Université catholique de Louvain (LEW), Faculté de Médecine
lambda phage
(SBIM 2520 January 2001)

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(workshop of molecular genetics SBIM 2520)
Dr Jean-Pierre HERVEG, Dr Alain AMAR-COSTESEC, et Philippe VERDOOT
herveg@bian.ucl.ac.be , amar@bchm.ucl.ac.be and p.verdoot@student.md.ucl.ac.be

plan:

1. a phage (bacteriophage) is a bacterial virus
2.
lambda phage has a head and tail
3. lambda phage DNA is either linear, circular, or concatenated.
4. it's a
temperate phage: being either lytic or lysogenic

definitions you should know:

prophage: a phage DNA integrated as a part of a bacterial chromosome.
lytic pathway: the type of bacteriophage infection involving phage multiplication (± 100) and release after lysis of the host cell..
lysogenic pathway: the type of bacteriophage infection involving integration of phage DNA into a host chromosome.
icosahedron is a 20-face 3-D picture. The head of the lambda phage is an icosahedron.
concatemer is a DNA sequence made of the succession of several genomes during phage replication. Each phage head receives one genome. Lambda phage concatemers are used as size markers in PFGE (Pulse Field Gel Electrophoresis)
multiplicity of infection
is the ratio of phages to bacteria.
plaque is a zone of clearing on a lawn of bacteria caused by the lysis of the cells by infecting bacteriophages

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History

Bacterial viruses were discovered in by TW Wort and F d'Hérelle (1). The authors had been under attack by a well known Belgian scientist (2). Reading one his paper gives an idea of how contemptuous people could have been in the beginning of the last century.

1. Discussion on the Bacteriophage (Bacteriolysin), F. d'Hérelle,
Br Med J, 2 (3216), 289-297 August 19, 1922
2. Concerning the theories of the so-called "bacteriophage", J. Bordet,
Br Med J, id.

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A phage (bacteriophage) is a bacterial virus.
The picture below represents the lambda phage of the bacterium E.coli.

Lambda derived phages were constructed and used as cloning vectors to generate gDNA and cDNA libraries.
website are devoted to phages and to phage history.

http://www.biosci.ohio-state.edu/~mgonzalez/Micro521/Lambda/phage_intro.html

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2. lambda phage has a head and tail.

The head has 20 faces. A 20-faces 3-D picture is called an icosahedron. The head is made of protein of several types, and contains a gDNA long of 46,500 bp.

The tail, consisting of 35 stacked discs. It ends in a fiber. Upon adsorption on the lamb receptor of a host cell, lambda gDNA is injected trough the tail, which forms a hollow tube through which the DNA passes. A connecting structure joins the head to the tail.

A phage infects E. coli by injecting its DNA into the host.

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3. lambda phage DNA is either linear, circular, or concatenated.

 

The linear form is found in the head of the phage.
Upon entering a bacterial cell, the DNA become circular. The circular DNA can either integrate in the host circular chromosomes, or stay free and circular within the cytosol.


Phage replication produces a continuous double strand DNA, a concatenate made of the succession of several genomes and known as a concatemer. During phage multiplication, each head receives one genome.

Concatemers containing various numbers of phage genomes are used as markers in pulse field gel electrophoresis.

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4. it's a temperate phage: being either lytic or lysogenic.

Two options are open to lambda phage upon infection of its host bacterium, E. coli. The lambda DNA may behave in a lytic or intemperate manner and enter the lytic cycle; that is to say, phage genes are expressed and phage DNA is replicated to reproduce many phage particles (± 100), which are eventually released by the lysis of the cell induced by the phage.

Lambda may exist in harmony with its host cell in a temporary noninfectious manner known as lysogeny. Lysogenic (temperate) bacteria carry a phage DNA in the form known as prophage, when the phage genome is integrated as a part of the bacterial chromosome. The integrated prophage is inherited in exactly the same manner as bacterial genes; but the prophage may be induced, for example, by ultraviolet irradiation, to enter the lytic cycle, in which case the phage DNA is released from the bacterial chromosome. A prophage is equivalent to a provirus.

The choice of whether a phage should enter the lytic cycle or form a lysogen upon infecting a host cell depends upon the conditions of infection and the genotypes of phage and bacterium.

Infection of a bacterial culture with an excess of a phage to which it is sensitive results in lysis of the bacteria. The ratio of phage particles to bacteria is usually described as the multiplicity of infection. With lytic phages, which are able only to enter the lytic cycle and cannot establish lysogeny, addition of a phage suspension (at a high multiplicity of infection, that is > 1) to a culture of sensitive bacteria growing in liquid medium results in a clearing of the culture after all the bacteria have been infected and lysed.

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After prolonged incubation, the usual turbidity may be regained; this is due to the presence in the population of bacteria resistant to the phage, which were not susceptible to infection and thus can reproduce to repopulate the culture. With temperate phages, however, the clearing of the culture is only transient and is rapidly overtaken by the reappearance of turbidity, in this case due to the presence of cells in which lysogeny was established; because of their immunity to subsequent lytic infection, the lysogenic bacteria reproduce and thus restore the turbidity. The activity of a phage preparation is usually assayed by examining its ability to form plaques in a bacterial culture grown on agar. Phage particles are mixed with an excess of bacteria in suspension (that is, at a very low multiplicity of infection), so that each phage particle infects a single bacterium (this bacterium is surrounded by numerous uninfected ones). When the mixture is poured onto an agar plate, it hardens so that the bacteria are immobilized in fixed positions. Lytically infected bacteria burst at the end of the lytic cycle to release a large number of progeny phage particles; these particles infect neighboring bacteria, which suffer the same ultimate fate of lysis. The spread of phage generations through neighboring cells finally is halted by a decline in bacterial metabolism, the result being the production of clearings in the confluent bacterial culture; these clearings are known as plaques. you can learn how to pick plaques.

 

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(Atelier de Génétique Moléculaire SBIM 2520)
Dr Jean-Pierre HERVEG, Dr Alain AMAR-COSTESEC, et Philippe VERDOOT
herveg@bian.ucl.ac.be , amar@bchm.ucl.ac.be and p.verdoot@student.md.ucl.ac.be


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Université catholique de Louvain (LEW), Faculté de Médecine

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