Subcellular localization of DNA polymerase gamma and changes in its activity in sea urchin embryos

1. Subcellular localization and changes in the activity of DNA polymerase gamma were examined in sea urchin eggs and embryos. 2. The enzyme was shown to be localized predominantly in mitochondria by differential and isopycnic centrifugation. 3. During embryogenesis, the enzyme activity per embryo remained constant until blastula stage, and thereafter increased. 4. Similarly mitochondrial DNA per embryo increased, indicating that mitochondrial DNA replication starts during embryogenesis. 5. The gamma-activity per mitochondrial DNA remained constant during embryogenesis. 6. These results suggest that mitochondria contain a constant amount of replicative enzyme (DNA polymerase gamma) regardless of mitochondrial DNA replication, which differs from the case of nuclear DNA replication.     

花生种子萌发早期胚轴细胞DNA合成的特点

花生种子吸胀6h后胚轴DNA中有~3H-胸苷掺入。咖啡因和羟基脲均对6～12h的~3H—胸苷掺入具强烈的抑制作用;当12～24h时,咖啡因的抑制作用较大;但30h以后,羟基脲的抑制作用超过咖啡因。双链DNA放射性从种子吸胀9h后迅速上升,单链DNA放射性在吸胀12h后出现一个明显的峰。但在吸胀12h后,单链DNA形成和存在的时间是短暂的。     

Effect of Ca2+, Mg2+-dependent deoxyribonuclease on DNA synthesis in cell nuclei from embryos of the sea urchin Strongylocentrotus intermedius

The sea urchin embryo nuclei which retained their ability to maintain the DNA synthesis in an in vitro system were isolated. The DNA synthesis isolated nuclei was shown to be an ATP-dependent process which is inhibited by low concentrations of actinomycin D, a polymerase alpha araCTP inhibitor. The newly synthesized DNA is represented by short fragments of about 4S. After addition of Ca2+, Mg2+-dependent DNAase to sea urchin embryo nuclei, the synthesis of short DNA fragments is enhanced. This stimulating effect of Ca2+, Mg2+-dependent DNAase is ATP-dependent and is observed only within a narrow range of enzyme concentrations (of the order of 1-5 units of DNAase activity per ml of incubation sample). The increase in the enzyme concentration to 10 or more units of activity results in the depression of DNA synthesis. It is concluded that DNA replication in sea urchin embryo nuclei depends on the presence of active DNAases as well as on the number of accessible initiation sites of DNA replication.     

GROWTH REGULATION BY ETHYLENE IN FERN GAMETOPHYTES. V. ETHYLENE AND THE EARLY EVENTS OF SPORE GERMINATION

Early events during the germination of spores of the fern Onoclea sensibilis were studied to determine the time during germination when ethylene had its greatest inhibiting effect. Water imbibition by dry spores was rapid and did not appear to be inhibited by ethylene. During normal germination DNA synthesis occurred about four hours before the nucleus moved from a central position to the spore periphery. Following nuclear movement, mitosis and cell division occurred, partitioning the spore into a small rhizoid cell and a large protonemal cell. Cell division was complete approximately six hours after nuclear movement. Ethylene treatment of the spores blocked DNA synthesis, nuclear movement, and cell division. The earliest DNA replication in uninhibited spores was observed after 14 hours of germination, and the maximal rate of spore labeling with ³H-thymidine was between 16 and 20 hours. Spores were most sensitive to ethylene, however, during the stages of germination prior to DNA synthesis, and it was concluded that ethylene did not directly inhibit DNA replication but blocked germination at some earlier fundamental step. The effects of ethylene were reversible. since complete recovery from inhibition of germination was possible if ethylene was released and the spores were kept in light. Recovery was much slower in darkness. It was hypothesized that light acted photosynthetically to overcome the ethylene inhibition of germination. Consistent with this, it was shown that spores exhibit net photosynthesis after only two hours of germination.     

Changes of enzymes and factors involved in DNA synthesis during wheat embryo germination

We have previously purified and characterized wheat germ DNA polymerases A and B. To determine the role played by DNA polymerases A and B in DNA replication, we have measured the level of their activities during wheat embryo germination. The level of cellular proteins known to be associated with DNA synthesis such as PCNA and DNA primase were also investigated. The activity of DNA polymerase A gradually increased reaching a maximal level at 12 h after germination. Three days later, only a residual activity was detected. DNA polymerase B showed the same pattern during germination with very similar changes in activity. Our results indicate a striking correlation between maximal activities of DNA polymerase A, DNA polymerase B and optimal levels of DNA synthesis. These results support a replicative role of these enzymes. The activity of wheat DNA primase that copurifies with DNA polymerase A also increases during wheat germination. Taking together all its properties, and in spite of its behaviour with some inhibitors, DNA polymerase A may be considered as the plant counterpart of animal DNA polymerase . Concerning DNA polymerase B we have previously shown that PCNA stimulates its processivity. Besides studying the changes of DNA polymerases A and B and DNA primase we have also studied changes in PCNA during germination. We show that PCNA is present in wheat embryos at a constant relatively high level during the first 24 h of germination. After 48 h, the absence of PCNA is concomitant with an important decrease in DNA polymerase B activity. In this report we confirm the behaviour of DNA polymerase B as a -like activity.Département de Biologie, Université de Drah-Lmraz,Fez, Maroc     

GA and ABA Regulation on the Cell Cycle in Germination of Tomato Seeds

Flow cytometric determination of ploidy levels in embryos of GA-deficient, ABA-deficient mutant and isogenic wild type tomato (Lycopersicon esculentum Mill. cv. Moneymaker) seeds revealed that, large amount of 2C DNA signals existed both in wild type and GA-deficient mutant seeds, showing that most cells had arrested in the cell cycle at presynthesis Gl, whereas a relative amount of 4C proportion which is a sign of seed germination was found in ABA-deficient mutant seeds, indicating that endogenous ABA play a role in regulating the switch from development to germination in seeds. DNA replication was stimulated 1 d after the seed was imbibed in water and a visible germination occurred subsequently either in wild type GA-deficient mutant seeds. But it was not the case for ABA-deficient mutant seeds unless an exogenous GA was supplemented. This demonstrated that DNA replication in embryo root tips cells was subjected to be a compulsory factor for seed germination, whereas endogenous GA triggered DNA synthesis. It was evident that exogenous ABA could inhibit seed germination not by suppressing DNA synthesis but by bloking the route leading to mitosis since a great amount of 4C proportion was found in the germinating wild type and GA-deficient mutant seeds in the ABA solution when visible ger mination did not occur. Finally a simple mode of hormonal regulation on cell cycle in high plants was hypothesized.     

RNA synthesis required for DNA replication in Vicia seed embryos

The synthesis of DNA and RNA during germination of Vicia seedswas examined. Incorporation of ³H-thymidine into DNA reacheda maximum at about 32 hr after the beginning of imbibition,and RNA synthesis was shown to precede DNA replication. Sedimentationanalyses of ³H-uridine-labeled RNAs indicated that the embryossynthesize all types of rRNA, heterodisperse RNA and 4–5SRNA before and also during the phase of DNA replication. Actinomycin-treatments at lower concentrations (50 or 100 µg/ml)resulted in the specific inhibition of rRNA synthesis. Suchinhibition did not lead to a large reduction in ³H-thymidineincorporation during the replication phase. However, DNA synthesiswas drastically inhibited by a higher level (200 µg/ml)of actinomycin D. The results strongly suggest the involvementof synthesis of heterodisperse RNA in DNA replication. (Received May 28, 1976; )     

Initiation of DNA replication in Bacillus subtilis. IV. The effect of an intercalating dye, ethidium bromide

Summary The effects of an intercalating dye, ethidium bromide (EtBr), on the initiation of chromosome replication in Bacillus subtilis were studied. Spores of a thymine requiring mutant acquired the ability to initiate one round of replication in the absence of RNA and protein synthesis (initiation potential) during germination in a thymine starved medium. When EtBr was added after the initiation potential was fully established, initiation of replication was completely inhibited. This inhibition was reversible, and initiation was resumed when the drug was removed. The recovery of initiation occurred in the absence of protein synthesis but did require RNA synthesis and an active dna gene product.During germination both a DNA-protein complex and a DNA-membrane complex were formed at the replication origin in parallel with the establishment of initiation potential. EtBr destroyed both of these complexes at the concentration which inhibited initiation.The first round of replication of a plasmid DNA, pSL103, during spore germination was also prevented by EtBr. However a higher concentration was required to inhibit plasmid replication. It was found that the plasmid formed two complexes identical to the S- and M-complex of the chromosome origin. Compared to the chromosome complexes the plasmid complexes were less sensitive to EtBr. The loss of sensitivity was equivalent to that for the initiation of the plasmid compared to the chromosome. These results indicate that the target of EtBr is the DNA in the S- and M-complexes whose conformation is essential for the initiation of chromosome and plasmid replication.III of this series is Murakami et al. 1976     

Biochemical Changes During Osmopriming of Leek Seeds

Osmotic priming treatments reduced both the mean time to germinationand the spread of germination for two leek seed-lots of highviability but differing vigour. In addition the differencesin germination performance between these two seed-lots was abolishedby the priming treatments. In the unprimed seed-lots, differencesin germination performance were reflected in differences inrates of protein biosynthesis in leek embryo tissue during germination.Osmopriming treatments abolished these differences upon subsequentgermination of osmotically primed seed and furthermore inducedhigh levels of protein biosynthesis in embryo tissue. DNA synthesiswas detectable in leek embryos during the priming period inthe absence of any cell division and was followed by a five-foldincrease in the rate of DNA synthesis in embryo tissue upongermination following priming at which time the rates of DNAsynthesis in these leek embryos was significantly greater thanthat found at any time over the first 4 d of germination inembryos of unprimed leek seeds. The increases in rates of bothprotein and DNA synthesis observed upon germination of primedseed occurred only after a 6–12 h lag period during whichtime there is little increase in these rates above those foundat the end of priming Analysis of nucleotide and nucleotide sugar levels in leek embryosboth during and after priming showed that only traces of GTPand CTP and low levels of ATP and UTP were present in embryosduring priming. After a 6 h lag period following the end ofpriming these levels increased sharply, probably via de novosynthesis. A similar pattern was found for UDP glucose levelsduring priming and subsequent germination. These results indicatethat there is considerable biochemical activity during primingand that the significant benefits in germination performanceof primed leek seeds is accompanied by marked increases in protein,DNA and nucleotide biosynthesis after a lag period of 6–12h following the end of the priming period Allium porrum, leek, seed, osmopriming, germination, protein synthesis, nucleic acids, nucleotides, nucleotide sugars     

Modulation of deoxyribonucleic acid polymerase III level during the life cycle of Bacillus subtilis.

Deoxyribonucleic acid (DNA) polymerase III is not detectable in Bacillus subtilis spores; the enzyme activity appears 20 to 30 min after spore activation and rapidly increases just before the onset of the first round of DNA replication (30 min later); the level of polymerase III further increases and reaches its maximum (on a per-genome basis) when the cells enter the vegetative phase of growth; this level is six- to eightfold higher than the one observed during germination. In the stationary phase, the polymerase III drops to levels comparable to those found in germinating spores at the first round of replication. On the contrary, DNA polymerase I is present at appreciable levels in the dormant spore; it increases during vegetative growth by a factor of three and, during the stationary phase, reaches its maximum level which is sixfold higher than that observed in the spores. The block of protein synthesis during vegetative growth does not cause an appreciable reduction of the two enzymes (in absolute terms), showing that the regulation of their levels is probably not due to a balance between synthesis and breakdown. These results indicate that polymerase III is probably one of the factors controlling the initiation of DNA synthesis during spore germination.     

Characterization of the early synthesized DNA in germinating Triticum aestivum embryos

A radioactive DNA preparation was isolated from the post-mitochondrial supernatant fraction of thymidine-[¹⁴C] fed wheat embryos. The isolated sDNA preparation was similar to cytoplasmic non-mitochondrial DNA of other eukaryotic cells. The buoyant density and frequency of pyrimidine nucleotide clusters found for the sDNA were, essentially, the same as those found for the nuclear DNA. In contrast to DNA that can be leaked from nuclei or other DNA-containing organelles, the sDNA is firmly bound to a protein component. At an early germination stage (6–12 hr), the sDNA is the only newly-synthesized DNA fraction that can be isolated from the embryo homogenate. Considerable synthesis of nuclear and organellar DNA starts 18 hr after the beginning of germination, just prior to the first maximum of the cell divisions. It is concluded that wheat embryo cells contain cytoplasmic non-mitochondrial DNA and are able to resume its synthesis at an early germination stage, prior to the first post-dormant round of nuclear DNA replication.     

赤霉素与脱落酸对番茄种子萌发中细胞周期的调控

利用细胞流检仪检测番茄（Ｌｙｃｏｐｅｒｓｉｃｏｎ ｅｓｃｕｌｅｎｔｕｍ Ｍｉｌｌ．） ＧＡ－缺陷型、ＡＢＡ－缺陷型和相应的正常品种（野生型）成熟种子胚根尖细胞倍性水平时发现：ＧＡ－缺陷型和野生型种子绝大多数细胞ＤＮＡ 水平为２Ｃ,而ＡＢＡ－缺陷型种子则含有较多的４Ｃ细胞。在标准发芽条件下,ＡＢＡ－缺陷型和野生型种子浸种１ ｄ 后胚根尖细胞ＤＮＡ 开始复制,随后胚根突破种皮而发芽。然而ＧＡ－缺陷型种子除非加入外源ＧＡ,否则既不发生细胞ＤＮＡ 复制,也不发芽。这说明内源ＧＡ 是启动番茄种子胚根尖细胞ＤＮＡ 复制的关键因素,同时也说明番茄根尖细胞ＤＮＡ 复制是种子发芽的必要条件。实验证明：ＡＢＡ 不抑制细胞ＤＮＡ 合成,但阻止Ｇ２ 细胞进入到Ｍ 期。外源ＡＢＡ处理野生型种子与渗控处理结果相似,可以大幅度提高胚根尖４Ｃ／２Ｃ细胞的比例,但抑制种子的最终发芽     

Deoxyribonucleic Acid Synthesis During Microcyst Germination in Myxococcus xanthus

Deoxyribonucleic acid (DNA) synthesis was measured during microcyst germination in Myxococcus xanthus by radioactive thymidine incorporation, autoradiography, and chemical analysis. Microcysts contained an average of 6.6 conserved units of DNA, corresponding to 3 to 4 chromosomes per cell. Correlation of the DNA content and chromosome number of microcysts indicated that the molecular weight of the nonreplicating M. xanthus chromosome is 4.9 x 10(9) daltons. DNA synthesis was initiated 3.5 to 4 hr after induction of germination. From 4 to 6 hr, the rate of synthesis was constant and the accumulation was linear. After a lag period (6 to 6.5 hr), the rate of DNA synthesis increased, reaching a second plateau at 9 hr. From 9 to 11 hr, the rate was again constant and the accumulation was linear. Cellular division during germination showed an unusual kind of synchrony. A model is presented that accounts for chromosomal replication and cell division during microcyst germination.     

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.     

Early growth of wheat embryonic axes and the synthesis of RNA and DNA

The requirement for the synthesis of RNA and DNA in early germination of wheat (Triticum aestivum var Newana) embryonic axes has been studied by incubating embryos in the presence of appropriate inhibitors and monitoring both embryo growth and the rates of specific metabolic processes. Experiments with 5-fluorouridine showed that both rRNA and DNA synthesis could be curtailed by 60 to 70% without affecting embryo growth to 24 hours. Similarly, the presence of mitomycin C and methotrexate inhibited DNA synthesis 70%, with only a small effect on growth. Experiments with a range of concentrations of cordycepin and α-amanitin indicated that mRNA synthesis could be curtailed by 30 to 40% within the first 8 hours of germination with only a small effect on embryo growth. Thus, at least the initial phases of seed embryo germination are not closely linked to the synthesis of mRNA, rRNA, or DNA. Maximal sensitivity of embryo growth was obtained with cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamide, supporting the idea that protein synthesis is the macromolecular process most closely linked to early germination.