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The Units of DNA Replication in Drosophila melanogaster
Chromosomes
Alan B. Blumenthal, Henry J. Kriegstein and David S. Hogness
Cold Spring Harb Symp Quant Biol 1974 38: 205-223
Access the most recent version at doi:10.1101/SQB.1974.038.01.024
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Copyright © 1974 Cold Spring Harbor Laboratory Press
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The Units of DNA Replication in
alihposorD retsagonalem
Chromosomes
ALAN B. *,LAHTNEMULB HENRY J. KRIEGSTEIN, DNA DAVID S. SSENGOH
tnemtrapeD of ,yrtsimehcoiB Stanford University loohcS of Stanford, California Medicine, 50349
The DNA which must be replicated in a chromo- the electron microscope because of the much lower
some of Drosophila melanogaster appears to exist as fork frequency, and we have therefore used the
a single molecule of double-stranded DNA, which, technique of radioautography to examine this state.
for the largest chromosomes, has a length of about A situation in which multiple forks cooperate in
2.1 cm, or 62,000 kb I (Kavenoff and Zimm, 1973). the replication of a single, duplex molecule is
We have studied both the topography of the units depicted by configuration A in Figure 1, where two
of replication in this chromosomal DNA and the topologically related ways of replicating a large
rate of replication per unit in two different classes of amount of DNA in a short time are indicated. Con-
Drosophila nuclei which exhibit very different S figuration A is similar to that suggested by
phases. Our purpose is to define the factors which Huberman and Riggs (1968) to account for the
determine the overall replication rate for these radioautographic patterns produced by replicating
giant DNA molecules. DNA from mammalian chromosomes. Here each of
The two classes are the rapidly replicating many origins (oi) in a single DNA molecule
cleavage nuclei and the slowly replicating nuclei in generates two replication forks, which move in op-
cell cultures. At 24~ the cleavage nuclei divide posite directions to create a serial array of "eye
every 9.6 min in the syncytium of the egg for a forms." Configuration B consists of a set of circular
period of about two hours after fertilization DNA molecules, each of which contains a single
(Rabinowitz, 1941). Since interphase occupies origin with this same capacity to generate bi-
3.4 min of this doubling time, we presume that each directional replication. The resulting "0 forms" are
chromosomal DNA molecule is replicated within typical of the replicating configurations of many
this short period. By contrast, the nuclei in cell prokaryotic chromosomes and have been described
cultures at 25~ exhibit an S phase of about in considerable detail for bacteriophage ~ (SchnSs
600 min (Dolfini et al., 1970), more than two orders and Inman, 1970; Inman and SchnSs, 1971). Since
of magnitude greater than that for cleavage nuclei. configuration A can be formed from B by cutting
The molecular rate of replication for the DNA in each circular DNA at a position other than i o and
the largest chromosomes in the cleavage nuclei joining the cut ends of adjacent circles, the topo-
must then be equal to or greater than 18,000 kb. logical aspects of replication are much the same in
rain -1 (cid:12)9 molecule -1. This extremely rapid molecular the two configurations.
replication is about four orders of magnitude However, the two configurations exhibit an
greater than the upper estimates for the rate of important difference for our present consideration.
movement of a DNA replication fork in animal Whereas each origin in B must be activated to
chromosomes (Huberman and Riggs, 1968; Callan, replicate all oft he DNA in the set, the activation of
this volume) and would therefore require the co- only one origin is sufficient for that purpose in A.
operative action of thousands of these forks per The time required to replicate all DNA in A can
molecule. This condition has allowed an examina- therefore be controlled by regulating the distribu-
tion of both the structure and the topography of tion of those origins which are activated, without
replication forks in cleavage nuclei by electron
A
microscopy. Forks in the nuclei from cell cultures ~ ~ 1 + ~+ 2
would, however, be extremely tedious to study in
* Present address: Laboratory of Radiobiology, University B 0 6 6
of California, San Francisco, California .22149
.... + + + + v +.-.
1 Abbreviations and notation: kb, kilo bases, a unit of
length equal to one thousand bases or base pairs in single- Figure .1 Two schemes for replicating a large amount of
or double-stranded nucleic acids, respectively. DNA in a short time.
205
Downloaded from symposium.cshlp.org on March 13, 2012 - Published by Cold Spring Harbor Laboratory Press
206 BLUMENTHAL, KRIEGSTEIN, AND HOGNESS
recourse to any change in the rate of fork move- labeled by the addition of enidimyht]Ha-lyhtem[
ment. Thus, if a unit of DNA replication is defined (45 to 56 Ci/mmole; New England Nuclear) to a
as that segment which is replicated by the forks concentration of 200 #Ci per ml of medium. Incor-
which emanate from a single origin, then the length poration of the [3H]thymidine into DNA proceeded
of a unit, though fixed in B, can be varied in A. linearly for a period of at least 2 hr with no detect-
The replicating DNA in both classes of Droso- able lag. In most cases, the [aH]thymidine was
phila nuclei examined here exhibits the configura- added directly to the above culture medium, and
tion given in A. In spite of the great difference in S the pulse terminated by addition of 2 volumes of
phase between these classes, no significant differ- ice-cold medium supplemented with unlabeled thy-
ence in the rate of fork movement was observed midine at 0.1 mM. In a few early experiments, the
between them. Distinct differences in the distribu- culture medium was replaced by a modified medium
tions of active origins were, however, observed. (the culture medium minus the yeast hydrolysate,
These observations have been used to construct a bactopeptone, and serum) containing the
general model for the topography of active origins [aH]thymidine. No significant differences in the
during S phase which accounts for its duration in radioautographie patterns given by the two
the different nuclei. methods of labeling were observed, and the data
from both have been combined here. A
[3H]thymidine pulse can be effectively chased (i.e.,
Methods
all tracks have sharp ends and do not exhibit the
"tailing" described in Huberman and Riggs, 1968,
Electron Microscopic Measurements on DNA
for pulse-chase experiments with Chinese hamster
from Cleavage Nuclei
cells) by replacing the labeled culture medium
Fertilized eggs of D. retsagonalem (Oregon R) laid with fresh culture medium supplemented with
over a 40-min interval at 25~ were collected and thymidine at 0.1 raM.
incubated for an additional 15 min before prepara-
Radioautography. Labeled cells from spinner
tion of cleavage nuclei. Egg collection, preparation
cultures were rinsed twice with ice-cold Schneider's
of cleavage nuclei, and isolation of the chromosomal
medium (without the bactopeptone and serum) by
DNA by equilibrium sedimentation in CsC1
centrifugation in a Sorvall HB4 rotor at 1000 rpm
gradients are described in another article (Kriegstein
for 5 min and resuspending to 2-6 (cid:141) l0 s cells per
and Hogness, 1973), which also contains a de-
ml. Surface cultures were directly suspended in the
scription of the methods used to prepare the DNA
ice-cold medium. Cells (3 (cid:127) 2 (cid:141) 401 per slide) were
for electron microscopy. Contour length measure-
lysed, and their DNA spread on subbed slides ac-
ments were made with a Hewlett-Packard 9864A
cording to the method of Lark et al. (1971), except
Digitizer and 9810A Calculator with a fully
that the lysis medium consisted of 2 o~ sodium
smoothed, length-calculation program giving an
dodecyl sulfate in 2 Mm Na EDTA. Dry slides were
accuracy of (cid:127) Lengths in kb units were
fixed for 30 rain in ~ 5 cold TCA and rinsed in 95
obtained by comparison to the lengths of reference
ethanol, after which they were coated with AR-10
phage DNAs. The single- and double-stranded
stripping film or NTB-2 liquid emulsion (Kodak),
references were M13 (6.6 kb; Marvin and Hohn,
dried, and stored at 4~ for 3-6 months. Slides
1969) and PM2 (9.9kb--determined from the
were developed for 2 min (NTB-2) or for 5 or l0 min
PM2/2 length ratio of 0.213 and the 46.5 kb length
(AR-10) in Kodak D-19 at 20~ Grain tracks were
of 2 DNA; Davidson and Szybalski, 1971).
observed under oil without a cover glass with a
Zeiss Planapo (40 (cid:141) using either bright or dark
Radloautographic Measurements on DNA
field illumination, and fields of approximately
from Cell Cultures
300/z were photographed with Panatomic X film
Cell line and culture. D. retsagonalem cells of for analysis.
Schneider's line #2 were grown at 25~ in Schnei-
Grain track measurements. Photographic
der's meclium (Grand Island Biological Co. #172)
negatives were projected onto the Hewlett-Packard
supplemented with baetopeptone and 15% heat-
Digitizer, and the lengths of grain tracks and gaps
inactivated fetal calf serum, as described in
between tracks in the linear arrays measured as
Schneider (1972), except that spinner as well as
describedf or the electron micrographs. Distances in
surface cultures were used. The cells were diploid,
microns were obtained by normalization to a
contained two X chromosomes, and exhibited a
micron grating photographed under the same
doubling time of about 24 hr at 25~
conditions and then converted to kb by dividing by
[3H]thymidine labeling of the DNA. Asyn- 0.34. The terminal grain tracks in a linear array
chronous cultures in exponential growth were pulse were never included in the analysis, as they may
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DNA REPLICATION IN CHROMOSOMES 207
contain broken ends. Grain densities were deter- served. Since the structure of these eye forms and
mined to set criteria for the following three classes the analysis of the single-stranded regions in their
of tracks: (a) one daughter duplex containing one forks are reported in detail elsewhere (Kriegstein
labeled strand (density = D); (b) a pair of such and Hogness, 1973), only a brief summary of these
daughter duplexes created by a single fork and subjects is given in this section. That the eye
drawn together when the DNA is stretched during forms result from DNA replication is indicated by
spreading (density= 2D); and (c) aggregated three kinds of observations. First, the two seg-
DNAs (density > 2D), which were eliminated from ments that form an eye are invariably of the same
the sample. To be scored, gap lengths had to be length and are doubIe-stranded, except for a small
greater than 1 tt and 2 # for 2D and 1D tracks, region at the fork which is described in the next
respectively. No significant differences in the dis- paragraph. Second, the two segments in each eye
tribution of grain-track lengths and of distances exhibit partial denaturation maps that indicate
between the centers of adjacent tracks were they contain related sequences of base pairs. These
detected after 2, 3, or 4 months of exposure. two characteristics are those expected for repli-
cated daughter segments. Finally, the frequency
of eye forms exhibited by DNA isolated from the
Results and Discussion slowly replicating nuclei of cell cultures is less than
that observed for cleavage nuclei by about two
Electron Microscope Observation of Repli- orders of magnitude.
cating DNA from Cleavage Nuclei Most forks (63 ~) have single-stranded regions
associated with them (Fig. 3A, B). These single-
The structure of "eye forms" and the stranded forks can be divided into the same two
mechanism of replication. When the DNA classes (SSGx and SSG2) observed for the replication
isolated from cleavage nuclei is examined in the forks in 2 and T7 DNAs (Inman and SchnSs, 1971;
electron microscope, molecules containing multiple Wolfson and Dressier, 1972). For this reason, and
eye forms, such as that shown in Figure 2, are ob- because they have the configuration and orientation
Figure 2. Replicating DNA chromosomal from cleavage nuclei. The portion of the molecule chromosomal shown here is
911 kb in length and contains 32 eye forms. This electron micrograph si reproduced from Kriegstein and Hogness ,)3791(
should which be consulted for the methods and conditions.
Downloaded from symposium.cshlp.org on March 13, 2012 - Published by Cold Spring Harbor Laboratory Press
208 BLUMENTHAL, KRIEGSTEIN, AND HOGNESS
eye forms in which (a) both forks contain single-
A 1 & SSG PRODUCTS -- 62% stranded regions (42~), (b) one fork is single-
stranded and one is all duplex 14( ~), and (c) both
forks are all duplex (16~o) are not significantly
different from the frequencies expected for in-
dependently occurring pairs of forks; i.e., the
WHISKER--SSG 1 WHISKER expected frequencies are (a) (0.63) ,2 or 40 ,o~ (b)
5.6% 11.4%
2(0.63)(0.37), or 47~o, and (c) (0.37) ,3 or 14~o,
1 - SSG 45% PRODUCTS OF BRANCH MIGRATION - 17%
respectively. These are the characteristics expected
C LLA XELPUD - %73 if both forks in an eye are replication forks.
B 2 - SSG 1%
Eye lengths and eye-to-eye distances. The
histograms given in Figure 4 indicate the distribu-
tions of eye lengths and of eye-to-eye distances. The
mean length of 439 eyfeo rms is 4.1 kb and the mean
of 316 eye-to-eye distances is 9.7 kb. The eye
lengths were taken from the same population of
trans CONFIGURATION OF 1 SSG PAIR IN EYES - 100%
molecules used for the eye-to-eye distances and
were therefore limited to those molecules con-
taining multiple eye forms. When all scorable eye
forms in each field were measured, the length
distribution of 279 eye forms was very similar to
that shown in Figure 4 but exhibited a slightly
Figure 3. The structure ofr eplication forks in the chromo- higher mean of 5.6 kb.
somes of D. melanogaster (Kriegstein and Hogness, 1973).
The percentages in (A), (B), and (C) derive from a popula- Mean origin-to-origin distance. The dis-
tion of 360 forks that were examined in the electron micro-
tance between origins in any given segment may be
scope after spreading DNA from cleavage nuclei in 40
formamide to allow visualization of both single- and different from the observed eye-to-eye distance;
double-stranded DNA. In (A), whiskers are shown to arise
from forks with one single-stranded gap (SSG1) by branch
migration that is assumed to occur while preparing the 25- I I 1 I I _=--/I
DNA. The lengths of the single-stranded regions in SSG~
and whisker forks yield equivalent distributions with
means of 0.22 kb and 0.21 kb, respectively. The arrows in 20 EYE LENGTHS
the forks indicate the 3'-OH termini in the newly replicated N=439
strands. The fact that the 3' --+ 5'-spoeifie E. coli exonu- ,z, MEAN = 4.1 kb
clease I destroys the whiskers confirms the orientation 15
given in the figure. This orientation is also compatible
with the property of DNA polymerases to add nucleotide
W
subunits only to 3'-OIt termini, which in turn forces syn-
thesis to be discontinuous in the daughter, where chain
elongation proceeds away from the fork. The SSG 1 forks
are a prediction of such a discontinuous synthesis, as are 5
forks with two single-stranded gaps (SSG~). The ratio of
SSG2 to SSG~ forks will depend on the rates of fork move- ~ i..i =.tr_ ~..p m-- ,,,..~.P
ment and of chain extension and will be small, as observed 0 (cid:127) "
here, if the fork movement is much slower than chain ex- 15 r T--'t------T--I --z'-
tension. The percentage in (D) refers to the fact that all of
the 37 eye forms in which both forks were SSG 1 exhibited
the trans configuration shown. These 37 are part of a total
EYE-TO-EYE DISTANCE
population of 128 eye forms in which each daughter seg- 10 N =316
ment could be unambiguously traced from one fork to
MEAN = 9.7 kb
another. The percentages for the different classes of eye
forms given in the text derive from this population.
Ill
O.
5
(see legend, Fig. 3) predicted from models which are
based on the properties of DNA polymerases, DNA
ligases, and Okazaki fragments, they are presumed
0 5 10 15 20 25 20>30
to represent replication forks.
kb
An analysis of the distribution of forks in the eye
Figure 4. Distributions of eye lengths and eye-to-eye
forms indicates that the eyes are created by bi- distances in the replicating DNA from cleavage nuclei. The
directional replication. All eye forms in which both eye length is the measured contour length of either
daughter segment in an eye. The eye-to-eye distance is
forks are SSG t exhibit the trans configuration
measured from the center of one eye to the center of the
shown in Figure 3D. The observed frequencies of adjacent eye.
Downloaded from symposium.cshlp.org on March 13, 2012 - Published by Cold Spring Harbor Laboratory Press
DNA REPLICATION IN CHROMOSOMES 209
fi§ ?i§247 §io, intervals and the mean value for the eye-to-eye
1 ~ + 1
I I I I I distance determined for each. These mean values
i I
O- i i I I have been plotted against the midpoint of their
tl , I i I I I respective class intervals to give the upper curve
shown in Figure 6. The mean values drop precipi-
tously to a plateau, which is reached when about
20-30 ~ of the segment is replicated. This plateau
value is maintained until about 70 to 80 o~ replica-
tion, when the mean values rise again. The simplest
interpretation of this curve is that initiation events
are highly synchronized and occur during the first
30 ~ of replication. The eyes formed by these events
then grow bidirectionally, with little merging until
I-
ca. 70 o~ replication, when mergers become a domin-
z 5
ant factor. The plateau value of 7.9 kb would
then represent the mean origin-to-origin distance.
Rate of fork movement. If we adopt the
above interpretation, that the plateau represents a
period when no new eyes are initiated and no
existing eyes merge, then the linear increase in
mean eye length observed during this period
(Fig. 6) can be used to determine the average rate of
fork movement. It is necessary to make a trans-
formation to units of time on the abscissa of Figure
6 for this determination. The diagram in Figure 5
01
illustrates this transformation. The line at the top of
Figure 5. The distribution of a segment of chromosomal the diagram represents a segment in the chromo-
DNA in a random population of cleavage nuclei repre- somal DNA just after nuclear division. Activation
senting all stages in the 10-min nuclear cycle. The single
line at the top of the figure (zero time) represents the of origins first occurs at time tl, and replication is
segment in nuclei which have just divided. Replication of completed by time Q, yielding two segments per
the segment begins in nuclei at 1 t and is just completed in
nucleus in place of one until the nucleus divides at
nuclei at t2, so that nuclei at later times in the cycle contain
two copies of the segment. 10 min (Rabinowitz, 1941). We are interested in
calculating the time it takes to replicate the
segment, or (t 2 -- tl), since this will represent the
either because there are origins in the segment that
interval 0-100~ replicated on the abscissa of
have not been activated by the time of sampling but
Figure 6.
would be activated later or because two or more eye
The population of segments examined in the
forms have merged before sampling. The effect of
electron microscope derive from a random popula-
these factors is depicted in Figure 5 between times
tion of cleavage nuclei in which nuclei from any
tl, when the first origins in a segment are activated,
given time interval in the nuclear cycle are equally
and t ,2 when the replication of the segment is
represented. The fraction of segments which are
complete. It is seen that the eye-to-eye distance at
observed to contain one or more eye forms, fe, is
first decreases, as more origins are activated and no
therefore given by
mergers take place. At later times, the effect of
mergers becomes dominant, and the eye-to-eye fe = (t2 tl)/[t2 -b 2(10 -- t2)],
distance should increase from a minimum value. It --
is this minimum value which best approximates the from which we derive
origin-to-origin distance in the segment.
We have obtained this minimum value by analyz- (t 2 -- tl) = f.(20 -- t~). (1)
ing the eye-to-eye distances in the following
manner. The data for the eye-to-eye distances was The value of fe for a random population of seg-
grouped into ten classes according to the fraction of ments having the same mean length as that for
the segment containing the eyes that had been the population used to obtain the data in Figure 6
replicated. That fraction equals the sum of the eye (see legend Fig. 6) is 0.07. Substituting this value
lengths in the segment divided by the total length in equation (1) yields
of the segment. The range between 0 and 100~o
replication was thereby divided into l0 equal class 2 t 1.4 -- 0.07t .2 (2)
-- t 1 =
Downloaded from symposium.cshlp.org on Ma r!chI 13, 2012 - Published by Cold Spring Harbor Laboratory Press
210 BLUMENTHAL, KRIEGSTEIN, AND HOGNESS
2,,t I i, i i, i, plateau interval). Since the eyes expand bidirec-
tionally, the rate off ork movement is one-half this
value, or 2.6 kb. rain -1.
Given the fork rate and the time required to
~/ E Y E - O T - E Y E
replicate a segment, one can then arrive at an
20 independent estimate of the mean origin-to-origin
distance, as this should equal twice the fork rate
16
multiplied by the replication time. The resuIting
value of 6.6 kb is in reasonable agreement with the
12 plateau value of 7.9 kb exhibited in Figure 6.
Distribution of active origins in cleavage
nuclei. According to the arguments given in the
preceding sections, the distribution of origin-to-
origin distances will be closely approximated by the
distribution of eye-to-eye distances in segments
that fall into the plateau region observed in Figure
6. The eye-to-eye distributions for segments which
r , 5 15 T N E C25 R E P D E35 T A45 C I L P E R 55 65 75 85 95 have experieneed 30 (~ 10), 40 ((cid:127) 10), 50 (q- 10), and
60(~:10)~o replication are given in Figure 7A-D,
and Figure 7E gives the distribution for a range of
I I I I I I
segments that includes most of the plateau region
0 0.25 0.50 0.75 1.00 1.25
(30 to 70 o~ replicated). The striking characteristie
TIME (MIN)
of these distributions is the periodic nature of the
F|gure 6. Mean eye.to-eye distances and mean eye lengths
modes that are observed. Five significant modes
as a function of the fraction of the segment that is repli-
cated. The data are the same as that given in Figure 4, but appear at 3.5, 7.5, 10.5, 13.5, and 16.5 kb in Figure
classified according to the extent of replication in each 7E and are usually seen in the four distributions
segment that was examined. The fraction of a segment that
with smaller samples. These modal values exhibit a
is replicated (upper scale on the abscissa) is defined as the
sum of the eye lengths divided by length of the segment, good fit to the integral multiples of 3.4 kb: i.e., 6.8,
where the boundaries of the segment are the midpoints of 10.2, 13.6, and 17.0 kb. There appears to be a sixth
the two terminal eyes (i.e., one-half of the length of each
of the two terminal eyes and the entire length of all other mode about 3 kb from the fifth, and there is a sug-
eyes are included in the sum of eye lengths). The segments gestion of a seventh mode about 3 kb from the
were divided into ten equal class intervals according to the
sixth.
percent replicated, and the mean value for each class was
plotted at the midpoint of the interval. The number of eye These curves indicate that the origins are not
lengths contributing to the mean for each class varied from randomly distributed but tend to be spaced at dis-
21 to 66 and averaged 44; the number of eye-to-eye
tances equal to 3.4a kb, where a is an integer. One
distances contributing to each class mean varied from 13 to
51 and averaged 32. The mean length of all segments was explanation for this spacing is that specific se-
25 kb. This is less than the mean length of 90 kb observed quences determine the positions of origins, that
for the molecules in the preparation, because the boundaries
of the field in the electron microscope were allowed to these origin-specific sequences tend to be spaced at a
restrict the size of the segments that were analyzed here. distance of 3.4 kb, and that the probability, P, that
a given sequence is activated to form an origin is
less than one. The probability of activation can be
Since t, is assumed to be less than or equal to the
estimated from the mean eye-to-eye distance for
interphase interval, or 3.4 min (Rabinowitz, 1941),
those distributions in which each eye is derived
then the minimum value for tz -- 1 t is 1.2 min. The
from a single origin (i.e., before mergers begin)
maximum value for 2 t --t I is 1.3rain and is
according to
calculated from equation (2) by substituting
2 t ~-- 1.2, since 1.2 < 2 t -- 1 < t tz. This small range
of values for ~t -- ~t allows us to use its midpoint, or P = (3.4)/(mean eye-to-eye distance).
1.25min, as the time interval covered by the
abscissa in Figure 5 without introducing errors The mean for the plateau region is essentially
greater than those involved in determining fe or the constant at 7.9 kb (Fig. 6), from which we estimate
nuclear doubling time (Rabinowitz, 1941). P ~--0.43. However, the mean steadily decreases
The rate at which the length of the eye forms with increasing replication in the preplateau region
increases during the plateau period can then be (0 to 30 o~ replication), indicating the progressive
determined from the slope of the eye length-versus- increase in P that is given in Table 1.
time curve (Fig. 6), which is 5.3kb.min -1 In this model, the frequency of origin-to-origin
(determined by method of least squares over the distances in the first peak (a = 1) will be P, that in
Downloaded from symposium.cshlp.org on March 13, 2012 - Published by Cold Spring Harbor Laboratory Press
2 3 4 5 6 7 2 3 4 5 6 7
20 I i i i I i i I i I i
i i i i i I i i I i i i
i ~ i I i I - i I I i ~ i _
15
I A. EYE-TO-EYE DISTANCES (cid:12)9 EYE-TO-EYE DISTANCES
30 (+10)% REPLICATED 60 (+10)% REPLICATED
Iz - I I N = 99 I N = 75 I
I= JJ MEAN = 8.3 kb MEAN = 8.0 kb
10 [ i I i- t I i -
441
I t i I I l t
xi i I I _ I I I
I/% I
I= I
I I , , I ,
I I I I I I I 1 '
15 i I i I I
V B. EYE-TO-EYE DISTANCES E. EYE-TO-EYE DISTANCES
40 (+-10)% REPLICATED 30-70% REPLICATED
I N =95 N = 170
I'-
/ MEAN = 7.8 kb I i MEAN = 7.9 kb
10
i i I i I I I i -
t,LI I I I I I I I
r
I )i ' I
, I't I I I /
I
0 L
25 I I I I | ] i i i i
i I i I I F. EYELENGTNS I L
1 i J J TOTAL POPULATION I -
20
N = 439, MEAN = 4.1 kb I
I t i i
C. EYE-TO-EYE DISTANCES
50 (+10)% REPLICATED N = 47, MEAN = 10.6 kb
15
I-- N =81 I t I
z
i.u MEAN = 7.8 kb ]
r I I I
1 I
10 I I
I [
i I
0
0 2.5 7.5 12.5 17.5 22.5 0 2.5 7.5 12.5 17.5 22.5
kb
Figure 7. Distribution of eye-to-eye distances in the plateau period. The eye-to-eye distances in segments that were
classified with respect to the extent of replication as described in Figure 6 are plotted in (A) through (D) for different
parts of the plateau period, and in (E) for the entire period, using 1-kb class intervals. Although the data for adjacent
distributions overlap, those used in (A) are independent from those in (C), and those in (B) are independent from those
in (D). Eye-to-eye distances greater than 25 kb, which are not shown, are: (A) 30-32 kb, 2.0~; (B) 30-32 kb, 1.1 ~;
(C) 32-34 kb, 1.2 ~ and 38-40 kb, 1.2 ~; (D) 32-34 kb, 1.3 ~ and 38-40 kb, 1.3 ~; (E) 30-32 kb, 32-34 kb, and 38-40 kb,
0.6 ~. Eye lengths in segments from the postplateau period are plotted in (F), where the distribution for the total population
is also shown (taken from Fig. 4) for comparative purposes. Eye lengths at 26-28 kb (2.1 ~o), 28-30 kb (6.3 ~o), 36-38 kb
(2.1 ~), and 50-54 kb (2.1 ~) in the 90 (cid:127) ( 10) ~ replicated segments are not shown.
Downloaded from symposium.cshlp.org on March 13, 2012 - Published by Cold Spring Harbor Laboratory Press
212 BLUMENTHAL, K_RIE~STEIN, AND HOGNESS
Table 1. Frequency Distributions of Origin-to-Origin Distances During Replication
Frequency of Origin-to.origin Distances in Peaks
Percent Probability 1st Peak 2nd Peak All Other Peaks
Replication P obs.a calc. obs. a calc. obs. a ealc.
Preplateau
10 (4-10) 0.220 0.22 0.220 0.20 0.172 0.58 0.608
15 (cid:127) ( 15) 0.280 0.28 0.280 0.23 0.202 0.50 0.518
20 (cid:127) ( 10) 0.343 0.34 0.343 0.20 0.225 0.46 0.432
25 ((cid:127) 0.382 0.39 0.382 0.20 0.236 0.41 0.382
Plateau
30, 40, 50, & 60 0.430 0.43 0.430 0.30 0.245 0.285 0.325
(cid:127) b (cid:127) b 4-0.035 b
a The observed values are taken from the eye-to-eye distances in the indicated distributions, which for the plateau region, are shown in
Figure 7A-D.
b These values give the range of frequencies observed for the four distributions in the plateau region, Figure 7A-D.
the second peak, P(1 -- P), and generally, that in apart (this should occur only late in replication of
the nth peak, P(1 -- p)n-1. We have calculated the the segment, since it will take 1.3 min at a fork rate
expected frequencies for the first and second peaks of 2.6 kb (cid:12)9 min -1 (cid:12)9 fork-l); and (d) five eyes from
and the sum of the frequencies for all other peaks origins separated by four 3.4-kb spacings will yield
[1-- P-- P(1-- P)---- 1--2Pq- p2]inthe pre- an eye 17.0 kb in length, as will three eyes from
plateau and plateau distributions and compared adjacent origins spaced 3.4 and 6.8 kb apart. These
these values with the observed values in Table .1 four length classes, which are also multiples of
There is reasonable agreement between the ob- 3.4 kb, are closely approximated by the positions of
served and calculated values, although in the peaksi n the distribution of eyel engths for segments
plateau region, the second peak is consistently that have been 90(-t-10)~ replicated (Fig. 7F).
larger than expected, and the third peak (Fig. 7) The largest peak, at 4.5 kb in Figure 7F, is the ex-
smaller. Part of this discrepancy may be due to a pected result of eye growth from single origins, with
low frequency of mergers taking place during the no mergers. The synchrony of activation, which
plateau period. limits the formationo f most if not all origins to the
It is evident from the relative constancy of the range 51 q- 51 o~ replication, and the bidirectional
eye-to-eye distributions in the plateau period growth of eyes with a fork rate of 2.6 kb (cid:12)9 -mIi n (cid:12)9
(Fig. 7 and Table )1 that mergers, like activations, fork -1 should yield a peak of eye lengths at 4.9 kb:
are infrequent between 30 and 60 o~ replication. i.e., (0.9 -- 0.15)(1.25 rain)(2 forks)(2.6 kb (cid:12)9 rain -1 (cid:12)9
However, the distributions of eye-to-eye distances fork -1) ~-- 4.9kb. Were there no mergers, this
for segments exhibiting greater extents of replica- would be the only peak expected; mergers decrease
tion indicate that mergers rapidly become a both the frequency of this peak and its modal value.
dominant factor. There is a shift toward greater We can estimate that this peak contains only 61 o~
distances, which increases the mean (Fig. 6), and a of all origins in this distribution and that about
loss or "blurring" of the peaks present in the 18 o~ of the origins are in eyes that result from
plateau distribution; indeed, it is this blurring effect mergers (the remaining 3 o~ are in the eyes of the
of mergers which makes it difficult to recognize the small peak seen at 1.5 kb in Figure 7F and may
3.4-kb spacing in the distribution of unclassified represent a low frequencY of activation that occurs
eye-to-eye distances shown in Figure 4. during the plateau and postplateau periods).
These considerations lead to the expectation that The distribution of eye lengths in segments that
the lengths of many or most eyes in postplateau exhibit progressively lesser extents of replication
segments will be determined by mergers. Thus, (a) are consistent with these interpretations. The fre-
two eyes from origins that are 3.4 kb apart will yield quency of origins in the peak attributable to eyes
an eye that is 6.8 kb in length at merger; (b) three that have not merged increases, and its modal value
eyes from adjacent origins separated by two 3.4-kb decreases with decreasing extent of replication;
spacings will yield an eye 10.2 kb in length; (c) four whereas the frequency otfh e length classes that can
eyes from origins separated by three 3.4-kb be attributed to mergers progressively decreases.
spacings will yield an eye 13.6 kb in length, as will For instance, at 40 (q-10) o~ replication, the peak of
two eyes from adjacent origins that are 6.8 kb nonmerged eyes contains 89 o~ otfh e origins and has
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DNA REPLICATION IN CHROMOSOMES 213
a modal value between 1.5 and 2.5 kb, whereas the is due to the absence of one or more activation
7.5- and 13.5-kb length classes that result from factors, except during a narrow time interval at the
mergers contain only 7 and 4% of the origins, beginning of replication. However, we have
respectively. observed small eyes (e.g., 0.5 kb) next to largere yes
(e.g., 3.3 kb) where the eye-to-eye distance was
Comparison of the number of activable about equal to the unit spacing of 3.4 kb. This
sequences in cleavage nuclei with the number topography is most simply explained by the
of chromomeres. The data presented in the formation oft he origin creating the small eye after
preceding section are qualitatively consistent with the origin-specific sequence in the larger eye has
the model that the aetivable, origin-specific been replicated.
sequences are repeated along the chromosomal We reserve a more detailed consideration of the
DNA with a spacing that is rather tightly centered concept of activable and nonactivable sequences
about a mean value of 3.4 kb, and that, in cleavage until after the following data on DNA replication in
nuclei, these sequences are randomly activated to cell culture nuclei has been presented.
produce origins with a probability that approaches
Radioautographic Observations of Replicat-
one-half over a short time interval (ca. 0.4 min).
ing DNA from Nuclei in Cell Cultures
The haploid genome of 165,000 kb (Rudkin, 1964,
1972; Rasch et al., 1971) would then contain about Experimental plan. Although the average
50,000 of these sequences. This is an upper limit, DNA segment is replicated in 1.2 to 1.3 min in
since our data are equally compatible with the cleavage nuclei, the S phase appears to include
possibility that the distribution of activable most, if not all, of the estimated 3.4 min of inter-
sequences is directly represented by the eye-to-eye phase (Rabinowitz, 1941). This conclusion is
distributions in Figure 7A-E (a model for this pos- inferred from the observation that the largest eye-
sibility is presented further on in this paper). In this to-eye distance that is consistently present in
case, the activation probability would approach distributions where it is unlikely to have arisen
one, and the mean distance between these sequences from mergers or to be erased by the formation of
would then be 7.9 kb. This leads to a mininmm new origins is 19.5 kb (Fig. 7B, C); and the time
number of 20,000 activable sequences per haploid required to replicate this interval between two
genome in cleavage nuclei. origins at the average fork rate is 3.7 min. The
The slightly more than 5000 chromomeres, 600 min devoted to S phase in cell cultures (Dolfini
counted by Bridges as bands in polytcne chromo- et al., 1970) at the same temperature (25~ is
somes, account for 78~o of the haploid genome therefore about 2 (cid:141) 10S-fold larger than in cleavage
(Rudkin, 1972). The number of activable sequences nuclei.
in this fraction of the genome is therefore between This increase in S phase could result from (a) a
16,000 and 40,000, or an average of three to eight decrease in the fork rate, (b) an increase in the
sequences per ehromomere. If there is validity to distances separating adjacent origins, and/or (c) an
the notion that individual chromomeres correspond increase in the time period during which the origins
to individual units of replication (see the review by are activated. In this initial study of the factors
Rudkin, 1972), then it would appear that the controlling the overall rate of chromosomal DNA
chromomeric structure somehow restricts the num- replication in Drosophila, we shall compare the fork
ber of these sequences that are available for rates and the distribution of origins in the two kinds
activation and that this aspect of the chromomeric of nuclei. A cursory examination oft he DNA from
structure is absent from cleavage nuclei. cell culture nuclei in the electron microscope
In regard to the possibility that the class of revealed a very much lower frequency of replicating
origin-specific sequences is divisible into activable forks than in DNA from cleavage nuclei. This
and nonactivable subclasses according to the observation indicated that a decreased fork rate
chromosomal state, we note that among the more cannot be the sole cause of the increased S phase
than one-thousand eye forms that we have ex- and that electron microscopy would be too time-
amined, no instance of an eye within an eye--i.e., consuming a method for the analysis of this DNA.
the reactivation of an origin within a daughter We therefore turned to radioautography, whereby
segment~has ever been observed. This observation the replicating DNA can be selectively examined
indicates that once replicated, a DNA sequence (Fig. 8).
cannot be activated to an origin until the next cycle The problems associated with the analysis of the
of replication. Such a restriction could result from linear series of grain tracks produced in the radio-
changes during the cycle in the DNA itself (e.g., autographs are summarized in Figure 9. Proceeding
methylation), or, more generally, in the chromatin from left to right in this figure, we see that individ-
structure. One can also imagine that this restriction ual grain tracks formed from eyes which exist at
