DNA, THE GENETIC MATERIAL

Several lines of indirect evidence have long suggested that DNA contains the genetic information
of living organisms. Most important, result obtained using several different experimental
procedures showed that most of the DNA is located in the chromosomes, whereas DNA and
protein are also abundant in the cytoplasm. Moreover, a precise correlation exists between the
amount of DNA per cell and the number of sets of chromosomes per cell. That is, most somatic
cell of diploid organisms, for example, contain exactly twice the amount of DNA as the haploid
germ cells or gametes of the same species. Finally, the molecular composition of the DNA in all of
the different cell of an organism is the same (with rare exceptions), whereas the compositon of
RNA and protein varies both qualitatively and quantitatively from one cell type to another.
Although these correlations strongly suggest that DNA is the genetic material, they by no means
prove it. Fortunately, direct evidence has established that the genetic information is encoded in
DNA.

Transformation in Pneumococcus

The first direct evidence showing that the genetic material is DNA rather than protein or RNA was
published by O. T. Avery, C. M. Macleod, and M. McCarty in 1944. They demonstrated that the
component of the cell responsible for the phenomenon of transformation in the bacterium
Diplococcus pneumoniae ( pneumococcus) is DNA. Transformation is a mode of recombination
(exchange or transfer of genetic information between organism or for one organism to another)
occuring in several, but not all, species of bacteria. It does not involve direct contact between the
bacteria cells or mediation by any vector such as a virus.

The phenomenon of transformation was discovered by Frederick Griffith in 1928. It should be

emphasized that although Griffith’s experiments demonstrated the occurrence of transformation
in pneumococcus and thus set the stage for the work of Avery, MacLeod, and McCarty, they
provided no evidence that DNA was involved in any way.

Pneumococci, like all other living organisms, exhibit genetic variability that can be recognized by
the existence of different phenotypes (table 5.1). the two phenotypic characteristics of
importance in Griffith’s demonstration of transformation were

1) The presence or absence of a surrounding polysaccharide (complex sugar polymer)

capsule and
 2) The type of capsule, that is, the spesific molecular composition of the polysaccharides
present in the capsules.

When grown on appropriate media (such as blood agar) in petri dishes, pneumococci with a
capsule form large, smooth colonies and are thus designated Type S. Such encapsulated
pneumococci are quit phatogenic to most mammals ( e.g., causing pneumonia in humans). These
virulent ( disease causing) Type S pneumococci mutate to a nonvirulent ( or nonphatogenic) form
that has no polysaccharide capsule (at a frequency of about one cell in 107). Such
nonencapsulated, nonvirulent pneumococci form small, rough-surfaced colonies when grown on
blood agar medium and are thus designated Type R ( Table 5.1 ). ( The polysaccharide capsule is
required for virulence since it protects the bacterial cell against phagocytosis by leukocytes). When
a capsule is present, it may be of several different antigenic types ( Type II, III, etc.), depending on
the specific molecular composition of the polysaccharides and, of course, ultimately on the
genotype of the cell.

The different capsule types can be identified immunologically. If Type II cells are injected into the
bloodstream of rabbits, the immune system of the rabbits will produce antibodies (a specific set
of large proteins whose function is to protect the organism against foreign substances such as
macromolecules, viruses, and bacteria; see Chapter 16) that react specifically with Type II cells.
Such Type II antibodies will agglutinate Type II pneumococci but not Type III pneumococci, and
vice versa.

Griffith’s unexpected discovery was that if he injected heat-killed Type IIIS pneumococci (virulent
when alive ) plus live Type IIR pneumococci ( nonvirulent ) into mice, many of the mice succumbed
to pneumonia, and live Type IIIS cells were recovered from the carcasses (Fig. 5.1). When mice
were injected with heat-killed Type IIIS pneumococci alone (Fig. 5.1, top), none of the mice dead.
The observed virulence was therefore not due to a few Type IIIS cells that survived the heat
treatment. It is critical to note that the live virulent pneumococci recovered from the carcasses
were of polysaccharide Type III, since it is known that nonencapsulated Type R cells can mutate
back to virulent encapsulated Type S cells. When such a mutation occurs in a Type IIR cell,
however, the resulting cell will be Type IIS, not Type IIIS. Thus, the “transformation” of nonvirulent
Type IIR cells to virulent Type IIIS cells cannot be explained by mutation, rather some component
of the dead Type IIIS cells (the “transforming principle”) must convert living Type IIR cells to Type
IIIS.
Subsequent experiments showed that the phenomenon described by Griffith, now called
transformation, was not mediated in any way by a living host. The same phenomenon occurred
in the test tube when live Type IIR cells were grown in the presence of dead Type IIIS cells or
extracts of Type IIIS cells. Since it was clearly shown that the new phenotype, Type IIIS, was
hereditary, that is, was due to a permanent inherited change in the genotype of the cells, the
demonstration of transformation neatly set the stage for determining the chemical basis of
heredity in pneumococcus. What remained was to determine what component of the cell extract
was responsible for transformation.

Proof That the “Transforming Principle “ Is DNA

The “transforming principle” was shown to be DNA in 1944 when Avery, MacLeod, and McCarty
published the results of a set of extensive and laborious experiments. They showed that if highly
purified DNA from Type IIIS pneumococci was present with Type IIR pneumococci, some of the
pneumococci were transformed to Type IIIS (fig. 5.2). But how could one be sure that the DNA
was really pure? Proving the complete purity of any macromolecular substance is extremely
difficult. Maybe the DNA preparation contained a few molecules of protein and these
contaminating proteins were responsible for the observed transformation. The most definitive
experiments in Avery, MacLeod, and McCarty’s “proof” that DNA was the transforming principle
involved the use of enzymes (proteins that catalyze specific metabolic reactions) that degrade
DNA, RNA, or protein. In separate experiments, highly purified DNA from Type IIIS cells was treated
with

1) Deoxyribonuclease (“Dnase,” which degrades DNA),

2) Ribonuclease (“Rnase,” which degrades RNA), or

3) Proteases (which degrade proteins)

And then tested for its ability to transform Type IIR cells to Type IIIS. Only Dnase had any effect on
the transforming activity of the DNA preparation; it totally eliminated all transforming activity (fig.
5.2).

Although the molecular mechanism by which transformation occurred remained to be worked out
in subsequent investigations, the results obtained by Avery and coworkers clearly established that
the genetic information in pneumococcus was present in DNA. We now know that the segment of
the DNA in the chromosome of pneumococcus that caries the genetic information specifying the
synthesis of a Type III capsule is physically integrated into the chromosome of the Type IIR
recipient cell by a specific recombination process occurring during transformation (see chapter 8)

The “Hershey-Chase Experiment”

Additional direct evidence indicating that DNA is the genetic material was published in 1952 by A.
D. Hershey (1969 Nobel Prize winner) and M. Chase. These experiments showed that the genetic
information of a particular bacterial virus (bacteriophage T2) was present in DNA. Their results,
although probably less definitive than the results of Avery, MacLeod, and McCarty, had a great
impact on the acceptance by sciencetists of DNA as the genetic material. This large impact
undoubtedly was the result of the elegant simplicity of the so-called “Hershey-Chase experiment”.

Viruses are the smallest living organisms; they are living at least in the sense that their
reproduction is controlled by genetic information stored in nucleic acid via the same processes as
in cellular organisms. Viruses, however, are acellular obligate parasites that can reproduce only in
appropriate host cells. Their reproduction is totally dependent on the metabolic machinery
(ribosomes, energy-generating systems, etc.) of the host. Viruses have been extremely useful in
studying many genetic processes because of their simple structure and chemical composition
(many contain only proteins and nucleic acids) and their very rapid reproduction (15-20 minutes
for some bacterial viruses under optimal conditions).

Bacteriophage T2, which infects the common colon bacillus Escherichia coli, is composed of about
50 percent DNA and about 50 percent protein (fig. 5.3). Experiments prior to 1952 had shown that
all Bacteriophage T2 reproduction takes place within E. coli cells. Therefore, when Hershey and
Chase showed that the DNA of the virus particle entered the cell, whereas most of the protein of
the virus remained adsorbed to the outside of the cell, this strongly implied that the genetic
information necessary for viral reproduction was present in DNA. The basis for the Hershey-Chase
experiment is that DNA contains phosphorus but no sulfur, whereas proteins contain sulfur but no
phosphorus. Thus, Hershey and Chase were able to specifically label either

1) The phage DNA by growth in a medium containing the radioactive isotope of phosphorus,
32
P, in place of the normal isotope, 31P, or
 2) The phage protein coats by growth in a medium containing radioactive sulfur, 35S, in place
of the normal isotope, 32S (fig. 5.3).

When T2 phage particles labeled with 35S were mixed with E. coli cells for a few minutes and were
then subjected to shearing forces by placing the infected cells in a Waring blender, it was found
that most of the radioactivity (and thus the proteins) could be removed from the cells without
affecting progeny phage production. When T2 phage in which the DNA was labeled with 32P were
used, however, essentially all the radioactivity was found inside the cells, that is, it was not subject
to removal by shearing in a blender. The sheared-off phage coats were separated from the
infected cells by low-speed centrifugation which pelets (sediments) cells while leaving phage
particles suspended. These results indicated that the DNA of the virus enters the host cell,
whereas the protein coat remains outside the cell. Since progeny viruses are produced inside the
cell, Hershey and Chase’s results indicated that the genetic information directing the synthesis of
both the DNA molecules and the protein coats of the progeny viruses must be present in the
parental DNA. Moreover, the progeny particles were shown to contain some of the 32P, but none
of the 35S of the parental phage.

However, the Hershey-Chase experiment did not provide unambiguous proof that the genetic
material of phage T2 is DNA. A significant amount of 35S (and thus protein) was found to be injected
into the host cells with the DNA. Thus, one could always argue that this small fraction of the phage
proteins contained the genetic information. More recently, however, it has been possible to
develop conditions in which protoplasts (cells with the walls removed) of E. coli can be infected
with pure phage DNA. Normal infective progeny phage are produced in these experiments, called
transfection experiments, proving that the genetic material of such bacterial viruses is DNA.

RNA as Genetic Material in Small Viruses

As more and more viruses were identified and studied, it became clear that many of them contain
RNA and proteins, but no DNA. In all cases studied to date, it is clear that these “RNA viruses”
store their genetic information in nucleic acid rather than in proteins just like all other organisms,
although in these viruses the nucleic acid is RNA. One of the first experiment that established RNA
as the genetic material in RNA viruses was the so-called reconstitution experiment of H. Fraenkel-
Conrat and B. Singer, published in 1957. Fraenkel-Conrat and Singer’s simple, but definitive,
experiment was done with tobacco mosaic virus (TMV), a small virus composed of a single
molecule of RNA encapsulated in a protein coat. Different strains of TMV can be identified on the
basis of differences in the chemical composition of their protein coats.

By using the appropriate chemical treatments, one can separate the protein coats of TMV from
the RNA. Moreover, this process is reversible; by mixing the protein and the RNA under
appropriate conditions, “reconstitution” will occur, yielding complete, infective TMV particles.
Fraenkel-Conrat and Singer took two different strains of TMV, separated the RNAs from the
protein coats, and reconstituted “mixed” viruses by mixing the proteins of one strain with the RNA
of the second strain, and vise versa. When these mixed viruses were used to infect tobacco leaves,
the progeny viruses produced were always found to be phenotypically and genotypically identical
to the parent strain from which the RNA had been obtained (fig. 5.4). Thus, the genetic information
of TMV is stored in RNA, not in protein.

DNA Structure

The genetic information of all living organism, except the RNA viruses, is stored in DNA. What,
then, is the structure of DNA, and in what form is the genetic information stored? What features
of the structure of DNA allow for the transmission of genetic information from generation to
generation?

Nucleic acid, first called “nuclein” because they were isolated from cell nuclei by F. Miescher in
1869, are macromolecules composed of repeating subunit called nucleotides. Each nucleotide is
composed of

1) A phosphate group

2) A five-carbon sugar (or pentose), and

3) A cyclic nitrogen-containing compound called a base (fig. 5.5).

In DNA, the sugar is 2-deoxyribose (thus the name deoxyribonucleic acid); in RNA, the sugar is
ribose (thus ribonucleic acid). There are four different base commonly found in DNA: adenine,
guanine, thymine, and cytosine. RNA also usually contains adenine, guanine, and cytosine,…

PLASMID AND EPISOMES

In the introductory section of this chapter, we stated that the genetic material of bacteria is carried
in one main chromosome plus, in many cases, from one to several extrachromosomal DNA
molecules or “ minichromosomes” called plasmids. By definition, a plasmid is a replicon (unit of
genetic material capable of independent replication) that is (stably inherited (maintained without
specific selection) in an extrachromosomal state. Most, but not all, plasmids are dispensable, that
is, they are not required for survival of the cell in which they reside. In many cases, however, they
are essential under certain environtment condition, such as in the presence of an antibiotic.

The importance of plasmids has become increasingly recognized during the last two decades.
Plasmids have been identified in almost all strains of bacteria tested. They are known to have
major practical significance in two areas:

1) The spread of multiple antibiotic and drug resistance in pathogenic bacteria and

2) The instability of industrially important microorganisms.

Multiple antibiotic and drug resistance will be discussed in some detail in chapter 9. In
Streptococcus lactis and related bacteria used in cheese processing, multiple plasmids have been
identified and shown to carry genes coding for enzymes important in the fermentation processes
involved in making chesses. These observations explain in part why the cheese “starter cultures”
of these bacteria are unstable and frequently must be discarded, at considerable expense to the
cheese-making industry (see chapter 24).

Three major types of bacterial plasmids have been extensively studied:

1) F and F’ plasmids, the conjugation fertility factors previously discussed;

2) R plasmids (previously called RTF, or resistance transfer factors), plasmids carrying genes
for resistance to antibiotics or other antibacterial drugs; and

3) Col plasmids (previously called colicinogenic factors), plasmids coding for colicins, which
are proteins that kill sensitive E. coli cells.

Plasmids are also known in bacteria that encode bacteriocins other than colicins. For example,
plasmids are known that code for vibriocins; these are proteins that kill sensitive Vibrio cholerae
cells. They are appear to be analogous to Col plasmids.