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.     

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.     

EFFECTS OF BORON DEFICIENCY ON MITOSIS AND INCORPORATION OF TRITIATED THYMIDINE INTO NUCLEI OF SUNFLOWER ROOT TIPS

Boron deprivation has multiple effects upon root growth within 6 hr after this essential micronutrient is withheld. Root elongation is inhibited and this response has been attributed to a cessation of mitosis and DNA synthesis. Our preliminary results using an autoradiographic analysis of sunflower roots labeled with [³H]-thymidine demonstrated no difference in label distribution between +/-B root tips. We found that mitosis in inhibited in -B roots but does not completely cease. Scintillation counting of whole root tips shows that boron-deficient roots up to 72 hr of treatment incorporate radioactive label at a level comparable to that of the controls. Because mitosis and presumably DNA synthesis are affected by prolonged boron deficiency, these results may be brought about by a change in membrane integrity or permeability. We propose that effects of boron deprivation on DNA synthesis and mitosis in sunflower are secondary and that primary events involve alterations in cellular membranes.     

Metabolism of Linoleic Acid or Mevalonate and 6-Pentyl-α-Pyrone Biosynthesis by Trichoderma Species

The understanding of the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species was achieved by using labelled linoleic acid or mevalonate as a tracer. Incubation of growing cultures of Trichoderma harzianum and T. viride with [U-¹⁴C]linoleic acid or [5-¹⁴C]sodium mevalonate revealed that both fungal strains were able to incorporate these labelled compounds (50 and 15%, respectively). Most intracellular radioactivity was found in the neutral lipid fraction. At the initial time of incubation, the radioactivity from [¹⁴C]linoleic acid was incorporated into 6-pentyl-α-pyrone more rapidly than that from [¹⁴C]mevalonate. No radioactivity incorporation was detected in 6-pentyl-α-pyrone when fungal cultures were incubated with [1-¹⁴C]linoleic acid. These results suggested that β-oxidation of linoleic acid was a probable main step in the biosynthetic pathway of 6-pentyl-α-pyrone in Trichoderma species.     

DURATION OF THE NUCLEAR CYC LE IN TRADESCANTIA PALUDOSA ROOT TIPS AS MEASURED WITH H3-THYMIDINE

Wimber , Donald E. (Brookhaven National Lab., Upton, N. Y.) Duration of the nuclear cycle in Tradescantia paludosa root tips as measured with H³-thymidine. Amer. Jour. Bot. 47(10): 828–834. Illus. 1960.—The duration of the nuclear cycle and its various subdivisions were measured in Tradescantia root tips by autoradiographic techniques. H³-thymidine was used as a nuclear label and was supplied to the roots for 0.5 hr. After labeling, the roots were allowed to grow in the absence of label for periods up to 38 hr. By determining the percentage of divisions labeled at the various times of fixation, a reconstruction of the nuclear cycle could be made. The average cycle was determined as 20 hr. in duration, DNA synthesis 10.8 hr., presynthetic interphase 4 hr., postsynthetic interphase 2.7 hr., prophase 1.6 hr., metaphase 0.3 hr. and anaphase-telophase 0.6 hr. Approximate standard deviations for the duration of some of the subdivisions were calculated.     

Effects of Decay of Incorporated H3-Thymidine on Bacteria

The killing efficiency due to the decay of incorporated H³-thymidine in three mutants of E. coli strain 15: 15_T-, 15_T-L-, and 15_T-U- has been determined. This efficiency is comparable to that previously determined by others for P³² decay. The killing efficiency has been determined as a function of H³-thymidine specific activity, storage media and storage temperature. We have observed a latent killing effect that causes lethality under certain conditions. The kinetics of latent killing have been examined at several temperatures. Finally, mutation production induced by H³-thymidine decays was shown to occur. The results are consistent with the idea that inactivation and mutations may be caused by a process in the nuclear transmutation that is not associated with β-particle ionization damage.     

Observation of Cytoplasmic and Vacuolar Malate in Maize Root Tips by C-NMR Spectroscopy

The accumulation of malate by maize (Zea mays L.) root tips perfused with KH¹³CO₃ was followed by ¹³C nuclear magnetic resonance spectroscopy. In vivo nuclear magnetic resonance spectra contained distinct signals from two pools of malate in maize root tips, one at a pH ~5.3 (assigned to the vacuole) and one at a pH > 6.5 (assigned to the cytoplasm). The ratio of cytoplasmic to vacuolar malate was lower in 12 millimeter long root tips than in 2 millimeter root tips. The relatively broad width of the signals from C1- and C4-labeled vacuolar malate indicated heterogeneity in vacuolar pH. During the 3 hour KH¹³CO₃ treatment, ¹³C-malate accumulated first primarily in the cytoplasm, increasing to a fairly constant level of ~6 millimolar by 1 hour. After a lag, vacuolar malate increased throughout the experiment.     

INCORPORATION OF H3-THYMIDINE INTO CHLOROPLAST DNA OF MARINE ALGAE

The chloroplasts of three genera of marine algae, Dictyota, Padina, and Bryopsis, were labeled with tritiated-thymidine for various time periods during culture in "Erd-Schreiber's" solution. Autoradiographs were prepared from both smeared and sectioned material. They revealed that almost all of the radioactivity was in the cytoplasm and associated with the chloroplasts, as detected in the overlying silver halide crystals. Deoxyribonuclease, ribonuclease, and hot trichloracetic acid treatments indicated that the loss of radioactivity corresponded to the removal of DNA and not RNA. Quantitative studies of silver grain distribution suggested that the radioactivity of the labeled DNA originated from the edge of the pyrenoids on either side in the longitudinal direction of Bryopsis chloroplasts. Nuclei did not incorporate H³-thymidine even though cells were dividing rapidly in the three genera examined. It is postulated that the enzyme, thymidine kinase, is absent as a coding sequence of nuclear DNA in algae, but is present in chloroplast DNA. When the chloroplasts of Dictyota and Padina in various stages of division were scored for labeling, there appeared to be a DNA synthesis period, analogous to S period in cell division. This chloroplast-labeling period occurred just previous to fission. Many of the criteria seem to have been satisfied to establish the self-reproducing and semi-autonomous nature of chloroplasts, especially when combined with the chemical, genetic, and morphological evidence.     

Incorporation of Tritium from Thymidine-methyl-H3 into DNA,RNA, Lipids and Protein in Logarithmic and Stationary Phase Tetrahymena pyriformis,Strain W*

SYNOPSIS. Numerous reports may be found in the literature on cytoplasmic and non-DNA utilization of tritium from H³-thymidine. Such reports underscore the need to clarify the metabolic fate of H³-thymidine. This investigation outlines the fate of thymidinemethyl-H³ (TMH³) in logarithmic phase and stationary phase Tetrahymena pyriformis, strain W. Isotope identification by liquid scintillation spectrometry in chemically derived fractions of log phase cultures grown thruout the initial 48 hours of population growth with TMH³ revealed the majority of the radioactivity (90% of intracellular recovery) to be in the DNA fraction. The remainder of the intracellular label was recovered in the acid soluble fraction, lipid fraction, and a small amount in the RNA and cell residue. On chromatographs, tritium appeared only in the thymine moiety of the nucleic acid derivatives. Hence in dividing cells, thymidine-methyl-H³ is “essentially” specific for DNA at the dosage used although some incorporation into other compounds was detected. Fractionation of the lipid extract from the above experiment on a florisil column localized most of the label to the triglyceride and phospholipid fractions with some recovery in the cholesterol-esters. Similar scintillation counting of the various fractions of early stationary phase cells incubated for the last 48 hours of culture with TMH³ revealed limited tritium distribution in all fractions.     

SYNTHETIC CAPACITIES OF CHROMOSOME FRAGMENTS CORRELATED WITH THEIR ABILITY TO MAINTAIN NUCLEOLAR MATERIAL

Onion (Allium cepa) and bean (Vicia faba) root tip cells containing many micronuclei, derived from x-ray-induced chromosome fragments, were exposed to H³-thymidine and H³-cytidine to determine the ability of such fragments to undergo DNA and RNA synthesis. Only a few micronuclei in onion and many in bean roots synthesize nucleic acid simultaneously with their main nuclei. A few micronuclei labeled with H³-thymidine undergo mitotic chromosome condensation along with the main nuclei, while the unlabeled ones never do so. The onset of nucleic acid synthesis as well as mitosis in micronuclei appears to be under generalized cellular control. Although all chromosomes and chromosome fragments at telophase give a positive reaction for a silver stainable nucleolar fraction, in the subsequent interphase only some micronuclei, derived from such chromosome fragments, are found to maintain nucleoli; others lose them with time. Those micronuclei which maintain nucleoli, perhaps due to the presence of specific chromosomal regions, are also active in DNA and RNA synthesis. These results are compatible with the concept that nucleoli and associated chromosome regions play an important role in the primary biosynthetic processes of the cell.     

CO2 and malate metabolism in starch-containing and starch-lacking guard-cell protoplasts

Isolated, purified mesophyll and guard-cell protoplasts of Vicia faba L. and Allium cepa L. were exposed to ¹⁴CO₂ in the light and in the dark. The guard-cell protoplasts of Vicia and Allium did not show any labeling in phosphorylated products of the Calvin cycle, thus appearing to lack the ability to reduce CO₂ photosynthetically. In Vicia, high amounts of radioactivity (35%) appeared in starch after 60-s pulses of ¹⁴CO₂ both in the light and in the dark. Presumably, the ¹⁴CO₂ is fixed into the malate via PEP carboxylase and then metabolized into starch as the final product of gluconeogenesis. This is supported by the fact that guard-cell protoplasts exposed to malic acid uniformly labeled with ¹⁴CO₂ showed high amounts of labeled starch after the incubation, whereas cells labeled with [4-¹⁴C]malate had minimal amounts of labeled starch (1/120).In contrast, the starch-deficient Allium, guard-cell protoplasts did not show any significant ¹⁴CO₂ fixation. However, adding PEP to an homogenate stimulated ¹⁴CO₂ uptake, thus supporting the interpretation that the presence of starch as a source of PEP is necessary for incorporating CO₂ and delivering malate. With starch-containing Vicia guard-cell protoplasts, the correlation between changes in volume and the interconversion of malate and starch was demonstrated. It was shown that the rapid gluconeogenic conversion of malate into starch prevents an increase of the volume of the protoplasts, whereas the degradation of starch to malate is accompanied by a swelling of the protoplasts.Abbreviations GCPs guard-cell protoplasts - MCPs mesophyll cell protoplasts - PEP phosphoenolpyruvate - DTT dithiothreitol - 3-PGA 3-phosphoglyceric acid - RiBP ribulose 1,5 bisphosphate - MDH malate dehydrogenase - MES 2-(N-morpholino)ethane sulfonic acid - CAM crassulacean acid metabolism     

Increased Fatty Acid beta-Oxidation after Glucose Starvation in Maize Root Tips

The effects of glucose starvation on the oxidation of fatty acids were studied in excised maize (Zea mays L.) root tips. After 24 hours of glucose starvation, the rate of oxidation of palmitic acid to CO₂ by the root tips was increased 2.5-fold. Different enzyme activities were tested in a crude particulate fraction from nonstarved root tips and those starved for 24 hours. The activities of the β-oxidation enzymes crotonase, hydroxyacyl-coenzyme A (CoA) dehydrogenase, and thiolase and those of catalase, malate synthase, and peroxisomal citrate synthase were higher after starvation. However, no isocitrate lyase activity was detected, thus suggesting that the glyoxylate cycle does not operate. The overall β-oxidation activity was assayed as the formation of [¹⁴C]acetyl-CoA from [¹⁴C]palmitic acid after high-performance liquid chromatography analysis of the CoA derivatives. An activity was detected in sugar-fed root tips, and it was increased by two-to fivefold in starved roots. Because the recovery of enzyme activities is only marginally better in starved roots compared with nonstarved roots, these results indicate that the β-oxidation activity in the tissues is increased during sugar starvation. This increase is probably an essential part of the response to a situation in which lipids and proteins replace carbohydrates as the major respiratory substrates. These results are discussed in relation to the metabolic changes observed in senescing plant tissues.     

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.     

H3-THYMIDINE DERIVATIVE POOLS IN RELATION TO MACRONUCLEAR DNA SYNTHESIS IN TETRAHYMENA PYRIFORMIS

The formation of a soluble H³-thymidine derivative pool has been examined in Tetrahymena pyriformis as a function of macronuclear DNA synthesis during the cell life cycle. An autoradiographic technique which allows the detection of water-soluble materials within a cell has shown that these cells do not take up and retain exogenous H³-thymidine during G1 or G2. Uptake of H³-thymidine is restricted to the S period of the cell cycle. Additional autoradiographic experiments show, however, that a soluble pool of H³-thymidine derivatives persists from the end of one DNA synthesis period to the beginning of the next synthesis period in the subsequent cell cycle. Since this persisting pool cannot be labeled with H³-thymidine, the pool does not turn over during non-S periods.     

Comparative Killing Efficiencies for Decays of Tritiated Compounds Incorporated into E. coli

The killing efficiencies due to the decay of incorporated H³-thymidine, H³-uridine, and H³-histidine in E. coli 15_T-L- have been determined. Decays from H³-thymidine are 2.0 times as effective in producing lethality as those from H³-uridine and 2.5 times as effective as those from H³-histidine. Therefore, it seems that the greater part of damage from H³-thymidine decays is due to chemical changes associated with nuclear transmutation.     

Transport and metabolism of [3H]iso-pentenyladenine following application to the tips of intact and decapitated tap roots of Pinus pinea

[³H]iso-Pentenyladenine ([³H]iP) was fed for 24 h to the tips of intact and root tip-decapitated Pinus pinea seedlings. Twelve and 24 h after application to the roots of intact plants most of the applied radioactivity (±60%) was transported to the shoot. Root tip removal increased transport of the applied radioactivity to the shoot, but the overall pattern of distribution of radioactivity in the seedling did not change. Large amounts of radioactivity were recovered from the elongation zone of the root. Some radioactivity also accumulated in the older part of the root with well-developed lateral roots. When [³H]iP was applied one day after decapitation, no significant changes in the pattern of radioactivity distribution were found between the intact and decapitated root systems. However, when applied 7 days after decapitation there was a significant increase of radioactivity in the region of the root where lateral roots were emerging. HPLC separation of extracts from the different root sections showed that [³H]iP was extensively metabolized in the root. Six peaks of radioactivity, which co-chromatographed with authentic cytokinin standards, were detected.Abbreviations ABA abscisic acid - ADE adenine - IAA indole-acetic acid - iP iso-pentenyladenine - HPLC high performance liquid chromatography - [OG]DHZ O-glycosyldihydrozeatin - [9R-MP]DHZ ribosyldihydrozeatin monophosphate - [9G]iP iso-pentenyladenine-9-glucoside - [9R]Z ribosylzeatin - [9R]iP iso-pentenyladenosine - TLC thin layer chromatography     

Effect of Manganese on Lignin Degradation by Pleurotus ostreatus during Solid-State Fermentation

Lignin degradation by Pleurotus ostreatus was studied under solid-state fermentation (SSF) in chemically defined medium containing various levels of Mn. Degradation of [¹⁴C]lignin prepared from cotton branches to soluble products, as well as its mineralization to ¹⁴CO₂, was enhanced by the addition of Mn. The effect of malonate on lignin mineralization was most marked during the first 10 days of SSF, in a treatment amended with 73 μM Mn. A high concentration of Mn (4.5 mM) caused inhibition of both fungal growth and mineralization rates during the first 2 weeks of incubation. Addition of malonate reversed this effect because of chelation of Mn. Mn was found to precipitate in all treatments, with or without the addition of malonate. α-Keto-γ-methiolbutyric acid cleavage to ethylene, an indication of ^. OH production, was observed as early as 3 days of incubation in all treatments.     

Preparation, Characterization, and Microbial Degradation of Specifically Radiolabeled [14C]Lignocelluloses from Marine and Freshwater Macrophytes

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [¹⁴C]phenylalanine, [¹⁴C]tyrosine, and [¹⁴C]cinnamic acid as precursors. Specifically radiolabeled [¹⁴C-polysaccharide]lignocelluloses were prepared by using [¹⁴C]glucose as precursor. The rates of microbial degradation varied among [¹⁴C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [¹⁴C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [¹⁴C]phenylalanine and [¹⁴C]tyrosine were found associated with protein, although very little (3%) radioactivity from [¹⁴C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [¹⁴C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [¹⁴C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to ¹⁴CO₂; during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.     

Metabolism of Abscisic Acid in Guard Cells of Vicia faba L. and Commelina communis L

Metabolism of abscisic acid (ABA) was investigated in isolated guard cells and in mesophyll tissue of Vicia faba L. and Commelina communis L. After incubation in buffer containing [G-³H]±ABA, the tissue was extracted by grinding and the metabolites separated by thin layer chromatography. Guard cells of Commelina metabolized ABA to phaseic acid (PA), dihydrophaseic acid (DPA), and alkali labile conjugates. Guard cells of Vicia formed only the conjugates. Mesophyll cells of Commelina accumulated DPA while mesophyll cells of Vicia accumulated PA. Controls showed that the observed metabolism was not due to extracellular enzyme contaminants nor to bacterial action.

Metabolism of ABA in guard cells suggests a mechanism for removal of ABA, which causes stomatal closure of both species, from the stomatal complex. Conversion to metabolites which are inactive in stomatal regulation, within the cells controlling stomatal opening, might precede detectable changes in levels of ABA in bulk leaf tissue. The differences observed between Commelina and Vicia in metabolism of ABA in guard cells, and in the accumulation product in the mesophyll, may be related to differences in stomatal sensitivity to PA which have been reported for these species.

     

Effect of l-Canavanine on Nitrate Reductase in Corn Roots

l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 μm canavanine was effective in root tips whereas only 50 μm was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO₃ resulted in a loss of NRA. NO₃⁻ protected nitrate reductase from this inactivation in both root tip and mature root sections.