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 mitochondria ofPolytoma papillatum at two different stages of the vegetative cell cycle

Summary The examination of adjacent sections ofPolytoma papillatum cells by the electron microscope enabled us to construct three dimensional models of the chondriomes. These reconstructions show that in two predivision cells the chondriome primarily consists of one large and highly convoluted mitochondrion. One and three small ovoid mitochondria also exist. Examination of two new daughter cells reveales 30 and 47 mitochondria of various shapes and sizes. This indicates that during the growth phase ofPolytoma (vegetative cell cycle) preponderantly one mitochondrion is formed by fusion of those numerous mitochondria found after cell cleavage. Our observations will be compared with results on this subject in other organisms.We are greatly indebted to Prof. Dr. C. G.Arnold (Erlangen) for his support in this work.     

Single-stranded molecules in DNA preparations from cultured mammalian cells at different moments of cell cycle

Long single-stranded DNA molecules have been observed at electron microscope in DNA preparations from synchronized Chinese hamster cells. The amount of single strandedness in parental DNA increases following a prolonged block of DNA synthesis by hydroxyurea as judged by the results obtained using an improved hydroxyapatite chromatography (Hanania et al., 1975). As far as newly replicated DNA is concerned, an increase of the single strand amount has been observed in DNA preparations from cells actively synthesizing DNA.     

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.     

Lethal effects of fluorodeoxyuridine on cultured mammalian cells at various stages of the cell cycle

The lethal damage induced by the exposure of synchronized Chinese hamster cells to various concentrations of 5-fluoro-2′deoxyuridine (FUdR) was not selectively restricted to cells exposed during the period of DNA synthesis S. The colony survival fraction observed after treatment for one hour with 5 × 10^?5 M FUdR was very low (0.0001–0.0003) whether the drug was administered during early G₁, late G₁, early S or in middle S. The survival of cells treated with the same concentration of FUdR during mitosis, however, was significantly higher (0.62) showing that mitotic cells were less sensitive to FUdR. Administration of 10^?7M thymidine or “conditioned” medium for one hour reversed the lethal effect of FUdR or improved the survival, depending on the time after removal of the FUdR at which these substances were given.     

Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.     

FTIR spectroscopy demonstrates biochemical differences in mammalian cell cultures at different growth stages

We have observed differences in the infrared spectra of viable fibroblast cells depending on whether the cells were in the exponential (proliferating) or plateau (nonproliferating) phase of growth. The spectral changes were observed even after correcting for cell number and volume, ruling out these trivial explanations. Several of the changes occurred for both transformed and normal cell lines and were greater for the normal cell line. The biochemical basis of the spectral changes was estimated by fitting the cell spectra to a linear superposition of spectra for the major biochemical components of mammalian cells (DNA, RNA, protein, lipids, and glycogen). The ratios of RNA/lipid and protein/lipid increased when the cells were in the exponential phase compared to the plateau phase of growth. The fits of cell spectra to individual biochemical components also demonstrated that DNA is a relatively minor spectroscopic component as would be expected biochemically. Contrary to other reports in the literature, our data demonstrate that determining DNA content or structure using Fourier transform infrared spectroscopy data is difficult due to the relatively small amount of DNA and the overlap of DNA bands with the absorption bands of other biochemical components.     

Transformations of sperm nuclei incorporated into sea urchin (Arbacia punctulata) embryos at different stages of the cell cycle

In order to test the hypothesis that regulators of male pronuclear development may have a more general role, sharing some relation to factors involved with the cell cycle, Arbacia zygotes and 2- to 8-cell stage embryos were inseminated during different phases of the cell cycle and examined by light and electron microscopy. Differences in the development and morphology of fertilization cones and sperm asters were observed in embryos inseminated during different stages of the cell cycle. Extremely large fertilization cones, approximately four times the length of those found in fertilized eggs, formed in embryos inseminated during metaphase to telophase. Sperm asters developed only in embryos inseminated during prophase to anaphase. These variations are believed to reflect changes in the status of the cortex and cytoskeletal system of the embryo. Although sperm nuclei underwent morphological changes subsequent to incorporation, in general, they failed to develop into male pronuclei. There was a consistent correlation in sperm nuclear transformations and the cell cycle which was expressed in two patterns of morphogenesis: (1) sperm nuclei incorporated into embryos just prior to prophase and at telophase failed, for the most part, to disperse and transformed into aggregations of chromatin granules approximately 40 nm in diameter; and (2) sperm nuclei incorporated into prometaphase-anaphase embryos dispersed and then condensed into chromatin masses, morphologically similar to chromosomes of the embryo. Evidence is discussed which indicates that following the normal period of fertilization changes occur in the zygote, rendering it unable to fully support the transformation of sperm nuclei into male pronuclei.     

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.