Download genetic recombination-unit-2-study material- 2012

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Genetic Recombination
According to the current state of research, at least three different mechanisms are recognised by which the
DNA transferred into a bacterial cell can be recombined with the recipient bacterial chromosome (or with a
plasmid) in vivo: (1) a general, homologous recombination; (2) a site.specific recombination; and (3) a
non-homologous recombination.
1. General homologous recombination. Homologous recombination comprises the mechanism by which the
DNA that has been transferred into the recipient cell recombine with the resident DNA by reciprocal
exchange of DNA sections. The recombining DNA partners must have more or less the same base sequence,
that is, exhibit maximal homology with the exception of any mutational differences. Homologous
recombination is under the control of the recA gene; mutants with a defect in this gene (rec) have lost the
capacity for homologous recombination. There are several models for the mechanism, but in all it is assumed
that base pairing takes place between unwound, single- stranded segments of both double-stranded DNA
partners. The second strand may then arise by replication or by repair mechanisms.
2.Site-specific recombination. Site-specific recombination is independent of homologous recombination and
can occur in rec mutants. It consists of the integration of a small, double-stranded DNA segment at a specific
site of a large double-stranded DNA. The small partner loses its autonomy by this integration. The model
example for site-specific recombination is the integration of the bacteriophage A Genetic experiments show
that in the transition of A to the prophage state, it becomes inserted at a certain site on the host chromosome.
namely between the gal and bio operons . The insertion is initiated by the strands being in close contact.
However, the homology of these segments is only slight; the process actually involves a phage-coded protein,
the so-called integrase. This catalyses a break at a specific site (att B) in the A DNA and another break at a
specific site of the host DNA (att A) and the subsequent crossed reunion of the phage and the host genomes.
Fig: Site –specific
recombination –
Integration of λ phage into
chromosome of host cell.
The circular phage
chromosome attaches, via
a protein ,with its attachment region attB to the att λ region of the host chromosome, between the bio and gal
operons. The phage chromosome is then integrated by means of breakage and crosswise rejoining of the
Site-Specific Recombination
Site-specific recombination differs from general recombination in that short specific sequences which are
required for the recombination, are the only sites at which recombination occurs. These reactions invariably
require specialized proteins to recognize these sites and to catalyze the recombination reaction at these sites.
The steps and features of the general recombination reaction, however, still apply:
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strand exchange
formation of a Holliday intermediate
branch migration
resolution.
Because they involve specific sites, there are really only two types of site-specific recombination reaction.
Inverted repeats
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If the two sites at which recombination will take place are oriented oppositely to one another in the same
DNA molecule then the following illustrates the sequence of events that will take place:
The net result is that the segment of DNA between the two recombinogenic sites has inverted with respect
to the rest of the DNA molecule.
In other words, recombination at inverted repeats causes an inversion.
Direct repeats
If the two sites at which recombination will take place are oriented in the same direction in the same DNA
molecule then the following illustrates the sequence of events:
The net result is that the segment of DNA between the two recombinogenic sites has been deleted from the
rest of the DNA molecule and appears as a circular molecule.
In other words, recombination at direct repeats causes a deletion.
Site-specific recombination reactions provide an unusual but important mechanism for regulating gene
expression. Since the order of the genes in an organism will change as a result of site-specific recombination,
the affected genes can be kept in a silent state until after the recombination has taken place. This type of
control occurs in the regulation of gene expression in a differentiating cell.
Examples of Site-Specific Recombination
Integration of bacteriophage lambda
When bacteriophage lambda infects E. coli, the phage chromosome circularizes (due to annealing at the cos
sites), and the phage must decide whether to follow a lytic growth pathway or a lysogenic growth pathway.
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In order for the lambda prophage to exist in a host E. coli cell, it must integrate into the host chromosome
which it does by means of a site-specific recombination reaction.
The E. coli chromosome contains one site at which lambda integrates. The site, located between the gal and
bio operons, is called the attachment site and is designated attB since it is the attachment site on the bacterial
chromosome. The site is only 30 bp in size and contains a conserved central 15 bp region where the
recombination reaction will take place.
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double strands.
3.Non-homologous recombination. Recombination events between DNA segments without recognisable
genetic homology are classified as non- homologous recombination. This represents an integrative form of
recombination, similar to that in the site-specific process, involving addition of DNA, rather than exchange.
This non-homologous recombination is also independent of rec A. The following types of DNA are able to
take part in such recombination: (1) insertion sequences (IS elements); (2) transposons (Tn elements); (3)
bacteriophage Mu. It was discovered that certain spontaneous mutants of E. coli are due to the insertion of
extraneous DNA (alien DNA). Such mutations can occur in structural and regulatory genes anywhere on the
chromosome. The extraneous DNA consists of so- called insertion sequences (IS elements), which occur in
bacterial chromosomes and in plasmids. They have 800—1400 base pairs but do not code for any
recognisable phenotypic characters. Little is known about their function, but their mutagenic action is due to
their insertion in DNA and consequent errors in transcription. It can be assumed that the IS elements play a
significant part in the re-orientating and joining of genetic material.
Transposons are DNA sequences that can be integrated into the genome at a number of places, though not
randomly. They can integrate from a plasmid to a bacterial chromosome, to another plasmid, or to a temperate
phage. Transposons contain genes that determine recognisable characters such as resistance to antibiotics
(penicillin, tetracyclines, or kanamycin). They are therefore more easily recognised than IS sequences. The
resistance genes in a transposon are flanked by two DNA segments with repeated base sequences, either in the
same direction or inverted. In some transposons the inverted repeats are almost identical to known IS elements.
The bacteriophage Mu has the same unusual integration behaviour as do transposons and IS elements. It has
typical phage properties and could be regarded as a giant transposon.
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