RNA synthesis in germinating embryonic axes of soybean and wheat

The rate of synthesis of RNA during early germination of wheat and soybean embryos was investigated by ascertaining the incorporation of radioactive uridine into RNA. In wheat embryos, where the lag period preceding rapid growth is 5.5 hours, there is a 2-fold increase in RNA synthesis between 1.5 and 5.5 hours, with half of the increase occurring by 3.5 hours. In soybean axes, where the lag period is 9.5 hours, the increased rate of RNA synthesis is 5.5-fold between 1.5 and 9.5 hours, with three fourths of this increase occurring after 4 hours.     

A novel effect of 6-azauridine on growth and protein synthesis in wheat embryonic axes

Growth and the rate of protein synthesis in germinating wheat embryonic axes are inhibited by the analog 6-azauridine via a mechanism which is independent of the usual effect of this compound as an inhibitor of de novo synthesis of UTP. The effects on growth and protein synthesis can be separated from that on UTP biosynthesis by analyses of the kinetics by which each effect is maximized following a 1.5-h pulse with 6-azauridine, and by saturation of the responses at different doses of the analog. The inhibitions of growth and protein synthesis are apparently not mediated through the rate of poly A(+) RNA synthesis (reduced as little as 8%), but rather by an effect on translation. Since cordycepin reduces the azauridine inhibitions of growth and protein synthesis, it is suggested that these latter effects of 6-azauridine may depend upon the synthesis of an inhibitory azauridyl-RNA.     

Early growth of wheat embryonic axes and the synthesis of RNA and DNA

The requirement for the synthesis of RNA and DNA in early germination of wheat (Triticum aestivum var Newana) embryonic axes has been studied by incubating embryos in the presence of appropriate inhibitors and monitoring both embryo growth and the rates of specific metabolic processes. Experiments with 5-fluorouridine showed that both rRNA and DNA synthesis could be curtailed by 60 to 70% without affecting embryo growth to 24 hours. Similarly, the presence of mitomycin C and methotrexate inhibited DNA synthesis 70%, with only a small effect on growth. Experiments with a range of concentrations of cordycepin and α-amanitin indicated that mRNA synthesis could be curtailed by 30 to 40% within the first 8 hours of germination with only a small effect on embryo growth. Thus, at least the initial phases of seed embryo germination are not closely linked to the synthesis of mRNA, rRNA, or DNA. Maximal sensitivity of embryo growth was obtained with cycloheximide and 2-(4-methyl-2,6-dinitroanilino)-N-methyl propionamide, supporting the idea that protein synthesis is the macromolecular process most closely linked to early germination.     

RNA synthesis in embryo axes of germinating pea seeds

RNA synthesis during germination was investigated by labelingpea embryo axes or seedling roots with radioactive uridine oradenosine. The results indicated that all RNA species of pre-rRNAs(ribosomal precursor RNAs), rRNAs, heterodisperse-type RNA and4–5S low molecular weight RNA were synthesized from the6th to 64th hour of the period examined. At the very early stageof germination, some conspicuous labeling of the heterodisperse-typeRNA was observed after pulse-labeling. There was no great differencein the labeling patterns of various RNA species with regardto other later stages. When embryo axes were labeled for 1 hrwith ³H-adenosine from the 16th hour, about 25% of the labeledwhole cell RNA was retained on the membrane filter. The ratioof labeled poly(A)-containing RNA, however, decreased as germinationproceeded. The poly (A)-containing RNA sedimented heterodisperselywith a mean value of about 20S in a sucrose density gradient;this size-distribution did not vary throughout germination. (Received January 16, 1979; )     

Ethanolamine incorporation into membranes of pea embryonic axes during germination

Changes in the ethanolamine pool of the embryonic axes of pea seeds exposed to different temperatures during imbibition and germination were followed. The ethanolamine pool decreased except during imbibition at 25°. Label from ethanolamine was incorporated almost entirely into phosphatidylethanolamine with incorporation into phosphatidylcholine being observed only after imbibition and germination at 25°. The incorporation of ethanolamine was apparently less sensitive to temperature than that of choline and glycerol, previously reported. Preliminary results also show an effect of the imbibition temperature on some of the membrane proteins, but most did not seem to be affected.     

In vivo 19F-NMR of 5-fluorouracil incorporation into RNA and metabolites in Escherichia coli cells

19F resonances from RNA with 5-fluorouracil incorporated could be observed in intact Escherichia coli cells, as well as in tRNA isolated from the cells. 19F-NMR signals from the metabolic breakdown products of the fluorinated RNA were also detected in vivo. By observing the 19F-NMR spectrum, variations in the metabolic disposition of administered 5-fluorouracil could be monitored as a function of time and be compared when the cells were deprived of oxygen and other nutrients, subjected to ethidium bromide treatment, or grown in the presence of mitomycin C.     

Purine nucleotide metabolism of germinating soybean embryonic axes

Isolated soybean (Glycine max L. cv. Kent) embyronic axes metabolized [¹⁴C]glycine to ATP within the 1 hour of imbibition. Radioactivity was not detected in GTP until the 3rd hour. Throughout most of the first 24 hours of germination about 10 to 26 times as much label from [¹⁴C]glycine appears in ATP as GTP. About five times as much [¹⁴C]hypoxanthine and [¹⁴C]inosine was converted into GTP as into ATP in embryonic axes. Two independent pools of IMP appear to be used in purine nucleotide synthesis of soybean axes.     

Benzyladenine reversal of abscisic Acid inhibition of growth and RNA synthesis in germinating bean axes

The effect of benzyladenine on growth, ATP pool size and specific radioactivity, and the rate and amount of RNA synthesis in aseptically cultured axes of Phaseolus vulgaris during the first 24 hours of germination were measured in experiments where the duration of benzyladenine application and its concentration were varied. Maximum promotion of growth (25%) occurs at 10(-5)m benzyladenine. Maximum promotion of RNA synthesis (44%) occurs at 10(-5)m benzyladenine. Benzyladenine has little effect on the size or specific radioactivity of the ATP pool. Benzyladenine can completely counteract abscisic acid inhibition of growth and RNA synthesis, and these reversals are measurable in 2 hours. GA(1) and GA(3) do not promote growth or counteract abscisic acid inhibition of growth in germinating bean axes in these experiments.     

In vitro mutagenesis by incorporation of N4-aminodeoxycytidine 5'-triphosphate

We have investigated the possibility of introducing a new way to carry out in vitro mutagenesis. N4-Aminodeoxycytidine 5'-triphosphate was used in the Klenow enzyme-catalyzed chain elongation of a primer oligonucleotide hybridized to a lacZ alpha region of M13mp2 viral single strand DNA, and the possibility of inducing an efficient, randomly distributed point mutations into this particular genomic region was explored. On transfection of the resulting DNA into E. coli, mutant phages emerged at frequencies up to 1%. Analysis of the DNA sequences of the mutants has shown that single transitions, either A to G or G to A, were induced in a random fashion, thus providing data to show the possibility of using this method for production of mutant proteins having various single amino-acid changes in a defined domain.     

Direct incorporation of guanosine 5'-diphosphate into microtubules without guanosine 5'-triphosphate hydrolysis

Using highly purified calf brain tubulin bearing [8-14C]guanosine 5'-diphosphate (GDP) in the exchangeable nucleotide site and heat-treated microtubule-associated proteins (both components containing negligible amounts of nucleoside diphosphate kinase and nonspecific phosphatase activities), we have found that a significant proportion of exchangeable-site GDP in microtubules can be incorporated directly during guanosine 5'-triphosphate (GTP) dependent polymerization of tubulin, without an initial exchange of GDP for GTP and subsequent GTP hydrolysis during assembly. The precise amount of GDP incorporated directly into microtubules is highly dependent on specific reaction conditions, being favored by high tubulin concentrations, low GTP and Mg2+ concentrations, and exogenous GDP in the reaction mixture. Minimum effects were observed with changes in reaction pH or temperature, changes in concentration of microtubule-associated proteins, alteration of the sulfonate buffer, or the presence of a calcium chelator in the reaction mixture. Under conditions most favorable for direct GDP incorporation, about one-third of the GDP in microtubules is incorporated directly (without GTP hydrolysis) and two-thirds is incorporated hydrolytically (as a consequence of GTP hydrolysis). Direct incorporation of GDP occurs in a constant proportion throughout elongation, and the amount of direct incorporation probably reflects the rapid equilibration of GDP and GTP at the exchangeable site that occurs before the onset of assembly.     

Development of RNase activity in embryonic axes of germinating pea seeds

RNase activity in embryonic pea axes increased in parallel withthe rise of RNA synthesis as germination proceeded. The developmentof this enzymatic activity was modified antagonistically byapplication of GA₃ and ABA and inhibited severely by treatmentwith CH. Sedimentation analysis of ³H-adenosine-labeled RNAindicated that the synthesis of all types of RNA species isuniformly stimulated by GA₃ and inhibited by ABA. However, 5-FUtreatments, which severely inhibited the synthesis of rRNA,with a slight effect on that of mRNA, had no appreciable effecton the development of RNase activity in the axes. These resultsindicate that active RNA synthesis during germination is independentof the development of RNase activity and that the de novo synthesisof RNases may be controlled by the synthesis of their specificmRNAs. Among the three types of RNase (RNase I, II and III) detectedin the embryonic axes, RNase III showed a sharp increase inactivity with embryo growth and the activity of this enzymewas mainly associated with the endoplasmic reticulum. (Received June 5, 1978; )     

Differential response of antioxidative enzymes in embryonic axes and cotyledons of germinating lupine seeds

Germination of lupine (Lupinus luteus L.) seeds was accompanied by an increase in concentration of free radicals with g ₁ and g ₂ values of 2.0056 ± 0.0003 and 2.0033 ± 0.0005, respectively. The highest intensity of free radical signal was observed in embryo axes immediately after radicle protruded through the seed coat. Hydrogen peroxide accumulated in embryonic axes and cotyledons during imbibition before the onset of germination in the seed population. The activities of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6) rose progressively in embryo axes. In cotyledons SOD activity did not change significantly, while that of CAT increased during germination. The enhancement of Cu, Zn-SODs and Mn-SOD isoforms in embryonic axes was observed. A new isoform of catalase was synthesized, suggesting that it plays a relevant role during germination. SOD and CAT activities were detected in dry seeds. Free radical generation and response of antioxidative enzymes differed between embryo axes and cotyledons during the germination timecourse.     

Specific incorporation of 5-fluorocytidine into Escherichia coli RNA

RNAs isolated from Escherichia coli B grown in the presence of 5-fluorouracil have high levels of the analog replacing uridine and uridine-derived modified nucleosides. Cytidine has also been shown to be replaced in these RNAs by 5-fluorocytidine, a metabolic product of 5-fluorouracil, but to a considerably lesser extent. When 5-fluorocytidine is added to cultured of E. coli B little 5-fluorocytidine (0.20 mol%) is incorporated into cellular RNAs because of the active cytosine/cytidine deaminase activities. Addition of the cytidine deaminase inhibitor tetrahydrouridine (70 micrograms/ml) increases 5-fluorocytidine incorporation to about 3 mol% in tRNAs, but does not eliminate 5-fluorouridine incorporation. E. coli mutants lacking cytosine/cytidine deaminase activities are able to more than double the extent of 5-fluorocytidine incorporation into their transfer and ribosomal RNAs, replacing cytidine with no detectable 5-fluorouridine incorporation. Levels of 5-methyluridine, pseudouridine and dihydrouridine in tRNAs are not affected. These fluorocytidine-containing tRNAs show amino acid-accepting activities similar to control tRNAs. Fluorocytidine was found to be quite susceptible to deamination under alkaline conditions. Its conversion to primarily 5-fluorouridine follows pseudo-first-order reaction kinetics with a half-life of 10 h in 0.3 M KOH at 37 degrees C. This instability in alkali probably explains why 5-fluorocytidine was not found earlier in RNAs isolated from cells treated with 5-fluorouridine, since most early RNA hydrolyses were carried out in alkali. It may also explain the mild mutagenic properties observed in some systems following 5-fluorouridine treatment. Initial 19F-NMR measurements in fluorocytidine-containing tRNAs indicate that this modified tRNA may be useful in future structural studies of tRNAs and in probing tRNA-protein complexes.     

In vivo incorporation of cytidine-monophosphate-ciliatine into rat liver lipids.

1. In vivo this investigation was carried out in order to compare the incorporation into rat lipids of free [1,2-minus 14C]-ciliatine and CMP-[1,2-minus 14C]-ciliatine which is the precursor in phosphonolipid biosynthesis. 2. The incorporation of the radioactivity from CMP-[1,2-minus 14C]-ciliatine took place more rapidly than that from free [1,2-minus 14C]-ciliatine in both liver and kidney. The amount of radioactivity from the CMP-[1,2-minus 14C]-ciliatine incorporated into total liver lipids was about 5 times higher than that incorporated into total liver lipids of rat two hrs after injecting free-[1,2-minus 14C]-ciliatine. 3. The amount of [1,2-minus 14C]-ciliatine incorporated into total liver lipids was 15 and 21 times higher than that incorporated into total kidney lipids of rat two and four hrs after injecting free [1,2-minus 14C]-ciliatine. 4. If the main pathway for the phosphonolipid biosynthesis is via CMP-ciliatine, the rate of phosphonolipid formation from CMP-ciliatine must therefore be higher than that from free-ciliatine. The results obtained here indicate therefore that the main pathway for phosphonolipid biosynthesis is a pathway involving CMP-ciliatine. 5. An unknow compound was detected in the water soluble fraction of the acid hydrolyzate of liver phosphonolipids. This material migrated with the N-trimethyl-derivative of ciliatine on the thin-layer chromatogram. The result shows that there is therefore a possibility of methylation of exogenous ciliatine to the phosphonate analogue of choline in the mammalian body.