BIOCHEMISTRY Nucleic Acids

Mutations
A mutation is an error base sequence in a gene that is reproduced during DNA replication.
Such errors alter the genetic information that is passed on during transcription. The altered
information can cause changes in amino acid sequence during protein synthesis. Sometimes such
changes have a profound effect on an organism.

Two common types of mutations are (1) a point mutation and (2) a frameshift mutation. A point
mutation is a mutation in which one base in a DNA base sequence is replaced with another base.
Such a mutation is often also called a substitution mutation.

The effect of a point mutation can vary from no effect to a change in primary protein structure to
termination of protein synthesis, as is illustrated in Example 5-10.

Example: Predicting the Effect of a Point Mutation

Predict the change that occurs in amino acid identity when each of the following point mutations
occur on 3' -to- 5' DNA base segments.
a. GAG is point mutated to GAA.
b. ATA is point mutated to ATT.
c. AAA is point mutated to AAT.

Solution
The same analysis applies to each of the three parts of this example.
1. Determine the DNA informational strand base sequence that is complementary to the given
DNA template strand base sequence.
2. Determine the mRNA base sequence using the informational strand base sequence.
3. They will be the same except that the RNA base U has replaced the DNA base T.
4. Using the genetic code, determine the amino acid that is specified by the mRNA base
sequence (codon).

a. Unmutated base sequence:. GAG (template strand) -> CTC (informational strand) -> CUC
(mRNA) -> Leu (amino acid)
Mutated base sequence: GAA (template strand) -> CTT (informational strand) -> CUU
(mRNA) -> Leu (amino acid)

b. This is silent mutation. Amino Acid identity is not affected, as both mRNA codons code for
the same amino acid.
Unmutated base sequence: AAA (template strand) -> TTT ( informational strand) -> UUU
(mRNA) -> Phe (amino acid)
Mutated base sequence: AAT (template strand) -> TTA (informational strand) -> UUA
(mRNA) -> Leu (amino acid)

The point mutation has produced a codon that codes for a different amino acid. Newly
formed dipeptide at the P site, and the third codon of mRNA is now available, at site A, to accept a
tRNA molecule whose anticodon complements this codon (Figure 5-24d). The movement of a
ribosome along an mRNA molecule is called translocation. Translocation is the part of translation in
which a ribosome moves down an mRNA molecule three base positions (one codon) so that a new
codon can occupy the ribosomal A site.

Now a repetitious process begins. The third codon, now at the A site, accepts an incoming tRNA
with its accompanying amino acid, then the entire dipeptide at the P site is transferred and bonded
to the A site amino acid to give a tripeptide (see Figure 5-24e). The empty tRNA at the P site is
released, the ribosome shifts along the mRNA, and the process continues.

The transfer of the growing peptide chain from the P site to the A site is an example of an acyl
transfer reaction. Figure 5-25 shows the structural detail for such transfer when Met is the amino acid
at the P site and Gly is the amino acid at the A site.

A frameshift mutation is a mutation that inserts or deletes a base in a DNA molecule base
sequence. Such change affects not only the base triplet located at the insertion or deletion point
but also all triplets that follow in the sequence. This contrasts with a point mutation where only one
triplet is affected. Figure 5-27 shows the effects that a frameshift mutation can have on an amino
acid sequence in a protein.

A mutagen is a substance or agent that causes a change in the structure of a gene. Radiation
and chemical agents are two important types of mutagens. Radiation, in the form of ultraviolet light,
X-rays, radioactivity and cosmic rays has the potential to be mutagenic. Ultraviolet light from the sun
is the radiation that causes sun burn and can induce changes in the DNA of skin cells. Sustained
exposure to ultraviolet light can lead to skin cancer problems.

Chemical agents can also have mutagenic effects Nitrous acid (HNO2) is a mutagen that causes
deamination of heterocyclic nitrogen bases For example, HNO2, can convert cytosine to uracil.

Deamination of a cytosine that was part of an mRNA codon would change the codon: for example.
CCG would become UGG.

Variety of chemicals--including nitrites, nitrates, and nitrosamines can form nitrous acid in the
body. The use of nitrates and nitrites as preservatives in foods such as bologna and hot dogs. is a
cause of concern because of their conversion to nitrous acid to the body and subsequent possible
damage to DNA.

Fortunately, the body has repair enzymes that recognize and replace altered bases. Normally,
the vast majority of altered DNA bases are repaired, and mutations are avoided Occasionally,
however, the damage is not repaired, and the mutation persists.

The focus of relevancy features Chemical Connections 5D-Erythropoietin (EPO), Red Blood
Cells Mutations and Athletic Performance-discusses a situation where a mutation involving a protein
hormone that regulates red blood cell production became an advantage for an individual.

Nucleic Acids and Viruses

Viruses are very small disease-causing agents that are considered the lowest order of life.
Indeed, their structure is so simple that some scientists do not consider them truly alive because they
are unable to reproduce in the absence of other organisms Figure 5-28 shows an electron
microscope image of an influenza virus.

A virus is a small particle that contains DNA or RNA (but not both) surrounded by a coat of
protein and that cannot reproduce without the aid of a host cell. Viruses do not possess the
nucleotides enzymes, amino acids, and other molecules necessary to replicate their nucleic acid or
to synthesize proteins. To reproduce, viruses must invade the cells of another organism and cause
these host cells to carry out the reproduction of the virus. Such an invasion disrupts the normal
operation of cells, causing diseases within the host organism. The only function of a virus is
reproduction: viruses do not generate energy.

There is no known form of life that is not subject to attack by viruses. Viruses attack bacteria,
plants, animals, and humans. Many human diseases are of viral origin. Among them are the common
cold, mumps, measles, smallpox, rabies, influenza, infectious mononucleosis, hepatitis, and AIDS.

Viruses most often attach themselves to the outside of specific cells in a host organism. An
enzyme within the protein overcoat of the virus catalyzes the breakdown of the cell membrane,
opening a hole in the membrane. The virus then injects its DNA or RNA into the cell. Once inside, this
nucleic acid material is mistaken by the host cell for its own, whereupon that cell begins to translate
and/or transcribe the viral nucleic acid. When all the virus components have been synthesized by
the host cell, they assemble automatically to form many new virus particles. Within 20 to 30 minutes
after a single molecule of viral nucleic acid enters the host cell, hundreds of new virus particles have
formed. So many are formed that they eventually burst the host cell and are free to infect other cells.

If a virus contains DNA, the host cell replicates the viral DNA in a manner similar to the way it
replicates its own DNA. The newly produced viral DNA then proceeds to make the proteins needed
for the production of protein coats for additional viruses.

An RNA-containing virus is called a retrovirus. Once inside a host, such viruses first make viral
DNA. This reverse synthesis is governed by the enzyme reverse transcriptase. The template is the
viral RNA rather than DNA. The viral DNA so produced then produces additional viral DNA and the
other proteins necessary for the protein coats.
 The AIDS (acquired immunodeficiency syndrome) virus is an example of a retrovirus. This virus
has an affinity for a specific type white blood cell called a helper T cell, which is an important part
of the body's immune system. When helper T cells are unable to perform their normal functions as a
result of such viral infection, the body becomes more susceptible to infection and disease.

A vaccine is a preparation containing an inactive or weakened form of a virus or bacterium.

The antibodies produced by the body against these specially modified viruses or bacteria effectively
act against the naturally occurring active forms as well. Thanks to vaccination programs, many
diseases, such as polio and mumps (caused by RNA-containing viruses) and smallpox and yellow
fever (caused by DNA-containing viruses), are now seldom encountered.

Recombinant DNA and Genetic Engineering

Ever-increasing knowledge about DNA molecules and how they function under various
chemical conditions has opened the door to an increasingly important field of technology known
by several names, including genetic engineering, genetic modification, and bioengineering. The
term genetic engineering will be used in this text to refer to this developing field.

Genetic engineering is the process whereby an organism is intentionally changed at the

molecular (DNA) level so that it exhibits different traits. The first organisms to be genetically
engineered were bacteria in 1973 and mice in 1974. Insulin-producing bacteria were
commercialized in 1982, and genetically modified food crops have been available since 1994.
Genetically modified forms of foods and fibers now dominate several major crops in the United
States, as is shown by the data in Figure 5-29.

For the plant crops listed in FIGURE 5-29, the most common genetic modification involves
introduction of a herbicide-tolerance trait. A gene is inserted that allows the crops to be sprayed
with the weed killer glyphosate (also known as Roundup) without harm to the plants. These crops
also frequently have an insect-resistant trait that is obtained from the presence of a gene obtained
from the soil bacteria. Bacillus thuringiensis. This gene's presence causes the plants to produce their
own pesticide. Plantings of insect-resistant corn and cotton are common and research continues on
tomato plants that carry this gene.

In 2011, the United States Department of Agriculture (USDA) gave approval for planting corn
that is genetically modified to produce the enzyme a-amylase, an enzyme that rapidly breaks down
starch into glucose (Section 1-6). For corn destined for ethanol production, the presence of this
amylase improves the economics of the production process.

Two additional examples, still in the developmental stage, of the direction that genetic
engineering can take are:
1. Genetically engineered tomato plants with a longer shelf life now exist. In this case, gene
insertion is not needed. Instead, a gene naturally present in the tomato plant that is involved
in the ripening process, resulting in less over ripening, softening, and overall deterioration.
2. A strawberry gene is introduced into a mustard plant variety that is very susceptible to attack
spider mites. The gene produces a chemical attractant for predator mites that eat the spider
mites, thus solving a "mitey" problem.

Bacteria are now routinely used as "protein factories". These genetically engineered bacteria,
which contain genes for human proteins, can produce large quantities of designated proteins
because of their rapid production rate. Human proteins in short supply that are produced in this
manner include insulin (see Chemical Connections 3-A-- Substitutes for Human Insulin), erythropoietin
(see Chemical Connections 5-D--Erythropoietin (EPO): Red blood cells, mutations, and Athletic
performance), and human growth hormone. Table 5-3 gives additional examples of human proteins
used in therapeutic medicine that have become available through the use of genetic engineering
technology.

Principles and Procedures of Genetic Engineering

Genetic engineering procedures involve a type of DNA called recombinant DNA.
Recombinant DNA is DNA that contains genetic material from two different organisms. The notation
rDNA is often used to designate recombinant DNA.

The bacterium E. coli, which is found in the intestinal tract of humans and animals, is the
organism most often used in recombinant DNA experiments. Yeast cells are also used, with increasing
frequency, in this research.
 In addition to their chromosomal DNA, E. coli (and other bacteria) contain DNA in the form of
small, circular, double-stranded molecules called plasmids. These plasmids, which carry only a few
genes, replicate independently of the chromosome. Also, they are transferred relatively easily from
one cell to another. Plasmids from E. coli are used in recombinant DNA work.

Step 1: Cell membrane dissolution. E. coli cells of a specific strain are placed in a solution that
dissolves cell membranes, thus releasing the contents of the cells.
Step 2: Isolation of plasmid fraction. The released cell components are separated into fractions, one
fraction being the plasmids. The isolated plasmid fraction is material used in further steps.
Step 3: Cleavage of plasmid DNA. A special enzyme, called a restriction enzyme, is used to cleave
the double-stranded DNA of a circular plasmid. The result is a linear (non-circular) DNA
molecule.
Step 4: Gene removal from another organism. The same restriction enzyme is then used to remove a
desired gene from a chromosome of another organism.
Step 5: Gene-plasmid splicing. The gene (from Step 4) and the opened plasmid (from Step 3) are
mixed in the presence of the enzyme DNA ligase. which splices the two together. This splicing,
which attaches one end of the gene to one end of the opened plasmid and attaches the
other end of the gene to the other end of the plasmid, results in an altered circular plasmid
(the recombinant DNA).
Step 6: Uptake of recombinant DNA. The altered plasmids (recombinant DNA) are placed in a live E.
coli culture, where they are taken up by the E. coli bacteria. The E. coli culture into which the
plasmids are placed need not be identical to that from which the plasmids were originally
obtained.

Note in Step 3 that the conversion of a circular plasmid into a linear DNA molecule requires a
restriction enzyme. A restriction enzyme is an enzyme that recognizes specific base sequences in
DNA and cleaves the DNA in predictable manner at these sequences. The discovery of restriction
enzymes made genetic engineering possible.

Restriction enzymes occur naturally in numerous types of bacterial cells. Their function is to
protect the bacteria from invasion by foreign DNA by catalyzing the cleavage of the invading DNA.
The term restriction relates to such enzymes placing a "restriction" on the type of DNA allowed into
the bacterial cells.

To understand how a restriction enzyme works consider one that cleaves DNA between G
and A bases in the 5'-to-3' direction in the sequence G-A-A-T-T-C. This enzyme will cleave the double-
helix structure of a DNA molecule in the manner shown in Figure 5-31.

Note that the double-helix is not cut straight across; the individual strands are cut at different
points, giving a staircase cut. (Both cuts must be between G and A in the 5'-to-3' direction) This
staircase cut leaves unpaired bases on each cut strand. These ends with unpaired bases are called
"sticky ends" because they are ready to "stick to" (pair up with) a complementary section of DNA if
they can find one.

If the same restriction enzyme used to cut a plasmid is also used to cut a gene from another
DNA molecule, the sticky ends of the gene will be complementary to those of the plasmid. This
enables the plasmid and gene to combine readily, forming a new, modified plasmid molecule. This
modified plasmid molecule is called recombinant DNA. In addition to the newly spliced gene, the
recombinant DNA plasmid contains all of the genes and characteristics of the original plasmid. Figure
5-23 shows diagrammatically the match between sticky ends that occurs when plasmid and gene
combine.

Step 6 involves inserting the recombinant DNA (modified plasmids) back into E. coli cells. The
process is called transformation. Transformation is the process of incorporating recombinant DNA
into a host cell.

The transformed cells then reproduce, resulting in large numbers of identical cells called
clones. Clones are cells with identical DNA that have descended from a single cell. Within a few
hours, a single genetically altered bacterial cell can give rise to thousands of clones. Each clone has
the capacity to synthesize the protein directed by the foreign gene it carries.
 Researchers are not limited to selection of naturally occurring genes for transforming bacteria.
Chemists have developed non-enzymatic methods of linking nucleotides together such that they
can construct artificial genes of any sequence they desire. In fact, benchtop instruments are now
available that can be programmed by a microprocessor to synthesize any DNA base sequence
automatically. The operator merely enters a sequence of desired bases, starts the instrument, and
returns later to obtain the product. Such flexibility in manufacturing DNA has opened many doors,
accelerated the pace of recombinant DNA research, and redefined the term designer genes.

The Polymerase Chain Reaction

The Polymerase chain reaction (PCR) is a method for rapidly producing multiple copies of a
DNA nucleotide sequence. Billions of copies of a specific DNA sequence (gene) can be produced
in a few hours via this reaction. The PCR is easy to carry out, requiring only a few chemicals, a
container, and a source of heat. (In actuality, the PCR process is now completely automated.)

By means of the PCR process, DNA that is available only in very small quantities can be
amplified to quantities large enough to analyze. The PCR process, devised in 1983, has become a
valuable tool for diagnosing diseases and detecting pathogens in the body. It is now used in the
prenatal diagnosis of a number of genetic disorders, including muscular dystrophy and cystic fibrosis,
and in the identification of bacterial pathogens. It is also the definitive way to detect the

The PCR process has also proved useful in certain types of forensic investigations. A DNA
sample may be obtained from a single drop of blood or semen or a single strand of hair at a crime
scene and amplified by the PCR process. A forensic chemist can then compare the amplified
samples with DNA samples taken from suspects. Work with DNA in the forensic is often referred to as
DNA fingerprinting.

DNA polymerase, an enzyme present in all living organisms, is a key substance in the PCR
process. It can attach additional nucleotides to a short starter nucleotide chain, called a primer,
when the primer is bound to a complementary strand of DNA that functions as a template. The
original DNA is heated to separate its strands and then primers, DNA polymerase, and
deoxyribonucleotides are added so that the DNA polymerase can replicate the original strand. The
process is repeated until, in a short time, millions of copies of the original DNA have been made.