REUTILIZATION OF LYMPHOCYTE DNA BY CELLS OF INTESTINAL CRYPTS AND REGENERATING LIVER

Lymphoid cells from mice injected 54 hours and 30 hours earlier with ³H-thymidine were washed and transfused into isogenic recipients at 29 to 30 hours after partial hepatectomy. The recipients were killed 28 to 30 hours later, and liver, intestine, Peyer''s patch, spleen, and the transfused cells were examined in autoradiographs exposed 6 months. Approximately 80 per cent of the labeled transfused cells were classed as lymphocytes. The labeled DNA contained in the transfused cells was partitioned to about 14 times as many recipient liver and intestinal cells, appearing in 72 to 78 per cent of hepatocyte nuclei, in 30 to 35 per cent of liver reticuloendothelial nuclei, and in 90 to 95 per cent of intestinal crypt nuclei. The label was not comparably widespread in the lymphoid organs, but was limited to a few intensely labeled lymphocytes and a somewhat larger number of very weakly labeled cells. When heat-killed cells rather than living cells were transfused, intensely labeled lymphocytes were absent from the lymphoid organs, but the labeling of cells in the recipients was otherwise identical. The results suggest that (a) reutilized DNA is derived from dead cells, (b) reutilized DNA is mainly degraded to nucleosides and nucleotides, the usual immediate de novo DNA precursors, before reincorporation into DNA, and (c) DNA reutilization may occur in the lymphoid organs, but on a less active scale than in intestine or regenerating liver.     

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.     

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.     

Carbon Transport and Root Respiration of Split Root Systems of Phaseolus vulgaris Subjected to Short Term Localized Anoxia

The influence of anoxia on carbon transport and root respiration was evaluated by applying [U-¹⁴C]sucrose to the foliage. Translocation patterns to the root systems of two dry edible bean genotypes (Phaseolus vulgaris L.) were examined after a 3-day exposure to aerated and nonaerated environments. Localized anoxia of root systems was simulated by growing roots in split configurations and exposing half of the system to anoxic conditions. Anoxia of the root system for 72 hours reduced the movement of ¹⁴C label into the roots with concurrent accumulations in the hypocotyl region. The translocation of ¹⁴C label to anoxic roots was less than 50% of the aerated controls of both genotypes. Most of the ¹⁴C label translocated to anoxic root systems was excluded from respiratory metabolism during the 3-hour pulse/chase period and was an order of magnitude less than the aerated controls. These observations suggest that the bulk of ¹⁴C label which entered the root during the anoxic period was unavailable for metabolism by the enzymes of glycolysis and/or was diluted by a relatively large metabolite pool. A higher percentage of ¹⁴C label was translocated to the aerated half of the localized anoxia treatment relative to the half of the aerated controls. The proportion of ¹⁴C label translocated to the root system in the aerated control was 20 and 16% compared to 28 and 25% in the aerated localized anoxia treatment for the genotypes Seafarer and line 31908, respectively. Line 31908 partitioned a greater percentage of ¹⁴C-labeled compounds to the actively growing fraction of the root system in the localized anoxia treatment than did Seafarer. This suggests a greater reliance on previously stored carbohydrate for immediate root growth in Seafarer than in line 31908.     

Heat-induced alterations in the localization of HSP72 and HSP73 as measured by indirect immunohistochemistry and immunogold electron microscopy.

The heat shock proteins are a family of stress-inducible proteins that act as molecular chaperones for nascent proteins and assist in protection and repair of proteins whose conformation is altered by stress. HSP72 and HSP73 are two major cytosolic/nuclear stress proteins of mammalian cells, with extensive sequence homology. HSP73 is constitutively expressed, whereas HSP72 is highly stress-inducible. However, it is unclear why two isoforms are expressed and whether these two proteins have different functions in the cell. To assist in the delineation of function, we have completed a detailed study of the localization of HSP72 and HSP73 in the cell before and after heat stress, using two different methods of detection. By indirect immunohistochemistry, the localization of these two proteins is similar, cytoplasmic and nuclear in nonstressed cells with a translocation to nucleoli immediately after heat. By the more sensitive immunogold electron microscopy technique, differences in localization were noted. In nonstressed cells, HSP72 was primarily nuclear, localized in heterochromatic regions and in nucleoli. HSP73 was distributed throughout the cell, with most cytoplasmic label associated with mitochondria. Mitotic chromosomes were also heavily labeled. After stress, HSP72 concentrated in nuclei and nucleoli and HSP73 localized to nuclei, nucleoli, and cytoplasm, with increased label over mitochondria. These differences in localization suggest that the HSP72 and HSP73 may associate with different proteins or complexes and hence have different but overlapping functions in the cell.     

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.     

ELECTRON MICROSCOPIC EXAMINATION OF THE SITES OF NUCLEAR RNA SYNTHESIS DURING AMPHIBIAN EMBRYOGENESIS

The site of H³-uridine incorporation and the fate of labeled RNA during early embryo-genesis of the newt Triturus pyrrhogaster were studied with electron microscopic autoradiography. Isolated ectodermal and mesodermal tissues from the embryos were treated in H³-uridine for 3 hours and cultured in cold solution for various periods before fixation with OsO₄ and embedding in Epon. At the blastula stage, the only structural component of the nucleus seen in electron micrographs is a mass of chromatin fibrils. At the early gastrula stage, the primary nucleoli originate as small dense fibrous bodies within the chromatin material. These dense fibrous nucleoli enlarge during successive developmental stages by the acquisition of granular components 150 A in diameter, which form a layer around them. Simultaneously larger granules (300 to 500 A) appear in the chromatin, and they fill the interchromatin spaces by the tail bud stage. Autoradiographic examination has demonstrated that nuclear RNA synthesis takes place in both the nucleolus and the chromatin, with the former consistently showing more label per unit area than the latter. When changes in the distribution pattern of radioactivity were studied 3 to 24 hours after immersion in isotope at each developmental stage, the following results were obtained. Labeled RNA is first localized in the fibrous region of the nucleolus and in the peripheral region of chromatin material. After longer culture in non-radioactive medium, labeled materials also appear in the granular region of the nucleolus and in the interchromatin areas. Further incubation gives labeling in cytoplasm.     

Deoxyribonucleic Acid synthesis in root cap cells of cultured roots of convolvulus

Isolated cultured roots of Convolvulus arvensis L. were incubated in 0.2 microcurie per milliliter methyl-³H-thymidine for 14 hours, for 64 hours, or for 14 hours followed by transfer to fresh nutrient medium without tritiated thymidine. Autoradiographs of serial, longitudinal sections of roots which were continuously incubated with tritiated thymidine showed that cells of the root cap columella did not undergo DNA synthesis after their formation from the root cap initials. In roots pulse-labeled with tritiated thymidine, the movement of labeled cells through the root cap columella was followed. Labeled cells were displaced at a constant rate of 72 microns per day over a period of 6 to 9 days before they were sloughed off from the root cap. The specialized role of the root cap cells in relation to their distinctive metabolism and longevity is discussed.     

Incorporation of a kinin, N, 6-benzyladenine into soluble RNA

Kinin requiring tobacco and soybean tissues incubated on a medium containing N,6-benzyladenine-8-C¹⁴ incorporated C¹⁴ into several RNA components including adenylic and guanylic acids. About 15% of the label taken up by the tissues appeared in RNA while the remainder was distributed among several metabolites in the soluble, nonpolynucleotide fraction. Tissue grown on a kinin labeled in the side chain (N,6-benzyladenine-benzyl-C¹⁴) also incorporated a small, but nevertheless repeatable, amount of radioactivity into minor RNA components.

Ultracentrifugation studies and methylated albumin chromatography indicated that the bulk of the label from benzyladenine-benzyl-C¹⁴ is in soluble RNA. Approximately 50% of the C¹⁴ in soluble RNA is in a component which has chromatographic properties like that of benzyladenine.

It is suggested that the biological action of the kinins may hinge on their providing substituted bases in RNA in tissues which through differentiation no longer synthesize RNA-methylating enzymes. As an alternative it was hypothesized that a small amount of benzyladenine was incorporated into a m-RNA, acting there as a derepressing agent, perhaps by preventing its normal repressing function.

     

Quantitation of Rates of Transport, Metabolic Fluxes, and Cytoplasmic Levels of Inorganic Carbon in Maize Root Tips during K Ion Uptake

Our aim was to determine whether fixation of inorganic carbon (C_i), due to phosphoenolpyruvate carboxylase activity, is limited by the availability of C_i in the cytoplasm of maize (Zea mays L.) root tips. Rates of C_i uptake and metabolism were measured during K₂SO₄ treatment, which stimulates dark C_i fixation. ¹³C_i uptake was followed by ¹³C-nuclear magnetic resonance (NMR); 5 millimolar K₂SO₄ had no significant effect on ¹³C_i influx. The contribution of respiratory CO₂ production to cytoplasmic HCO₃⁻ was measured using in vivo ¹³C-NMR and ¹H-NMR of cell extracts; K₂SO₄ treatment had no effect on respiratory CO₂ production. The concentration of cytoplasmic HCO₃⁻ was estimated to be approximately 11 millimolar, again with K₂SO₄ having no significant effect. These experiments allowed us to determine the extent to which extracellularly supplied ¹⁴C_i was diluted in the cytoplasm by respiratory CO₂ and thereby measure phosphoenolpyruvate (PEP) carboxylase activity in vivo using ¹⁴C_i. PEP carboxylase activity in root tips was enhanced approximately 70% over controls within 12 minutes of the addition of 5 millimolar K₂SO₄. The activity of carbonic anhydrase, which provides PEP carboxylase with C_i, was determined by saturation transfer ¹³C-NMR to be more than 200 times that of PEP carboxylase in vivo. The regulation of PEP carboxylase in K₂SO₄-treated roots is discussed.     

Sorbitol,a precursor of L-guluronic acid in alginic acid biosynthesis

Radioactive D-[U-¹⁴C]sorbitol 6-phosphate injected into young growing Sargassum muticum tips is transformed within a few hours into radioactive L-guluronic acid. A C₅-epimerase intervenes reversibly to balance the ratio of mannuronic and guluronic acids in the newly synthesized alginic acid.     

OBSERVATIONS ON SYNAPTONEMAL COMPLEXES IN PREMEIOTIC INTERPHASE OF WHEAT

Synaptonemal complexes (SCs) were found in stage 3, premeiotic (S phase) pollen mother cell (PMC) nuclei of wheat which were labeled with ³H-thymidine. Three nucleoli are present in PMC nuclei at the beginning of stage 3, premeiotic interphase (S3). During S3, nucleoli move toward the nuclear envelope and fuse to form one nucleolus near the end of the stage. PMC nuclei labeled with ³H-thymidine were serially sectioned to show that more than one nucleolus was present and that SCs were also present in these DNA synthetic nuclei. Entire S3 PMC nuclei were serially sectioned to show the presence of SCs and all three nucleoli. Entire leptotene nuclei were also serially sectioned and segments of SCs were found. It is concluded that the association of homologous chromosomes in S3 of wheat is an early step in SC formation which proceeds through leptotene and is completed in zygotene and pachytene. Thus there is evidence that the continuum of chromosome pairing in wheat starts much earlier than was once thought.     

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.     

Purine biosynthesis and catabolism in soybean root nodules: incorporation of 14C from 14CO2 into xanthine

Nodulated root systems of soybean plants were exposed to ¹⁴CO₂ in the presence and absence of allopurinol. After 5 h about one-fifth of the label in the perchloric acid-soluble fraction of the nodules was found to be in xanthine in the allopurinol-treated plants. Control plants contained much lower levels of xanthine, but with similar specific activity. Hypoxanthine was not detected in either control or allopurinol-treated plants, even though it would be expected to accumulate in the latter. Degradation of labeled xanthine from allopurinol-treated plants using xanthine oxidase and uricase resulted in the loss of most of the label. The preferential incorporation and accumulation of ¹⁴C from ¹⁴CO₂ into C₆ of xanthine in allopurinol-treated plants is consistent with the involvement of phosphoribosylaminoimidazole carboxylase in the de novo synthesis of purines. The accumulation of xanthine and absence of hypoxanthine in nodules of allopurinol-treated plants confirms earlier observations. In addition, the similar specific activities of ¹⁴C in xanthine in allopurinol-treated and control plants indicate that the xanthine which accumulates in allopurinol-treated plants is the product of de novo purine biosynthesis.     

The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves: III. A Comparison between Some C(3) and C(4) Plants

The chlorophyll-based specific activity of cytochrome oxidase and three exclusively mitochondrial enzymes of the tricarboxylic acid cycle showed little variation between leaves of C₃ and C₄ plants or between mesophyll and bundle sheath cells of Atriplex spongiosa and Sorghum bicolor. However, a large, light-dependent transfer of label from intermediates of the tricarboxylic acid cycle to photosynthetic products was a feature of leaves of C₄ plants. This light-dependent transfer of label was barely detectable in leaves of C₃ plants and in leaves of F₁ and F₃ hybrids of Atriplex rosea (C₄) and Atriplex patula spp hastata (C₃). The light-dependent transfer of label to photosynthetic products in leaves of C₄ plants was inhibited by the tricarboxylic acid cycle inhibitors malonate and fluoroacetate. The requirement for continued tricarboxylic acid cycle activity was also indicated in experiments with specifically labeled succinate-¹⁴C. These experiments, together with the distribution of ¹⁴C in glucose prepared from sucrose-¹⁴C formed during the metabolism of succinate-2,3-¹⁴C, confirmed that the photosynthetic metabolism of malate and aspartate derived from the tricarboxylic acid cycle, and not the refixation of respiratory CO₂, was the main path of carbon from the cycle to photosynthesis.     

Effects of Copper on Root Growth, Cell Division, and Nucleolus of Zea Mays

The effects of different concentrations (10^–5 – 10^–2 M) of copper sulfate on root growth, cell division and nucleoli in root tip cells of Zea mays L. were investigated. 10^–5 M Cu stimulated root growth, but at higher concentrations (10^–4 – 10^–2 M) inhibited it. Cu had toxic effects on chromosomal morphology: c-mitosis, anaphase bridges, and chromosome stickiness were induced. Some nuclei had irregular shape and particles extruded from nucleoli to nuclei and finally from the nuclei into the cytoplasm.     

Changes in RNA-, DNA- and proteinsynthetic activity during the formation of analfin processes in ethisterone-treated females of Oryzias latipes

The incorporations of uridine-³H, thymidine-³H, and leucine-³H were studied in the process-forming regions of the anal-fin rays of the ethisterone-treated females of Oryzias latipes. The activity of alkaline phosphatase was also studied. The increased incorporation of uridine-³H was detected between 12 and 24 hours of ethisterone treatment, attaining the maximum at 24 hours. The percentage of thymidine-³H labeled nuclei increased rapidly between 48 and 84 hours. The incorporation of leucine-³H was found to increase during the first 12 hours, attaining a constant level at 24 hours. An additional increase in incorporation of leucine-³H took place at 60 hours, the incorporation coming up to the maximum at 72 hours. In the horny substance secreted by the scleroblast mass, grains in the autoradiograph were detected at and after 72 hours. Alkaline phosphatase activity was manifested between 48 and 72 hours. These results seem to correspond to the histological changes, such as the appearance of the precursor cells of scleroblasts at 48 hours, the formation of scleroblast mass during the next 24 hours, and the initiation of horny substance secretion at 72 hours.     

Localization of repetitive DNA in cereals byin situ hybridization: Cross hybridization among wheat,rye, barley,and oat

³H-RNA, complementary to repetitive DNA of wheat, rye, barley, and oat, was hybridizedin situ to root tip or pollen mother cells of the species mentioned. The cRNAs hybridized best with the DNA in cell nuclei of the species from which they were prepared. Cross hybridization with cells of the other related species resulted in a significant but diminished labelling. Wheat, rye, and barley hybridized better to each other than to oat, andvice versa, in agreement with the usual taxonomical classification. Over the interphase nuclei the label was distributed unevenly; not all regions of dense chromatin were labelled, and little label was found over the nucleoli. On chromosomes, the repetitive DNA was located somewhere along the chromosome arms or near the centromers in wheat, barley, and oat. Only in rye, most of the label was located near the telomers, probably over the large heterochromatin areas.     

Biochemistry of Suberization: Incorporation of [1-C]Oleic Acid and [1-C]Acetate into the Aliphatic Components of Suberin in Potato Tuber Disks (Solanum tuberosum)

Biosynthesis of the aliphatic components of suberin was studied in suberizing potato (Solanum tuberosum) slices with [1-¹⁴C]oleic acid and [1-¹⁴C]acetate as precursors. In 4-day aged tissue, [1-¹⁴C]oleic acid was incorporated into an insoluble residue, which, upon hydrogenolysis (LiA1H₄), released the label into chloroform-soluble products. Radio thin layer and gas chromatographic analyses of these products showed that ¹⁴C was contained exclusively in octadecenol and octadecene-1, 18-diol. OsO₄ treatment and periodate cleavage of the resulting tetraol showed that the labeled diol was octadec-9-ene-1, 18-diol, the product expected from the two major components of suberin, namely 18-hydroxyoleic acid and the corresponding dicarboxylic acid. Aged potato slices also incorporated [1-¹⁴C]acetate into an insoluble material. Hydrogenolysis followed by radio chromatographic analyses of the products showed that ¹⁴C was contained in alkanols and alkane-α,ω-diols. In the former fraction, a substantial proportion of the label was contained in aliphatic chains longer than C₂₀, which are known to be common constituents of suberin. In the labeled diol fraction, the major component was octadec-9-ene-1,18-diol, with smaller quantities of saturated C₁₆, C₁₈, C₂₀, C₂₂, and C₂₄-α,ω-diols. Soluble lipids derived from [1-¹⁴C]acetate in the aged tissue also contained labeled very long acids from C₂₀ to C₂₈, as well as C₂₂ and C₂₄ alcohols, but no labeled ω-hydroxy acids or dicarboxylic acids were detected. Label was also found in n-alkanes isolated from the soluble lipids, and the distribution of label among them was consistent with the composition of n-alkanes found in the wound periderm of this tissue; C₂₁ and C₂₃ were the major components with lesser amounts of C₁₉ and C₂₅. The amount of ¹⁴C incorporated into these bifunctional monomers in 0-, 2-, 4-, 6-, and 8-day aged tissue were 0, 1.5, 2.5, 0.8, and 0.3% of the applied [1-¹⁴C]oleic acid, respectively. Incorporation of [1-¹⁴C]acetate into the insoluble residue was low up to the 3rd day of aging, rapid during the next 4 days of aging, and subsequently the rate decreased. These changes in the rates of incorporation of exogenous oleic acid and acetate reflected the development of diffusion resistance of the tissue surface to water vapor. As the tissue aged, increasing amounts of the [1-¹⁴C]acetate were incorporated into longer aliphatic chains of the residue and the soluble lipids, but no changes in the distribution of radioactivity among the α-ω-diols were obvious. The above results demonstrated that aging potato slices constitute a convenient system with which to study the biochemistry of suberization.