extrachromosomal circular DNA – can be unstable though

70
Q

what is the lytic/lysogeny switch

A

Decision to follow either the lytic or the lysogenic pathway is made early after infection (involves early genes)
Determined by the balance between cI and cro proteins

71
Q

if cro more than cI what happens

A

lytic cycle

72
Q

if cI more than cro what happens

A

lysogenic cycle

73
Q

what does the cI repressor do

A

expressed early from PM
switches off PL and PR leading to down regulation of Cro
Promotes LYSOGENIC cycle

74
Q

what does the cro (anti-cl repressor) do

A

expressed early from PR
antagonises cI due to much weaker repression of PL and PR
Promotes LYTIC cycle

75
Q

what are the additional genes that are activated after pathway chosen

A

delayed early genes

76
Q

what happens if lysogeny is followed

A

cII and cIII become expressed, which increase cI repressor expression and help establish and stabilise the lysogenic state

77
Q

what mechanism are lysogeny and lytic cycle

A

Dominance of either cro or cI will further increase their expression: positive feedback

78
Q

what does DNA damage cause

A

induction of phage Lambda

79
Q

what happens when DNA is damaged

A

accumulation of activated RecA (SOS repair)
cI is broken down
Cro accumulates
DNA replication activated for the lytic cycle

80
Q

what are the T4 phage ends like

A

has homologous ends

81
Q

what happens in homologous recombination

A

homologous recombination between them catalysed by RecA allows circularisation to replicative form

82
Q

what happens in phage T4 replication

A
  1. RecA-mediated homologous recombination makes circular RF
  2. Theta form replication makes RF copies
  3. rolling circle replication makes concatamers pf phage genome for packaging new phage particles
83
Q

what happens in phage T4 terminal redundancy

A
  1. RCR makes linear concatemers of dsDNA genomes
  2. genome packaged into empty capsid heads until they become full - more than one whole genome so ends are duplicated
  3. concatamers cut and next portion of DNA packaged into another phage head
84
Q

what does RCR do in phage T4

A

makes linear concatamers of ds DNA genome

85
Q

what is the terminal redundancy

A

170kbp linear packaged genome with 2 ends having identical sequences of 1.6kbp

86
Q

what is circularly permuted

A

in linear form each phage genome may start and end with different genes

87
Q

what are the common features to all 4 phages

A

dsDNA circular
need to be in a host cell to replicate
theta form - initial copies of their genome
RCR

88
Q

what does cro protein do

A

action promotes phage Lambda lytic cycle

89
Q

what does int protein do

A

catalyse recombination of attB and attP

90
Q

what does cI (c1) protein do

A

action promotes phage Lambda lysogenic cycle

91
Q

what does Xia protein do

A

assists Int to catalyse recombination of attL and attR

92
Q

what does RecA protein do

A

accumulates upon phage Lambda induction in response to DNA damage, promotes DNA recombination

93
Q

what is cos

A

unpaired gene sequence found at both ends of Lambda phage genome

94
Q

what are the bacteriophage applications

A

phage display
transduction
phage-based therapies

95
Q

what are the useful applications in phage display - M13

A

Protein-protein interactions
Drug discovery
Monoclonal antibody discovery

96
Q

what happens to the phage genes to be packed into M13 cloning vectors

A

Some phage genes can be deleted and other genes can be cloned in, which will be packaged into phage particles

97
Q

what happens to the cloned sequences - M13 cloning vectors

A

cloned sequences are fused to capsid protein resulting in their display on the surface of the phage particle

98
Q

what is biopanning used for

A

identify phage peptides with desired binding properties

99
Q

what is the phage display selection cycle

A

phage displaying antibody
selective binding using an immobilised antigen
wash off unbound phage
elute bound phage
amplification of phage in E. coli
next screening round - repeat for enrichment

100
Q

what are the two types of transduction

A

generalised

specialised

101
Q

what can phages mediate between bacterial cells

A

horizontal gene transfer between bacterial cells

102
Q

what is generalised transduction

A

Segment of DNA is carried from one bacterial cell to another by a bacteriophage
Bacteriophage attaches to bacterial host cell and injects its nucleic acid
Phage enzyme made that breaks down host DNA to smaller fragments
Phage DNA is replicated, phage coat proteins are made
During formation of the mature phage particles, a few phage heads may surround fragments of bacterial DNA instead of phage DNA
Phage particle carrying bacterial DNA infects another cell, transferring bacterial DNA to the new cell, when bacterial DNA introduced into new cell, can become integrated into the bacterial chromosome, so transferring genes to recipient
Cell then multiplies

103
Q

what is specialised transduction

A

transfer of a few species genes using a phage
Phage injects DNA into bacteria host cell
This integrates into a specific site in the host chromosome
When an E.coli culture carrying the Lambder phage is induced, phage particles are produced
On rare occasions part of the bacterial DNA is taken that is attached to the phage DNA and some phage DNA is left behind. The phage are ineffective as they do not carry the whole phage genome, but can still infect other cells
The defective phage can attach to another bacterial cell and the DNA can be injected and integrated into the new host chromosome
Only bacterial genes located near the integration site can be transduced

104
Q

what is phage therapy an alternative to

A

antibiotics

Applied Genetics (20 decks)

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