Learning Outcomes:

At the end of this lecture, students will be able to: Explain the structure and function of selected phages, particularly in regard to replication mechanisms. Describe how bacteriophage vectors are used to clone DNA Give examples of vectors used to clone long pieces of DNA, and evaluate the strengths and weaknesses of each type

BACTERIOPHAGES
- are viruses that infect bacteria; - simple in structure - DNA (or RNA) molecule with some genes - surrounded by a protective coat (capsid) made up of protein molecules. - 2 main types of phage structure head and tail (eg. ) filamentous (eg. M13)

Structure of T4 bacteriophage

BACTERIOPHAGES
Has 2 developmental pathways: Lytic (productive cycle) Lysogenic (non-productive cycle)

Lytic pathway
Lytic or virulent phages are phages which can only multiply on bacteria and kill the cell by lysis at the end of the life cycle. Infection is a 3 step process:- attachment to bacterium and injection of DNA into the cell - the phage DNA is replicated - new phage particles are assembled and released from the bacterium. Lysis of the host and releasing ~ 100 progeny particles/cell

Assay for Lytic Phage

Plaque assay - Lytic phage are enumerated by a plaque assay. A plaque is a clear area which results from the lysis of bacteria. Each plaque arises from a single infectious phage. The infectious particle that gives rise to a plaque is called a pfu (plaque forming unit).

Lysogenic pathway
Phage DNA inserted into the host chromosome at a specific site The integrated form of phage DNA is called prophage (provirus). The host cell containing a prophage is called a lysogenic bacterium / lysogen Most phage functions are switched off Phage DNA molecule can be retained in the host bacterium for many-many cell divisions. Entrance and exit of the DNA from the chromosome are site-specific genetic recombination events catalysed by the lambda integrase protein. Can be induced to undergo lytic development

Lambda ( ) bacteriophage
Complex, extensively studied virus of E. coli is a typical example of a head and tail Its DNA is a double stranded linear molecule of about 49 kb. The ends of the genomic DNA are single-stranded and are cohesive, i.e. they are complementary to one another. The two cohesive ends - known as cos sites - are 12 nucleotides in length. When the linear DNA is injected into the host, they circularise via the cohesive termini (cos sites). A temperate phage - Lysogenic or temperate phages are those that can either multiply via the lytic cycle or enter a lysogenic cycle

- a temperate phage

 bacteriophage
The cos sites are important during infection cycle because: allow linear DNA injected into host to be circularised act as recognition sequence for an endonuclease to produce complete single genomes

Rolling circle replication

To begin rolling circle replication, a cut is made in a double stranded circle of DNA. This produces a 3'OH and 5' P end . Nucleotides are added to the 3' end to synthesize the DNA, causing the 5' end to pull out of the circle and produce a linear double stranded DNA molecule. When the 3' OH end is extended, the 5' end can be displaced in a manner analogous to the strand displacement reaction. Synthesis on this strand is also analogous to leading strand synthesis. The displaced strand can, in turn, serve as an template for replication as long as a suitable primer is available. Synthesis on this strand is analogous to lagging strand synthesis.

Lambda genome packaging

Genome size 48.5-50 kb Only about 60% of the phage genome is necessary for lytic growth

 All the genes coding for components of the capsid are grouped together in the left hand third of the molecule. Genes controlling integration into host genome are clustered in the middle To the right are genes concerned with gene regulation and immunity to superinfection (N, cro, cI) followed by DNA synthesis (O, P), late function regulation (Q) and host cell lysis. Clustering of related genes is very important for the control of the genome because it allows genes to be switched on and off as a group.