chapter 4

THE SUBSTRUCTURE
OF GENES

A: we pointed out in Chapter 1, most evolutionists
feel that all levels of evolution can be interpreted in terms of a single
basic process, the natural selection of organisms whose phenotypes
have been modified in some favorable way by gene mutation. Dur-
ing the earliest stages of evolutionary development, the sudden ap-
pearance of new genes must have been a frequent and important
occurrence. However, it is, perhaps, not too heretic to suggest that
the major proportion of evolution has been the result of a continual
process of modification, and integration into new systems of organi-
zation, of genetic potentialities already present in our extremely dis-
tant ancestors. (We shall consider the evidence for the antiquity of
some of our own genes in a later chapter.) Although evolution of the
sort undergone by the Gryphaea (page 7) is generally referred to as
microevolution, such a process is many orders of magnitude morc
complex than the process that can be demonstrated in favorable test
objects like bacteriophages. With bacteriophage we may detect
“ultramicroevolutionary” changes caused by even extremely infre-
quent single-gene mutations. The morphology and general physiol-
ogy of these most elementary “organisms” are discussed in the fol-
lowing pages, together with a consideration of the progress of “fine-

67
68

    
 

~ 71012

71020

   

  

of ge" °

fase ca i

r 1019

 

 

THE MOLECULAR BASIS OF EVOLUTION

structure genetics” which has been so stimulated by work on bac-
teriophages and on their bacterial cell hosts.

Bacteriophages were discovered by D’Herelle in 1917. He demon-
strated that these were extremely small particles, invisible by ordi-
nary microscopy, which multiply parasitically within their host and
escape in a burst when the bacterial cell wall ruptures. Later
studies by a number of investigators, among whom Delbriick,
Demerec, Luria, and Burnet deserve particular mention, showed that
the bacteriophage bacterial cell microcosm exhibited many elegant ex-
amples of evolutionary adaptation.

A particularly cogent example is presented by the case of the r
mutants of bacteriophage T4. Each wild-type T4 particle produces
a small, fuzzy plaque (Figure 34), when grown on agar containing
E. coli of strain B. This plaque type is one of the phenotypic char-
acters of this particular phage. From time to time these wild T4
undergo a mutation which introduces a new phenotypic character.
Thus we may isolate, from individual plaques of wild T4, mutants
that produce quite a different sort of plaque, nine of which are shown
in Figure 34. In spite of the identical appearance of plaques pro-
duced by all these r mutants on E. coli B, we may easily distinguish
several different sorts of r mutants by employing a new bacterial
host. Thus, as shown in Figure 35, when the nine r mutants are
plated on E. coli K rather than B, three subgroups become evident;
two of the mutants still form r-type plaques, one forms wild-type
plaques, and six do not multiply at all. Whereas on strain B all the
mutants produce a visible effect, on K some are visible (the r
plaques), some are “innocuous” (wild type), and some are lethal.
Escherichia coli B, therefore, supplies something which is not sup-
plied by K, at least for the latter r mutants. In a sense, K may be
thought of as an ecological niche which is not attainable by these
“species” of T4 bacteriophages until a mutation has occurred which
makes the “something” in B no longer of importance.
<4 __
Figure 34. Plaques formed on E. coli B by wild-type bacteriophage T4 (left)
and by nine independently arising r mutants, As described in the text, wild-
Type T4 particles infect and multiply within E. coli cells of strain B to produce
the small plaques shown. The progeny infect and lyse neighboring bacterial
cells to produce plaques, which are actually areas in the E. coli B-agar mixture
that have become translucent owing to lysis of the bacterial suspension; r mu-
tants produce the large plaques shown in the nine photographs at the right of the
figure. The plaques may easily be distinguished from those produced by the

wild-type bacteriophage particles by their characteristic plaque morphology.
From S. Benzer, The Chemical Basis of Heredity, Johns Hopkins Press, 1957.

 

THE SUBSTRUCTURE OF GENES 69
Another point of evolutionary interest can be illustrated by these
observations. The r mutants which behave like wild-type phage on
E. coli K are not wild type as is shown by their behavior on B. In
terms of evolutionary adaptation, these particles are very much analo-
gous to the reptiles which we discussed in Chapter 1. The reader
will recall that changes occurred in the structure of the jaw of
reptiles which were not of apparent adaptive value until millions
of years later when certain of the bones of the reptilian jaw became
important units in the assembly of the characteristic mammalian ear.
These changes (call them “aristogenic” if you like) could not be
taken advantage of until the proper ecological situations made their
presence of great value to the budding mammal. The “false” wild-
type phage mutants, in a similar manner, are the possessors of muta-
tions which are, at least superficially, neither “detrimental” nor “ad-
vantageous” in a world of E. coli K, but which facilitate a marked
change in phenotype in a world of E. coli B. These r mutations
might be preserved for a long time, to become of critical importance
when environmental (host cell) conditions accidentally changed from
K to B. More likely, the abnormal gene would eventually back-
mutate to the normal allelic form and be forgotten as an evolutionary

ripple.

r 1014
r 1020

 

r 1013
r1019

  

The Chemistry and Enzymology of Bacteriophages

Although a number of different sorts of bacteriophages have been
studied with respect to their chemical composition, morphology, and
genetic constitution, we shall restrict our discussion to the so-called
T-even (and particularly T2) phages since these have been most
commonly chosen for study by virologists and are by far the best
understood. These viruses, which infect and kill cells of E. coli
B, may be divided into major groups on the basis of their ability

r 1015
r 1018

  

 

®<¢ * or inability to invade E. coli B cells of certain types. Thus E. coli
é Qe oa wm * © ‘ B6 will support growth of T2 and T4 viruses, B4 will support T2 and
* ss ° 5 T6 but not T4, and so on. They may be further distinguished on the
Xess ©. oth ° = basis of their plaque morphology, the rate at which such plaques

'e * #6 o <
“ 9 . ° Figure 35. When the nine r mutants, shown in Figure 34, are plated on E. coli
‘ Oo os K rather than on strain B, three subgroups become distinguishable. Two mutants

‘ continue to form plaques similar to those formed on E, coli B, one forms wild-
type plaques, and six do not multiply at all. From S. Benzer, The Chemical
Basis of Heredity, Johns Hopkins Press, 1957.

70 THE MOLECULAR BASIS OF EVOLUTION THE SUBSTRUCTURE OF GENES 71
 

(a) (b)

Figure 36. Electron photomicrographs of (a) bacteriophage T2 and of (b)
“ghosts” prepared by osmotic shock. These photographs were obtained through
the kindness of Dr. Roger Herriott. From R. M. Herriott and J. L. Barlow.
J. Gen. Physiol., 40, 809 (1957).

become visible under standard conditions of agar plate culture, and
their specific serological characteristics.

The virus particles can be visualized in the electron microscope,
and the morphology of T2 coliphage, which is more or less typical
for all the T phages, is shown in Figure 36. The particles are made
up of a rounded hexagonal head and a tail. The tip of the tail is
slightly expanded into a knob-like structure. This can be deduced
from the outlines of some of the shadows in the electron micrograph.

Intact phage particles consist of approximately 60 per cent protein
and 40 per cent DNA, together with traces of lipid material. The
nucleic acid component is entirely of the DNA variety and, as dis-
cussed earlier, contains the unusual pyrimidine 5-hydroxymethylcyto-
sine partly in glucosidic combination with glucose. It was believed
for some time that the DNA and protein were tightly conjugated in
nucleoprotein form. It can be shown, however, that the DNA is
actually physically enclosed within a protein shell, the so-called
bacteriophage “ghost,” and can be released from the interior of
the particle by suitable treatment, whereupon it becomes digestible

72 THE MOLECULAR BASIS OF EVOLUTION

by deoxyribonuclease. In Figure 36 is shown a collection of such
ghosts which have been prepared by submitting virus particles to
“osmotic shock,” that is, rapid dispersion into distilled water after
prior equilibration with strong salt solution. The ghosts may be
prepared in a form essentially devoid of polynucleotide by proper
washing. The separability of protein and DNA is elegantly demon-
strated by the fact that, when phages are doubly labeled with P*2
and S%5, the P82 is found entirely in the DNA released by “shock”
and the S#5 in the material of the ghosts.

Doubly labeled phage have also been used by Hershey and Chase
to study the fate of the various components of phage during infection
and rupture of bacterial cells. These investigators allowed the
labeled particles to infect E. coli cells and, after a brief period, ex-
posed the mixture to rapid agitation in a Waring Blendor. The
P8?-labeled DNA was found entirely within the infected bacterial
cells, whereas a large part of the protein was sheared off. The small
amount of $%5-labeled protein which adhered to the bacterial cells
after the agitation procedure did not contribute to the structure of
the progeny phage produced when the infected cells were allowed to
incubate and rupture spontaneously. Since the infected cells which
were exposed to the blender produced a normal yield of progeny,
it may be concluded that DNA alone is capable of directing the
synthesis of new particles, genotypically identical with the parent
phage, and that the protein shell around the DNA makes no contri-
bution to the process of genetic information transfer. Although lack-
ing any genetic role, the protein ghost is responsible for many
features of bacteriophage infection in addition to its function as an
enclosure for DNA. Ghosts can attach to compatible host cells or
cell walls, that is, they bear the host specificity of the intact virus (see
Chapter 8). Cells which have been “infected” with ghosts are
killed and subsequently lyse, although no progeny phage are pro-
duced. Ghosts also have the mysterious ability to cause inhibition
of RNA synthesis and protein synthesis within the host cell.

It has been known for some time that small molecular fragments
of the bacterial cell wall are released shortly after infection by an
enzyme present in both intact viruses and ghosts. This material has
been identified as a mucopeptide derivative having the interesting
structure shown in Figure 37. Now, although the nature of the
natural substrate for the enzyme, lysozyme, is not known with pre-
cision, a number of observations suggest that the function of lysozyme
is to attack mucopolysaccharide or mucoprotein compounds. It will
degrade chitin, for example, which is a long-chained_poly-N-acetyl
glucosamine, although it will not attack this substance after de-

THE SUBSTRUCTURE OF GENES 73
N-acetylglucosamine portion

0
ll
| pew ~~~
0 4
Z

of N-acetylmuramic
acid)

O—(To next residue of

|

|

|

|

|
(To preceding residue-|-

|

|

I

|

| N-acetylglucosamine)

 

oO
CH3;—-CH-—C— (Peptide containing
alanine, glutamic acid,
and diaminopimellic
acid, or lysine) 4

Figure 37. A proposed structure for the fragment which is produced from the
cell walls of certain Gram-positive bacteria by digestion with lysozyme. The
arrows indicate the probable site of attack by the enzyme. Redrawn from
W. Brumfitt, A. C. Wardlaw, and J. T. Park, Nature, 181, 1783 (1958).

acetylation. The chemical nature of the fragment split off from the
cell wall is sufficiently reminiscent of chitin to suggest that the viral
enzyme responsible for bacterial penetration is, indeed, of the lyso-
zyme variety. Dreyer and Koch? have recently compared the action
of crystalline egg white lysozyme and virus ghosts on bacterial cell
walls and have found that a very similar cleavage product is released
by both catalytic agents. Further, the enzymatic activity in the
ghosts distributes in the same way as the egg white enzyme during
purification with the specific adsorbant, Bentonite, and can be
chromatographed on the ion exchange resin, XE-64, to give a similar
elution pattern. Chemical comparison of the two lysozymes has not
yet been made, so we cannot state categorically that the virus enzyme
has structural features in common with the egg white enzyme. The
chromatographic properties and similar molecular size of the phage
enzyme (slowly dialyzable through cellophane membranes) makes
this similarity rather likely, however, and sequential analysis should
give very interesting results. It will be of obvious importance, in
connection with the “age” of genes and with the evolutionary
process in general, to determine whether or not an enzyme designed

74 THE MOLECULAR BASIS OF EVOLUTION

THE SUBSTRUCTURE OF GENES

to digest cell wall material is an essential component even in “semi-
living” biological systems like the coliphages.

Morphology of T Phages

Some idea of the number of morphological components making up
the bacteriophage particle may be obtained by electron microscopic

 

Figure 38. An electron photomicrograph of a crude lysate of bacteriophage T2
prepared by treatment of infected E. coli cells with chloroform. The photo-
graph shows intact bacteriophage particles and a collection of “heads,” rods, and

filaments. This photograph was obtained through the kindness of Dr. E. Kellen-
berger of the University of Geneva.

75
 

Figure 39. An electron photomicrograph of partially purified rods from the lysate
shown in Figure 38. Many of these rods may be seen to terminate in a brush-
like arrangement. This photograph was obtained through the kindness of Dr.
E, Kellenberger of the University of Geneva.

examination of a phage which has been subjected to various degrada-
tive treatments (peroxide, cadmium cyanide, N-ethylmaleimide)
which split off from the body of the phage certain parts of the tail
structure. We may also examine the contents of artificially lysed
bacterial cells which are still in the process of synthesizing phage.
The electron micrograph shown in Figure 38, for example, was made
by Kellenberger and Sechaud on the crude lysate of T2 prepared by
treatment of infected E. coli cells with chloroform. The photograph
shows, in addition to intact phages, a collection of empty heads,
rods, and filaments, the latter very likely being strands of DNA
which have not yet been wrapped in their protein envelope. These
phage components may be concentrated as shown in Figure 39. The
rods, which must constitute a large part of the tail structure, are
seen in many cases to terminate in a wire-brush-like arrangement.
In some electron micrographs we can distinguish rod-like structures
which are somewhat smaller in diameter than the intact viral tail

76 THE MOLECULAR BASIS OF EVOLUTION

and which have been interpreted as a sort of plug in the tail which
confines the DNA to the head of the phage. Two other phage com-
ponents, not visualized by the electron microscope, are the pene-
tration enzyme (lysozyme?) and the protein material responsible
for host range specificity. The latter may be associated with one
of the morphologically distinguishable tail components.

The drawing in Figure 40 summarizes, in a schematic way, the
subunits of structure which have been detected microscopically.
A slightly different reconstruction has been proposed by Kozloff,
Lute, and Henderson.?__ This diagram helps to point out one of the
major puzzles of bacteriophage biosynthesis, namely, the problem of
how the DNA strand or strands are gotten into the preformed heads,
if these are indeed synthesized before DNA is. We might visualize
that either 'an enzyme system, rich in genetic information, is able to

  
   

Head
protein coat

Tail sheath
(contractile?)

    
 

“Core” with hole
through center

    
   
 
  
 

Lysozyme activity
presumably in
distal tail region

   
  

Tail fiber
(Some coiled around
tail spike and

some unwound;
bears host-range
specificity?)

Figure 40. A schematic reconstruction of the morphology of a bacteriophage
particle. The dimensions shown are approximate and are averaged from several
sources (see, for example, the excellent work of R. C. Williams and D. Fraser
Virology, 2, 289 (1956).

THE SUBSTRUCTURE OF GENES 77
 

carry out DNA synthesis within the head (perhaps in cooperation
with the protein of the head itself) or that DNA can thread itself
into the head after synthesis on the outside. The possibility must not
be overlooked, however, that phage fragments really represent abor-
tive attempts at synthesis and that the protein shell and tail of com-
pleted phage particles are assembled around a preformed mass of
DNA. The latter possibility is supported by a certain amount of
experimental evidence and certainly makes the best sense, meta-
bolically and mechanically. For example, Hershey has shown with
isotopic tracer methods that the DNA of phage is synthesized before
the protein precursors and that the small amount of protein synthesis
which occurs before DNA synthesis may form some essential enzymes
(perhaps for the synthesis of the unique pyrimidine, or for the for-
mation of a ribonucleic acid “template”) which are not otherwise
present in the bacterial host.

The Molecular Heterogeneity of Coliphage DNA

The total phosphorus content of a single T2-phage particle is
about 210-7 grams. If all this phosphorus resides in a single
DNA molecule, we may calculate the total molecular weight of this
DNA to be approximately 110 million. Should the Watson-Crick
type of structure apply in phage, we are faced with the problem of
how each enormously long polynucleotide strand becomes replicated
during phage multiplication and how the two intertwined strands in
the double helix unwind during, or in preparation for, this event.

This problem has been somewhat simplified by the observations of
Levinthal and Thomas.* Their experiments indicate that the DNA of
phage may not be in a single large unit but may be made up of
a number of smaller parts, the largest one of which makes up about
40 per cent of the DNA. The technique employed by them is a
particularly elegant example of simplicity combined with sensitivity
and precision. Levinthal and Thomas embedded bacteriophage par-
ticles, previously labeled with P32, in an electron-sensitive emulsion
in which the passage of fast electrons, such as are emitted by P#?
during radioactive decay, causes the formation of a track of silver
grains. A single phage particle, acting as a “point source,” can thus
cause the formation of a “star” in the emulsion, each arm of which
is produced by the decay of a single atom of phosphorus within
the DNA of the particle (Figure 41). The sensitivity of the detect-
ing method is such that the radioactivity of a phage particle, emitting

78 THE MOLECULAR BASIS OF EVOLUTION

 

Figure 41. Photomicrograph of a “star” produced by radioactive decay of labeled
phosphorus within the DNA of a phage particle embedded in a photographic
emulsion. This photograph was obtained through the kindness of Professor Cyrus
Levinthal, Massachusetts Institute of Technology.

only about fifteen disintegrations per month, can be estimated. In
uniformly labeled electron sources (e.g. the DNA of a single phage
particle or fragments thereof) the number of tracks per star gives a
measure of the molecular size of the emitting source, relative to the
intact phage particle.

The number of tracks per star was determined for intact phage and
for first- and second-generation progeny before and after osmotic
shock. The results for the various fractions are shown in Table 2,
expressed in terms of the number of P#? atoms in the star-forming
particle, normalized to 100 for the original uniformly labeled phage.
(Put in another way, if each star formed by intact parent phage
particles has 100 tracks, each star produced by particles in suspen-
sions of “shocked” phage, having half the content of DNA, will show
50 tracks.) The results indicate that (a) there is a single large
piece of DNA comprising about 40 per cent of the total DNA (i..,

THE SUBSTRUCTURE OF GENES 79
about 45 million in molecular weight), (b) after one generation in
nonradioactive bacteria there are, among the progeny, a few particles
in which this large piece of DNA has approximately half the P*?
atoms of the large piece in the parent phage, and (c) after a second
generation the size of this large piece does not change (the same
number of tracks per star is observed). Since some 100 to 200
progeny are formed from each parental phage, the number of labeled
particles in second generation shockates was too few to permit accu-
rate detection and track counting.

TABLE 2
The Number of Arms per “Star” for Various Suspensions of Intact or

Ruptured Phage Particles, Normalized to 100 for the Original, Intact

Particles®

 

Intact After

Phage Osmotie Shock
Uniformly labeled parental phage 100 40 +4
First-generation progeny 2443 3 +3
Second-generation progeny 26+3 Too dilute for study

 

The simplest and most appealing interpretation of these data is
that the large piece represents the “chromosome” of the phage and
that the two halves of its double helical structure serve as templates
for the synthesis of new DNA in the progeny. This wishful picture
is completely analogous to the scheme growing out of the Meselson
and Stahl experiments on E. coli DNA. There is, however, no com-
pelling evidence to indicate that the DNA of the T phages is in
the form of a double helix, although this is generally assumed in
most discussions of the subject for the obvious reason that it makes
discussion possible. .

The Levinthal-Thomas experiments suggest a “conservative mecha-
nism of replication during which the structure of the large piece of
DNA is not degraded or involved in exchange with external sources
of nucleotides. —_

It would have been pleasant, in connection with this discussion of
these experiments, to have been able to present some evidence for
the chromosome-like nature of the “big piece.” Attempts have been
made to show that, during the production of progeny phage, genetic
markers, chosen at random along the genetic strand, always accom-
pany the large star-forming fragment. The early experiments along

80 THE MOLECULAR BASIS OF EVOLUTION

this line looked quite encouraging but, of late, the picture has become
rather murky and, after consultation with some of the investigators in
the field, it appears that the subject is best avoided at the present
time. There are, for example, several lines of evidence which
suggest rather strongly that the “40 per cent piece” is not a single
molecule but that various mild treatments can cause this aggregate to
dissociate into still smaller units having molecular weights in the
neighborhood of 12 to 15 million. The results of the original “star”
experiments seem to be unequivocal and, indeed, chromatographic
separation on special columns of ion exchange resin have also shown
the presence in T2 DNA of an entity containing about 40 per cent
of the total phosphorus. Nevertheless, any attempt to relate the
chemical structure of phage DNA to genetic behavior must be de-
ferred until much more data have been obtained.

The Life Cycle and Biosynthesis of T Phages

It may be useful at this point to consider the events that occur
following infection of E. coli with T phages. Most of the metabolic
consequences of infection are summarized in Figure 42, which is a
slightly modified form of that given by R. Hotchkiss in his excellent
review of 1954, and in F igure 43, taken from an article by L. Kozloff
in the Cold Spring Harbor Symposia for 1953. Figure 42 gives a
condensed view of the fates of various morphological components of
the infecting phage and the host cell and also indicates, in a rough
way, the manner in which the metabolism of the combined DNA,
protein, and ribonucleic acids of the infected cell is altered. It is
assumed that the RNA of the host is, in some way, involved in the
biosynthesis of phage protein. This assumption is based on certain
general observations on protein synthesis which we shall discuss in
more detail in Chapter 10.

Kozloff’s pictorial summary of the origins of phage DNA and pro-
tein (Figure 43) emphasizes that the major share of phage protein is
derived from the nutrient medium. Of the newly synthesized phage
DNA, it would appear that most of the phosphorus is also exogenous
in origin, but that a considerable amount of host DNA is reutilized
as a source of purines and pyrimidines.

The kinetics of the various metabolic processes that commence after
phage infection are of particular importance in connection with the
elucidation of the way in which the genetic information in phage
DNA is translated into the chemical structure of progeny phage.

THE SUBSTRUCTURE OF GENES 81
Phage

Dit

- DNA

Protein

Not digested by deoxyribonuclease
until phage is damaged; 40-50%
of weight of phage.

Contains all phage sulfur, host
range specificity, penetration en-
zyme, killing power.

 

 

 

DNA Nuclear DNA of host.

RNA About three times as much as
Host DNA
cell is .

s Protein Enzymes of normal cell. Synthe-
sis of new enzymes may be induced
by proper substrates.

-DNA Phage DNA now accessible to
DNase if bacteria are ruptured;
40-50% goes to progeny.
:
--Protein 80% or more removed by Waring
O Blendor.
Infected Cell Cytologically disorganized. Syn-
cell Q | DNA _ thesis stops.
Cell Normal synthesis stops. New sys-
~~
protein tems develop.
Cell Only slight turnover after infec-
RNA tion.
YDNA 20-35% of P derived from host
! @ @) DNA |
rroseny, Protein Mostly from endogenous sources
150-300 particles of amino acids. Same host range
as parent phage.
+

 

Ge ar

 

82

Phage ghost still attached.

THE MOLECULAR BASIS OF EVOLUTION

When the host cell becomes infected, its metabolic machinery ap-
pears to become completely devoted to phage synthesis. Protein
synthesis goes on but produces a new set of proteins. After a short
delay, DNA synthesis proceeds actively. However, if the initial pro-
tein synthesis is prevented by the addition of a suitable inhibitor
such as chloramphenicol, the synthesis of DNA does not take place.
On the other hand, if the inhibitor is added after protein synthesis
has gotten under way, DNA synthesis can proceed. These observa-
tions suggest that an essential set of enzymes must first be formed,
perhaps for the synthesis of the unique 5-hydroxymethylcytosine or
its glucose conjugate, before DNA assembly is possible.

Stent and his colleagues have carried out interesting experiments‘
which have a direct bearing on the problem of the order of events
during information transfer. They infected P32-labeled cells with
P32-labeled phage, both having a sufficiently high level of isotope
to cause chemical modification of a significant fraction of the DNA
when the phosphorus atoms decayed to form sulfur atoms. The in-
fected cells were incubated in a P82 medium so that the progeny
phage were also heavily labeled. Samples of infected cells were
frozen at —196°C. at various times after infection and were stored
in this state of suspended animation. During this time P%? decay
took place. The surprising result was obtained that, in those samples
frozen approximately 10 minutes after infection, there was no de-
tectable change in the ability of the infected cells to produce infective
phage progeny in spite of the destruction of a considerable portion
of the DNA by radioactive “suicide.” The results have been in-
terpreted in several ways, the most provocative being the hypothesis

that, early in the synthesis of progeny, information in the DNA of

the infecting phage particle is transferred to some substance, perhaps
protein in nature, which is not susceptible to radioactive decay. An-
other speculation which could explain the results would require that
the structure of DNA be such that, in spite of occasional breaks in the
sugar-phosphate backbone of the molecule caused by P82 decay, hy-
drogen bonds hold the macromolecule intact and in a configuration
adequate for purposes of replication (Figure 44).

The possible conflicts in logic that might arise from a comparison
of the P82-decay data with the experiments of Levinthal and Thomas
or with the mass of evidence supporting the central role of a specific

4
<q

Figure 42. The course of infection of E. coli with bacteriophages of the T series.
Redrawn after R. D. Hotchkiss, The Nucleic Acids, volume 2, Academic Press,
1954.

THE SUBSTRUCTURE OF GENES 83
 
 

Bacterium

 
     

Liquid medium New virus

 

Paippo;) 70-80%

 

  

Cigiucosey 75-85% Synthetic

mechanisms

  
   

Nau?) 75-85%

 

 

Protein

Figure 43. Schematic representation of the origin of the phosphorus, nitrogen,
and carbon of bacteriophages T2, T4, and T6. Percentages show origin of
material found in viral offspring. Redrawn from L. N. Kozloff, Cold Spring
Harbor Symposia Quan. Biol., 18, 209 (1953).

DNA structure in the determination of heredity are not, to my
knowledge near resolution. Fortunately for the following discussion
of genetics in phage, we do not need an answer to the fascinating
problem of phage replication. Although the behavior of phage dur-
ing “mating” may only superficially resemble the process as it occurs
in more respectable organisms, the phenomena of recombination and
segregation, and of linkage, are exhibited in qualitatively the same
way.

Genetic Mapping in Bacteriophage

In most organisms it is not possible to construct a single gene
map, but instead we find that the various genes fall into several link-
age groups (see Chapter 2). However, in bacteriophage T4 and T2,
for which quite a few “genes” have been mapped, only a single

84 THE MOLECULAR BASIS OF EVOLUTION

linkage group seems to be present, and these phages behave geneti-
cally as an organism with a single haploid chromosome. The sort
of gene map obtained depends, naturally, on the strain of bacteria
chosen as host. As we have already shown, the plaque characteristics
of mutants that are r type on E. coli B may be quite different when
the mutants are plated in E. coli K. The map obtained with a par-
ticular host cell, therefore, includes only the loci that cause a detect-
able change in phenotype in this chosen environment.

The construction of a genetic map for bacteriophage involves more
or less the same manipulations that have been used for such or-
ganisms as Drosophila or peas. A wild-type strain is chosen arbi-

 

 

 

 

 

 

  

“Nonlethal decay”

Figure 44. A schematic representation
of how breaks in the chains of the DNA
dougle helix, caused by radioactive de-
cay of P32, might occur and still permit
the maintenance of a relatively intact
structure through the forces of hydrogen
bonding between complementary pairs
of purine and pyrimidine bases.

 

 

 

THE SUBSTRUCTURE OF GENES 85
trarily. Mutants are then chosen on the basis of some easily recog-
nized phenotypic character such as plaque morphology on a particular
strain of E. coli. The relative position of two mutant loci on the
“chromosome” of the phage may then be established by “mating”
the mutants and observing the proportion of the progeny that differ
from the two parental mutants. True mating does not, of course,
occur with phage. Instead, bacterial cells are mixedly infected with
the two phages in question, and recombination is allowed to take
place within the cell by some obscure process that has the superficial
characteristics of crossing over in higher organisms. As we discussed
earlier, conventional crossing over results in the production of the
two possible recombinants, the wild type and the double recombinant
containing both mutant loci. It has been shown that, in phage, such
reciprocal crossover does not take place, but that in any single re-
combination event only one or the other recombinant is formed
[perhaps because of a “copy-choice” replication (Figure 45)]. How-
ever, since a single phage particle gives rise to several hundred
progeny, and since there appears to be about the same probability
for the formation of the two kinds of recombinants, the usual statisti-
cal treatment may be applied.

Seymour Benzer* has, over the past few years, been in the process
of mapping a very large number of r mutants of bacteriophage T4.
These studies represent the first real attempt to determine the ulti-
mate limits of recombination and involve “running the map into the
ground,” to use an expression attributed by Benzer to Max Delbriick.
The detection and mapping of mutations in bacteriophages are facili-
tated by several factors, including the sensitivity of the technique for
detecting mutants and the occurrence of certain mutations which
involve the “deletion” of a considerable portion of the map. The
latter factor, as we shall see, has permitted Benzer to group many
point mutations into “families,” thus greatly decreasing the number
of crosses required.

In any population of T4 there occurs from time to time a spon-
taneous mutation which leads to the production of an r-type plaque
on E. coli B agar plates. Since an astronomical number of phage
particles may be plated on a single Petri dish surface, the presence
of a single mutant in as many as 10° particles is easily observed by
its r phenotype. When a suitable number of such mutants has been
accumulated, they may be further subdivided into three subgroups
as mentioned earlier, by plating on E. coli K. By this trick (see page
69) we may distinguish those that still show r morphology on K,
those that show the wild-type phenotype, and those that fail to grow

86 THE MOLECULAR BASIS OF EVOLUTION

i+

   
 
  

    
  

PURE PORN Pea Ne CIRO
oe TUL ‘tt SAAS RFA BOLLE ‘cD

NECA (0177/7 RHUL ALUM ee LUC TCU
BDL CCRT) TCA Le A ER

  
 

1+
1+

A @)

Fe saree AN Aha ko Deck an Ded OU Rn einer Ler om it Sher bean ma hentia eo Ned w ea re ie eee a
ae caslites beet ee ee eee ee rE ed tee er a ee

   
 

1+

    
 
 

 

 

Coe TANS MUH CUO
SAAN TIPee cecce ces

   
 

 

CUO NL
OSTA URAL eC

b

   

   

AL Aer

Figure 45. Three possible mechanisms of genetic recombination: A and B repre-
sent two genetic loci and a and b their alleles. (1) Fragmenting crossing over;
the two parental duplexes synapse and break between the loci in question, and
heterologous pieces rejoin. (2) Nonfragmenting copy choice. (3) Fragmenting
copy choice; parent duplexes unwind and semiconservative replication occurs
along the single strands. A switchover occurs between loci A and B as the
parent chains break, and the daughter chains continue to grow complementary to
a single chain of the second duplex. Redrawn from M. Delbriick and G. Stent
The Chemical Basis of Heredity, Johns Hopkins Press, 1957.

THE SUBSTRUCTURE OF GENES 87
Bacterial
host rl r

1 ru
strain WEA ——— [7 —-—-— +7.

Figure 46. The order of the three subunits of the r region in the genetic material
of bacteriophage T4. Since rII and rlII are farther apart than rII and rl, the
frequency of recombination between rII and rlII mutants should be greater than
that between rII and rl mutants,

at all. These three groups, termed the rI, rII, and rIII categories,
respectively, may be arranged in the proper linear order by perform-
ing mixed-infection crosses. As may be deduced from the scheme
in Figure 46, the frequency of recombination between members of
the rII and rIII groups would be greater than between mutants of
the rII and rl varieties, making the usual assumption of a linear ar-
rangement of “genes.”

Benzer has chosen to concentrate mainly on the riI region for his
genetic analysis. The rII mutants have one clear advantage from
the point of view of detection, in that they do not grow on K at all
and therefore give an unequivocal phenotypic test. Some thousand
mutants of the rll type could thus easily be selected for the mapping
project.

The rII mutants were further subdivided on the basis of the cis-trans
test. It will be recalled that this genetic trick enables us to de-
termine whether or not two mutants which show a similar phenotypic
abnormality are concerned with the same functional unit of heredity
or with two separate functions which interact, cooperatively, in pro-
ducing the change. As we discussed in connection with the “lozenge”
genes of Drosophila (Chapter 2), three closely linked genetic loci all
appeared to be part of the same functional unit since crosses between
double heterozygotes bearing both mutant loci on the same strand
of the chromosome duplex (i.e., in the cis arrangement) produced
wild-type recombinants, whereas crosses between double heterozy-
gotes in the trans arrangement did not. The adequacy of the cis
arrangement was explained on the assumption that the one, un-
marred, strand of the chromosome supplied the required unit of
physiological function in sufficient quantity to satisfy the needs of the
organism even in the presence of one “nonfunctional” chromatid.

In phage, although the genetic material behaves as though the
organism were haploid, the cis-trans test can still be applied since
mixed infection with two mutants appears to simulate the more con-
ventional diploid situation in higher organisms. Thus, when a mutant

88 THE MOLECULAR BASIS OF EVOLUTION

phage containing an r mutation is introduced into an E. coli K host
cell along with a wild-type phage particle, the cells are lysed and
both kinds of parent phage are liberated. The presence of the wild-
type presumably supplies the function missing in the r mutant (the
rll mutation is “recessive”). Although the test has not been made,
it is also assumed that mixed infection with a wild type, and with
a double r mutant bearing both mutations on the same strand, would
permit the formation of both sorts of progeny, and for the same
reason. When two r mutants are used, however, lysis of host cells
and progeny production will take place only if the two mutants are
deficient in different functional units. The various situations are
schematized in Figure 47. When two r mutants fail to lyse E. coli K
following mixed infection, the mutations are said to belong to the
same “cistron” (the name derived from the test employed). When
they supplement one another and cause lysis, they are said to belong
to different cistrons. By this sort of technique, Benzer divided the
rll mutants into two functionally different groups which he names
the A and B cistrons (Figure 48). (We might visualize that each
of the various phage-synthesizing enzymes, whose formation is in-
duced in bacteria by phage infection, is represented by a different
cistron. )

Now it is clear that to map a large number of mutant loci, even
after such further subdivision, would require an impossible number
of crosses, and Benzer has taken advantage of the fact that some of
the mutations appear to involve a change in much more than a point
locus. These unusual mutations are detected by the fact that, when

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

~ ++— + ——++-
! ! ——
Active Active Presumed active
4 v t i t
vet 1 © i
+) ta i |
vy if 1
Inactive Inactive Active

Figure 47. Schematic diagram showing the results of cis and trans configurations
of double “heterozygotes” bearing two rII mutations. Active means extensive
lysis of the doubly infected cells. Inactive means very little lysis. The presence
of one “unmarred” strand is pictured as being sufficient to supply the required
unit of physiological function.

THE SUBSTRUCTURE OF GENES 89
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

m
42
[
t
ve
a
a
we
oo ~~
7 7
oo A segment we
B segment
—_— _—_ _______-______>
~ ~~ a oo yA — ——
ao oN
“oo mS
a NL
“ ~~
a sw
“ +N
“eo ~s
a L ___164 Ps
“173 240 279 271 274 201 106
| { | Hue |
T I I } TH J
“7 ‘
a“ “
“7 ~N
“7 “
” NX
“7 NN
“7 NN
“7 ~
ae “Y
335 209
131
219 \\ 274 /7 333 155
2005)
0.014%
rt 0.017%
ECL hy 0.0000%
et
ae
EL 0.000%
rE
tH m = minute plaque mutant
MT r = rapid lysis mutant
tu = turbid plaque mutant

Figure 48, A partial linkage map of T4 bacteriophage. The successive drawings
indicate increasing orders of magnification of the rII region as revealed by the
fine-structure analysis of this region. This figure is highly schematic, and for de-
tails of its construction the reader should consult the article by S. Benzer in
“Mutation,” Brookhaven Symposia in Biol., No. 8 (1956). Redrawn by permis-
sion of the author.

90 THE MOLECULAR BASIS OF EVOLUTION

they are crossed with several other mutants which give wild-type
recombinants when crossed with one another, no detectable wild-
type recombinants are observed. The situation is explainable on the
basis of the chart shown in Figure 49. If mutant 6 represents a
“deletion” type of mutation, and 1 through 5 represent various other
ordinary point mutations, crosses between 6 and either 2, 3, or 4
will not yield wild-type recombinants since 6 is deficient in the same
properties that are missing in the other three. Wild-type recom-
binants could, however, be obtained from crosses between 6 and
either of 1 and 5 since there is no overlap. Using “deletion” mutants
for further screening within each of the two cistrons, Benzer was
able to simplify the task of mapping since it was then necessary to
perform crosses in all combinations only with the mutants that fell
within any given deletion region, rather than with all the mutants
in each cistron. We shall not describe, in detail, this part of the
mapping project. In spite of the shortcuts devised, the job was ob-
viously extremely laborious. The schematic map shown in Figure
50 was constructed from crossover data obtained on some of the
923 r mutants and shows, in addition to the location of “point” muta-
tions, the location of various of the deletion mutants (the horizontal
bars or lines) which were of such value in mapping. The map also

 

Figure 49. Schematic diagram showing the behavior of an “anomalous” mutant.
Mutant 6 fails to give wild-type recombinants with mutants 2, 3 and 4 located
within the same segment of the genetic strand. Wild-type recombinants are
given, however, with 1 and 5.

THE SUBSTRUCTURE OF GENES 91
92

B cistron

A cistron

 

287

271

320

205

145

168

638

 

w
(3)
rfl

ee

 

 

 

 

 

~
gia
me ON
~~ *x
ns 2
nN
— « a
«
a
ee
x
x Is
g = |*
&

 

al

 

nS
a
~
a
©
a

 

™
fo)
AY onl a] oy
‘SRI
i pe

vt
uw] [io fied ss
BRABER
tft ON LO

Se”

(27)
734

at

~~

i

%

)

 

47

 

 

386

 

 

eos] [168]
799
a

 

aS 2
mo
ae
sy %
zg =
Ea =
= oy
=

SEPREROSE EES ant
ee

FO as

665

199(Ry)r11

all of which are probably at the same locus
nternal order was not established. To appre-

Some regions of the genetic material appear to
e Chemical Basis of Heredity, Johns Hopkins

Parentheses indicate groups in which i

re A genetic map showing the locations of a number of rII mutants.
be “hot spots,” and at such localities a large series of mutants may be isolated

(e.g. the series which begins with 102).
ciate this map properly the reader should consult the article by S. Benzer in Th

Press, 1957.

Figure 50,

THE MOLECULAR BASIS OF EVOLUTION

incorporates the observation, made by Benzer, that a great many
separate mutations turn out to be in the same location. In other
words, some regions of the genetic material of the rII zone appeared
to possess a much higher mutability than others. The word “ap-
peared” is used advisedly since it must be remembered that the
detectability of a mutant depends on the phenotypic test. We have
seen, for example, that some r mutants behave on E. coli K as
though the mutation were lethal to the phage in this environment.
Others behave like wild-type phage. These classes are, of course,
not associated with the rII region as was just discussed. However,
we may postulate (and the postulation is supported in part by certain
observations which we shall discuss in Chapter 6) that the substances
whose synthesis is controlled by one or the other of the two cistrons
in the rII region contain some structural features of greater im-
portance than others. A mutation leading to modifications in the
functionally less critical parts of structure in a phenotypic protein
might be much less frequently detected since its influence on the
“r-ness” of an r mutant growing on K might be slight in comparison
with the effects of the mutations that cause a complete inability to
grow on K; that is, those that have caused modifications in the chem-
istry of a phenotypic protein of a magnitude which completely
abolishes function.

The cistron, conceived of by Benzer as the genetic unit of func-
tion, is clearly a very sophisticated structure. It contains many dis-
tinguishable genetic subunits as is indicated by the number of indi-
vidual loci, within its length, that can be detected by recombination.
Benzer, who like Levinthal is a converted physicist, has coined names
for the operational subunits within the cistron that have a delight-
fully physical ring. The recon is defined as the smallest element in
the one-dimensional array that is interchangeable, but not divisible,
by recombination. The muton is defined as the smallest element of
the cistron that, when altered, gives rise to a mutant form of the
organism.

The size of the recon can be estimated by isolating and mapping
so large a number of mutants within any given region that the dis-
tance between individual points on the map begin to approach the
indivisible unit. The recon is thus an empirical unit smaller than or
is equal to the smallest nonzero interval between pairs of mutants.
The length of the muton is determined by estimating the discrep-
ancy in map distances between closely linked sets of mutants. As
shown in Figure 51, the “length” of mutation 2 is equal to the dis-
crepancy between the long distance and the sum of the two short dis-

THE SUBSTRUCTURE OF GENES 93
 

 

 

 

 

 

To LL
L?
{3}
1
x2
1x3 ;
2x3
+=

 

 

 

Figure 51. Determining the “length” of a mutation. The discrepancy between the
long distance and the sum of the two short distances measures the “length” of the
central mutation.

tances. The muton would also be measurable by determining the
maximum number of mutations, separable by recombination, that can
be packed into a portion of the map.

It must be emphasized that the genetic units proposed by Benzer
are completely operational in meaning and origin. They serve the
important function of pointing up the complexity of the word “gene”
by making a clear distinction between the several operational com-
ponents of this word, namely, recombination, mutation, and function.
Although it is far too early to suggest that the observations made on
bacteriophages may be directly applicable to higher organisms, the
results at least give a rational basis for the consideration of such
phenomena as pseudoallelism. More important, the high density of
mapping attained and the indications therefrom of the size of the
recon now make it possible to discuss chemical structure and genetics
in the same breath. By making assumptions about the chemical
length of the genetic strand of DNA in phage (perhaps on the order
of 80,000 nucleotide pairs, in a double helix) and the total “genetic”
length (perhaps on the order of 800 recombination units), Benzer
is able to suggest that a single nucleotide pair might correspond to
about 0.01 recombination units. Since this distance is of the same
magnitude as that observed between some of the most closely linked
loci on the map (about 0.02 units), it is possible that the techniques

94 THE MOLECULAR BASIS OF EVOLUTION

employed have, indeed, been able to distinguish the genetic effect of
a modification in as few as two pairs of nucleotides in a DNA mole-
cule. If, as we may hope, the details of protein structure in an or-
ganism are the reflections of the chemical structure of its genetic
material, these calculations would suggest that a mutation may be
detectable in the phenotype when as little as a single amino acid re-
placement has occurred. The reader must view these speculations
with absolute candor. The hypothesis is under active experimental
test, and we must now rely on the protein chemist to help close the
gap between genetic speculation and chemical fact.

Before leaving the subject of genetic fine structure, brief mention
must be made of another technique for mapping the substructure of
genes. This technique, discovered by Zinder and Lederberg® in
1952, relies on a phenomenon known as transduction, in which ge-
netic information in bacterial chromosomes is transmitted from cell
to cell by bacteriophage particles. Certain bacteria may be in-
habited by so-called temperate bacteriophages which exist in a sym-
biotic arrangement with their host, wherein they only rarely lyse the
bacterial cell. When such lysis does occur, the liberated phage par-
ticles can infect new host cells and, in the process, carry with them
some of the genetic peculiarities of the original host. Only a single
phenotypic character is generally transduced at a time, as though
the phage were carrying with it a single gene. However, there is
occasional transfer of more than one genetic marker in a bundle, and
such events enable the investigator to determine the closeness of link-
age between two or more functional units of heredity.

The explanation might be offered for transduction in general that
parts of the “chromosome” of the phage have exchanged with parts
of the bacterial chromosome and that, following the establishment of
a new symbiosis, this newly acquired information is unloaded on the
new host. Many characteristics of transduction are highly reminis-
cent of transformation. In both, transfer of genetic information ap-
pears to depend on the physical transfer of DNA, either packaged as
in phage or free. Both phenomena, although generally involving
only a single genetic marker, may occasionally involve several.
Transduction has been employed by a number of investigators for
the mapping of genetic material in several bacterial species and per-
mits a degree of discrimination comparable to that achieved by
Benzer through bacteriophage crossing. However, since the con-
cept of “fine-structure genetics” is quite elegantly illustrated by the
bacteriophage approach, we shall defer to the excellent reviews listed
below and proceed to the next item of business, the protein molecule.

THE SUBSTRUCTURE OF GENES 95
REFERENCES ©

1. W. J. Dreyer and G. Koch, Virology, 6, 291 (1958).

2. L. M. Kozloff, M. Lute, and K. Henderson, J. Biol. Chem., 228, 511 (1957).

3. Discussed by C. Levinthal and C. A. Thomas in The Chemical Basis of Heredity.
See Suggestions for Further Reading,

4. Summarized in The Chemical Basis of Heredity. See Suggestions for Further
Reading.

5. N. D. Zinder and J. Lederberg, J. Bacteriology, 64, 679 (1952).

SUGGESTIONS FOR FURTHER READING

The Chemical Basis of Heredity (W. D. McElroy and B. Glass, editors), Johns
Hopkins Press, Baltimore, 1957.

Cold Spring Harbor Symposia on Quantitative Biology, 21, “Genetic Mecha-
nisms: Structure and Function” (1956). ,

Cold Spring Harbor Symposia on Quantitative Biology, 17, “Viruses” (1953).

96 THE MOLECULAR BASIS OF EVOLUTION