J. Hoi.
Biol. (1976) 94,127-132
The Replication Time of the Escherichia cob K12 Chromosome as a Function of Cell Doubling Time The relative frequency of two chromosomal markers in a strain of Escherichia coli K12 haa been measured as a function of cell doubling time by DNA:DNA hybridization. The results indicate that the replication time of the chromosome is constant and independent of the doubling time of the cells.
The pattern of chromosome replication in bacteria changes as a function of the doubling time (T) of the cells. In fast-growing cells DNA synthesis is continuous and the chromosome contains multiple growing points (Yoshikawa et al., 1964). As the doubling time is increased the mean number of growing points per chromosome decreases and gaps in DNA synthesis during the cell cycle may be observed (Lark, 1966; Helmstetter, 1967; Kubitschek et al., 1967). This changing pattern of DNA synthesis had led to the suggestion that the replication time of the chromosome, the time between initiation and termination of a round of replication, is similar in cells growing with different doubling times and that the overall rate of DNA synthesis is determined primarily by the frequency with which new rounds of replication are initiated (see e.g., Ma&e & Kjeldgaard, 1966). Helmstetter & Cooper (1968) used cultures synchronized by the membrane selection technique (Helmstetter & Cummings, 1963) to show that in Escherichia wli B/r cultures growing with doubling times between 22 and 40 minutes, the replication time (C) is constant at approximately 41 minutes. These measurements were later extended to a wider range of growth rates (Helmstetter et al., 1968); the authors concluded that the replication time remains constant up to cell doubling times of about 60 minutes but, that above this, the replication time becomes proportional to the cell doubling time (C = 2/3~). S’imilar conclusions have been reached by Gudas t Pardee (1974) for various E. coli strains synchronized by size selection following sucrose gradient centrifugation. Kubitschek & Freedman (1971), however, have measured DNA content per cell in exponentially growing batch cultures and have concluded that the replication time of the E. coli B/r chromosome remains constant, at about 47 minutes up to doubling times of at least 50 hours. In an attempt to resolve this question by a new approach, we have evaluated the chromosome replication time in batch cultures of a strain of E. coli K12 as a function of the doubling time of the cultures, by measuring the relative frequency of two chromosomal genea in an exponentially growing population of cells. This relative frequency is dependent on the mean number of growing points per chromosome (Sueoka, & Yoshikawa, 1965) and on the relative position of the two genes on the chromosome. The mean number of growing points per chromosome is, in turn, dependent on the frequency with which new growing points are introduced onto the chromosome and on the replication time. By definition, initiation of a new round of replication in cells under conditions of balanced growth must occur once per generation on the average, and thus the frequency of initiation is defined by the doubling 127
128
M. CHANDLER,
R. E. BIRD
AND
L.
CAR0
time of the culture. The relation between the frequency of two chromosomal markers (a and b) which are located on the same replication unit of the chromosome, the doubling time (T) and the mean replication time (C) is: a/b = 2CA’T (Chandler & Pritchard, 1975),
(1)
where A is the relative distance between the two chromosomal markers as a fraction of the replication unit. Measurement of the relative frequency of two known genes in a culture growing with a known doubling time will thus allow determination of the mean replication time. In our experiments, the relative frequency of two genes was measured by the method of Bird et al. (1972), in which the relative number of copies of two bacteriophages (Mu-l and h) integrated into the host chromosome at known positions is determined by DNA:DNA hybridization. The strain Mx213 used in this study is E. coli K12 WI485 thi thy A leu ilv (Louarn et al., 1974). It requires low concentrations of thymine for growth. This strain carries bacteriophage Mu-l integrated within the ilv locus (76 min on the standard E. coli genetic map; Taylor & Trotter, 1972) and is lysogenic for Airad- (inserted at a$tA, 17 min). DNA extracted from cultures growing at different rates was denatured, immobilized on membrane filters and the Mu-l/h ratios determined by hybridization with a mixture of 3H-labelled Mu-l DNA and 14C-labelled X DNA. The results are shown in Figure 1 (solid symbols). These values represent the ratio of the [3H](Mu-1) to [‘“C](X) counts bound, divided by the ratio obtained from parallel hybridizations of DNA preparations from a culture growing in glucose/Casamino acids and subsequently starved for amino acids. It has previously been shown that amino acid starvation of this strain, under these conditions, results in a Mu-l/h ratio of unity (Bird et al., 1972). This operation therefore acts as a normalization in all hybridization results. The solid curve (Fig. 1) represents the theoretical behaviour of the Mu-l/h ratio as a function of the doubling time, assuming that the replication time is a constant of 40 minutes and that replication is bidirectional and symmetric with an origin close to the ilv region. The value for A (eqn (1)) evaluated from the map of Taylor & Trotter (1972) is 32.5145 or 0.72. The Mu-l/h ratio predicted by the model of Helmstetter et al. (1968) for E. coli B/r is also shown in Figure 1 (dotted line). The data are clearly in agreement with the first model. It is possible that the cell densities attained by slow-growing cells at A450 = 0.2 may in some way affect the balanced growth of the culture. For example, the culture could be entering its stationary phase. This possibility was tested by extracting DNA from the slow-growing cultures at an A450 = O-1. The results, included in Figure 1 (A), show that there is no significant difference between the Mu-l/h ratio in DNA extracted from cells harvested at an A450 of O-1 and that extracted from cells harvested at an A,,, of O-2. The mean values of the replication time calculated from the data in Figure 1 are given in Table 1. The replication time calculated in this way (eqn (1)) is constant up to doubling times of at least 3.5 hours. The strain used in this study was a thymine auxotroph requiring low levels of thymine for growth. It has been shown that the intracellular levels of thymine in several such strains of E. coli affect the chromosome replication time (Pritchard & Zaritsky, 1970; Zaritsky & Pritchard, 1971).
LETTERS
u
TO THE
EDITOR
129
Doubling timc(r)(minl
Pm. 1. The ratio of Mu-l/A es a function of cell doubling time. Strain Mx213 (0, A) was grown in the following media, etloh supplemented with thymine to 80 pg/ml: L-broth (Lennox, 1966) (r = 22 mm); M9 (Adams, 1969) oontaining the following carbon sources at 1 o/, : glucose (T = 68 min) ; glucose with 0.4% oasein hydrolysate (T = 36 min) ; suooinate (r = 73 mm); glyoerol (7 = 30 mm); espartste (7 = 130 min); end proline (T = 229 min). Mx249 (Mx213 thy-4 +) (0, A) was grown in gluoose medium with 0.4% casein hydrolysem (T = 36 mm) and in M9 eapartam (7 = 160 min) in the absence of exogenous thymine. All minimal media were supplemented with isoleucme, v&line and leuoine to 20 &ml. The differenoe in doubling times of the aspartate-grown Mx213 and Mx249 wee real and persisted for at least 0 generations of balanoed growth in each experiment. The oells were grown between an A,,, of 0.1 and 0.2 with twofold dilutions once per generation into fresh warmed medium. Samples were removed et intervals for turbidity measurements ad, once per generation, SO-ml samples were removed when the cells reaohed an &,J of 0.2 (0, 0) and plaoed into ioe-cold M9 medium oontaming 10% pyridine for DNA extraction. In the 0888 of the aspertate and proline-grown cultures, s sample was also taken at an -4~s of 0.1 (A, A). AS a oontrol in the subsequent hybridizations, DNA was prepared from a culture in which rounds of replioation had been terminated by amino mid starvation. The DNA extra&ion and purifloation proaedures used were those desoribed by Louam et al. (1974), and the concentrations of 811 DNA ssmplea were determined using the diphenylamine method (Burton, 1966). The preparation of W-labelled h and 3H-labelled Mu-l DNA has been desoribed previously (Bird et al., 1972). The level of free phage was less than 1 in 10’ oells under 8ll conditions. Hybridizations were carried out on Sartorius membrane filters (MFSO) in the presenoe of 2 x SSC (SSC is O-16 ar-N&l, 0.016 r&sodium citrate) snd 60% formamide as desoribed by Kourilsky et al. (1971). The DNA concentrations were 2 H of oold DNA on filters hybridized with 0.2 rg of eaoh radioactive DNA, and hybridizations were carried out at 42’C for 6 days.
In consequence all the growth media used in this study were supplemented with a high concentration of thymine; it is then possible that the intracellular thymine pools are maintained at an artificially high level under these conditions, resulting in the maintenance of a fixed (minimum) replication time. It could be postulated that in a thymine prototroph, growing in the absence of exogenous thymine, the intracellular thymidine triphosphate pools may become rate limiting for cells growing with doubling times in excess of 60 minutes. This could lead to an increase in replication time as the doubling time is increased beyond this value. In order to test this possibility the strain Mx213 was made thyA+ by transduction and the Mu-l/X ratio measured in DNA samples obtained from cultures growing in the absence of exogenous thymine. The results obtained from two growth rates are presented in Figure 1 (open circles). It can be seen that there is no difference between
130
M. CHANDLER,
R. E. BIRD
AND
L.
CAR0
TABLE 1
The replication time of the E. coli K12 chromosome as a function of cell doubling time Time W-4
Strains
22 35 68 73 80 130 220 36 160
Mx213 Mx213 Mx213 Mx213 Mx213 Mx213 Mx213 Mx249 Mx249
No. of DNA samples
6
MU-l/l
(min)
39.0
2.43*0.05 1.74kO.04 1.42&O-04 1.34kO.02 1~29~0~01 1.18&0.02 1.11&0.07 1.70*0~05 1~11~0*04
38.7 39.8 42.7 40.7 43.0 45.9 37.1 31.3
The n~an replication time (6) has been calculated according to equation (1). Mean Mu-l/h ratios are given with their standard errors.
the results obtained from Mx213 and those obtained from its thyA+ derivative. It should be noted however that the thy+ strain still contains the drm or &a mutation either or both of which were present in the parental strain. In the experiments presented here we have considered the segment ilv-atth, representing approximately two thirds of the upper “replicon” of the bidirectionally replicating chromosome, and we have measured the average number of growing forks present between the two markers in exponential cultures at various growth rates. The results show that fork velocity remains constant, in that region of the chromosome, at all growth rates studied. They are entirely consistent with the conclusion that the replication time, in E. coli K12 W1485, is constant and independent of cell doubling time. The assumptions implicit in this conclusion, (1) nonselective isolation of DNA from various chromosomal regions, (2) constancy of average fork velocity over the entire replicon, (3) symmetry of the two forks produced in bidirectional replication, have been thoroughly tested only for some of the growth conditions used, and mostly for the fast growth rates (Bird et al., 1972). Our results agree with those of Kubitschek & Freedman (1971) who had measured the DNA content per cell in steady-state cultures, grown in the presence of various carbon sources or in glucose-limited chemostats, and who had concluded that the replication time remains constant irrespective of cell doubling time. A number of other experiments, however, have led to different conclusions. Estimates
of the replication
time from observations
of the relative
time of replica-
tion of various markers during a round of replication synchronized by amino-acid and thymine starvation (Bird et al., 1972) indicate that the replication time is dependent on the growth medium used. However, as the authors atate, this behaviour may be due to differential recovery of the cells in the different media following the synchronization procedure. Replication
times
have
also been measured
in synchronous
either by the membrane elution technique (Helmstetter
cultures
obtained
et al., 1968; Helmstetter,
LETTERS
TO THE
EDITOR
131
1974) or by selecting small cells from a sucrose gradient (Gudas & Pardee, 1974). The results from both types of experiments have been interpreted to show a proportionality between the replication time and the generation time for cells growing with generation times in excess of 60 minutes. Although strain differences cannot be entirely ruled out (Helmstetter, 1974) as an explanation for the disparity between the various results, there axe alterrmtive explanations. All methods of synchronization can reasonably be suspected of introducing metabolic changes which may affect replication time. More subtle changes, such as an increased spread in the timing of initiation or in fork velocity or the selection of a slightly abnormel class of cells, for example, could also affect the estimate of replication time in synchronized cultures (R. H. Pritchard, personal communication). Data obtained with synchronized cultures cannot, therefore, be assumed to reflect accurately the situation found in steady-state growth. A crude estimate of replication time can be given by an autoradiographic determination of the number of cells labelled, during steady-state growth, by a short pulse of [3H]thymine. Experiments of this type (Lark, 1966; Urban & Lark, 1971; Chai & Lark, 1970; M. Chandler t L. Caro, unpublished experiments) have, in general, results which indicated a replication time longer than 40 minutes in cells growing with long doubling times. The discrepancy between these results and our conclusions could be due to a number of possible factors: lack of symmetry in initiation or velocity of the two replication forks at slow growth rates, high levels of repair replication, residual incorporation of [3H]thymine pool after the presumed end of a pulse, irregular cell division, etc. Any one of these could affect the interpretation of autoradiograph experiments. In summary, our experiments show that, at cell doubling times between 22 and 220 minutes, average replication fork velocity remains constant during the duplication of the chromosomal segment situated between ilv and atth. That it remains constant over the entire chromosome is an assumption which requires further proof. One of us (M. C.) was the recipient of a Royal Society European Fellowship. was supported by grant no. 3.810.72 from the Fonds National Suisse. Deppertement de Biologie Moleculaire 30, quai Ernest-Ansermet CR- 12 1I-Geneve 4, Switzerland Received 2 September
This work
M. CHANDLER R.E. BIRD L. CARO
1974, and in revised form 3 February
1975
REFERENCES
Interscience Publishers, Inc., New York. Bird, R. E., Louarn, J., Martuscelli, J. t Care, L. (1972). J. Mol. Biol. 70, 549-500.
Adams, M. H. (1959). Editor
of Bacteriophages,
Burton, K. (1966). BiochmnGtmJ, 62, 316-323. Chai, N. & Lark, K. G. (1970). J. BacterioZ. 104, 401-409. Chandler, M. & Pritchard, R. H. (1975). Mol. Gen. @en&. In the press.
Gudas, L. J. & Pardee, A. B. (1974). J. BuctwioZ. Hehnstetter, Helm&et&r, Helmstetter, Helmstetter,
C. C. C. C.
E. E. E. E.
117, 1216-1223. 24, 417-427. (1974). J. Mol. 84, 1-19. & Cooper, S. (1968). J. Mol. BioZ. 81, 607-518. & Cummings, D. J. (1963). Proo. Not. Acad. Sci.,
(1967). J. Mol.
Bill. Biol.
U.S.A.
50, 767-774.
132
M. CHANDLER,
Hehnstetter,
C. E., Cooper,
S., Pierucci,
R. E. BIRD
0. & Revelas,
AND
L.
CAR0
E. (1968). CoZdSpring Harbor Symp.
Quunt. Biol. 33, 809-822. Kourilsky, P., Leidner, J. & Tremblay, G. Y. (1971). Biochimk, 58, 1111-1114. Kubitschek, H. E. & Freedman, M. L. (1971). J. Baderiol. 107, 95-99. Kubitschek, H. E., Bendigkeit, H. E. & Loken, M. R. (1967). Proc. Nat. Acad. Sci., U.S.A. 57, 1611-1617. Ltuk, C. (1966). Biochim. Biophya. Acta, 119, 517-525. Lennox, E. S. (1955). Virology, 1, 190-206. Louern, J., Funderburgh, M. & Bird, R. E. (1974). J. Bacterial. 120, l-5. Synthesis, W. A. BenjaMalee, 0. & Kjeldgaard, N. 0. (1966). Control of MacrmoZecuZa~ min, Inc., New York. Pritchard, R. H. & Zaritsky, A. (1970). Nature (London), 226, 126-131. Sueoke, N. & Yoshikawe, H. (1965). cfenet&, 52, 747-757. Rev. 36, 504-524. Taylor, A. L. & Trotter, C. D. (1972). Bactetil. Urban, J. E. & Lark, K. G. (1971). J. Mol. Biol. 58, 711-724. Yoshikawa, H., O’Sullivan, A. & Sueoka, N. (1964). Proc. Nut. Ad. Sci., U.S.A. 52,
973-980. Zaritsky,
A. t Pritchard,
R. H. (1971). J. Mol. BioZ. 60, 65-74.
Biol. (1976) 94,127-132
The Replication Time of the Escherichia cob K12 Chromosome as a Function of Cell Doubling Time The relative frequency of two chromosomal markers in a strain of Escherichia coli K12 haa been measured as a function of cell doubling time by DNA:DNA hybridization. The results indicate that the replication time of the chromosome is constant and independent of the doubling time of the cells.
The pattern of chromosome replication in bacteria changes as a function of the doubling time (T) of the cells. In fast-growing cells DNA synthesis is continuous and the chromosome contains multiple growing points (Yoshikawa et al., 1964). As the doubling time is increased the mean number of growing points per chromosome decreases and gaps in DNA synthesis during the cell cycle may be observed (Lark, 1966; Helmstetter, 1967; Kubitschek et al., 1967). This changing pattern of DNA synthesis had led to the suggestion that the replication time of the chromosome, the time between initiation and termination of a round of replication, is similar in cells growing with different doubling times and that the overall rate of DNA synthesis is determined primarily by the frequency with which new rounds of replication are initiated (see e.g., Ma&e & Kjeldgaard, 1966). Helmstetter & Cooper (1968) used cultures synchronized by the membrane selection technique (Helmstetter & Cummings, 1963) to show that in Escherichia wli B/r cultures growing with doubling times between 22 and 40 minutes, the replication time (C) is constant at approximately 41 minutes. These measurements were later extended to a wider range of growth rates (Helmstetter et al., 1968); the authors concluded that the replication time remains constant up to cell doubling times of about 60 minutes but, that above this, the replication time becomes proportional to the cell doubling time (C = 2/3~). S’imilar conclusions have been reached by Gudas t Pardee (1974) for various E. coli strains synchronized by size selection following sucrose gradient centrifugation. Kubitschek & Freedman (1971), however, have measured DNA content per cell in exponentially growing batch cultures and have concluded that the replication time of the E. coli B/r chromosome remains constant, at about 47 minutes up to doubling times of at least 50 hours. In an attempt to resolve this question by a new approach, we have evaluated the chromosome replication time in batch cultures of a strain of E. coli K12 as a function of the doubling time of the cultures, by measuring the relative frequency of two chromosomal genea in an exponentially growing population of cells. This relative frequency is dependent on the mean number of growing points per chromosome (Sueoka, & Yoshikawa, 1965) and on the relative position of the two genes on the chromosome. The mean number of growing points per chromosome is, in turn, dependent on the frequency with which new growing points are introduced onto the chromosome and on the replication time. By definition, initiation of a new round of replication in cells under conditions of balanced growth must occur once per generation on the average, and thus the frequency of initiation is defined by the doubling 127
128
M. CHANDLER,
R. E. BIRD
AND
L.
CAR0
time of the culture. The relation between the frequency of two chromosomal markers (a and b) which are located on the same replication unit of the chromosome, the doubling time (T) and the mean replication time (C) is: a/b = 2CA’T (Chandler & Pritchard, 1975),
(1)
where A is the relative distance between the two chromosomal markers as a fraction of the replication unit. Measurement of the relative frequency of two known genes in a culture growing with a known doubling time will thus allow determination of the mean replication time. In our experiments, the relative frequency of two genes was measured by the method of Bird et al. (1972), in which the relative number of copies of two bacteriophages (Mu-l and h) integrated into the host chromosome at known positions is determined by DNA:DNA hybridization. The strain Mx213 used in this study is E. coli K12 WI485 thi thy A leu ilv (Louarn et al., 1974). It requires low concentrations of thymine for growth. This strain carries bacteriophage Mu-l integrated within the ilv locus (76 min on the standard E. coli genetic map; Taylor & Trotter, 1972) and is lysogenic for Airad- (inserted at a$tA, 17 min). DNA extracted from cultures growing at different rates was denatured, immobilized on membrane filters and the Mu-l/h ratios determined by hybridization with a mixture of 3H-labelled Mu-l DNA and 14C-labelled X DNA. The results are shown in Figure 1 (solid symbols). These values represent the ratio of the [3H](Mu-1) to [‘“C](X) counts bound, divided by the ratio obtained from parallel hybridizations of DNA preparations from a culture growing in glucose/Casamino acids and subsequently starved for amino acids. It has previously been shown that amino acid starvation of this strain, under these conditions, results in a Mu-l/h ratio of unity (Bird et al., 1972). This operation therefore acts as a normalization in all hybridization results. The solid curve (Fig. 1) represents the theoretical behaviour of the Mu-l/h ratio as a function of the doubling time, assuming that the replication time is a constant of 40 minutes and that replication is bidirectional and symmetric with an origin close to the ilv region. The value for A (eqn (1)) evaluated from the map of Taylor & Trotter (1972) is 32.5145 or 0.72. The Mu-l/h ratio predicted by the model of Helmstetter et al. (1968) for E. coli B/r is also shown in Figure 1 (dotted line). The data are clearly in agreement with the first model. It is possible that the cell densities attained by slow-growing cells at A450 = 0.2 may in some way affect the balanced growth of the culture. For example, the culture could be entering its stationary phase. This possibility was tested by extracting DNA from the slow-growing cultures at an A450 = O-1. The results, included in Figure 1 (A), show that there is no significant difference between the Mu-l/h ratio in DNA extracted from cells harvested at an A450 of O-1 and that extracted from cells harvested at an A,,, of O-2. The mean values of the replication time calculated from the data in Figure 1 are given in Table 1. The replication time calculated in this way (eqn (1)) is constant up to doubling times of at least 3.5 hours. The strain used in this study was a thymine auxotroph requiring low levels of thymine for growth. It has been shown that the intracellular levels of thymine in several such strains of E. coli affect the chromosome replication time (Pritchard & Zaritsky, 1970; Zaritsky & Pritchard, 1971).
LETTERS
u
TO THE
EDITOR
129
Doubling timc(r)(minl
Pm. 1. The ratio of Mu-l/A es a function of cell doubling time. Strain Mx213 (0, A) was grown in the following media, etloh supplemented with thymine to 80 pg/ml: L-broth (Lennox, 1966) (r = 22 mm); M9 (Adams, 1969) oontaining the following carbon sources at 1 o/, : glucose (T = 68 min) ; glucose with 0.4% oasein hydrolysate (T = 36 min) ; suooinate (r = 73 mm); glyoerol (7 = 30 mm); espartste (7 = 130 min); end proline (T = 229 min). Mx249 (Mx213 thy-4 +) (0, A) was grown in gluoose medium with 0.4% casein hydrolysem (T = 36 mm) and in M9 eapartam (7 = 160 min) in the absence of exogenous thymine. All minimal media were supplemented with isoleucme, v&line and leuoine to 20 &ml. The differenoe in doubling times of the aspartate-grown Mx213 and Mx249 wee real and persisted for at least 0 generations of balanoed growth in each experiment. The oells were grown between an A,,, of 0.1 and 0.2 with twofold dilutions once per generation into fresh warmed medium. Samples were removed et intervals for turbidity measurements ad, once per generation, SO-ml samples were removed when the cells reaohed an &,J of 0.2 (0, 0) and plaoed into ioe-cold M9 medium oontaming 10% pyridine for DNA extraction. In the 0888 of the aspertate and proline-grown cultures, s sample was also taken at an -4~s of 0.1 (A, A). AS a oontrol in the subsequent hybridizations, DNA was prepared from a culture in which rounds of replioation had been terminated by amino mid starvation. The DNA extra&ion and purifloation proaedures used were those desoribed by Louam et al. (1974), and the concentrations of 811 DNA ssmplea were determined using the diphenylamine method (Burton, 1966). The preparation of W-labelled h and 3H-labelled Mu-l DNA has been desoribed previously (Bird et al., 1972). The level of free phage was less than 1 in 10’ oells under 8ll conditions. Hybridizations were carried out on Sartorius membrane filters (MFSO) in the presenoe of 2 x SSC (SSC is O-16 ar-N&l, 0.016 r&sodium citrate) snd 60% formamide as desoribed by Kourilsky et al. (1971). The DNA concentrations were 2 H of oold DNA on filters hybridized with 0.2 rg of eaoh radioactive DNA, and hybridizations were carried out at 42’C for 6 days.
In consequence all the growth media used in this study were supplemented with a high concentration of thymine; it is then possible that the intracellular thymine pools are maintained at an artificially high level under these conditions, resulting in the maintenance of a fixed (minimum) replication time. It could be postulated that in a thymine prototroph, growing in the absence of exogenous thymine, the intracellular thymidine triphosphate pools may become rate limiting for cells growing with doubling times in excess of 60 minutes. This could lead to an increase in replication time as the doubling time is increased beyond this value. In order to test this possibility the strain Mx213 was made thyA+ by transduction and the Mu-l/X ratio measured in DNA samples obtained from cultures growing in the absence of exogenous thymine. The results obtained from two growth rates are presented in Figure 1 (open circles). It can be seen that there is no difference between
130
M. CHANDLER,
R. E. BIRD
AND
L.
CAR0
TABLE 1
The replication time of the E. coli K12 chromosome as a function of cell doubling time Time W-4
Strains
22 35 68 73 80 130 220 36 160
Mx213 Mx213 Mx213 Mx213 Mx213 Mx213 Mx213 Mx249 Mx249
No. of DNA samples
6
MU-l/l
(min)
39.0
2.43*0.05 1.74kO.04 1.42&O-04 1.34kO.02 1~29~0~01 1.18&0.02 1.11&0.07 1.70*0~05 1~11~0*04
38.7 39.8 42.7 40.7 43.0 45.9 37.1 31.3
The n~an replication time (6) has been calculated according to equation (1). Mean Mu-l/h ratios are given with their standard errors.
the results obtained from Mx213 and those obtained from its thyA+ derivative. It should be noted however that the thy+ strain still contains the drm or &a mutation either or both of which were present in the parental strain. In the experiments presented here we have considered the segment ilv-atth, representing approximately two thirds of the upper “replicon” of the bidirectionally replicating chromosome, and we have measured the average number of growing forks present between the two markers in exponential cultures at various growth rates. The results show that fork velocity remains constant, in that region of the chromosome, at all growth rates studied. They are entirely consistent with the conclusion that the replication time, in E. coli K12 W1485, is constant and independent of cell doubling time. The assumptions implicit in this conclusion, (1) nonselective isolation of DNA from various chromosomal regions, (2) constancy of average fork velocity over the entire replicon, (3) symmetry of the two forks produced in bidirectional replication, have been thoroughly tested only for some of the growth conditions used, and mostly for the fast growth rates (Bird et al., 1972). Our results agree with those of Kubitschek & Freedman (1971) who had measured the DNA content per cell in steady-state cultures, grown in the presence of various carbon sources or in glucose-limited chemostats, and who had concluded that the replication time remains constant irrespective of cell doubling time. A number of other experiments, however, have led to different conclusions. Estimates
of the replication
time from observations
of the relative
time of replica-
tion of various markers during a round of replication synchronized by amino-acid and thymine starvation (Bird et al., 1972) indicate that the replication time is dependent on the growth medium used. However, as the authors atate, this behaviour may be due to differential recovery of the cells in the different media following the synchronization procedure. Replication
times
have
also been measured
in synchronous
either by the membrane elution technique (Helmstetter
cultures
obtained
et al., 1968; Helmstetter,
LETTERS
TO THE
EDITOR
131
1974) or by selecting small cells from a sucrose gradient (Gudas & Pardee, 1974). The results from both types of experiments have been interpreted to show a proportionality between the replication time and the generation time for cells growing with generation times in excess of 60 minutes. Although strain differences cannot be entirely ruled out (Helmstetter, 1974) as an explanation for the disparity between the various results, there axe alterrmtive explanations. All methods of synchronization can reasonably be suspected of introducing metabolic changes which may affect replication time. More subtle changes, such as an increased spread in the timing of initiation or in fork velocity or the selection of a slightly abnormel class of cells, for example, could also affect the estimate of replication time in synchronized cultures (R. H. Pritchard, personal communication). Data obtained with synchronized cultures cannot, therefore, be assumed to reflect accurately the situation found in steady-state growth. A crude estimate of replication time can be given by an autoradiographic determination of the number of cells labelled, during steady-state growth, by a short pulse of [3H]thymine. Experiments of this type (Lark, 1966; Urban & Lark, 1971; Chai & Lark, 1970; M. Chandler t L. Caro, unpublished experiments) have, in general, results which indicated a replication time longer than 40 minutes in cells growing with long doubling times. The discrepancy between these results and our conclusions could be due to a number of possible factors: lack of symmetry in initiation or velocity of the two replication forks at slow growth rates, high levels of repair replication, residual incorporation of [3H]thymine pool after the presumed end of a pulse, irregular cell division, etc. Any one of these could affect the interpretation of autoradiograph experiments. In summary, our experiments show that, at cell doubling times between 22 and 220 minutes, average replication fork velocity remains constant during the duplication of the chromosomal segment situated between ilv and atth. That it remains constant over the entire chromosome is an assumption which requires further proof. One of us (M. C.) was the recipient of a Royal Society European Fellowship. was supported by grant no. 3.810.72 from the Fonds National Suisse. Deppertement de Biologie Moleculaire 30, quai Ernest-Ansermet CR- 12 1I-Geneve 4, Switzerland Received 2 September
This work
M. CHANDLER R.E. BIRD L. CARO
1974, and in revised form 3 February
1975
REFERENCES
Interscience Publishers, Inc., New York. Bird, R. E., Louarn, J., Martuscelli, J. t Care, L. (1972). J. Mol. Biol. 70, 549-500.
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