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© 2001 Nature Publishing Group http://structbio.nature.com
© 2001 Nature Publishing Group http://structbio.nature.com
book review
The 1966 Phage and the origins of molecular
biology6, edited by John Cairns, Gunther
Stent, and James Watson, offered a genealogy of molecular biology with Delbrück as
founding father. The book’s implicit message that molecular biology derived from
the phage group drew fire from some. John
Kendrew argued that structural biologists
(such as those in his unit at Cambridge)
had also played a crucial role in the founding of molecular biology7.
Gunther Stent subsequently offered a
conciliatory view of the origins of molecular biology that combined the ‘informational’ school of phage genetics with the
‘structural’ school of X-ray crystallographers, a meeting of the minds that was
conveniently personified by Watson and
Crick8. This enlarged family tree still did
not satisfy everyone; Origins of molecular
biology: A tribute to Jacques Monod9,
stressed the early importance of French
microbiologists. Nonetheless, historical
interpretations that emphasized these various ‘origins’ tended to demarcate molecular biology firmly from pre-existing
fields, especially biochemistry (Delbrück
famously disparaged biochemistry).
By all these accounts, Hershey makes an
odd revolutionary. He is described by
friends as a “biochemist’s biochemist” as
well as a pioneering molecular geneticist.
His research defied any dichotomy between
informational and structural approaches,
and he retained a skeptical attitude towards
the supremacy of nucleic acids. Attention to
Hershey as one of the original molecular
biologists complicates the story, showing
the limitations of views that emphasize a
single approach, dogma, or charismatic
leader. By portraying a trailblazing biologist
who combined structural, biochemical, and
genetic methods in his quest to understand
life at the molecular level, We can sleep later
cautions us to keep a broad view of molecular biology’s past — and its future.
Angela N. H. Creager is in the Department
of History and Program in History of
Science, Princeton University, Princeton,
New Jersey 08544, USA. email:
[email protected]
1. As quoted in Judson, H.F. The eighth day of
creation: The makers of the revolution in biology,
275 (Simon and Schuster, New York; 1979).
2. Hershey, A.D. & Rotman, R. Genetics 34, 44–71
(1949).
3. Hershey, A.D. & Chase, M. J. Gen. Physiol. 36, 39–56
(1952).
4. Avery, O.T., MacLeod, C.M. & McCarty, M. J. Exp.
Med. 79, 137–158 (1944).
5. Hershey, A.D., ed. The bacteriophage lambda. (Cold
Spring Harbor Laboratory Press, Cold Spring
Harbor, New York; 1971).
6. Cairns, J., Stent, G.S. & Watson, J.D. Phage and the
origins of molecular biology. (Cold Spring Harbor
Press, Cold Spring Harbor, New York; 1966).
7. Kendrew, J. Sci. Am. 216, 141–43 (1967).
8. Stent, G.S. Science 160, 390–395 (1968).
9. Lwoff, A. & Ullmann, A. Origins of molecular
biology: A tribute to Jacques Monod. (Academic
Press, New York; 1979).
history
Phage facts
Often the simplest experiments lead to
the most remarkable insights. So it was
with the famous fluctuation experiments
of Luria and Delbrück and the Waring
blender experiments of Hershey and
Chase for which they were awarded the
Nobel Prize in Physiology or Medicine in
1969. While the results of these experiments are permanently etched into every
first year biology student’s brain, it is
worth recalling what was known at the
time these experiments were conducted
and the conclusions the authors drew.
In the early 20th century, the role of
chance mutations in the genetic variation
of higher organisms was generally accepted. However, bacterial cultures seemed to
be plastic. Exposed to adverse conditions,
cultures of bacteria could give rise to resistant variants that remained resistant “to
the action of the virus even if subcultured
through many generations in the absence
of the virus”1. At the time, many scientists
believed that the “virus by direct action
induced the resistant variants”1. Others
believed that the “resistant bacterial variants are produced by mutation in the culture prior to the addition of virus. The
virus merely brings the variants into
prominence by eliminating all sensitive
bacteria”1. These two alternative theories
— induced immunity and selection of
spontaneous mutations — were rigorously tested both theoretically and experimentally by Max Delbrück and Salvadore
E. Luria, respectively, in 1943.
The two hypotheses lead to different predictions regarding the distribution of resistant bacteria in a series of parallel cultures.
The hypothesis of acquired or induced
immunity predicts that the number of
mutants in each culture would be clustered
around the mean with little variation
between cultures. According to the selection
hypothesis, resistant bacteria could arise at
any point in the life of the culture. This
would result in very different numbers of
mutants per culture and thus the variability
from culture to culture would be high.
Luria found that in every experiment the
“fluctuation of the numbers of resistant
bacteria is tremendously higher than could
be accounted for by the sampling errors, in
striking contrast to the results of plating
from the same culture and in conflict with
the expectations from the hypothesis of
acquired immunity”1. Luria and Delbrück
concluded that the “resistance to virus is
due to a heritable change of the bacterial cell
which occurs independently of the action of
the virus”1. In other words, the mutation
was there before the virus was added.
nature structural biology • volume 8 number 1 • january 2001
Later, when Alfred D. Hershey and
Martha Chase began their experiments,
little was known about the early steps of
phage infection. What was known was
that after the phage adsorbed to the bacteria there was a latent period of ∼10
minutes after which time infectious virus
particles were made, ultimately leading
to host cell lysis and phage release.
Hershey and Chase reasoned that if they
knew the fate of the viral protein and the
nucleic acid at the beginning of phage
infection they would understand more
about the nature of those early steps.
They labeled the phage with either 35S or
32P, allowed them to adsorb to the bacteria
and then the cells were separated from the
unadsorbed material by centrifugation.
The cells were resuspended and the suspension was spun in a Waring blender to
separate the phage from the infected bacteria. They found that ∼80% of the 35S label
was removed from the bacteria whereas
only ∼20% of the 32P label was removed.
They concluded that “most of the phage
DNA enters the cell, and a residue containing at least 80% of the sulfur-containing
protein of the phage remains at the cell
surface”2. Together with the 1944 experiments of Oswald Avery3 in which nucleic
acid, not protein, was shown to have
“transforming” properties, these findings
persuaded most scientists that DNA carried the genetic information. Curiously,
however, Hershey himself remained skep19
© 2001 Nature Publishing Group http://structbio.nature.com
history
© 2001 Nature Publishing Group http://structbio.nature.com
tical. Hershey and Chase concluded their
paper by saying “the sulfur-containing
protein has no function in phage multiplication, and the DNA has some function”2.
Talk about an understatement. More bons
mots from and about Hershey can be
found in “We can sleep later: Alfred D.
Hershey and the origins of molecular
biology” which is reviewed on page 18 of
this issue.
Boyana Konforti
1. Luria, S.E. & Delbrück, M. Genetics 28, 491–511
(1943).
2. Hershey, A.D. & Chase, M. J. Gen. Physiol. 36, 39–56
(1952).
3. Avery, O.T., MacLeod, C.M. & McCarty, M. J. Exp.
Med. 79, 137–156 (1944).
picture story
A DNA wormhole
The bacteriophage φ29 is a virus that
infects the bacterium Bacillus subtilis. The
mature φ29 contains a head region that
encloses its genome and a tail region that
contacts the cell surface to initiate the
infectious cycle. In the late stage of the maturation process from which functional
phage particles emerge, the phage genome
— a linear double-stranded DNA of
∼19,000 base pairs — is packaged into a
preassembled empty head (called the prohead). This process requires condensing
the viral genome, which would span
∼6,400 Å if it were completely extended,
into the phage head, which is only ∼500 Å
in length. How the phage performs this
remarkable task is not yet fully understood.
At one end of the phage head is a small
opening through which the viral genome
enters and exits. A protein complex called
the ‘connector’ — so named because the
tail of the phage is connected to the head
through this complex — sits at the opening and participates in the DNA packaging
process. To understand the structural
basis of how the connector regulates DNA
packaging, Simpson et al. (Nature, 408,
745–750; 2000) have determined its high
resolution structure.
The connector complex consists of 12
identical subunits (top and middle panels,
one monomer is colored red to illustrate
how the subunits are assembled in the
complex). The overall shape of the complex is like a bottle stopper (side view;
middle panel) with a central channel (top
20
view; top panel). The narrowest part of
the channel is ∼36 Å in diameter; a double
stranded DNA could easily pass through
the channel without any steric problems.
To characterize how the connector fits
into the phage head, Simpson et al. generated a three-dimensional reconstruction
of the prohead from cryo-electron
microscopy data and then fit the crystal
structure into the electron density. The
result shows that the narrow part of the
connector protrudes outside of the viral
shell (bottom panel, a thin section across
the center of the prohead along the axis of
the connector central channel is shown;
the red mesh wire represents the electron
density from the prohead and the DNA is
modeled in the superposition for illustration purposes). This region of the connector interacts with a phage-encoded
RNA (marked by green mesh wire) and
possibly a viral ATPase (not present in
this reconstruction), both of which had
been shown to be important for DNA
packaging.
With the translocating DNA as the
movable central shaft, the connector, the
viral RNA and ATPase appear to constitute a ‘motor’, with the viral ATPase
powering conformational changes in the
connector complex. The energy of ATP
hydrolysis is converted into a translational motion of DNA. This study thus
provides a glimpse of the initial event in
the DNA packaging process of bacteriophage φ29.
Hwa-ping Feng
Adapted from Simpson et al.
nature structural biology • volume 8 number 1 • january 2001