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    Lecture 3

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    MuhammadFakhriAimi
    The document summarizes key information about bacteriophages and the lambda phage: 1) It explains the structure and life cycles of bacteriophages, distinguishing between lytic and lysogenic pathways. The lytic pathway kills the host cell while the lysogenic pathway integrates viral DNA into the host chromosome. 2) It describes the structure and genome of the lambda phage, noting it can enter either the lytic or lysogenic cycle. The lambda genome uses rolling circle replication and packages its DNA using a head and tail structure. 3) It discusses how lambda vectors are used to clone DNA fragments and gives examples of vectors used to clone long DNA pieces, highlighting their strengths and weaknesses.

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    Lecture 3

    Uploaded by

    MuhammadFakhriAimi
    69 views17 pages
    The document summarizes key information about bacteriophages and the lambda phage: 1) It explains the structure and life cycles of bacteriophages, distinguishing between lytic and lysogenic pathways. The lytic pathway kills the host cell while the lysogenic pathway integrates viral DNA into the host chromosome. 2) It describes the structure and genome of the lambda phage, noting it can enter either the lytic or lysogenic cycle. The lambda genome uses rolling circle replication and packages its DNA using a head and tail structure. 3) It discusses how lambda vectors are used to clone DNA fragments and gives examples of vectors used to clone long DNA pieces, highlighting their strengths and weaknesses.

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    The document summarizes key information about bacteriophages and the lambda phage: 1) It explains the structure and life cycles of bacteriophages, distinguishing between lytic and lysogenic pathways. The lytic pathway kills the host cell while the lysogenic pathway integrates viral DNA into the host chromosome. 2) It describes the structure and genome of the lambda phage, noting it can enter either the lytic or lysogenic cycle. The lambda genome uses rolling circle replication and packages its DNA using a head and tail structure. 3) It discusses how lambda vectors are used to clone DNA fragments and gives examples of vectors used to clone long DNA pieces, highlighting their strengths and weaknesses.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    69 views17 pages

    Lecture 3

    Uploaded by

    MuhammadFakhriAimi
    The document summarizes key information about bacteriophages and the lambda phage: 1) It explains the structure and life cycles of bacteriophages, distinguishing between lytic and lysogenic pathways. The lytic pathway kills the host cell while the lysogenic pathway integrates viral DNA into the host chromosome. 2) It describes the structure and genome of the lambda phage, noting it can enter either the lytic or lysogenic cycle. The lambda genome uses rolling circle replication and packages its DNA using a head and tail structure. 3) It discusses how lambda vectors are used to clone DNA fragments and gives examples of vectors used to clone long DNA pieces, highlighting their strengths and weaknesses.

    Copyright:

    Attribution Non-Commercial (BY-NC)
    Download as PPT, PDF, TXT or read online from Scribd
    Download as ppt, pdf, or txt
    of 17

    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.

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