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TA Brown Chapter 2 Notes

Plasmids are circular DNA molecules that exist independently in bacteria and often carry genes that provide useful traits to the host. Common plasmid types include fertility plasmids, resistance plasmids, colicin plasmids, degradative plasmids, and virulence plasmids. Bacteriophages are viruses that infect bacteria and have lifecycles that involve attaching to the host, injecting DNA, replicating, and releasing new phage particles which may cause cell lysis or integrate into the genome. Lysogenic phages like lambda can integrate their DNA and remain dormant for many cell divisions. Viruses have also been used as cloning vectors for eukaryotic organisms like humans, insects

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44 views7 pages

TA Brown Chapter 2 Notes

Plasmids are circular DNA molecules that exist independently in bacteria and often carry genes that provide useful traits to the host. Common plasmid types include fertility plasmids, resistance plasmids, colicin plasmids, degradative plasmids, and virulence plasmids. Bacteriophages are viruses that infect bacteria and have lifecycles that involve attaching to the host, injecting DNA, replicating, and releasing new phage particles which may cause cell lysis or integrate into the genome. Lysogenic phages like lambda can integrate their DNA and remain dormant for many cell divisions. Viruses have also been used as cloning vectors for eukaryotic organisms like humans, insects

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Chapter 2 of TA Brown

Vectors for Gene Cloning: Plasmids and Bacteriophages

2.1 Plasmids

Plasmids are circular molecules of DNA that lead an independent existence in the
bacterial cell (Figure 2.1). Plasmids almost always carry one or more genes, and
often these genes are responsible for a useful characteristic displayed by the host
bacterium. For example, the ability to survive in normally toxic concentrations of
antibiotics such as chloramphenicol or ampicillin is often due to the presence in the
bacterium of a plasmid carrying antibiotic resistance genes. In the laboratory,
antibiotic resistance is often used as a selectable marker to ensure that bacteria in a
culture contain a particular plasmid (Figure 2.2).

2.1.3 Plasmid classification

The most useful classification of naturally occurring plasmids is based on the main
characteristic coded by the plasmid genes. The five major types of plasmid
according to this classification are as follows:

Fertility or F plasmids carry only tra genes and have no characteristic beyond the
ability to promote conjugal transfer of plasmids. A well-known example is the F
plasmid of E. coli.

Resistance or R plasmids carry genes conferring on the host bacterium resistance


to one or more antibacterial agents, such as chloramphenicol, ampicillin, and
mercury. R plasmids are very important in clinical microbiology as their spread
through natural populations can have profound consequences in the treatment of
bacterial infections. An example is RP4, which is commonly found in Pseudomonas,
but also occurs in many other bacteria.

Col plasmids code for colicins, proteins that kill other bacteria. An example is ColE1
of E. coli.

Degradative plasmids allow the host bacterium to metabolize unusual molecules


such as toluene and salicylic acid, an example being TOL of Pseudomonas putida.
Virulence plasmids confer pathogenicity on the host bacterium; these include the
Ti plasmids of Agrobacterium tumefaciens, which induce crown gall disease on
dicotyledonous plants.

Figure 2.3 Replication strategies for (a) a non-integrative plasmid, and (b) an
episome.

The smaller plasmids make use of the host cell’s own DNA replicative enzymes in
order to make copies of themselves A few types of plasmid are also able to replicate
by inserting themselves into the bacterial chromosome (Figure 2.3b). These
integrative plasmids or episomes may be stably maintained in this form through
numerous cell divisions, but always at some stage exist as independent elements.

The two main types of phage structure: (a) head-and tail (e.g. λ); (b)
filamentous (e.g. M13).

Bacteriophages, or phages as they are commonly known, are viruses that


specifically infect bacteria.

2.2.1 The phage infection cycle

The general pattern of infection, which is the same for all types of phage, is a three-
step process (Figure 2.6):
1. The phage particle attaches to the outside of the bacterium and injects its DNA
chromosome into the cell.

2 The phage DNA molecule is replicated, usually by specific phage enzymes coded
by genes in the phage chromosome.

3 Other phage genes direct synthesis of the protein components of the capsid, and
new phage particles are assembled and released from the bacterium.
2.2.2 Lysogenic phages

Lambda phage - lysogenic infection is characterized by retention of the phage DNA


molecule in the host bacterium, possibly for many thousands of cell divisions. The
integrated form of the phage DNA (called the prophage) is quiescent, and a
bacterium (referred to as a lysogen) that carries a prophage is usually physiologically
indistinguishable from an uninfected cell.
A limited number of lysogenic phages follow a rather different infection cycle. When
M13 or a related phage infects E. coli, new phage particles are continuously
assembled and released from the cell. The M13 DNA is not integrated into the
bacterial genome and does not become quiescent. With these phages, cell lysis
never occurs, and the infected bacterium can continue to grow and divide, albeit at a
slower rate than uninfected cells. Figure 2.8 shows the M13 infection cycle.
2.2.3 Viruses as cloning vectors for other organisms

Most living organisms are infected by viruses and it is not surprising that there has
been great interest in the possibility that viruses might be used as cloning vectors for
higher organisms. This is especially important when it is remembered that plasmids
are not commonly found in organisms other than bacteria and yeast. Several
eukaryotic viruses have been employed as cloning vectors for specialized
applications: for example, human adenoviruses are used in gene therapy (p. 259),
baculoviruses are used to synthesize important pharmaceutical proteins in insect
cells (p. 240), and caulimoviruses and geminiviruses have been used for cloning
in plants (p. 120). These vectors are discussed more fully in Chapter 7.

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