Considerable information is embedded in the precise order of nucleotides
in DNA. For life to exist with stability, it is essential that
the nucleotide sequence of genes is not disturbed to any great extent.
However, sequence changes do occur and often result in altered
phenotypes. These changes are largely detrimental but are
important in generating new variability and contribute to the
process of evolution. Microbial mutation rates also can be increased,
and these genetic changes have been put to many important
uses in the laboratory and industry.
Mutations [Latin mutare, to change] were initially characterized
as altered phenotypes or phenotypic expressions. Long before
the existence of direct proof that a mutation is a stable, heritable
change in the nucleotide sequence of DNA, geneticists predicted that
several basic types of transmitted mutations could exist. They believed
that mutations could arise from the alteration of single pairs of
nucleotides and from the addition or deletion of one or two nucleotide
pairs in the coding regions of a gene. Clearly, mutations may
be characterized according to either the kind of genotypic change
that has occurred or their phenotypic consequences.

              Mutations can alter the phenotype of a microorganism in several different
ways. Morphological mutations change the microorganism’s
colonial or cellular morphology.  Lethal mutations, when expressed result in the death of the microorganism. Since the microorganism
must be able to grow in order to be isolated and studied, lethal mutations
are recovered only if they are recessive in diploid organisms
or conditional (see the following) in haploid organisms. 
Conditional mutations are those that are expressed only under
certain environmental conditions. For example, a conditional
lethal mutation in E. coli might not be expressed under permissive
conditions such as low temperature but would be expressed
under restrictive conditions such as high temperature. Thus the
hypothetical mutant would grow normally at the permissive temperature but would die at high temperatures. 

A resistant mutant is a particular type of biochemical mutant
that acquires resistance to some pathogen, chemical, or antibiotic.
Such mutants also are easy to select for and very useful in microbial genetics.

Mutations occur in one of two ways. (1) Spontaneous mutations
arise occasionally in all cells and develop in the absence of
any added agent. (2) Induced mutations, on the other hand, are the
result of exposure of the organism to some physical or chemical agent is called a mutagen.

Although most geneticists believe that spontaneous mutations
occur randomly in the absence of an external agent and are
then selected, observations by some microbiologists have led to a
new and controversial hypothesis. John Cairns and his collaborators
have reported that a mutant E. coli strain, which is unable to
use lactose as a carbon and energy source, regains the ability to
do so more rapidly when lactose is added to the culture medium
as the only carbon source. Lactose appears to induce mutations
that allow E. coli to use the sugar again. It has been claimed that
these and similar observations on different mutations are examples
of directed or adaptive mutation—that is, some bacteria
seem able to choose which mutations occur so that they can better
adapt to their surroundings. Many explanations have been offered
to account for this phenomenon without depending on bacterial
selection of particular mutations. One of the most
interesting is the proposal that hypermutation can produce such
results. Some starving bacteria might rapidly generate multiple
mutations through activation of special mutator genes. This
would produce many mutant bacterial cells. In such a random
process, the rate of production of favorable mutants would increase,
with many of these mutants surviving to be counted.
There would appear to be directed or adaptive mutation because
many of the unfavorable mutants would die. There is support for his hypothesis. Mutator genes have been discovered and do  cause hypermutation under nutritional stress. Even if the directed the mutation hypothesis is incorrect, it has stimulated much valuable
research and led to the discovery of new phenomena. 

Spontaneous Mutations: 

Spontaneous mutations arise without exposure to external agents.
This class of mutations may result from errors in DNA replication,
or even from the action of transposons. 
Generally replication errors occur when the base of a template
nucleotide takes on a rare tautomeric form. Tautomerism is
the relationship between two structural isomers that are in chemical
equilibrium and readily change into one another. Bases typically
exist in the keto form. However, they can at times take on either
an imino or enol form (. These tautomeric
shifts change the hydrogen-bonding characteristics of the bases,
allowing purine for purine or pyrimidine for pyrimidine substitutions

that can eventually lead to a stable alteration of the nucleotide sequence. Such substitutions are known 
as transition mutations and are relatively common, although

most of them are repaired by various proofreading functions. 
In transversion mutations, a purine is substituted
for a pyrimidine, or a pyrimidine for a purine. These mutations
are rarer due to the steric problems of pairing purines with

purines and pyrimidines with pyrimidines. 
Spontaneous mutations also arise from frameshifts, usually
caused by the deletion of DNA segments resulting in an altered
codon reading frame. These mutations generally occur where
there is a short stretch of the same nucleotide. In such a location,
the pairing of template and new strand can be displaced by the
distance of the repeated sequence leading to additions or deletions

of bases in the new strand.

Spontaneous mutations originate from lesions in DNA as well
as from replication errors. For example, it is possible for purine nucleotides
to be depurinated—that is, to lose their base. This results
in the formation of an apurinic site, which will not base pair normally
and may cause a transition type mutation after the next round
of replication. Cytosine can be deaminated to uracil, which is then
removed to form an apyrimidinic site. Reactive forms of oxygen

such as oxygen free radicals and peroxides are produced by aerobic metabolism. 
These may alter DNA bases and cause
mutations. For example, guanine can be converted to 8-oxo-7,8-dihydrodeoxyguanine,

which often pairs with adenine rather than cytosine during replication. 

Finally, spontaneous mutations can result from the insertion
of DNA segments into genes. This results from the movement of
insertion sequences and transposons, and usually
inactivates the gene. Insertion mutations are very frequent in

E. coli and many other bacteria. 

Induced Mutations: 
Virtually any agent that directly damages DNA, alters its chemistry,
or interferes with repair mechanisms  will induce
mutations. Mutagens can be conveniently classified according
to their mechanism of action. Four common modes of
mutagen action are incorporation of base analogs, specific mispairing,
intercalation, and bypass of replication.
Base analogs are structurally similar to normal nitrogenous
bases and can be incorporated into the growing polynucleotide
chain during replication. Once in place, these compounds typically
exhibit base pairing properties different from the bases
they replace and can eventually cause a stable mutation. A
widely used base analog is 5-bromouracil (5-BU), an analog of
thymine. It undergoes a tautomeric shift from the normal keto
form to an enol much more frequently than does a normal base.
The enol forms hydrogen bonds like cytosine and directs the incorporation
of guanine rather than adenine . The

mechanism of action of other base analogs is similar to that of 5-bromouracil.

Specific mispairing is caused when a mutagen changes a
base’s structure and therefore alters its base pairing characteristics.
Some mutagens in this category are fairly selective; they
preferentially react with some bases and produce a specific

kind of DNA damage. An example of this type of mutagen is
methyl-nitrosoguanidine, an alkylating agent that adds methyl
groups to guanine, causing it to mispair with thymine (figure 11.27).
A subsequent round of replication could then result in a GC-AT transition.
DNA damage also stimulates error-prone repair mechanisms.
Other examples of mutagens with this mode of action are the alkylating
agents ethylmethanesulfonate and hydroxylamine. Hydroxylamine
hydroxylates the C-4 nitrogen of cytosine, causing it to base
pair like thymine. There are many other DNA modifying agents that

can cause mispairing.

Intercalating agents distort DNA to induce single nucleotide
pair insertions and deletions. These mutagens are planar
and insert themselves (intercalate) between the stacked bases of
the helix. This results in a mutation, possibly through the formation
of a loop in DNA. Intercalating agents include acridines such
as proflavin and acridine orange.
Many mutagens, and indeed many carcinogens, directly damage
bases so severely that hydrogen bonding between base pairs is
impaired or prevented and the damaged DNA can no longer act as a
template. For instance, UV radiation generates cyclobutane type
dimers, usually thymine dimers, between adjacent pyrimidines. Other examples are ionizing radiation and carcinogens
such as aflatoxin B1 and other benzo(a)pyrene derivatives. Such
damage to DNA would generally be lethal but may trigger a repair
mechanism that restores much of the damaged genetic material, although

with considerable error incorporation. 

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