Genetics of Viruses

• Life cycles of viruses & bacteriophage

• Mechanisms of gene transfer
• Recombination mapping in viruses using phage mutants
- recombination & deletion mapping
- one-factor & two-factor crosses
- models of genetic recombination

1
 Genetic Analysis of Bacterial Viruses
Bacterial viruses = Bacteriophages
Phages are easy to analyze genetically.
Reasons:
1. Phages have small chromosomes.
2. Phages replicate rapidly, during which they undergo frequent
recombination events.
3. Easy to examine very large populations and therefore easy to
detect rare recombination.

2
Phages are:
- Nucleoprotein complexes in the mature form
which is found extracellularly
- Capsid
• Icosahedral
• Prolate (head-tail)
• Filamentous
- Nucleic acid component is either DNA or RNA
- DNA viruses: linear duplex DNA or circular single-
stranded DNA
- Carry between 3 – 170 genes 3
The life cycle of bacteriophage T4. 4
An electron micrograph of an E. coli cell infected with T4.
5
 Two Main Types of Viral Infection
1. Virulent phages – Lytic Cycle of Infection
e.g. T4; fX174 & R17 phages

2. Temperate phages
Lytic Cycle of Infection
OR
Lysogenic Cycle of Infection
e.g. lambda phage integrates into the host genome
(chromosome) à becomes a lysogen

6
 Third Type of Infection
Non Lytic
e.g. M13 phages replicate without destroying the host
cell and are released (extruded) from the cell without
damaging the cell envelope.

7
Non Lytic

A cutaway diagram of the influenza virion. HA and NA spikes are embedded in a lipid bilayer that forms
the virion’s outer envelope. Matrix protein M1 coats the under side of this membrane. The virion core
contains eight single-stranded RNA segments that comprise its genome in complex with the proteins NP,
PA, PB1, & PB2 to form helical structures named nucleocapsids.
8
Life cycle of Influenza viruses

9
The development of new strains of flu viruses

Antigenic Drift Antigenic Shift 10

The life cycle of bacteriophage T4.

11
The formation of phage plaques on a lawn of bacteria
Bacterial lawn Bacterial cells
on petri plate

Phage
capsid

Phage
DNA

plaque Each infected

cell lyses and
releases phages
Infected
cell

that infect
nearby cells. Phages

Nearby cells lyse,

infecting more cells.
Infected cell lysing and releasing
new phages that infect nearby cells

Plaque

Process
continues.

Plaque is a clear area

where the bacterial lawn
has been destroyed. 12
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
Schematic illustration of the plaque
assay for bacteriophage analysis.
Serial dilutions of a bacterial culture
infected with bacteriophages are first
made. Three phage dilutions (10-3, 10-5,
& 10-7) are analyzed using the plaque
assay technique. In each case, 0.1ml
of the diluted phage is used. Each
plaque represents the initial infection
of one phage particle.

13
 Eclipse Maturation
Adsorption period

Early Protein
Relative virus enzymes Nucleic coats
count (plaque- acid Cell lysis is
forming units, induced by a
pfu) virus-
encoded
Virus added Assembly enzyme
and release (lysozyme)
Latent period

0 30 60
Time

The one-step growth curve of virus replication. This graph displays the results of a single round of viral multiplication in a population of cells.
Following adsorption, the infectivity of the virus particles disappears, a phenomenon called eclipse. This is due to the uncoating of the virus
particles. During the latent period, replication of viral nucleic acid & synthesis of protein coat occurs which is followed by the maturation
period, where viral nucleic acid & proteins are assembled into mature virus particles. At this time, if the cells are broken up, active virus can
be detected. Finally, release occurs, either with or without cell lysis. The timing of the one-step growth cycle varies with the virus & host.
With many bacterial viruses, the whole cycle may be complete in 30-60 minutes, whereas with animal viruses 12-24 hours are usually required
for a complete cycle. 14
 Plaque Morphology of r Mutants Plated on Three E.coli Strains
Phage Strain E.coli B K12S K12(l)l

Wild Wild Wild Wild

rI r r r
T4
rII r Wild -*
mutants
rIII r Wild Wild
* A few rII mutants are able to multiply somewhat on K12, producing visible tiny plaques.
r:: rapid-lysis, large plaques
Wild-type: small, fuzzy plaques Wild-type Rapid-lysis

Early studies to determine Maps of phage chromosomes are done by Recombination

Mapping.

Chromosome map = order of genes on chromosome

To observe viral DNA recombination, host cells must be simultaneously infected by at

least two viruses that contain genetically distinct markers.

r mutants (rI, rII and rIII) are classified according to their plaque morphology when
plated on E. coli 12S and E. coli K12(l).
15
 Genetic mapping
Intergenic mapping
gene A gene B
DNA

Distance between gene A and gene B

Intragenic mapping (fine-structure mapping)

gene C (allele 1)

gene C (allele 2)

Distance between 2
mutations in gene C
16
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 Complementation Test
rII strain 1 rII strain 2 rII strain 3 rII strain 4

E. coli K12 (λ) E. coli K12 (λ)

rII strain 1 rII strain 2 rII strain 3 rII strain 4

(gene A is defective, (gene A is defective, (gene A is defective, (gene A is normal,
gene B is normal) gene B is normal) gene B is normal) gene B is defective)
gene A gene B gene A gene B gene A gene B gene A gene B

Coinfect E. coli K12 (λ) Coinfect E. coli K12 (λ)

Plate and observe if Plate and observe if

plaques are formed. plaques are formed.

No plaques Viral plaques

No complementation occurs, because the coinfected cell is unable to make Complementation occurs, because the coinfected cell is able to make normal
the normal product of gene A. The coinfected cell will not produce viral products of gene A and gene B. The coinfected bacterial cell will produce
particles, thus no bacterial cell lysis and no plaque formation. viral particles that lyse the cell, resulting in the appearance of clear plaques.

Noncomplementation Complementation 17
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 Intragenic recombination

E. coli K12 (λ)

gene A gene A

rll mutation rll mutation

Coinfection
gene A

Rare crossover

gene A gene A

Double mutant Wild type

18
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 Intragenic mapping in the rII region
r103 r104

gene A
gene A

Isolate 2 different (noncomplementing)

Phage Strain E.coli B K12S K12(l)l
rII phage mutants, r103 and r104. Mix
the 2 phages together. Coinfect E. coli B.
A new population of phages will be made. Wild Wild Wild Wild
The E. coli B cells will eventually lyse.
rI r r R
rII r Wild -*
rIII r Wild Wild
Phage * A few rII mutants are able to multiply somewhat on K12, producing visible tiny plaques.
E. coli B

Isolate this new population of phages. It will primarily

contain nonrecombinant phages, but it will occasionally
contain intragenic recombinants of wild-type and double mutant
phages (depicted in white and black, respectively).
The phage preparation can contain several billion phages
per milliliter.
Nonrecombinant
phages
Wild-type
phage

Double
mutant
phage

Take some of the phage preparation,

dilute it greatly (10–8 ), and infect
E. coli B. Also, take some of the phage
preparation, dilute it somewhat (10–6 ),
–8
10 10
–6 and infect E. coli K12 (λ).

Phage Plate the cells and observe the Phage

number of plaques. The number
E. coli B of plaques observed from the E. coli K12 (λ)
E. coli B infection provides a measure
of the total number of phages in the
population. The number of plaques
observed from the E. coli K12 (λ)
infection provides a measure of
the wild-type phage produced by
intragenic recombination.

19
66 plaques 11 plaques
Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 r103 r104

gene A
gene A

Isolate 2 different (noncomplementing)

rII phage mutants, r103 and r104. Mix
the 2 phages together. Coinfect E. coli B.
A new population of phages will be made.
The E. coli B cells will eventually lyse.

Phage
E. coli B

Isolate this new population of phages. It will primarily

contain nonrecombinant phages, but it will occasionally
contain intragenic recombinants of wild-type and double mutant
phages (depicted in white and black, respectively).
The phage preparation can contain several billion phages
per milliliter.
Nonrecombinant
phages
Wild-type
phage

Double
mutant
phage

Take some of the phage preparation,

dilute it greatly (10–8), and infect
E. coli B. Also, take some of the phage
10–8 10–6 preparation, dilute it somewhat (10–6), 20
and infect E. coli K12 (λ).
 Isolate this new population of phages. It will primarily
contain nonrecombinant phages, but it will occasionally
contain intragenic recombinants of wild-type and double mutant
phages (depicted in white and black, respectively).
The phage preparation can contain several billion phages
per milliliter.
Nonrecombinant
phages
Wild-type
phage

Double
mutant
phage

Take some of the phage preparation,

dilute it greatly (10–8), and infect
E. coli B. Also, take some of the phage
10–8 10–6 preparation, dilute it somewhat (10–6),
and infect E. coli K12 (λ).

Phage Plate the cells and observe the Phage

number of plaques. The number
E. coli B E. coli K12 (λ)
of plaques observed from the
E. coli B infection provides a measure
of the total number of phages in the
population. The number of plaques
observed from the E. coli K12 (λ)
infection provides a measure of
the wild-type phage produced by
intragenic recombination.

66 plaques 11 plaques
21
 Intragenic mapping in the rII region
r103 r104

gene A
gene A

Isolate 2 different (noncomplementing)

Phage Strain E.coli B K12S K12(l)l
rII phage mutants, r103 and r104. Mix
the 2 phages together. Coinfect E. coli B.
A new population of phages will be made. Wild Wild Wild Wild
The E. coli B cells will eventually lyse.
rI r r R
rII r Wild -*
rIII r Wild Wild
Phage * A few rII mutants are able to multiply somewhat on K12, producing visible tiny plaques.
E. coli B

Isolate this new population of phages. It will primarily

contain nonrecombinant phages, but it will occasionally
contain intragenic recombinants of wild-type and double mutant
phages (depicted in white and black, respectively).
The phage preparation can contain several billion phages
per milliliter.
Nonrecombinant
phages
Wild-type 2×𝑊𝑇 𝑝𝑙𝑎𝑞𝑢𝑒𝑠 (𝐸. 𝑐𝑜𝑙𝑖 𝐾12 𝜆 )
phage
𝐹𝑟𝑒𝑞𝑢𝑒𝑛𝑐𝑦 𝑜𝑓 𝑟𝑒𝑐𝑜𝑚𝑏𝑖𝑛𝑎𝑛𝑡𝑠 =
𝑇𝑜𝑡𝑎𝑙 𝑛𝑜. 𝑜𝑓 𝑝𝑙𝑎𝑞𝑢𝑒𝑠 (𝐸. 𝑐𝑜𝑙𝑖 𝐵)
Double
mutant
phage 2×(11×106)
=
Take some of the phage preparation,
dilute it greatly (10–8 ), and infect 6.6×109
E. coli B. Also, take some of the phage
preparation, dilute it somewhat (10–6 ),
–8 –6 and infect E. coli K12 (λ).
= 3.3 ×10 − 3
10 10

= 0.0033
Phage Plate the cells and observe the Phage
number of plaques. The number
E. coli B of plaques observed from the E. coli K12 (λ)
E. coli B infection provides a measure
of the total number of phages in the
population. The number of plaques
observed from the E. coli K12 (λ)
infection provides a measure of
3.3 recombinants / 1000 phages
the wild-type phage produced by
intragenic recombination.

22
66 plaques 11 plaques
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 Fine Mapping
Map distances are calculated from the frequency of
recombination i.e. number of rII progeny in relation to
total progeny by the following relationship:

No. of rII progeny

Map Units = x 2 x 100
Total no. of progeny

N.B. Total no. of progeny determined by plating on E. coli strain B.

No. of WT recombinants is determined by plating on E. coli
K12.

Factor 2 in Equation (1) compensates for the fact that there

should be 2 types of recombinants in the cross – WT
recombinants & double mutants, which are not detected on E.
coli K12.
23
 Deletion Mutant Crosses (Mapping)
2 1 2 3
1 0 0 +
1 3 2 0 0 0
3 + 0 0

0 + = presence of WT
recombinants
+
0 = absence of WT
0 recombinants

If 2 mutants have deletions that do not overlap, WT recombinants are

produced when they are crossed. 2 mutants that have deletions that
overlap do not produce WT recombinants because there is no way of
compensating for the missing genetic region.
24
 Deletion Mutant Mapping
A

B Point mutant here will give no

recombination with A and B but WT
C recombinants with C, D, or E.

Gene

Deleted region in A deletion mutant

If 2 mutants have deletions that do not overlap, WT recombinants are produced when they are
crossed. 2 mutants that have deletions that overlap do not produce WT recombinants
because there is no way of compensating for the missing genetic region.
Use of deletion mutants for determining the approximate location of a point mutation. Point
mutant is crossed with a family of ordered deletion mutants. On one side of the point mutation
WT recombinants are obtained. On the other side no recombinants are observed. Dividing
line determines location of point mutation.
25
 Deletion Mutant Mapping
Deletion Wild-type recombinants
strain when coinfected with r103?

1272 Deleted region No

1241 Deleted region No

J3 Deleted region No

PT1 Deleted region No

PB242 Deleted region Yes

A 105 Deleted region Yes

638 Del. reg. Yes

A1 A2 A3 A4 A5 A6 B1–B10
gene rIIA gene rIIB
26
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 Deletion Mutant Mapping
Hot spot

Start of
rIIA gene A1a A1b1 A1b2 A2a A2c A2e A2f A2g
A2b A2d A2h1

A2h2
A4d A4c A4b A4a A3i A3h A3g A3f A3e A3a–d A2h3

A4e
Hot spot
A4f

A4g
A5a A5b A5c1 A5c2 A5d A6a1 A6a2 A6b

A6c

B6 B5 B4 B3 B2 B1 A6d

B7
Hot spots Hot spots Hot spots
End of Start of End of
rIIB gene rIIB gene rIIA gene

B8 B9a B9b B10

27
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 One-Factor Crosses Involving One Mutant Type & Wild Type (+)

Parents Progeny

s x + 2050 s 2340 +

Lambda mutants co1 x + 761 co1 707 +

based on differences
in plaque mi x + 923 mi 736 +
morphology
c x + 5900 c 6600 +

co2 x + 2100 co2 1500 +

s = small; mi = minute; c = clear

Results of the above cross infection:

Progeny phenotypes were similar to the 2 parental phenotypes.

Interpretation:
1. WT & mutant grew equally well.
2. No new phenotypes emerged, indicating the original/individual
mutants were single mutants. 28
 Two-Factor Crosses Involving Two Mutant-Type Phage l
Crosses Recombination
Parents Progeny
No. %

I co1,+ x +,mi 5162 co1,+ 6510 +,mi 311 +,+ 341 co1,mi 5.3
459 398 17 25 4.7
720 672 44 46 6.1
co1,mi x +,+ 36 30 795 620 4.4
74 56 1005 956 6.2

II s,+ x +, co1 7101 s,+ 5851 +, co1 145 +,+ 169 s, co1 2.4
s, co1 x +,+ 46 53 1615 1774 2.8

III s,+ x +,mi 647 s,+ 502 +,mi 65 +,+ 56 s,mi 9.5
s,mi x +,+ 1024 1155 13083 13253 7.6

IV s,+ x +,c 808 s,+ 566 +,c 19 +,+ 20 s,c 2.8

V c,+ x +,mi 1213 c,+ 1205 +,mi 84 +,+ 75 c,mi 6.2
VI c,+ x +, co1 6000 c,+ 6000 +, co1 14 +,+ - c, co1* 0.1
VII co2,+ x +,mi 1477 co2,+ 1949 +,mi 109 +,+ 131 co2,mi 6.6

* type c, co1 not distinguishable from c, +. 29

Two Factor Crosses
Results:
1. Approximately equal numbers of either parental phenotype were observed, and
equal numbers of recombinant phenotypes.
2. Each cross involving 2 different factors gave a different value for the
percentage of recombination.
* Assuming that the percentage of recombination is greater (larger) for 2 factors
(markers) that are located farther apart, a genetic map can be derived:

2.6 5.3
s co1 mi
(a)
7.9
Inspection of cross III;
OR the % of recombination
for s & mi = 8.5 favours
2.6 2.7 (a) as the correct order.
co1 s mi
(b)
5.3

30
 Recombination
• Homologous recombination
– DNA segments are similar or identical to each other
– Occurs when chromosomes cross over during meiosis
– Enhance genetic diversity
– Repair DNA and ensure proper segregation of chromosomes

• Site-specific recombination
– Non-homologous DNA segments at specific sites
– Certain viruses integrate their genomes into host cell DNA
– Within genes that encode antibody polypeptides

31
 Homologous Recombination
A A A A

A crossover occurs
between identical
chromatids.

B B B B

(a) Sister chromatid exchange

A A a a A A a a A a Two with
Meiosis is parental
A crossover occurs completed genotype
between homologous to yield 4 B b
chromatids. haploid cells.
A a
B B b b B b B b Two with
recombinant
b B genotype
(b) Recombination between homologous chromosomes during meiosis

Homologous recombination

32
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 Site-specific Recombination Organization of domains in the precursor gene for the k light chain
λ DNA
V1 V2 V3 V 4 . . . . V 300 J3 J4 C
Integrase recognizes J1 J2

RAG1 and RAG2 recognize recombination

the core sequences signal sequences and catalyze the
Integrase
and brings them attP core sequence breakage at the end of a variable domain
and beginning of a joining domain. In this
close together. GC T T T T T T T A C T A A example, it occurs at the end of V78 and
CG A A A A A A A A T G A T
T . . . . V 300 beginning of J2.
J1

V1 V2 V3 V 4 . . . . V 78 J2 J3 J4 C

CTTTTTTAT CT The intervening DNA is lost. NHEJ

G A A A A A A A AGA A A
G T T TT
C proteins catalyze the joining of the
Note: The E. coli
chromosome is
last V domain and first J domain in
attB core sequence
actually much the remaining DNA.
larger than the V1 V2 V3 J4 C
V 4 . . . . V 78 J 2 J3
λ DNA.
The gene is transcribed into a
pre-mRNA starting at the last
Integrase makes 4 cuts E. coli chromosome variable domain.
as indicated by the arrows. 5¢ V 78 J2 J3 J4 C 3¢

The strands are exchanged The region between the first joining
domain and constant domain in the
and then ligated together. pre-mRNA is spliced out.
5¢ 3¢
V 78 J2 C

The mRNA is translated into a

polypeptide containing 1 variable,
1 joining, and 1 constant domain.
NH 2 V 78 C COOH
J2
GC T T T T T T T A C T A A
CG A A A A A A A A T G A T T Two light-chain polypeptides and 2
heavy-chain polypeptides assemble to
form a functional antibody protein.
CTTTTTTAT CT
G A A A A A A A AGA A A
G T T TT
Heavy-chain
C polypeptide

COOH A functional
antibody
COOH made in
1 B cell

λ DNA integrated into

E. coli chromosome
33
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 A B
A B Sister chromatids
Holliday model
Homologous chromatids

a b

A a b B
5¢ 3¢
3¢ 5¢
3¢ 5¢
5¢ 3¢
a b
Both chromatids are nicked at
identical locations.
A B

Nicks

a b
The DNA strands to the left of the nicks
invade the homologous chromosomes and
attach to the strands to the right of the nicks.
A B

Holliday junction

a b © Dr. John D. Cunningham/Visuals Unlimited.

The Holliday junction migrates from left to (b) Micrograph of a Holliday junction
right. This is called branch migration. It
creates 2 heteroduplex regions.
A B

a b
Two heteroduplex regions that
have a few base mismatches
In this next step, the figure is simply redrawn by bending
the ends labeled A and B upwards, and bending the ends labeled
a and b downwards. This makes it look more like a true Holliday End result
junction. The Holliday junction is viewed in two different planes. A A B
A A
A B
The strands are
B B connected to create B
Short
nonrecombinant heteroduplex
The strands that
chromosomes with a region
were originally a b
nicked are broken. short heteroduplex region.
a b
Nonrecombinant chromosomes
b b b with a heteroduplex region

a Viewed in a different plane a

a
(when rotated 180°)
A A A A B
A b
The strands are connected
B B to create recombinant B
The strands that Short
were not originally chromosomes with a short heteroduplex
nicked are broken. heteroduplex region. region
a B

a b
b b b Recombinant chromosomes
with a heteroduplex region

a a a 34
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Holliday Model
This explains the facts about genetic recombination.
1. First step in crossing over requires making single-strand breaks at
identical or nearly identical points in the homologous DNA duplexes.
2. The cut strands migrate and base-pair with homologous regions on
the opposing unbroken DNA strands.
3. The crossover strands become covalently linked to the opposing
strands to form a stable bridge structure. This bridge between the
duplexes is known as the Holliday Junction.
4. Further exchange of the single strands between the interacting
chromosomes is mediated by bridge migration. Bridge migration is
very fluid i.e., it is able to migrate up and down the interacting
duplexes, making and breaking base-pair linkages.
Summary
For this model, heteroduplexes are formed by symmetrical strand exchange
and bridge migration – resulting in different genetic products depending on
whether they are corrected before mitosis or left uncorrected. 35
 Double-strand break model
Double-strand
break
A Z
5¢ 3¢
3¢
3¢
5¢
5¢
Homologous
chromosomes The Szostak double-strand break repair model
5¢ 3¢
a z
Strand degradation occurs at the
double-strand break site to
(1) A double strand cut is made in one duplex, a
A
yield single-stranded ends.

Z
gap flanked by 3’ single strands is formed by the
action of exonuclease.
a z (2) One 3’ end invades a homologous duplex,
displacing a D-loop.
Strand invasion causes
D-loop formation.

D-loop
A Z

(3) The D-loop is enlarged by repair synthesis until

a z
the other 3’ end can anneal to complementary
Gap repair synthesis fills in
the vacant regions.
single-stranded sequences in the D-loop.
A Z
(4) Repair synthesis from the second 3’ end
completes the process of GAP repair, and
a z
Branch migration and resolution
branch migration can take place at the two
can produce recombinant or
nonrecombinant chromosomes.
Holliday junctions. Resolution of the 2 junctions
or by cutting either inner or outer strands leads to 2
A Z A z possible non-crossover configurations and 2
Nonrecombinant Recombinant
possible crossover configurations.
chromosomes chromosomes

36
a z a Z
Crossover site Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.
 Gene Conversion
5¢ 3¢
G G C C
C C G G Strand invasion causes
The top chromosome carries the recessive D-loop formation.
3¢ 5¢
b allele, while the bottom chromosome
Branch migration carries the dominant B allele.

5¢ 3¢

G A C C
b
5¢ 3¢
C T G G
3¢ 5¢ 3¢ 5¢
3¢ 5¢
Branch migration travels
over a region that has a
5¢ 3¢
minor sequence difference. B
B
Heteroduplexes Gap repair synthesis uses the strands
5¢ 3¢
A double-strand break occurs from the dominant B allele to fill in the
within the recessive gene.
G G C C
region.
C G G
T b B
3¢ 5¢

5¢ 3¢

G A C C B B
C C G G
3¢ 5¢ A region adjacent to the double-
strand break is digested away, The intertwined
which eliminates the recessive b allele strands are resolved.
DNA mismatch repair yields
4 possible combinations.
B

G G C C G G C C G A C C G A C C
C C G G C C G G C T G G C T G G

B
B

Both chromosomes carry the B allele.

G G C C G A C C G G C C G A C C
C C G G C T G G C C G G C T G G

Gene No gene No gene Gene

conversion conversion conversion conversion

DNA Mismatch Repair DNA Gap Repair 37

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 Transposition
• First identified by Barbara McClintock in the early
1950s from her classic studies with corn plants
• Involves the integration of small segments of DNA into
the chromosome
– Simple transposition (bacterial and eukaryotic species)
– Replicative transposition (only in bacterial species)
– Retrotransposition (retrotransponson)
• The DNA segments that transpose themselves
– Transposable elements (TEs)/transposons
– Jumping genes
38
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 Transposition
Simple transposition

Transposon

Transposon

• ‘Cut-and-paste’ mechanism
• Widely found in bacterial and eukaryotic species
39
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 Transposition
Replicative transposition
Replication

Transposon

Transposon Transposon

• Replication of the TE and insertion of the newly made copy

into a second site
• Relatively uncommon and found only in bacterial species 40
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 Transposition
Retrotransposition

Retrotransposon
Transcription

DNA
RNA Reverse
transcriptase

Retrotransposon Retrotransposon

• Commonly found in eukaryotic species

• Transposable elements termed retrotransposons
• Reverse transcriptase 41
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 Transposition

Transposase
gene

• Direct repeats (DRs)

› Target-site duplications
Transposase
gene
Antibiotic-
resistance

› Identical nucleotide sequences gene

› Same direction and repeated

› Adjacent to both ends of the elements
• Inverted repeats (IR)
› Identical (or very similar) but run in opposite directions
Transposase Resolvase
gene gene

› Range from 9 to 40 bp in length

• May contain a central region that encodes the enzyme transposase
42
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 Transposition
Transposase
gene

Composite transposons
Transposase Antibiotic- • Contain additional genes that are not necessary for
gene resistance
gene transposition per se
• Confer a selective advantage to the organism under
certain growth conditions
• Prevalent in bacteria
Replicative transposons
Transposase Resolvase
• Similar to insertion sequences except that replicative
gene gene
transposons have a resolvase gene between the
inverted repeats
• Both transposase and resolvase are needed
Retrotransposons
• Categorizes based on their evolutionary relationship to
retroviral sequences (Retroviruses make a DNA copy
Reverse Integrase
transcriptase gene that integrates into the host’s genome)
gene
• Long terminal repeats (LTRs) retrotransposons –
relate to known retroviruses and encode virally related
proteins that are needed for the retrotransposition
process. Typically a few hundred nucleotides in length
Reverse
transcriptase/
endonuclease
• Non-LTR retrotransposons – evolutionarily derived
gene from normal eukaryotic genes. E.g. Alu family of
repetitive sequences is derived from a single ancestral
gene known as the 7SL RNA gene
43
• Approximately 1,000,000 copies
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 Simple Transposition TE— The TE is actually a few hundred to
several thousand base pairs in length.
3¢

IR IR Transposase recognizes the inverted

repeats and cleaves at both ends of
the transposable element, releasing it
from its original site.

Transposase

TE

IR IR
Transposase carries the TE to a new site and
cleaves the target DNA at staggered sites.
A T G C T Target
A A
T C G DNA

The transposable element is

inserted into the target site.
T
A T G C
A A
T C G

IR IR

Transposable
element DNA gap
repair synthesis
T T
A T G C A T G C
A A A A
T C G T C G

IR IR
Transposable
element

Direct
repeats
44
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 Simple Transposition

Transposon

Transposon

45
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 Simple Transposition
Transposition

TE

DNA replication proceeds past the

TE
point where the TE has been
inserted. The top copy of the TE
then transposes ahead of the fork,
where it is copied again.

TE

TE TE

The bottom copy of DNA has 2 TEs.

46
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 Replicative Transposition The gaps are filled in by DNA
polymerase and sealed by DNA
ligase to produce a cointegrant.

Cointegrant
Transposable
+ element
Target
sequence

Target Resolvase catalyzes recombination between

DNA The gaps are filled the 2 elements. This resolves the cointegrant
in by DNA into 2 separate structures, each with a copy
Transposase makes polymerase and of the transposable element.
cuts at the arrows and sealed by DNA
the strands are ligase to produce a
exchanged and cointegrant.
ligated together.

47
 Retrotransposition

RNA
Reverse
transcriptase
Integrase
DNA

Transcription

Ty

Ty Ty Ty
48
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 Transposition
• Greater genetic variability
• Survival advantage such as
antibiotics-resistance gene in
bacterial TEs
• Outcomes are likely to be harmful
• Highly regulated processes
• Occurs only in a few individuals
under certain conditions such as
radiation, chemical mutagens, and
hormones stimulation
49
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Plos One Transposition
The emergence of plasmid-mediated carbapenemases,
such as NDM-1 in Enterobacteriaceae is a major public
health issue. Since they mediate resistance to virtually all
β-lactam antibiotics and there is often co-resistance to
other antibiotic classes, the therapeutic options for
infections caused by these organisms are very limited.

a 55 kb backbone which shared 97% homology with

pEL60 originating from the plant pathogen, Erwina
amylovora in Lebanon and a 28.9 kb variable region.

The 28.9 kb region has a composite transposon-like structure which includes intact or
truncated genes associated with resistance to β-lactams (blaTEM-1, blaNDM-1, ΔblaDHA-1),
aminoglycosides (aacC2, armA), sulphonamides (sul1) and macrolides (mel, mph2). It
also harbors the following mobile elements: IS26, ISCR1, tnpU, tnpAcp2, tnpD,
ΔtnpATn1 and insL. 50
Transposition

51
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)

CRISPR Antiviral Defense System 52

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