Gene transfer

1. Molecularbiology
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Gene transfer
 Viruses: are extremely small parasites, existing at a level between living things and
nonliving molecules incapable of replication, transcription or translation outside of a host
cell. Virus particles (virions) are sub-microscopic essentially comprises a nucleic acid
genome and protein coat or capsid. The complex of genome and capsid is known as the
nucleocapsid .Some viruses have a lipoprotein outer envelope, and some also contain
nonstructural proteins essential for transcription or replication soon after infection.
Virus genomes: Virus particles (virions) can replicate only inside a host cell. All viruses
rely entirely on the host cell for translation, and some viruses rely on the host cell for
various transcription and replication factors as well. Unlike the genomes of true
organisms, the virus genome can consist of DNA or RNA but not both, which may be
double - or single-stranded. In some viruses, the genome consists of a single molecule of
nucleic acid, which may be linear or circular.
Bacteriophages, or phages, any virus that infects bacteria; many (perhaps most, or all)
bacteria are susceptible to one or more types of phage. A given phage may specifically infect
bacteria of only one species or strain, or it may have a wider host range.
Phages are often said to be virulent or temperate. A virulent phage lyses (kills) the bacterial
host cell. A temperate phage can form a more or less stable relationship with a bacterium; in
most cases the phage genome (the prophage) integrates in the bacterial chromosome, but in
some cases (e.g. phage P1) it remains a circular, extrachromosomal ‘plasmid’.
Under certain conditions a temperate phage may be induced to enter the lytic cycle – in
which case virions are formed and cell lysis occurs; thus, a prophage may retain the potential
for virulence, and in a population of lysogenic bacteria (i.e. bacteria which are hosts to a
temperate phage), spontaneous induction may occur in a small number of cells.

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 Plasmids are small, extrachromosomal DNA molecules that are stably inherited from one
generation to another in the extra-chromosomal state. Usually closed circles of either
single-stranded or double-stranded DNA (mostly). Plasmids are widely distributed
throughout prokaryotes and range in size from approximately 1500 bp to over 300 kbp.
Plasmids are commonly dispensable, i.e. not essential to their host cells, and not all cells
contain plasmids; for example, plasmids are commonly absent in bacteria of the genera,
Brucella and Rickettsia.
The replication of the plasmid is often coupled to that of the host cell in which it is
maintained, with plasmid replication occurring at the same time as the host genome is
replicated. They contain an origin of replication (ori), which enables them to be replicated
independently, although this normally relies on polymerases and other components of the
host cell’s machinery.
 Features encoded by plasmids: Collectively, the plasmids encode a vast range of
functions. In many cases they encode resistance to particular antibiotics or groups of
antibiotics and/or to other inimical agents – such as mercury (or other heavy metal)
ions. In certain pathogenic bacteria (e.g. Bacillus anthracis, causal agent of anthrax)
the toxin(s) and/or other virulence factors are plasmid-encoded. (In some pathogens
the virulence factors are phage-encoded.)
Some plasmids encode products (such as particular enzymes) which enhance the
metabolic potential of a cell; for example, the Cit plasmid in some strains of
Escherichia coli enables those strains to use citrate as the sole source of carbon and
energy (an ability which is lacking in wild-type strains of E. coli). Again, the TOL
plasmid confers on certain strains of Pseudomonas the ability to metabolize toluene
and xylene. Certain plasmids (e.g. ColE1) encode a colicin or other type of bacteriocin.
In the archaean Halobacterium, structural components of gas vacuoles are plasmid-
encoded.
 Size: In size, plasmids typically range from about several kilobases to several hundred
kilobases. In DNA technology, the small circular plasmids can be inserted into cells by
transformation more readily than large plasmids; moreover, small plasmids are less
susceptible to damage by shearing forces. In general, linear plasmids are poorly
transformable e.g. in normal cells of E. coli – although linear forms of DNA can be
transformed by using a method such as the lambda (λ) red recombination system.
 Copy number: The copy number is characteristic for a given plasmid, in a particular
host cell, under given conditions. Plasmids are referred to as ‘low-copy-number’ if they
occur singly, or in a few copies, in each cell; the f plasmid is one example.
Multicopy plasmids are normally present in appreciably higher numbers – e.g. >10
copies; the ColE1 plasmid (often 10–30 copies) is one example. Factors which
influence copy number include the plasmid’s specific type of replication control system
and its mode of partition (segregation to daughter cells during cell division).
 Compatibility: Inc groups: Different types of plasmid can occur in the same cell:
those plasmids which have different modes of replication and partition are said to
exhibit compatibility; such plasmids are able to co-exist – stably – within the same cell.
On the other hand, plasmids with similar or identical systems of replication/partition
are incompatible: they are not able to co-exist, stably, in the same cell. This is the
basis of the incompatibility groups (the so-called Inc groups): a given Inc group
consists of those plasmids which have similar or identical replication/partition systems
and which cannot co-exist, stably, in the same cell.

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 Experimental elimination of plasmids: In some cases there is a need for plasmid-
free cells. Various approaches have been used to eliminate plasmids from a bacterial
population: e.g. curing.
 Plasmid stability: Plasmid-borne features are often lost from a population at a higher
frequency than would be expected for the normal processes of mutation. The extent of
this instability varies from one plasmid to another. Naturally-occurring plasmids are
usually (but not always) reasonably stable. Artificially constructed plasmids on the
other hand are often markedly unstable.
Naming genes and DNA: Traditionally, recombinant plasmids tend to bear the initials of
their creator(s) followed by a number that may indicate the numerical order in which the
plasmids were produced, or perhaps has some deeper meaning. For example, the name
of the plasmid pBR322 can be dissected into the following components: p – Plasmid, BR –
named by Paco Bolivar and Ray Rodrigues, who developed the plasmid and 322 – the
number of the plasmid within their stock collection.
Plasmids and phages provide an important extra dimension to the flexibility of the
organism’s response to changes in its environment, whether those changes are hostile
(e.g. the presence of antibiotics) or potentially favorable (the availability of a new
substrate). This extra dimension therefore consists of characteristics that are peripheral to
the replication and production of the basic structure of the cell – they are the optional
extras. Their role in contributing these additional characteristics is particularly significant
because of the relative ease with which they can be transferred between strains or
between different species
Many DNA sequences can replicate independently of the rest of the genome. Such
sequences have widely different degrees of independence from their host cells. Of these,
virus chromosomes are the most independent because they have a protein coat that
allows them to move freely from cell to cell. The viruses are closely related to plasmids
and transposable elements, which are DNA sequences that lack a coat and are therefore
more host cell- dependent and confined to replicate within a single cell and its progeny.
Transposable elements are DNA sequences that differ from viruses in being able to
multiply only in their host cell and its progeny; like plasmids, they cannot exist stably
outside of cells. Unlike plasmids, they normally replicate only as an integral part of a
chromosome. Some transposable elements, however, are closely related to retroviruses
and can move from place to place in the genome by the reverse transcription of an RNA
intermediate. Both viruses and transposable elements can be viewed as parasites.
Gene transfer: The concept of the re-assortment of characteristics through sexual
reproduction in animals and plants was a familiar one long before Mendel put it on a scientific
footing. Not only do the features of individuals represent a combination of those of their
parents (or grandparents), but the phenomenon has been used over the centuries to
establish new strains of plants and animals that combine the best characteristics of different
strains. How can we apply the same concept to organisms such as bacteria that do not
exhibit sexual reproduction?

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Bacteria do exchange genetic information, not only in the laboratory but also in nature. There
are three fundamentally distinct mechanisms by which such genetic transfer can occur.
 Transformation, in which a cell takes up isolated ‘naked’ DNA molecules from the
medium surrounding it.
 Conjugation, which involves the direct transfer of DNA from one cell to another.
 Transduction in which the transfer is mediated by bacterial viruses (bacteriophages).
Not all bacterial species exhibit all of these modes of genetic transfer. Conjugation is most
readily demonstrated in Gram-negative bacteria but does occur in some Gram-positive
genera such as Streptomyces and Streptococcus. Although some bacterial species are
naturally transformable, in many other species transformation is only readily demonstrated
after some form of artificial pre-treatment of the cells and therefore probably does not occur
naturally in those organisms.
These mechanisms differ from true sexual reproduction in two main respects: there is no link
with reproduction and the genetic contribution from the parents is unequal. The parents are
thus referred to as donor and recipient cells; the recombinant progeny resemble the original
recipient strain in most characteristics.
Transformation
 Some bacteria can take up fragments of DNA from the external medium. The source of
the DNA can be other cells of the same species or cells of other species. In some cases
the DNA has been released from dead cells; in other cases the DNA has been secreted
from live bacterial cells. The DNA taken up integrates into the recipient’s chromosome. If
this DNA is of a different genotype from the recipient, the genotype of the recipient can
become permanently changed, a process aptly termed transformation.
 Important in genetic analysis of some species and play a key role in gene cloning.

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 Natural transformation is of limited usefulness for artificial genetic modification of bacteria,
mainly because it works best with linear DNA fragments rather than the circular plasmid
DNA that is used in genetic modification.
 For introducing foreign genes into a bacterial host, various techniques are used to induce
an artificial state of competence. Alternatively, a mixture of cells and DNA may be briefly
subjected to a high voltage which enables the DNA to enter the cell (a process known as
electroporation). Although the mechanisms involved are quite different, they all share
the characteristic feature of the uptake of ‘naked’ DNA by the cells and are therefore also
referred to as transformation.
Transduction
 Transduction is the phage-mediated transfer of genetic material. The key step is the
packaging of DNA into the phage heads during lytic growth of the phage.
 Normally highly specific for phage DNA, but with some phages, errors can be made and
fragments of bacterial DNA (produced by phage-mediated degradation of the host
chromosome) are occasionally packaged by mistake leading to phage-like particles that
contain a segment of bacterial genome. These transducing particles are capable of
infecting a recipient cell, since the information necessary for attachment and injection of
DNA is carried by the proteins of the phage particle, irrespective of the nucleic acid it
contains. The transduced segment of DNA will therefore be injected into the new host cell.
 Not all bacteriophages are capable of carrying out transduction. The basic requirements
of an effective transducing phage are that infection should result in an appropriate level of
degradation of the chromosomal DNA to form suitably sized fragments at the right time for
packaging and that the specificity of the packaging process should be comparatively low.

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Conjugation
o Conjugation: is the direct transmission of DNA from one bacterial cell to another. In most
cases, this involves the transfer of plasmid DNA, although with some organisms
chromosomal transfer can also occur.
o In the simplest of cases, conjugation is achieved in the laboratory by mixing the two
strains together and after a period of incubation to allow conjugation to occur, plating the
mixture onto a medium that does not allow either parent to grow, but on which a
transconjugant that contains genes from both parents will grow. For example, in the
experiment illustrated in Figure 6.1, one strain (the donor) carries a plasmid that confers
resistance to ampicillin, while the second strain does not have a plasmid but has a
chromosomal mutation that makes it resistant to nalidixic acid. After incubating the mixed
culture, a sample is plated onto a medium containing both antibiotics. Neither parent can
grow on this medium, so the colonies that are observed are due to the transfer of a copy
of the plasmid from the donor to a recipient cell.
Recombination
 A common feature of all the forms of gene transfer between bacteria, except for
the transfer of plasmids (which can replicate independently), is the requirement
for the transferred piece of DNA to be inserted into the recipient chromosome
by breaking both DNA molecules, crossing them over and rejoining them. This
process, known as recombination. (homologous recombination and site-specific
recombination)