DNA synthesis during the division cycle of rapidly growing Escherichia coli

Helmstetter, Charles E.; Cooper, Stephen

doi:10.1016/0022-2836(68)90424-5

Cited by 458 publications

(276 citation statements)

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“…These data are consistent with the existence of multiple growing forks for about 10 Conclusion.-The results of the experiments reported here indicated that in rapidly growing E. coli B/r, the chromosome contains multiple growing points. Since these experiments essentially "count" the number of growing points present at various cell ages, the possibility that the variation in the rate of DNA synthesis with cell age observed by Helmstetter and Cooper'0 is due to a change in the rate of synthesis of a single growing point may be ruled out.…”

supporting

confidence: 91%

EVIDENCE FOR MULTIPLE POINTS ON THE GENOME OF RAPIDLY GROWING E. coli B/r

Ward

Glaser

1969

Proc. Natl. Acad. Sci. U.S.A.

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Abstract.-Synchronized cultures of rapidly growing E. coli B/r cells were starved for a required amino acid at various cell ages to allow ongoing chromosome replication to be completed without initiation of new replication cycles. It has been found that when such synchronized cultures are exposed to the mutagen nitrosoguanidine, genetic markers just in the process of replication show a markedly higher mutation rate than markers elsewhere on the chromosome. The number of growing points on the chromosome at each cell age can then be determined by observing the nitrosoguanidine-induced mutation rates for specific genetic markers on the genome. These experiments indicate that there exist multiple growing points on the genome for about ten minutes during the life cycle when the cells are growing with a doubling time of 28 minutes.Introduction.-The E. coli chromosome is thought to be a single circular doublestranded molecule of DNA which, in slowly growing cells, is replicated semiconservatively by a single Y-shaped growing fork.1-6 Maal0e and Kjeldgaard7 have suggested that the rate of DNA synthesis per fork at a given temperature is independent of the cell growth conditions provided there are no substrate or energy limitations. One prediction of this model is that cells growing with a doubling time less than the time required for one fork to traverse the length of the genome will have multiple growing points for at least a portion of the life cycle. Evidence for such multiple growing points in B. subtilis has been reported.8 9Helmstetter and Cooper10 have examined the variation with cell age of the rate of DNA synthesis in rapidly growing E. coli and found it to be consistent with the existence of multiple growing points. However, such measurements do not eliminate the possibility that the rate of synthesis per fork, rather than the number of forks per genome, changes during the life cycle, leaving the number of forks constant. The present work was performed to distinguish between these two

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supporting

confidence: 91%

EVIDENCE FOR MULTIPLE POINTS ON THE GENOME OF RAPIDLY GROWING E. coli B/r

Ward

Glaser

1969

Proc. Natl. Acad. Sci. U.S.A.

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“…(iv) The probability of initiation of a cell to spore formation is proportional to the fraction of T, target sites unoccupied at any particular instant, i.e. From this, by means of equations ( I ) and (22) Lark, 1966, andHelmstetter &Cooper, 1968).…”

Section: Te+t + Ttementioning

confidence: 99%

Sporulation in Bacillus subtilis. Theoretical and Experimental Studies in Continuous Culture Systems

Dawes

Thornley

1970

Journal of General Microbiology

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A theoretical treatment of growth and sporulation of Bacillus subtilis Marburg strain 168 in continuous culture is given. Sporulation is considered as a probability event, and a specific rate constant for its initiation in vegetative cells is introduced. The processes of growth and sporulation have been assumed to compete with each other in the vegetative organism. Equations are given which allow calculation of the rate constants characterizing growth and sporulation in steady-state continuous-flow systems. The relationship between the specific rate of initiation of sporulation and the growth rate of vegetative cells was determined with glucose-limited cultures and was approximately linear ; the spore initiation rate increased with decreasing growth rate. This result was used to obtain equations relating the incidence of refractile spores to the dilution rate of a continuous culture maintained in a steady state. A number of possible models of the process of spore initiation have been considered; two were consistent with the linear relationship between spore initiation rate and growth rate determined by experiment.

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“…Escherichia coli B/r F65 (provided by Alan Leonard) containing the minichromosome pAL49 (7.6 kb, Kanr; provided by A. C. Leonard) and the R6K plasmid (Ampr Strr; provided by D. R. Helinski) or the mini-R6K plasmid pMF26 (Ampr; provided by D. R. Helinski) was grown for six to nine generations in 100 ml of C medium (3) supplemented with the amino acids histidine (42 ,ug/ml), proline ( a 37°C incubator. Fractions were collected for an interval of time equivalent to the labelling period.…”

mentioning

confidence: 99%

Replication of the R6K plasmid during the Escherichia coli cell cycle

1992

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The cell-cycle replication pattern of the R6K plasmid has been investigated by using the membrane-elution technique to produce cells labeUled at different times during the division cycle and scintillation counting for quantitative analysis of radioactive plasmid DNA. The high-copy plasmid R6K replicates exponentially in a cell-cycle-independent manner. A mini-R6K plasmid deleted for the onra origin of replication also replicates, exponentially in a cell-cycle--independent manner. Plasmid R6K is a relatively large, high-copy, conjugative resistance plasmid with three replication origins (7). In vivo, replication initiates from orio about 50% of the time, from oril3 about 35% of the time, and from ori-y about 15% of the time (1). R6K is unique in that most large, self-transmissible plasmids exist at low copy number and are DNA polymerase I independent, whereas most high-copy plasmids are small, nonconjugative, and DNA polymerase I dependent (6).Recently, we showed that the low-copy F plasmid and P1 prophage replicate in a cell-cycle-specific manner and that replication occurs when a constant mass per origin is achieved (4, 5). In contrast, the high-copy ColEl-type plasmids replicate in a cell-cycle-independent manner (9, 14). Considering copy number alone, one might expect R6K to replicate in a cell-cycle-independent manner. Taking into account structural and functional characteristics of the plasmid, one might expect R6K to replicate in a cell-cyclespecific manner. The replication pattern has not been studied previously. In order to solve this dilemma, we have now investigated the replication patterns of the R6K plasmid and mini-R6K plasmid pMF26 (deleted for oria) by using the membrane-elution technique and quantitative determination of label incorporated into plasmid DNA.Escherichia coli B/r F65 (provided by Alan Leonard) containing the minichromosome pAL49 (7.6 kb, Kanr; provided by A. C. Leonard) and the R6K plasmid (Ampr Strr; provided by D. R. Helinski) or the mini-R6K plasmid pMF26 (Ampr; provided by D. R. Helinski) was grown for six to nine generations in 100 ml of C medium (3) supplemented with the amino acids histidine (42 ,ug/ml), proline (46 ,g/ml), and thymine (20 jig/ml) and with glucose (0.4%) or succinate (0.4%) at 37°C in order to obtain exponential growth prior to starting a membrane-elution experiment. The exponentially growing cells (100 ml, 108 cells per ml) were labelled with[3H]methyl thymidine (60 to 80 Ci/mmol, 10 RCi/ml) for 5% of the doubling time and filtered onto a nitrocellulose membrane at the end of the labelling period. The cells were washed with medium of the same composition containing cold thymidine (100 ,ug/ml). The membrane apparatus was inverted, and newborn cells were eluted from the membrane with prewarmed C medium pumped at a rate of 2 ml/min in * Corresponding author. t Present address: Department of Chemical Engineering, University of California, Berkeley, CA 94720. a 37°C incubator. Fractions were collected for an interval of time equivalent to the labelling period. Ce...

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DNA synthesis during the division cycle of rapidly growing Escherichia coli

Cited by 458 publications

References 12 publications

EVIDENCE FOR MULTIPLE POINTS ON THE GENOME OF RAPIDLY GROWING E. coli B/r

EVIDENCE FOR MULTIPLE POINTS ON THE GENOME OF RAPIDLY GROWING E. coli B/r

Sporulation in Bacillus subtilis. Theoretical and Experimental Studies in Continuous Culture Systems

Replication of the R6K plasmid during the Escherichia coli cell cycle

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