THE RELATIONSHIP BETWEEN DEOXYRIBONUCLEIC ACID REPLICATION AND CELL DIVISION IN HEAT-SYNCHRONIZED TETRAHYMENA

The effect of supraoptimal temperature on macronuclear DNA synthesis in Tetrahymena was studied by radioautography during prolonged heat and heat-shock synchronization treatments. Prolonged heat treatments (34°C) delayed the initiation of S, but did not appreciably delay DNA synthesis in progress. Return to optimal temperature (28°C) 50 or 100 min later resulted in initiation of S, in delayed cells, at a rate greater than in controls. During the synchronization treatment, most cells were unable to enter S during a heat shock, but initiated S with a slight delay during the following intershock period. These cells were not appreciably delayed in completion of S by subsequent heat shocks. Supraoptimal temperature appears to affect the DNA synthetic cycle near the G₁ to S transition. Cells subjected to the heat-shock treatment in early G₁ all participated in one S period, and many underwent a succession of two S periods. DNA synthesis occurred in about 50% of the cells between EST and the first synchronous division, with the likelihood of DNA synthesis becoming greater the longer the interval between these two events. In some cells no detectable DNA synthesis occurred between EST and the second synchronous division. It was concluded that a precise temporal alternation of DNA replication and cell division is not obligatory in Tetrahymena.     

PROLIFERATIVE RESPONSE IN THE RAT KIDNEY INDUCED BY A SINGLE, CARCINOGENIC DOSE OF DIMETHYLNITROSAMINE

With the application of autoradiography, the pattern of DNA synthetic activity in the rat kidney following a carcinogenic dose of DMN was traced during the 3 weeks following treatment. Depending on cell class and location, DNA synthesis was constantly depressed for the first 1 or 2 days throughout all zones of the kidney. Mesenchymal cells of the cortex and outer band of the outer medulla commenced a wave of stimulated activity at the third day with a peak of response at the sixth day. Epithelial cells of these same two zones were slower to respond, attaining a peak of DNA synthetic activity at the tenth day. By the start of the third week, activity had returned to normal or near-normal levels. The observations are discussed in relation to the known morphological events which occur in the kidneys of DMN-treated rats prior to the development of neoplasia.     

THE INCORPORATION OF TRITIUM FROM THYMIDINE INTO PROTEINS OF THE MOUSE

Tritium from methyl-H³-thymidine was found to be incorporated into proteins in mice. This incorporation in the mouse as a whole represented between 1 and 10% of the injected tritium. Tritiated water was not an intermediate. Transmethylation reactions are proposed as a means whereby certain amino acids might have acquired the tritium from thymidine at some stage of its catabolism. The initial (2 hr) ratios of DNA to protein tritium activities per milligram of wet tissue ranged from 5 in two tissues of low DNA synthetic activity (pancreas, liver) to 35 to 40 in two tissues of high DNA synthetic activity (spleen, small intestine). Labeled nuclear protein was coincident with labeled DNA in nuclei, where it constituted less than 2.5% of the total tritium. The significance of the findings is discussed.     

The nuclear matrix continues DNA synthesis at in vivo replicational forks

Alkaline cesium chloride gradient analysis of in vivo [3H]bromodeoxyuridine-labeled and in vitro [alpha-32P]dCTP-labeled DNA was used to determine whether in vitro DNA synthesis in regenerating rat liver nuclei and nuclear matrices continued from sites of replication initiated in vivo. At least 70 and 50% of the products of total nuclear and matrix-bound in vitro DNA synthesis, respectively, were continuations of in vivo initiated replicational forks. The relationship of the in vitro DNA synthetic sites in total nuclei versus the nuclear matrix was examined by using [3H]bromodeoxyuridine triphosphate to density label in vitro synthesized DNA in isolated nuclei and [alpha-32P]dCTP to label DNA synthesized in isolated nuclear matrix. A minimum of about 40% of matrix-bound DNA synthesis continued from sites being used in vitro by isolated nuclei. Furthermore, nuclear matrices prepared from in vitro labeled nuclei were 5-fold enriched in DNA synthesized by the nuclei and were several-fold enriched, compared to total nuclear DNA, in a particularly high density labeled population of DNA molecules.     

七星瓢虫雌成虫咽侧体的活性

以短期体外放射化学测定法测定了七星瓢虫雌成虫的咽侧体(CA)活性。结果表明,七星瓢虫的CA在TC 199培养液中培养时活性最高。在最适培养条件下CA合成保幼激素(JH)的速度在1—4小时呈直线增加。 七星瓢虫雌虫生殖期CA的活性变化与卵黄发生有相关性。羽化初期CA活性很低,羽化后4—8天CA活性增加,卵母细胞内卵黄沉积开始增多;羽化后8—12天CA活性高峰出现,此时卵母细胞内有大量卵黄沉积;羽化后12—15天CA活性下降,卵完全成熟并陆续出现产卵个体。 食料对七星瓢虫成虫的卵黄发生的影响:取食人工饲料雌虫的CA活性增长缓慢,直至羽化后15天CA活性仍很低,因而抑制了卵黄原蛋白的合成,使卵巢发育缓慢。此种雌虫中CA活性高峰的出现比取食蚜虫的延迟约2倍,前者的产卵前期也较后者延长约2倍。     

CHANGES IN THE DNA SYNTHESIS PATTERN OF PARAMECIUM WITH INCREASED CLONAL AGE AND INTERFISSION TIME

The clonal age in paramecia refers to the total number of vegetative divisions a clone has undergone since its origin at autogamy (self-fertilization). As clonal age increases, the interfission time usually increases. The DNA synthesis pattern of cells of different ages was compared by autoradiographic analysis of the DNA synthesis of synchronized cells at various time intervals during the cell cycle (from one division to the next). The study showed that the G₁ period (the lag in DNA synthesis post division) was constant, irrespective of interfission time or clonal age; but the duration of the DNA synthesis period increased with increased interfission time or clonal age. Therefore, we have shown for the first time that the G₁ period is fixed, and the S period is increased in a eukaryotic unicellular organism as a function of interfission time and clonal age.     

DIFFERENCES IN REUTILIZATION OF THYMIDINE IN HEMOPOIETIC AND LYMPHOPOIETIC TISSUES OF THE NORMAL MOUSE

Swiss Albino mice received a single i.v. injection of ³H-thymidine (TdR) or of ¹²⁵I-deoxyuridine (IUdR). Bone marrow, thymus, spleen and mesenteric lymph node were examined for the efficiency of precursor incorporation into DNA, and for DNA renewal from day 1 to day 8.
TdR is 5–8 times more efficiently incorporated by the different organs in vivo and in vitro than is IUdR. This indicates that the discrimination against IUdR occurs at the level of DNA synthesizing cells.
A diminished DNA turnover rate measured with ³H-TdR in comparison with ¹²⁵I-UdR is interpreted to indicate reutilization of TdR.
TdR reutilization was observed in bone marrow and spleen from at least day 1 on, and in the thymus from day 3 on, following pulse labeling of DNA synthesizing cells. The degree of TdR reutilization appears higher in the thymus (67%) than the bone marrow (43%) and spleen (38%). The mesenteric lymph node indicates either no, or a very low efficiency of TdR reutilization. The data are also consistent with a reutilization equally efficient for TdR and IUdR.
It is suggested that the TdR salvage pathway in hemopoietic tissues is largely localized to single organs which have immediate access to TdR made available by catabolism of DNA. The contribution of TdR from systemic reutilization to the organs studied falls within the limits of error of measurements. Moreover, the TdR salvage pathway especially in the lymph node may involve other DNA breakdown products than nucleosides.     

QUANTITATIVE CHANGES WITH AGE IN THE DNA CONTENT OF METHYLAZOXYMETHANOL-INDUCED MICROENCEPHALIC RAT BRAIN1

Abstract— DNA synthesis in methylazoxymethanol (MAM)-treated foetal brain was reduced during the first 3 days after the injection of the compound into the mother rat. The MAM-treated brain grew at almost the normal rate after this period, but the reduction in DNA persisted through maturity of the animal. This difference in DNA content between normal and microencephalic brain was restricted to the cerebral hemispheres. The major increase in DNA content of prenatal brain occurred in the cerebrum, whereas the postnatal increase took place in the cerebellum. jH-Labelled MAM was incorporated more extensively into foetal brain DNA than into RNA. The half-life of the MAM-modified nucleic acids was 4–5 days. We suggest that removal of necrotic cells from the brain may account for the rapid loss of label from nucleic acids.     

EVIDENCE FOR AN ESSENTIALLY CONSTANT DURATION OF DNA SYNTHESIS IN RENEWING EPITHELIA OF THE ADULT MOUSE

Tritiated thymidine autoradiography has been applied to several renewing epithelial tissues of the adult mouse in order to determine (a) the average time required for DNA synthesis; and (b) the temporal relationship of the synthesis period to the progenitor cycles of these populations. The average duration of DNA synthesis has been computed from curves describing the rates of appearance and disappearance of labeled metaphase figures in epithelia of colon, ileum, duodenum, esophagus, and oral cavity, in both normal and colchicine-treated animals. In general, application of colchicine does not significantly influence the derived values for DNA synthesis duration. The DNA synthetic time is remarkably similar in the tissues examined, despite wide differences in the times required for completion of the progenitor cycle (and for tissue renewal). Synthesis of DNA in these epithelial cells of the mouse requires approximately 7 hours. Agreement between this value and those derived by other investigators for mammalian cells in vivo and in vitro indicates that DNA synthetic time may be a temporal constant, of considerable potential utility to studies of cell proliferation. The advantages and shortcomings of this experimental approach to problems of cell population kinetics in vivo are discussed.     

AGE-DEPENDENT TOXIC PROPERTIES OF ACTINOMYCIN D AND X-RAYS IN CULTURED CHINESE HAMSTER CELLS

A comparative study was made of the toxic properties of actinomycin D and X-rays using synchronized populations of Chinese hamster cells cultured in vitro. X-irradiated cells are most resistant in the latter half of the DNA synthetic period (late S). While cells treated with actinomycin D appear to go through a survival maximum at the same age, they are most resistant after the completion of DNA synthesis; i.e. in G₂ (or G₂-mitosis). In spite of these differences, we found that actinomycin D damage in late S cells interacts with X-ray damage. Thus, a common locus for the site of actions of both agents is suggested which may be in or around the genome of a cell in view of the well-known DNA binding properties of actinomycin D.     

THE RELATIONSHIP BETWEEN DIURNAL VARIATION OF THE NUMBER OF CELLS IN MITOSIS AND OF THE NUMBER OF CELLS SYNTHESIZING DNA IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH

1. The numbers of cells in mitosis and in DNA synthesis in the epithelium of the hamster cheek pouch have been studied at different times of the day and night. 2. By accumulation of mitotic cells using colcemid, both the rate of entry of cells into mitosis and the duration of mitosis have been estimated at two different times of day. 3. A diurnal variation has been demonstrated in both the mitotic index and in the tritiated thymidine labelling index. Although these variations are of different amplitude and timing, the experimental data fit closely to the hypothesis that the diurnal mitotic variation is the result of a partially synchronous population moving through the DNA synthetic period. No direct action on the mitotic process need be postulated. 4. From the results of mitotic accumulation, it is clear that the rate of entry of cells into mitosis depends on the time of day at which this is studied. There is also the possibility that the duration of mitosis is slightly longer when the mitotic index is high. 5. It is concluded that, at least in the epithelium of the hamster cheek pouch, the diurnal rhythm in the number of mitoses present is a reflection of the diurnal variation in the number of cells synthesizing DNA at some time earlier. Small fluctuations in the mitotic pattern imposed by this partially synchronous population moving from S into mitosis, could be caused by slight variations in the duration of mitosis.     

LIMITED DNA SYNTHESIS IN THE ABSENCE OF PROTEIN SYNTHESIS IN PHYSARUM POLYCEPHALUM

Actidione (cycloheximide), an antibiotic inhibitor of protein synthesis, blocked the incorporation of leucine and lysine during the S phase of Physarum polycephalum. Actidione added during the early prophase period in which mitosis is blocked totally inhibited the initiation of DNA synthesis. Actidione treatment in late prophase, which permitted mitosis in the absence of protein synthesis, permitted initiation of a round of DNA replication making up between 20 and 30% of the unreplicated nuclear DNA. Actidione treatment during the S phase permitted a round of replication similar to the effect at the beginning of S. The DNA synthesized in the presence of actidione was replicated semiconservatively and was stable through at least the mitosis following antibiotic removal. Experiments in which fluorodeoxyuridine inhibition was followed by thymidine reversal in the presence of actidione suggest that the early rounds of DNA replication must be completed before later rounds are initiated.     

EVIDENCE FOR ASEXUAL RESTING CYSTS IN THE LIFE CYCLE OF THE MARINE PERIDINOID DINOFLAGELLATE,SCRIPPSIELLA HANGOEI1

Scrippsiella hangoei (Schiller) Larsen is a peridinoid dinoflagellate that grows during winter and spring in the Baltic Sea. In culture this species formed round, smooth cysts when strains were mixed, indicating heterothallic sexuality and hypnozygote production. However, cysts of the same morphology were also formed in clonal strains exposed to slightly elevated temperature. To better understand the role of cysts in the life cycle of S. hangoei, cyst formation and dormancy were examined in culture experiments and the cellular DNA content of flagellate cells and cysts was compared in clonal and mixed strains using flow cytometry. S. hangoei exhibited a high rate of cyst formation in culture. Cysts produced in both clonal and mixed strain cultures were thick‐walled and underwent a dormancy period of 4 months before germinating. The S. hangoei flagellate cell population DNA distributions consisted of 1C, intermediate, and 2C DNA, indicative of respective eukaryotic cell cycle phases G1, S, and G2M. The majority (>95%) of cysts had a measured DNA content equivalent to the lower 1C DNA value, indicating a haploid nuclear phase and an asexual mode of cyst formation. A small percentage (<5%) of cysts produced in the mixed strain culture had 2C DNA, and thus could have been diploid zygotes. These findings represent the first measurements of dinoflagellate resting cyst DNA content, and provide the first quantitative evidence for dinoflagellate asexual resting cysts. Asexual resting cysts may be a more common feature of dinoflagellate life cycles than previously thought.     

The effect of vinyl chloride monomer,chloroethylene oxide and chloracetaldehyde on DNA synthesis in regenerating rat liver

Vinyl chloride monomer used in the manufacture of polyvinyl chloride is a chemical of increasing industrial importance but has recently been incriminated as a carcinogen, producing a mutagenic effect after being metabolized to active metabolites. The initial effect of vinyl chloride monomer and two of its presumed metabolites, chloracetaldehyde and chloroethylene oxide, on DNA synthesis was investigated in vivo in regenerating rat liver. The established control curve for the DNA synthesis rate after partial hepatectomy demonstrated two waves of synthetic activity at 21 and 30 h. Vinyl chloride, injected intravenously immediately on completion of the operation, depressed the first wave of DNA synthesis by 49.6%. The second peak of DNA synthetic activity was similar to that of the control. Chloracetaldehyde and chloroethylene oxide both produced similar effects on the first wave of DNA synthesis after partial hepatectomy, inhibiting the DNA synthesis rate by approx. 50%. After a regenerating period of 27 h, however, they produced very different effects, chloroethylene oxide raising the control DNA synthesis rate at 30 h by 49% while chloracetaldehyde tended to desynchronize the well-defined second peak of the control. The test compounds have been compared to literature reports of the inhibitory effects of various carcinogens on DNA synthesis.     

Genetic Relatedness Studies with Adenovirus-associated Viruses

Adenovirus-associated viruses (AAV) contain double-stranded deoxyribonucleic acid (DNA). DNA from each of the four AAV serotypes was used as template for the in vitro synthesis of complementary (3)H-ribonucleic acids(RNA). An estimation of genetic interrelatedness was made on the basis of hybridization reactions between synthetic AAV RNA and AAV DNA. Heterologous reactions were 27 to 37% of homologous reactions, suggesting that the AAV serotypes are related to about the same extent. AAV-1 synthetic RNA was also reacted with DNA from helper adenovirus types 2, 7, and SV15. Very low levels of RNA binding were observed, but it is not likely that these reactions represent AAV-adenovirus genetic relatedness.     

马铃薯Y病毒外壳蛋白基因的克隆及序列分析

本文报道应用聚合酶链式反应(PCR)技术,在体外扩增马铃薯 Y 病毒外壳蛋白基因及其克隆和序列分析的结果。病毒 RNA 从马铃薯 Y 病毒感染的烟草叶片中提取,用合成的PCR 3引物及 AMV 逆转录酶合成了单链的 cDNA。利用 PCR 技术,经30个循玎的扩增。得到了一特异的0.8kb 片段。克隆后对此片段进行了限制性内切酶物理图谱分析,并测定了其全序列。实验结果证明,我们克隆到的是完整的马铃薯 Y 病毒的外壳蛋白基因。与国外报道的马铃薯 Y 病毒 N 株相比,其核苷酸序列及推测的氨基酸序列的同源率分别为97.8%和97%。将该基因导入马铃薯以期获得抗 Y 病毒马铃薯的工作正在进行。本文还对 PCR 技术用于扩增植物 RNA 病毒的方法以及用基因工程方法培育抗病毒作物新品种的可行性等进行了讨论。     

Amplification and characterization of a beta-globin gene synthesized in vitro.

Full-length, double-stranded globin DNA was synthesized in vitro starting from rabbit globin mRNA. Several restriction endonuclease cleavage sites with known recognition sequences were mapped on this DNA as a means of assessing the accuracy of in vitro synthesis. By comparing this map with the nucleotide sequences known or predicted from the amino acid sequences of alpha-and beta-chain rabbit hemoglobin, it was possible to show that the synthetic globin DNA is a faithful copy of beta-globin mRNA. Amplification of the synthetic globin DNA was achieved by inserting the molecule into the plasmid PMB9 using the poly(dA)-(dT) joining procedure, and transforming E. coli with the hybrid DNA. Transformants carrying beta-globin DNA were identified by colony hybridization using purified 125I-beta-mRNA probe. Comparison of the restriction maps of the synthetic and inserted globin DNAs showed that the entire synthetic globin DNA molecule was amplified without sequence rearrangements. Both the synthetic and the cloned DNA include the entire coding sequence of the beta-globin gene plus a substantial portion of the untranslated regions flanking the structural gene.     

THE PROLIFERATION OF LYMPHOID CELLS IN GUINEA-PIG BONE MARROW

A double isotope DNA labelling method has been used to determine the duration of DNA synthesis (S) in bone marrow lymphoid cells classified by their nuclear diameters in smears. Incorporation of ³H-thymidine was confined almost entirely to marrow lymphoid cells of 8·0-15·0 μm nuclear diameter (large lymphoid cells). After exposure to ³H-thymidine in vivo and ¹⁴C-thymidine 40-104 min later in vitro , the proportion of cells labelled with ³H alone to those labelled with ¹⁴C(±³H) in radioautographic smears, plotted against time indicated the efflux from S per hour. Collectively, 28·3 ± 1·1% of all large lymphoid cells were in S and the efflux from S was 15·1% per hour. With decreasing cell size (nuclear diameter) the efflux fell progressively from 28·3% per hour (11·0 μm) to 9·2% per hour (8·0-8·9 μm) and the proportion of cells in S declined from 54·9 ± 2·3% to 14·8 ± 1·6%. Influx into S, measured in vitro by reversing the sequence of isotopes, closely resembled the corresponding efflux values in vivo relative to cell size. Most DNA synthesizing marrow large lymphoid cells belonged to a subgroup with deeply basophilic cytoplasm. The results demonstrate that basophilic large lymphoid cells in the marrow are actively proliferating and have a mean S phase duration of 6·6 hr. The largest marrow lymphoid cells (11·0 μm) proliferate most rapidly (S phase, 3·5 hr; maximum cell cycle time, 6·4 hr) while S duration is prolonged progressively to 10·9 hr for the smaller cells (8·0-8·9 μm).     

STUDIES ON THE REGULATION OF LYMPHOCYTE PRODUCTION IN THE MURINE THYMUS AND SOME EFFECTS OF A CRUDE THYMUS EXTRACT

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0–8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering Go/Gi cells into DNA synthesis, the inhibition of G₂ efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.