GENES ,were first detected and analyzed bv ‘Ich

del, and subsequently many other scientis by

following their patterns of transmission from genera

tion to generation (Chapter 2). These studies, while

greatly elucidating the nature of inheritance in living

organisms, provided no insight into the structure o

molecular composition of genes.

e precise corre

lation between the patterns of transmission of genes

from generation to generation (segregation and independent assortment) and the behavior of chromo-

some during sexual reproduction, specifically the

reduction division of meiosis and fertilization (Chap-

ter 3). These and related experiments provided strong

early evidence that genes are usually located on chromosomes. Thus, in posing questions about the
chem

ical basis of heredity, scientists began by probing the

biochemical composition of chromosomes.

Whatever its chemical composition, it was clear

even in Mendel's time that the genetic material had to

fulfill two key requirements.

1. The genotype function or replication. The genetic

material must be capable of storing genetic information

and transmitting this information faithfully from parents

to progeny, generation after generation (although, as

we will see in Chapter 11, the genetic material does

undergo occasional heritable changes called mutations).

2. The phenotype function or gene expression. The genetic material must control the development of
the

phenotype of the organism, be it a virus, a bacterium, a

plant, or an animal such as a human being. That is, the

genetic material must dictate the grow th and differentiation of the organism from the single-celled
zygote to

the mature adult. To control this complex process, the

genetic material must not only express itself accurately

but each gene must act at the proper time and piace to

guarantee that the liver is made up of liver cells, the

nervous system of nerve cells, and so on (see Chapters

10 and 15).

Chromosomes are composed of two types cf large

organic molecules (macromolecules) called proteins

and nucleic acids. The nucleic acids are of two types:

deoxyribonucleic acid (DNA) and ribonucleic acid

(RNÁ). For many years, there was considerable dis-

agreement among scientists as to which of these three

macromolecules carries the genetic information. Dur-

ing the 1940s and early 1950s, several elegant experiments were carried out that clearly established that
the

genetic information resides in the nuclei: acids rather

than in proteins. More specifically, these experiments

showed that the genetic information resides in DNA.

In a few simple viruses, however, RNA carries the

genetic information; these particular viruses contain

no DNA.)

DNA, THE GENETIC MATERIAL

Se era lines of indirect evidence have long suggested DNA contains the genetic information of living

organisms. Most important, results obtained using sev

era different experimental procedures showed that

most of the DNA is located in the chromosomes,

whereas RNA and proteins are also abundant in the

Cytoplasm. Moreover, a precise correlation exists be

tween the amount of DNA per cell and tt.e number of

sets of chromosomes per cell. That is, most somatic

cells 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 cells of

an organism is the same (with rare exceptions),

whereas the composition of RNA and proteins varies

th qualitatively and quantitatively from one cell type

to another. Although these correlations strongly sug.

gest 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. Macleode, and M.
McCarty in

1944. They demonstrated that the component of the

cell responsible for the phen menon of transformation in the bacterium Diplococcus pneumoniae
(pneumococcus) is DNA. Transformation is a mode of

recombination (exchange or transfer of genetic in-

formation between organisms or from one organism

to another) occurring in several, but not all, species of

bacteria. It does not involve direct contact between the

bacterial cells or mediation by any vector such as a

virus (see Chapter 8, pp. 206-208).

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 Ave

MacLeod, and McCarty, they provided no evidence that

DNA was involved in ar.y way

Pneumococci, like all other living organisms, en-

hibit 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 pres

ence or absence of a surrounding polysaccharide

(complex sugar polymer) capsule and (2) the type of

capsule, that is, the specific 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 quite pathogenic

most mammals (e.g., causing pneumonia in humans

These virulent (disease causing) Type S pneumococci

mutate to a nonvirulent (or nonpathogenic) form that

has no polysaccharide capsule (at a frequency of abour

one cell in 10). Such nonencapsulated, nonvirulent

pneun.ococci 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 differ-

ent 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 im

munologically. 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 specif

ically 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 in

jected heat-killed Type IIIS pneumococci (virulent

when alive) plus live Type IIR pneumococci (nonviru

lent) into mice, many of the mice succumbed to pneu

monia, 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 died. The observed virulence was

therefore not due to a few Type IIS 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 "trans

formation" 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 conrert living

Type IIR cells to Type IIIS.

Subsequent experiments showed that the phe-

nomenon-described by Griffith, now called transfor

mation, was not mediated in any way by a living host.

The same phenomenon occurred in the test tube when

live Type IR 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 transformatio 1.

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 bigbly purified DNA from Type

ills pneumococci was present with Type IIR pneu

mococci some uf the pneumococci were trans-

formed 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 difficul: Maybe the DNA preparation con-

tained a few molecules of protein and these contami-

Figure 5.1 Grifith's derronstration of transiormatio i

eumococcus. When heat-killed encapsulated (designated

S for smooth colony formation) Type III pneumococci were

injected into mice, the mice did not develop pneumon

Similarly, when living nonencapsulated (designated R for

rough colony formation) Type Il cells were injected into

mice, the mice showed no ill effects. Injection of living Type

IIS pneumococci resulted in severe pneumonia ad the

death of many of the mice. Surprisingly, the injection of

heat-killed Type IlIS cells (virulent if alive) together with

living Type IIR cells (nonvirulent) caused the death of many

of the mice.

nating proteins were responsible for the observed

transformation. The most definitive experiments in

Avery, MacLeod, and McCurty's "proof" that DNA was

the transforming principle involved the use of en-

zymes (proteins that catalyze specific metabolic reac-

tions) that degrade DNA, RNA, or protein. In separate

experiments, highly purified DNA from Type II'S 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 IIS. Only DNase had any effect
on the transforming activity of the DNA preparaion, it

totally eliminated all transforming activity (Fig. 5.2).

Although the molecular mechanism by which

transformation occurred remained to be worked out

in subsequent investig ations, the results obtained by

Avery and coworkers clearly established that tt.e genetic information in pneumococcus was present in

DNA. We now know that the segment of the DNA in the

chromosome of pneumococcus that carries the genetic information specifying the synthesis of a Type Ill

capsule is physically integrated into the chromosome

of the Type IIR recipient cell by a specific recombina-

tion 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. ler-

shey (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, Macleode, and Mc-

Carty, had a great impact on the a

tists 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

acids 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 thè metabolic

machinery (ribosomes, energy-generating systems

etc.) of the host. Viruses have been extremely useful in

studying many genetic processes because of their sim-

ple structure and chemical composition (many con-

tain 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 co-

lon bacillus Escherichia coli, is composed of about 50

pei cent DNA and about 50 percent protein (Fig. 5.3).

E: periments prior to 1952 had shown that all bacte-

riophage T2 reproduction takres place within E. coli

cells. Therefore, when Hershey and Chase showed that

the DNi rof beviris partide emered the cdi, wherens

most of the protein of the virus remained adsorbed to

the outside of the cell, this strongly impliod that the

genetic information necessary for viral reprod i tion

was present in DNA. The basis for the Hers e -Cl ase
Figure 5.2 Avery, Macleode, and McCarty's proof that the

transforming principle" is DNA. Transformation of T pe lIIR

pneumococci to Type IIIS could be demonstrated using

highly purified DNA from Type IIIS cells as well as using

heat-killed Type IIIS cell.i. Proof that the active c

was DNA and not small amounts of cor aminating RNA or

protein was accomplished by treating the purified DNA with

the enzymes DNase, RNase, and trypsin (a protease), which

very specifically degrade DNA, RNA, and protein, resped

tively. Treatment with RNase or protease had no eff ct on the

ability of the purified DNA preparation to transform Type IIR

cells to Type IIIS. DNase treatment destroyed the transform-

ing activity of the DNA preparation.

experiment is that DNA contains phosphorus but no

sulfur, wbereas proteins contain sulfur but no phos

phorus. Thus, Hershey and Chase were able to specif-

ically label either (1) the phage DNA by growh in a

edium containing the radioactive isotope of phos-

phorus, 32P, in place of the normal isotope, 51P, or (2)

the phage protein coats by growth in a medium con

taining radioactive sulfur, 35S, in place of the normal

isotope, 32s (Fig. 5.3). When T2 phage particles labeled

with 3 S 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 D

was labeled with 2P 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 pel

lets (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 5SS

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 prcteins contained the

genetic information. More recently, however, it has

been possible to develop conditions in which proto-

plasts (cells with the walls removed) of E. coli can be

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 in-

formation in nucleic acids

like all other organisms, although in these viruses the

nucleic acid is RNA. One of the first experiments 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

rather than in proteins just

Figure 5.3 The "Hershey.-Chase experiment": evidence

that DNA is the genetic material in bacteriophage T2. Eschr

ichia coli cells were infected with 32P-labeled phage (D

labeled), and after being allowed time for infection, they

were agitated in a blender, which sheared off the phage

coats. The phage coats and the infected cells were then

separated by centrifugation. Radioactivity was measurea in

the cell pellet (the sediment) and in the phage coat suspen

sion. Most of the radioactivity was found in the cells. Wher.

the same experiment was done using phage with 5"S tabeled

proteins, the results were very different. Most of the radio-

activit, was found in the suspension of phage coats; very litle

rtered the host cells. Since phage reproduction (both DNA

synthe sis and new-coat protein synthesis) occurs inside the

infected ce ls, and since only the phage DNA enters the host

cell, the DNA, not the protein, must carry the genetic infor-

maticn. (Based on R. Sagar and F. J. Ryan, Cell Heredity

Vil-y, New York, 1961.)

experinent 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 ide ntified 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

proteins and the RNA under appropriate conditions,

"reconstitution" will occur, yielding complete, infec-

tive 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 vice versa. When these mixed vi-

ruses were used to infect tobacco leaves, the progeny

viruses produced were always found to be phenotypi

cally 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.

The ge etic information of all living organisms, except

the RNA viruses, is stored in DNA. What, then, is the

structure of DNA, and in what form is the genetic

information storea? What features of the structure of

DNA allow for the transmission of genetic information

from generation to ge eration?

Nucleic acids, first-called "nuclein" because they

were isolated from cell nuclei by F. Miescher in 1869,

are macromolecules composed of repeating subunits

called nucleotides. Each nucleotide is composed of

(1) a phosphate group, (2) a five-carbon sugar (or

pentose), and (3) a cyclic nitrogen-containing com-

pound called a base (Fig. 55). 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 bases commonly found in

DNA: adenine, guanine, thymine, and cytosine. RNA

also usually contains adenine, guanine, and cytosine,

Figure 5:4 Proof that the genetic material of tobac

saic virus (TMV) is RNA, not protein. The RNA r olecules and

the protein coats of two different strains (A and B) of TMV

were separated biochemically. The RNA of strałi A wa t.en

mixed with the protein coats of strain B unler conditit.as

where complete, infective virus particles are reconstitute

When the reconstituted viruses were rubbed cnto live to-

virus particles are reconstitute

bacco leaves, the prog ny viruses were phenotypically and

genotypically identiçal to strain A from which the RNA was

obtained and unlike strain B from which the protein was

obtained When the reconstitvred viruses contained RNA of

type B and protein of type A, the progeny were of type l

(After II. Fracnkel-Conrat and B. Singer, Biochinm. Biophys

Acta 24: 540, 1957.)