Incorporation of different labelled precursors into chloroplast RNA of Chlorella

Summary Radioactive uridine is incorporated by Chlorella strain 211-8b/p into ribosomal subunits and their rapidly labelled RNA comigrates with chloroplast RNA on polycrylamide gels.Ribosomal particles which can be labelled by short pulses of orotic acid cosediment with the particles labelled by uridine pulses and contain the same RNA species as these when separated either on sucrose gradients or on polycrylamide gels. This incorporation is, like that of uridine, sensitive to rifampin and chloramphenicol, but insensitive to cycloheximide.A comparative study of short-time incorporation of uridine, orotic acid and guanosine into the RNA of Chlorella showed that all three precursors were incorporated mainly into RNA of chloroplastic origin. However, guanosine was also partly incorporated into cytoplasmic rRNA. Nitrogen-deficient cells always incorporated part of all three precursors into cytoplasmic rRNA, but the proportions of these were different among the different precursors.These results are consistent with the hypothesis that the described differences in the incorporation of the above mentioned precursors into RNA of different cellular compartments are largely attributable to effects of pool sizes.     

DNA and RNA Syntheses by Intraerythrocytic Stages of Plasmodium knowlesi*

Incorporation of the nucleic acid precursors, orotic acid, adenosine, thymidine, and uridine, was studied in various stages of intraerythrocytic Plasmodium knowlesi from infected rhesus monkeys. Incubation of the parasitized erythrocytes with the precursors was for 3 hr periods using a plasma-free culture medium. The samples containing primarily rings, early trophozoites, or late trophozoites incorporated orotic acid, adenosine, and uridine into RNA; however, these stages exhibited negligible or very low levels of incorporation of any of the precursors into DNA. The sample containing late trophozoite and schizont stages incorporated orotic acid, adenosine, and uridine into RNA, and orotic acid, adenosine, and very low levels of thymidine into DNA. These results indicate that DNA synthesis (the S phase of the cell cycle) occurs very close to the time of nuclear division, and that either the G1 or G2 phase is very short in P. knowlesi. It was also observed that adenosine and orotic acid, 2 precursors which are incorporated into both DNA and RNA, are utilized differently by the intraerythrocytic parasites. Incorporation of orotic acid into RNA and DNA and adenosine incorporation into DNA were continuous for the entire incubation period, whereas incorporation of adenosine into RNA was very low during the last 2 hr of each period. It was further demonstrated that the parasites utilized exogenous uridine for synthesis of RNA, and that the older parasite stages incorporated thymidine into DNA.     

Effect of SV40 infection on [3H]uridine incorporation.

More [3H]uridine was incorporated into RNA of SV40-infected than into uninfected cells 31 h after infection. When the specific activity of the uridine triphosphate pools in infected and uninfected cells was equated by the addition of appropriate amounts of exogenous unlabelled uridine, no difference in the rate of [3H]uridine incorporation into RNA was observed. Although no difference in [3H]uridine entry or phosphorylation was demonstrable, the apparently smaller pools of endogenous RNA precursors in infected cells resulted in less isotope dilution and thus to synthesis of uridine triphosphate and RNA of higher specific activity.     

Effect of sodium selenite on RNA synthesis in loach embryos

The effects of sodium selenite on the ribonucleic acid synthesis in normal diploid embryos and isolated nuclei of the loach Misgurnus fossilis were studied. The relative rate of synthesis was determined from incorporation of [3H]uridine into the total RNA, taking into consideration the incorporation of the label into the total acid-soluble fraction and into phosphorylated derivatives of uridine. It was shown that sodium selenite inhibits the RNA synthesis in loach embryos at the gastrula stage. It was also found that sodium selenite exerts an inhibiting effect on the activity of form I of DNA-dependent RNA-polymerase in isolated loach nuclei without affecting the activity of the enzyme form II.     

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 puromycin on synthesis, processing, and nucleocytoplasmic translocation of rRNA in Tetrahymena pyriformis.

1. Treatment of Tetrahymena pyriformis with various concentrations of puromycin results in a more pronounced inhibition of [3H]uridine accumulation in stable RNA than of protein synthesis. 2. At a concentration of 500 micrograms/ml, which is almost completely inhibitory to [3H]uridine incorporation in vivo, puromycin has no influence on the incorporation of [3H]UTP into RNA in isolated macronuclei. Pretreatment of the cells with the antibiotic, however, reduces the activity of RNA polymerases in isolated nuclei to less than 30%. 3. In puromycin-treated cells a small amount of pre-rRNA is synthesized but not processed into cytoplasmic rRNAs. 4. Puromycin reduces the nucleocytoplasmic translocation of pre-existing RNA to about 25% of the control rate within 5 min, resulting in an accumulation of relatively stable rRNA precursor molecules in the macronucleus.     

Free pyrimidine nucleotide pool of Ehrlich ascites-tumour cells. Compartmentation with respect to the synthesis of heterogeneous nuclear RNA and precursors to ribosomal RNA.

The incorporation of [14C]orotate and [14C]uridine into UMP residues of hnRNA (heterogeneous nuclear RNA) and pre-rRNA (precursors to rRNA) of Eharlich ascites-tumour cells was compared: orotate was incorporated at a markedly higher rate into hnRNA. On the other hand, the rate of incorporation of uridine into pre-rRTNA was even somewhat higher than into hnRNA. The ratio of specific radioactivities of CMP to UMP residues in pre-rRNA and hnRNA was studied. At all times of labelling this ratio was similar for both RNA species independently of the precursor used. On addition of excess unlabelled uridine, the CMP/UMP labelling ratio in both pre-rRNA and hnRNA rose. However, this increase was much more pronounced with hnRNA. It is concluded that nuclear pyrimidine nucleotide pool for RNA synthesis is compartmentalized. The synthesis of hnRNa is supplied preferentially by the large and the small compartment, respectively. A detailed model for the cellular compartmentation of uridine nucleotide precursors to RNA is proposed.U     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

RNA SYNTHESIS IN THE MOUSE OOCYTE

RNA synthesis in the oocyte and granulosa cell nuclei of growing follicles has been studied in the mouse ovary. The RNA precursor [³H]uridine was administered intraperitoneally to adult mice and the amount of label incorporated into ovarian RNA was quantitated autoradiographically using grain-counting procedures. Uridine incorporation into the nucleus is low in oocytes of small, resting follicles but increases during follicle growth and reaches a peak prior to the beginning of antrum formation. Thereafter uptake rapidly declines and is very low in the oocytes of maturing follicles. Uridine incorporation into granulosa cell nuclei, in contrast to that found in the oocyte, increases gradually during most of the period of follicle growth. Qualitative studies of the activity of endogenous, DNA-dependent RNA polymerases have also been made in fixed oocytes isolated from follicles at different stages of growth. Polymerase activity is demonstrable in the nucleolus and nucleoplasm of oocytes from growing follicles, but is absent from maturing oocytes of large follicles.     

Incorporation of exogenous precursors into uridine and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo

The possibility of compartmentation of UTP in vivo was investigated in the renal cortex of unanaesthetized rats. In addition, liver and spleen were studied in order to compare tissues with different utilization of precursors for pyrimidine nucleotide synthesis. After continuous 2h infusions of [(3)H]uridine or [(3)H]orotate, their incorporation into UTP, UDP-sugars and RNA was quantified. Rates of RNA synthesis were calculated by dividing the incorporation of precursor into RNA by the average specific radioactivity of the UTP pool. Although similar RNA-synthesis rates might have been expected with the two precursors, higher rates were found with uridine than with orotate. The relative incorporation into UDP-sugars of these precursors was also different. Similar results were obtained in the liver. In the spleen, equal amounts of both precursors were incorporated into UTP, but [(3)H]orotate incorporation did not lead to labelling of RNA. To evaluate the heterogeneity of cells with respect to the metabolism of pyrimidines, precursor incorporation was studied in isolated glomeruli and by radioautography. Incorporation into glomeruli was qualitatively similar to but quantitatively different from results in the renal cortex. Although there is obvious tissue heterogeneity, compartmentation of UTP pools is the most credible explanation for the results obtained with the renal cortex and liver. Consequently RNA and UDP-sugars may originate from two different UTP pools. Tissue heterogeneity is the likely explanation for the results obtained in the spleen. Studies of synthesis of pyrimidine and RNA, particularly in relation to growth and regeneration, must take into consideration the precursor used, the apparent existence of UTP compartmentation and the degree of cellular heterogeneity.     

Non-specific incorporation of nucleic acid precursors in Blastomyces dermatitidis and Histoplasma capsulatum

Incorporation of thymidine, thymidine monophosphate (TMP), thymidine triphosphate (TTP), uridine and orotic acid into DNA, RNA and protein in Blastomyces dermatitidis and Histoplasma capsulatum was studied utilizing a specific acid hydrolysis technique developed for these fungi. Thymidine was incorporated to the greatest extent (approximately 0.5 % of added label) followed by uridine, orotic acid, TMP and TTP. In Blastomyces, uridine and orotic acid labeled primarily RNA. TMP and TTP labeled RNA, DNA and protein at nearly the same level. In Histoplasma RNA was labeled poorly by any of these precursors. TMP and TTP labeled DNA predominately and protein to a slightly lower level. Deoxyadenosine or uridine media supplements of 250 g/ml did not enhance incorporation. All precursors tested were found to be nonspecific in that RNA, DNA and protein were labeled. All data indicate that neither RNA nor DNA synthesis can be specifically measured in whole cells or acid precipitates by any of these precursors. Specific radiometric monitoring with these isotopes therefore requires the separation of these macromolecules.     

Qualitative and quantitative studies on DNA and RNA synthesis in Loxodes striatus nuclei.

In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei and differentiated morphologically into a clearly recoginzable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a 3H-labeled precursor ([3H]thymidine for DNA and [3H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [3H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [3H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macronucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components synthesis can occur in adult macronuclei. The possible explantion of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.     

Ribonucleic acid synthesis in vitro in primary spermatocytes isolated from rat testis

The incorporation of [(3)H]uridine into RNA was studied quantitatively (by incorporation of [(3)H]uridine into acid-precipitable material) and qualitatively (by phenol extraction and electrophoretic separation of RNA in polyacrylamide gels) in preparations enriched in primary spermatocytes, obtained from testes of rats 26 or 32 days old. The rate of incorporation of [(3)H]uridine into RNA of isolated spermatocytes was constant during the first 8h of incubation, after which it decreased, but the decreased rate of incorporation was not reflected in a marked change in electrophoretic profiles of labelled RNA. In isolated spermatocytes, [(3)H]uridine was incorporated mainly into heterogeneous RNA with a low electrophoretic mobility. Most of this RNA was labile, as shown when further RNA synthesis was inhibited with actinomycin D. Spermatocytes in vivo also synthesized heterogeneous RNA with a low electrophoretic mobility. A low rate of incorporation of [(3)H]uridine into rRNA of isolated spermatocytes was observed. The cleavage of 32S precursor rRNA to 28S rRNA was probably retarded in spermatocytes in vitro as well as in vivo. RNA synthesis by preparations enriched in early spermatids or Sertoli cells was qualitatatively different from RNA synthesis by the spermatocyte preparations. It is concluded that isolated primary spermatocytes maintain a specific pattern of RNA synthesis, which resembles RNA synthesis in spermatocytes in vivo. Therefore isolated spermatocytes of the rat can be used for studying the possible regulation of RNA synthesis during the meiotic prophase.     

A Method for the Isolation of Macronuclei from Paramecium aurelia

SYNOPSIS. A method is described for the isolation of macronuclei from Paramecium aurelia. It has also been successfully employed with Didinium nasutum. The yield is up to 50% with P. aurelia. Some results on the DNA, RNA and protein contents are given. The isolated macronuclei of P. aurelia are able to incorporate ATP and UTP into acid-insoluble material, a process which is probably RNA synthesis mediated by RNA polymerase. The macronuclei were thus highly purified, and retained a measure of biosynthetic activity.     

Incorporation of Radioactive Precursors into DNA and RNA of Plasmodium knowlesi in vitro

SYNOPSIS. Cultures of the intra-erythrocytic stages of Plasmodium knowlesi incubated in vitro utilized all the pre-formed radioactive purines tested (adenine, adenosine, deoxvadenosine, guanine, guanosine and hypoxanthine) but none of the pyrimidines (thymine, thymidine, uracil, uridine, cytidine and deoxycytidine). They did, however, utilize the pyrimidine precursor orotic acid.
All precursors analysed, including deoxyadenosine, were incorporated into both DNA and RNA (in the ratio of ∼1:3) but 19% was incorporated into other unidentified compounds. ³Hadenosine was incorporated into adenine and guanine residues of both DNA and RNA.
No unambiguous evidence was obtained for any periodicity in the synthesis of DNA or RNA in our cultures, even tho cultures remained as synchronous in vitro as they are in vivo. An estimate is presented of the amount of DNA made during one cycle in vitro.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

Bevorzugter Einbau markierten Uridins in die Vorläufer von Chloroplasten-Ribosomen in Algenzellen

Summary After short time pulses with 5-[3H]uridine have been given to Chlorella cells, most of the radioactivity of the ribosome fractions is neither in the polysomes nor in the cytoplasmic ribosomes. Peaks with sedimentation of about 50 S and 30 S are found which are comparable in sedimentation to ribosomal subunits of Escherichia coli. During chase treatment with the one-hundred-fold amount of unlabelled uridine, the radioactivity shifts into the 70 S region. The RNA of the rapidly labelled 50 S and 30 S particles is shown to have 23 S, 14 S and 5 S, respectively.In contrast to this, radioactive inorganic phosphate and amino acids are mainly incorporated into the cytoplasmic ribosomes with 80 S and into, their polysomes.The chloroplast-damaged mutant of Chlorella, Nr.125 of Schwarze, shows no uridine incorporation into particles of 50 S and of 30 S, but some very weak labelling of the 80 S cytoplasmic monosomes.Nitrogen deficient Chlorella cells also incorporate uridine mainly into the 50 S and 30 S particles. When chase treatment with unlabelled uridine is performed under recovering conditions, the label shifts into the 70 S particles as well as into the 80 S cytoplasmic ribosomes.The results indicate that in Chlorella, uridine is incorporated into chloroplast ribosome precursors rather than into particles of nuclear origin.     

Separate pyrimidine-nucleotide pools for messenger-RNA and ribosomal-RNA synthesis in HeLa S3 cells.

Kinetic analyses of mRNA and 28-S RNA labeling [3H]uridine revealed distinctly different steady-state specific radioactivities finally reached for uridine in mRNA and 28-S RNA when exogenous [3H]uridine was kept constant for several cell doubling times. While the steady-state label of (total) UTP and of uridine in mRNA responded to the same extent to a suppression of pyrimidine synthesis de novo by high uridine concentrations in the culture medium, uridine in 28-S RNA was scarcely influenced. Similar findings were obtained with respect to labeling of cytidine in the various RNA species due to an equilibration of UTP with CTP [5-3H]Uridine is also incorporated into deoxycytidine of DNA, presumably via dCTP. The specific radioactivity of this nucleosidase attained the same steady-state value as UTP, uridine in mRNA and cytidine in mRNA. The data indicate the existence of two pyrimidine nucleotide pools. One is a large, general UTP pool comprising the bulk of the cellular UTP and serving nucleoplasmic nucleic acid formation (uridine and cytidine in mRNA, deoxycytidine in DNA). Its replenishment by de novo synthesis can be suppressed completely by exogenous uridine above 100 muM concentrations. A second, very small UTP (and CTP) pool with a high turnover provides most of the precursors for nucleolar RNA formation (rRNA). This pool is not subject to feedback inhibition by extracellular uridine to an appreciable extent. Determinations of (total) UTP turnover also show that the bulk of cellular RNA (rRNA) cannot be derived from the large UTP pool.     

Qualitative and Quantitative Studies on DNA and RNA Synthesis in Loxodes striatus Nuclei*

SYNOPSIS. In this study the characteristics of the synthesis of DNA and RNA in the nuclei of Loxodes were investigated. Loxodes striatus is a primitive ciliate with 2 pairs of structurally differentiated diploid nuclei, the macro- and micronuclei. The macronuclei are differentiated morphologically into a clearly recognizable central core and an outer zone. To determine DNA and RNA synthesis, individual organisms were analyzed by autoradiography after incubating groups of cells with a ³H-labeled precursor ([³H]thymidine for DNA and [³H]uridine for RNA). The following observations were made: (A) All portions of macro- and micronuclei appeared to contain DNA as judged by the localizations of incorporated [³H]thymidine. (B) The macro- and micronuclei did not synthesize DNA at the same time; moreover, the duration of DNA synthesis in the former was much longer than of the latter nucleus. (C) Replication of DNA in the inner core and outer zone of the macronucleus occurred at separate times with little if any overlap. (D) All of the detectable [³H]uridine incorporation was found in the macronucleus and none in the micronucleus. Within the macro-nucleus the central core was more heavily labeled. (E) The quantitative differences in the label of the different components of the nuclear complex were investigated. (F) Contrary to the previously reported information our results suggest that DNA synthesis can occur in adult macronuclei. The possible explanation of these results is discussed in the context of the nuclear evolution of ciliates and of recent information on nuclear differentiation.