Selective determination of mRNA specific radioactivity in HeLa cells without the use of inhibitors

Polysomes from (³H)-uridine pulse-labeled HeLa cells were isolated and the specific radioactivity of polysome-associated mRNA was determined by selective enzymic hydrolysis at 0°C of the $\text{inter}$ribosomal mRNA sections. $\text{Intra}$ribosomal mRNA protected from hydrolysis during ribonuclease treatment and subsequently isolated by the proteinase K method (1) exhibited the same specific radioactivity as the interribosomal mRNA split products.When labeled polysomes were subjected to ribonuclease treatment at 25°C instead of 0°C a higher specific radioactivity of the interribosomal split products resulted, while intraribosomal sections still exhibited the same values as after 0°C treatment. The labeled polysomes used as substrate exhibited one single A₂₆₀ and radioactivity peak in CsCl density gradients. No RNP material banding at ? = 1.35 ? 1.45 could be detected. However, the radioactivity maximum banded at slightly lower densities than the A₂₆₀ peak (? = 1.55 versus 1.57). The shift appears to be caused by a contaminant RNA. These findings as well as the radioactivity pattern of pulse-labeled polysomes in sucrose gradients may indicate the presence of newly synthesized mRNA associated with monosomes (and oligosomes) protected from ribonuclease action at 0°C by (transport?) proteins.     

Physical aspects and cytoplasmic distribution of messenger RNA in mouse kidney.

As a prerequisite to examining mRNA metabolism in compensatory renal hypertrophy, polyadenylated RNA has been purified from normal mouse kidney polysomal RNA by selection on oligo(dT)-cellulose. Poly(A)-containing RNA dissociated from polysomes by treatment with 10 mM EDTA and sedimented heterogeneously in dodecyl sulfate-containing sucrose density gradients with a mean sedimentation coefficient of 20 S. Poly(A) derived from this RNA migrated at the rate of 6-7 S RNA in dodecyl sulfate-containing 10% polyacrylamide gels. Coelectrophoresis of poly(A) labeled for 90 min with poly(A) labeled for 24 h indicated the long-term labeled poly(A) migrated faster than pulse-labeled material. Twenty percent of the cytoplasmic poly(A)-containing mRNA was not associated with the polysomes, but sedimented in the 40-80 S region (post-polysomal). Messenger RNA from the post-polysomal region had sedimentation properties similar to those of mRNA prepared from polysomes indicating post-polysomal mRNA was not degraded polysomal mRNA. Preliminary labeling experiments indicated a rapid equilibration of radioactivity between the polysomal and post-polysomal mRNA populations, suggesting the post-polysomal mRNA may consist of mRNA in transit to the polysomes.     

Evidence for the attachment of RNA to pulse-labeled DNA in the slime mold, Physarum polycephalum

When $\text{Physarum}\text{polycephalum}$ is pulse-labeled for up to 20 minutes with ³H-thymidine and the shortest labeled DNA strands are partially purified by sedimentation through a neutral aqueous sucrose gradient and then through a formamide-sucrose gradient, these short strands band in Cs₂SO₄ isopycnic density gradients at a density greater than that of bulk single-stranded DNA. Their density is brought partially or nearly completely back to that of single-stranded DNA by hydrolysis with pancreatic RNase A or alkali, respectively. Therefore the dense material attached to the short pulse-labeled DNA strands consists at least partially of RNA.     

Glycosylation of nascent polypeptide chains in Aspergillus niger

Polysomes were isolated from Aspergillus niger and were characterized on sucrose gradients in several ways. First, they were found to be susceptible to degradation by treatment with RNase or EDTA. Second, they were labeled after treating mycelia with short pulses of [³H]uridine or [³H]leucine prior to polysome isolation. Third, they were capable of stimulating incorporation of [³H]leucine into trichloroacetic acid-precipitable material in a chick reticulocyte cell-free protein-synthesizing system. When isolated [³H]leucine pulse-labeled polysomes were treated with either EDTA-RNase or puromycin, 80–90% of the radioactivity was released, indicating that only the nascent polypeptide chains were labeled. After exposing mycelia for 1 min to [¹⁴C]mannose, the polysomes were exclusively labeled, indicating that initial glycosylation takes place on nascent polypeptide chains. Preincubation of mycelia with 2-deoxyglucose followed by pulse-labeling with [³H]leucine and [¹⁴C]mannose showed that 2-deoxy-d-glucose inhibits both protein synthesis and glycosylation. However, similar preincubation with tunicamycin caused an 80% drop in [¹⁴C]mannose label in the polysomes, but only a 10–20% drop of [³H]leucine label, suggesting that glycosylation of nascent chains in A. niger involves an oligosaccharide-lipid intermediate, since it has been shown that tunicamycin inhibits the synthesis of such an intermediate. When isolated polysomes were placed into an in vitro glycosylating mixture containing Mn²⁺, GDP-[¹⁴C]mannose, and smooth membranes from A. niger nascent chains were labeled. This reaction was shown to be dependent on addition of polysomes to the mixture and was not inhibited by 2-deoxy-d-glucose or tunicamycin. Both in vivo and in vitro glycosylated nascent chains were found to have about the same size range, and so it is suggested that in vitro no new oligosaccharide chains were synthesized, but preexisting chains were extended.     

Protein synthesis in halophytes: The influence of potassium,sodium and magnesium in vitro

The amino acid (³⁵S-methionine) incorporating activity of an in vitro wheat germ translation system was found to be maximal in 80 to 125 mol m^–3 K with 2 to 4 mol m^–3 Mg both as the acetate. Substitution of Na for K, or chloride for acetate at concentrations above 80 mol m^–3 inhibited incorporation. When the K acetate concentration was raised to 200 mol m^–3, no incorporation of radioactive methionine occurred.Translation by polysomes extracted from leaf tissue of S. maritima, supplemented with postribosomal supernatant from wheat germ, showed activity which was optimal in the presence of 225 mol m^–3 K acetate and 8 mol m^–3 Mg acetate. However, the translation system was not directly comparable with the wheat germ system, as studies with an initiation inhibitor, aurintricarboxylic acid, suggested that the S. maritima system was essentially elongation-dependent, while initiation occurred in the wheat germ system.Elongation-dependent polysomal preparations were extracted from leaves of the glycophytes Pisum sativum, Triticum aestivum, Oryza sativa and Hordeum vulgare, and from the halophytes Atriplex isatidea and Inula crithmoides. Translation by polysomes from the salt-tolerant plants was optimal at higher K and Mg concentrations, than by polysomes from the glycophytes. Furthermore, NaCl was better able partially to substitute for the role of K in polysomal preparations from halophytes than glycophytes.     

The effects of different thymidine concentrations on DNA replication in pea-root cells synchronized by a protracted 5-fluorodeoxyuridine treatment

Single-cell and DNA fiber autoradiography, cytophotometry and velocity sedimentation in alkaline sucrose gradients were used to analyse DNA replication and nascent replicon maturation in 5-fluorodeoxyuridine (FUdR)-synchronized cells of Pisum sativum. The replicon size was not significantly changed by the protracted FUdR treatment. When the synchronized cells were released from the inhibitor, labeled with [³H]TdR for 30 min, and chased in medium containing 1 × 10⁻⁶ M or lower concentrations of cold thymidine, DNA replication stopped after approx. 25% of the genome had replicated, and the nascent strands failed to grow above 9–12 × 10⁶ D single-stranded (ss) DNA. When the cells were chased in medium with 1 × 10⁻⁵ M cold thymidine, the DNA content of the labeled cells steadily increased with time and the size of the nascent molecules grew continuously until replicon size was achieved; then they were accumulated at replicon size until the cells arrived in late S or G2. When the FUdR-synchronized cells were chased in medium containing 1 × 10⁻⁴ M cold thymidine, the size of the nascent strands increased continuously with time, indicating that some neighbouring nascent replicons were joined as soon as they completed their replication. These observations led us to postulate that in FUdR-synchronized cells the rates of chain elongation, cell progression through the S phase and nascent replicon maturation are controlled by thymidine availability.     

Messenger ribonucleoprotein particles in developing sea urchin embryos

Messenger RNA entering polysomes during early development of the sea urchin embryo consists of both oogenetic and newly transcribed sequences. Newly transcribed mRNA enters polysomes rapidly while oogenetic mRNA enters polysomes from a pool of stable, nontranslatable messenger ribonucleoprotein particles (mRNPs) derived from the unfertilized egg. Protein content may relate to differences in the regulation of newly transcribed and oogenetic mRNAs. Oogenetic poly(A)⁺ mRNA was found to be present in both polysomal and subpolysomal fractions of cleavage stage and early blastula stage embryos. This mRNA was found to be present in subpolysomal mRNPs with a density of 1.45 g/cm³ in Cs₂SO₄. Poly(A)⁺ mRNPs released from polysomes of embryos cultured in the presence of actinomycin D sedimented in a broad peak centered at 55 S and contained RNA of 21 S. The density of these particles was sensitive to the method of release; puromycin-released mRNPs had a density of 1.45 g/cm³, while EDTA caused a shift in density to 1.55 g/cm³, indicating a partial loss of protein. The results with newly synthesized mRNAs contrast sharply. Newly transcribed mRNA in subpolysomal mRNPs had a density of 1.55–1.66 g/cm³, a density approaching that of deproteinized RNA. Messenger RNA released from polysomes either by EDTA or puromycin was examined to determine the possible existence of polysomal mRNPs. When [³H]uridine-labeled mRNA was released from late cleavage stage embryo polysomes by either technique, and centrifuged on sucrose gradients, two broad peaks were found. One peak centered at 30 S contained 21 S mRNA while the other at 15 S contained 9 S histone mRNA. When these fractions were fixed with formaldehyde, they banded on Cs₂SO₄ gradients at a density of 1.60–1.66 g/cm³, very similar to that of pure RNA. We conclude that the newly transcribed mRNA may be present in stable mRNPs containing up to 10% protein in either subpolysomal or polysomal fractions. These mRNPs are clearly distinguishable from the protein-rich mRNPs containing oogenetic mRNAs.     

Messenger RNA for isocitrate lyase from Chlorella fusca var. vacuolata

Chlorella fusca cultures growing in the light and adapting to acetate in the dark were labelled with adenine-³H and adenine-¹⁴C, respectively. Poly(A)-containing RNA from the mixed cultures was analysed for ¹⁴C/³H ratio after polyacrylamide gel electrophoresis in 98% formamide. The RNA from acetateadapting C. fusca cells contained excess label migrating in the gels at a position equivalent to about 0.85×10⁶ mol.wt. Partially purified anti-isocitrate lyase serum linked to p-aminobenzoyl-cellulose bound 3.5–13% of polysomes from acetate-adapting C. fusca, containing 5–10% of polysomal poly(A)-containing RNA. The antibody-bound poly(A)-containing RNA fraction showed a unimodal size distribution with a mean size of about 0.85×10⁶ mol.wt. after electrophoresis on 4% polyacrylamide gels in 98% formamide. Cell-free translation assays showed a three-fold enrichment of isocitrate lyase mRNA after antibody selection of polysomes and indicated that isocitrate lyase mRNA was abundant in acetate-adapting C. fusca cells.Abbreviations A ₂₆₀ unit The amount of material in 1.0 ml giving an absorbance of 1.0 at 260 nm in a 1 cm light path - PAB-cellulose p-aminobenzoyl-cellulose - SDS sodium lauryl sulphate To whom offprint requests are to be sent     

The purification of beta-galactosidase-specific polysomes by affinity chromatography.

Affinity chromatography on a β-galactosidase substrate analog-Sepharose column was used to purify β-galactosidase-specific polysomes from E. coli. The purification was monitored by hybridization of [³H]uridine pulse-labeled RNA extracted from polysomes to p lac 5 DNA. A purification of at least 12-fold was obtained. Binding of lac polysomes to the column required the presence of Sepharose-bound substrate analog; salt and pH conditions favorable to β-galactosidase binding; and intact polyribosomes. It was calculated that 40–50% of the labeled mRNA recovered was lac RNA.     

Nerve-dependent changes in content of ribosomes, polysomes, and nascent peptides in newt limb regenerates.

Regeneration of a newt limb requires a constant supply of adequate amounts of a neuronal contribution at the amputation site. Denervation during the early stages of regeneration precludes its growth and morphogenesis. It has been reported that denervation of a regenerating limb lowers the efficiency of incorporation of radioactive amino acids to 60% of contralateral control levels. To gain more insight into the mechanism responsible for this decrease, we examined the effects of denervation on the size distribution and quantity of regenerate polysomes. We characterized the [³⁵S]methionine-labeled nascent peptidyl-tRNA from polysomes by hydroxyapatite chromatography. Moreover, we show that the labeled nascent peptides on polysomes can serve as a measure to quantitate the relative amounts of ribosomes on polysomes and the relative size of the translational machinery. Thus, we report that [³⁵S]methionine-labeled nascent polypeptides on polysomes from denervated regenerates contain about 48% less radioactivity than those from controls. Despite decreased incorporation of [³⁵S]methionine into nascent peptides, the relative distribution of radioactivity across linear sucrose gradients is not significantly altered by denervation. Studies of polysomes labeled with [³H]uridine prior to denervation indicate that ribosome content is depressed by denervation. Our results suggest that the nerve-dependent decrease in protein synthesis is mediated by decreasing the number of ribosomes active in protein synthesis. In addition, similarities in the ratios of free monosomes to polysomes and the relative size distribution of polypeptides between denervated and innervated regenerates indicate that in denervated regenerates the number of translatable mRNA molecules decreases in a coordinate manner with the number of ribosomes active in protein synthesis.     

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.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.     

Incorporation of 13C,15N-labeled nucleosides and measurement of RNA synthesis and turnover in sea urchin embryos

The uptake of nucleosides into sea urchin embryos and their subsequent incorporation into RNA increases with increasing external nucleoside concentration. When embryos are incubated with high concentrations of ¹³C,¹⁵N-labeled nucleosides, newly synthesized RNA becomes sufficiently labeled with heavy isotope to be separated from unlabeled RNA on cesium formate equilibrium gradients. High concentrations of nucleosides do not affect development of embryos or rates of RNA synthesis. The extent of density-labeling of precursor pools increases with incubation time, and only levels off after many hours. During incubations with high concentrations of nucleosides, ATP pools expand up to twofold. Using density-labeling to circumvent precursor pool measurements, a method is presented to study the synthesis and decay of pulse-labeled RNA. The instantaneous rate of synthesis of total RNA at the blastula stage is 9.3 × 10^?15 mol of total nucleotide/embryo per minute and the average half-life of total RNA is 23 minutes.     

Myxococcus xanthus synthesizes a stabilized messenger RNA during fruiting body formation

Initial characterization of the unstable 5S-to-16S RNA fraction from developingMyxococcus xanthus cells reveals that it is rapidly labeled with radioactive RNA precursor and is associated with polyribosomes and released by puromycin from polyribosomes. The total unstable RNA fraction from 10-min pulse-labeled developing cells has a half-life of 13 min, compared with a 4-min half-life for unstable RNA (presumptive mRNA) from vegetative cells pulse-labeled for 2 min. We conclude that this developmental 5S-to-16S RNA contains messenger RNA and that this mRNA is stabilized compared with that in vegetative cells.     

Mobilization of newly synthesized RNAs into polysomes inXenopus laevis embryos

Summary The mobilization of newly synthesized 18S and 28S rRNAs, 4S RNA and poly(A)⁺ RNA into polysomes was studied in isolated cells ofXenopus laevis embryos between cleavage and neurula stages. Throughout these stages, 4S RNA and poly(A)⁺ RNA were mobilized immediately following their appearance in the cytoplasm. 18S rRNA however, stayed in the ribosomal subunit fraction for about 30 min until the 28S rRNA appeared, when the two rRNAs were mobilized together at an equimolar ratio. This mobilization, at a 1:1 molar ratio, appeared to be realized at initiation monome formation. Thus, the efficiency of the mobilization of two newly synthesized rRNAs, shortly after their arrival at the cytoplasm, differed considerably but difference disappeared once steady state was reached.The contribution of newly synthesized 18S and 28S rRNAs to polysomes remains small throughout early development. around 3% of newly synthesized 4S RNA is polysomal which is the same distribution observed for unlabeled 4S RNA. Less than 10% of the newly synthesized cytoplasmic poly(A)⁺ RNA was mobilized into polysomes during cleavage, but in later stages the proportion increased to around 20%–25%. These results show that newly synthesized RNAs are utilized for protein synthesis at characteristic rates soon after they are synthesized during early embryonic development. On the basis of the data presented here and elsewhere we discuss quantitative aspects of the utilization of newly synthesized and maternal RNAs during early embryogenesis.     

Size distribution of poly(A)-containing messencer RNA from free polysomes of HeLa cells

The sedimentation properties of pulse-labeled and long-term labeled mRNA from highly purified HeLa cell free-polysomes, selected for poly(A) content by two successive passages through poly(T)-cellulose columns, were analyzed under native and denatured conditions. The sedimentation profile of the mRNA on both sodium dodecyl SO₄-sucrose gradients and formaldehyde-sucrose gradients showed a broad distribution of components with estimated molecular weights ranging from 2 × 10⁵ to 5.5 × 10⁶ daltons and a weight-average molecular weight of 8.5 × 10⁵ daltons.     

Characterization of large mammalian DNA species sedimented in a reorienting zonal rotor

The characteristics of a Beckman-designed slow acceleration unit for the reorientation of alkaline sucrose gradients in a Ti-15 zonal rotor are described. The large DNA species (> 250S) obtained from cultured rat brain tumor cells with this system sediment linearly with time, have virtually no [³H]leucinelabeled or covalently bonded [³H]choline-labeled material sedimenting with them, sediment independently of smaller single-stranded DNA molecules (? 165S) and are 60–80% degraded by the single-strand-specific S1 nuclease. Therefore, it is postulated that these species are collapsed, partially denatured DNA molecules or a collapsed form of single-stranded DNA. When cells were labeled with [¹⁴C]TdR, then frozen and stored at ? 79°C, this system could detect radiation-induced DNA damage from decay of the incorporated label at accumulated doses as small as 18–126 rads.