Repair of mutations
Most organisms possess various ways to repair mutations. Generally, when a base substitution occurs, it is only on one of the two strands in the duplex. Therefore, the mispairing creates a bulge (distortion) in the helix, which allows an organism to recognise the mutation. There are two main ways that the bug rectifies this:
a) photoreactivation - here when the DNA contains thymine dimers, and the organism remains in the light, an enzyme is activated that breaks the bond between the thymine dimer.
b) error-prone repair - here the organism senses that there is a mismatch due to a bulge, and and endonuclease comes along and breaks the DNA chain near the dimer. Another enzyme comes along and then chews away the strand leaving a gap. This gap is then filled in by DNA polymerase and repair is complete.
c) methyl-directed mismatch repair - Occasionally, mutations arise due to mistakes during replication (i.e. the wrong base is inserted for some reason). Now the problem for the cell is to know which strand contains the real sequence so that the correct base can be removed. What the bug does, is to methylate its DNA as it is being replicated. Consequently, older DNA is methylated on both strands, whereas, newly replicated DNA is hemimethylated due to the mechanism of semi-conservative replication (i.e.one strand - the template strand - is methylated, whereas the nascent strand is non-methylated). Therefore, the cell recognises these methylation patterns and determines that the methylated strand contains the correct sequence and brings in a bunch of enzymes to remove the non-methylated strand. Once removed, DNA polymerase then resynthesises the strand using the methylated strand as template.
d) recombinational repair - here genetic recombination is used to repair mutations. If an extreme mutation has occured within a cell (e.g. an insertion or deletion), then invariably that cell has multiple chromosomes within it. Consequently, there are several copies of the correct sequence around to correct the mutation. These extra templates are then used to correct the mutation via genetic recombination (see later).

Ames test
This is a genetic test to determine whether a substance is a carcinogen. Many seemingly natural things are carcinogens if given is large enough quantitites e.g. carrots. The basis of the test is that carcinogens tend to be mutagenic. Therefore, what Ames did was create an auxotrophic Salmonella strain that is super-sensitive for the detection of mutations. The strain in question is auxotrophic for histidine synthesis (i.e. requires histidine to be supplied for growth). The test is based on the fact that a second gene, when mutated, allows the organism to synthesise histidine. The test therefore monitors mutations in this second gene. This is a quantitative experiment - the greater the mutagenic capacity of a substance the more histidine reversion to prototrophy occurs. Essentially, all you have to do is grow the organism in the presence of the potentially mutagenic substance.

Mechanisms of Gene Transfer
There are three mechanisms of gene transfer employed by bacteria
i) conjugation
ii) DNA transformation
iii) transduction

Conjugation
Plasmids are the most frequently transferred DNA unit via conjugation. They carry specialised functions that allow transfer of the plasmid DNA. Two types of cell are needed (see Figure 14:1):

F+ (donor cells) and F- (recipient cells) (called F because these cells contain the fertility factor)

Among the information carried by the F plasmid are genes encoding the F pilus (see Figure 14:2) which forms a bridge from the F+ cell to the F-. A copy of the F+ DNA is then made in the donor cells and then transferred to the F-. DNA is transferred as a single strand, and once in the recipient the DNA is replicated to reconstitute the plasmid molecule.

Important types of plasmids that are transferred by conjugation are the R factors. There are plasmids that carry drug resistance genes. This is an important route by which antibiotic drug resistance is spread through nature.

Occasionally, the F plasmid is incorporated into the chromosome of the donor cell. These cells are called Hfr cells (high frequency of recombination). This time, instead of mobilizing a plasmid DNA molecule between cells, the donor chromosome is mobilised into the recipient (see Figure 14:3).

The importance of conjugation is that it contributes to genetic variation, and is important for increasing genetic diversity. Conjugation is generally genus specific (primarily due to the specifics of the F pilus). Therefore, gram-negative bacteria can conjugate with themselves, as do gram-positive bacteria within themselves.

DNA transformation
Historically, DNA transformation is important in that it was the first example of genetic transfer between bacteria (Streptococcus pneumonaie). Basically, with transformation, naked DNA is taken up by recipient bacteria. Recipient bacteria can only take up DNA if they have become competent (see Figure 14:4). There are several bacteria that are naturally competent for DNA transformation: Neisseria gonorrhoeae, Haemophilus influenzae, Bacillus subtilis, and Streptococcus pneumoniae. O

nce the DNA is taken up the DNA recombines with the host chromosome.

Although mainly a laboratory phenomenon, transformation does occur in nature at reasonably high frequencies. This occurs when dead organisms release their DNA into the environment and recipient cells then take up this DNA and recombine it into their chromosomes. If there are useful genetic characteristics associated with this DNA then the ÒtransformedÓ bacteria can assimilate them.

Transduction
Unlike transformation where naked DNA is taken up by bacteria, bacteriophage (viruses; more later) mediate transduction (see Figures 14:5 and 14:6). Bacteriophage are viruses of bacteria. Consequently, they need bacteria to grow and propagate. There are two types of phage:

a) lytic phage - lytic phage produce many copies of themselves. This causes the cell to burst open and release the new phage particles. For these new phage to be able to survive they must then reinfect new cells so that they can replicate again.
b) lysogenic phage - this is an alternative strategy employed by phage. Here they incorporate themselves into the bacterial genome and are replicated as the chromosome is replicated. Phage that are incorporated into the chromosome are called prophage. Lysogenic phage are important because when they excise, they can sometimes take a small segment of the chromosome with them. Therefore, once they infect a new bacterium they take these small pieces of DNA with them into the new bacterium - called cotransduction.

The most important transduction process when it comes to gene transfer between bacteria is generalised transduction. Here a phage infects a cell, but this time during its replication process, phage enzymes digest the host chromosome into small pieces. The small pieces of DNA are then packaged into headfuls. Once the DNA is packaged, the phage becomes infectious and can then inject the DNA into a new host. Note: the phage doesnÕt care whether it packages its own DNA or host DNA. It just needs a headful.

Host defenses
i) restriction enzymes
ii) genetic incompatibility

Restriction enzymes (restriction endonucleases - i.e. enzymes that cut DNA) allow bacteria to distinguish their own DNA from foreign DNA. These are enzymes that recognize specific DNA sequences (from 4 to 13 bases). Many restriction systems also have a modification function associated with the nuclease function - modification is in the form of methylation of DNA. The idea is that host DNA is methylated by the modification component. Foreign DNA that enters the cell does not carry the correct modification signals and is therefore cleaved by the endonuclease component. Consequently invading DNA is destroyed before it can recombine with the host chromosome.

Genetic incompatibility
This is a mechanism to exclude plasmid DNAs. Because plasmids possess all the necessary information to replicate within their new host, this makes them relatively easy to establish provided they can overcome DNA restriction. However, there are special sequences at plasmid origins of replication that recognise certain effector molecules. If the effector molecules cannot recognise the origin, then they are saidto be incompatible. Generally, this mechanism is operating in cells that already have plasmids and donÕt want any more.

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