Effect of Growth Conditions on the Formation of the Relaxation Complex of Supercoiled ColE1 Deoxyribonucleic Acid and Protein in Escherichia coli

Colicinogenic factor E1 (ColE1) is present in Escherichia coli strain JC411 (ColE1) cells to the extent of about 24 copies per cell. This number does not appear to vary in situations which give rise to twofold differences in the amount of chromosomal deoxyribonucleic acid (DNA) present per cell. If cells are grown in the absence of glucose, approximately 80% of the ColE1 molecules can be isolated as strand-specific DNA-protein relaxation complexes. When glucose is present in the medium, only about 30% of the plasmid molecules can be isolated as relaxation complexes. Medium shift experiments in which glucose was removed from the medium indicate that within 15 min after the shift the majority (>60%) of the plasmid can be isolated as relaxation complex. This rapid shift to the complexed state is accompanied by a two- to threefold increase in the rate of plasmid replication. The burst of replication and the shift to the complexed state are both inhibited by the presence of chloramphenicol. Inhibition of protein synthesis in log cultures by the addition of chloramphenicol or amino acid starvation allows ColE1 DNA to continue replicating long after chromosomal replication has ceased. Under these conditions, noncomplexed plasmid DNA accumulates while the amount of DNA that can be isolated in the complexed state remains constant at the level that existed prior to treatment. In the presence of chloramphenicol, there appears to be a random dissociation and association of ColE1 DNA and “relaxation protein” during or between rounds of replication.     

Probable synchronous replication of mitochondrial DNA in cultures of chick embryo fibroblasts

Cultures of chick embryo fibroblasts were synchronized using a procedure previously described. The profile of incorporation of tritiated thymidine showed a main peak of nuclear DNA replication followed by a small peak between 18 and 24 hr after induction of the cell division, and representing 10 to 25% of the main peak. To identify this small peak, cells were treated with ethidium bromide(EB) chloramphenicol (CAP) or 9-B-D arabinofuranosyl adenine (Ara-A). When EB (1 mug ml-1) and CAP(25mug ml-1) were added at time of induction of mitosis (T0) or 14 hr later (T14) the small peak was suppressed whereas the main peak was not decreased. On the contrary, only the main peak was suppressed when Ara-A was added at T0 or T14. These results suggest that the peak might correspond to the synchronous replication of the mitochondrial DNA during the G2 and M phases of the cell division cycle.     

Replication of colicinogenic factor E 1 DNA: evidence for a discontinuous replication mechanism

The mechanism of Col E 1 DNA replication was investigated in a plasmolysed cell system prepared from chloramphenicoltreated E. coli JC 411 (Col E 1). After pulse-labelling with (3)H-dTTP a considerable fraction of the newly synthesized DNA was recovered as single-stranded fragments. Upon alkali denaturation the pulse label was found in DNA chains sedimenting slower than unit length Col E 1 strands with a prominent peak at 5 S. During a chase with unlabeled precursors the label is transferred nearly completely into supercoiled Col E 1 DNA. DNA ligase appears to be required for the joining of the 5 S pieces since in the absence of NAD an accumulation of short fragments is observed.     

Analysis of a recessive plasmid copy number mutant: evidence for negative control of Col E1 replication.

The Col E1-derivative copy number mutant plasmid pOP1Δ6 has been used to investigate the control of plasmid replication. pOP1Δ6 normally exists at about 200 copies per chromosome, while the wild-type plasmid from which it was derived (pBGP120) exists at about 15 copies per chromosome. We have observed that in E. coli containing both pOP1Δ6 and pBGP120, the copy number of pOP1Δ6 is lowered to 4–6 copies per chromosome. Thus the mutation in pOP1Δ6 is recessive. The association between the two plasmids is stable in E. coli, indicating that incompatibility properties as well as replication control characteristics have been altered in pOP1Δ6. Co-residence of the unrelated plasmid pSC101 with pOP1Δ6 has no detectable effect on pOP1Δ6 copy number. These results suggest that a plasmid-specific, diffusible repressor may act negatively to control plasmid copy number, and that pOP1Δ6 produces a defective repressor or is altered in repressor synthesis. We have constructed in vitro a plasmid which is identical in size to pQP1Δ6 but contains a replication origin region derived from pBGP120. Since this plasmid, pNOP1, exists stably (like pBGP120) at 10–15 copies per chromosome, the high copy number of pOP1Δ6 is not related to its reduced size relative to pBGP120. To localize the mutation in pOP1Δ6 responsible for DNA overproduction, we have cloned fragments of pBGP120 into pOP1Δ6 and selected for plasmids with wild-type copy number. We find that a 2.0 kb region of pBGP120 DNA surrounding the origin of plasmid DNA replication is capable of suppressing the DNA overproducer phenotype of pOP1Δ6. The 2.0 kb fragment is capable of independent self-replication or can integrate into pOP1Δ6 in vivo to form a composite plasmid with two origins of replication. The overproducer phenotype of pOP1Δ6 is suppressed in either configuration.     

Changing rates of histone mRNA synthesis and turnover in Drosophila embryos

The rates of synthesis and turnover of histone mRNA in Drosophila embryos were determined by hybridization of in vivo and in vitro labeled embryonic RNA to Drosophila histone DNA of the recombinant plasmid cDm500. There is a large store of maternal histone mRNA, equivalent to at least 7 X 10(7) copies of each of the five classes of histone mRNA per embryo. Embryonic synthesis of histone mRNA begins at 90 min after oviposition, making the histone genes among the first to be transcribed by embryonic nuclei. Embryonic histone mRNA accumulates rapidly during the blastoderm and gastrula stages. The peak in the rate of histone mRNA synthesis per embryo coincides with the peak in the rate of DNA synthesis per embryo, which occurs at 6 hr after oviposition. After 6 hr, as the rate of DNA synthesis per embryo decreases, the rate of histone mRNA synthesis and the total mass of histone mRNA per embryo both drop sharply. The rate of histone mRNA synthesis per gene falls more than 60 fold in the first 13 hr after oviposition, from 1.3 -2.5 copies per gene-min at 2 hr to 0.02-0.03 copies per gene-min at 13 hr. From measurements of the mass of histone mRNA per embryo and of the rate of accumulation of newly synthesized histone mRNA at a number of stages of early embryogenesis we determined that the cytoplasmic half-life of histone mRNA decreases approximately 7 fold during early Drosophila development, from 2.3 hr at blastoderm to 20 min by the end of gastrulation. Thus the level of expression of histone genes in Drosophila development is controlled not only by the size of the maternal mRNA pool and changes in the rate of histone mRNA synthesis, but also by changes in the rate of histone mRNA turnover.     

Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation.

Physiocochemical evidence presented indicates plasmid deoxyribonucleic acid (DNA) can associate with host chromosome without linear insertion of the former into the latter. This conclusion is based on the observation that covalently closed circular (CCC) plasmid DNA can cosediment with undegraded host chromosome in a neutral sucrose gradient. When F plus bacteria are lysed under conditions that preserve chromosome, approximately 90% of CCC F sex factor plasmid (about 1% of the total DNA) is found in folded chromosomes sedimenting at rates between 1,500 and 4,000s. The remaining 10% of the CCC F DNA sediments at the rate (80S) indicative of the free CCC plasmid form. Reconstruction experiments in which 80S, CCC F DNA is added to F plus or F minus bacteria before cell lysis show that exogenous F DNA does not associate with folded chromosomes. In F plus bacteria, F plasmid is harbored at a level of one or two copies per chromosomal equivalent. In bacteria producing colicin E1, the genetic determinant of this colicin, the Col E1 plasmid, is harbored at levels of 10 to 13 copies per chromosomal equivalent; yet, greater than 90% of these plasmids do not cosediment with the 1,800S species of folded chromosome. However, preliminary evidence suggests one or two Col E1 plasmids may associate with the 1,800S folded chromosome. Based on evidence presented in this and other papers, we postulate F plasmid can link to folded chromosome because the physicochemical structure of the plasmid resembles a supercoiled region of the chromosome and, therefore, is able to interact with the ribonucleic acid that stabilizes the folded chromosome structure. Implications of this model for F plasmid replication and segregation are discussed.     

Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.     

Effects of cycloheximide on virus RNA replication in an inducible line of polyoma-transformed rat cells.

In this article, we describe two distinct effects of cycloheximide (CH), a potent inhibitior of protein synthesis, on the replication of polyoma virus (PV) DNA in an inducible line of PV-transformed rat cells (LPT cells). Exposure of LPT cells to CH causes up to an 8 fold increase in the cellular concentration of PV DNA determined by molecular hybridization. The same treatment inhibits cell division and chromosomal DNA replication. However, the amount of chromosomal DNA per cell is not affected by the drug. In LPT cells treated with mitomycin C (MMC), PV DNA replication is enhanced after 7 hr. During the period extending from 7 hr to 24 hr, the concentration of virus DNA increases at least 100 fold. CH added to the cells 0-7 hr after treatment with MMC inhibits the replication of PV DNA by 90-100%. The inhibition is less effective in cells exposed to CH from 7 hr and on. The inhibitory effect is reversible: virus DNA synthesis is resumed after removal of CH from the growth medium. Thus CH acts as an inducer of virus DNA synthesis in cells whose resident viral genome is repressed, but inhibits the autonomous replication of the activated genome following induction with MMC.     

Effects of rifampicin, streptolydigin and actinomycin D on the replication of Col E1 plasmid DNA in Escherichia coli

We recently reported (Clewell et al., 1972) on an inhibitory effect of rifampicin on Col E1 plasmid replication. The present study represents a further characterization of this phenomenon as well as a study of the effects of two other known inhibitors of RNA synthesis, Streptolydigin and actinomycin D.During treatment of cells with chloramphenicol the colicinogenic factor E₁ (Col E1) continues to replicate for more than ton hours. During this time 4 to 5 S RNA is also synthesized. When varying concentrations of rifampicin were included during chloramphenicol treatment, inhibition of plasmid DNA synthesis correlated very closely with inhibition of cellular RNA synthesis. Similar experiments testing the effects of Streptolydigin and actinomycin D (during chloramphenicol treatment) showed that cellular RNA synthesis was at least 100 times more sensitive to these drugs than was plasmid DNA synthesis.When actively growing cells (i.e. cells not treated with chloramphenicol) were treated with a high concentration of rifampicin (250 μg/ml), chromosomal DNA synthesis continued to an extent representing about a 50% increase in DNA, while plasmid DNA synthesis appeared to stop abruptly.     

Direct Inhibition of <Emphasis Type="Italic">Col</Emphasis> E<Subscript>1</Subscript> Plasmid DNA Replication in <Emphasis Type="Italic">Escherichia coli</Emphasis> by Rifampicin

COLICINOGENIC factor E₁ (Col E₁) is a small bacterial plasmid (4.2×10⁶ daltons) present in colicinogenic strains of Escherichia coli¹ to the extent of about twenty-four copies per cell (Clewell and Helinski, unpublished results), which continues to replicate in the presence of high levels of chloramphenicol, a specific inhibitor of protein synthesis, although the chromosome only completes current rounds of replication and ceases (Clewell and Helinski, unpublished results). The average rate of Col E₁ semiconservative replication in the absence of protein synthesis is, in certain conditions, faster than (as much as eight times) the normal rate of synthesis (Clewell, unpublished results). Replication continues for 10–15 h after the addition of chloramphenicol, resulting in nearly 3,000 copies of Col E¹ DNA per cell. We are taking advantage of this system to study the effects of a number of antibiotics on DNA replication and now report evidence that rifampicin (an active semisynthetic derivative of rifamycin B)², an antibiotic known specifically to inhibit bacterial DNA dependent RNA polymerase^3–6, has a dramatic inhibitory effect on Col E¹ DNA replication.     

Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans

The complexity of the genome of Micrococcus radiodurans was determined to be (2.0 +/- 0.3) X 10(9) daltons by DNA renaturation kinetics. The number of genome equivalents of DNA per cell was calculated from the complexity and the content of DNA. A lower limit of four genome equivalents per cell was approached with decreasing growth rate. Thus, no haploid stage appeared to be realized in this organism. The replication time was estimated from the kinetics and amount of residual DNA synthesis after inhibiting initiation of new rounds of replication. From this, the redundancy of terminal genetic markers was calculated to vary with growth rate from four to approximately eight copies per cell. All genetic material, including the least abundant, is thus multiply represented in each cell. The potential significance of the maintenance in each cell of multiple gene copies is discussed in relation to the extreme radiation resistance of M. radiodurans.     

Deoxyribonucleic Acid Synthesis During Microcyst Germination in Myxococcus xanthus

Deoxyribonucleic acid (DNA) synthesis was measured during microcyst germination in Myxococcus xanthus by radioactive thymidine incorporation, autoradiography, and chemical analysis. Microcysts contained an average of 6.6 conserved units of DNA, corresponding to 3 to 4 chromosomes per cell. Correlation of the DNA content and chromosome number of microcysts indicated that the molecular weight of the nonreplicating M. xanthus chromosome is 4.9 x 10(9) daltons. DNA synthesis was initiated 3.5 to 4 hr after induction of germination. From 4 to 6 hr, the rate of synthesis was constant and the accumulation was linear. After a lag period (6 to 6.5 hr), the rate of DNA synthesis increased, reaching a second plateau at 9 hr. From 9 to 11 hr, the rate was again constant and the accumulation was linear. Cellular division during germination showed an unusual kind of synchrony. A model is presented that accounts for chromosomal replication and cell division during microcyst germination.     

Segregation into and Replication of Plasmid Deoxyribonucleic Acid in Chromosomeless Segregants of Escherichia coli

The progeny cells of Escherichia coli strain P678-54, which normally do not contain deoxyribonucleic acid (DNA), were found to carry DNA when the parental cell carried colicin factor E1 (Col E1). The DNA found in the normally DNA-less segregants was shown to be Col E1 DNA, which is present primarily as a covalently closed circular molecule that can undergo more than one complete cycle of replication.     

Incompatibility properties of Col E1 and pMB1 derivative plasmids: Random replication of multicopy replicons

The incompatibility properties of Col E1-like plasmids have been examined in Rec⁺ and RecA^? bacteria. Two Col E1- (or two pMB1-) derivative plasmids coreplicated in the same clone for many cell doublings, irrespective of the rec genotype of host bacteria. Their kinetics of segregation were found to be consistent with models that assume a random choice of template molecule for each plasmid replication event, but with models based on a single (master) template molecule per cell. In contrast, minimal coreplication of a Col E1- and a pMB1-derivative plasmid occurred, with the latter type rapidly excluding the former. We suggest here that the pMB1 derivatives, pMB9 and pBR322, are less sensitive than Col E1 derivatives to the putative inhibitor that regulates plasmid replication, due to base sequence differences in their target for the inhibitor, and consider one mechanism whereby the duplication of Col E1-like plasmids might be regulated.     

Replication of Simian Virus 40 Deoxyribonucleic Acid: Analysis of the One-Step Growth Cycle

The time course of replication of simian virus 40 deoxyribonucleic acid (DNA) was investigated in growing monolayer cultures of subcloned CV1 cells. At multiplicities of infection of 30 to 60 plaque-forming units (PFU)/cell, first progeny DNA molecules (component 1) were detected by 10 hr after infection. During the following 10 to 12 hr, accumulation of virus DNA proceeded at ever increasing rates, albeit in a non-exponential fashion. The rate of synthesis then remained constant, until approximately the 40th hour postinfection, when DNA replication stopped. Under these conditions, the duration of the virus growth cycle was approximately 50 hr. The time needed for the synthesis of one DNA molecule was found to be approximately 15 min. At multiplicities of infection of 1 or less than 1 PFU/cell, the onset of the linear phase of DNA accumulation was delayed, but the final rate of DNA synthesis was the same, independent of the input multiplicity. This was taken as a proof that templates for the synthesis of viral DNA multiply in the cell during the early phase of replication. However, the probability for every replicated DNA molecule to become in turn replicative decreased constantly during that phase. This could be accounted for by assuming a limited number of replication sites in the infected cell.     

Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state

Thymidylate synthase (TS) activity is very low in resting mouse 3T6 fibroblasts but increases sharply in growth-stimulated cells at about the same time the cells enter S phase. To study the mechanism responsible for the increase in TS level, we isolated a 5-fluorodeoxyuridine (5-FdUrd)-resistant cell line (LU3-7) that overproduces TS and its mRNA about 50-100-fold. In this paper we show that the LU3-7 cells were able to rest in the G0 state of the cell cycle when maintained in medium containing 0.5% serum. When the serum concentration was increased to 10%, the resting cells reentered the cell cycle and began DNA replication about 12 hr later. TS activity remained at the resting level until DNA replication began, then increased at later times. The increase was not affected when the cells were stimulated in the presence of DNA synthesis inhibitors. The rate of synthesis of TS (as determined in a pulse-labeling experiment) remained at the resting level for the first 10 hr following stimulation, then increased 8-9-fold by 25 hr following serum stimulation. The half-life of TS in growing LU3-7 cells was measured in a pulse-chase experiment and found to be greater than 24 hr. Therefore the increase in TS activity was primarily due to an increase in the rate of synthesis of the enzyme. Since TS gene expression appears to be regulated in a similar manner in LU3-7 cells and in the parental 3T6 cells, the LU3-7 cells should be a good model system for detailed analysis of the mechanism for regulating TS gene expression in mammalian cells.     

DNA replication and the development of spontaneous mutations in the somatic cells of mammals. I. The relationship between the outcome of gene mutations and the duration of cell life]

The reduction of serum concentration in culture medium to 0,1% was proposed as a method of slowing division and DNA replication in Chinese hamster cells in vitro. Under these conditions the rate of DNA replication was reduced by more than one order of magnitude, while cell viability remained high enough (about 10%) for a long period up to 30 days. The spontaneous mutation rate to 6-mercaptopurine resistance, as calculated per day in cells with reduced DNA replication rate, was comparable to that in normally dividing cells [(1,2 +/- 0,3) X 10(-5) and (3,1 +/- 0,5) X 10(-5) per cell per day respectively]. This proves that a considerable fraction of spontaneous mutants arise independently on DNA replicative synthesis.