Inhibition by adenine compounds of the induced formation of photosynthetic pigments in Rhodopseudomonas spheroides cells under dark-semiaerobic conditions

Effects of adenine, adenosine, AMP, ADP and ATP on the inducedformation of bacteriochlorophyll and carotenoids in cell suspensionsof dark-aerobically grown Rhodopseudomonas spheroides were examinedunder dark-semiaerobic conditions where no significant cellgrowth occurred. Pigment formation was strongly inhibited by3 mM adenine, adenosine, AMP or ATP, but less strongly by ADP.Inhibition by either adenosine or ATP was completely reversible.Addition of 3 mM adenosine resulted in complete inhibition ofpigment formation, while inhibition by more than 10 min ATPdid not exceed 80%. No accumulation of any precursor-like pigmentsof either bacteriochlorophyll or carotenoids was observed incells incubated in the presence of adenine compounds. Amountsof exogenously-added adenine, adenosine, or AMP decreased significantlyduring incubation, whereas the amount of exogeneously-addedATP or ADP did not appreciably decrease. Addition of 3 mM ATPor adenosine also significantly suppressed ³H-leucine incorporationinto bacterial proteins. Nucleosides other than adenosine wereineffective in inhibiting the induced formation of photosyntheticpigments, indicating that the inhibitory action is specificto adenine compounds. It was assumed that both adenosine andATP inhibit chromatophore formation rather than a particularstep(s) in the biosynthetic pathways of the photosynthetic pigment,and that ATP exerts its effect from outside the cells, whereasadenosine does so after being taken up by the cells. (Received July 24, 1972; )     

Induced formation of ribulose 1,5-diphosphate carboxylase in Rhodopseudomonas spheroides with particular concern to its relation with chromatophore formation

A comparative study was made on features of the induced synthesisof RuDP carboxylase in three strains of R. spheroides with differentbiochemical properties. In strains Sb and Sa, which were able to grow under either light-anaerobicor dark-aerobic conditions, activities of RuDP carboxylase inthe light-grown cells were much higher than those in dark-growncells. The level of RuDP carboxylase activity in dark-growncells of the Sb strain (wild type strain) increased two to threetimes in the dark by incubating the heavy cell suspension underlow aeration, but, for a further increase in enzyme activity,a light-anaerobic condition was required. This is in contrastto the induced formation of bacteriochlorophyll which has beenshown to proceed actively in the dark as well as in the light.On the other hand, with dark-grown cells of the Sa strain, whichhad possible partial defects in the chlorophyll synthesis system,the induced synthesis of RuDP carboxylase under the light-anaerobiccondition was markedly retarded as compared to that with theSb strain. RuDP carboxylase formation was not induced in L-57(a colorless mutant) under any of these conditions. The induced formation of RuDP carboxylase, as well as of bacteriochlorophyll,under the light-anaerobic condition was considerably suppressedby hydroxyurea and mitomycin C. This suggests that the geneticcontrol systems of RuDP carboxylase synthesis may be closelyrelated with those for the formation of the photosynthetic apparatus. ¹This work was supported in part by Public Health Research GrantAM 08016 from the National Institute of Arthritis and MetabolicDiseases, U.S.A. (G. K.). ²Present address: Laboratory of Radioisotope Experiment, TohokuUniversity School of Medicine, Sendai, Japan. (Received September 6, 1968; )     

Inhibition of Cell Division and DNA Synthesis by Gibberellin in Isolated Zinnia Mesophyll Cells

A culture system of isolated mesophyll cells of Zinnia eleganswas used to examine the action of gibberellic acid (GA) on celldivision. Isolated Zinnia mesophyll cells cultured in a mediumcontaining auxin and cytokinin reinitiated cell division ina partly synchronized manner. When mesophyll cells isolatedfrom 21-day-old seedlings were used, GA added to the culturemedium at concentrations of 1 x 10^–6 M or higher suppressedthe initial rise in the number of divided cells. Tracer experimentswith [³H]-dThd revealed that GA treatment inhibited the incorporationof [³H]-dThd into DNA in the nucleus without inhibiting theuptake of [³H]-dThd into the cells, indicating that GA inhibitedDNA synthesis. GA applied at 48 h inhibited the incorporationof [³H]-dThd into DNA during the following 24 h, but GA appliedat 72 h did not inhibit the incorporation during the subsequent24 h. This suggests that GA affects the process of reinitiationof DNA synthesis, but does not affect DNA synthesis once cellshave become proliferative. (Received January 14, 1986; Accepted March 31, 1986)     

Heterogeneous profile in thermal denaturation of DNA preparation from the non-sulfur purple photosynthetic bacterium Rhodopseudomonas spheroides

Thermal denaturation profiles of DNA preparations from fourstrains of Rhodopseudomonas spheroides were comparatively studied.All the melting curves in 0.1 ? SSC displayed Tm at 82 ?0.5?and a faint bimodal transition near 78?. The differential meltingrates as a function of temperature exhibited several peaks,suggesting that inter- or intra-molecular structural heterogeneitymay be present in R. spheroides DNA. ¹ Present address: Biological Institute, Faculty of Science,Tohoku University, Sendai, Japan. (Received February 15, 1975; )     

Influence of ethylene on nucleic acid synthesis in etiolated Pisum sativum

Ethylene applied to intact etiolated seedlings of Pisum sativumcv. Alaska inhibits incorporation of ³H-thymidine into DNA insubsequently excised plumular and subapical tissue segmentsbut has no influence on incorporation of ³H-uridine into RNA.The effect on DNA synthesis begins about 2 hr after ethyleneis applied, and intensifies progressively. A similar inhibitionof DNA synthesis occurs when ethylene is applied directly toplumular sections cut from control plants, but not with subapicalsegments under these conditions. Inhibition of DNA synthesisby ethylene is reversed by benzyl adenine in plumular sections.Brief exposure of dark grown seedlings to red light causes asubsequent increase in DNA synthesis in plumular tissue. Thechanges in DNA synthesis in tissues exposed to ethylene, benzyladenine and red light are correlated with the effects of thesetreatments on the mitotic index. (Received March 12, 1973; )     

Heterogeneity of the DNA preparation from light-anaerobically grown Rhodopseudomonas spheroides cells as revealed by methylated albumin kieselguhr column chromatography

Biochemical properties of DNA preparations obtained from light-anaerobicallygrowth R. spheroides cells and from dark-aerobically grown cellswere examined comparatively using methylated albumin kieselguhrcolumn chromatography. The DNA preparation from light-growncells, which formed chromatophores, exhibited considerable heterogeneityin its size or conformation. ¹ Present address: The Third Department of Medicine, TohokuUniversity School of Medicine, Sendai, Japan. (Received August 2, 1974; )     

The Effect of Cycloheximide (Actidione) on Protein and Nucleic Acid Synthesis by Chlorella

Incorporation of ¹⁴C-phenylalanine, ¹⁴C-carbon dioxide, ¹⁴C-glucose,and ¹⁴C-glycine into the protein of Chlorella is inhibited bycycloheximide. A concentration of 2.5 µg per ml inhibitsincorporation by about 80 per cent; increasing the concentrationup to 10 µg per ml does not increase the degree of inhibition.The incorporation of ¹⁴C-adenine into ribonucleic acid (RNA)and deoxyribonucleic acid (DNA), and of ¹⁴C-glucose into polysaccharideis also inhibited. Unlike inhibition of protein synthesis, thatof nucleic acid and polysaccharide synthesis is observed onlyafter some delay. The delay is shortest for DNA synthesis andlongest for polysaccharide synthesis. Inhibition of ¹⁴C-glycineincorporation into DNA and RNA follows a similar pattern tothat obtained with ¹⁴C-adenine but the delay is much shorter.Cycloheximide also inhibits the formation of isocitrate lyasc(isocitrate-glyoxylate lyase, EC 4.1.3.1 [EC] ) when autotrophicallygrown cells are supplied with acetate.     

Aerobiosis and the hemoprotein content of photosynthetic bacteria

Summary Rhodospirillum rubrum and Rhodopseudomonas spheroides, grown under various degrees of illumination, aeration, and iron deprivation, have been assayed for their content of cytochrome c, RHP, catalase, total iron, bacteriochlorophyll, and carotenoids.Concentrations of bacteriochlorophyll and carotenoids were consistent with the findings of Cohen-Bazire et al. (1957).Total iron content, which ranged from 0.017 to 0.04% of the dry weight, reflected the content of the principal hemoproteins but exceeded the amount of iron in these hemoproteins.The catalase content of R. rubrum, on a dry weight basis, was 0.0005% for cells grown anaerobically in the light, and 0.0028% for cells grown in darkness with vigorous aeration; that of Rps. spheroides was 0.006% and 0.25%, respectively. The catalase content in both species rose with increasingly vigorous aeration.Cytochrome c in both species, and RHP in R. rubrum, attained the same levels in cells grown under vigorous aeration as in cells grown anaerobically in the light. In cells grown under limited aeration the levels of these substances were about 50% higher. In Rps. spheroides the RHP content was greatest in cells grown anaerobically, falling under gentle aeration and declining further under more vigorous aeration.Iron deficiency caused a decrease in the catalase content of cells grown anaerobically in the light but not in cells grown aerobically. The content of cytochrome c and of RHP was diminished by iron depletion in aerobic cultures, but not in anaerobic cultures.operated by Union Carbide Corporation for the U.S.Atomic Rnergy Commission.     

The Inhibitory Effect of (2-Chloroethyl)-trimethylammonium Chloride on Chlorophyll and Protein Synthesis in Lettuce Cotyledons, and its Reversal by Potassium

Germinated seeds of Lactuca sativa (L.) were placed in Petri-dishesin (2-chloroethyl)-trimethyl-ammonium chloride (CCC; 0.005–0.05M), KN0₃ (0.01 M), and KC1 (0.01 M) solutions, and incubatedfor 2 or 5 days under continuous light. CCC strikingly arrestedchlorophyll accumulation, and retarded cotyledon growth relativelylittle. The retardant inhibited ¹⁴C-leucine incorporation intobulk proteins of the cotyledons. KN0₃ and KC1 promoted cotyledongrowth and chlorophyll synthesis per cotyledon by about 150per cent, and about doubled protein synthesis. Potassium saltscompletely reversed the inhibitory effects of CCC on chlorophylland protein synthesis. It is suggested that the inhibition ofgreening by CCC is dependent on a prior inhibition of proteinsynthesis.     

Changes in the Synthesis of RNA and Protein during Tracheary Element Differentiation in Single Cells Isolated from the Mesophyll of Zinnia elegans

Cycloheximide (CH) prevented tracheary element (TE) differentiationand cell division in a culture of single cells isolated fromthe mesophyll of Zinnia elegans at the concentrations whichinhibited incorporation of [¹⁴C]-leucine into protein. Whenthe cells were pulse-treated with this inhibitor for 12 h atvarious times of culture, TE formation was inhibited most stronglyby the treatments made between 24 and 60 h of culture. Incorporationof [¹⁴C]-leucine into protein showed a high level during thisperiod. The inhibitory effect of actinomycin D (Act-D) on TEdifferentiation was also marked when it was administered from24 to 60 h of culture when incorporation of [¹⁴C]-uridine intonucleic acid was at a high level. These results indicate thatRNA and protein syntheses are prerequisites for cytodifferentiationto TE and that the syntheses between 24 and 60 h of cultureare closely associated with cytodifferentiation. Studies of qualitative changes in proteins using two-dimensionalelectrophoresis revealed that approximately 400 polypeptidesextracted from [³⁵S]-methionine-labeled cells could be reproduciblyresolved and that most of them were synthesized in both differentiatingand non-differentiating cells. During TE differentiation, however,the synthesis of two polypeptides was shut off and two otherpolypeptides were newly synthesized between 48 and 60 h of culture,preceding the morphological changes. The relationship betweenTE differentiation and the synthesis of RNA and protein is discussed. (Received November 20, 1982; Accepted February 18, 1983)     

Events surrounding the early development of Euglena chloroplasts 13. Photocontrol of protein synthesis

Protein synthesis measured as leucine incorporation was followedduring the early hours of light exposure of dark-grown cellsof wild type cells of Euglena gratilis var. bacillaris and ofbleached mutants W₃BUL and W₁₀SmL which lack detectable plastidDNA. In all strains, linear rates of leucine incorporation wereobserved in dark-grown resting cells and on exposure to light,this rate increased. After about 3 hr light exposure in wildtype cells and somewhat later in the mutants, the rate of proteinsynthesis sharply declined below that of the dark-grown anddark-incubated cells. Experiments in wild type cells showedthat leucine uptake was not rate limiting for protein synthesisalthough light exposure decreased the rate of uptake. The changesin rate found during continuous labeling of wild type cellswere verified by pulse-labeling experiments in continuous light.Exposure of dark-grown wild type cells to a two hour pulse oflight produced a transient increase in the rate of leucine incorporationwhich subsequently returned in darkness to the level of thedark-grown cells which received no light; thus the changes inrate of leucine incorporation are light-dependent. Since theeffects of light on leucine incorporation can be reproducedin mutants lacking detectable plastid DNA, the photoreceptormachinery involved cannot be coded in plastid DNA, and probablyoriginates in nuclear DNA. The role of light in programmingprotein synthesis and turnover in early chloroplast developmentis discussed. ¹Supported by Grant Number GM-14595 from the National Institutesof Health. ²Microbiology trainee of the National Institutes of Health,Grant Number GM1586. Portions of the material in this paperwere taken from a dissertation submitted by S. D. S. to theGraduate Faculty of Brandeis University in partial fulfillmentof the requirements for the Ph.D. degree. Present address: Schoolof Life Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska68588, U. S. A. ³Abraham and Etta Goodman Professor of Biology and Director,Institute for Photobiology of Cells and Organdies, BrandeisUniversity, Waltham, MA, U. S. A. 02154, to whom reprint requestsshould be sent. (Received February 8, 1979; )     

ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells

In human osteoblast-like MG-63cells, extracellular ATP increased [³H]thymidineincorporation and cell proliferation and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-induced[³H]thymidine incorporation. ATP-induced[³H]thymidine incorporation was mimicked by thenonhydrolyzable ATP analogs adenosine5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2purinoceptor antagonist suramin, suggesting involvement of P2purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptorantagonist reactive blue 2 did not affect [³H]thymidineincorporation, whereas the P2X receptor antagonist pyridoxalphosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced[³H]thymidine incorporation, suggesting that ATP-inducedDNA synthesis was mediated by P2X receptors. RT-PCR analysis revealedthat MG-63 cells expressed P2X₄, P2X₅,P2X₆, and P2X₇, but not P2X₁,P2X₂, and P2X₃, receptors. In fura 2-loadedcells, not only ATP, but also UTP, increased intracellularCa²⁺ concentration, and inhibitors for severalCa²⁺-activated protein kinases had no effect on ATP-inducedDNA synthesis, suggesting that an increase in intracellularCa²⁺ concentration is not indispensable for ATP-induced DNAsynthesis. ATP increased mitogen-activated protein kinase activity in aCa²⁺-independent manner and synergistically enhancedplatelet-derived growth factor- or insulin-like growth factor I-inducedkinase activity. Furthermore, the mitogen-activated protein kinasekinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors byactivating a mitogen-activated protein kinase pathway.

     

Estradiol-17beta stimulates proliferation of mouse embryonic stem cells: involvement of MAPKs and CDKs as well as protooncogenes

Although the importance of estradiol-17 (E₂) in many physiological processes has been reported, to date no researchers have investigated the effects of E₂ on embryonic stem (ES) cell proliferation. Therefore, in the present study, we have examined the effect of E₂ on the DNA synthesis of murine ES (ES-E14TG2a) cells and its related signaling pathways. The results of this study show that E₂ (10^–9 M) significantly increased [³H]thymidine incorporation at >4 h and that E₂ (>10^–12 M) induced an increase of [³H]thymidine incorporation after 8-h incubation. Moreover, E₂ (>10^–12 M) also increased 5'-bromo-2'-deoxyuridine (BrdU) incorporation and cell number. Indeed, E₂ stimulated estrogen receptor (ER)- and - protein levels and increased mRNA expression levels of protooncogenes (c-fos, c-jun, and c-myc). Tamoxifen (antiestrogen) completely inhibited E₂-induced increases in [³H]thymidine incorporation. In addition, estradiol-6-O-carboxymethyl oxime-BSA (E₂-BSA; 10^–9 M) increased [³H]thymidine incorporation at >1 h, and E₂-BSA (>10^–12 M) increased [³H]thymidine incorporation after 1-h incubation. E₂-BSA-induced increase in BrdU incorporation also occurred in a dose-dependent manner. Tamoxifen had no effect on E₂-BSA-induced increase of [³H]thymidine incorporation. Also, E₂ and E₂-BSA displayed maximal phosphorylation of p44/42 MAPKs at 10 and 5 min, respectively. E₂ increased cyclins D1 and E as well as cyclin-dependent kinase (CDK)2 and CDK4. In contrast, E₂ decreased the levels of p21^cip1 and p27^kip1 (CDK-inhibitory proteins). Increases of these cell cycle regulators were blocked by 10^–5 M PD-98059 (MEK inhibitor). Moreover, E₂-induced increase of [³H]thymidine incorporation was inhibited by PD-98059 or butyrolactone I (CDK2 inhibitor). In conclusion, estradiol-17 stimulates the proliferation of murine ES cells, and this action is mediated by MAPKs, CDKs, or protooncogenes. cyclin-dependent kinase; mitogen-activated protein kinase     

EFFECTS OF LIGHT ON THE DEOXYRIBONUCLEIC ACID FORMATION AND CELLULAR DIVISION IN CHLORELLA PROTOTHECOIDES

1. It has been demonstrated previously that when Chlorella protothecoidesis grown in a medium rich in glucose and poor in nitrogen source(urea), chlorophyll-less cells with markedly degenerated plastids—called "glucose-bleached" cells—are produced eitherin the light or in darkness. When the glucose-bleached cellsare incubated in a medium enriched with the nitrogen sourcebut without added glucose, normal green cells with fully organizedchloroplasts are obtained in the light, and pale green cellswith partially organized chloroplasts in darkness. During theseprocesses of chloroplast development in the glucose-bleachedcells, there occurs, after a certain lag period, an active DNAformation followed by a more or less synchronous cellular division.In the present study the effects of light on the DNA formationand cellular division were investigated in the presence of CMUor under aeration of CO²-free air to exclude the interveninginfluence of photosynthetic process.
2. It was revealed thatlight severely suppresses the DNA formationand cellular divisionof the glucose-bleached cells while enhancingremarkably theirgreening. The suppression was saturated atthe light intensityof about 1,000 lux. Blue light was mosteffective, being followedby green, yellow and red light inthe order of decreasing effectiveness.
3. Further experiments unveiled that light exerts two apparentlyopposing effects on the DNA formation depending upon the timeof application during the incubation of algal cells. When thealgal cells were illuminated only during the lag period beforethe active DNA synthesis, there occurred an enhancement of theDNA synthesis occurring during the subsequent dark incubation.When, on the other hand, the cells were transferred to the lightfrom darkness at or after the start of the DNA synthesis, itcaused an almost complete abolition of the subsequent synthesisof DNA in the algal cells. No such effects of light were observedwith RNA and protein (total)
4. These findings were discussedin relation to the process ofchlorophyll formation occurringconcurrently in the algal cells.

(Received August 10, 1967; )     

Organelle DNA Synthesis in the Quiescent centre of Arabidopsis thaliana (Col.)

The behaviour of cell nuclei and organelle nucleoids (organellenuclei) was studied in the root apical meristem of 3-d-old seedlingsof Arabidopsis thaliana (Col.). Samples were embedded in Technovit7100 resin, cut into thin sections and stained with 4'-6-diamidino-2-phenylindole(DAPI) for observation of DNA. DNA synthesis in cell nucleiand organelle nucleoids was investigated using the incorporationof [³H] thymidine or 5-bromo-2'-deoxyuridine (BrdU). Incorporated[³H] thymidine and BrdU were detected by microautoradiographyor immunofiuorescence microscopy, respectively. Central cellsand cells just above the central cells of the quiescent centre(QC) showed an extremely low activity of DNA synthesis. However,DNA synthesis occurred in at least one organelle nucleoid ofall cells in the QC within 24 h. This suggests the cells inthe QC are quiescent with regard to nuclear DNA synthesis, butnot with regard to the organelle nucleoids. Key words: Arabidopsis thaliana, quiescent centre, root apical meristem, mitochondrial nucleoid (nuclei), plastid nucleoid (nuclei)     

Variability and predictability of the empirical conversion factor for converting 3H-thymidine uptake into bacterial carbon production for a eutrophic lake

To assess the variability and predictability of the empiricalconversion factor (CF) for converting the uptake of ³H-thymidine(TDR) to bacterial carbon production rates in eutrophic environments,we performed 11 dilution culture experiments and results wererelated to a set of important concomitantly recorded environmentalvariables. TDR incorporation data from a field study were thenconverted to carbon production with the established empiricalCF and a theoretical CF and compared to production estimatesderived from leucine incorporation. Mean empirical CF variedbetween 0.5 and 7.0 x 10⁶ cells (pmol TDR)^–1 over theyear and showed highly significant negative correlation withTDR incorporation rate and a highly significant positive correlationto temperature. Values of carbon production derived with thevariable empirical CF were lower than the values obtained bythe use of the theoretical CF of 1 x 10⁶ cells (pmol TDR)^–1.They showed less seasonal variation than values obtained byleucine incorporation, and periods of uncoupling were observed.However, when the empirical CF was calculated from the multipleregression equation including TDR incorporation and temperature,the resulting carbon production rates showed a high correspondencewith the leucine-derived production rates. Results of the analyseswere interpreted as an indication that under favourable conditions(resulting in high TDR incorporation) bacteria may be able tooptimize DNA duplication over protein synthesis as a possiblestrategy to persist and to maintain their potential to divideunder limiting conditions (e.g. decrease in temperature andsubstrate availability). In unfavourable conditions (resultingin low incorporation rates), bacteria may then use already producedDNA copies for rapid growth, when the environmental conditionsturn favourable again. Thus, an experimental set-up causingnutrient enrichment of the sampled water by autoclaving andfiltering, as generally used for dilution culture experiments,does not always reflect in situ situations, especially duringperiods of low nutrient concentrations.     

Further studies on ribulose-l,5-diphosphate carboxylase from Rhodopseudomonas spheroides and Rhodospirillum rubrum

Some enzymic properties of RuDP carboxylase isolated from Rhodopseudomonasspheroides and Rhodospirillum rubrum, grown under autotrophic,semi-autotrophic and heterotrophic conditions were studied.Regardless of the growing condition, the nature of the enzymefrom each respective bacterium remained unchanged. The molecularweight of R. spheroides and R. rubrum RuDP carboxylase was estimatedto be approximately 2.4x10⁵ and 8.3x10⁴, respectively. Bothenzymes require Mg⁺⁺. Their kinetic properties were also examined. ¹Part XII. Structure and Function of Chloroplast Proteins ²Supported in part by research grants from the Ministry of Educationof Japan (No. 8719), USPHS (AM. 10792-03) and the Asahi Press(Tokyo) (Received March 18, 1970; )     

Photosystem I and photophosphorylation-dependent leucine incorporation in the blue-green alga Anacystis nidulans

L-Leucine uptake and incorporation in the blue-green alga Anacystisnidulans were measured during illumination with monochromaticlight of 630 and 717 nm. With near as well as far red light,an enhanced uptake of ¹⁴C-L-leucine was observed. In far redlight, the leucine uptake depended on light intensity and pHvalue. After the first few minutes, the uptake remained constantfor more than one hour. The rate of uptake in light was thesame in air as in nitrogen. The incorporation of ¹⁴C-leucinein the soluble fraction decreased in the presence of chloramphenicolwhich prevents protein synthesis. In far red light, its incorporationwas insensitive to DCMU (5 ? 10^–6 M) but was depressedby uncouplers like CCCP or desaspidin. These effects are takenas evidence that leucine incorporation under the conditionsused is dependent on photosystem I reactions and cyclic photophosphorylation.DBMIB and KCN in high concentrations decrease the leucine incorporationin far red light and indicate that plastoquinone and plastocyaninare members of the cyclic electron flow also in intact cellsof Anacystis. Antimycin A has no inhibitory effect. The inhibitionby other less specific inhibitors like salicylaldoxime, desaspidinand DSPD is discussed. (Received August 19, 1978; )     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA