The effects of abscisic acid,kinetin and 5-fluorouracil on ribonucleic acid and protein synthesis in senescing radish leaf disks

Summary The effects of abscisic acid and kinetin on RNA synthesis in senescing radish leaf disks were investigated using the improved resolution afforded by polyacrylamide gel electrophoresis. Kinetin stimulated and abscisic acid inhibited incorporation of radioactivity into cytoplasmic ribosomal RNA and soluble RNA. Chloroplast ribosomal RNA synthesis appeared to be confined to the period of leaf expansion and was not detected in fully mature leaves. The effects of kinetin in retarding and of abscisic acid in accelerating leaf senescence were not altered by the inhibition of cytoplasmic ribosomal RNA synthesis with 5-fluorouracil. Following inhibition of cytoplasmic ribosomal RNA synthesis with 5-fluorouracil, kinetin stimulated and abscisic acid inhibited incorporation of radioactivity into polydisperse RNA. These results are discussed in relation to the possible mode of action of kinetin and abscisic acid in senescing leaf tissue.     

Effect of 5-fluorouracil post-treatment on UV-resistance ofEscherichia coli cells

The effect of 5-fluorouracil (5-FU) and uracil starvation on the UV-sensitivity ofEscherichia coli 15 was followed in a 60 minutes period after the irradiation. The presence of 5-FU caused a significant increase of radioresistance when compared with the control, on the other hand under conditions of uracil starvation the cells were slightly more sensitive than in the control sample. The DNA synthesis was highly inhibited under conditions of uracil starvation, but the same effect was observed in the presence of 5-FU. Maximum inhibition of protein synthesis was observed also when uracil was omitted from the cultivation medium and approximately 50% of inhibition was noticed in the sample with 5-FU. The increase of resistance caused by the presence of 5-FU in the 60 minutes period after UV-irradiation cannot be explained by prolonging the sensitive postirradiation phase. It seems probable that in the presence of 5-FU the conditions are favourable for a better course of the repair processes.     

Studies on frost hardiness in Chlorella ellipsoidea II. Effects of inhibitors of RNA and protein synthesis and surfactants on the process of hardening

Chlorella ellipsoidea cells at an intermediate stage in theripening phase of the cell cycle were brought into contact withinhibitors of RNA and protein synthesis, and surfactants duringor after hardening at 3°C. Cycloheximide, actinomycin D and 5-fluorouracil inhibited thedevelopment of frost hardiness in the algal cells. In contrastwith cycloheximide, chloramphenicol did not cause complete inhibitionof hardiness increase, even at a high concentration. These resultssuggest that protein synthesis on cytoplasmic ribosomes is indispensablefor the development of frost hardiness, but protein synthesison chloroplast ribosomes has little or no involvement in thehardening process. Triton X-100 and sodium dodecyl sulfate inhibited the developmentof frost hardiness, but sodium cholate and sodium deoxycholatedid not. Hardened cells were more susceptible to sodium dodecylsulfate, in contrast to sodium cholate, sodium deoxycholateand Triton X-100, than unhardened cells. From the results, considerablealterations in both lipids and proteins constituting cellularmembranes appear to be involved in the process of hardening. (Received December 3, 1975; )     

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.     

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.     

Effect of Purine and Pyrimidine Analogues on Growth and RNA Metabolism in the Soybean Hypocotyl-the Selective Action of 5-Fluorouracil

The effects of several base analogues and cycloheximide on RNA synthesis, protein synthesis, and cell elongation were studied in excised soybean hypocotyl. None of the pyrimidine analogues tested affected growth or protein synthesis; only 5-fluorouracil appreciably inhibited RNA synthesis. 8-Azaguanine and 6-methylpurine markedly inhibited RNA and protein synthesis and cell elongation. Cycloheximide effectively inhibited both cell elongation and protein synthesis.The results show that 5-fluorouracil selectively inhibited ribosomal and soluble RNA synthesis without affecting the synthesis of D-RNA. These results indicate that the requirement for RNA synthesis to support continued protein synthesis and cell elongation is restricted to the synthesis of D-RNA.5-Fluorouracil was incorporated into all classes of RNA in a form believed to be 5-fluorouridylic acid.Cycloheximide markedly inhibited the accumulation of ribosomal RNA, but the results indicate that CH did not inhibit, per se, the synthesis of ribosomal RNA. The accumulation of newly synthesized D-RNA was only slightly affected by cycloheximide. These results show that the inhibition of cell elongation by cycloheximide correlates with the inhibition of protein synthesis, but not with the effect on RNA metabolism.     

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)     

Studies on ribonucleic acids from Chlorella protothecoides with special reference to the degradation of chloroplast RNA during the process of "glucose-bleaching"

By growing Chlorella protothecoides under certain nutritionaland light conditions the following three different types ofalgal cells were obtained: (i) normal "green" cells grown ina medium rich in a nitrogen source (urea) and poor in glucoseunder illumination, (ii) "etiolated" cells cultivated in thesame medium in darkness, and (iii) "glucose-bleached" cellsgrown, in the light or in darkness, in a medium rich in glucoseand poor in the nitrogen source. The "glucose-bleached" cellscontain profoundly degenerated plastids, and the "etiolated"cells have only partially organized plastids. From these algalcells RNA was extracted by the cold phenol method, and fractionatedby MAK column chromatography and sucrose density gradient centrifugation,making use of ³²P-labelled E. coli RNA as the internal marker.It was found that in comparison with the green cells that arerich in chloroplast ribosomal RNA as well as in nonchloroplastic("cytoplasmic") ribosomal RNA, the etiolated cells possess acomparable amount of "cytoplasmic" rRNA but a significantlylesser amount of chloroplast rRNA. Both types of rRNA existat extremely low levels in the glucose-bleached cells. During the process of bleaching (chloroplast degeneration) ofthe green cells induced by the addition of a high concentrationof glucose, marked changes were observed in the patterns offractionation of RNA as followed by the above procedures. Itwas disclosed that the chloroplast rRNA is rapidly degradedduring an early phase of the bleaching process, while the quantityof "cytoplasmic" rRNA remained almost unaltered. ¹Part of this work was reported at the Symposium on Cell Differentiationsponsored by the Institute of Applied Microbiology, Universityof Tokyo, in November 1965, and at the Symposium on Biogenesisof Subcellular Particles, the 7th Internatl. Congress of Biochemistry,Tokyo, 1967. ²Present address: Faculty of Pharmaceutical Sciences, Universityof Hokkaido, Sapporo.     

Synthetic rates of chloroplast and cytoplasmic ribosomal ribonucleic acids during the cell cycle of Chlorella

By feeding radioisotopic precursors of RNA ([5-³H]uracil and[5-³H]uridine) to cells of Chlorella ellipsoidea at variousstages in the cell cycle effected by autotrophic synchronousculture, we examined synthetic rates of the chloroplast andthe cytoplasmic ribosomal ribonucleic acids (chl-rRNA and cyt-rRNA,respectively). The net incorporation of the precursors intochl-rRNA was higher than that into cyt-rRNA in the early stagesof the cell cycle, and vice versa in the late stages. The specificactivity of chl-rRNA was extremely high, and this phenomenonwas likely to be intrinsic to small cells at the start of thecell cycle under autotrophic conditions, namely, cell-cyclestagespecific. We conclude that algal cells grown autotrophicallysynthesize chl-rRNA at a distinctly higher rate than cyt-rRNAin the early stages of the cell cycle. (Received July 21, 1978; )     

Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil

Reich, Melvin (The George Washington University School of Medicine, Washington, D.C.), and H. George Mandel. Dissociation of cellular functions in Bacillus cereus by 5-fluorouracil. J. Bacteriol. 91:517-523. 1966.-5-Fluorouracil (FU) produced a marked inhibition of growth and deoxyribonucleic acid (DNA) synthesis in Bacillus cereus 569H. Protein and ribonucleic acid (RNA) synthesis were not specifically inhibited, and proceeded at the rate of turbidometric increase of the cells. Cell-wall synthesis, respiration, and penicillinase production continued in the presence of FU at essentially the control rate. The addition of equimolar concentrations of uracil and FU prevented growth inhibition but did not restore DNA synthesis. The addition of thymidine with FU did not relieve growth inhibition but did restore the DNA content to normal. Thymidine supplementation also increased the quantity of FU, but not uracil, incorporated into RNA and the acid-soluble fraction. The data indicate that inhibition of growth can be dissociated from inhibition of DNA synthesis and that more DNA is present in normal cells than is needed for growth and reproduction.     

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.     

The Effect of Light and Inhibitors on Chloroplast and Cytoplasmic RNA Synthesis

Chloroplast RNA is synthesized in dark-grown radish cotyledons at about one-third the rate of that in the light. The synthesis, however, continues for longer in the dark and the percentage of chloroplast RNA can approach that in light-grown tissue. Light stimulates the synthesis and accumulation of both cytoplasmic and chloroplast RNA, but shows a 4-fold greater stimulation of the chloroplast RNA. Chloramphenicol, streptomycin and cycloheximide inhibit the synthesis of chloroplast RNA with little effect on cytoplasmic RNA. 5-Fluorouracil inhibits the synthesis of cytoplasmic more than chloroplast RNA. Synthesis of the 0.56 x 10(6) mol wt chloroplast RNA is inhibited much less than the other ribosomal RNA components by actinomycin D.     

EFFECTS OF CHLORAMPHENICOL ON CHLOROPLAST AND MITOCHONDRIAL ULTRASTRUCTURE IN OCHROMONAS DANICA

The effect of chloramphenicol (CAP) on cell division and organelle ultrastructure was studied during light-induced chloroplast development in the Chrysophyte alga, Ochromonas danica. Since the growth rate of the CAP-treated cells is the same as that of the control cells for the first 12 hr in the light, CAP is presumed to be acting during that interval solely by inhibiting protein synthesis on chloroplast and mitochondrial ribosomes. CAP markedly inhibits chloroplast growth and differentiation. During the first 12 hr in the light, chlorophyll synthesis is inhibited by 93%, the formation of new thylakoid membranes is reduced by 91%, and the synthesis of chloroplast ribosomes is inhibited by 81%. Other chloroplast-associated abnormalities which occur during the first 12 hr and become more pronounced with extended CAP treatment are the presence of prolamellar bodies and of abnormal stacks of thylakoids, the proliferation of the perinuclear reticulum, and the accumulation of dense granular material between the chloroplast envelope and the chloroplast endoplasmic reticulum. CAP also causes a progressive loss of the mitochondrial cristae, which is paralleled by a decline in the growth rate of the cells, but it has no effect on the synthesis of mitochondrial ribosomes. We postulate that one or more chloroplast ribosomal proteins are synthesized on chloroplast ribosomes, whereas mitochondrial ribosomal proteins are synthesized on cytoplasmic ribosomes.     

Isolation of active polyribosomes from the cytoplasm, mitochondria and chloroplasts of Euglena gracilis

1. A procedure is described for the isolation of intact polyribosomes from the cytoplasm, chloroplasts and mitochondria of Euglena gracilis. 2. All three polyribosomal preparations incorporated labelled amino acids in a system in vitro. The cytoplasmic system was inhibited by chcloheximide but not by chloramphenicol. Both the chloroplast and the mitochondrial systems, however, were inhibited by chloramphenicol but not by cycloheximide. It is shown that mitochondrial polyribosomes, like the polyribosomes from cytoplasm and chloroplasts, can participate directly in protein synthesis without supplementary mRNA being added to the synthesizing system, as in previously reported instances. 3. Sedimentation coefficients were measured for the ribosomes, ribosomal subunits, and rRNA of the cytoplasm, chloroplasts and mitochondria. 4. The G+C content was 55% for cytoplasmic rRNA, 50% for chloroplast rRNA, and 29% for mitochondrial rRNA. 5. The cytoplasmic ribosomal subunits contained a ribonuclease activity that was inhibited by heparin.     

Effect of 5-fluorouracil and cycloheximide on the early development of Phycomyces blakesleeanus spores and the activity of N-acetylglucosamine synthesizing enzymes

The development of germinating Phycomyces spores was not inhibited by 5-fluorouracil (1 mM) until the emergence of the germination tube. Fluorouracil was incorporated into RNA as efficiently as uracil; it did not inhibit the synthesis of proteins and the increase in respiratory activity during early develpment. Cycloheximide inhibited development as well as the increase in respiration and protein synthesis. This suggested that protein synthesis or some other cycloheximide dependent process, but no mRNA synthesis, was needed for the first developmental stages. The activity of two enzymes involved in the synthesis of N-acetylglucosamine increased markedly during germination. This increase was inhibited by both 5-fluorouracil and cycloheximide; this suggested that those enzymes were synthesized on mRNA formed during germination.     

Effect of 5-Fluorouracil on Growth and Morphogenesis of Tissue Cultures of Nicotiana sylvestris

Tissue cultures of Nicotiana sylvestris treated with increasingconcentration of 5-fluorouracil (5-FU) showed a differentialeffect of the drug on growth and morphogenesis. In the rangeof concentrations tested, 5-FU did not inhibit increases infresh weight, but it inhibited the production of shoots. Theeffect of 5-FU on shoot production was reversible and dependedon the time of adding 5-FU to the tissue culture. Uracil andthymine did not counteract the effect of 5-FU on morphogenesis,whereas uracil partially reduced the inhibition of growth undersome culture conditions. (Received July 16, 1985; Accepted April 5, 1986)     

Processing of the precursor to a chloroplast ribosomal protein made in the cytosol occurs in two steps, one of which depends on a protein made in the chloroplast.

In pulse-chase experiments in which log-phase cells of Chlamydomonas reinhardtii were labeled in vivo for 5 min with H2(35)SO4, fluorographs of immunoprecipitates from whole cell extracts revealed that chloroplast ribosomal proteins L-2, L-6, L-21, and L-29, which are made in the cytosol and imported, appeared in their mature forms. However, in the case of chloroplast ribosomal protein L-18, which is also made in the cytoplasm and imported, a prominent precursor with an apparent molecular weight of 17,000 was found at the end of a 5-min pulse. This precursor was processed to its mature size (apparent molecular weight of 15,500) within the first 5 min of the subsequent chase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the precursor to L-18 formed in vivo was 1.5 kilodaltons smaller than the primary product detected in translations of Chlamydomonas polyadenylated RNA in vitro. Upon a 10-min incubation with a postribosomal supernatant from Chlamydomonas, the 18,500-dalton precursor detected in vitro could be partially converted into a polypeptide that comigrated with the 17,000-dalton precursor detected in extracts of cells labeled in vivo. Under conditions in which the total amounts of chloroplast proteins had been reduced and cells were made to synthesize ribosomes rapidly, the apparent half-life of the 17,000-dalton precursor was extended over that seen in log-phase cells. When chloroplast protein synthesis was inhibited with lincomycin for 3 h before labeling under these conditions, the 17,000-dalton L-18 precursor but not the mature form was found, and the precursor was slowly degraded during a 60-min chase. When cells were placed in the dark for 3 h before labeling, processing of this precursor to the mature form appeared unaffected, but the chloroplast-synthesized ribosomal protein L-26 was detected, indicating that chloroplast protein synthesis was still occurring. We interpret these results to indicate that the maturation of protein L-18 in vivo involves at least two processing steps, one of which depends on a protein made on chloroplast ribosomes.