Absence Of Deoxyribonucleic Acid Synthesis Research Articles

Yeast-phase cell cycle of the polymorphic fungus Wangiella dermatitidis.

The yeast-phase cell cycle of Wangiella dermatitidis was studied using flow microfluorimetry and the deoxyribonucleic acid (DNA) synthesis inhibitor hydroxyurea (HU). Exposure of exponential-phase yeastlike cells to 0.1 M HU for 3 to 6 h resulted in the arrest of the cells in DNA synthesis and produced a nearly homogeneous population of unbudded cells. Treatment of the yeast-phase cells with HU for 9 h or longer resulted in the accumulation of the cells predominantly as budded forms having either a single nucleus in the mother cell or a single nucleus arrested in the isthmus between the mother cell and the daughter bud. Exposure of unbudded stationary-phase cells to 0.1 M HU resulted in the accumulation of the cells in the same phenotypes. Analysis by flow microfluorimetry and cell counts of HU-inhibited mithramycin-stained cells indicated that the eventual progress of HU-inhibited cells from unbudded to the two budded forms was due to the limited continuation of the growth sequence of the cell cycle even in the absence of DNA synthesis, nuclear division, and in some cases nuclear migration. On the basis of these observations and the results of flow microfluorimetric analysis of exponential-phase cells, a map of the yeast-phase cell cycle was constructed. The cycle appears to consist of two independent sequences of events, a budding growth sequence and a DNA division sequence. The nuclear division cycle of yeast-phase cells growing exponentially with a 4.5-h generation time is composed of a G1 interval of 148 min, as S phase of 16 min, and a G2 plus M interval of 107 min.

Anucleate cell production and surface extension in a temperature-sensitive chromosome initiation mutant of Bacillus subtilis.

At 45 C, in a temperature-sensitive initiation mutant (TsB134) of Bacillus subtilis 168 Thy- tryp-, growing in a glucose-arginine minimal medium, chromosome completion occurred over a period of 80 to 90 min, after which there was no further nuclear division. Normal symmetrical cell divisions continued for a generation afterwards, so that nuclei were segregated into separate cells. During this period asymmetric divisions started to occur. Septa appeared at 25 to 30% from one end of the cell, giving a small anucleate cell and a larger nucleate cell. During inhibition of deoxyribonucleic acid (DNA) synthesis by thymine starvation under the restrictive conditions, asymmetrical division also occurred until there was approximately one nucleus per cell (about one generation time). Asymmetric division, giving anucleate cells, then occurred. Similar results were obtained when DNA synthesis was inhibited by nalidixic acid. After 3 h at 45 C, the rate of anucleate cell production in the presence and absence of thymine was constant at one division per 85 min per chromosome terminus present when DNA synthesis stopped. In the absence of DNA synthesis (during thymine starvation) at 35 C, growth in cell length was linear (i.e., the rate was constant), but at 45 C during thymine starvation the rate gradually increased by more than twofold. It is suggested that this was due to the establishment of new sites of growth associated with anucleate cell production. In the presence of thymine at 45 C, the rate of length extension increased by more than fourfold, which it is suggested was caused by the appearance of new growth zones as a result of chromosome termination and a contribution associated with anucleate cell production. If the mutant was incubated at 45 C for 90 min, both in the presence and absence of thymine, then anucleate cell formation could continue on restoration to 35 C in the absence of thymine...

Integration and repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Haemophilus influenzae.

The extent of association between donor transforming deoxyribonucleic acid (DNA) and recipient DNA in Haemophilus influenzae as a function of ultraviolet (UV) dose to the transforming DNA has been measured by isopycnic analysis of lysates of (3)H-labeled recipient cells exposed to DNA labeled with (32)P and heavy isotopes. Except for doses above 15,000 ergs/mm(2), the results of these measurements are in good agreement with previous estimates made by another technique. Experiments with a mutant temperature sensitive for DNA synthesis and another mutant defective in excision of pyrimidine dimers suggest that the discrepancy between the methods of high doses results from DNA synthesis, in which portions of the associated donor DNA containing pyrimidine dimers are excised and broken down, and the components are reutilized for synthesis. Repair of UV-irradiated, transforming DNA during incubation of recipient cells is observed as an increase in transforming ability when fractions from CsCl gradients of cell lysates are assayed on excision-deficient cells. When transforming DNA containing markers of different UV sensitivities is used, repair of the UV-resistant nov marker by excision proficient cells takes place exclusively in the donor DNA that is associated with recipient DNA, and this repair is observed even in the absence of DNA synthesis. However, no repair is observed in the case of the more UV-sensitive str marker, possibly because excision events may remove a large fraction of the integrated str markers in addition to repairing a small fraction of the integrated DNA containing this marker.

Effects of isoleucine deficiency on nucleic acid and protein metabolism in cultured Chinese hamster cells. Continued ribonucleic acid and protein synthesis in the absence of deoxyribonucleic acid synthesis.

Effects of isoleucine deficiency on nucleic acid and protein metabolism in cultured Chinese hamster cells. Continued ribonucleic acid and protein synthesis in the absence of deoxyribonucleic acid synthesis.

Mitomycin C and Temperature Induction of Colicin B in the Absence of Deoxyribonucleic Acid Synthesis

An Escherichia coli K-12 strain and its mutant, temperature-sensitive in deoxyribonucleic acid (DNA) synthesis, were used as hosts for two different ColB factors (ColB3-K166 and ColB4-K98). Induction of either colicin occurred in both hosts in the presence of mitomycin C at 42 C. Induction of the temperature-sensitive colicinogenic hosts also occurred without mitomycin C at 42 C at which temperature no DNA synthesis was observed. Colicin synthesis was a nonlethal event in ColB4-K98(+) cultures, whereas in cultures of ColB3-K166(+) induction led to death of a large fraction of the population.

Regulation of deoxyribonucleic acid replication and cell division in Escherichia coli B-r.

Synchronous cultures of Escherichia coli strain B/r were used to investigate the relationship between deoxyribonucleic acid (DNA) replication and cell division. We have determined that terminal steps in division can proceed in the absence of DNA synthesis. Inhibition of DNA replication with nalidixic acid prior to the start of a new round of replication does not stop cell division, which indicates that the start of the round is not essential in triggering cell division. Inhibition of DNA replication at any time prior to the termination of a round of replication completely blocks cell division, which suggests that there may be a link between the end of the replication cycle and the commitment of the cell to divide. Studies that use a temperature-sensitive mutant which is unable to synthesize DNA at the nonpermissive temperature are in complete agreement with those that use nalidixic acid to inhibit DNA synthesis. This adds support to the idea that the treatments employed limit their action to DNA synthesis. Investigation of minicell production indicates that the production of minicells is blocked when DNA synthesis is inhibited with nalidixic acid. Although nuclear segregation is not required for cell division, DNA synthesis is still required to trigger division. The evidence presented suggests strongly that (i) DNA synthesis is essential for cell division, (ii) the end of a round of replication triggers cell division, and (iii) there is considerable time lapse (one-half generation) between the completion of a round of DNA replication and physical separation of the cells.

Cell division during inhibition of deoxyribonucleic acid synthesis in Escherichia coli.

When cultures of Escherichia coli B/r growing at various rates were exposed to ultraviolet light, mitomycin C, or nalidixic acid, deoxyribonucleic acid (DNA) synthesis stopped but cell division continued for at least 20 min. The chromosome configurations in the cells which divided were estimated by determining the rate of DNA synthesis during the division cycle. The cultures were pulse-labeled with (14)C-thymidine, and the amount of label incorporated into cells of different ages was found by measuring the radioactivity in cells born subsequent to the labeling period. The cells which divided in the absence of DNA synthesis were those which had completed a round of chromosome replication prior to the treatments. It was concluded that completion of a round of replication is a necessary and sufficient condition of DNA synthesis for cell division.

Genetic Recombination of Transforming Deoxyribonucleic Acid Molecules with the Recipient Genome and Among Themselves in Protoplasts of Bacillus subtilis

Hirokawa, Hideo (Southwest Center for Advanced Studies, Dallas, Tex.), and Yonosuke Ikeda. Genetic recombination of transforming deoxyribonucleic acid molecules with the recipient genome and among themselves in protoplasts of Bacillus subtilis. J. Bacteriol. 92:455-463. 1966.-Re-extraction of transforming deoxyribonucleic acid (DNA) from protoplasts of Bacillus subtilis is much more efficient than from intact competent cells. This facilitated the detection of physical recombination between donor and recipient DNA molecules, as indicated by a high cotransfer index of ind(+) and his(+) markers which were originally located in exogenous and endogenous DNA molecules, respectively. This recombinant DNA was extracted after 30 min of incubation of ind his(+) protoplasts with ind(+)his DNA, previously extracted from a corresponding mutant strain of B. subtilis. The intracellular formation of recombinant molecules (ind(+)his(+)) bearing markers from two different exogenous DNA species was also detected 15 min after exposure of ind his recipient protoplasts to a mixture of ind(+)his and ind his(+) donor DNA molecules. The unity of the recombinant molecule was ascertained by dilution experiments and by its being resistant to ribonuclease and trypsin treatment (but being sensitive to deoxyribonuclease). The formation of recombinant molecules showed an inverse kinetics to that of the intracellularly induced loss of linkage between the corresponding markers in the wild-type DNA, thus suggesting a breakage and reunion process which is also favored by the absence of DNA synthesis in the protoplasts and the effect of some specific inhibitors.

Rise in deoxyribonucleic acid polymerase activity in the absence of deoxyribonucleic acid synthesis in cultured kidney cells.

IN the liver cells of the partially hepatectomized rat and in kidney cells cultured directly from the rabbit, the stimuli to cell multiplication are not followed immediately by DNA synthesis. Instead, there is a long presynthetic period (12–18 h in rat liver1, 30 or more hours for the cultured cells2) during which metabolic changes occur that endow the cells with the ability to replicate DNA. In both the liver3 and kidney cells4 a marked increase in DNA polymerase (EC 2.7.7.7) activity occurs, beginning at about the same time as DNA replication. This temporal coincidence could mean that DNA synthesis begins after the rise in level of enzymes involved in its synthesis or, conversely, that initiation of DNA synthesis indirectly stimulates the manufacture of higher levels of enzymes and substrates needed for replication.