Developmental changes in ribosomal ribonucleic Acid and fraction I protein in wheat leaves

In light-grown wheat (Triticum aestivum L.) seedlings, the amount of chloroplast and cytoplasmic ribosomal RNA increased to a maximum in the first leaf near the end of its growth and declined by about 60% in the following 3 days. While total ribosomal RNA was declining, labeled uracil was still incorporated into cytoplasmic ribosomal RNA, but the rate of incorporation into chloroplast ribosomal RNA fell by more than 80%, as did the incorporation of labeled leucine into fraction I protein. Either there is greater replacement of cytoplasmic ribosomal RNA than chloroplast ribosomal RNA in mature leaves, or chloroplasts are able to repress the incorporation of exogenous precursor when there is no net synthesis of RNA.     

Effects of two TMV strains on the synthesis and stability of chloroplast ribosomal RNA in tobacco leaves

Summary Multiplication of TMV-strains vulgare (light-green/dark-green mosaic symptoms) and flavum (severe yellow/green mosaic) had different effects on the ribosomal RNA of tobacco leaf chloroplasts. Vulgare inhibited chloroplast ribosomal RNA synthesis while having no effect on cytoplasmic ribosomal RNA synthesis (Fig. 2). Flavum inhibited chloroplast ribosomal RNA synthesis more severely than vulgare, and caused an earlier degradation of chloroplast ribosomal RNA than in control or vulgare-infected leaves (Fig. 1). Flavum also inhibited cytoplasmic ribosomal RNA synthesis. A connection between these differing effects on chloroplast ribosomal RNA metabolism and severity of visible symptoms is suggested, and discussed in relation to a possible influence on symptoms of denatured virus coat protein.Abbreviations TMV Tobacco Mosaic Virus - RNA Ribonucleic acid - DNA Deoxyribonucleic acid - m millions (in molecular weight values)     

Comparison of the action of light on ribosomal RNA synthesis in cabbage and mustard seedlings

We have compared the action of light on ribosomal RNA synthesis in mustard and cabbage seedlings, two of the most frequently used systems for the studies of anthocyanin synthesis. The level of RNA (both t-RNA and r-RNA) “stored” in mustard dry seeds is much lower than in cabbage dry seeds. The kinetics of RNA synthesis in cabbage and mustard seedlings exposed to light are very different: In cabbage seedlings, light produces no apparent stimulation of cytoplasmic r-RNA synthesis, while it does increase plastid r-RNA synthesis. On the other hand, in mustard seedlings, light promotes both cytoplasmic and plastid ribosomal RNA synthesis. Streptomycin, which inhibits chlorophyll formation and chloroplast development while having no effect (mustard) or enhancing (cabbage) anthocyanin synthesis in these two systems, is in both cases an effective inhibitor of plastid r-RNA synthesis, but not of cytoplasmic r-RNA synthesis.     

THE EFFECTS OF INHIBITORS OF RNA AND PROTEIN SYNTHESIS ON CHLOROPLAST STRUCTURE AND FUNCTION IN WILD-TYPE CHLAMYDOMONAS REINHARDI

Wild-type cells of the unicellular green alga Chlamydomonas reinhardi have been grown for several generations in the presence of rifampicin, an inhibitor of chloroplast DNA-dependent RNA polymerase, spectinomycin and chloramphenicol, two inhibitors of protein synthesis on chloroplast ribosomes, and cycloheximide, an inhibitor of protein synthesis on cytoplasmic ribosomes. The effects of cycloheximide are complex, and it is concluded that this inhibitor cannot give meaningful information about the cytoplasmic control over the synthesis of chloroplast components in long-term experiments with C. reinhardi. In the presence of acetate and at the appropriate concentrations, the three inhibitors of chloroplast protein synthesis retard growth rates only slightly and do not affect the synthesis of chlorophyll; however, photosynthetic rates are reduced fourfold after several generations of growth. Each inhibitor produces a similar pattern of lesions in the organization of chloroplast membranes. Only rifampicin prevents the production of chloroplast ribosomes.     

Estimation of rRNA synthesis and degradation rates in senescing wheat leaves

Changes in cytoplasmic and chloroplast rRNA content and rates of rRNA synthesis and degradation of detached wheat leaves were determined. It was found that rRNA loss is proportionally higher in chloroplasts than in cytoplasm. Rates of synthesis were measured by incorporation of large amounts of [3H]orotic acid into rRNA. This approach overcame size differences between pyrimidine pools of cells under different physiological status. Furthermore, these pools reached nearly the same specific radioactivity as that of the administered solution. Rates of degradation were estimated either as the difference between synthesis and net variation of rRNA or by disappearance of radioactivity from 32P-labeled rRNA. Results indicated a decrease in the net rRNA synthesis capacity of leaves after 48 h of detachment. However, the fractional rates of rRNA synthesis were maintained in both cytoplasm and chloroplasts. Ribosomal RNA degradation rates were 2.5-fold higher in chloroplast than in cytoplasm. The observed chloroplast rRNA loss is due to an increased degradation rate which is 15-fold higher than the synthesis rate 48 h after detachment.     

Cell-free translation of chloroplast RNAs in the wheat germ system

Spinach, tobacco and Euglena chloroplast RNAs (cp RNA) can be successfully translated in the wheat germ cell-free system. The in vitro translation products obtained from spinach cp RNA in the wheat germ and in the Escherichia coli system are similar to each other and to that of in organello synthesis, if analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Translation of mixtures of chloroplast and total RNA of leaves reveals that under conditions of mRNA competition the cytoplasmic type of RNA is preferentially translated in the wheat germ system.     

Inhibition of chloroplast protein synthesis by lincomycin and 2-(4-methyl-2,6- dinitroanilino)-N-methylpropionamide

Selective effects of lincomysin and cycloheximide in detached shoots of Pisum sativum on the synthesis of photosystem I and II proteins, and a chloroplast membrane protein of molecular weight 32000, confirm results obtained from studies of protein synthesis by isolated chloroplasts. A model is proposed in which one role of chloroplast ribosomes is to synthesize membrane proteins required for the immobilization of chloroplast components, such as photosystem I protein, which are synthesized by cytoplasmic ribosomes. 2-(4-Methyl-2,6-dinitroanilino)-N-methylpropionamide rapidly inhibits the synthesis of both the large and small subunits of Fraction I protein in greening detached pea shoots. This observation can be reconciled with the site of synthesis of the large subunit being in the chloroplast by a model which proposes that the small subunit is a positive initiation factor for the synthesis or translation of the messenger RNA for the large subunit.     

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     

Effect of benzyladenine on RNA and protein synthesis in intact bean leaves at various stages of ageing

Primary leaves of intact bean plants ( Phaseolus vulgaris L.) were treated with benzyladenine (BA) at different stages of ageing, BA promoted the synthesis of RNA, and soluble and insoluble proteins. The effects of BA stimulation differed depending on the age at which the leaf received the hormone treatment. In leaves attached to the plant, BA appeared to stimulate the rate of synthesis more than the rate of decomposition of RNA and protein, resulting in a net increase in RNA and protein. Both chloroplast and cytoplasmic ribosomes were still observed in intact yellowish green leaves. Polysomes in the cytoplasm increased remarkably when BA treatment was begun at late stages.     

Determination of the site of synthesis of some Euglena cytoplasmic and chloroplast ribosomal proteins.

Ribosomal protein synthesis during chloroplast development in Euglena gracilis has been studied by using inhibitors specific for either chloroplast or cytoplasmic protein syntheses. Fifty proteins of cytoplasmic and 39 of chloroplast ribosomes have been examined. Synthesis of all cytoplasmic ribosomal proteins is strongly inhibited by cycloheximide. Lincomycin (LIN) seems to have no effect on the synthesis of these proteins. In contrast, formation of 12 chloroplast ribosomal proteins is inhibited by cycloheximide (CHI), that of 9 by lincomycin, and that of 6 by both of these antibiotics; the technique used in this study did not permit definite determination of the sites of synthesis of the remaining proteins.     

A method for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves

A method is described for extracting intact chloroplast and cytoplasmic ribosomal RNA from leaves of two higher plant species. Sodium dodecyl sulfate (1%) and 25 mM magnesium ions are required to inhibit ribonuclease action during RNA purification by phenol deproteinization. The ethanol-precipitated RNA product, including 23s chloroplast ribosomal RNA, is completely stable during electrophoresis in the absence of magnesium ions, even in the presence of EDTA. The $\text{in}$$\text{vivo}$ mole fraction of chloroplast ribosomes relative to cytoplasmic ribosomes is estimated. Bentonite is shown to cause preferential losses of chloroplast RNA during extraction.     

The Chloroplast and Cytoplasmic Ribosomes of Euglena: II. Characterization of Ribosomal Proteins

Cytoplasmic and chloroplast ribosomal proteins were isolated from Euglena gracilis and analyzed on polyacrylamide gels. Cytoplasmic ribosomes appear to contain 75 to 100 proteins ranging in molecular weight from 10,200 to 104,000, while chloroplast ribosomes appear to contain 35 to 42 proteins with molecular weights ranging from 9,700 to 57,900. This indicates that the cytoplasmic ribosomes are similar in composition to other eucaryotic ribosomes, while chloroplast ribosomes have a protein composition similar to the 70S procaryotic ribosome. The kinetics of light-induced labeling of cytoplasmic ribosomal proteins during chloroplast development has been determined, and the results are compared with the kinetics of ribosomal RNA synthesis.     

Sites of synthesis of chloroplast ribosomal proteins in Chlamydomonas

Cells of Chlamydomonas reinhardtii were pulse-labeled in vivo in the presence of inhibitors of cytoplasmic (anisomycin) or chloroplast (lincomycin) protein synthesis to ascertain the sites of synthesis of chloroplast ribosomal proteins. Fluorographs of the labeled proteins, resolved on two-dimensional (2-D) charge/SDS and one-dimensional (1-D) SDS-urea gradient gels, demonstrated that five to six of the large subunit proteins are products of chloroplast protein synthesis while 26 to 27 of the large subunit proteins are synthesized on cytoplasmic ribosomes. Similarly, 14 of 31 small subunit proteins are products of chloroplast protein synthesis, while the remainder are synthesized in the cytoplasm. The 20 ribosomal proteins shown to be made in the chloroplast of Chlamydomonas more than double the number of proteins known to be synthesized in the chloroplast of this alga.     

Chloroplast elongation factor Tu: Evidence that it is the product of a chloroplast gene in Euglena

The chloroplast protein synthesizing factor responsible for the binding of aminoacyl-tRNA to ribosomes (EF-Tu_chl) has been identified in extracts of Euglena gracilis. This factor is present in low levels when Euglena is grown in the dark and can be induced more than 10-fold when the organism is exposed to light. The induction of the chloroplast EF-Tu by light is inhibited by streptomycin, an inhibitor of protein synthesis on chloroplast ribosomes, indicating that protein synthesis within the chloroplast itself is required for the induction of this factor. The induction of the chloroplast EF-Tu by light is also inhibited by cycloheximide, a specific inhibitor of protein synthesis on cytoplasmic ribosomes. The effect of cycloheximide probably results from the inhibition of chloroplast ribosome synthesis which requires the synthesis of many proteins by the cytoplasmic translational system. Chloroplast EF-Tu cannot be induced by light in an aplastidic mutant (strain W₃BUL) of Euglena which has neither significant plastid structure nor detectable chloroplast DNA. These data strongly suggest that the genetic information for chloroplast EF-Tu resides in the chloroplast genome and that this protein is synthesized within the organelle itself.     

Characterization of cytoplasmic and chloroplast 5S ribosomal ribonucleic acid from broad-bean leaves

Green leaves of the broad bean (Vicia faba) contain two 5S RNA components that can be separated from each other by polyacrylamide-gel electrophoresis. The major component is located in the larger subunit of cytoplasmic ribosomes, whereas the minor component occurs in the larger subunit of chloroplast ribosomes. Their electrophoretic mobilities relative to those of Escherichia coli 5S RNA (120 nucleotides) and plant 4S RNA (78 nucleotides) suggest that they consist of 118 and 122 nucleotide residues respectively. Thermal ;melting' profiles of plant cytoplasmic and chloroplast 5S RNA species at 260nm indicate the similarity of their secondary structures, not only to each other, but also to those of E. coli and mammalian 5S RNA species. The base compositions of the two plant 5S RNA species have more in common with each other than with the corresponding molecules from either E. coli or mammalian cells.     

Synthesis and Stability of Chloroplast Ribosomal-RNA's

The chloroplast ribosomal-RNAs (1.1 × 10⁶ and 0.56 × 10⁶ mol wt) are synthesized in the normal ratio of 2:1. The non-ribosomal distribution observed after extraction and fractionation results from the lability of the 1.1 × 10⁶ component, and a correction for this breakdown can be applied in certain cases. Newly synthesized 1.1 × 10⁶ RNA is more stable than the older accumulated 1.1 × 10⁶ RNA. Accumulation of the chloroplast RNA during growth of radish cotyledons occurs at a later time than the accumulation of cytoplasmic RNA, and its turnover is much less than that of the cytoplasmic ribosomal-RNA.     

Euglena gracilis Chloroplast DNA Codes for Polyadenylated RNA

Polyadenylated RNA, isolated from total cellular RNA of photoautotrophically grown Euglena gracilis, comprised 2.1% of the total cellular RNA and contained 6.2% polyadenylic acid. Polyadenylated RNA, labeled in vitro with ¹²⁵I, hybridized at saturating levels to an average 7.7% of the chloroplast DNA. In the presence of excess chloroplast rRNA, hybridization of polyadenylated RNA was reduced, but was still observed at a level corresponding to 2.8% of the chloroplast DNA. Polyadenylic acid was not detected in mRNA prepared from chloroplast polyribosomes, indicating a level of less than 0.1% polyadenylic acid in mature chloroplast mRNA. Of the total RNA isolated from cytoplasmic polyribosomes, 2.0% contained polyadenylic acid. This latter polyadenylated RNA did not hybridize to chloroplast DNA.