Viral Genetics

All organisms—plants, animals, fungi, and bacteria—are

infected by viruses.

virus is a simple replicating structure

made up of nucleic acid surrounded by a protein coat
 Viral Genetics

Viruses have simple structure comprising usually

nucleic acid packed into a protein head.

They lack a metabolic machinery for isolated

multiplication and must invade a host cell in order
to reproduce.

This parasitic lifestyle gives rise to some

interesting reproductive life cycles.
 Viral Genetics
Bacteriophages (phages) have played a central role
in genetic research since the late 1940s.

They are ideal for many types of genetic research

because they have small and easily manageable
genomes, reproduce rapidly, and produce large
numbers of progeny.
 Viral Genetics
Bacteriophages have two alternative life
cycles:

the lytic and the lysogenic cycles.

 Viral Genetics lytic cycle
In the lytic cycle, a phage attaches to a receptor on the
bacterial cell wall and injects its DNA into the cell.

Inside the host cell, the phage DNA is replicated,

transcribed, and translated, producing more phage DNA
and phage proteins.
 Viral Genetics lytic cycle
New phage particles are assembled from these
components. The phages then produce an enzyme that
breaks open the host cell, releasing the new phages.

Virulent phages reproduce strictly through the lytic cycle

and always kill their host cells.
 Viral Genetics lysogenic cycle
The lysogenic cycle begins like the lytic cycle but, inside
the cell, the phage DNA integrates into the bacterial
chromosome, where it remains as an inactive prophage.

The prophage is replicated along with the bacterial DNA

and is passed on when the bacterium divides.

Certain stimuli can cause the prophage to dissociate from

the bacterial chromosome and enter into the lytic cycle,
producing new phage particles and lysing the cell.
Viral Genetics
 Viral Genetics viral structure
A virus is an obligate intracellular parasite.
It must invade a host cell in order to reproduce.
An isolated virus can not replicate itself or carry on
metabolism because it lacks many of the enzymes and
structures necassary for reproduction, protein synthesis
and ATP generation. Therefore it invades and takes
control of host’s metabolic machinery in order to multiply.

Viruses are extremely small ranging in 20 to 14000 nm in

length.
 Viral Genetics viral structure
Viruses come in a great variety of
shapes and Sizes .
Some have DNA as their genetic
material, whereas others have
RNA
The nucleic acid may be double
stranded or single stranded, linear
or circular.
Not surprisingly,
viruses reproduce in a number of different ways.
 Viral Genetics viral structure
The protein coat or capsid surrounds the nucleic acid and
protects it from the environment.

A capsid is composed of repeating units called capsomeres.

The protein making up the capsomere is determined by the

viral nucleic acid.

A virus may also be covered by an envelope composed of

lipids, proteins and carbohydrates.

The envelope may also have spike projecting from the

surface.
 Viral Genetics host range
A virus can only infect and reproduce within certain host cells.

Viruses identify their hosts by specific receptor molecules on

the outside of the host cells.

Some viruses have broad host ranges and can infect different
types of cells.

Other viruses have extremely narrow ranges and can infect

only very specific cells.
 Viral Genetics general life cycle of virus
General life cycle of virus can be described:

Recognition of host
Genetic material enters host
Replication using host nucleotides
Protein synthesis using host enzymes, ribosomes,
tRNA, ATP etc.
Self assembly of capsids and packaging of
genome.
Release from host
 Viral Genetics general life cycle of virus
The Phage T4 and the phage lambda both infect E.
Coli.

The phage T4 mutliply by the lytic cycle and kills the

host cell.

Whereas the phase lambda is multiplied by lysogenic

cycle and doesn’t kill the host cell. In lysogeny, the
phage DNA remain latent in the host cell until it
breaks out into a lytic cycle.
 Viral Genetics lytic cycle
Attachment
Penetration
Biosynthesis
Maturation
Release
 Viral Genetics lytic cycle
Attachment:
T4 phage virus uses tail fibres to attach to complementary
receptor sites on E. Coli cell wall.
Weak chemical bonds form between the attachment and
receptor site adhering the virus to the host cell.

Penetration:
T4 injects it’s DNA into E. Coli by releasing the enzyme, phage
lysozyme, which breaks down a portion of the cell wall.
T4 contracts it’s tail sheath, drive viral DNA into bacterial cell.
Capsid remains outside the bacterial cell.
 Viral Genetics lytic cycle
Biosynthesis:
Host/bacterial protein synthesis is stopped by viral – caused -
degradation of host (bacterial) DNA & interference of host
transcription and translation occurs.
T4 uses host DNA to replicate it’s DNA and uses host
ribosomes, enzymes and amino acids to make it’s enzymes
and proteins.
During biosynthesis, no complete phage is found in the host
and it is called the eclipse period.
Maturation:
Spontaneous assembly of capsids and packaging of DNA
inside the head takes place & tails fibers joins the complex.
 Viral Genetics lytic cycle
Release:
The viral enzyme lysozyme breaks cell wall of E. Coli.

The E. Coli dies.

Bursting time is the period from attachment of virus to

bacterial cell wall to lyses and release of the new phage
particles which ranges from 20-40 minutes.

The number of viruses released from bacterial cell is called

bursting size which ranges from 50- 200 viruses.
 Viral Genetics lysogenic cycle
When lambda phage enters a lysogenic cycle, it’s DNA
recombines with E. Coli’s chromosome.

The inserted phage DNA is called a prophage.

Every time the bacterial chromosome is replicated, prophage

DNA is replicated as well.

The prophage can remain latent in the bacterial chromosome

for many generations.

A spontaneous event at any time may cause the virus to break

out of it’s latent state and enter the lytic cycle.
 Viral Genetics Techniques to study
Viruses reproduce only within host cells; so
bacteriophages must be cultured in bacterial cells.

To do so, phages and bacteria are mixed together and

plated on solid medium on a petri plate.

A high concentration of bacteria is used so that the

colonies grow into one another and produce a continuous
layer of bacteria, or ―lawn,‖ on the agar.
 Viral Genetics Techniques to study
An individual phage infects a single bacterial cell and
goes through its lytic cycle.

Many new phages are released from the lysed cell

and infect additional cells; the cycle is then repeated.
 Viral Genetics Techniques to study
The bacteria grow on solid medium; so the diffusion
of the phages is restricted and only nearby cells are
infected.

After several rounds of phage reproduction, a clear

patch of lysed cells, or plaque, appears on the plate.
Viral Genetics Techniques to study
 Viral Genetics Techniques to study
Each plaque represents a single phage that multiplied
and lysed many cells.

Plating a known volume of a dilute solution of phages on

a bacterial lawn and counting the number of plaques that
appear can be used to determine the original
concentration of phage in the solution.
 Viral Genetics Gene mapping in bacteriophages
For mapping genes in bacteriophages,

Crosses are made between viruses that differ in two

or more genes, and recombinant progeny phages are
identified and counted.

The proportion of recombinant progeny is then used

to estimate the distances between the genes and
their linear order on the chromosome.
 Viral Genetics Gene mapping in bacteriophages
Rates of recombination were examined between genes in
two strains of the T2 bacteriophage that differed in plaque
appearance and host range (Hershey and Rotman,
1949).
 Viral Genetics Gene mapping in bacteriophages
One strain was able to infect and lyse type B E. coli
cells but not B/2 cells (normal host range, h) and
produced an abnormal plaque that was large with
distinct borders (r).

The other strain was able to infect and lyse both B

and B/2 cells (mutant host range, h) and produced
normal plaques that were small with fuzzy borders (r).
 Viral Genetics Gene mapping in bacteriophages
Hershey and Rotman crossed the h+ r- and h- r+
strains of T2 by infecting type B E. coli cells with a
mixture of the two strains.

They used a high concentration of phages so that most

cells could be simultaneously infected by both strains.

Homologous recombination occasionally took place

between the chromosomes of the different strains,
producing h+ r + and h- r - chromosomes, which
were then packaged into new phage particles.
 Viral Genetics Gene mapping in bacteriophages
When the cells lysed, the recombinant phages were
released, along with the nonrecombinant h+ r- phage and
h- r+ phage.

The genotypes of these progeny phages could

therefore be determined by the appearance of the
plaque.
 Viral Genetics Gene mapping in bacteriophages

In this type of phage cross, the recombination

frequency (RF) between the two genes can be
calculated by using the following formula:
 Viral Genetics Gene mapping in bacteriophages

Recombination frequencies can be used to determine the

distances between genes and their order on the phage
chromosome
Viral Genetics Gene mapping in bacteriophages
Viral Genetics Gene mapping in bacteriophages
 Viral Genetics Transduction: Using phages to
map bacterial gene
Three mechanisms of gene transfer were identified:
conjugation, transformation, and transduction. Let’s take a
closer look at transduction, in which genes are transferred
between bacteria by viruses.

In generalized transduction, any gene may be transferred.

In specialized transduction, only a few genes are

transferred.
 Viral Genetics Transduction: Using phages to
map bacterial gene
Study of Lederberg and Zinder in 1952 on Salmonella
typhimurium, mixing the strain of phe+ trp+ tyr+ met-
His- with a strain that was phe- trp- tyr- met+ his+

and plated them on minimal medium. A few prototrophic

recombinants (phe+ trp+ tyr+ met+ his+) appeared,
suggesting that conjugation had taken place.
 Viral Genetics Transduction: Using phages to
map bacterial gene
However, when they tested the two strains in a U-shaped
tube similar to the one used by Davis, some phe+ trp+ tyr+
met+ his+ prototrophs were obtained on one side of the
tube.

This apparatus separated the two strains by a filter with

pores too small for the passage of bacteria; so how
were genes being transferred between bacteria in
the absence of conjugation?
 Viral Genetics Transduction: Using phages to
map bacterial gene
The results of subsequent studies revealed that the agent
of transfer was a bacteriophage.
Viral Genetics Transduction: Using phages to
map bacterial gene
Viral Genetics Transduction: Using phages to
map bacterial gene
 Viral Genetics Transduction: Using phages to
map bacterial gene
Genes can be transferred from one bacterium to other
through generalized transduction.
Viral Genetics Transduction: Using phages to
map bacterial gene
Viral Genetics Transduction: Using phages to
map bacterial gene