Cell division and DNA synthesis in uvrA recA double mutants of E. coli K12

Summary Cell division and incorporation of ³H-thymidine into acid-insoluble fraction were investigated for three uvrA recA double mutants of E. coli K12 irradiated with UV at 1.5 ergs/mm², producing about ten pyrimidine dimers per genome (about 0.01% survival). Cell division was measured both in M9 medium and in the same medium which was made very viscous by the addition of Metlose (the same product as Methocel used by Lin et al., 1971). It was found that a major fraction of irradiated bacteria continues to divide once or twice and stops thereafter. Incorporation of ³H-thymidine proceeded at a considerable rate for a short period following irradiation and then stopped. During subsequent incubation, the incorporation gradually decreased and after 4 h incubation most of the early incorporated radioactivity disappeared from the acid-insoluble fraction. These results indicate that cell division occurs after irradiation without parallel DNA synthesis as in a recA thy mutant of E. coli K12 deprived of thymine (Inouye, 1971). These results suggest that UV irradiation increases lethal sectoring due to the reckless cell division without parallel DNA synthesis. Since DNA synthesis took place only for a short period after irradiation, it may be assumed that the recA gene normally has at least a dual function; 1. elimination of damage induced by UV to support elongation or initiation of DNA, and 2. maintenance of coordination between DNA synthesis and cell division.     

Cell division and nucleolar ribonucleic acid synthesis in synchronous cultures

Summary Nucleolar RNA synthesis is inhibited and cell division delayed in synchronous cultures of mouse fibroblasts (strain L-929) treated with actinomycin D (0.04 μg per ml). The gradual loss of actinomycin D from the cells during a 2-hr period following incubation is accompanied by an increase in the rate of nucleolar RNA synthesis to the control level. Following this the rate of protein synthesis is decreased by 25% for approximately 9 hr. The length of time that nucleolar RNA and protein synthesis are inhibited accounts for the delay in mitosis 1 1/2 cell cycles later. These data support the contention that certain proteins produced during one interphase are prerequisite for division in a subsequent cycle.     

Cell division and nucleolar ribonucleic acid synthesis in synchronous cultures

Summary Nucleolar RNA synthesis is inhibited and cell division delayed in synchronous cultures of mouse fibroblasts (strain L-929) treated with actinomycin D(0.04 μ per ml). The gradual loss of actinomycin D from the cells during a 2-hr period following incubation is accompanied by an increased in the rate of nucleolar RNA synthesis to the control level. Following this the rate of protein synthesis is decreased by 25% for approximately 9 hr. The length of time that nucleolar RNA and protein synthesis are inhibited accounts for the delay in mitosis 1 1/2 cell cycles later. These data support the contention that certain proteins produced during one interphase are prerequisite for division in a subsequent cycle.     

The mechanism of killing of mouse fibroblasts by the amino acid analogue 5-fluorotryptophan

The mechanism of killing of A9 fibroblasts by 5-fluorotryptophan has been studied. L-tryptophan competitively relieves the growth inhibition caused by 5FT. After incubation with 5FT, 3H-5FT was incorporated into protein, replacing tryptophan residues. During the initial hours of incubation with 5FT, a specific inhibition was observed of the incorporation of L-tryptophan into protein; later this inhibition was followed by a general inhibition of protein synthesis and cell division. However, nuclear division continued after cell division had ceased. While 5FT was observed to be incorporated into protein after a 1 hour period in MEM + 0.40 mM 5FT in A9, no 3H-5FT was incorporated into protein in a mutant isolated by its resistance to killingy by 5FT. These results support the hypothesis that cell death occurs due to malfunctioning proteins which contain 5FT residues.     

Division Delay Induced in Escherichia coli by Near-Ultraviolet Radiation

Beams of near-ultraviolet radiation at several principal emission lines of a mercury arc were isolated with a grating monochromator and directed upon cell suspensions. During subsequent incubation at room temperature in Nutrient Broth, the population was studied by removing samples and obtaining cell numbers and cell size distributions with an electronic cell counter. Division delay without lethality was observed. The shapes of the dose-response curves for induction, the doses of near-ultraviolet radiation required, and the action spectrum for division delay were found to be similar to those for growth delay (in broth) and for photoprotection. These findings indicate that all three effects, division delay, growth delay, and photoprotection, are induced by a common type of critical event. Changes in cell size distribution in the culture during incubation in Nutrient Broth after near-ultraviolet irradiation are very similar for control and irradiated populations, although these changes occur at a much later time in the irradiated population. This indicates that, in Nutrient Broth, the population recovers completely from the inhibition of growth and division, thus justifying use of the term "delay," and suggesting that the damage is nongenetic.     

Physiological Effects of Growth of an Escherichia coli Temperature-Sensitive dnaZ Mutant at Nonpermissive Temperatures

The physiological effects of incubation at nonpermissive temperatures of Escherichia coli mutants that carry a temperature-sensitive dnaZ allele [dnaZ(Ts)2016] were examined. The temperature at which the dnaZ(Ts) protein becomes inactivated in vivo was investigated by measurements of deoxyribonucleic acid (DNA) synthesis at temperatures intermediate between permissive and nonpermissive. DNA synthesis inhibition was reversible by reducing the temperature of cultures from 42 to 30 degrees C; DNA synthesis resumed immediately after temperature reduction and occurred even in the presence of chloramphenicol. Inasmuch as DNA synthesis could be resumed in the absence of protein synthesis, we concluded that the protein product of the dnaZ allele (Ts)2016 is renaturable. Cell division, also inhibited by 42 degrees C incubation, resumed after temperature reduction, but the length of time required for resumption depended on the duration of the period at 42 degrees C. Replicative synthesis of cellular DNA, examined in vitro in toluene-permeabilized cells, was temperature sensitive. Excision repair of ultraviolet light-induced DNA lesions was partially inhibited in dnaZ(Ts) cells at 42 degrees C. The dnaZ(+) product participated in the synthesis of both Okazaki piece (8-12S) and high-molecular-weight DNA. During incubation of dnaZ(Ts)(lambda) lysogens at 42 degrees C, prophage induction occurred, and progeny phage were produced during subsequent incubation at 30 degrees C. The temperature sensitivity of both DNA synthesis and cell division in the dnaZ(Ts)2016 mutant was suppressed by high concentrations of sucrose, lactose, or NaCl. Incubation at 42 degrees C was neither mutagenic nor antimutagenic for the dnaZ(Ts) mutant.     

Process of cellular division in Escherichia coli: physiological study on thermosensitive mutants defective in cell division

Thermosensitive fts mutants of Escherichia coli belonging to seven previously identified genetic classes (ftsA to ftsG) were studied from a physiological standpoint. These mutants immediately stopped dividing and formed multinucleated filaments when the temperature was shifted to 41 C. Macromolecular syntheses (deoxyribonucleic acid), ribonucleic acid, cell mass, and murein) continued exponentially for at least 40 to 120 min. The number of surviving bacteria remained constant during the time of incubation, and this number began to decrease exponentially, as the rate of cell mass increase leveled off from the initial rate. The recovery of cell division at 30 C in these filamentous cells was studied after 60 min of incubation at 41 C. The existence of three types of mutants was shown. The ftsA and ftsE mutants resumed cell division without new protein synthesis; ftsD mutants resumed cell division only if new protein synthesis occured, while ftsB, C, F and G mutants did not resume cell division at all. No alteration in the cell envelope was detected by the method used here, although the ftsA, B, D, F and G mutations, in contrast with ftsC and E, caused an increased resistance to penicillin G. It was also shown that the recA mutation did not suppress the effect of the fts mutations and that none of the lysogenic fts mutants induced prophage multiplication while forming filaments. The effects of osmotic pressure and salts which rescue the mutant phenotype is described.     

Macromolecular synthesis, differentiation and cell division in Tetrahymena pyriformis, mating type I variety 1

In Tetrahymena pyriformis, mating type I, variety 1, cycloheximide rapidly and completely inhibited incorporation of ¹⁴C-L-leucine into protein. Actinomycin D (25 μg per ml) inhibited incorporation of ¹⁴C-uracil into cold-TCA-insoluble material, after a 5–10 minute lag. Frequently a subsequent decline in the amount of radioactivity was observed. Protein synthesis continued in actinomycintreated cultures for a variable time after cessation of RNA synthesis. Oral development was affected by cycloheximide virtually immediately, and by actinomycin D after a 10–15 minute lag. Cells affected by either drug before the onset of oral membranelle formation were permanently arrested in the stomatogenic field phase. Cells affected in the early and middle stages of membranelle formation completed development of membranelles, but did not invariably complete cell division. Cycloheximide, when added at the beginning of membranelle formation, brought about arrest or resorption of membranelles after they were completed. Actinomycin did not elicit resorption, but sometimes brought about blockage during cell division. Cells affected by either drug after membranelles were fully formed (and cell division was just beginning) completed oral development, nuclear divisions, and cell division. These results suggest that concurrent RNA and protein synthesis are essential for the initiation but not for the completion of membranelle differentiation. The results also suggest that a specific messenger RNA(s) with a very short half-life is required for the synthesis of proteins involved in the initiation of membranelle differentiation.     

A model for restriction point control of the mammalian cell cycle

Inhibition of protein synthesis by cycloheximide blocks subsequent division of a mammalian cell, but only if the cell is exposed to the drug before the "restriction point" (i.e. within the first several hours after birth). If exposed to cycloheximide after the restriction point, a cell proceeds with DNA synthesis, mitosis and cell division and halts in the next cell cycle. If cycloheximide is later removed from the culture medium, treated cells will return to the division cycle, showing a complex pattern of division times post-treatment, as first measured by Zetterberg and colleagues. We simulate these physiological responses of mammalian cells to transient inhibition of growth, using a set of nonlinear differential equations based on a realistic model of the molecular events underlying progression through the cell cycle. The model relies on our earlier work on the regulation of cyclin-dependent protein kinases during the cell division cycle of yeast. The yeast model is supplemented with equations describing the effects of retinoblastoma protein on cell growth and the synthesis of cyclins A and E, and with a primitive representation of the signaling pathway that controls synthesis of cyclin D.     

Late division kinetics in relation to modification of protein synthesis in mouse eggs blocked in the G2 phase after X-irradiation

Mouse zygotes (BALB/c) blocked in the G2 phase of the first cell cycle after X-irradiation were allowed to develop in culture medium. Delayed cleavage occurred at the same time in embryos exposed to 1 or 2 Gy and late division coincided with the second division in controls. Two-dimensional electrophoresis showed that blocked irradiated embryos underwent the same modifications in protein synthesis as control embryos of the same age, except during first mitosis, for three polypeptide sets of 30, 35 and 45 kilodaltons molecular weight. The most remarkable difference between them was the appearance in cleaving controls of three spots at 35 kilodaltons that were absent in blocked irradiated embryos. It is assumed that blocked embryos 'missed' some signal necessary for cell division, but remained ready to cleave when a second signal occurred. Eggs from the BALB/c strain were particularly susceptible to this effect of X-irradiation but it was also found in eggs from other strains, irradiated with much higher doses.     

GROWTH OF THE BARLEY COLEOPTILE. I. ITS RELATIONSHIP TO CELL NUMBER AND LENGTH IN NORMAL AND γ-IRRADIATED SEEDLINGS

The number of cells per vertical column in barley coleoptiles differs in various growth classes; it is highest in tall coleoptiles, intermediate in medium ones, and lowest in short ones. In those that elongate early and grow rapidly, cells per column increased from 88–218 between 12 and 44 hr after the seeds were placed on water; in short coleoptiles they increased to only 85 per column after 115 hr because elongation and division are restricted in these. Cell number does not increase in coleoptiles from seeds irradiated with 250 krad. Variation in the growth pattern of irradiated coleoptiles was similar to that of normal ones, although the range in lengths was reduced. Although the number of cells is much higher in tall controls than in irradiated coleoptiles, the latter can become tall; therefore an increase in cell number during germination does not seem to be a prerequisite for tallness. Coleoptiles 32 mm long, from irradiated seeds, have the same number of cells per column as the shortest ones (6 mm) after 119 hr.     

Sensitivity of the proliferation of normal and tumor cells to cytochalasin D

The effect of cytochalasin D, which is known to disrupt specifically actin cytoskeleton, on DNA replication was studied. The incubation of cultured mouse embryonic fibroblasts (MEF), cells of Balb/3T3 line and cells of minimally transformed clones 12 MC and 6 st/T CAK-7 line with cytochalasin D leads to inhibition of DNA synthesis. A complete inhibition of labeled index in MEF culture was observed after an 8 day incubation in cytochalasin D. Part of cells of clones 12 MC and 6 st/T were insensitive to cytochalasin D and continued to enter to S-phase even after a 10 day incubation. The transfer of cells into a fresh medium leads to a rapid restoration of DNA synthesis. Strongly transformed L cells were almost insensitive to cytochalasin D. Thus, the reorganization of actin cytoskeleton caused by cytochalasin D can inhibit the cycle of normal and minimally transformed cells. In the course of neoplastic progression, in the transformed cells there is a loss of dependence of cell proliferation on microfilament system.     

Cell cycle dependence of transformation expression in mouse cells transformed by a thermosensitive mutant (Ts-121) of polyoma virus.

Change in division capability as a phenotypic expression of cellular transformation was investigated by using one of the temperature-sensitive (ts) mutants of the polyoma virus-transformed cell line, the 121-6-5 cells of BALB/3T3. When contact -inhibited cells were treated with hyaluronidase at 39 degrees C, a single round of cell division was induced after which cell growth was inhibited by cell density. However, if the cells were incubated at 35 degrees C, after the enzyme treatment, density-inhibition block disappeared and the cells entered a second division. This indicates that the release of cells from density-inhibition depends on the low temperature incubation. The ability of cells to complete a second division was examined by shifting the cells from 39 degrees C to 35 degrees C during different phases of the first division cycle after the enzyme-treatment. A 6-hour incubation of S phase cells at 35 degrees C resulted in a second cycle of division, while the 24-hour incubation of G1 cells at 35 degrees C did not induce a second round of division. These results suggest that expression of the transformed phenotype in 121-6-5 cells is clearly dependent upon both the temperature and the phase of the division cycle.     

Inhibition of lateral wall elongation by mecillinam stimulates cell division in certain cell division conditional mutants of Escherichia coli.

The effect of mecillinam, a beta-lactam antibiotic that specifically binds penicillin-binding protein 2 of Escherichia coli, causes transition from rod to coccal shape, and inhibits cell division in sensitive cells, has been tested on three different E. coli temperature-sensitive cell division mutants. At the nonpermissive temperature, the antibiotic allows an increase in cell number for strains BUG6 and AX655 but not for AX621. In strain AX655, the cell division stimulation was observed only if the antibiotic was added immediately after shifting to the nonpermissive temperature, whereas in BUG6, the rise in cell number was observed also when mecillinam was added after 90 min of incubation at the nonpermissive temperature. In all cases, cell division began occurring 30 min after addition of the antibiotic. Mecillinam had no effect on division of dnaA, dnaB temperature-sensitive mutants or on division of BUG6 derivatives made resistant to this antibiotic. Other beta-lactam antibiotics such as penicillin, ampicillin, cephalexin, and piperacillin and non beta-lactam antibiotics such as fosfomycin, teichomycin, and vancomycin that inhibit cell wall synthesis did not show any effect on cell division for any of the mutants. The response of the three cell division mutants to mecillinam is interpreted in terms of a recently proposed model for shape regulation in bacteria.     

Relationship between cellular autolytic activity, peptidoglycan synthesis, septation, and the cell cycle in synchronized populations of Streptococcus faecium.

Synchronized, slowly growing (TD = 70 to 80 min) cultures were used to study several wall-associated parameters during the cell cycle: rate of peptidoglycan synthesis, septation, and cellular autolytic activity. The rate of peptidoglycan synthesis per cell declined during most of the period of chromosome replication (C), but increased during the latter part of C and into the period between chromosome termination and cell division (D). An increase in cellular septation was correlated with the increased rate of peptidoglycan synthesis. Cellular autolytic capacity increased during the early portion of C, reached a maximum late in C or early in D, and declined during D. Inhibition of DNA synthesis during C prevented the decline in autolytic capacity at the end of the cell cycle, caused a slight reduction in the rate of peptidoglycan synthesis, delayed but did not prevent septation, and prevented the impending cell division by inhibiting cell separation. Inhibition of DNA synthesis during D did not prevent the increase in autolytic capacity during the next C phase, but, once again, prevented the decline at the end of the subsequent cycle. Thus, increased autolytic capacity at the beginning of the cell cycle did not seem to be related to chromosome initiation, whereas decreased autolytic capacity at the end of the cell cycle seemed to be related to chromosome termination. The data presented are consistent with the role of autolytic enzyme activity in the previously proposed model for cell division of S. faecium (G.D. Shockman et al., Ann. N.Y Acad. Sci. 235:161-197, 1974).     

Reactivation of Ribonucleic Acid and Protein Synthesis During Germination of Blastocladiella Zoospores and the Role of the Ribosomal Nuclear Cap

Freshly harvested zoospores of Blastocladiella emersonii begin to germinate about 15 min after inoculation into a defined growth medium at a density of 10(6) zoospores per ml. Flagellum retraction accompanies encystment, and dispersal of the ribosomal nuclear cap takes place shortly thereafter. The primary rhizoid begins to emerge at 25 to 30 min and starts to branch at ca. 60 min. The first nuclear division occurs between 120 and 190 min. The dry weight per cell increases linearly after 60 min, whereas the deoxyribonucleic acid per cell doubles between 120 and 240 min. A linear increase in total ribonucleic acid (RNA) is detectable beginning at 40 to 45 min, and in total protein beginning at 80 min; neither process is interrupted during nuclear division. Encystment and nuclear cap disorganization are associated with a sharp rise in the rates of precursor incorporation into RNA and protein. Cycloheximide at 20 mug/ml prevents leucine incorporation at all stages and inhibits development beyond the earliest encystment stage. Actinomycin D at 25 mug to 50 mug/ml prevents uracil incorporation, but it has no effect on leucine incorporation or development until 40 to 45 min. At the latter stage, actinomycin D causes a sharp developmental arrest and begins to inhibit leucine incorporation. It is concluded that early protein synthesis must occur on the ribosomes formed during the prior growth phase and conserved through the zoospore stage in the nuclear cap. The results further indicate that this synthesis is dependent upon messenger RNA already present in the zoospore before germination.     

Behavior of Chloroplast Nucleus during Chloroplast Development and Degeneration in Chlamydomonas reinhardii

Changes in morphology of chloroplast nuclei (cp-nuclei), totalcp-DNA content, number of cp-nuclei, oxygen-evolution activityand chlorophyll (a and b) content were examined during the degenerationand development of chloroplasts, using Chlamydomonas reinhardiicells which had been incubated on solid medium for various periods. Under 4'-6-diamidino-2-phenylindole (DAPI) epifluorescence microscopy,each cell that had been incubated for 7 days had one cell nucleus,one cup-shaped chloroplast and about 10 small, dispersed cp-nucleiin the chloroplast. One day after incubation of these cellson fresh medium, the cell volume and cp-nuclei increased insize 2-3 fold, but rapidly decreased in size after cell division.After about 7 days of incubation, cells ceased to divide andcp-nuclei began to associate with each other. At about 20 daysthey formed a ring-shaped structure surrounding the pyrenoid,followed by condensation into one cp-nuclear particle near thepyrenoid. When 41-day-old cells, having only one cp-nucleus,were reinoculated on fresh solid medium, the cp-nucleus increasedin size 2–3 fold, divided into several cp-nuclear particlesand then dispersed into the chloroplast, forming a bead-likestructure, before cell division. From microscopic fluorometry,a 4-fold increase in total cp-DNA content per chloroplast, withoutan increase in the number of cp-nuclear particles per chloroplast,occurred one day after the start of the experiment and one dayafter reinoculation of 41-day-old cells onto fresh medium. Theprocess of condensation of dispersed cp-nuclear particles intoone cp-nucleus during degeneration of the chloroplast was notaccompanied by any change in total cp-DNA content per chloroplast.A large peak of oxygen-evolution (0.6–0.9 pmoles/cell/hour)was seen one day after inoculation and reinoculation of thecells. The chlorophyll content (a+b) was high (1.2–2.2pg/cell) during the first week of incubation, after which itgradually decreased. (Received December 18, 1985; Accepted April 2, 1986)