Chromosomal radiosensitivity of ataxia telangiectasia cells at different cell cycle stages

Summary X-ray induced chromosomal aberrations in peripheral blood lymphocytes as well as in skin fibroblasts from ataxia telangiectasia patients, and from normal individuals were studied. At all stages of cell cycles—namely G₀, G₁, and G₂, more aberrations were induced in AT cells than in normal cells. In addition, AT cells were sensitive to induction of chromosomal aberrations by tritium beta rays from incorporated radioactive thymidine. Possible reasons for the increased sensitivity of AT cells for induction of chromosomal aberrations by ionizing radiations are discussed.     

Chromosomal radiosensitivity of Down syndrome lymphocytes at different stages of the cell cycle

Summary Data on 151 non-mosaic 47,XXY males from Sardinia, previously reported by Filippi (1986), were analysed for associations with parental ages at birth, sib order and sex ratio among siblings. The results confirm those of earlier Scottishbased studies in that: (1) there was a significant increase in risk of 47,XXY livebirths at advanced parental ages; (2) maternal age, and maternal age alone, was sufficient to explain the effect; (3) there were no independent effects of paternal age or sib order once maternal age had been taken into account; (4) there was no evidence of any distortion of the sex ratio among siblings. Estimates of relative risk at different maternal ages were compatible with those from the Scottish studies, and pooled estimates are therefore derived. They suggest, for example, that the risk at maternal age 40 years is 2–3 times that at age 30 years. In 33 cases, the parental origin of the supernumerary X chromosome was determined by analysing the segregation of genetic markers. The mean parental ages of 19 maternal cases were significantly raised above those of controls, whereas those of 14 paternal cases were slightly, and marginally significantly, reduced. The conclusions were essentially unaffected by whether the Sardinian population, the siblings of cases or a group of 94 unrelated Sardinian males were used as controls.     

Kinetics of nuclease digestion of Physarum polycephalum nuclei at different stages of the cell cycle

The kinetics of nuclease digestion of Physarum polycephalum nuclei by staphylococcal nuclease and DNase I has been studied at different stages of the cell cycle. Significant differences in the digestion behaviour of nuclei from metaphase and interphase have been detected with DNase I but not with staphylococcal nuclease. Furthermore the structure of newly replicated DNA in S phase differs from the bulk in that it is more easily degraded to acid-soluble products by either staphylococcal nuclease or by DNAase I. At least four types of chromatin structure can be distinguished by our digestion kinetics experiments.     

Studies of Schizosaccharomyces pombe DNA polymerase alpha at different stages of the cell cycle.

The status of Schizosaccharomyces pombe (fission yeast) DNA polymerase alpha was investigated at different stages of the cell cycle. S.pombe DNA polymerase alpha is a phosphoprotein, with serine being the exclusive phosphoamino acid. By in vivo pulse labeling experiments DNA polymerase alpha was found to be phosphorylated to a 3-fold higher level in late S phase cells compared with cells in the G2 and M phases, but the steady-state level of phosphorylation did not vary significantly during the cell cycle. Tryptic phosphopeptide mapping demonstrated that the phosphorylation sites of DNA polymerase alpha from late S phase cells were not the same as that from G2/M phase cells. DNA polymerase alpha partially purified from G1/S cells had a different mobility in native gels from that from G2/M phase cells. The partially purified polymerase alpha from G1/S phase cells had a higher affinity for single-stranded DNA than that from G2/M phase cells. Despite the apparent differences in cell cycle-dependent phosphorylation, mobility in native gels and affinity for DNA, the in vitro enzymatic activity of the partially purified DNA polymerase alpha did not appear to vary during the cell cycle. The possible biological significance of these cell cycle-dependent characteristics of DNA polymerase alpha is discussed.     

The fate of DNA injected into mammalian oocytes and zygotes at different stages of the cell cycle.

The stability of exogenous DNA microinjected into the cytoplasm at different stages of the meiotic cycle and after pronuclear formation was examined in ungulate species. Metabolism of the injected 1201 base pair (bp) DNA was examined by Southern blotting. Similar levels of metabolism of the injected DNA were detected in pig, sheep and bovine oocytes before germinal vesicle breakdown, in which about 30-40% of detected DNA was ligated into higher-molecular-weight forms. Porcine metaphase oocytes and pronuclear zygotes showed a reduced ability to ligate the exogenous DNA. In contrast, sheep and bovine metaphase oocytes and zygotes showed increased levels of ligation and, at the pronuclear stage, generated significant amounts of extremely large (greater than 15 kbp) ligation products. These results are discussed in the context of maternal precursors and metabolic activities in the egg.     

Configural changes in the plastidome ofPolytoma papillatum after completion of cytokinesis and during fusion of the gametes

Summary Changes in plastidome morphology in several definite stages of both the vegetative (asexual) and generative (sexual) life cycle ofPolytoma papillatum were examined by means of serial sectioning technique. Just after completion of cytokinesis daughter cells contain a simple through-shaped, bulky and scarcely perforated leucoplast whose opening faces the original plane of cell cleavage (=lateral part of the new daughter cell). In the course of progressive reorganization the trough-shaped leucoplast is first transformed into a hollow oviform meshwork whose reticulated frame is irregular in shape, size and distribution and whose interior is traversed at the base by two sheets, thus forming separate chambers. This then becomes the typical cup-shaped leucoplast of interphase cells, whose elongated cylindrical lateral part is thin and highly perforated and whose cup-opening faces the apical part of the cell. The changes in leucoplast morphology, probably due to reshuffling and reorganization processes, are correlated to leucoplast areas characterized both by starch and membrane breakdown and by frequent appearance at times of upheaval during leucoplast differentiation. Fusion of two leucoplasts to again form a large, highly perforated cup was also investigated; union of both gametic leucoplasts in such a spherical quadriflagellate zygote is demonstrated by a reconstructed model. Finally the relevancy of these morphological data to extranuclear inheritance is discussed.     

The two chromosomes of Vibrio cholerae are initiated at different time points in the cell cycle

The bacterium Vibrio cholerae, the cause of the diarrhoeal disease cholera, has its genome divided between two chromosomes, a feature uncommon for bacteria. The two chromosomes are of different sizes and different initiator molecules control their replication independently. Using novel methods for analysing flow cytometry data and marker frequency analysis, we show that the small chromosome II is replicated late in the C period of the cell cycle, where most of chromosome I has been replicated. Owing to the delay in initiation of chromosome II, the two chromosomes terminate replication at approximately the same time and the average number of replication origins per cell is higher for chromosome I than for chromosome II. Analysis of cell-cycle parameters shows that chromosome replication and segregation is exceptionally fast in V. cholerae. The divided genome and delayed replication of chromosome II may reduce the metabolic burden and complexity of chromosome replication by postponing DNA synthesis to the last part of the cell cycle and reducing the need for overlapping replication cycles during rapid proliferation.     

Effect of caffeine on the frequency of radiation-induced chromosome aberrations at different stages of the cell cycle

The effect of caffeine on the frequency of chromosome aberrations in human lymphocytes irradiated at different stages of the cell cycle was studied. Caffeine appeared to nearly double the frequency of chromosome aberrations induced by irradiation at S and G2 stages and did not influence the effect of irradiation at G0 and G1 stages.     

Endocytosis of EGF-receptor complexes at various cell cycle stages

Parameters of EGF-receptor complex endocytosis have been studied in the early and late G1 phase and in mitosis. As a model, mouse mammary epithelial cells HC11 were used, whose growth depends on EGF presence in the medium. The Scatchard analysis has demonstrated that the surface receptors are represented by two receptor populations: 4800 high affinity (KD = 10(-11) M) receptors, and 73,000 low affinity (KD = 4.10(-9) M) receptors. Incubation of cells with the growth factor (5 ng/ml) resulted in a decrease in 125I-EGF binding, with its level being low until entering the S-phase. Under these conditions, receptors disposed on the plasma membrane presented a homogeneous population (KD = 8.10(-11) M, 14,000 receptors per cell). No reliable difference was revealed between the EGF-receptor complexes, internalized in early and late G1 phases, in respect to the internalization rate, level of recycling, degradation, and dynamics of compartmentalization. However, endocytosis of EGF-receptor complexes was found to be completely blocked in mitosis at the stage of internalization.     

Ovarian superstimulation after follicular wave synchronization with GnRH at two different stages of the estrous cycle in cattle

The objective of this study was to evaluate superovulatory programs based on synchronization of follicular waves with GnRH at 2 different stages of the estrous cycle. Sixteen Holstein cows were randomly assigned to 1 of 3 groups and administered GnRH (Cystorelin, 4 ml i.m.) between Days 4 and 7 (Groups 1 and 3) or between Days 15 and 18 (Group 2) of the estrous cycle (estrus = Day 0). Four days after GnRH treatment, > or = 7-mm follicles were punctured in Groups 1 (n = 6) and 2 (n = 6) or were left intact in Group 3 (n = 4). All cows were superstimulated 2 d later (i.e., from Days 6 to 10 after GnRH treatment) with a total of 400 mg NIH-FSH (Folltropin-V) given twice daily in decreasing doses. The GnRH treatment caused a rapid disappearance of large follicles (P < 0.005), rapid decrease in estradiol concentrations (P < 0.003), and increase in the number of recruitable follicles (4 to 6 mm; P < 0.04), indicative of the emergence of a new follicular wave within 3 to 4 d of treatment. Between 4 and 6 d after GnRH treatment, the mean number of 4- to 6-mm follicles decreased (4.7 +/- 1.8 to 1.5 +/- 3.3) in the nonpunctured group but increased (3.9 +/- 1.0 to 7.3 +/- 1.9) in the punctured group of cows (P < 0.05). In response to FSH treatment, the increase in the number of > or = 7-mm follicles was delayed by approximately 2 d in the nonpunctured group (P < 0.006). Moreover, the mean number of > or = 7-mm follicles at estrus was higher (16.9 +/- 1.7 vs 11.5 +/- 3.0; P < 0.1) in the punctured than the nonpunctured group. The increase in progesterone concentration after estrus was delayed in the nonpunctured group (P < 0.1) compared with the punctured follicles. Mean numbers of CL as well as freezable (Grade 1 and 2) and transferable (Grade 1, 2 and 3) embryos were similar (P > 0.1) in punctured and nonpunctured groups. Spontaneous estrus did not occur prior to cloprostenol-induced luteolysis in any group, and stage of the estrous cycle during which GnRH was given did not affect (P > 0.1) hormonal and follicular responses in the punctured groups. In conclusion, GnRH given at different stages of the estrous cycle promotes the emergence of a follicular wave at a predictable time. Puncture of the newly formed dominant follicle increases the number of recruitable follicles (4 to 6 mm) 2 d later and, in response to superstimulation with FSH, causes a greater number and faster entry of recruitable follicles into larger classes (> or = 7 mm) and a faster postovulatory increase in progesterone concentrations.     

Synchronization of human diploid fibroblasts at multiple stages of the cell cycle

Because of the scarcity of techniques for synchronizing the growth of cultured human diploid fibroblasts at multiple stages within the cell cycle, efforts were expended in this report to establish a set of protocols that would permit synchronization of cells at several different points throughout the cycle. The protocols that were developed to synchronize the growth of HSF-24 and HSF-55 cells, human foreskin-derived fibroblast cultures, were modifications of procedures employed to synchronize the growth of cultured rodent cells. Optimization of synchrony induction was directed by consideration of both the biochemical properties of the synchronized populations (determined via three-parameter flow cytometric measurements of DNA, RNA, and protein contents) and their kinetic behavior following reversal of the synchronization-inducing blockade (determined via combined flow cytometric analysis of DNA content, [3H]thymidine autoradiography, and measurement of increase in cell number). The conditions judged to yield the best results for studying events associated with production of a G0 block or for maintaining cells for prolonged periods in G0 were those in which the cells were grown to confluency in D-MEM supplemented with 10% fetal bovine serum. Procedures producing the best results for studying processes associated with the G0 to G1 transition, G1 events, and operations accompanying the transition from G1 to S, employed subconfluent growth for 48 h in alpha-MEM + 0.1% fetal bovine serum (alpha-MEM0.1F) followed by resuspension in alpha-MEM containing 10% fetal bovine serum (alpha-MEM10F). When the goal was to obtain cells in which to study very early S-phase events, satisfactory results were achieved by combining a 48-h period of subconfluent growth in alpha-MEM0.1F, followed by treatment for 24 h in alpha-MEM10F containing 5 micrograms/ml aphidicolin. For study of events occurring in mid- to late-cycle, acceptable results were achieved by combining a 48-h block in alpha-MEM0.1F with resuspension for 24 h in alpha-MEM10F containing 10(-3) M hydroxyurea followed by resuspension in drug-free alpha-MEM10F. The best results were obtained with these latter synchronization procedures (i.e., low-serum/high-serum + APC or HU/high serum) when the fetal calf serum was replaced with heat-inactivated calf serum. The success achieved in synchronizing the growth of these human diploid fibroblasts compared favorably/exceeded the results obtained with synchronized cultures of Chinese hamster ovary cells.     

Lactobacillus lactis protoplasts at different stages of cell cultivation

Abstract Protein profiles of both protoplasts and cell homogenates of Lactobacillus lactis prepared in the course of cell multiplication were compared. The sodium dodecyl sulfate acrylamide gel electrophoretograms of whole cell homogenates and protoplasts show almost the same complexity. Protoplast protein profiles after 4 hours of growth exhibit new bands in both the low and high molecular mass region of the gel. The changes in protein composition during conversion of the cells into protoplasts are predominantly quantitative in nature.