Observations on the Feeding and Symptomatology of Xiphinema and Longidorus on Selected Host Roots

In vitro feeding of Xiphinema brevicolle, X. index and Longidorus africanus on roots of host seedlings is described. Both Xiphinema spp. fed mainly along roots rather than at tips and up to several days at a single site. Feeding of L. africanus was confined to root tips and lasted up to 15 min. No visible short term reaction of roots parasitized by the Xiphinema spp. could be discerned, but both swelling and cessation of growth of root tips were observed within 20 hr after feeding by L. africanus. Long-term (12-month) symptoms on roots of several host plants caused by cultured populations of X. brevicolle, X. index, X. italiae, L. africanus and L. brevicaudatus are described. All the Xiphinema spp. caused a thinning and distinct darkening of root systems and, at some sites, a breakdown of the cortex. Both species of Longidorus caused stubby and swollen root tips. Root symptom severity was in proportion to nematode population levels.     

Biochemical Changes in Terminal Root Galls Caused by an Ectoparasitic Nematode,Longidorus africanus: Phenols,Carbohydrates and Cytokinins

Biochemical changes occurred in roots of bur marigold and grapevine infected with Longidorus africanus. Phenols of infected root tips differed quantitatively from those of healthy root tips (optical density of 0.68 and 0.32, respectively). Column chromatography of the phenol extracts resulted in more fractions in the infected than in the healthy root extract. Of the enzymes involved in phenol metabolism, three were tested. No polyphenol oxidase activity was detected and no difference in catalase activity was found between healthy and infected roots. Peroxidase activity, however, was much higher in infected roots. The extra peroxidase was associated mainly with the cortical cells of the infected root tips.Galled roots contained twice the amount of simple sugars and inulin as did the healthy roots. A cytokinin-active fraction was found only in the noninfected roots.     

DNA and RNA Syntheses by Intraerythrocytic Stages of Plasmodium knowlesi*

Incorporation of the nucleic acid precursors, orotic acid, adenosine, thymidine, and uridine, was studied in various stages of intraerythrocytic Plasmodium knowlesi from infected rhesus monkeys. Incubation of the parasitized erythrocytes with the precursors was for 3 hr periods using a plasma-free culture medium. The samples containing primarily rings, early trophozoites, or late trophozoites incorporated orotic acid, adenosine, and uridine into RNA; however, these stages exhibited negligible or very low levels of incorporation of any of the precursors into DNA. The sample containing late trophozoite and schizont stages incorporated orotic acid, adenosine, and uridine into RNA, and orotic acid, adenosine, and very low levels of thymidine into DNA. These results indicate that DNA synthesis (the S phase of the cell cycle) occurs very close to the time of nuclear division, and that either the G1 or G2 phase is very short in P. knowlesi. It was also observed that adenosine and orotic acid, 2 precursors which are incorporated into both DNA and RNA, are utilized differently by the intraerythrocytic parasites. Incorporation of orotic acid into RNA and DNA and adenosine incorporation into DNA were continuous for the entire incubation period, whereas incorporation of adenosine into RNA was very low during the last 2 hr of each period. It was further demonstrated that the parasites utilized exogenous uridine for synthesis of RNA, and that the older parasite stages incorporated thymidine into DNA.     

Molecular specificity of 2-aminopurine in Saccharomyces cerevisiae

The rates of DNA, RNA and protein synthesis were investigated by incorporation of radioactive precursors into the excised root tips of V. faba. 2-h exposure to 0.1% caffeine resulted in inhibition of protein synthesis to about 60% of the control rate. RNA synthesis was reduced in the range of 20–30%. The same concentration of caffeine did not affect the rate of DNA synthesis even during 12-h incubation, but concentrations higher than 1% caused a significant decrease in [³H]thymidine incorporation.     

Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

Der Einfluß von Rifampicin,Chloramphenicol und Cycloheximid auf den Uridin-Einbau in chloroplastidäre Ribosomenvorstufen von Chlorella

Summary The unicellular green alga Chlorella incorporates labeled uridine mainly into the precursors of chloroplast ribosomes. After treatment with rifampicin for 60 min, the uridine incorporation into the particles is completely inhibited. Chloramphenicol treatment results in the same complete inhibition. In constrast, cycloheximide (actidione) slightly stimulates the incorporation of uridine into the chloroplast ribosome precursors.Short-time incorporation of inorganic phosphate into the ribosome fractions is nearly unaffected by rifampicin and chloramphenicol, but it is strongly inhibited by cycloheximide.Isolation and chromatographic separation of nucleic acids after treatment of cells with rifampicin shows that uridine incorporation into RNA is completely inhibited. Chloramphenicol causes only partial inhibition of uridine labeling in the high molecular weight RNA. Here again, cycloheximide stimulates the uridine incorporation.The results indicate that uridine is preferentially incorporated by Chlorella cells into the chloroplast ribosome precursors. Inorganic phosphate is introduced both into cytoplasmic and into chloroplasmic RNA, but because of the quantitative distribution, the cytoplasmic ribosomes are more extensively labeled. Since only inhibitors of bacterial and chloroplasmic RNA-and protein synthesis affect the formation of uridine-labeled ribosomes, this synthesis must take place in the chloroplast itself.

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Abkürzungen DNA Desoxyribonucleinsäure - RNA Ribonucleinsäure - MAK-Säule Säule aus methyliertem Albumin mit Kieselgur - Bis-MSB bis-(O-Methylstyryl)-Benzol - PPO 2,5 Diphenyloxazol - Tris Trimethylaminomethan     

Comparison of Compatible and Incompatible Response of Potato to Meloidogyne incognita

One susceptible (D6) and two resistant (E2 and N4) clones of Solanum sparsipilum × (S. phureja × haploid of S. tuberosum) were used to study the responses of potato roots and tubers to race 1 of Meloidogyne incognita (Kofoid &White) Chitwood. The compatible response was characterized by rapid penetration of large numbers of second-stage juveniles (J2) into roots, cessation of root growth, and occasional curving of root tips. The life cycle of M. incognita in the susceptible clone was completed in 25 days at 23-28 C. The incompatible response was characterized by penetration of fewer J2 into roots, necrosis of feeding sites within 2-7 days, and lack of nematode development. There were no differences in response of tubers from resistant and susceptible clones to nematode infection. Small numbers of J2 were detected in tubers, but they did not develop.     

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.     

Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5 % of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 g/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.     

Vertical dye-buoyant density gradients for rapid analysis and preparation of plasmid DNA

A rapid and easy method for the isolation of plasmid DNA, both in analytical and preparative scale, is described. Using dye-buoyant density-gradient centrifugation in a vertical rotor, separation of covalently closed, supercoiled plasmid DNA from relaxed circular and linear DNA is completed within 1 to 2 h.     

Incorporation of different labelled precursors into chloroplast RNA of Chlorella

Summary Radioactive uridine is incorporated by Chlorella strain 211-8b/p into ribosomal subunits and their rapidly labelled RNA comigrates with chloroplast RNA on polycrylamide gels.Ribosomal particles which can be labelled by short pulses of orotic acid cosediment with the particles labelled by uridine pulses and contain the same RNA species as these when separated either on sucrose gradients or on polycrylamide gels. This incorporation is, like that of uridine, sensitive to rifampin and chloramphenicol, but insensitive to cycloheximide.A comparative study of short-time incorporation of uridine, orotic acid and guanosine into the RNA of Chlorella showed that all three precursors were incorporated mainly into RNA of chloroplastic origin. However, guanosine was also partly incorporated into cytoplasmic rRNA. Nitrogen-deficient cells always incorporated part of all three precursors into cytoplasmic rRNA, but the proportions of these were different among the different precursors.These results are consistent with the hypothesis that the described differences in the incorporation of the above mentioned precursors into RNA of different cellular compartments are largely attributable to effects of pool sizes.     

Effect of trifluralin on growth, morphology, and nucleic Acid synthesis

Roots and shoots of corn seedlings (Zea mays L. var. Dixie 18) germinated in trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine) solutions are characterized by radial enlargement of the cortical cells and by multinucleate cells in the meristematic regions. Trifluralin inhibits elongation of Avena coleoptile sections at concentrations of 0.1 μm to 10 μm. Synthesis of DNA, RNA, and protein is suppressed in the root tips while no significant effect is noticeable in the shoots of corn germinated in trifluralin. A ³²P time-course study of 48, 72, and 96 hours utilizing phenol extraction and MAK column separation of corn root and shoot nucleic acids showed suppression of ³²P incorporation in the treated roots; however, the 72 and 96 hour treated shoots incorporated a much greater amount than the control with most of the increased incorporation found in the sRNA and DNA fractions. The increased activity in the DNA may be due to a high G-C type DNA. No selective suppression or enhancement of any particular RNA species was noticed in the treated plants.     

NEWLY SYNTHESIZED RNA IN NUCLEI ISOLATED FROM NERVE AND GLIAL CELLS

—Nuclear RNA from neurones, astrocytes and other glial cells was pulse-labelled in vivo with [³H]uridine and analysed by sucrose density-gradient centrifugation after various periods of incorporation. Thirty min after the injection of the isotope, rapidly-labelled RNA appeared in all three cell types, a heterogeneous fraction sedimenting above 30S, the others at 25 and 12S. The transformation rate of the two latter components was equally rapid in all three types of nuclei studied. These components are assumed to be of messenger nature. The heavy fractions underwent transformations which in other cells have been described to lead to rRNA formation. The temporal pattern as well as the sequence of changes were similar in nuclei from neurones and astrocytes, the only difference being that a 35S intermediate was found in the former and a 32S in the latter. In non-astrocytic glial nuclei, synthesis and transformation of the 45S component were delayed as compared to the other cell types and the processing of this component may involve both a 32S and a 35S intermediate. Moreover, the radioactivity incorporated in all the nuclear RNA species was always lower in these cells.     

Biochemical Changes in Terminal Root Galls Caused by an Ectoparasitic Nematode,Longidorus africanus: Amino Acids

The amino acids of terminal root galls caused by Longidorus africanus on bur marigold (Bidens tripartita L.) and grapevine (Vitis vinifera L.) were studied. The galled roots of bur marigold contained 73% more cell-wall protein and 184% more free amino acids. The main changes among the free amino acids of the galled tissue were a large increase (1900%) in proline and a decrease in aspartic acid (56%) compared with the respective check tissue. Hydroxyproline decreased in the wall protein fraction from 5.6% in the healthy tissue to 3.6% in the infected tissue.Percent of hydroxyproline in total amino acids of the wall protein fraction of grapevine roots decreased from 0.7% in the healthy tissue to 0.3% in the galled tissue, and total proteins of this fraction decreased from 9.5 mg to 4.5 rag, respectively. Total protein in the protoplasmic fraction also decreased from 3.0 mg in healthy to 1.0 mg in infected roots. No change was noticed in total proteins in the free amino acids fraction but free proline decreased 40% in the infected roots.The relationship of these differences to the specific reactions of the hosts to nematode feeding is discussed.     

Metabolic Requirement of Cucurbita pepo for Boron

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [³H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [¹⁴C]leucine into protein and respiration as measured by O₂ consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O₂ consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.     

Cytochalasin B. VI. Competitive inhibition of nucleoside transport by cultured Novikoff rat hepatoma cells

Cytochalasin B competitively inhibits the transport of uridine and thymidine by Novikoff rat hepatoma cells growing in suspension culture with apparent K_i''s of 2 and 6 µM, respectively, but has no effect on the intracellular phosphorylation of the nucleosides. Choline transport is not affected by cytochalasin B. Results from pulse-chase experiments indicate that cytochalasin B has no direct effect on the synthesis of RNA, DNA, or uridine diphosphate-sugars. The inhibition of uridine and thymidine incorporation into nucleic acids by cytochalasin B is solely the consequence of the inhibition of nucleoside transport.     

Activities of DNA polymerases and RNA polymerases detected in situ in growing and differentiating cells of root cortex.

Activities of DNA polymerases and RNA polymerases were studied by autoradiographic methods in growing and differentiating root cortex cells of Zea mays - a species in which endomitosis occurs - and Tulipa kaufmanniana - in which this process does not occur. In Tulipa kaufmanniana, the highest activity of DNA polymerase appears in the nuclei of meristematic zone during the S phase of the cell cycle. In Zea mays, endomitotic replication of DNA occurs in all growth and differentiation zones and the activity of DNA polymerase in the nuclei is similar to that in the meristematic zone. In both species, nuclear RNA synthesis, measured with 3H uridine incorporation, is highest in the meristematic zone and declines steadily with development. Activity of nuclear RNA polymerase is present in all developmental zones in both species and is similar to that in the meristematic zone. 3H uridine incorporation into nucleoli decreases markedly in both species, whereas the activity of nucleolar RNA polymerase remains at a high level in all root segments in Zea mays and decreases slightly in Tulipa kaufmanniana. It is argued that the differences between the incorporation of 3H uridine and that or 3H UMP may be caused by a reduction of the pool of endogenous ribonucleoside triphosphates. Marked activities of DNA polymerase and RNA polymerase in cytoplasm are possibly related to the growth and division of plastids and mitochondria.     

Drosophila polyribosomes. The characterization of two populations by cell fractionation and isotopic labeling with nucleic acid and protein precursors

Two populations of polyribosomes have been isolated from third instar larvae of D. melanogaster. One population appeared to be soluble while the second seemed membrane-bound. Short-term labeling of the two RNP fractions with radioactive nucleic acid and protein precursors was achieved by using a feeding stimulant. RNA was extracted from both polyribosomal fractions following 25, 40, and 60 min of in vivo uridine-³H incorporation. Soluble polyribosomes exhibited more rapid uptake of uridine into ribosomal and heterogeneous RNA fractions than did membrane-bound polyribosomes at comparable time periods. In vivo amino acid incorporation into the two polyribosomal populations was examined after 10, 20, 40, 60, and 80 min of incubation in leucine-³H. In this case, the membrane-bound polyribosomes reached a higher specific activity than did the soluble ones. These functional differences confirmed the observation, based on cellular fractionation studies, that the two classes of polyribosomes represented functionally distinct populations. These data have been compared with those from studies on other metazoan systems. In addition, dithiothreitol has been demonstrated to be a powerful ribonuclease inhibitor.     

Inhibition of mitosis in pisum root meristems by continuous gamma radiation: the influence of temperature on the synthesis of DNA, RNA and protein during inhibition

Tritiated precursors of DNA, RNA and protein were used to measure synthesis at 10 and 20C in root meristem cells of Pisum after they were mitotically arrested by continuous irradiation with gamma rays. The experiments were designed to determine if the arrested cells accumulated in a certain part of interphase, to determine the effect on DNA, RNA and protein synthesis, to find out if the effects were temperature dependent, and finally to reveal possible relationships between growth inhibition and altered synthesis. The results showed that the incorporation of DNA and RNA precursors was impaired by irradiation and that decreased temperature further increased radiation impairment of DNA synthesis. Protein synthesis on the other hand was not impaired by irradiation at either temperature. Irradiation at 20C reduced the number of DNA-synthesizing cells; at 10C this number was reduced to near zero. Although irradiated cells synthesizing RNA showed a reduction in grain counts when compared to the controls, they still retained the ability to incorporate tritiated uridine at 10C. It was hypothesized that the combination of reduced DNA and RNA synthesis and unaffected protein synthesis resulted in precocious maturation of the arrested meristem cells. Growth which occurred in the absence of cell division was attributed to meristematic cells which precociously matured and cells which were in the region of elongation.     

Biosynthesis of lectin in roots of germinating and adult cereal plants

Root tips of wheat, rye, barley and rice seedlings contain lectins which are identical to the respective embryo lectins with respect to their molecular weight, sugar-specificity and serological properties. Using in vivo labelling techniques, it could be demonstrated that lectin is synthesized de novo in these tissues. The presence of lectin mRNA in seedlings was confirmed by in-vitro synthesis of lectin in root-tip extracts. Lectin synthesis occurs both in primary and first adventitious roots and is confined to the apical part (2mm) of the root. As seedling development proceeds, lectin synthesis in root tips gradually decreases. Adventitious roots of adult (five to six months old) wheat, rye and barley, but not rice, plants also contain lectins which are indistinguisable from the embryo lectins by the above-mentioned criteria. These lectins are synthesized in vivo in isolated root tips (5 mm) with labelled cysteine and in vitro in cell-free extracts prepared from root tips. Synthesis of lectin in roots of adult plants is also confined to the apical (2 mm) tip of the roots. At the molecular level, root lectin synthesis is very similar to that in embryos. All root lectins are synthesized as 23 000-M_r precursors which are post-translationally converted into the mature 18 000-M_r polypeptides. The observation that seedling roots and adventitious roots of six-month-old plants actively synthesize lectins strongly indicates that lectin genes are expressed in these tissues. In addition, since the root lectins are indistinguishable from the embryo lectins, we postulate that the same lectin genes are expressed.Abbreviations ABA abscisic acid - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - WGA wheat-germ agglutinin