Axonal migration of various ribonucleic acid species along the optic tract of the chick

—The avian visual system has been used to study the axonal transport of RNA and protein. After monocular injection of radioactive uridine into 1-day-old chicks, a considerable amount of labelled RNA migrated along the optic tract to the optic tectum contralateral to the injected eye. This RNA was largely ribosomal, although it was contained in several subcellular fractions. The migration of RNA appeared to be a slow process. However, following monocular injection of radioactive proline, the migration of ribosomal protein was rapid. This discrepancy was resolved by examination of the kinetics of labelling of RNA and protein within the retina after intraocular injection of a mixture of labelled uridine and proline. Cytoplasmic RNA was labelled much more slowly than cytoplasmic protein. This lag in labelling of RNA could account for the delayed arrival of RNA at the contralateral optic lobe and suggests that ribosomes may travel rapidly along the axon. In other experiments, eyes were removed 4 days after the injection of labelled precursors. After a further 14 days, the remaining radioactivity in RNA and protein of contralateral optic lobes was 5–15% of that attributable to migration along the axon in control, unenucleated birds. Thus, the survival of the bulk of migrating macromolecules depends on the integrity of synaptic terminals. This observation suggests that both RNA and protein migrate within the axon rather than extra-axonally, and that they remain largely within the nerve cells along the axons of which they are transported.     

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.     

Axoplasmic transport of RNA

The transport of RNA from the ganglion cell bodies within the retina to the contralateral optic tectum has been studied in the chick following intraocular injection of radioactive uridine. By tracing the appearance of labeled RNA at the proximal end of the optic nerve as it leaves the eyeball and comparing this to the time of arrival of RNA within the optic tectum, the migratory velocity of axonal RNA has been calculated to be around 12 mm per day. The continuation of RNA migration to the optic tectum in the presence of intracerebrally injected actinomycin-D but not in the presence of the intraocularly injected drug, suggests a retinal site of synthesis of the excess RNA found in the tectum innervated by the injected eye. A study of the rate of disppearance of radioactivity of the transported RNA in the optic lobes, suggested that this RNA turns over more rapidly than the bulk of tectal RNA. The destination of migrating RNA within the optic tectum has been autoradiographically examined. Most radioactive RNA is found in the outer tectal layers in which are found the afferent fibers of the optic tract and most of their synaptic terminations. Label is not confined to these areas however but is also present in the deeper layers of the optic tectum which are not known to contain any primary synapses of the axons from retinal ganglion cells.     

The regulation of protein synthesis in the liver of rats. Mechanisms of dietary amino acid control in the immature animal

Weanling (23-day-old) rats were fed either on an amino acid-deficient diet (6% of casein, which in effect represents an `amino acid-deficient' diet) or on a diet containing an adequate amount of protein (18% of casein) for 28 days. The hepatic cells from the animals fed on the low-protein diet were characterized by low amino acid content, almost complete inhibition of cell proliferation and a marked decrease in cell volume, protein content and concentration of cytoplasmic RNA compared with cells from control rats. The lower concentration of cytoplasmic RNA was correlated with a decreased ribosomal-RNA content, of which a larger proportion was in the form of free ribosomes. The protein-synthetic competence and messenger-RNA content of isolated ribosomes from liver cells of protein-deprived animals were 40–50% of those noted in controls. At 1hr. after an injection of radioactive uridine, the specific radioactivity of liver total RNA was greater in the group fed on the low-protein diet, but the amount of label that was associated with cytoplasmic RNA or ribosomes was significantly less than that noted in control animals. From these data it was concluded that dietary amino acids regulate hepatic protein synthesis (1) by affecting the ability of polyribosomes to synthesize protein and (2) by influencing the concentration of cytoplasmic ribosomes. It is also tentatively hypothesized that the former process may be directly related to the concentration of cellular free amino acids, whereas the latter could be correlated with the ability of newly synthesized ribosomal sub-units to leave the nucleus.     

Origin of axoplasmic RNA in the squid giant fiber

The origin of axoplasmic RNA in the squid giant fiber was investigated after exposure of the giant axon or of the giant fiber lobe to [3H]uridine. The occurrence of a local process of synthesis was indicated by the accumulation of labeled axoplasmic RNA in isolated axons incubated with the radioactive precursor. Similar results were obtained in vivo after injection of [3H]uridine near the stellate nerve at a sizable distance from the ganglion. Exposure of the giant fiber lobe to [3H]uridine under in vivo and in vitro conditions was followed by the appearance of labeled RNA in the axoplasm and in the axonal sheath. While the latter process is attributed to incorporation of precursor by sheath cells, a sizable fraction of the radioactive RNA accumulating in the axoplasmic is likely to originate from neuronal perikarya by a process of axonal transport.     

Axonal transport of RNA during regeneration of the optic nerves of goldfish.

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of 3H-uridine has been studied during various stages of optic nerve regeneration. 3H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Five were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, 14C-proline was also injected into the right eye as a marked of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of 3H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microsocpic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be 3H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of 3H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikaprya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.     

Axonal transport of RNA during regeneration of the optic nerves of goldfish

The distribution of radioactive RNA and RNA precursors in the goldfish optic tecta following intraocular injection of ³H-uridine has been studied during various stages of optic nerve regeneration. ³H-uridine was injected into the posterior chamber of the right eye 17, 30, or 60 days after both optic nerves were crushed. Fish were sacrificed at time intervals ranging from 0.5 to 21 days after injection. One day prior to sacrificing, ¹⁴C-proline was also injected into the right eye as a marker of fast axonal protein transport. Seventeen to 23 days after crushing, the approximate time of nerve reconnection, the amount of radioactive RNA appearing in the left optic tectum was increased by more than ten times control values. Approximately 30 days after crushing the nerve, when the reconnected nerve is maturing, RNA values were still elevated, but significantly decreased from the earlier stage. By 60 days after crushing the optic nerve, the amounts of RNA in the left tectum was close to normal. Evidence suggesting that, at least, some of the radioactive RNA in the tectum originated from RNA transported along optic axons rather than from RNA synthesized locally in the tectum was provided by autoradiographic experiments. Autoradiograms of paraffin sections taken from the goldfish optic tecta after the intraocular injection of ³H-uridine showed a distribution of grains in a linear pattern, suggesting a distribution over the incoming fibers during the reconnection stage of regeneration. Electron microscopic autoradiography of glutaraldehyde fixed epoxy sections confirmed that a significant number of grains (shown to be ³H-RNA) were, in fact, over regenerating optic axons. Intracranial injection of ³H-uridine, during the same stage of regeneration, on the other hand, resulted in a distribution of grains, specifically over cell perikarya. These experiments suggest that during the reconnection phase of nerve regeneration, large amounts of RNA may be carried within regenerating optic axons as they enter the optic tectum.     

Synthesis of RNA by dissociated cells of the sea urchin embryo

The incorporation of radioactive uridine into RNA by micromeres, mesomeres and macromeres of sea urchin embryos was studied, employing methods for separating the cell types in pure suspension. At the 16-cell stage, the 3-cell types, on a per genome basis, synthesized RNA at approximately the same rate although on a per mg protein basis the micromere-RNA synthetic rate was considerably higher than either mesomeres or macromeres. At the 32-cell stage, incorporation of radioactive uridine by micromeres decreased relative to mesomeres and macromeres. It was demonstrated that radioactive uridine could not be effectively washed or diluted out of the cells of 16-cell stage embryos. Experiments on reaggregating cells did not detect any transfer or transport of radioactivity from micromeres to the other cells. Possible explanations for these findings versus the disparate results of previous investigators were presented.     

Problems associated with the labelling of RNA with radioactive precursors in vivo (author's transl)]

The radioactivity of RNA, DNA and proteins in the liver, muscles and cerebrum of 30-day-old rats after labelling with [3H]uridine, [14C]uridine, [3H]cytidine or [3H]orotic acid was measured. It was found that after administration of [3H]uridine, the proteins were 5 - 10 times more radioactive than the RNA. After administration of [14C]uridine, the proteins were 1 - 2 times more heavily labelled than the RNA. Hydrolysis of the proteins followed by chromatography of the amino acids revealed that the protein labelling was mostly due to [3H]glutamate. In the liver, [3H]orotic acid produced very specific labelling of the RNA. The radioactivity of the proteins is very slight. However, the specific labelling of the RNA in the muscles and cerebrum is not so pronounced with this precursor. [3H]Cytidine is an ideal precursor for RNA. The labelling of protein in all three organs examined is very slight, and furthermore, the specific activity of the RNA is 10 - 20 times higher than after labelling with uridine. We were also able to show that after labelling with radioactive uridine, the method of isolation of RNA by alkaline hydrolysis gives incorrect results, because [3H]amino acids interfere with the measurement of the specific activity of the RNA. The heavy labelling of proteins by [3H]-uridine must also be taken into account in histoautoradiography, because our experiments showed that in liver, the proteins in the cell nucleus are 3 times as radioactive as the nucleic acids. The particulate components of the cytoplasm are even 20 times more radioactive than the nucleic acids.     

Isolation and properties of polyribosomes and fragments of the endoplasmic reticulum from rat liver

1. A centrifugation method for the fractionation of the postmitochondrial fraction from rat-liver homogenates is described. The technique, in which no detergent is used, may be used as a tool to discriminate between two classes of ribosomes. One class is firmly bound to membranes and the other consists either of free polysomes or of ribosomes attached by weaker forces to the membranes of the endoplasmic reticulum. 2. Electron-micrograph studies revealed that the polysomes were not contaminated with bound ribosomes or with membranous fragments. 3. The separated fractions were characterized by their RNA, protein, ribonuclease and phospholipid content. 4. The influence of starvation on the RNA and protein contents of the different fractions was investigated. 5. Labelling of the various centrifugal fractions in vivo revealed no difference in uptake of radioactive amino acid between the two classes of ribosomes. 6. Incorporation of radioactive leucine in vitro and the polyuridylic acid-directed phenylalanine incorporation were similar for both classes of ribosomes.     

Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

Axoplasmic Transport of Transfer RNA in the Chick Optic System

It has previously been shown that 4S RNA is transported in the optic nerve of the chick, but that no movement of rRNA can be detected. The 4S component behaved as though it were composed mainly of transfer RNA (tRNA), but the possibility remained that it could contain significant amounts of material resulting from RNA degradation. The transport of this 4S component has been examined in more detail to determine its nature. In addition, the transported material was examined to establish whether the transport of tRNA is a general phenomenon or that there are only a limited number of species involved. This was done using the same principles applied in the previous study; i.e., the specific activities of separated 4S RNA species appearing in the optic tectum 4 days after intraocular injection of [3H]uridine were compared with that of 5S RNA, a nontransported species. The separation was accomplished using 2.8-5-10-17% slab polyacrylamide gels, and 18 separate regions of 4S species could be identified. The results show that at least most, if not all 4S RNA species are transported. In a separate series of experiments the 4S RNA was aminoacylated and again separated on slab gels. In this instance, the RNA was labelled with [3H]uridine and the aminoacyl component with [14C]amino acids. Gel profiles of these dual-labelled components showed excellent correspondence between the two labels, demonstrating that 4S RNA species could be aminoacylated and were therefore tRNA species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of messenger ribonucleic acid in excised pea-seedling root segments. Separation of the messenger from microsomes by electrophoresis

1. Electrophoresis on cellulose acetate membrane in a tris-pyrophosphate buffer was used to separate microsomal fractions into three components: (1) the lipoprotein; (2) the nucleoprotein (termed the beta-band); (3) traces of free RNA (termed the alpha-band). In tris buffer containing Mg(2+) the alpha-band was not obtained. 2. The incorporation of uridine and phosphate into RNA by excised pea-seedling root segments was studied by using this electrophoretic technique. 3. It was shown that after a short (;pulse') incubation in the radioactive precursor and a longer (;chase') incubation in the non-radioactive precursor most of the incorporation was into the RNA of the alpha-band and little into that of the beta-band. Previous work showed that in roots of whole seedlings the incorporation is mostly into the ribosomal RNA, corresponding to the material in the beta-band. 4. A pulse-labelled RNA has also been found; this seems to be a cell fraction distinct from the microsomes or ribosomes. 5. The apparent base compositions of labelled RNA in the alpha-band and small amounts of labelled RNA in the beta-band and of unfractionated RNA were very different from the composition of ribosomal or transfer RNA, and somewhat like that of DNA. 6. It is suggested that the excised root segment synthesizes a messenger-RNA fraction labelled after a pulse incubation and a distinct messenger RNA labelled after a pulse and chase incubation, but no ribosomal or transfer RNA. The system is thus similar to the ;step-down' culture conditions in bacteria.     

Protein synthesis by membrane-bound and free ribosomes of the developing rat cerebral cortex

1. Rates of RNA and protein synthesis were measured in rat cerebral-cortex slices, and compared with amino acid incorporation into protein by membrane-bound and free ribosomes from the same tissue, in the first 3 weeks of life. 2. A rapid age-dependent decline in the incorporation of labelled precursors into both RNA and protein was observed, which was more marked for amino acid incorporation into protein. 3. Although membrane-bound ribosomes comprise only a small fraction of total ribosomes, they were more active in incorporating amino acids into protein than were free ribosomes, especially immediately after birth. The decline in activity with age was more marked in the membrane-bound fraction than in free ribosomes. This loss of activity was largely independent of alterations in soluble factors or endogenous mRNA content and appeared to involve some alteration of the function of the ribosome itself, with relatively small alterations in the ratio of membrane-bound to free ribosomes. 4. Thyroidectomy, performed soon after birth, had no effect on the incorporation of radioactive precursors into RNA or protein by either slices or the cell-free preparations during the first 3-4 weeks of life.     

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.     

POLYADENYLIC ACID-CONTAINING RNA FROM RAT BRAIN SYNAPTOSOMES

Abstract— Synaptosomal RNA of rat brain was labelled in vivo by intracranial injection of tritiated uridine. The change in the specific activity of this material with time was similar to that of polysomal RNA. The percent of the radioactive synaptosomal RNA which bound to oligo(dT)-cellulose columns decreased with time after intracranial labelling. The percent of the total synaptosomal RNA which bound to oligo(dT)-cellulose was greater than that of polysomes. The length of the polyadenylate (poly(A)) sequence of synaptosomal RNA was approximately one-half that of polysomal RNA, and about the same as that from mitochondria. Investigation of synaptosomal RNA using sucrose gradients and polyacrylamide gel electrophoresis indicated that there were several distinct species present, and that they were similar to those from the mitochondria. The poly(A)-containing RNA isolated from synaptosomes stimulated the incorporation of radioactive leucine into TCA-precipitable material in a cell-free protein synthesis system. Isolation of RNA from subsynaptosomal components indicated that most, if not all, of the synaptosomal messenger activity was localized in the synaptic mitochondria.     

A precursor of globin messenger RNA

The size of pulse-labeled globin messenger RNA nucleotide sequences was investigated, to determine whether newly transcribed globin mRNA molecules are larger than steady-state globin mRNA. Molecular hybridization techniques were used to compare directly the sedimentation of steady-state (unlabeled) and pulse-labeled (radioactive) globin mRNA sequences in the same analytical sucrose gradient. In gradients containing 98% formamide, radioactive globin mRNA sequences from mouse fetal liver cells labeled for 15 to 20 minutes with [³H]uridine sediment in a broad band with a peak at approximately 14 S, while steady-state globin mRNA sediments at 10 S. The large radioactive RNA can be recovered from one gradient and recentrifuged in a second gradient, in which it again sediments in a broad band with a peak at 14 S. The large radioactive RNA is cleaved to 10 S during a 75-minute “chase” with either actinomycin D or unlabeled uridine plus cytidine. The estimated half-life of the precursor is 45 minutes or less under these conditions. A covalent RNA precursor larger than 18 S with a similar turnover rate is not observed.     

In vitro effects of ACTH on uridine incorporation into rapidly labelled adrenal RNA of normal and dexamethasone-suppressed rats

Adrenals from control and dexamethasone suppressed rats were incubated in a continuous flow system and exposed to ACTH and radioactive uridine. The adrenal response to ACTH was measured by corticosterone determination in the effluent medium. Cytoplasmic RNA was extracted and fractionated by polyacrylamide gel electrophoresis. ACTH increased the incorporation of labelled uridine into total RNA and all RNA species on the gels of normal and suppressed adrenals, but with a preferential incorporation into the 18S fraction.     

Pyrimidine-rich oligonucleotides from rat-liver ribosome surface.

The distribution of oligonucleotides which are released from rat liver ribosomes by treatment with pancreatic ribonuclease has been studied. Rat liver monoribosomes lost from 15 to 17% of their nucleotides by treatment with pancreatic ribonuclease. This quantity was highly reproducible and did not depend significantly on the temperature (0-20 degrees C) and time (10-120 min) of incubation or on the concentration of enzyme (1:5000-1:50). Whereas the amounts of oligonucleotides liberated was 16%, it was shown by column chromatography that they consisted of 71% mononucleotides, 16% dinucleotides, 6% trinucleotides, 4% tetranucleotides and 2% pentanucleotides and that these oligonucleotides were enriched in uridine, containing approximately half of the uridine residues present in the high-molecular-weitht ribosomal RNA. The high molecular weight of the RNA from ribonuclease-treated ribosomes was preserved until it was heated; after heating, RNA fragments having sedimentation coefficients of 5 S and less were present. It is inferred that the olignucleotides are derived from pyrimidine-rich clusters located in single-stranded "hairpin" loops on the outside surface of the ribosome.