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J. Mol. Biol. (1963) 6, 208-213
The Bacterial Chromosome and its Manner of Replication
as seen by Autoradiography
JOHN CAIRNS
Department of Microbiology, Australian National University,
Canberra, A.C.T., Australia
(Received 19 November 1962)
In order to determine the form of replicating DNA, E. coli B3 and K12 Hfr
were labelled for various periods with [3H]thymidine. Their DNA was then
extracted gently and observed by autoradiography. The results and conclusions
can be summarized as follows.
(I) The chromosome of E. coli consists of a single piece of two-stranded
DNA, 700 to 900 p.long.
(2) This DNA duplicates by forming a fork. The new (daughter) limbs of the
fork each contain one strand of new material and one strand of old material.
(3) Each chromosome length of DNA is probably duplicated by one fork.
Thus, when the bacterial generation time is 30 min, 20 to 30 p. of DNA is
duplicated each minute.
(4) Totally unexpected was the finding that the distal ends of the two
daughter molecules appear to be joined during the period of replication. The
reason for this is obscure. Conceivably the mechanism that, in vivo, winds
the daughter molecules lies at the point of their union rather than, as commonly
supposed, in the fork itself.
(5) The chromosomes of both B3 (F-) and Kl2 (Hfr) appear to exist as a
circle which usually breaks during extraction.
1. Introduction
The semiconservative nature of DNA replication, predicted on structural grounds
by Watson & Crick (1953), was demonstrated experimentally first for bacterial
DNA (Meselson & Stahl, 1958), but how the two strands of the double helix come to
separate during replication is not known. There must be formidable complexities to
any process which, however feasible energetically (Levinthal & Crane, 1956), at once
unwinds one molecule and winds two others. So a study was undertaken of the shape
and form of replicating DNA.
Bacteria, despite their complexity, promised to be the most accessible source of
replicating DNA. Each bacterium in an exponentially growing culture of E. coli
makes DNA for more than 80% of the generation time (McFall & Stent, 1959;
Schaechter, Bentzon & Maal0e, 1959); if, as seems likely, the bacterial chromosome
is a single molecule of DNA, this molecule must be engaged in replication most of
the time. A method had already been devised for extracting this DNA with little
degradation (Cairns, 1962a) and it seemed probable that, with more care, the chromosome could be isolated intact and, caught in the act of replication, its DNA be displayed by autoradiography.
208
AUTORADIOGRAPHY OF BACTERIAL CHROMOSOMES
209
2. Materials and Methods
Bacteria. Since the chromosomes of F- and Hfr bacteria differ in the type of their
genetic linkage (Jacoh & Wollman, 1958) and in the manner of their duplication (Nagata,
. 1962), two strains of E. coli were used, B3 (F-) (Brenner) and K12 3000 thy- B 1 - (Hfr).
Both strains require thymine or thymidine.
Medium. The A medium of Meselson & Weigle (1961) was used. To this was added
3 mg /rnl. casein hydrolysate, which had first been largely freed of thymine by steaming
with charcoal. In this medium, supplemented with 2 fLgjml. TDR, t both strains have a
generation time of 30 min.
Preparation of labelled bacteria for autoradiography. The bacteria were grown with
aeration to 1o8jml., centrifuged and resuspended in an equal volume of medium containing 2 pgjml. [3H]TDR (9 ejm.mole). In pulse-labelling experiments, incorporation of
label was stopped by diluting the bacteria either 50-fold into medium containing 20 fLgjml.
TDR or 250-fold into cold 0,15 ~1-NaCI containing 0-01 M·KCN and 0·002% bovine
serum albumin. In long-term experiments, the bacteria were labelled for two generations
(1 hr) so that roughly half of the DNA would be fully labelled and half would be a hybrid
of labelled and unlabelled strands.
Lysis of bacteria. Only in a few minor respects has the procedure been altered from
that already published (Cairns, 1962a). Labelled bacteria are lysed after dilution to a
final concentration of about 1Q4jml. Since it was important in certain experiments to be
sure that DNA synthesis did not continue beyond the time the bacteria were sampled,
0·01 M-KCN was added to the lysis medium (1,5 M-sucrose, 0·05 M-NaCI, 0·01 M-EDTA).
Various types of cold carrier DNA (4-7 fLgjml. calf thymus, E . coli or T2 DNA) were
used at various times without apparently influencing the results. As before, lysis was
obtained by dialysis against 1% Duponol C (Dupont, Wilmington, Delaware, U.S.A.) in
lysis medium for 2 hr at 37°C. The Duponol was then removed by dialysis for 18 to 24 hr
against repeated changes of 0·05 M·NaCl, 0·005 M-EDTA. As before, the DNA was collected
on the dialysis membrane (VM Millipore filter, Millipore Filter Corporation, Bedford,
Mass., U.S.A.).
Autoradiography. As before, Kodak ARlO stripping film was used and the exposure was
about 2 months.
Thymidine incorporation experiments. To determine whether incorporation was delayed
following transfer to a medium containing [3H]TDR, bacteria were grown in cold medium
to 5 x 10 Bjml., centrifuged and resuspended in medium containing 2 fLgjml. [3H]TDR
(1 ejm-mole). Samples were then removed into cold 5% TCA and washed on Oxoid
membrane filters (average pore diameter 0·5 to 1·0 p, Oxo Ltd, London, England) with
cold TCA and finally with 1 % acetic acid. The filters were dried, placed in scintillator
fluid (0'4% 2,5-diphenyloxazole, 0'01% 1,4-bis-2[5-phenyloxazolyl]-benzene in toluene)
and counted in a scintillation counter.
3. Results
In interpreting autoradiographs of extracted DNA certain assumptions are necessary. These can be stated at the outset.
(1) It is not clear why some molecules of bacterial DNA choose to untangle whereas
others do not. However, the few that do are assumed to be a fairly representative
sample; specifically we assume that they do not belong to some special class that is
being duplicated in some special way.
(2) The ratio of mass to length for this untangled DNA is taken to be at least that
of DNA in the B configuration, namely 2 x 106 daltonsju (Langridge, Wilson, Hooper,
Wilkins & Hamilton, 1960). We assume that single-stranded DNA will not be found
in an extended state and so, even if present, will not contribute to the tally of untangled DNA.
t
Abbreviation used: TDR stands for thymidine.
210
J. CAIRNS
(3) The density of grains along these labelled molecules is assumed to be proportional to the amount of incorporated label. Specifically we assume that if one
piece of DNA has twice the grain density of another this shows that it is labelled in
twice as many strands.
(a) Pulse labelling experiments
Simple pulse-labelling experiments could tell much about the process of DNA
replication. As pointed out already, DNA synthesis in E. coli is virtually continuous.
If, therefore, the bacterial chromosome is truly a single piece of two-stranded DNA
and if, at any moment, duplication is occurring at only a single point on this molecule,
then the length of DNA labelled by a short pulse will be just that fraction of the
total length of the chromosome that the duration of the pulse is a fraction of the
generation time; if there are several points of simultaneous duplication on the single
molecule, or several molecules which are duplicated in parallel, then the length of
DNA labelled will be appropriately less. Further, from such pulse experiments it'
should be possible to determine whether one or two new strands are being made in
each region of replication.
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FIG. 1. Incorporation of aH into cold TCA-insoluble material, following transfer of E. coli B3
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However, first it was necessary to find out how quickly [3H]TDR gets into DNA
when the bacteria are transferred from a medium containing cold TDR. The result
of such an experiment with E. coli B3 is shown in Fig. 1 (see also Materials and
Methods). A similar result was obtained for E. coli K12. So it seems that, under these
particular conditions, the error in timing a pulse will be less than 1 minute.
Autoradiographs of E. coli B3 DNA were prepared following (i) a 3 minute pulse
of [3H]TDR, (ii) a 3 minute pulse, followed by 15 minutes in cold TDR, and (iii) a
6 minute pulse, followed by 15 minutes in cold TDR. Examples of what was found
are shown in Plate 1. Similar results were obtained using E. coli K12.
From these experiments the following conclusions can be drawn.
(1) Immediately after a 3 minute pulse of label (Plate I(a), (b) and (c)) the
labelled DNA consists of two pieces lying in fairly close association. Fifteen
minutes later (Plate Itd), (e) and (f)) these pieces have moved apart and can be
photographed separately. It seems therefore that two labelled molecules are being
formed in the region of replication.
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PLATE 1. Autoradiographs of E. coli B3 DNA labelled by a pulse of [3HJTDR. Exposure time
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(a), (b), (c): Immediately after a 3 min pulse; (d), (e), (f): 15 min after a 3 min pulse; (g):
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PLATE II. Autoradiographs of E. coli B3 (a), (b), (c) and E, coli KI2 (d) DNA following incorporation of [3HJTDR for a period of I hr (two generations). The arrows show the point of
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PLATE III. Autoradiographs of E. coli B3 (a), (b), (c) and E. coli K12 (d) DNA following
incorporation of [3H]TDR for a period of 1 hr. In (b), (c), and (d), the postulated break is marked
x - x • Exposure time 61 days. The scales show 100 p..
AUTORADIOGRAPHY OF BACTERIAL CHROMOSOMES
211
(2) There is approximately 1 grain per micron in these labelled regions. Since similarly
labelled T2 and A bacteriophage DNA, both of which are known to be largely twostranded, show about twice this grain density (Cairns, 1962b), the two molecules
being created at the point of duplication must each be labelled in one strand.
(3) A 3 minute pulse labels two pieces of DNA each 60 to 80 p. long (Plate I(a) to
(f»; a 6 minute pulse labels about twice this length (Plate I(g». In one generation time
(30 minutes) the process responsible for this could cover 600 to 800 p., or slightly
more than this if the duration of the pulse is being over-estimated (see Fig. 1). There
is unfortunately no precise estimate for the DNA content of the E. coli chromosome.
When the generation time is 1 hour, each cell contains 4 x 109 daltons DNA (Hershey
& Melechen, 1957). If each cell contained only one nucleus, this value would have to
be divided by 1·44 (lfln 2) to correct for continuous DNA synthesis. However, as
such cells are usually multinucleate (see Schaechter, Maalee & Kjeldgaard, 1958),
this corrected value of 2·8 x 109 daltons (or 1400 p. DNA) must be too high. There is
therefore no marked discrepancy between the total length of DNA that has to be
duplicated « 1400 p,) and the distance traversed by the replication process in one
generation (at least 600 to 800 p.). This suggests that one or at the most two regions
of the chromosome are being duplicated at any moment.
These various conclusions are reinforced by the results reported in the next section.
(b) Two-generation labelling experiments
One conspicuous feature of the pulse experiments is not apparent from the
photographs and was not listed. The proportion of the labelled DNA that had
untangled, and could therefore be measured, was far lower immediately after the
3 minute pulse than 15 minutes later. Thus the replicating region of a DNA molecule
is apparently less readily displayed than the rest. It was not surprising therefore that
considerable search was necessary before untangled and replicating molecules were
found in the two-generation experiments. Since the products of such searches are
generally somewhat suspect, the guiding principles of this particular search will be
given.
As pointed out earlier there are reasons for assigning the hypothetical DNA
molecule of E. coli a length of less than 1400 p,. Therefore no molecules were accepted
whose length, presumably through breakage, was much less than 700 p.. No replicating
(forking) molecules were accepted unless both limbs of their fork were the same length.
Lastly many extended n:.olecules were excluded because of the complexity of their
form; although perhaps interpretable in terms of a known scheme they could not
be used to provide that scheme. Samples of what remained are shown in Plate II.
They have been selected to illustrate what appear to be various stages of DNA
replication.
First, the rough agreement between the observed length of E. coli DNA (up to
900 p.) and the estimated DNA content of its chromosome « 1400 JL) supports the
conclusion, arising from the pulse experiments, that this chromosome contains a piece
of two-stranded DNA.
Second, it seems clear that this DNA replicates by forking and that new material
is formed along both limbs of the fork. This latter was shown by the pulse experiments
and is confirmed here. In each of the forks shown, one limb plainly has about twice
the grain density of the other and of the remainder of the molecule. The simplest
hypothesis is that we are watching the conversion of a molecule of hybrid (hot-cold)
212
J. CAIRNS
DNA into one hybrid and one fully-substituted (hot-hot) molecule. It is not surprising that, nominally after two generations of labelling, the process is seen at
various stages of completion. In Plate Uta), duplication has covered about a sixth of
the visible distance in what appear to be two sister molecules. In Plate II(b) and
(c), duplication has gone about a third and three-quarters of the distance, respectively. In Plate II(d), duplication is almost complete, about 800 J.I. of DNA having
been replicated. So these pictures support what seemed likely from the pulse
experiments-namely, that the act of replication proceeds from one end of the
molecule to the other.
FIG. 2. The consequence of uniting the ends of the replicating fork. The arrows show the
direction of rotation, as the parent molecule unwinds and the two daughter molecules are formed
(modified from Delbriick & Stent, 1957).
The most conspicuous and totally unexpected feature of these pictures has been
left for discussion last. In the case of each replicating molecule, the ends of the fork
are joined. This complication seems to have been taken one stage further in Plate
II(a); here the two limbs of the fork may be joined to each other but they also appear
to be joined to their opposite numbers which are being formed from the sister molecule.
Conceivably such terminal union of the new double helices (which must be alike in
their base sequence) is the artificial consequence of a freedom to unite that only
comes with lysis ; this union may not exist inside the. bacterium. Alternatively,
terminal union may be the rule during the period of replication. If so, whatever
unites the two ends must have the freedom to rotate so that the new helices can
rotate as they are formed (Fig . 2). This uniting structure, or swivel, could in fact
be the site of the mechanism that, in vivo, spins the parent molecule and its two
daughters.
(c) The bacterial chromosome
The primary object of this work was to determine the form of DNA when it is
replicating, not the form of the entire bacterial chromosome. The pictures presented
so far give little indication of the latter as they show molecules that are either broken
A UTORADIOGRAPHY OF BACTERIAL CHROMOSOMES
213
(Plate II(c» or partly tangled (Plate II(a) and (h)). It seemed possible, however' that
out of all the material that had been collected some model for the shape of the whole
chromosome might emerge.
In searching for such a model that could account for all the kinds of structure seen,
the premise was adopted that since excess cold carrier DNA was invariably present
these structures must be related to the model by breakage, if needs be, but not by
end-to-end aggregation. Granted this premise, there seems to be only one model that
Flo. 3. Two stages in the duplication of a circular chromosome. (B) and (D) mark the positions
of the breaks postulated to have producedthe structures shownin Plate III(b) and (d), respectively.
could generate every structure merely by breakage. This model supposes that the
chromosome exists as a circle. Duplication, as in Fig. 2, proceeds by elongation of a
loop at the expense of the remainder of the molecule; since, however, the distal end
of the molecule is also attached to the swivel, duplication creates a figure 8 each half
of which ultimately constitutes a finished daughter molecule (Fig. 3). Depending on
how this structure breaks at the time of extraction, it may form a rod with a terminal
loop (Plate II), a rod with a subterminal loop (Plate III(c) and (dj), or a circle
(Plate III(a) and (bj}; in the case of a circle, the circumference may be up to twice
the length of the chromosome. AIl structures seen, including circles of varying
circumference, can be readily derived from this model whereas they do not conform
to any other obvious scheme. It is, however, possible that the process of duplication
may vary; that, for example, the structure shown in Plate II(a) was genuinely an
exceptional case . In any event, here as elsewhere no significant difference was detected
between the chromosomes of E. ooli B3 (F-) and E. coli K12 (Hfr).
I am greatly indebted to Dr . A. D. Hershey and Professor Max Delbriick for helpful
criticism and to Miss Rosemary Henry for able technical assistance.
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McElroy & B. Glass. Baltimore: Johns Hopkins Press.
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