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Genetic Recombination
Genetic recombination is the process by which a strand of genetic material (usually DNA; but
can also be RNA) is broken and then joined to a different DNA molecule. In eukaryotes
recombination commonly occurs during meiosis as chromosomal crossover between paired
chromosomes. This process leads to offspring having different combinations of genes from
their parents and can produce new chimeric alleles.
Enzymes called recombinases catalyze natural recombination reactions. RecA, the
recombinase found in E. coli, is responsible for the repair of DNA double strand breaks
(DSBs).
In yeast and other eukaryotic organisms there are two recombinases required for repairing
DSBs. The RAD51 protein is required for mitotic and meiotic recombination and the DMC1
protein is specific to meiotic recombination.
Type of Recombination :
Chromosomal Crossover
Refers to recombination between the paired chromosomes inherited from each of one's
parents, generally occurring during meiosis. During prophase I the four available
chromatids are in tight formation with one another. While in this formation, homologous
sites on two chromatids can mesh with one another, and may exchange genetic information.
Because recombination can occur with small probability at any location along chromosome,
the frequency of recombination between two locations depends on their distance. Therefore,
for genes sufficiently distant on the same chromosome the amount of crossover is high
enough to destroy the correlation between alleles.
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Gene Conversion
In gene conversion, a section of genetic material is copied from one chromosome to
another, but leaves the donating chromosome unchanged.
Nonhomologous Recombination
Recombination can occur between DNA sequences that contain no sequence homology.
This is referred to as Nonhomologous recombination or nonhomologous end joining.
In B Cells
B cells of the immune system perform genetic recombination, called immunoglobulin class
switching. It is a biological mechanism that changes an antibody from one class to another,
for example, from an isotype called IgM to an isotype called IgG.
Chiasma
(plural: chiasmata), in genetics, is thought to be the point where two homologous nonsister chromatids exchange genetic material during chromosomal crossover during meiosis
(sister chromatids also form chiasmata between each other, but because their genetic
material is identical, it does not cause any change in the resulting daughter cells). The
chiasmata become visible during the prophase I of meiosis. When each bivalent, which is
composed of two pairs of sister chromatids, begins to split, the only points of contact are at
the chiasmata. The chiasmata referes to the actual break of the phosphodiester bond during
crossing over. The larger the number of map units between the genes, the more crossing
over occurs.
Holliday model
One of the first plausible models to account for the preceding observations was
formulated by Robin Holliday. The key features of the Holliday model are the formation
of heteroduplex DNA; the creation of a cross bridge; its migration along the two
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heteroduplex strands, termed branch migration; the occurrence of mismatch repair;
and the subsequent resolution, or splicing, of the intermediate structure to yield different
types of recombinant molecules. The model is depicted in Figure 1.
Fig 1.hollidy model of recombination.
Enzymatic cleavage and the creation of heteroduplex DNA
Looking at Figure 1a, we can see that two homologous double helices are aligned,
although note that they have been rotated so that the bottom strand of the first helix
has the same polarity as the top strand of the second helix (5′ → 3′ in this case). Then
a nuclease cleaves the two strands that have the same polarity (Figure 1b). The free
ends leave their original complementary strands and undergo hydrogen bonding with
the complementary strands in the homologous double helix (Figure 1c). Ligation
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produces the structure shown in Figure 1d. This partially heteroduplex double helix is
a crucial intermediate in recombination, and has been termed the Holliday structure.
Branch migration
The Holliday structure creates a cross bridge, or branch, that can move, or migrate,
along the heteroduplex (Figure 1d and e). This phenomenon of branch migration is a
distinctive property of the Holliday structure. Figure 2 portrays a more realistic view
of this structure as it might appear during branch migration.
Fig 2. Branch migration, the movement of the crossover point between DNA
complexes.
Resolution of the Holliday structure
The Holliday structure can be resolved by cutting and ligating either the two
originally exchanged strands (Figure 1f, left) or the originally unexchanged strands
(Figure 1f, right). The former generates a pair of duplexes that are parental, except for
a stretch in the middle containing one strand from each parent. If the two parents had
different alleles in this stretch, as indicated here, then the DNA will be heteroduplex.
The latter resolution step generates two duplexes that are recombinant, with a stretch
of heteroduplex DNA. The Holliday model also postulated that the heteroduplex DNA
mismatches can be repaired by an enzymatic correction system that recognizes
mismatches and excises the mismatched base from one of the two strands, filling in
the excised base with the correct complementary base. The resulting molecules will
carry either the wild-type or the mutant allele, depending on which allele is excised.
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Fige 3. (a) The Holliday structure shown in an extended form. (b) The rotation of the
structure shown in part a can yield the form depicted in part c. Resolution of the structure
shown in part c can proceed in two ways, depending on the points of enzymatic cleavage,
yielding the structures shown in part d. The dotted lines show which segments will rejoin to
form recombinant strands for each particular cleavage scheme.
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Section #2
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Making recombinant DNA
How does recombinant DNA technology work? The organism under study, which will
be used to donate DNA for the analysis, is called the donor organism. The basic
procedure is to extract and cut up DNA from a donor genome into fragments
containing from one to several genes and allow these fragments to insert themselves
individually into opened-up small autonomously replicating DNA molecules such as
bacterial plasmids. These small circular molecules act as carriers, or vectors, for the
DNA fragments. The vector molecules with their inserts are called recombinant DNA
because they consist of novel combinations of DNA from the donor genome (which
can be from any organism) with vector DNA from a completely different source
(generally a bacterial plasmid or a virus). The recombinant DNA mixture is then used
to transform bacterial cells, and it is common for single recombinant vector molecules
to find their way into individual bacterial cells. Bacterial cells are plated and allowed
to grow into colonies. An individual transformed cell with a single recombinant
vector will divide into a colony with millions of cells, all carrying the same
recombinant vector. Therefore an individual colony contains a very large population
of identical DNA inserts, and this population is called a DNA clone. A great deal of
the analysis of the cloned DNA fragment can be performed at the stage when it is in
the bacterial host. Later, however, it is often desirable to reintroduce the cloned DNA
back into cells of the original donor organism to carry out specific manipulations of
genome structure and function. Hence the protocol is often as follows:
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Inasmuch as the donor DNA was cut into many different fragments, most colonies
will carry a different recombinant DNA (that is, a different cloned insert). Therefore,
the next step is to find a way to select the clone with the insert containing the specific
gene in which we are interested. When this clone has been obtained, the DNA is
isolated in bulk and the cloned gene of interest can be subjected to a variety of
analyses, which we shall consider later in the chapter. Notice that the cloning method
works because individual recombinant DNA molecules enter individual bacterial host
cells, and then these cells do the job of amplifying the single molecules into large
populations of molecules that can be treated as chemical reagents. In figure below
gives a general outline of the approach.
The term recombinant DNA must be distinguished from the natural DNA
recombinants that result from crossing-over between homologous chromosomes in
both eukaryotes and prokaryotes. Recombinant DNA in the sense being used in this
chapter is an unnatural union of DNAs from nonhomologous sources, usually from
different organisms. Some geneticists prefer the alternative name chimeric DNA, after
the mythological Greek monster Chimera. Through the ages, the Chimera has stood as
the symbol of an impossible biological union, a combination of parts of different
animals. Likewise, recombinant DNA is a DNA chimera and would be impossible
without the experimental manipulation that we call recombinant DNA technology.
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Isolating DNA
The first step in making recombinant DNA is to isolate donor and vector DNA.
General protocols for DNA isolation were available many decades before the advent
of recombinant DNA technology. With the use of such methods, the bulk of DNA
extracted from the donor will be nuclear genomic DNA in eukaryotes or the main
genomic DNA in prokaryotes; these types are generally the ones required for analysis.
The procedure used for obtaining vector DNA depends on the nature of the vector.
Bacterial plasmids are commonly used vectors, and these plasmids must be purified
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away from the bacterial genomic DNA. A protocol for extracting plasmid DNA by
ultracentrifugation is summarized in figure below. Plasmid DNA forms a distinct
band after ultracentrifugation in a cesium chloride density gradient containing
ethidium bromide. The plasmid band is collected by punching a hole in the plastic
centrifuge tube. Another protocol relies on the observation that, at a specific alkaline
pH, bacterial genomic DNA denatures but plasmids do not. Subsequent neutralization
precipitates the genomic DNA, but plasmids stay in solution. Phages such as λ also
can be used as vectors for cloning DNA in bacterial systems. Phage DNA is isolated
from a pure suspension of phages recovered from a phage lysate.
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Cutting DNA
The breakthrough that made recombinant DNA technology possible was the discovery
and characterization of restriction enzymes. Restriction enzymes are produced by
bacteria as a defense mechanism against phages. The enzymes act like scissors,
cutting up the DNA of the phage and thereby inactivating it. Importantly, restriction
enzymes do not cut randomly; rather, they cut at specific DNA target sequences,
which is one of the key features that make them suitable for DNA manipulation. Any
DNA molecule, from viral to human, contains restriction-enzyme target sites purely
by chance and therefore may be cut into defined fragments of a size suitable for
cloning. Restriction sites are not relevant to the function of the organism, and they
would not be cut in vivo, because most organisms do not have restriction enzymes.
Let's look at an example: the restriction enzyme EcoRI (from E. coli) recognizes the
following six-nucleotide-pair sequence in the DNA of any organism:
This type of segment is called a DNA palindrome, which means that both strands
have the same nucleotide sequence but in antiparallel orientation. Many different
restriction enzymes recognize and cut specific palindromes. The enzyme EcoRI cuts
within this sequence but in a pair of staggered cuts between the G and the A
nucleotides.
This staggered cut leaves a pair of identical single-stranded “sticky ends.” The ends
are called sticky because they can hydrogen bond (stick) to a complementary
sequence.
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Dozens of restriction enzymes with different sequence specificities have now been
identified, some of which are shown in Table below. All the target sequences are
palindromes, but, like EcoRI, some enzymes make staggered cuts, whereas others
make flush cuts. Even flush cuts, which lack sticky ends, can be used for making
recombinant DNA.
Table 1. Note in the table the m(modification site).
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DNA can also be cut by mechanical shearing. For example, agitating DNA in a
blender will break up the long chromosome-sized molecules into flush-ended clonable
segments.
Joining DNA
Most commonly, both donor DNA and vector DNA are digested with the use of a
restriction enzyme that produces sticky ends and then mixed in a test tube to allow the
sticky ends of vector and donor DNA to bind to each other and form recombinant
molecules. Figure a shows a plasmid vector that carries a single EcoRI restriction site;
so digestion with the restriction enzyme EcoRI converts the circular DNA into a
linear molecule with sticky ends. Donor DNA from any other source (say,
Drosophila) also is treated with the EcoRI enzyme to produce a population of
fragments carrying the same sticky ends. When the two populations are mixed, DNA
fragments from the two sources can unite, because double helices form between their
sticky ends. There are many opened-up vector molecules in the solution, and many
different EcoRI fragments of donor DNA. Therefore a diverse array of vectors
carrying different donor inserts will be produced. At this stage, although sticky ends
have united to generate a population of chimeric molecules, the sugar-phosphate
backbones are still not complete at two positions at each junction. However, the
backbones can be sealed by the addition of the enzyme DNA ligase, which create
phosphodiester bonds at the junctions (Figure b). Certain ligases are even capable of
joining DNA fragments with blunt-cut ends.
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Amplifying recombinant DNA
The ligated recombinant DNA enters a bacterial cell by transformation. After it is in
the host cell, the plasmid vector is able to replicate because plasmids normally have a
replication origin. However, now that the donor DNA insert is part of the vector's
length, the donor DNA is automatically replicated along with the vector. Each
recombinant plasmid that enters a cell will form multiple copies of itself in that cell.
Subsequently, many cycles of cell division will take place, and the recombinant
vectors will undergo more rounds of replication. The resulting colony of bacteria will
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contain billions of copies of the single donor DNA insert. This set of amplified copies
of the single donor DNA fragment is the DNA clone .
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