Principles of Genetics Lecture 16

Phage and Viruses

Phage and viruses have used similar strategies for infection and replication in
hosts
They both show host specificity
They both use host machinery for their propagation, to reproduce as they
dont have all the genes required for independent growth
They can be temperate (latent) or virulent
They can have DNA or RNA as their genetic material
Single stranded mammalian RNA viruses are also known as retroviruses
Viruses can be as diverse as phage in terms of their various shapes, genetic
material and genome

Single stranded RNA viruses

Single stranded mammalian RNA viruses are also known as retroviruse

Retroviruses differ dramatically from ssRNA phages

The retroviral RNA encodes three genes, and the termini consists of long
terminal repeats (LTRs). The 3 genes consist of: gag responsible for diverse
functions in the control of the host, pol codes for polymerase which serves
to replicate viral genome and env which is involved in the function of the
envelope protein, making the envelope for the mature virus. These 3 genes
are then framed by LTRs.

Reverse transcriptase (RNA dependent DNA polymerase) produces a

complementary DNA strand from the template RNA. Following the synthesis
of the complementary DNA strand, the degradation of the original RNA strand
allows for the production of the second complementary DNA strand
synthesised by DNA dependent polymerase which the host provides, the
dsDNA is then integrated into host DNA.

The dsDNA form (RF) inserts at random locations into the host genome and
the inserted viral DNA is called a provirus, these viruses are often called slow
viruses (latent) because upon infection, they show no symptoms but then
leads to sudden increase in severity of condition

When it is activated, the provirus leaves the latent state. The genes are
expressed to make multiple produces. Transcription also occurs to produce
copies of ssRNA that can be packaged into progeny heads.

Virus of this type are often called slow viruses as inserts they no longer show
the acute phase of infection, but can re appear if they excise and undergo the
rest of the infectious cycle, an example being HIV. Extrinsic factors will
influence when the virus will express its virulent characteristics, just like there

are extrinsic factors that determine when prophages hop out of the host
genome and undergo the lytic cycle.
Retrotransposons may be defective retroviruses
Transposons are DNA elements that are often similar and scattered throughout the
genome, it is suspected that retrotransposons may have been derived from
retroviruses.

Summary

Phage can exist in a number of different forms and their mode of replication
reflects these differences
dsDNA phage can be either virulent or temperate
ssDNA phages require a dsDNA replicative intermediate
ssRNA phages have dsRNA replicative intermediates and infect only male
bacteria
The same is true of mammalian viruses
Both dsDNA and ssRNA viruses exist but the replication of ssRNA viruses is
VERY different from ssRNA phage requiring a dsDNA intermediate.

Cloning Vectors and Gene Therapy

Phage vectors have been used, phage, cosmid and artificial chromosomes. Viral
vectors are also used in gene therapy techniques. Such means are used to insert
desired DNA into a phage and into viruses which then multiples the copies of the
target gene.
Cloning Vectors
All genes associated with the Lysogenic cycle are dispensable, thus their removal
doesnt influence the phages ability to replicate viably. If ou were to rid the phage
of all these genes, the phage would simply proceed down the lytic pathway but
remain to be viable.

packages DNA by recognising COS sites

Phage assembly can occur in vitro and does not have to proceed in a
bacterial cell

As a result, its possible to package foreign DNA flagged by COS sites at each
terminal and then have them packaged into phage heads, using the virus as a
vector.
Phage genetics

Phage are haploid (for the most part, see T4)

They have no nuclei and do NOT undergo mitosis or meiosis and so Mendels
laws dont apply to these organisms.

However recombination and complementation are both features of phage

that allow them to be used like any other genetic organism.
BUT to do genetic crosses we need to have genetic variation.

Genetic Variation in Phage - Unconditional and conditional mutants

Unconditional - they exhibit the phenotype all the time, regardless of the condition

Plaque morphology mutants - phage that have lost the ability to form
lysogens show plaques that do not have any lysogens growing in them; that
is they have lost their turbidity. Rapid lysis mutants (r-) of phage T4 these
mutants create a plaque on E. coli B that has a very sharp edge as compared
with a wild type fuzzy edged plaque. This type of mutant is an example of a
host range specific mutants. Rapid lysis mutants cant infect e. coli K strains.
T4 X e. coli B = wildtype plaque
T4 rapid lysis X e. coli B = rapid lysis
plaque
Wildtype X e. coli K = wildtype
T4 rapid lysis X e. coli K = nothing

clear plaque mutants

lysogen negative mutants

Conditional

Temperature-sensitive mutants = grow at 37C but not 42C - heat sensitive grow at 37C but not at 32C - cold sensitive, its possible that this may be
due to a single amino acid change thus affecting the conformational shape of
the protein at certain temperatures

Host range specific mutants sometimes it requires the host to have a

particular phenotype for it to survive, for example, the rII mutant phage are
unable to grow on e. coli K

Grow on E.coli B but not on E.coli K - r mutants

Grow on suppressor+ strains but not suppressor- strains. E.g. nonsense

suppressors

These are amber and ochre mutants.

Conditional mutants will grow under one set of condition known as the permissive
conditions. These mutants will not grow under another set of conditions known as
the non-permissive or restrictive conditions.

Gene mutations/changes that lead to the blockage of lysogenic cycle

4 causes of constitutively lysogenic bacteria
Loss of function mutation involving c1, thus c1 cant bind effectively to suppress
lytic genes, hence the phage is constitutively lytic
Mutated int gene, cant integrate phage DNA into the host DNA thus cant undergo
the lysogenic cycle
Mutation of the attachment site for the phage genome. A mutated attP site prevents
the occurrence of the effective recombination event that leads to integration of the
phage DNA into that of the host
C1 binding site on DNA is mutated and non functional, this is the equivalent of
knocking out c1
Complementation
Complementation suggests that the mutations are on different genes, so what you
do is set up experiments in which different mutants are mixed and plated at
restrictive conditions for both mutants and if plaques are observed, then
complementation has occurred because that would suggest that one mutant has
compensated the defects for another. Hence it may be concluded that the
mutations are on different genes. If no plaques are observed, then there has been
no complementation supporting the conclusion that the mutations are on the same
gene.
When setting up these experiments, we aim to minimise the effect of
recombination, we want little or no complementation.
Coinfection of host cell is ensured by using a high multiplicity of infection usually
5X as many phages as bacterium, making sure that each bacterium is infected by at
least one of each mutant. Coinfection creates a diploid state for the phage, if the
phage carries mutations in different genes, then within this cell all of the
components to make new phage will be present.
Complementation tests can identify deletion mutants a certain mutation which
complement 2 or more other mutations which are suggested to be on different
genes may be a deletion. In such a case, the results also indicate that these genes
are likely to be close to each other on the chromosome (or the deletion is extremely
large).

Recombination in phage
Whilst the complementation test tells you how many genes are involved,
recombination indicates how far genes are from one another regardless of whether
theyre on the same gene or not.
Recombination involves:

Coinfection of host cell using a high multiplicity of infection (MOI = 5 of each

parent) thus each bacterium is infected with at least one of each type of
mutant
Coinfection creates a diploid state for the phage
This permits recombination between two phage DNA molecules with different
mutations
The recombination event if it occurs will create 2 distinct phage DNA
molecules from those of the parents and thus the progeny phage will have
different genotypes to the parents and you are scoring the number of these
different phenotypes.
Recombination can occur early or late and even more than once

Mechanics of a cross

Coinfect bacteria with 2 mutants

Collect progeny phage
Plate progeny onto bacteria to score for recombination and total phage

RF = 2 X (WT recombinant progeny) / total progeny

Refer to case example at the end of lecture 16 lecture notes.

Recombination frequency is a measure of distance. If 2 genes are close then

RF is lower, clearly being able to assay for very low numbers of
recombinations means that finer maps can be constructed.