Dynamic aspects of membrane-bound DNA in Bacillus subtilis during the course of synchronous growth

Changes in the marker frequencies of membrane-bound DNA (M-DNA) were studied with special respect to the division cycle of $\text{B}\text{.}\text{subtilis}$ W23 cells. M-DNA was obtained by a sucrose density gradient centrifugation after a mild shearing of Brij-58 lysate from synchronized cells. It was found that the markers located in the replication point appeared in the M-DNA fraction successively in the order of the map position during the synchronous growth. The result suggests that the replication of DNA proceeds in the membrane-bound state during the whole course of the division cycle.     

Mitochondrial DNA synthesis in synchronous cultures of the yeast,Saccharomyces cerevisiae

The synthesis of mitochondrial DNA (mtDNA) has been investigated by three independent methods of analysis during consecutive synchronous cell cycles in the yeast, Saccharomyces cerevisiae. The rates of pulse-label incorporation indicate maximal [³H]adenine uptake into mtDNA at the time of nuclear DNA synthesis. In contrast, the relative concentrations of mtDNA as determined by both the ratio of mtDNA to total cellular DNA and by the kinetics of isotope dilution analysis were found to increase continuously during synchronous growth. We conclude that whereas nuclear DNA replicates discontinuously during the cell cycle, mitochondrial DNA is synthesized continuously during this time. The discontinuous pattern of pulse-label incorporation into mtDNA is not considered to reflect its true mode of replication during the cell cycle.     

Cloning of the replication origin from Drosophila virilis mitochondrial DNA.

Mitochondrial DNA from $\text{Drosophila}$ contains high “A+T”-rich region. Its DNA replication starts in the “A+T”-rich region and proceeds unidirectionally around the molecule. In order to determine precise location of the DNA replication origin and elucidate unique feature of its nucleotide sequence, the “A+T”-rich region of mitochondrial DNA from $\text{Drosophila}\text{virilis}$ has been cloned in $\text{Escherichia}\text{coli}$. The chimeric plasmid DNA containing the “A+T”-rich region stimulates $\text{in}\text{vitro}$ DNA replication system from $\text{Drosophila}\text{virilis}$ mitochondria about ten fold higher than the parental plasmid DNA, as does native mitochondrial DNA.     

DNA synthesis during the division cycle of three substrains of Escherichia coliBr

The relationship between chromosome replication and the bacterial division cycle has been examined in three substrains of Escherichia coli$\text{B}\text{r}$ obtained from different sources and designated $\text{B}\text{r}$ A, $\text{B}\text{r}$ F and $\text{B}\text{r}$ K. At growth rates greater than 1.0 doubling per hour (μ > 1.0), the time for a round of chromosome replication (C) was 42 minutes in all three substrains, but the time between the end of a round and cell division (D) was 22 minutes in $\text{B}\text{r}$ A, 16 minutes in $\text{B}\text{r}$ F and 14 minutes in $\text{B}\text{r}$ K. At slower growth rates C and D increased, but to significantly different extents in the three substrains. When μ = 0.5, C and D were approximately 80 and 40 minutes in $\text{B}\text{r}$ A, 60 and 20 minutes in $\text{B}\text{r}$ F, and 70 and 20 minutes in $\text{B}\text{r}$ K.As a consequence of the lengths of the C and D periods in the three stocks of E. coli$\text{B}\text{r}$, the patterns of chromosome replication during the division cycle differed. The most obvious difference was that E. coli$\text{B}\text{r}$ F and E. coli$\text{B}\text{r}$ K possessed periods devoid of DNA synthesis at both the beginning and the end of the division cycle during slow growth, whereas E. coli$\text{B}\text{r}$ A contained only one period devoid of DNA synthesis at the end of the cycle.     

Decreased excision of O6-methylguanine and N7-methylguanine during the S phase in 10T12 cells

The excision of N⁷-methylguanine (N⁷-meGua) and O⁶-methylguanine (O⁶-meGua) lesions in DNA caused by treatment of $\text{10T1}\text{2}$ cells with N-methyl-N′-nitro-N-nitrosoguanidine was evaluated as cells synchronously traversed the pre-S and S phases of the cell cycle. Proliferation of cells was arrested by growth to confluence, then cells were treated with MNNG and released into a synchronous cell cycle by replating at lower density. The frequency of the two methylated guanines (methylated guanines/10⁶ guanines) was determined at the time of replating, immediately prior to the onset of S phase and at the conclusion of S phase. During the pre-S interval N⁷-meGua and O⁶-meGua were lost at rates consistant with the reported biological half-lives of 26–28 hr and 20–21 hr, respectively. In contrast, when the reduction in frequency of methylated guanines was determined for the S phase it was found that the apparent decrease could be explained by the increased DNA content of the cultures resulting from DNA replication.     

Variation in periodic replication of the chromosome in Escherichia coli B/rTT.

A method using 5-bromouracil photolysis induction with 313 nm radiation was employed to estimate the variation in the period between successive rounds of DNA replication in rapidly growing cultures of Escherichia coli$\text{B}\text{rTT}$ The coefficient of variation of this period was 9.3%, which is significantly less than the corresponding value of about 20% reported for variation in the cell interdivision period. Thus chromosome replication is much more tightly controlled than is cell division. The reduced variability of the DNA replication cycle indicates that the period (D) between termination of a round of DNA replication and cell division and the following period ending in initiation of the next round of DNA replication (B) are riot independent of each other but tend to have compensatory variations. The results suggest that other events in the cell cycle are related more closely to DNA replication rather than to the much less regular event of cell division.     

A comparison of DNA content between two substrains of Escherichia coli B/r.

The average DNA content per cell was measured in steady-state cultures of two substrains of $\text{E. coli}\text{B}\text{r}$ growing at various rates at 37°C. The DNA content of substrain $\text{B}\text{r}$ F was consistently lower than that of substrain $\text{B}\text{r}$ A. It is suggested that the differences in DNA contents are consequences of strain-specific differences in the relationship between chromosome replication and the division cycle of $\text{E. coli.}$     

Preferential synthesis of yeast mitochondrial DNA in alpha factor-arrested cells

α factor is a diffusible substance produced by $\text{S. cerevisiae}$ cells of the $\text{α}$ mating type which inhibits cell division (1) and the initiation of nuclear DNA synthesis (2) in cells of the $\text{a}$ mating type. In this report, it is shown that mitochondrial DNA synthesis continues at a normal rate in $\text{a}$ cells for at least 6 hours in the presence of α factor, resulting in a 5-fold increase in the amount of mitochondrial DNA per cell. The continued synthesis of mitochondrial DNA in the absence of nuclear DNA synthesis allows specific labeling of yeast mitochondrial DNA.     

DNA replication in Physarum polycephalum: characterization of replication products made in isolated nuclei

Nuclei isolated from synchronous S-phase plasmodia of the myxomycete $\text{Physarum polycephalum}$ were competent in production of low molecular weight DNA replication intermediates. Furthermore, these nuclei showed some competence in joining these fragments into DNA of intermediate molecular weight. The DNA molecules made $\text{in vitro}$ could be correlated with products made $\text{in vivo}$.     

Initiation of chromosome replication in Escherichia coli. I. Requirements for RNA and protein synthesis at different growth rates

The effects of inhibition of protein and RNA synthesis on initiation of chromosome replication in Escherichia coli$\text{B}\text{r}$ were determined by measuring rates of DNA synthesis during the division cycle before and after addition of chloramphenicol and rifampicin. The ability of cells to initiate a round of replication depended upon the pattern of chromosome replication during the division cycle. Initiation in the presence of chloramphenicol (200 μ/ml) and rifampicin (100 gmg/ml) was observed only in slowly growing cells which normally initiated a new round between the end of the previous round and the subsequent division (i.e. in the D period of the division cycle). The cells that initiated were in the D period at the time of addition of the drugs. Rapidly growing cells which normally initiated before the D period and slowly growing cells which normally initiated after the D period did not initiate in the presence of the drugs. The contrasting effects of the drugs in cells possessing different chromosome replication patterns, and the coupling between septum-crosswall formation (the D period) and initiation suggest that the timing of initiation of chromosome replication in E. coli is controlled by the cell envelope.     

Mitochondrial DNA synthesis in cell cycle mutants of saccharomyces cerevisiae

Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with α factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.     

Conjugation in Tetrahymena: the relationship between the division cycle and cell pairing

Conjugation, a sexual stage in the life cycle of Tetrahymena, is marked by the pairing of two cells of opposite mating types. Pairing establishes cytoplasmic continuity between the two cells and initiates the complex of nuclear events involved in sexual exchange. After mixing cells of opposite mating types in nonnutrient medium, a 3-hr refractory period ensues before pairing begins.A wave of cell division occurs concurrently with the onset of pairing. However, although all cells pair, the population does not double. This indicates that some cells do not divide and yet are capable of pairing. Apparently division per se is not required for pairing but does occur in most of the cells.Autoradiographic analysis demonstrates that the cells that divide before pairing were at a stage in the cell cycle beyond the initiation of macronuclear replication at the time they were transferred to nonnutrient medium. Cells that did not divide were in G₁ at the time of shift-down. Thus, neither replication nor division is required to be able to fuse. However, since fusion occurs only in G₁ and most cells are not in G₁ at the time of shift-down, a traverse of the cell cycle is required.Shift-down induces G₁ arrest and preparations for the mating reaction. Mixing the cells induces a synchronous wave of division for cells beyond the $\text{G}{}_1\text{S}$ interface. Preparations for the mating reaction occur independently of but simultaneous with the preparations for cell division.     

Effects of netropsin on yeast mitochondria

Netropsin binds tightly to AT rich regions of DNA and correspondingly is an efficient inhibitor of mitochondrial DNA replication in $\text{Saccharomyces}\text{cerevisiae}$. Netropsin treatment does not cause formation of large populations of petite cells. However, a large portion of cells grown in cultures with ethanol as carbon source are killed by 1 μg/ml netropsin. When petite induction by berenil or ethidium bromide is carried out in the presence of netropsin, the petite cells are killed. This appears to be an effect of netropsin action on the cells during the process of petite formation.     

DNA synthesis and the cell cycle in cultures of normal and mutant (mdg/mdg) embryonic mouse muscle cells.

The relationship between cell fusion, DNA synthesis and the cell cycle in cultured embryonic normal and dysgenic ($\text{mdg}\text{mdg}$) mouse muscle cells has been determined by autoradiography. The experimental evidence shows that the homozygous mutant myotubes form by a process of cell fusion and that nuclei within the myotubes do not synthesize DNA or undergo mitotic or amitotic division. The duration of the total cell cycle and its component phases was statistically the same in 2-day normal and mutant ($\text{mdg}\text{mdg}$) myogenic cultures with the approximate values: T, 21.5 hr; G₁, 10.5 hr; S, 7.5 hr; and G₂, 2.5 hr. In both kinds of cultures, labeled nuclei appeared in myotubes 15–16 hr after mononucleated cells were exposed to [³H]thymidine, and the rate of incorporation of labeled nuclei into multinucleated muscle cells was comparable in control and dysgenic cultures. Thus, homozygous $\text{mdg}\text{mdg}$ muscle cells in culture are similar to control cells with respect to their mechanism of myotube formation and the coordinate regulation of DNA synthesis and the cell cycle during myogenesis.     

DNA postreplication repair gap filling in temperature-sensitive dnaG, dnaC, dnaA mutants of Escherichiacoli

Gaps in daughter-strand deoxyribonucleic acid (DNA) synthesized after exposure of wild-type $\text{Escherichia}\text{coli}$ to ultraviolet light are filled during reincubation. In this study the $\text{dnaG}\text{,}\text{dnaC}$, and $\text{dnaA}$ gene products have been examined for their role in postreplication repair. These gene products are unique in their specific control of certain types of DNA synthesis: initiation of rounds of replication and chain propagation. Initiation of rounds of replication is not essential to gap filling; however, chain propagation by short DNA piece initiation appears to be essential for gap filling.     

Catalytic function of thymidylate synthase is confined to S phase due to its association with replitase

Catalytic activity of thymidylate synthase, as measured $\text{in}\text{,}\text{vivo}$, is tightly linked to S phase of the cell cycle in Chinese hamster embryo fibroblast cells. This activity, as measured $\text{in}\text{,}\text{vitro}$, is found in all parts of the cell cycle. Thymidylate synthase activity in nuclear (karyoplast) extracts increased as the cells progressed from $\text{G}{}_0\text{G}{}_1$ to S phase. This enzymatic activity in the nuclei of S phase cells is associated with the multienzyme complex (replitase) that also contained DNA polymerase and other enzymes of DNA replication and precursor synthesis. The degree of association of thymidylate synthase with replitase, which increased co-ordinately as the cells progressed from $\text{G}{}_0\text{G}{}_1$ phase to S phase, coincided strongly with the level of $\text{in}\text{,}\text{vivo}$ activity of the enzyme.     

Time of origin of Mauthner's neuron in Xenopus laevis embryos

The time of the last DNA replication of the Mauthner's neuron precursor cell has been investigated using radioautography. Embryos of Xenopus laevis were labeled at different stages of early development by single microinjections of tritiated thymidine. Labeling times were designed to cover the entire period of development between gastrula and hatching stages. The embryos were fixed at later stages (41 to 44, according to Nieuwkoop and Faber, 1967), when the Mauthner neuron can be readily distinguished by its characteristically large size and large nucleolus.Mauthner neurons of embryos which received tritiated thymidine from stage 10 (beginning of gastrulation) to stage 12 (advanced gastrula, medium yolk plug) were always labeled. Those embryos which received the isotope at or after stage $\text{12}\text{1}\text{2}$ (advanced gastrula, small yolk plug) were never found labeled. These results imply that the last DNA replication of the cell destined to give rise to the Mauthner neuron occurs during the last gastrula stages. This last DNA replication immediately proceeds the time of the so-called “histogenetic determination” of the Mauthner neuron proposed to correspond to stage 13 (slit blastopore) by Stefanelli (1951).Therefore it appears that the developmental program of the Mauthner neuron involves a remarkably early cessation of DNA replication closely followed by histogenetic determination. This is the earliest known event of this type for a specific, well characterized neuron in the amphibian embryo.     

The influence of dna A and dna C mutations on the initiation of plasmid DNA replication

The dependence of the replication of several plasmids on the chromosome-determined initiation products, $\text{dna}$ A and $\text{dna}$ C, has been studied. The initiation of the replication of $\text{Col}$ E₁ DNA requires the chromosomal $\text{dna}$ A product. In contrast two de-repressed transfer factors ($\text{R 1 drd 16}$ and $\text{Hly}$₁₅₂) seem to determine a corresponding plasmid-specific factor. The $\text{dna}$ C-product is necessary for the ordered initation of all plasmids studied. The addition of low concentrations of chloramphenicol leads to a relaxed replication of $\text{Col}$ E₁ DNA at the restrictive temperature in $\text{dna}$ A-mutants, but not in $\text{dna}$ C-mutants.     

Variations in mitochondrial DNA concentration as a function of different growth conditions in yeast

The relative concentration of mitochondrial DNA in the yeast, $\text{Saccharomyces}\text{cerevisiae}$ has been examined under a variety of different growth conditions by means of an isotope dilution procedure which is shown to yield accurate estimates of mitochondrial DNA content in small samples of this yeast. Under a derepression scheme in which only limited cell proliferation occurs, mitochondrial DNA exhibited nearly a doubling in relative amount. The concentration of mitochondrial DNA was also observed to fluctuate depending upon the strain, growth phase and carbon source included in the growth media. Our results indicate that the relative proportion of mitochondrial DNA does indeed vary according to a variety of different conditions that the cells are subjected to.