Thursday, 21 February 2013

DNA is the genetic material of bacteria,viruses & animals


The idea that genetic material is nucleic acid had its roots in the
discovery of transformation in 1928. The bacterium Pneumococcus
kills mice by causing pneumonia. The virulence of the bacterium is
determined by its capsular polysaccharide. This is a component of the
surface that allows the bacterium to escape destruction by the host. Several
types (I, II, III) of Pneumococcus have different capsular
polysaccharides. They have a smooth (S) appearance.
Each of the smooth Pneumococcal types can give rise to
variants that fail to produce the capsular polysaccharide. These
bacteria have a rough (R) surface (consisting of the material
that was beneath the capsular polysaccharide). They are avirulent.
They do not kill the mice, because the absence of the polysaccharide
allows the animal to destroy the bacteria.
When smooth bacteria are killed by heat treatment, they lose
their ability to harm the animal. But inactive heat-killed S bacteria
and the ineffectual variant R bacteria together have a quite
different effect from either bacterium by itself. the
mouse dies as the result of a Pneumococcal infection. Virulent
S bacteria can be recovered from the mouse postmortem.
In this experiment, the dead S bacteria were of type III. The live R
bacteria had been derived from type II. The virulent bacteria recovered
from the mixed infection had the smooth coat of type III. So some property
of the dead type III S bacteria can transform the live R bacteria so
that they make the type III capsular polysaccharide, and as a result become
Figure 1.4 shows the identification of the component of the dead
bacteria responsible for transformation. This was called the transforming
principle. It was purified by developing a cell-free system, in which
extracts of the dead S bacteria could be added to the live R bacteria before
injection into the animal. Purification of the transforming principle
in 1944 showed that it is deoxyribonucleic acid (DNA).


Having shown that DNA is the genetic material of bacteria, the
next step was to demonstrate that DNA provides the genetic material
in a quite different system. Phage T2 is a virus that infects the bacterium E. coli. When phage particles are added to bacteria, they adsorb
to the outside surface, some material enters the bacterium, and
then -20 minutes later each bacterium bursts open (lyses) to release a
large number of progeny phage.
Figure 1.5 illustrates the results of an experiment in 1952 in which
bacteria were infected with T2 phages that had been radioactively labeled
either in their DNA component (with 32P) or in their protein component
(with 35S). The infected bacteria were agitated in a blender, and
two fractions were separated by centrifugation. One contained the
empty phage coats that were released from the surface of the bacteria.
The other fraction consisted of the infected bacteria themselves.
Most of the 32P label was present in the infected bacteria. The
progeny phage particles produced by the infection contained ~30% of
the original 32P label. The progeny received very little—less than
1%—of the protein contained in the original phage population. The
phage coats consist of protein and therefore carried the 35S radioactive
label. This experiment therefore showed directly that only the
DNA of the parent phages enters the bacteria and then becomes part
of the progeny phages, exactly the pattern of inheritance expected of
genetic material.


When DNA is added to populations of single eukaryotic cells
growing in culture, the nucleic acid enters the cells, and in some
of them results in the production of new proteins. When a purified DNA
is used, its incorporation leads to the production of a particular protein.
Although for historical reasons these experiments are described as
transfection when performed with eukaryotic cells, they are a direct
counterpart to bacterial transformation. The DNA that is introduced
into the recipient cell becomes part of its genetic material, and is inherited
in the same way as any other part. Its expression confers a new trait
upon the cells (synthesis of thymidine kinase in the example of the figure).
At first, these experiments were successful only with individual
cells adapted to grow in a culture medium. Since then, however, DNA
has been introduced into mouse eggs by microinjection; and it may become
a stable part of the genetic material of the mouse
Such experiments show directly not only that DNA is the genetic
material in eukaryotes, but also that it can be transferred between different
species and yet remain functional.
The genetic material of all known organisms and many viruses is
DNA. However, some viruses use an alternative type of nucleic acid, ribonucleic acid (RNA), as the genetic material. The general principle
of the nature of the genetic material, then, is that it is always nucleic
acid; in fact, it is DNA except in the RNA viruses.

DNA is a double helix

The observation that the bases are present in different amounts in
the DNAs of different species led to the concept that the sequence
of bases is the form in which genetic information is carried. By the
1950s, the concept of genetic information was common: the twin problems
it posed were working out the structure of the nucleic acid, and explaining
how a sequence of bases in DNA could represent the sequence
of amino acids in a protein.
Three notions converged in the construction of the double helix
model for DNA by Watson and Crick in 1953:
• X-ray diffraction data showed that DNA has the form of a regular
helix, making a complete turn every 34 A (3.4 nm), with a diameter
of ~20 A (2 nm). Since the distance between adjacent nucleotides is
3.4 A, there must be 10 nucleotides per turn.
• The density of DNA suggests that the helix must contain two
polynucleotide chains. The constant diameter of the helix can be
explained if the bases in each chain face inward and are restricted
so that a purine is always opposite a pyrimidine, avoiding partnerships
of purine-purine (too wide) or pyrimidine-pyrimidine (too
• Irrespective of the absolute amounts of each base, the proportion
of G is always the same as the proportion of C in DNA,
and the proportion of A is always the same as that of T. So the
composition of any DNA can be described by the proportion
of its bases that is G + C. This ranges from 26% to 74% for
different species.
Watson and Crick proposed that the two polynucleotide
chains in the double helix associate by hydrogen bonding between
the nitrogenous bases. G can hydrogen bond specifically
only with C, while A can bond specifically only with T. These
reactions are described as base pairing, and the paired bases (G
with C, or A with T) are said to be complementary.
The model proposed that the two polynucleotide chains
run in opposite directions (antiparallel), as illustrated in Figure
1.8. Looking along the helix, one strand runs in the 5'—>3' direction,
while its partner runs 3'—»5'.
The sugar-phosphate backbone is on the outside and carries
negative charges on the phosphate groups. When DNA is in solution
in vitro, the charges are neutralized by the binding of
metal ions, typically by Na+. In the cell, positively charged proteins
provide some of the neutralizing force. These proteins play an important
role in determining the organization of DNA in the cell.
The bases lie on the inside. They are flat structures, lying in pairs
perpendicular to the axis of the helix. Consider the double helix in terms of a spiral staircase: the base pairs form the treads, as illustrated
schematically in Figure 1.9. Proceeding along the helix, bases are
stacked above one another, in a sense like a pile of plates.
Each base pair is rotated ~36° around the axis of the helix relative to
the next base pair. So ~10 base pairs make a complete turn of 360°. The
twisting of the two strands around one another forms a double helix
with a minor groove (~12 A across) and a major groove (~22 A across),
as can be seen from the scale model of Figure 1.10. The double helix is
right-handed; the turns run clockwise looking along the helical axis.
These features represent the accepted model for what is known as the
It is important to realize that the B-form represents an average, not a
precisely specified structure. DNA structure can change locally. If it
has more base pairs per turn it is said to be overwound; if it has fewer
base pairs per turn it is underwound. Local winding can be affected by
the overall conformation of the DNA double helix in space or by the
binding of proteins to specific sites.

Genes are DNA

The hereditary nature of every living organism is defined by its
genome, which consists of a long sequence of nucleic acid that
provides the information needed to construct the organism. We use the
term "information" because the genome does not itself perform any active
role in building the organism; rather it is the sequence of the individual
subunits (bases) of the nucleic acid that determines hereditary
features. By a complex series of interactions, this sequence is used to
produce all the proteins of the organism in the appropriate time and
place. The proteins either form part of the structure of the organism, or
have the capacity to build the structures or to perform the metabolic
reactions necessary for life.
The genome contains the complete set of hereditary information for
any organism. Physically the genome may be divided into a number of
different nucleic acid molecules. Functionally it may be divided into
genes. Each gene is a sequence within the nucleic acid that represents a
single protein. Each of the discrete nucleic acid molecules comprising
the genome may contain a large number of genes. Genomes for living
organisms may contain as few as <500 genes (for a mycoplasma, a type
of bacterium) to as many as >40,000 for Man.
In this chapter, we analyze the properties of the gene in terms of its
basic molecular construction. Figure 1.1 summarizes the stages in the
transition from the historical concept of the gene to the modern definition
of the genome.
The basic behavior of the gene was defined by Mendel more than a
century ago. Summarized in his two laws, the gene was recognized as a
"particulate factor" that passes unchanged from parent to progeny.
A gene may exist in alternative forms. These forms are called alleles.
In diploid organisms, which have two sets of chromosomes, one
copy of each chromosome is inherited from each parent. This is the
same behavior that is displayed by genes. One of the two copies of each
gene is the paternal allele (inherited from the father), the other is the
maternal allele (inherited from the mother). The equivalence led to the
discovery that chromosomes in fact carry the genes.

Each chromosome consists of a linear array of genes. Each gene resides
at a particular location on the chromosome. This is more formally
called a genetic locus. We can then define the alleles of this gene as the
different forms that are found at this locus.
The key to understanding the organization of genes into chromosomes
was the discovery of genetic linkage. This describes the observation that
alleles on the same chromosome tend to remain together in the progeny
instead of assorting independently as predicted by Mendel's laws. Once
the unit of recombination (reassortment) was introduced as the measure
of linkage, the construction of genetic maps became possible.
On the genetic maps of higher organisms established during the first
half of this century, the genes are arranged like beads on a string. They
occur in a fixed order, and genetic recombination involves transfer of
corresponding portions of the string between homologous chromosomes.
The gene is to all intents and purposes a mysterious object (the
bead), whose relationship to its surroundings (the string) is unclear.
The resolution of the recombination map of a higher eukaryote is restricted
by the small number of progeny that can be obtained from each
mating. Recombination occurs so infrequently between nearby points
that it is rarely observed between different mutations in the same gene.
By moving to a microbial system in which a very large number of progeny
can be obtained from each genetic cross, it became possible to
demonstrate that recombination occurs within genes. It follows the same
rules that were previously deduced for recombination between genes.
Mutations within a gene can be arranged into a linear order, showing
that the gene itself has the same linear construction as the array of
genes on a chromosome. So the genetic map is linear within as well as
between loci: it consists of an unbroken sequence within which the
genes reside. This conclusion leads naturally into the modern view that
the genetic material of a chromosome consists of an uninterrupted
length of DNA representing many genes.
A genome consists of the entire set of chromosomes for any particular
organism. It therefore comprises a series of DNA molecules (one for
each chromosome), each of which contains many genes. The ultimate
definition of a genome is to determine the sequence of the DNA of each

The first definition of the gene as a functional unit followed from
the discovery that individual genes are responsible for the production of
specific proteins. The difference in chemical nature between the DNA
of the gene and its protein product led to the concept that a gene codes
for a protein. This in turn led to the discovery of the complex apparatus
that allows the DNA sequence of gene to generate the amino acid sequence
of a protein.
Understanding the process by which a gene is expressed allows us to
make a more rigorous definition of its nature. Figure 1.2 shows the
basic theme of this book. A gene is a sequence of DNA that produces another
nucleic acid, RNA. The DNA has two strands of nucleic acid, and
the RNA has only one strand. The sequence of the RNA is determined by
the sequence of the DNA (in fact, it is identical to one of the DNA
strands). In many, but not in all cases, the RNA is in turn used to direct
production of a protein. Thus a gene is a sequence of DNA that codes for
an RNA; in protein-coding genes, the RNA in turn codes for a protein.
From the demonstration that a gene consists of DNA, and that a
chromosome consists of a long stretch of DNA representing many
genes, we move to the overall organization of the genome in terms of its
DNA sequence. In 2 The interrupted gene we take up in more detail the
organization of the gene and its representation in proteins. In 3 The
content of the genome we consider the total number of genes, and in 4
Clusters and repeats we discuss other components of the genome and
the maintenance of its organization.