The pattern of protein and nucleic acid synthesis in germinating spores of Phycomyces blakesleeanus

Summary Synthesis of proteins, RNA and DNA is measured by incorporation of labelled precursors at different times during germination of Phycomyces spores.RNA and protein synthesis increases immediately after activation. DNa synthesis begins at a later stage (± 8 h) of germination when germ tubes are already present. Nuclear division occurs earlier in germination (±4–5 h) and is accompanied by a decrease in RNA synthesis. It can be concluded that at least most of the dormant spores are in the G2 phase of the cell cycle.Analysis of ribosomal RNA after pulse-chase labelling shows only three labelled compounds: a precursor molecule (2.25×10⁶ daltons) and the two mature ribosomal RNA compounds (1.4×10⁶ and 0.7×10⁶ daltons). This suggests that the two rRNAs are formed directly from the precursor molecule. Cycloheximide totally blocks the transformation of the ribosomal precursor molecule into mature rRNA.     

Ribonucleic acid synthesis and loss of viability in pea seed

Summary RNA synthesis and protein synthesis in embryonic axis tissue of viable pea (Pisum arvense L. var. N.Z. maple) seed commences during the first hour of germination. Protein synthesis in axis tissue of non-viable pea seed is barely detectable during the first 24 h after the start of imbibition. Nonviable axis tissue incorporates significant levels of [³H]uridine into RNA during this period but the level of incorporation does not increase significantly over the first 24 h of imbibition. In axis tissue of non-viable seed during the first hour of imbibition most of the [³H]uridine was incorporated into low molecular weight material migrating in advance of the 4S and 5S RNA species in polyacrylamide gels but some radioactivity was incorporated into a discrete species of RNA having a molecular weight of 2.7×10⁶. After 24 h, non-viable axis tissue incorporates [³H]uridine into ribosomal RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and a heterogeneous RNA species of molecular weight ranging from 2.2×10⁶ to 2.7×10⁶. No 4S or 5S RNA synthesis is detectable after 24 h of imbibition in non-viable axis tissue. Axis tissue of viable pea seed synthesises rRNA, 4S and 5S RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and the rRNA precursor species at both periods of germination studied. Loss of viability in pea seed appears to be accompanied by the appearance of lesions in the processing of rRNA precursor species and a significant loss of RNA synthesising activity.Abbreviations rRNA ribosomal RNA - TCA trichloroacetic acid - SLS sodium lauryl sulphate - PPO 2,5 Diphenyloxazole - POPOP 1,4-Bis-2-(4-methyl-5-penyloxazolyl)-benzene     

Increase in calmodulin level in the early phases of radish seed (Raphanus sativus) germination

Abstract Calmodulin (Cam), the heat-stable, ubiquitous, Ca²⁺-dependent regulator protein, has been purified to apparent homogeneity from germinating radish seeds (Raphanus sativus). The characteristics of radish Cam-molecular weight, absorption spectrum, Ca²⁺-dependent activation of brain phosphodiesterase (PDE)-are very similar to those described for Cam from other plant materials. Radish Cam, like other plant Cam, shows some differences to Cam of calf brain. The total amount of Cam in radish embryos at 24 h of germination is ca. 37 μg g⁻¹ fresh weight. Approximately 95% of the total amount of Cam is present in the soluble fraction (supernatant at 100,000 g). The level in the embryo axis strongly increases in the first 24 h of germination (+540%); this increase is strongly reduced when the germination is inhibited by abscisic acid (ABA). In the presence of Ca²⁺, no ‘free’ Cam (i.e. not bound to other structures) is present in the soluble fraction, suggesting that, during early germination, Cam level is a limiting factor for the activities of Ca²⁺ -Cam-dependent systems. These studies suggest that Cam plays an important role in the early phases of seed germination. An inhibitor of the Ca²⁺-Cam-dependent phosphodiesterase is present in the soluble fraction from radish embryos; this substance decreases during germination. A possible role of this inhibitor during the early germination phases is hypothesized.     

Rate of Synthesis of Non-Ribosomal RNA in Castrate Uterus after Oestradiol-17β Stimulation

OESTROGENS have been reported to stimulate preferentially the synthesis of ribosomal RNA in the castrate uterus^1–4. Thus it has been suggested that 50% or more of the RNA that is synthesized in the oestrogen-stimulated uterus is ribosomal precursor RNA^1,2,4. The concept is supported by the reports of enhanced ribosome formation during early oestrogen action^5,6. It has also been shown, however, that during the first 6 h after oestrogen administration there is no increase in total uterine RNA in the rat uterus^4,7 and also the castrate mouse uterus⁸. These findings seem to be incompatable with the idea that much of the RNA that is synthesized during this first 6 h is ribosomal precursor RNA, most of which accumulates as new stable rRNA. Determination of the absolute rates of total RNA synthesis in vivo should provide some insight into the amounts of various species of RNA that are synthesized after oestrogen administration. Data presented here for the rate of total RNA synthesis strongly suggest that all except a small portion of the RNA that is being synthesized at 4 h after oestrogen stimulation is unstable in vivo and hence is not ribosomal precursor RNA.     

Cell cycle analysis in asynchronous cultures using the BUdR-Hoechst technique.

During synchronized germination of spores of Dictyostelium discoideum, protein synthesis begins almost concomitantly with syntheses of messenger-like RNA (mlRNA) and 4–5S RNA (presumably tRNA) in the swollen spore stage and the initiation of ribosomal RNA (rRNA) synthesis is somewhat delayed. DNA synthesis occurs in the early stages of the amoeba emergence phase. Cycloheximide (200 μg/ml) blocked spore germination as well as total protein synthesis, whereas actinomycin D (60 μg/ml) did not affect either. This concentration of actinomycin D selectively inhibited formation of rRNA but did not influence the synthesis of mlRNA. Examinations of RNA labeled with [¹⁴C]uracil during germination indicated that polysomes initially detectable in the course of the germination process contain ¹⁴C-labeled mlRNA. It was concluded that at least some of mRNA synthesized during germination of D. discoideum spores is involved in protein synthesis required for the germination.     

Colonization of growing radish plants by clinical and nonclinical isolates ofKlebsiella inoculated onto seeds

Klebsiella was found to multiply and colonize growing radish root bulb surfaces following the inoculation of seeds with 10¹–10⁴ cells. All 29 cultures ofKlebsiella originally isolated from 5 different sources were capable of growth to 10⁶–10⁷ colony-forming units/g of root bulb within 1 week after seed germination. Linear regression analysis illustrated differences inKlebsiella survival rates over 4 weeks of radish plant development. Analysis of covariance showed the survival ability wasKlebsiella from vegetables>mastitis>human, water, and pulp mill isolates. It was also shown thatKlebsiella species 2 had a significantly higher survival rate than the otherKlebsiella species. This finding correlates well with the observation thatKlebsiella species 2 is the most commonKlebsiella species isolated from vegetables. Average densities for allKlebsiella groups at plant harvest (5 weeks) ranged from 10³–10⁵ colony-forming units/g of radish plant. The possible health significance of these densities ofKlebsiella on vegetables consumed raw by humans is discussed.     

The nucleolus organizers of Plethodon and Aneides located by in situ nucleic acid hybridization with Xenopus 3H-ribosomal RNA

The main clusters of ribosomal genes, or nucleolus organizers, have been located by in situ nucleic acid hybridization of Xenopus laevis ³H labelled ribosomal RNA to mitotic chromosomes in squash preparations of intestinal epithelium from 7 species of Plethodon and 3 species of Aneides. The species used were chosen on account of having well known karyotypes and genome sizes. The Plethodon species covered a range of genome size of 20–69.4 pg. The locations of those nucleolus organizers that could be detected by autoradiography after in situ hybridization varied from species to species, and in Aneides there were differences between two populations of the same species. On the other hand, some distantly related species of Plethodon, with widely different genome sizes, had nucleolus organizers at corresponding positions. The results are discussed in relation to ideas on karyotype stability, homosequentiality and chromosome repatterning.     

Calmodulin levels in radish (Raphanus sativus L.) seeds germinating at low calcium availability induced by EGTA treatments

Incubation of radish (Raphanus sativus L.) seeds in the presence of 1 or Smol m^?3 Ca-EGTA, which increased Ca²⁺ activity in the incubation medium (c. 0.24 or 0.37 mol m^?3 at 24 h with respect to c. 0.13 mol m^?3 in the control), did not affect germination, the restoration of K⁺ net influx, the increase in DNA and RNA levels or protein synthesis. Incubation in 1 mol m^?3 Na-EGTA, which reduced Ca²⁺ activity in the incubation medium (20 mmol m^?3 at 24 h), decreased the total Ca²⁺ level in embryo axes (-21%), but only slightly inhibited the increase in fresh weight without affecting the restoration of K⁺ net influx, the increase in DNA and RNA levels or protein synthesis. In the presence of 5 mol m^?3 Na-EGTA (Ca²⁺ activity in the incubation medium was 0.6 mmol m^?3), the decrease in the total Ca²⁺ level was greater (c. -27%) and the increases in fresh weight, DNA and RNA were inhibited by about 50, 39 and 40%, respectively. These results indicate that increased Ca²⁺ availability does not affect germination and suggest that the effect of Na-EGTA, at least up to 5 mol m^?3, is a result of an induction of Ca²⁺ deficiency. The amount and specific activity of calmodulin (CaM) present in the soluble fraction (100 000g) of radish embryo axes greatly increased during the first 24 h of incubation (c. 5-fold and 7-fold, respectively). This increase was very similar in the Ca-EGTA-treated seeds but was inhibited (c. -38%) by 1 mol m^?3 Na-EGTA, even if the increases in DNA and RNA levels and protein synthesis were not significantly reduced. The lower amount of CaM after 24 h of incubation in 1 mol m^?3 Na-EGTA (c. -30%) was due to a reduction in the fraction of CaM bound to a proteinaceous CaM inhibitor present in radish seeds [M. Cocucci & N. Negrini (1988) Plant Physiology 88, 910–914] and not involved in the metabolic reactivation of the seed. These results suggest that the level of CaM is controlled by Ca²⁺ availability and that the CaM inhibitor has a role in controlling the amount of Ca-CaM available for the Ca-CaM-dependent enzymes.     

Novel class of rDNA repeat units in somatic hybrids between Nicotiana and Atropa

Summary Behavior of ribosomal RNA genes in the process of somatic hybridization was analyzed using hybrids Nicotiana tabacum + Atropa belladonna. Blothybridization of parental species DNAs to ³²P-rDNA specific probes revealed two classes of ribosomal repeats in both tobacco and nightshade; their length was 11.2 kb, 10.4 kb (tobacco) and 9.4 kb, 10.2 kb (night-shade). For analysis of hybrids, labelled ³²P rDNA specific probes were hybridized to DNA of parental species and somatic hybrids digested with restriction endonucleases EcoR1, EcoRV and BamH1. A new class of ribosomal DNA repeat, absent in parental species, was found in hybrid line NtAb-1. Possible mechanisms of appearence of a new rDNA class in the process of somatic cell fusion are discussed.     

Involvement of Ca2+-calmodulin in Cd2+ toxicity during the early phases of radish (Raphanus sativus L.) seed germination

The toxicity of Cd²⁺in vivo during the early phases of radish (Raphanus sativus L.) seed germination and the in vitro Cd²⁺ effect on radish calmodulin (CaM) were studied. Cd²⁺ was taken up in the embryo axes of radish seeds; the increase in fresh weight of embryo axes after 24 h of incubation was inhibited significantly in the presence of 10 mmol m^?3 Cd²⁺ in the external medium, when the Cd²⁺ content in the embryo axes was c. 1.1 μmol g^?1 FW. The reabsorption of K⁺, which characterizes germination, was inhibited by Cd²⁺, suggesting that Cd²⁺ affected metabolic reactivation. The slight effect of Cd²⁺ on the transmembrane electric potential of the cortical cells of the embryo axes excluded a generalized toxicity of Cd²⁺ at the plasma membrane level. After 24 h of incubation, Cd²⁺ induced no increase in total acid-soluble thiols and Cd²⁺-binding peptides able to reduce Cd²⁺ toxicity. Ca²⁺ added to the incubation medium partially reversed the Cd²⁺-induced inhibition of the increase in fresh weight of embryo axes and concomitantly reduced Cd²⁺ uptake. Equilibrium dialysis experiments indicated that Cd²⁺ bound to CaM and competed with Ca²⁺ in this binding. Cd²⁺ inhibited the activation of Ca²⁺-CaM-dependent calf-brain phosphodiesterase, inhibiting the Ca²⁺-CaM active complex. Cd²⁺ reduced the binding of CaM to the Ca²⁺-CaM binding enzymes present in the soluble fraction of the embryo axes of radish seeds. The possibility that Cd²⁺ toxicity in radish seed germination is mediated by the action of Cd²⁺ on Ca²⁺-CaM is discussed in relation to the in vivo and in vitro effects of Cd²⁺.     

Effect of antibiotics on ribosomal RNA and proteins from Streptococcus pyogenes

Summary Cells of Streptococcus pyogenes were prepared under rigid conditions. The microorganisms were then incubated for 3 hours in the presence or absence of chloramphenicol, actinomycin or puromycin. RNA, ribosomal fraction and ribosomal proteins were isolated from the cells. The materials were invesigated with the help of infra red spectroscopy using the potassium bromide pellet method. Quantitative differences in the 1750–1500 cm^-1 region were observed with materials treated with the antibiotics. Synthetic mixtures of ribosomal RNA with progressively larger amounts of ribosomal proteins show analogous changes, namely a progressive increase in the strength of the 1650 cm^-1 band relative to the 1685 cm^-1 band, and an increase in the 1535 cm^-1 band. The analytical results obtained with the ribosomal RNA isolated from S. pyogenes treated with antibiotics indicated increased amounts of proteins which could not be removed by the applied extraction method. The evidence presented suggests a change in the binding between ribosomal RNA and ribosomal proteins in the material isolated from the antibiotic treated microorganisms. The I. R. spectroscopy seems to be an useful tool in the investigation of some aspects of biological materials.     

In vitro allelopathic properties of wild rocket (Diplotaxis tenuifolia DC) extract and of its potential allelochemical S-glucopyranosyl thiohydroximate

Abstract

An aqueous extract of wild rocket (Diplotaxis tenuifolia DC) was tested for its allelopathic activity in vitro on radish germination and seedling growth in light and darkness. The extract caused a delay in the onset and a significant decrease in the rate of germination (40%) in the light. The photo-inhibition was accompanied by an inhibition of water uptake into the seed, and a decrease of protein content as well as an increase of peroxidase activity into the seedlings. Microscopic observations suggest that the extract markedly changes radish radicle development inducing a decreased imbibition and distension of seed cells. Consistent results were obtained with some species such as purslane, lambsquarter and tree of heaven present in the cultivated wild rocket field and with cultivated lettuce and barley. Finally, a potential allelochemical, biologically active, was isolated from the extract: S-glucopyranosyl thiohydroximate at concentration of 6.3×10^?4 M.     

Minor Components of Rat Liver Ribosomal RNA as Possible Intermediates in the Degradation of 28S RNA in Vivo

The minor RNA components in large ribosomal subunits of rat liver were analyzed by gel electrophoresis. Quantitative analysis showed that these minor components, whose apparent molecular weights ranged from 8.48 × 10⁵ to 11.4 × 10⁵, represented 10 to 13% of the total high-molecular-weight ribosomal RNA.

It was elucidated that they were not artifacts arose during the cell homogenization, sub-cellular fractionation, RNA preparation and gel electrophoresis.

Labeling experiment in vivo showed that they were preferentially present in “old” ribosomes. It was predicted that these minor components were the intermediates in the degradation of 28S ribosomal RNA in vivo.

In the regenerating liver, where the degradation of proteins was reported to be blocked, the relative amount of the minor RNA components was decreased to about a half of that of normal liver. There was, however, no increase in their relative amount in the long-term starved rat liver, where RNA degradation should be going very rapidly.     

Seed germination ecology and salt stress response in eight Mediterranean populations of Sarcopoterium spinosum (L.) Spach

This study aims to deepen the analysis of seed germination ecology and salinity tolerance of Sarcopoterium spinosum (Rosaceae). Germination tests were conducted to evaluate the effect of the fruit’s spongy tissue and the intraspecific variability in seed germination among eight populations of the species on responses to light and total darkness, constant and alternating temperatures, salt stress and germination recovery. The effect of the presence of the spongy tissue varied among populations, with significant results for seed germination. For all populations, optimum germination temperatures were observed in the range of 10–20°C, indicating that S. spinosum and its germination in the field might occur preferably in the period between autumn and early spring. The high water availability due to rainfall during this period could be a considerable advantage for the seed germination of this species. Seeds of S. spinosum showed the ability to germinate in up to 250 mM NaCl in the substrate, and their ability to recover after salt exposure may be interpreted as adaptation to the coastal habitats in which they generally grow. These results give this species a halo-tolerant character. Great inter-population variability is detected in this study in several aspects, which indicated that the Mediterranean populations of S. spinosum differ considerably and are adapted to their local conditions. This study provides new information about S. spinosum seed ecology, which could help to preserve and apply effective conservation measures for this species, which in several areas of its distribution range is endangered.     

Incorporation of mevalonic Acid into ribosylzeatin in tobacco callus ribonucleic Acid preparations

The incorporation of ¹⁴C-2-mevalonic acid into transfer RNA and ribosomal RNA (high molecular weight RNA) in rapidly growing, cytokinin-dependent tobacco (Nicotiana tabacum var. Wisconsin No. 38) callus cultures has been investigated. Approximately 40% of the label incorporated into transfer RNA was present in a ribonucleoside with chromatographic properties identical to those of cis-ribosylzeatin. The remainder of the label in the transfer RNA appears to be nonspecific incorporation resulting from degradation and metabolism of ¹⁴C-2-mevalonic acid by the tobacco callus tissue. Although the total radioactivity incorporated into ribosomal RNA was roughly the same as in transfer RNA, the specific radioactivity of the transfer RNA was about four times higher than that of the ribosomal RNA, and the ribosomal RNA labeling could be distinguished from the cytokinin labeling observed in transfer RNA. The distributions of the ¹⁴C-2-mevalonic acid label and cytokinin activity in tobacco callus transfer RNA fractionated by benzoylated diethylaminoethylcellulose chromatography indicate that at least two cytokinin-containing transfer RNA species are present in this tissue.     

Protein synthesis of binucleate and trinucleate pollen and its relationship to tube emergence and growth

Under humid conditions, both bi- and trinucleate pollen species incorporate, on the average, very low amounts of leucine, e.g., 0.4 pmol min^-1mg pollen^-1. During germination in vitro, however, the two types of pollens greatly differ in their capacity for protein synthesis.Binucleate pollen species such as Typha, which are characterized by slow respiration in humid air and prolonged lag periods during germination in vitro, contain large amounts of monoribosomes at dehiscence. Polyribosomes are formed soon after the pollen is wetted in the germination medium, and a considerable incorporation of leucine is initiated after 10–15 min. More rapidly respiring, binucleate pollen showing a short lag period, such as Tradescantia, may already contain many polysomes at dehiscence and incorporate leucine within 2 min of germination. However, rapidly respiring, trinucleate Compositae pollen contains very limited amounts of ribosomal material and never attains any substantial level of incorporation. Cycloheximide completely inhibits both protein synthesis and tube emergence and growth in the slowly respiring, binucleate pollen species. The more rapidly respiring types are less dependent on protein synthesis, while germination of the phylogenetically advanced, trinucleate Compositae pollen proceeds completely independently. It is concluded that the level of phylogenetic advancement of the male gametophyte is characterized by its overall state of metabolic development at dehiscence rather than by the number of its generative cells.Abbreviations BSA bovine serum albumin - CHI cycloheximide - EGTA ethyleneglycol-bis(-aminoethyl ether) N,N-tetraacetic acid - RH relative humidity - TCA trichloroacetic acid     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.