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Applied Genetics > Plasmid structure and replication > Flashcards

Plasmid structure and replication Flashcards

1
Q

how do bacterial plasmids form

A

self replicating

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2
Q

where is bacterial plasmids

A

Extrachromosomal not part of genomal material

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3
Q

are plasmids needed for bacteria survival

A

Dispensable to cells (in general bacteria can survive without plasmids)

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4
Q

why do some bacteria have plasmids

A

Carry functional genes – selective advantage

in some environments

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5
Q

what are the forms of plasmids

A

ds circular DNA (CCC, covalently closed circular)
ds linear DNA (Streptomyces)
ss circular DNA (Myxococcus)

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6
Q

what is the GC content % like in bacterial plasmid compared to host

A

% GC may be different to host (acquired not evolved)

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7
Q

what is cryptic bacterial plasmid

A

no known function, do not change the phenotype of the host cell

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8
Q

what is the phenotypic bacterial plasmid

A

gives the cell an additional property e.g antibiotic-resistance

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9
Q

what are phenotypic plasmids

A

Lots of genes held on plasmids that are not needed by the cell all of the time

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10
Q

what is catabolic gene - bacterial plasmid

A

using energy sources e.g. toulene degredation
Catabolic genes “break down” something. E.g. toluene - a colourless liquid hydrocarbon present in coal tar and petroleum and used as a solvent and in organic synthesis – harmful to animals/humans

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11
Q

what is antibiotic resistance genes - bacterial plasmid

A

fighting off competitors

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12
Q

what is virulence genes - bacterial plasmid

A

accessing new nutrient sources

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13
Q

how can plasmids be moved

A

Conjugation, Mobilisation
Transformation
Transduction

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14
Q

what is bacterial movement important for

A

bacterial evolution

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15
Q

how can plasmids be exchanged

A

Plasmids can be exchanged between cells to move these traits between cells

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16
Q

which cells can plasmids pass into

A

Plasmids can be exchanged between cells, not just cells of the same type, they can pass bacteria to different types of bacteria – move traits between cells

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17
Q

what is the physical structure of plasmids

A

Most commonly used plasmids are covalently closed circular (CCC) molecules
all bases are joined by phosphodiester bonds
B-form DNA there are 10.4 bp per turn of the molecule
This is termed “Relaxed” DNA

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18
Q

what is DNA like inside cells

A

supercoiled
tightly wound/twisted DNA with compact structure
Compact so it fits in the cell
Extent of supercoiling also helps regulate gene expression

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19
Q

what causes relaxed DNA to untwist

A

DNA gyrase

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20
Q

what causes supercoiled DNA to twist

A

DNA topoisomerases

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21
Q

what does DNA gyrase do

A

essential bacterial enzyme that catalyzes the ATP-dependent negative super-coiling of double-stranded closed-circular DNA

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22
Q

how is DNA supercoiling regulated

A

enzymes DNA gyrase and topoisomerase can introduce fewer or more twists into DNA structure

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23
Q

what is positive supercoiling

A

Positive supercoiling is a force on DNA in right hand direction, same direction of DNA double helix

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24
Q

what does supercoiling cause

A

causes DNA to form a more compact structure

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25
what does enzyme DNA gyrase or topoisomerase II do
enzyme DNA gyrase or topoisomerase II functions to add negative supercoils to bacterial DNA
26
what does DNA gyrase bind to in supercoiling, effect
DNA gyrase binds to circular, supercoiled DNA molecule | alleviates one positive supercoil. Gyrase first introduces a double-stranded break in DNA
27
what happens when segment of DNA once broken in DNA supercoiling
segment of DNA passes through break to opposite side of gyrase protein movement of DNA needs ATP hydrolysis by gyrase, and introduces a negative supercoil into DNA molecule
28
how is DNA gyrase released
break in strands repaired, gyrase is released from DNA
29
what effect does DNA gyrase do
DNA molecule with one positive supercoil now has one negative supercoil
30
how does plasmid DNA separate from chromosomal DNA
supercoiling of plasmid DNA allows us to separate it from chromosomal DNA by physical methods e.g. CsCl (Cesium Chloride density gradient centrifugation) density gradient
31
what happens to chromosomal DNA during cell lysis
Chromosomal dna is usually broken into fragments during cell lysis, but plasmids are usually small enough to be isolated intact in supercoiled form
32
what is CsCl
Cesium Chloride density gradient centrifugation
33
how do plasmids move in agrose gel
On agarose gels, supercoiled DNA runs faster than relaxed (or open circular) DNA
34
why does CCC DNA look smaller than relaxed DNA
Compact structure of CCC DNA makes it appear smaller than relaxed DNA
35
how do we know if DNA was cut from supercoiled
If try to cut DNA and doesn’t work will stay supercoiled and will therefore migrate differently on the gel to one that was cut
36
what is a replicon
segment of DNA to be replicated and the proteins needed in the process. Each replicon contains an origin of replication (oriV)
37
what is replicon responsible for
copy number incompatibility host range
38
what is oriV
plasmid origin of replication
39
what does plasmid replication require
some plasmid encoded genes and some chromosomally encoded genes
40
what is copy number in bacterial plasmids
how many copies of a plasmid there are in the cell
41
what is incompatibility in bacterial plasmids
whether certain plasmids can live together, if they’re compatible
42
what is host range in bacterial plasmids
which host cells can this plasmid live in, can survive in all, need certain features in host cell, some plasmids can be recognised by more hosts than others
43
what are the basic features of plasmids
phenotypic genes cryptic genes oriV
44
what are phenotypic genes
can give cell selective advantage
45
what are cryptic genes
no phenotype can be seen
46
what is oriV
origin of replication, allows the host cell to copy the plasmid DNA
47
what type of replication is DNA
semiconservative – half (semi) of the original DNA molecule is conserved during replication
48
what are the types of semiconservative replication
theta form | rolling circle replication
49
what is theta form replication
Used for replicating the bacterial chromosome and many plasmids
50
what is rolling circle replication (RCR)
Used to replicate some plasmids, bacteriophage genomes, and some circular RNA genomes of viroids
51
what happens in theta form replication
Unwinding of DNA DNA helicase remove supercoiling = single stranded DNA End up with a bubble, replication fork(s) Forms two double stranded products
52
what is bidirectional
If have two replication forks
53
what is unidirectional
If have one replication fork
54
which replication is fastest
Is quicker to copy if have two replication forks | bidirectional
55
what does primase do
makes each RNA primer
56
what happens in bidirectional DNA replication initiation
Form a bubble Proteins that bind to ssDNA When unwound by DNA gyrase, bubble have two pieces of ssDNA, want to stabilise and protect the ssDNA Primase comes in, the primers added on both sides and DNA polymerase can extend them to form new strands Leading strand 5’ to 3’ direction Lagging 3’ to 5’ so has to make it backwards in fragments then they are joined
57
what are the key features of theta replication
Requires a circular DNA template Can be uni- or bi-directional Produces 2 circular DNA molecules Requires an RNA primer (gets replication started)
58
how is the bacterial chromosome replicated in theta replication
bacterial chromosome is replicated similarly to plasmid DNA, but they are replicated independently and regulated differently
59
what molecules are needed in plasmid ColE1 replication
RNAII Rnase H RNAI Rop protein
60
what does RNAII molecule do in plasmid ColE1 replication
processed to form primer for DNA synthesis
61
what does Rnase H do in plasmid ColE1
cleaves 3’ end of RNAII molecule in region complementary to oriV sequence (Rnase H is a cellular enzyme that cleaves RNA paired with DNA)
62
what does RNAI do in plasmid ColE1 replication
can pair with RNAII (antisense RNA)
63
what does Rop do in plasmid ColE1 replication
protein stabilises RNAI/RNAII hybrid, thereby preventing cleavage/further replication when Rop is at high levels
64
what happens in plasmid ColE1 replication after cell division
concentration of Rop per cell decreases (cytoplasmic content shared between the 2 cells)
65
what effect does low Rop have in plasmid ColE1 replication
allows plasmid replication to initiate/start again
66
what effect does an increase in plasmid ColE1 replication
Rop increases again | plasmid replication stops again... process repeats
67
when is plasmid copy number constant
under constant physiological conditions
68
what is a high copy number
often 50-100 per cell
69
what is a medium copy number
5-20 per cell
70
what is a low copy number
1-2 per cell, generally large
71
what controls bacterial plasmid copy number
speed of replication initiation at oriV and how quickly it is completed
72
what does bacterial plasmid controls for copy number require
plasmid-encoded functions (rep or cop genes)
73
which plasmids have a low metabolic burden
Small plasmids have high copy number
74
which plasmids have a high metabolic burden
Large have small copy number
75
what can influence bacterial plasmid number
Copy number can be influenced by some of the plasmid encoded genes such as rep or cop and physiological conditions
76
what determines the host range of a plasmid
determined by the ability of the replicon to be recognised and activated by different host cells
77
what must plasmids do to allow their replication
must interact with host cell proteins to allow their replication (e.g. helicase, anchoring proteins, DNA polymerase, primase)
78
which cells do plasmids replicate in
plasmids are only replicated in cells which recognise their oriV site
79
what is the host range for gram-negative bacteria
bacterial plasmids can be either narrow or broad host range
80
what is the host range for gram-positive bacteria
bacterial plasmids are usually narrow host range
81
what are shuttle vectors (plasmids)
broad range plasmids that can replicate in Gram-positive AND Gram-negative bacteria
82
what are the eukaryotic and prokaryotic bacterial plasmid host range
shuttle vectors have two different origins and will replicate in two different cell types
83
what are eukaryotic/prokaryotic shuttle vectors useful for
useful for eukaryotic exogenous protein expression – prokaryotic replication for plasmid generation and amplification in bacteria. Eukaryotic replication for expression of cloned genes in eukaryotic cells
84
what happens in plasmid partitioning when low copy number
One generation to the next is vertical transmission Par genes regulate the process specifically for low copy number plasmids One of the par proteins can polymerase itself and keep adding monomers in till it forms a long chain which pushes the two plasmids apart – so one plasmid in each cell, quite physical process Ensure both daughter cells have a plasmid
85
what happens in plasmid partitioning when high copy number
random partitioning | Don’t need par process, wont necessarily have par proteins
86
what is vertical transmission
plasmids replicate and cells divide, plasmids are passed from generation to generation
87
why may plasmids be maintained
maintained in a population if there is either (a) a selective advantage, or (b) no disadvantage, to the host cell
88
what does cured mean
Cells that have lost a plasmid
89
what is plasmid incompatibility
inability of two particular plasmids to be maintained in the same host cell (competition) Used to classify bacterial plasmids - every plasmid belongs to an incompatibility (Inc) group
90
what causes plasmid incompatibility in low copy number plasmids
related partitioning systems
91
what causes plasmid incompatibility in high copy number plasmids
due to unequal partitioning

Applied Genetics (20 decks)

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