Chapter 46
VIRUS GENETICS: BACTERIOPHAGE

PRE-LECTURE ASSIGNMENT

1.

2.

Quickly review notes for the previous lec-
ture.

Suggested readings:

a. General genetics textbooks

Altenburg: Chap. 22, pp. 386-393.
Colin: Chap. 12, pp. 216-220.
Sinnott, Dunn, and Dobzhansky: Chap.
16, pp. 225-226; Chap. 23, pp. 317-
318; Chap. 27, p. 373.

Snyder and David: Chap. 27, pp. 412-
413.

Srb and Owen: Chap. 14, pp. 282-283.

Winchester: Chap. 23, pp. 327-328. Cc.
. Additional references

Anfinsen, C. B. 1959. The molecular
basis of evolution. 230 pp. New York:
John Wiley & Sons, Inc.

Brenner, 8. 1959. Physiological as-
pects of bacteriophage genetics. Adv.
Virus Res., 6: 138-158,

Burnet, F. M., and Stanley, W. M.
Editors, 1959. The viruses. Vol. 1,
General virology. 609 pp. New York:
Academic Press.

Luria, S. E. 1953. General virology.
427 pp. New York: John Wiley & Sons,
Inc.

LECTURE NOTES

A. Nucleotide pairs (n'its) in different organisms D.
Mouse 5 billion per cell
Drosophila 80 million per cel}
Bacterium 6 million per cell

260

 

If 2,000 n'its specify an average protein (see
Chap. 44), bacteria could have about 3, 000
different enzymes specified by its DNA con-
tent. It is surprising they could be free-living
with so low a number.

Bacteriophages like T2 or lambda contain E.

about 100, 000 n'its, which, on this view, per-

Lecturer—J. LEDERBERG

mit the manufacture of only about 50 different

proteins.

1. It is not surprising, then, these are not
free-living organisms.

2. The host cell provides most of the acces-
sory metabolism for synthesizing new vi-
rus. .

3. The virus, in adding elements unique to its
structure,

a. competes with the metabolic systems of
its host, and

b. produces the protective structures en-
closing it when outside its host.

Smaller viruses

1. These include animal viruses causing en-
cephalitic diseases and the exceptional
phage ¢X174.

2. These have only about 5,000 n'its, which
could encompass only one or a few genes.

3. Their nucleic acid is one-stranded,

a. Most small viruses of plants and ani-
mals contain RNA.

b. ¢X174 is exceptional in containing one-
stranded (otherwise typical) DNA.

4. It is remarkable that by probably producing
but a few distinctive proteins these viruses
can divert the host's metabolism so that
virus nucleic acid and protein are made
rather than the host's normal metabolic
products.

Bacteriophage structure

1. These bacteria-attacking viruses are.1 to
-2u long, about one-tenth the bacterial di-
ameter,

2. They have a somewhat-ecrystalline protein
coat within which is packed double-stranded
DNA (about 34 long when extended).

3. The protein coat has a tail, covered by a
spiral protein, with tail tip fibers.

Lytic cycle of a bacteriophage

1. Phage attaches to the bacterium tail first.
. The DNA enters the bacterium leaving a

shell of protein outside.

a, Hershey and Chase labeled the DNA by
P32 and the protein by 835.

b. The empty shells, containing all or al-
most all the phage protein, can be
sheared off the bacteria without changing
the fate of the infected cells.

c. The bacterium was innoculated, there-
fore, not with whole phage but with
phage's genetic element.

. An eclipse period follows during which no

infective phage can be demonstrated in the

recently infected bacterium.

a, During the first several minutes there is
replication of the phage DNA.

b. This vegetative phage forms a pool of
DNA units.

. From time to time this pool is sampled, in

that a fraction of it undergoes condensation
and is surrounded by a new skin (head and
tail) formed from a cycle of protein synthe-
sis and organization; as a consequence in-

fective phage is produced.

. Phage-infected bacteria produce endolysins

which, 20-40 minutes after infection, lyse
the cell wall and liberate infective phage in
the medium.

. The free infective phage can attach and

penetrate a sensitive bacterium, then

a. repeat its lytic cycle,

b. or, as in the case of lambda (Chaps. 44
and 45), establish a relationship with
the bacterial chromosome, so that the
bacterium is lysogenic and has the pro-
perty of having some of its descendents
produce some lytic phage.

F. Methods for assaying phage

1.

One method is to determine the time re-
quired for complete lysis of a liquid cul-
ture of sensitive bacteria.

If a few phage are added on top of an agar

medium recently seeded heavily with sensi-

tive bacteria,

a. the bacterial clones will form a continu-
ous and somewhat opaque lawn.

b. each phage particle that enters a bac-
terium will lyse it and release up to
several hundred daughter particles
which will attack bacteria near the ori-
ginal burst. This cycle will result in a
progressively increasing zone of lysis
which appears as a clearing or plaque
in the bacterial lawn.

c. each plaque will be a phage colony de-

rived from one ancestral particle.
d. Genetically different phages may pro-
duce different types of plaques.

What is unique to phage DNA?

 

1.

4,

It must be able to stop bacteria from mak-
ing their own protein and DNA.

. Even-numbered phages, T2, T4, and T6,

have hydroxymethyl-cytosine in their DNA
instead of cytosine.

. But in Tl or lambda there is no such pecu-

liarity in base composition,

a. yet Tl is virulent (causes lysis),

b, and lambda can act either temperately
(in a lysogenic bacterium) or lytically
(upon ultraviolet induction).

The complete answer is still unknown.

Genetic recombination in phage

 

1,

Given one phage strain mutant for both host
range, h, and plaque type, r, and one
strain that is wild-type.

. Sensitive bacteria are exposed to a mixture

of such large numbers of the two mutants
that some will have been multiply-infected.
Some doubly-infected cells will carry both
phage types.

The daughter phage from such mixedly-in-
fected cells may be of parental (4 r and
ht r+) and recombinational (h* r and h r*)
types.

Fine structure analysis of T4

 

1,

Since T4 contains about 100, 000 n'its,
there could be that number of mutational
sites,

One segment, r, is about 1% of the total
map length, or about 1, 000 n'its long.

. The wild-type, r*, grows on both strains B

and K-12 of E. coli, while the r mutants
grow on B but not K~12.

Benzer made mixed infections with different
r mutants, then easily detected the rare

(1 in 106 or 108) recombinant particles cap-
able of growing on K-12.

. Of 1,500 spontaneous r mutants Benzer

tested, 100 were different. Thus there are
at least 100 sites for mutation in the r seg-

ment.

. Certain mutants must have recurred fre-

quently, indicating certain sites in r were
"hot spots" of spontaneous mutation.

. The frequency of least recombination ob-

served between two mutants was .0001.
Since there are about 10 exchanges (mor-
gans) per phage per generation (lytic cycle)
over its whole linkage map, the total num-
ber of crossover sites in phage should be

261
10/. 0001 or 100, 000.

. Thus, crossover site number equals n'it

number; the genetic unit of recombination
equals the smallest meaningful chemical
unit -- the interval between two successive
niits.

- Benzer also found phenotypic interaction

between different r mutants growing in the

same bacterium.

a. By itself neither r mutant can lyse K-12.

b. The vegetative phage of both together, in
the same cell, can cause lysis.

c. Since the phage liberated were usually of
parental type, this phenotypic coopera-
tion did not require genetic recombina-
tion. ‘

d. The r region can be divided into A and B
sections or cistrons. Only a mixed in-
fection including one mutant from A and
one from B results in phenotypic coop-
eration.

e. It is concluded, therefore, Yr governs the
production of two separate protein ele-
ments.

f, Mutants which behave as deletions for
different regions in r have been obtained.
It is possible to arrange the spontaneous
point mutations in only one order which
will give a compatible superposition of
the various deletion types.

g. Even in the utmost details of its fine
structure the genetic material of phage
T4 is organized in a linear fashion.

POST-LECTURE ASSIGNMENT

262

1.

Ne]

Read the notes immediately after the lec-
ture or as soon thereafter as possible,
making additions to them as desired.

. Review the reading assignment.
. Be able to discuss or define orally or in

writing the items underlined in the lecture
notes.
Complete any additional assignment.
QUESTIONS FOR DISCUSSION

46. 1. What functions are attributed to the spiral
protein and tip fibers of the tail of a bacterio-
phage ?

46, 2. Distinguish a virus from a bacteriophage.

46. 3. Defend the statement: "Viruses are not
genes."
46. 4. What do you consider the most remarkable

and important feature of phage ¢X174? Why?

46. 5. What evidence proves or suggests that a
transducing virus cannot

a. carry a whole bacterial chromosome ?
b. simultaneously carry a viral genome
in addition?

46. 6. Design an experiment which would demon-
strate the "eclipse period" of phage.

46. 7. Discuss the phenotypic interactions be-
tween viral and bacterial genes.

46. 8. Are phages harmful to a bacterial species
as awhole? Explain.

46. 9. Discuss the statement: "The holes ina
bacterial lawn are contagious. "'

46.10. Why is it so important to learn the chemi-
cal nature of the proteins specified by phage?

46.11. From what is known about the chemical
nature of the hereditary material in different
organisms, discuss the evolutionary changes
which the gene itself may have undergone.

46.12, What preliminary experiments had to be
performed and what results obtained, do you
suppose, before Benzer accepted an rt geno-
type as the result of phage recombination?

46.13. Using different r mutants of phage T4, in-
dicate the requirements and procedures nec-
essary, in each case, to obtain

a. genetic recombination without pheno-
typic cooperation.

b. both phenotypic cooperation and genetic
recombination.

c. spontaneous reverse mutations to rt,

46.14. What hypotheses can you offer to explain
the occurrence of "hot spots" for spontane-
ous mutation in the r chromosome segment
of E. coli?

46.15. Of what significance is the fact that cross-
over percentages lower than .01% were not
found between r mutants, even though lower
values would have been detected readily ?

46.16. What bearing has Benzer's work to our
understanding of the gene as a unit?

46.17. Briefly describe the evidence that phage
DNA is organized linearly.

46.18. What attributes are common to all viruses?

46.19. Which do you suppose came first in evolu-
tion, bacterial-like or viral-like organisms?

Explain.

46.20. Compare viruses and antibiotics.

263