LA SYNTHÈSE DE L''ADN MITOCHONDRIAL CHEZ TETRAHYMENA PYRIFORMIS : Etude Radioautographique Quantitative au Microscope Èlectronique

Electron microscopic radioautography has been used to study the synthesis of mitochondrial DNA after incorporation of thymidine-³H by cultures in logarithmic phase of Tetrahymena pyriformis during periods ranging from 15 min to 12 hr. The great majority of silver grains are distributed over the macronuclei, the micronuclei, and the mitochondria. The intensity of the label over the entire mitochondrial population is a function of the length of the incubation period within the time interval considered. The intensity of the mitochondrial label was compared with that of the nuclear label. Mitochondria incorporate at the same rate whether the nuclei are synthesizing or not. This persistence of mitochondrial incorporation in the absence of nuclear incorporation excludes the hypothesis of a nuclear origin for mitochondrial DNA. We are not able to determine whether the apparent continuity of synthesis in the entire mitochondrial population of a cell actually represents a series of asynchronous discontinuities.     

DNA SYNTHESIS IN THE OOPLASM OF DROSOPHILA MELANOGASTER

Tritiated thymidine was injected into 2-day-old Drosophila melanogaster females, and tissue sections were prepared from the ovary for radioautography with both the light and electron microscopes. Besides the expected incorporation of H³-thymidine into nuclei of nurse cells and follicle cells, there was a relatively high level of incorporation of label into ooplasmic DNA. The highest level of incorporation occurred at stage 12. At the same time, the 15 nurse cell nuclei also incorporate thymidine in spite of the fact that they are breaking down and degenerating. The label in the ooplasm is not removed by extraction with DNase (although this removes nuclear label) unless extraction is preceded by a treatment with protease. Electron microscopic radioautography revealed that 36% of the silver grains resulting from decay of H³-thymidine are found over mitochondria, with a further 28% being located within 0.25 µ of these organelles. The remaining 36% of the silver grains was not found to be associated with any organelles, and it probably represents synthesis in the cytoplasm by the "storage DNA" characteristic of many eggs. It is suggested that one mechanism acting throughout the egg chamber is responsible for the synchronous synthesis of DNA in the degenerating nurse cells, in the mitochondria of the egg, and in the "storage DNA" of the ooplasm.     

DNA synthesis associated with a DNA-nuclear-membrane complex from rat liver

Summary Ultrastructural analysis of M-band from nuclei of rat liver showed small amounts of chromatin, fragments of inner nuclear membrane, some amorphous nuclear material, and nucleopores. The outer nuclear membrane with its associated ribosomes was removed by Sarkosyl during the preparation of the M-band sample. Morphological features of nucleopores and the inner nuclear membrane were confirmed by freeze-fracture technique. The gross chemical composition of the M-band was similar to that of nuclear membrane fractions prepared by other techniques. The M-band contained the greatest proportion of newly-labeled DNA and also supported DNA synthesis in vitro. Electron-microscopic autoradiography of the M-band showed localization of silver grains of thymidine-³H presumably over newly synthesized DNA. The DNA synthesis could not be attributed to spurious attachment of DNA polymerase to M-band during its isolation. It was partially removed from the M-band by treatment with 0.5 M KC1, phospholipase A or C; and completely, by the action of pancreatic DNase. DNA synthesis was greater in M-band fractions isolated from nuclei of 24-hour regenerating liver.Supported in part by a grant (CA 10298) from the National Cancer Institute     

EVIDENCE FOR CYTOPLASMIC DNA IN ROOT CELLS OF NICOTIANA

Sterile root cultures from Nicotiana tabacum were grown with H³-thymidine added to the medium for various intervals. Incorporation of the labeled nucleoside into nuclear DNA occurred in a fraction of the nuclei which increased with time. In addition, the cytoplasm of all cells incorporated enough tritium to be readily detected by autoradiography. The tritium was not removed by hydrolysis in 1 N HCl at 60°C for 10 minutes, but was removed by digestion in a DNase solution which also removed nuclear DNA. The amount of tritium in the cytoplasm increased during the first 2 hours, but did not appear to increase significantly during the following 5 hours. If the roots were transferred to unlabeled medium after 2 hours, the label was diluted faster than expected by growth without turnover of the labeled component. If FUdR was added to the unlabeled medium, the depletion occurred faster during the first 6 hours, but later appeared to level off so that at 10 hours these cultures did not differ from those incubated without FUdR. However, the addition of an excess of unlabeled carrier had no effect on the rate of depletion of the cytoplasmic label. Actinomycin D, which inhibited the incorporation of H³-cytidine into RNA in the root tips, had no effect on the incorporation of H³-thymidine into the cytoplasmic component. However, Mitomycin C or a high concentration of deoxyadenosine inhibited the incorporation of H³-thymidine into the cytoplasmic component as well as into the nuclear DNA. It is concluded that H³-thymidine is incorporated into a cytoplasmic fraction which has the characteristics of DNA, with a measurable rate of turnover. This fraction is synthesized regardless of whether or not the nucleus is synthesizing DNA. Although the function of cytoplasmic fraction is not yet known, it does not appear to be that of supplying precursors for the synthesis of the nuclear DNA.     

IN VIVO SPECIFIC LABELING OF CHLAMYDOMONAS CHLOROPLAST DNA

When Chlamydomonas reinhardi is supplied with (methyl-³H)-thymidine, radioactivity is incorporated specifically into chloroplast DNA Chromatographic analysis of the products of enzymatic hydrolysis of the DNA reveals that only thymidine monophosphate has been labeled. Use of thymidine-6-³H yields an identical result. If thymidine-³H monophosphate is supplied, a small amount of radioactivity is incorporated into both nuclear and chloroplast DNA in proportion to the abundance of these DNA components. These observations are consistent with earlier suggestions that algae lack cytoplasmic thymidine kinase, but that the enzyme is present within their chloroplasts.     

Cessation of Nuclear DNA Synthesis in Differentiating Naegleria*

SYNOPSIS. DNA synthesis during growth and differentiation in Naegleria gruberi strain NEG populations has been studied. Autoradiography of cells labeled with [³H]thymidine revealed that grains are concentrated over the nuclei in logarithmically growing populations of cells, whereas in differentiating cells, grains are scattered over the cytoplasm; i.e. no significant nuclear labeling is detectable. It was established by MAK chromatographic analysis that [³H]thymidine is incorporated into double-stranded DNA in Naegleria and that the actual amount of incorporation in the logarithmically growing populations of cells is 20 times greater than that in differentiating cells. These results suggest that nuclear DNA synthesis is reduced markedly soon after the initiation of differentiation, while cytoplasmic DNA synthesis continues. It was established from cell cycle analysis that the approximate intervals of G₁, S, G₂, and M phases were 180, 183, 90, and 28 min, respectively. Hence, the reduction in the nuclear DNA synthesis in differentiating cells is not due to the inhibition of initiation of DNA replication, but rather to the termination of the DNA replicating process. Thus DNA synthesis is curtailed in the presence of RNA and protein synthesis which are required for differentiation.     

LOW LEVEL INCORPORATION OF TRITIATED THYMIDINE INTO THE NUCLEAR DNA OF PURKINJE NEURONS OF ADULT MICE

Adult mice were pulse labeled with tritiated thymidine [³H]TdR and killed 9 hr later. A low level incorporation of [³H]TdR into the nuclear DNA of Purkinje neurons was found in autoradiographs. Enzymatic digestions with DNase and with RNase in combination with autoradiographic grain counts indicate that a portion of nuclear DNA is not stable in the Purkinje nucleus. These results are discussed in light of reports of the stable nature of DNA in Purkinje neurons of adult mice.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Nuclear and cytoplasmic DNA synthesis in cotton embryos: a correlated light and electron microscope autoradiographic study

Summary To investigate the possibility, implied by an earlier report, that large amounts of degradable DNA are probably present in the cytoplasm of young cotton embryos, an investigation was undertaken to establish the distribution, amount and metabolic stability of DNA in cotton embryos. Several sensitive cytochemical tests failed to detect any but small amounts of extranuclear DNA. Quantitative determination of the nucleic acid content of embryos during embryogenesis showed that the amounts of DNA and RNA remained fairly constant during embryogenesis, with a ratio of RNA to DNA of about 3.5 to 1. Quantitative autoradiography at both the light and electron microscope levels of sections from embryos pulse-labeled with ³H-thymidine showed that the grain density over the nucleus and cytoplasm did not change during a seven-hour period after labeling, nor did the distribution of label in the cytoplasm. Virtually all incorporation was eliminated by the inclusion of iododeoxy-uridine in the medium. Almost all of the nuclear label and at least 90% of the cytoplasmic label after ³H-thymidine incorporation was eliminated by deoxyribonuclease. It was concluded that there are no unusual features related to DNA distribution or metabolism in cotton embryo; i.e., that only small amounts of DNA are present in the cytoplasm and that all of the DNA is metabolically stable.Approximately 40% of the cytoplasmic grains after ³H-thymidine labeling were not associated with either plastids or mitochondria (i.e., were more than 0.1 micron distant). No fully satisfactory explanation for such an apparently high figure could be given.This work was supported by a Public Health Service fellowship 5-F2-GM-22,031-02 from the National Institute of General Medical Sciences, by NSF grant GB 3460, by NIH grant 5-R01-Ca0356-10 and by Miller Institute for Basic Science.     

Cytoplasmic DNA in rat corpora lutein cells : Effect of prolactin on [H]thymidine incorporation

Biochemical fractionation studies of homogenates of massively luteinized ovaries showed that DNA could be isolated from mitochondrial and microsomal fractions of this tissue and that prolactin administration enhanced [³H]thymidine incorporation into mitochondrial DNA in vivo. These findings were confirmed by autoradiographic analysis of tissue sections at the light and electron microscopic levels. Further support for the existence of microsomal DNA in situ was provided by the autoradiographic detection of acid-insoluble grains from [³H]thymidine over the cytoplasm of differentiating corpora lutein cells in the control and experimental groups. A significant effect on [³H]thymidine incorporation into microsomal DNA by prolactin could not be demonstrated in this experimental system.     

RADIOAUTOGRAPHIC LOCALIZATION OF DEOXYTHYMIDINE TRIPHOSPHATE IN TRADESCANTIA POLLEN GRAINS DURING DNA SYNTHESIS

Tradescantia pollen grains, isolated during the period of DNA synthesis in the generative cell, accumulate deoxythymidine triphosphate (dTTP)-³H after incubation with thymidine-³H in the presence of millimolar deoxyadenosine. Most of this dTTP-³H was found to resist extraction by the fixative, cold ethanol-acetic acid, and its location was investigated by radioautography with thin, dry emulsion. Substantial binding of dTTP-³H occurred as an artifact; but when nuclei were isolated from the fixed pollen grains by sonication, it was found that they were differentially labeled: generative nuclei contained dTTP-³H, vegetative nuclei did not. This observation is discussed and is interpreted as evidence supporting the idea that thymidine is phosphorylated only in the generative cell of the pollen grain.     

Studies on the release of barley aleurone cell proteins: Autoradiography

Summary Both uptake and incorporation of radioactivity from [³H]l-leucine into gibberellic-acid (GA₃)-treated aleurone layers of barley (Hordeum vulgare L.) was enhanced by pretreatment with 5 mM potassium bromate. The effect of 5 mM KBrO₃ on amino-acid incorporation was quantitative rather than qualitative and could be partly reversed by the addition of neutralized casein hydrolysate at 10 mg/ml. Autoradiographs of GA₃-treated aleurone cells pulsed with [³H]leucine showed distribution of silver grains predominantly over the endoplasmic reticulum (ER) and aleurone grains. After chasing with carrier l-leucine for 60 min, fewer silver grains were associated with the ER and aleurone grains while nearly half of the silver was associated with the ground cytoplasm of the cell. Autoradiographs were prepared from aleurone cells previously stratified by ultracentrifugation. After a 10-min pulse of label, the silver grains were found over the central ER zone of centrifuged cells; however, with an increase in duration of the chase, label was found distributed throughout the aleurone grain and spherosome region of the cell. The silver grains which were located over the central zone of centrifuged cells at the end of the pulse were almost exclusively associated with the ER. There is no evidence for association of label with dictyosomes or with vesicles derived from dictyosomes. The experimental evidence indicates that labelled amino acids are incorporated into aleurone cells on the ER and are released from these cells without the participation of a membrane-bound vesicle.     

Chloroplast activities of dark-imbibed and photoinduced spores of the fernOnoclea sensibilis

Summary Chloroplast activities of dark-imbibed (non-germinating) and photoinduced (germinating) spores of the sensitive fern,Onoclea sensibilis were compared to gain insight into the germination process. There were no changes in the number of chloroplasts or in the chlorophyll contents of the spore during dark-imbibition and during the early phase of germination. Levels of increase in the Chloroplast DNA content of dark-imbibed and photoinduced spores were nearly the same and were associated with autoradiographic incorporation of [³H]thymidine into the cytoplasm. However, incorporation of the label into the nucleus occurred only during photoinduction of spores. Analysis of Chloroplast and nuclear DNA contents by dot-blot hybridization with labeled gene-specific probes has confirmed that chloroplast DNA content of the spore increases during dark-imbibition and photoinduction, while increase in nuclear DNA occurs only in photoinduced spores. Chloroplasts isolated from dark-imbibed and photoinduced spores incorporated [³H]TTP into an acid-insoluble fraction identified as DNA. The results show that physiological activities of chloroplasts of dark-imbibed and photoinduced spores ofO. sensibilis are similar and support an exclusive role for nuclear DNA synthesis in spore germination.     

Autoradiographic Studies of H3-Thymidine Uptake in Entamoeba histolytica in CLG Medium*

SYNOPSIS. Entamoeba histolytica grown with H³-thymidine in CLG medium took up tritium into DNase-sensitive material in the nucleus and cytoplasm. The distribution of nuclear activity indicated that the entire nucleus, including the peripheral chromatin, may possess DNA; previous investigators reported DNA only in the endosome. The penicillin-inhibited bacterial associate (Bacteroides sp.) used in the CLG medium incorporated tritium from H³-thymidine into autoradiographically detectable DNase-sensitive material. Autoradiographs of amebae fed bacteria prelabeled with H³-thymidine also revealed some nuclear and cytoplasmic label. Thus, the amount of cytoplasmic label due to ingested, prelabeled bacterial DNA and/or actual biosynthesis of cytoplasmic DNA by the amebae themselves, is not known. Also, at least some of the nuclear DNA of amebae is synthesized from ingested bacteria, or, more likely, from bacterial degradation products.     

Evaluation of four methods for scoring cytoplasmic grains in the in vitro unscheduled DNA synthesis (UDS) assay

The in vitro unscheduled DNA synthesis (UDS) assay measures DNA repair (incorporation of [³H]thymidine) following in vitro treatment of rat primary hepatocytes. The autoradiographic method was used to detect UDS by counting developed silver grains in the photographic emulsion overlaying nuclei and cytoplasmic areas of the hepatocytes. In this communication we report results using 4 scoring methods: (1) the most heavily labeled cytoplasmic areas adjacent to the nucleus (our standard method), (2) the cytoplasmic area left of the nucleus, (3) the cytoplasmic areas left and right of the nucleus, and (4) 2 cytoplasmic areas whose positions were selected at random. Rat primary hepatocyte cultures treated with a medium control, a solvent control (dimethyl sulfoxide) and 5 known genotoxic chemicals (2-acetylamino-fluorene, dimethylnitrosamine, diethylnitrosamine, methyl methanesulfonate and ethyl methanesulfonate) were scored using these 4 methods. The average or maximum cytoplasmic grain count was subtracted from the nuclear grain count to yield net grains/nucleus (NG). In general, NG counts for Methods 2,3 and 4 were similar, although shifted about 3–10 grains higher than Method 1 for controls and most treated groups. Methods 2, 3 and 4 showed more experiment-to-experiment variability in sensitivity for detecting statistically significant increases in treated groups than did our standard method. Thus, the alternative methods afforded no consistent improvements in sensitivity or reduction of variability for this assay. Subtraction of the average or the highest cytoplasmic count had virtually no effect on the sensitivity of the assay, but simply requires an appropriate adjustment of the criteria for a positive response.     

Determining [3H]Thymidine Incorporation into Bacterioplankton DNA: Improvement of the Method by DNase Treatment

Determination of [³H] thymidine incorporation into bacterial DNA versus other macromolecules is usually achieved by NaOH and hot trichloroacetic acid hydrolysis. This procedure was found not to be specific enough. An alternative method founded on DNase treatment is proposed. Under the new method, the fraction of thymidine incorporated into DNA ranged from 10 to 83%.     

Cytoplasmic Distribution of DNA in a Strain of Hartmannellid Amoeba1, 2

SYNOPSIS. The distribution of cytoplasmic DNA as determined by H³-thymidine incorporation in the Fernald strain of Hartmannella rhysodes was studied by electron microscope autoradiography. Of a total of 1,313 silver grains counted over the cytoplasm in thin sections, 488, 271, 202 and 230 were attributed to DNA in mitochondria, cytoplasmic matrix, plasmalemma and ectoplasm, respectively. On the basis of the ratio of grains associated with the relative area occupied by the various compartments, the plasmalemma accounted for 3 times more grains than the mitochondria and about 20 times that attributed to DNA in cytoplasmic matrix and ectoplasm. These findings are interpreted to indicate the presence of endosymbionts in this strain of soil amoeba. Since no definitive microorganism could be seen in this cell, the most likely endosymbiont is defective DNA virus(es) or episome-like genetic element(s).     

Utilization of the label from bacterial [3H] DNA by isolated barley roots and embryos under different conditions

[³H] DNA fromEscherichia coli and [³H] thymidine were applied, in sterile conditions, on isolated barley embryos and on roots excised from these embryos, both cultivated in the liquid medium and on halves of barley seeds, through the endosperm bridge. In embryos and roots, the labelled compounds were applied in 1.5% sucrose + 0.2 SSC alone, or together with either unlabelled thymidine or DEAE-dextran. Similar labelling indices were found after [³H] thymidine and [³H] DNA treatment which shows that the activity of [³H] DNA is utilized during the S phase. After application of [³H] thymidine, only cell nuclei in S phase were labelled. After the application of [³H] DNA an extranuclear label, in addition to the labelling of nuclei in the S phase, was observed in some experimental variants. The density of label above labelled nuclei after [³H] DNA treatment sharply decreased when unlabelled thymidine or DEAE-dextran was added, while the density of label above nuclei labelled by [³H] thymidine decreased when unlabelled thymidine but not DEAE-dextran was added. The labelling of nuclei with the label from [³H] DNA is the result of degradation of exogenous DNA reutilization of low molecular weight products. Extranuclear labelling is most probably due to the polymerous or partly degraded DNA.     

In vivo uptake and metabolism of [3H]ecdysone in the vitellogenic follicles of Sarcophaga bullata (Diptera) and its localisation by autoradiography

Within 4 h after injection of [³H]ecdysone, almost all tritiated material has disappeared from the haemolymph, indicating that the uptake by the tissues is very fast. After only 15 min, 19% of the label was found in the ecdysterone fraction and 4% in the highly polar products (HPP) fraction. The uptake of [³H]ecdysone by the ovary (mid-vitellogenic) is almost complete within 1 h after injection. The pattern of [³H]ecdysteroids in the ovaries follows a well ordered sequence: firstly, [³H]ecdysone is the major component of the [³ H]ecdysteroids but it disappears within 2 h, next a peak value of [³H]ecdysterone was found at 1 h, whereafter this also disappeared, and from 2 h on, there was a considerable increase in HPP. The HPP consisted of 3 fractions (A, B and C). Glusulase treatment revealed that apparently only fraction B consisted of glucuronide and/or sulphate-conjugates of ecdysteroids. Autoradiographic experiments confirmed that the uptake of [³H]ecdysone was a very rapid process. In ovaries fixed 1 h after injection, the silver grains were abundant in the ooplasm but were also found in the follicle cell cytoplasm and in trophocytes. In follicles examined 16 h after injection, only a few silver grains were observed in the trophocytes and follicle cells. However, the cytoplasm of the oocyte was labelled. The border cells also accumulated label.

The major results indicate that all cell types of the follicle seem to be able to absorb ecdysone from the haemolymph and that there seems to be a rather selective uptake of ecdysone. In the ooplasm, ecdysone is converted to highly polar conjugates.