Axonal transport of 4S RNA in the chick optic system

The axonal transport of tRNA has been investigated in the chick optic system. Chicks were injected with [³H]uridine intraocularly or intracranially and the RNA of the retina, nerve complex, and tecta separated by polyacrylamide gel electrophoresis and then counted. The ratio of TRNA to rRNA specific activities increased with time in both the nerve complex and contralateral tectum. The ratio increased more rapidly in the nerve complex than the tectum. However, no increase was observed in the case of intracranially injected animals. This is consistent with the axonal flow of tRNA. When [methyl-³H]methionine was used as precursor, the preferential labeling of 4S RNA to rRNA which resulted more clearly showed a transport of 4S RNA from the retinal cells to the tectum. In conclusion, it was found that about 40% of the radioactive RNA observed within the optic tectum 4 days after an intraocular injection of [³H]uridine was accounted for by 4S RNA which had flowed from the retina. However, the migration of a methylated RNA molecule of size 4S, but unrelated to tRNA, cannot be entirely eliminated.     

Frequency of N-benzyladenine incorporation into major RNA fractions of tobacco cell suspensions grown at stimulatory or cytostatic cytokinin concentration

The frequency of incorporation of the cytokinin N⁶-[p-³H]benzyladenine into major RNA species of tobacco (Nicotiana tabacum cv W 38) cells steadily increased as a function of its concentration in the culture medium, up to a 10 micromolar cytostatic overdose. During a 55-hour incubation of cells with 0.4 micromolar benzyladenine (BA), which is the optimal concentration for cell division, the incorporation frequency increased to one BA per 1.5 to 2.0 × 10⁴ conventional bases in total RNA. Frequencies of BA incorporation into 18S and 25S rRNA and into RNA precursors were very similar, 2- to 3-fold higher than the frequency of BA incorporation into the 4S + 5S RNA fraction. In cells incubated with 10 micromolar BA, the rate of RNA synthesis between 24 and 55 hours was lower than at optimal growth conditions; 18S and 25S rRNA synthesis was depressed more than the synthesis of 4S + 5S RNA. At 55 hours, BA was incorporated into total RNA at the steady state frequency of one per 1,300 conventional bases. All major RNA species were BA-labeled to approximately the same level, except that the labeling of the RNA precursors was 2-fold higher than the labeling of mature RNA species. These results may reflect an alteration in the processing of the RNA precursors at supra-optimal cytokinin concentration.     

A method using 3-O-methyl-D-glucose and phloretin for the determination of intracellular water space of cells in monolayer culture.

A method is presented for determining the extent of methylation of tRNAs synthesized in mammalian and bacterial cell systems and is based upon determining the distribution of radioactivity associated with the guanine constituents of total cellular tRNA preparations previously labeled with [2-¹⁴C]guanosine and with [methyl]-³H or -¹⁴C]methionine. Whereas labeling with guanosine provides a means of assessing the extent of methylation of the [2-¹⁴C]guanine residues incorporated into tRNA, methionine labeling provides a measure of the percentage of [methyl-³H or -¹⁴C]methylated constituents that are methylated guanines. Analyses such as the above reveal that the tRNA of KB cells acquires approximately three times as many methyl groups as that of E. coli B tRNA. Coupled with the knowledge that both mammalian and bacterial tRNA preparations contain an average of 24 guanine residues per molecule, the above analyses further reveal that 7.2 and 2.4 methyl groups are incorporated into each tRNA molecule synthesized in exponentially growing KB- and E. coli B-cells, respectively. Additional information regarding the extent of formation of individual methylated constituents per tRNA molecule synthesized is presented.     

Ribosomal ribonucleic acid maturation in synchronous strain l cells

Summary The incorporation of [³H]-5-uridine into cytoplasmic 18S and 28S ribosomal ribonucleic acid (rRNA) was examined in Colcemid-synchronized strain L cells during G1 and S phases of the cell cycle in the presence of 5×10⁻⁵ m uridine, which was determined to be the saturating concentration for this system. The data show that in S phase a significant increase occurs in the level of [³H]-5-uridine incorporation into each rRNA species. During a 90-min exposure period, S phase cells incorporate 3.4 times as much [³H]-5-uridine into 18S rRNA and 1.9 times as much into 28S rRNA as do G1 cells. The time required for maturation of the ribosomal RNA species during G1 and during S phase is the same, with 18S rRNA appearing in the cytoplasm in 20 min and 28S rRNA in 40 min.     

Changes in rate of RNA synthesis and ribosomal gene number during oogenesis of Drosophila melanogaster.

A new method for separating Drosophila egg chambers into different developmental classes (Jacobs-Lorena and Crippa, 1977) made it possible to study changes in the rate of ribosomal RNA (rRNA), 5S RNA, and tRNA synthesis and the changes in ribosomal gene number during oogenesis. Synthesis of RNA was measured by [³H]uridine incorporation in vivo and subsequent analysis on sucrose gradients or gel electrophoresis. Specific radioactivity of nucleotide pools has also been determined. Ribosomal gene number has been measured by hybridization of egg chamber DNA to rRNA of high specific radioactivity. Our findings led us to conclude that in Drosophila melanogaster: (i) rRNA, 5S RNA, and tRNA are synthesized in all stages of oogenesis. (ii) In every stage, rRNA is the main RNA species synthesized. (iii) The rate of rRNA, 5S RNA, and tRNA synthesis increases greatly during oogenesis and is paralleled by a similar increase in ribosomal gene number resulting from the polyploidization of the nurse cell nuclei.     

Ribonucleic acid synthesis and loss of viability in pea seed

Summary RNA synthesis and protein synthesis in embryonic axis tissue of viable pea (Pisum arvense L. var. N.Z. maple) seed commences during the first hour of germination. Protein synthesis in axis tissue of non-viable pea seed is barely detectable during the first 24 h after the start of imbibition. Nonviable axis tissue incorporates significant levels of [³H]uridine into RNA during this period but the level of incorporation does not increase significantly over the first 24 h of imbibition. In axis tissue of non-viable seed during the first hour of imbibition most of the [³H]uridine was incorporated into low molecular weight material migrating in advance of the 4S and 5S RNA species in polyacrylamide gels but some radioactivity was incorporated into a discrete species of RNA having a molecular weight of 2.7×10⁶. After 24 h, non-viable axis tissue incorporates [³H]uridine into ribosomal RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and a heterogeneous RNA species of molecular weight ranging from 2.2×10⁶ to 2.7×10⁶. No 4S or 5S RNA synthesis is detectable after 24 h of imbibition in non-viable axis tissue. Axis tissue of viable pea seed synthesises rRNA, 4S and 5S RNA, the low molecular weight material migrating in advance of the 4S and 5S RNA peak in polyacrylamide gels and the rRNA precursor species at both periods of germination studied. Loss of viability in pea seed appears to be accompanied by the appearance of lesions in the processing of rRNA precursor species and a significant loss of RNA synthesising activity.Abbreviations rRNA ribosomal RNA - TCA trichloroacetic acid - SLS sodium lauryl sulphate - PPO 2,5 Diphenyloxazole - POPOP 1,4-Bis-2-(4-methyl-5-penyloxazolyl)-benzene     

Onset of nucleic acid synthesis during germination of Pisum sativum L.

Summary Measurments of total nucleic acid content of the embryonic axis indicated that massive net synthesis of both DNA and RNA was initiated at approximately 30 h after the onset of germination. The onset of net nucleic acid synthesis was marked by an increase in the rate of incorporation of [³H]thymidine into DNA, and of [³H]orotic acid and [³H]uridine into both DNA and RNA. rRNA was usually more heavily labelled than tRNA, but was not preferentially accumulated, suggesting a grater rate of turnover of rRNA than tRNA. Some incorporation of precursors occurred prior to the onset of net nucleic acid synthesis, particularly into RNA. This was taken to represent nucleic acid turnover. There was no evidence that the scavenging pathways for nucleotide biosynthesis were more important than the normal pathways in contributing precursors for net nucleic acid synthesis.     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

Rapidly-labelled RNA species isolated from cell suspensions ofDaucus carota

Summary Carrot cells in suspension culture were incubated during the log-phase of the culture transfer cycle for different periods with one of the following precursors of nucleic acid synthesis: [^32P]-orthophosphate, [5,6-³H]-uridine, and [2-¹⁴C]-uridine. Cells were gently broken by a short period of sonication, and the total RNA of the cells was extracted by a phenoldetergent method at pH 9.0. Subsequently, crude RNA was purified from contaminating substances like carbohydrates and nucleotides, and the pure RNA preparations were characterized by MAK-chromatography and constant velocity sedimentation in isokinetic sucrose gradients.Rapidly-labelled RNA-fractions were detected in the radioactive profiles obtained with both separation methods. These RNA-fractions showed a high specific incorporation rate, but almost no detectable UV-absorbance,i.e., they are RNA species with a high turnover rate and represent only a small part of the total RNA of the cell. With increasing periods of labelling and in a series of pulse-chase experiments high molecular weight RNA-fractions released by high-salt washing of MAK-columns exhibited a shift of the incorporated radioactivity from fractions with higher to those of lower molecular weights. Furthermore, in sucrose gradients a similar shift was observed for RNA-fractions with estimated sedimentation coefficients of 50 S, 40 S, 34 S and 22 S; the radioactivity was converted from these high to the low S-values of the 26 S and 18 S rRNAs, respectively. This parallel in the behaviour of the high molecular weight RNA-fractions from both separation methods indicates their putative role as precursors of rRNA-synthesis. Moreover, there is evidence that the high molecular weight RNA-fractions from the MAK-columns which were eluted after the 26 S rRNA consist not only of the precursors of rRNAs, but also of polydisperse RNA-fractions with S-values smaller than 18 S. These probably contain fractions of HnRNA and mRNA.     

Leishmania donovani: Autoradiographic evidence for molecular exchanges between parasites and host cells

Promastigotes of Leishmania donovani, 2S strain, or hamster peritoneal exudate cells, were pulse labeled in vitro with [³H]uridine or [³H]leucine. Washed labeled parasites were used to infect unlabeled macrophages in Leighton tube cultures. Washed labeled cells in Leighton tube cultures were also infected with unlabeled parasites. Cover slips were harvested at various times following infection, methanol fixed, and washed in cold trichloroacetic acid, dipped in NTB-3 nuclear emulsion (Kodak) and developed after 2 wk in the dark. Grain counts and photographs showed that when host cells were prelabeled with either compound then radioactive material accumulated in the parasite. Likewise, when parasites were prelabeled, radioactive material accumulated in the host cells. Experiments using [6-³H]uridine, RNAse, DNAse, and prelabeled macroghages indicated parasites were synthesizing DNA from host cell RNA precursors or precursor pool. The studies thus describe a system for investigating the molecular level relationships between Leishmania species and their host cells.     

Effect of hypoxia on nucleic acid and protein synthesis in different brain regions

The incorporation of [methyl-³H]thymidine into DNA, of [5-³H]uridine into RNA, and of [1-¹⁴C]leucine into proteins of cerebral hemispheres, cerebellum, and brainstem of guinea pigs after 80 hr of hypoxic treatment was measured. Both in vivo (intraventricular administration of labeled precursors) and in vitro (tissue slices incubation) experiments were performed. The labeling of macromolecules extracted from the various subcellular fractions of the above-mentioned brain regions was also determined. After hypoxic treatment the incorporation of the labeled precursors into DNA, RNA, and proteins was impaired to a different extent in the three brain regions and in the various subcellular fractions examined; DNA and RNA labeling in cerebellar mitochondria and protein labeling in microsomes of the three brain regions examined were particularly affected.     

Enhanced activity of tRNA-pseudouridine synthetase in Yoshida ascites sarcoma.

Five days after transplantation of Yoshida ascites sarcoma cells into a rat, specific activity of tRNA-pseudouridine synthetase was extremely high in the supernatant of tumor cells and moderately high in the tumor-bearing rat liver compared with normal rat liver. Enzyme assay was performed at 37°C by determining the release of tritium from heterogeneous [³H] tRNA extracted from $\text{E. coli}$ B grown in the presence of [5,6-³H]-uracil and resulting in the increased ratio of the amount of pseudouridine to uridine residues in [³H] tRNA. Neither [5-³H]-uridine, [5,6-³H]-UTP, nor [5,6-³H]-poly U released tritium in the present assay conditions.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

THE DIRECT MEASUREMENT OF THE AXOPLASMIC TRANSPORT OF INDIVIDUAL RNA SPECIES: TRANSFER BUT NOT RIBOSOMAL RNA IS TRANSPORTED

Analysis of chick retinal and tectal RNA revealed that in addition to the major cytoplasmic RNAs (rRNA and tRNA), a number of the small mol wt nuclear RNAs (snRNAs) can also be detected. Subfractionation data indicated that one of these molecules, DD′, is of at least 95% nuclear location within the retina. Thus, very little, if any, of the retinal DD′ is available for axoplasmic transport from the retina into the optic nerve and tectum. Following intraocular injection of [³H]uridine, considerable incorporation of isotope into DD′ was observed within the optic tectum after 4, 8 and 16 days. This result indicates the presence of considerable local (i.e. tectal) synthesis. The specific activities of 29S, 18S and 5S rRNA and 4s tRNA relative to that of DD′ were measured in the optic tectum 8 and 16 days after the intraocular introduction of [³H]uridine. The same measurements were also made in intracranially injected animals. While the 29S/DD′, 18S/DD′ and 5S/DD′ specific activity ratios obtained were independent of the injection route, the 4S/DD′ ratio obtained from intraocularly injected animals was significantly greater (at least 2-fold) than that obtained from intracranially injected animals. Similar analysis was also performed with the optic nerve complex at 16 days post-injection with identical results. These results demonstrate that tRNA, but not rRNA, is transported from the retina into the optic nerve and tectum in the 2-day-old chicken.     

Distribution of incorporated, synthetic cytokinins in ribosomal RNA preparations from tobacco callus

The distribution of incorporated synthetic cytokinins (N⁶-[8-¹⁴C]benzyladenine ([8-¹⁴C]bzl⁶Ade) and N⁶[8-¹⁴C]furfuryladenine ([8-¹⁴C]fr⁶Ade) in ribosomal RNA prepared from tobacco callus (Nicotiana tabacum L. var. Wis. No. 38) grown in the presence of one of these for 25 or 26 days has been studied. The rRNA of tissue supplied with [8-¹⁴C]bzl⁶Ade or [8-¹⁴C]fr⁶Ade was fractionated by methylated albumin-Kieselguhr column chromatography and preparative gel electrophoresis, respectively. In each case about 80% of the incorporated cytokinin was recovered as the ribonucleoside [8-¹⁴C]bzl⁶A or [8-¹⁴C]fr⁶A in the rRNA peak after the fractionations. [8-¹⁴C]fr⁶A was found associated with both the 18S and 25S rRNA components in quantities roughly proportional to their 260 nm absorbance. This pattern of apparently nonspecific association was not affected by prior denaturation of the RNA with formamide.     

The control of ribonucleic acid synthesis in bacteria. The synthesis and stability of ribonucleic acid in rifampicin-inhibited cultures of Escherichia coli

A study was made of the kinetics of labelling of the stable ribonucleic acids (rRNA+tRNA) and the unstable mRNA fraction in cultures of Escherichia coli M.R.E.600, inhibited by the addition of 0.1g of rifampicin/l. Labelling was carried out by adding either [2-¹⁴C]- or [5-³H]-uracil as an exogenous precursor of the cellular nucleic acids. From studies using DNA RNA hybridization, the kinetics of the synthesis and degradation of mRNA was followed in the inhibited cultures. Although a considerable proportion of the mRNA labelled in the presence of rifampicin decayed to non-hybridizable products, about 25% was stabilized beyond the point where protein synthesis had finally ceased. It therefore seems unwise to extrapolate the results of studies on mRNA stability in rifampicin-inhibited cultures to the situation existing in the rate of steady growth, where there appears to be little, if any, stable messenger. The kinetics of labelling of RNA in inhibited cultures indicated that the clapsed time from the addition of rifampicin to the point at which radioactivity no longer enters the total cellular ribonucleic acids is a measure of the time required to polymerize a molecule of rRNA. At 37°C, in culture grown in broth, glucose–salts or lactate salts media, exogenous [2-¹⁴C]uracil entered rifampicin-inhibited cells and was incorporated into RNA for 2 3min after the antibiotic was added. Taking this time as that required to polymerize a complete chain of 23S rRNA, the polymerization rate of this fraction in the three media was 25, 22 and 19 nucleotides added/s to the growing chains. Similar experiments in cultures previously inhibited by 0.2g of chloramphenicol/l showed virtually identical behaviour. This confirmed the work of Midgley & Gray (1971), who, by a different approach, showed that the polymerization rate of rRNA in steadily growing and chloramphenicol-inhibited cultures of E. coli at 37°C was essentially constant at about 22 nucleotides added/s. It was thus confirmed that the rate of polymerization of at least the rRNA fraction in E. coli is virtually unaffected by the nature of the growth medium and therefore by bacterial growth rate.     

Toxoplasma gondii: Growth in the absence of host cell protein synthesis

Host cell protein synthesis continues when cultured cells are infected by Toxoplasma gondii. In order to determine if this host function is necessary for the parasite we used two independent methods that specifically block cellular protein synthesis. In the first, we infected a temperature-sensitive Chinese hamster ovary cell mutant that has a thermolabile leucyl tRNA synthetase. At the restrictive temperature of 40 C, the mutant cells showed only negligible protein synthesis that was probably mitochondrial. At this temperature, the growth and nucleic acid synthesis of T. gondii proceeded normally and [³H]leucine was specifically incorporated into the parasite as demonstrated by autoradiography. A secpnd method for blocking protein synthesis by the host cell employed treatment of uninfected human fibroblast cells with muconomycin A, an inhibitor of initiation. Repeated washing of monolayer cultures reduced the free muconomycin A to an insignificant level while the cells remained incapable of protein synthesis. T. gondii infected and grew normally in the inhibited cells. Autoradiographic localization of the incorporation of [³H]leucine showed that it was almost exclusively in the intracellular parasites in the cells pretreated with muconomycin A. In the untreated control most of the [³H]leucine was incorporated by the host cell rather than the parasite. We conclude that de novo protein synthesis by the host cell is not required to support the growth of intracellular T. gondii.