Nucleic Acid Metabolism During Early Development of Pollinated and Auxin-induced Parthenocarpic Watermelon Fruits

Following pollination or induction of parthenocarpy by NAA¹treatment watermelon ovaries synthesize large quantities ofRNA. Hormone-treated ovaries increase their rate of RNA synthesisover untreated controls by 6 h after treatment whereas pollinatedovaries do so by 24 h. The lag period with pollinated ovarieshas been related to the growth of pollen tubes toward the ovulesand it is suggested that auxin secretion by the growing pollentubes may be the stimulus for increased RNA synthesis. Evidenceis presented that the cells in the flesh of developing ovariesbecome polyploid as they enlarge.     

In vitro protein synthesis with polysomes from unpollinated,cross- and self-pollinated Petunia ovaries

Summary From in-vitro protein synthesis studies it has been shown that in Petunia hybrida (clone W166K, incompatibility genes S₁S₂) the ovary is stimulated to synthesize proteins before the pollentubes reach the ovary. A difference in protein metabolism is detected after self- and cross-pollination. On the basis of this result has been concluded that a signal has to be sent from the stigma or the style towards the ovary which induces the changes in metabolic activity. The signal must be different for cross- and self-pollination.     

Localization of the 5S RNA genes in Zea mays by RNA-DNA hybridization in situ

5S RNA was extracted from Zea mays tissue and iodinated in vitro with ¹²⁵I to a high specific activity. Acrylamide gel electrophoresis of the ¹²⁵I-5S RNA, 11/2 weeks after iodination demonstrated that most of the 5S RNA molecules were degraded to half-size or smaller. In situ hybridization with this iodinated RNA to pachytene microsporocyte chromosomes showed that the 5S RNA cistrons are located near the end of the long arm of chromosome 2. No obvious association of the 5S locus with the nucleolus was seen during pachytene or later stages.     

Evidence that multiple species of aminoacylated transfer RNA are present in regenerating optic axons of goldfish

This study reports that 4S RNA present in regenerating optic axons of goldfish is likely to be transfer RNA. Evidence is also presented which indicates that this transfer RNA is similar to transfer RNA found in tectal cells and that its aminocylation is likely to occur both in retinal ganglion cells prior to axonal transport as well as in the axon itself. Fish with regenerating optic nerves received intraocular injections of [³H]uridine followed 4 days later by intracranial injections of [¹⁴C]uridine. Radioactive tectal 4S RNA was isolated 6 days after [³H]uridine injections and chromatographed by BD cellulose chromatography. Optical density as well as radioactivity profiles for both [¹⁴C]4S RNA (from tectal cells) and [³H]4S RNA (90% of which originated from regenerating optic axons) were found to be similar toE. coli transfer RNA optical density profiles, indicating that the intra-axonal 4S RNA is likely to be transfer RNA. Moreover, comparisons of³H/¹⁴C suggest that intra-axonal and cellular 4S RNAs are composed of similar species of transfer RNA. Results of other experiments indicated that aminoacylation of axonally transported tRNA occurs both in the retina and in optic axons subsequent to axonal transport.     

Autoradiographic Studies of Uridine-5-H3 Uptake in Entamoeba histolytica in the CLG Medium1

SYNOPSIS. Using uridine-5-H³, “long-term” labeling experiments over a 72 hr growth cycle were done with E. histolytica strain K9 grown in CLG medium with penicillin-inhibited Bacteroides. Autoradiographic analysis revealed that tritium occurs primarily in cytoplasm and rarely the nucleus of amebae. The most extensive cytoplasmic activity was observed during the initial 0–24 hr growth period of amebae as compared to later labeling periods. RNase or RNase followed by DNase extracted a large amount but not all label from amebae. These nucleases were least effective during the initial 24 hr period of growth. Thus it appears that tritium from uridine-5-H³ is not highly specific for RNA in amebae. However, the possibility that such label is associated with RNase-resistant RNA cannot be ruled out. More recent cytochemical studies do indicate the presence of RNase-resistant RNA in the cytoplasm of amebae. The activity found in penicillin-inhibited Bacteroides after uridine-5-H³ labeling and their reaction to the various digestive procedures was similar to amebae at corresponding labeling periods. Therefore at least some of the RNase-resistant material present in the cytoplasm of amebae may be derived from the ingested bacteria; this has been further found by appropriate experiments in which amebae were fed prelabeled bacteria. Nuclear activity when observed (always after 24 hrs growth) was associated either with the periphery of the nucleus and/or the endosome. It was not seen in the nuclear stroma. Some of this activity is RNase-resistant, perhaps representing double or multi-stranded RNA. It therefore appears that RNA is not distributed in the nuclear stroma in “long-term” labeling experiments.     

Extraction of ribosomal RNA from soil for detection of Frankia with oligonucleotide probes

Sequences of 16S rRNA of the nitrogen-fixing Frankia strain Ag45/Mut15 and the ineffective Frankia strain AgB1.9 were used to design a genus-specific oligonucleotide probe. Hybridization experiments of this Frankia probe and a second probe, specific for Nif⁺-Frankia strains only, were used to detect Frankia specific target sequences in RNA isolations from soil. A method is described for direct isolation of RNA from a loamy soil and a peat. Yields of about 10 ng RNA/g wet soil are obtained without detectable contamination with humic acids. Isolation of RNA after initial extraction of bacteria from soil resulted in significantly lower RNA yields, compared to the direct isolation procedure. Hybridization with both probes against rRNA isolations from Frankia-containing soil could detect target sequences within RNA isolations from 1 g wet soil with an estimated detection limit of 10⁴ cells.     

The disease resistance response in pea is associated with increased levels of specific mRNAs

A cDNA library was constructed using poly(A)⁺RNA fromPisum sativum which had been treated for 8 h with the fungusFusarium solani f. sp.phaseoli. Two thousand four hundred recombinant colonies were screened by differential colony hybridization using³²P-labelled cDNAs prepared from RNA extracted from either noninoculated or inoculated pea tissue. cDNA clones were then selected, which showed greater hybridization with cDNA prepared from pea RNA 8 h post-inoculation than with a cDNA probe from 0 h. Seven distinct hybridization classes were chosen for further study. Northern blot analyses of total cellular RNAs inoculated for 16 h with eitherF. solani phaseoli or water demonstrated that each cDNA clone selected represents an mRNA species which increases substantially in abundance during infection. Results of³H-uridine pulse-labelling experiments suggested that enhanced synthesis is at least partially responsible for the accumulation of the fungus-inducible mRNAs which hybridized with the clones.     

Liposome-Mediated transfer of bacterial RNA into carrot protoplasts

The uptake of liposome-encapsulated E. coli [³H]RNA by carrot (Daucus carota L.) protoplasts was examined. [³H]RNA extracted from protoplasts that had been incubated with [³H]RNA-containing, large, unilamellar lipid vesicles (liposomes) obtained by ether infusion, and examined by sucrose gradient centrifugation and formamide-polyacrylamide gel electrophoresis, appeared substantially degraded, with a total elimination of 23S RNA and a partial loss of 16S RNA. In contrast, no breakdown of the [³H]RNA was apparent in the liposomes after sequestration, even in the presence of externally added ribonuclease, or in the unfused liposomes remaining after incubation of protoplasts with liposomes. Thus, the degradation of the [³H]RNA extracted from the protoplasts must have occurred within the protoplasts and represents evidence for liposome-mediated RNA uptake. Naked RNA added to the protoplast culture was found to be totally degraded after incubation with the protoplasts. The uptake of liposome-sequestered RNA by protoplasts was demonstrated to be a function both of the lipid composition of the liposomal membrane and of the temperature of incubation of the liposomeprotoplast mixture. Furthermore, the mode of this uptake (fusion versus endocytosis) could be manipulated by adjusting the cholesterol content of the liposomal membrane. The implications of the ability to insert RNA into protoplasts without degradation by extracellular nucleases are discussed.     

Tsw gene-based resistance is triggered by a functional RNA silencing suppressor protein of the Tomato spotted wilt virus

As a result of contradictory reports, the avirulence (Avr) determinant that triggers Tsw gene-based resistance in Capsicum annuum against the Tomato spotted wilt virus (TSWV) is still unresolved. Here, the N and NSs genes of resistance-inducing (RI) and resistance-breaking (RB) isolates were cloned and transiently expressed in resistant Capsicum plants to determine the identity of the Avr protein. It was shown that the NSs^RI protein triggered a hypersensitive response (HR) in Tsw-containing Capsicum plants, but not in susceptible Capsicum, whereas no HR was discerned after expression of the N^RI/RB protein, or when NSs^RB was expressed. Although NSs^RI was able to suppress the silencing of a functional green fluorescence protein (GFP) construct during Agrobacterium tumefaciens transient assays on Nicotiana benthamiana, NSs^RB had lost this capacity. The observation that RB isolates suppressed local GFP silencing during an infection indicated a recovery of RNA silencing suppressor activity for the NSs protein or the presence of another RNA interference (RNAi) suppressor. The role of NSs as RNA silencing suppressor and Avr determinant is discussed in the light of a putative interplay between RNAi and the natural Tsw resistance gene.     

The concentrations of stable RNA and ribosomes in Rickettsia prowazekii

The obligate Intracellular parasite, Rickettsia prowazekii, is a slow-growing bacterium with a doubling time of about 10h. In the present study, DNA and RNA were obtained from the rickettsiae by two independent methods, i.e. simultaneous isolation of DNA and RNA from the same sample by phenol:chloroform extraction and CsCI gradient centrifugation. In addition, ribosomal RNA was obtained by sedimentation of partially purified ribosomes from the rickettsiae. The results demonstrated that, after correction for the cell volumes, the concentrations of stable RNA and ribosomes in R prowazekii, a slow-growing organism, were about 62fg μm⁻³ and 17000 per μm³, respectively, which were very simitar (66fg μm⁻³ and 21 000 per μm³) to those in Escherichia coli with a generation time of 40min. However, on a per cell basis, R. prowazekii had 5.6 fg of RNA and 1500 ribosomes per cell, which was only about 8% of the amount of both stable RNA (71.2 fg) and ribosomes (24000) per cell as was found in E. coli. These results indicated that R. prowazekii possesses a ribosome concentration greater than might have been predicted from its slow growth rate. This high concentration of ribosomes could be due to a large population of non-functioning ribosomes, a low efficiency of amino acid production, or a high rate of protein turnover. However, this study also demonstrated that the rickettsiae have very limited protein turnover. Knowledge of the kinetics and control mechanisms for protein synthesis in R. prowazekii remains to be established to determine the logic of the extra rickettsial ribosomes.     

Properties of RNA fractions from nuclei of brain cells which stimulate incorporation of amino acids by brain ribosomes

Abstract— The properties of RNA fractions from nuclei of brain cells which were capable of stimulating amino acid incorporation into proteins of an homologous ribosomal system were investigated. RNA was routinely prepared from crude nuclear preparations of rat brain by a method which involved treatment with sodium dodecyl sulphate and phenol at 65°. The capacity of this preparation to stimulate incorporation of radioactivity from a mixture of 15 l -[¹⁴C]amino acids was greatly enhanced by preliminary incubation of the ribosomal system from brain for 5–20 min. The response was markedly dependent upon the concentrations of ribosomes and of the pH 5 fraction. The optimal level of Mg²⁺ for basal incorporation of amino acids into protein was 8 mm ; however, incorporation in the presence of nuclear RNA was greater at higher concentrations of Mg²⁺. The response to nuclear RNA was also enhanced as the K⁺ concentration was increased from 25 to 100 mm . The stimulatory effect of nuclear RNA on incorporation of l -[¹²C]eucine was either unaltered or depressed by addition of a mixture of 19 l -[¹²C]amino acids each at concentrations, of 10^?8, 10^?2, or 10^?1 mm . Under appropriate conditions of incubation, basal rates of incorporation and rates of incorporation stimulated by nuclear RNA were linear for 30 min. The response was proportional to the concentration of nuclear RNA between 34 and 136 μg. RNA prepared from ribosomes of rat brain essentially failed to stimulate incorporation of amino acids over this range of concentrations. Fractionation of nuclear RNA by centrifugation in sucrose density gradients revealed that 75 per cent of the stimulatory activity was in the fraction which sedimented below 12 S and contained about 25 per cent of the total RNA. Most of the remaining activity was in the 18 S region. Less than 5 per cent of the RNA in the lightest fraction (< 12 S) exhibited amino acid-acceptor activity, The stimulatory action of nuclear RNA on incorporation of amino acids was readily destroyed by mild treatment with pancreatic ribonuclease, whereas amino acid-acceptor activity was relatively resistant to this treatment. The results suggest that the brain may contain low molecular weight RNA with properties of messenger RNA.     

In vitro translation of mRNA from Nostoc commune (Cyanobacteria)

RNA pools were extracted from cells of Nostoc commune UTEX 584 in exponential growth (liquid cultures) and from cells which had been immobilized and dried rapidly at -99.5 MPa. Levels of incorporation of ³⁵S-methionine, five- to sixfold higher than the endogenous level, were obtained after in vitro translation of the RNA preparations in a heterologous S30 cell-free system purified from Escherichia coli Q13. The levels of incorporation, obtained with a homologous N. commune UTEX 584 S30 system, were much lower. The requirement for magnesium in the heterologous system was 15–21 mM, translation of N. commune UTEX 584 RNA was inhibited when the RNA concentration was greater than 0.3 mg ml^–1, and translation was stimulated significantly by the presence of ammonium chloride. Few qualitative differences were observed between the pattern of proteins (SDS-PAGE) obtained after translation of the RNA pools from cells in exponential growth, and from those cells subjected to immobilization and rapid drying. The data suggest that short-term desiccation of N. commune UTEX 584 does not have a marked selective effect on the composition of the mRNA pool. In contrast, preparations of RNA from field materials of Nostoc commune HUN (desiccated for 5 years) were unable to drive high rates of translation in any of the systems tested and optimized for use in this study.     

VARIABILITY IN RATIOS OF PHYTOPLANKTON CARBON AND RNA TO ATP AND CHLOROPHYLL A IN BATCH AND CONTINUOUS CULTURES1,2

The feasibility of estimating phytoplankton carbon and RNA concentrations from measurements of ATP and chlorophyll a (chl a) concentrations was studied using chemostat populations of the marine diatom Thalassiosira weissflogii (Grunow) Fryxell & Hasle (= T. fluviatilis Hustedt). C:ATP and RNA:ATP ratios were studied for six additional marine species in batch culture representing five classes of phytoplankton. Statistical analyses revealed that both the growth rate and the factor limiting growth (NO₃^-, NH₄⁺, PO₄^3- or light) could alter C:ATP, RNA: ATP, C:chl a and RNA:chl a ratios by amounts which were large compared to measurement error. An analysis of variance of the batch culture results indicated that both species and the source of inorganic nitrogen (NO₃^-, or NH₄⁺) had a significant effect on C:ATP and RNA:ATP ratios. Light had less of an influence on C:ATP and RNA:ATP ratios than on C:chl a and RNA:chl a ratios, and for this reason we feel that phytoplankton C and RNA concentrations can be estimated with greater reliability from ATP than from chl a measurements. The range of C:ATP and RNA:ATP values found for T. weissflogii under a variety of growth conditions was similar to that for the six additional species grown in batch culture, suggesting that this range of values is indicative of the extremes likely to occur in living cells. Our results and additional data in the literature indicate that phytoplankton C and RNA concentrations can be estimated to within a factor of two by multiplying ATP concentrations by 311 and 35, respectively, in N limited systems, and by 341 and 36, respectively in PO₄^3- limited systems.     

Deficiency mapping of the 93D heat-shock locus in Drosophila melanogaster

The 93D heat shock locus was mapped relative to an overlapping series of deficiencies of the 93D region by three criteria: the ability of the deleted chromosomes to puff at 93D, the ability of the deleted chromosomes to synthesize RNA from the 93D region after a temperature shift and the presence of heat shock RNA sequences at 93D as assayed by in situ hybridization. The results are essentially the same by all three criteria. Chromosomes with deficiencies that did not extend distal to 93D4 puffed and incorporated ³H-uridine after a temperature shift, and were labelled at 93D following in situ hybridization of heat shock RNA from tissue culture cells. All the other deficiency chromosomes tested failed to puff and to incorporate ³H-uridine following a temperature shift and did not show hybridization in this region after in situ hybridization with heat shock RNA. The heat shock locus was mapped to the overlapping region of Df(3R)e ^Gp4and Df(3R)GC14 just outside the inverted region of In(3R)GC23.     

Biological and biochemical properties of an isolate of cherry rasp leaf virus from red raspberry

A mechanically transmissible virus obtained from symptomless plants of a red raspberry selection imported into Scotland from Quebec, Canada was indistinguishable serologically from a cherry isolate of cherry rasp leaf virus (CRLV). The raspberry isolate, CRLV-R, was graft transmitted to several virus indicator species and cultivars of Rubus without inducing noticeable symptoms. In Chenopodium quinoa sap, CRLV-R lost infectivity after dilution to 10^-5 or heating for 10 min at 60°C but was infective after 16 days (the longest period tested) at 18°, 4° or - 15°C. The virus particles are isometric, c. 28 nm in diameter, and were purified with difficulty from infected C. murale and C. quinoa plants. The particles comprise two nucleoprotein components with sedimentation coefficients of 89 and 115 S and are prone to aggregate during purification. When centrifuged to equilibrium in CS₂SO₄ solution, purified virus preparations formed two major components with p= 1·28 and 1·36 g/cm³. Virus particles contained two RNA species which, when denatured in glyoxal and electrophoresed in agarose gels, had estimated mol. wt of 2·56 × 10⁶ (RNA-1) and 1·26 × 10⁶ (RNA–2). Infectivity of CRLV-R RNA was abolished by treatment with proteinase K, suggesting that the RNA is linked to protein necessary for infectivity; RNA molecules contained polyadenylate. In reticulocyte lysates, CRLV-R RNA stimulated the incorporation of ³H-leucine, mainly into two polypeptides of estimated mol. wt 200 000 and 102 000. When electrophoresed in polyacrylamide gels, protein obtained from CRLV-R particles purified by centrifugation to equilibrium in Cs₂SO₄ separated into three bands with estimated mol. wt 26 000 , 23 000 and 21 000.     

Recovery of plastids from photooxidative damage: Significance of a plastidic factor

Glyoxysomal citrate synthase (gCS) was purified from crude extracts of watermelon (Citrullus vulgaris Schrad.) cotyledons, yielding a homogenous protein with a subunit MW of 48 kDa. The enzyme was selectively inhibited by 5,5-dithiobis-(2-nitrobenzoic acid), allowing quantification in the presence of the mitochondrial isoenzyme (mCS). Differences were also observed with respect to inhibition by ATP (k _i=2.6 mmol · l^-1 for gCS, k _i=0.33 mmol · l^-1 for mCS). The antibodies prepared against gCS did not cross-react with mCS. The immunocytochemical localization of gCS by the indirect protein A-gold procedure was restricted to the glyoxysomal membrane or the peripheral matrix of glyoxysomes. Other compartments, e.g. the endoplasmic reticulum, were not labeled. Xenopus oocytes were used for the translation of watermelon polyadenylated RNA (poly(A)⁺RNA). A translation product with a MW of 51 kDa was immunoprecipitated by the anti-gCS antibodies. It was absent in controls without poly(A)⁺RNA or with preimmune serum. A similar translation product was also immunoprecipitated after cell-free synthesis of watermelon poly(A)⁺RNA in a reticulocyte system, in contrast to the in-vivo labeled gCS (48 kDa). It was concluded that gCS is synthesized as a higher-molecular-weight precursor.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - gCS glyoxysomal citrate synthase - gMDH glyoxysomal malate dehydrogenase - k _i inhibitor constant - mCS mitochondrial citrate synthase - OAA oxaloacetate - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Differential display analysis of RNA accumulation in arbuscular mycorrhiza of pea and isolation of a novel symbiosis-regulated plant gene

Differential RNA display was used to analyze gene expression during the early steps of mycorrhiza development on Pisum sativum following inoculation with Glomus mosseae. Seven out of 118 differentially displayed cDNA fragments were subcloned and sequenced. One fragment corresponded to part of the fungal 25S ribosomal RNA gene and a second one showed similarity to a human Alu element. The others were derived from plant genes of unknown function. One of the fragments was used for the isolation of a full-length cDNA clone. It corresponded to a single-copy gene (psam1) which is induced during early symbiotic interactions, and codes for a putative transmembrane protein. Northern and RNA dot blot analyses revealed enhanced accumulation of psam1 RNA after inoculation with G. mosseae of wild-type pea and an isogenic mutant deficient for nodule development (Nod⁻, Myc⁺). Received: 3 March 1997 / Accepted: 12 May 1997