Effects of cordycepin on the synthesis of nuclear and cytoplasmic RNAs in embryonic cells of Xenopus laevis

Effects of cordycepin on the incorporation of [3H] guanosine into embryonic Xenopus cells were examined. Cordycepin inhibited the labeling not only of poly(A) + RNA, but of all the other major classes of RNAs. Cellular fractionation showed that this inhibition was much stronger in the labeling of cytoplasmic RNAs than of nuclear RNAs. [3H]Cordycepin was incorporated into both poly(A) + RNA and other RNA species.     

Evidence for a precursor-product relationship in the biosynthesis of ribosomal protein S20.

The kinetics of labeling ribosomal protein S20 of Escherichia coli strains H882 and H882 groE44 have been examined using partial reconstitution as a means of binding this and some other 30S subunit proteins selectively to 16S RNA from crude extracts prepared by acetic acid extraction of pulse-labeled whole cells. The rate of labeling of S20 during short pulses at 44 degrees C is less than 20% of that observed at 28 degrees C. S20 can be recovered from the cells labeled at the higher temperature if they are chased at 28 degrees C, but not at 44 degrees C, in the presence of excess sulfate prior to their extraction. These observations suggest that S20 is derived from a precursor whose processing is blocked at 44 degrees C. Among the proteins extracted from cells labeled at 44 degrees C capable of binding to 16S RNA is a novel polypeptide, p2, which is not normally present on the 30S subunit. The kinetics of its appearance at 44 degrees C, and its chasing at 28 degrees C, suggest a precursor-product relationship with S20. p2 contains a tryptic peptide with the chromatographic properties of the peptide Ser-Met-Met-Arg at position 25-28 in S20. A second methionine-containing peptide at positions 49-59 of S20 is missing from p2. In addition, the apparent molecular weight of p2 (8600) is less than that of S20 (9500). p2 may represent the product of degradation of a precursor to S20, yet retains the ability to bind to 16S RNA. It is much less likely that p2 is a bona fide precursor which is converted into S20 by fusion to some other polypeptide.     

Establishment of a C3Hf Mammary Tumor Cell Line Expressing Endogenous Mouse Mammary Tumor Virus: Antigenic and Genetic Relationships of This Virus with Highly Oncogenic Mouse Mammary Tumor Viruses

A single-cell clone of C3Hf mammary tumor cells (clone 14) was developed into a continuous cell line expressing high levels of endogenous mouse mammary tumor virus (MMTV) with less than 0.1% murine leukemia virus expression. Comparison of the C3Hf MMTV protein profile on sodium dodecyl sulfatepolyacrylamide gel electrophoresis with that of C3H MMTV revealed that the protein content of the two viruses was quite similar. However, oligonucleotide fingerprints obtained of MMTV 70S RNA revealed that approximately 20% of the large oligonucleotides examined were unique to each virus. The oligonucleotide fingerprint indicated that although the viruses were similar, they differed in their genetic content. The differences in the two viruses extended to immunological differences in the major envelope glycoprotein, gp52. C3Hf MMTV competed only partially in a homologous radioimmunoassay for gp52 of C3H MMTV, whereas C3H MMTV gave complete competition, indicating that gp52 of C3H MMTV contained type-specific determinants not present on gp52 of C3Hf MMTV. Comparison of C3Hf MMTV with highly oncogenic C3H, GR, and RIII MMTVs in a homologous C3H MMTV gp52 assay gave two patterns of reactivity: complete competition by GR and C3H MMTV and incomplete competition by C3Hf and RIII MMTV. Absorption of anti-C3H MMTV serum by either C3Hf MMTV or RIII MMTV removed all antibodies against both viruses but not against GR and C3H MMTVs. These results indicate that C3H and GR MMTVs are more closely related to each other than to RIII and C3Hf MMTVs.     

Synthesis of polyadenylic acid-containing ribonucleic acid during the germination of Neurospora crassa conidia.

Ribonucleic acid (RNA) synthesized during the first 1 h of conidial germination (15 to 20, 25 to 30, and 55 to 60 min) has been characterized by sucrose-sodium dodecyl sulfate gradient centrifugation, binding to polyuridylic acid filters, and oligo(dT)-cellulose chromatography. At all labeling periods examined, polyadenylic acid-containing RNA is synthesized, processed, and incorporated into polysomes. Approximately 40% of the labeled RNA sedimenting between 5 and 17S binds to polyuridylic acid filters. RNA which binds to oligo(dT)-cellulose displays a heterogeneous distribution in sucrose-sodium dodecyl sulfate gradients with a major, broad peak at 10-16S. In addition, some polyadenylic acid-containing RNA sediments beyond the 25S marker. Approximately 3% of the [3H]adenosine in pulse-labeled polysomal RNA is in polyadenylic acid segments resistant to pancreatic and T1 ribonucleases.     

Catabolism of Tritiated Thymidine by Aquatic Microbial Communities and Incorporation of Tritium into RNA and Protein

The incorporation of tritiated thymidine by five microbial ecosystems and the distribution of tritium into DNA, RNA, and protein were determined. All microbial assemblages tested exhibited significant labeling of RNA and protein (i.e., nonspecific labeling), as determined by differential acid-base hydrolysis. Nonspecific labeling was greatest in sediment samples, for which ≥95% of the tritium was recovered with the RNA and protein fractions. The percentage of tritium recovered in the DNA fraction ranged from 15 to 38% of the total labeled macromolecules recovered. Nonspecific labeling was independent of both incubation time and thymidine concentration over very wide ranges. Four different RNA hydrolysis reagents (KOH, NaOH, piperidine, and enzymes) solubilized tritium from cold trichloroacetic acid precipitates. High-pressure liquid chromatography separation of piperidine hydrolysates followed by measurement of isolated monophosphates confirmed the labeling of RNA and indicated that tritium was recovered primarily in CMP and AMP residues. We also evaluated the specificity of [2-³H]adenine incorporation into adenylate residues in both RNA and DNA in parallel with the [³H]thymidine experiments and compared the degree of nonspecific labeling by [³H]adenine with that derived from [³H]thymidine. Rapid catabolism of tritiated thymidine was evaluated by determining the disappearance of tritiated thymidine from the incubation medium and the appearance of degradation products by high-pressure liquid chromatography separation of the cell-free medium. Degradation product formation, including that of both volatile and nonvolatile compounds, was much greater than the rate of incorporation of tritium into stable macromolecules. The standard degradation pathway for thymidine coupled with utilization of Krebs cycle intermediates for the biosynthesis of amino acids, purines, and pyrimidines readily accounts for the observed nonspecific labeling in environmental samples.     

RNA synthesis in the ultrastructural and biochemical components of the nucleolus of Chinese hamster ovary cells

A correlated autoradiographic and biochemical study of RNA synthesis in the nucleoli of chinese hamster ovary cells has been made. Quantitative analysis of the labeling indicates that the fibrillar ribonucleoprotein (RNP) component is labeled faster than 80S RNP and 45S RNA molecules, but approaches simultaneously a steady-state 3H to 14C ratio or grains/mum2 after 30 min of [3H]uridine incorporation. On the other hand, the 55S RNP, the 36S + 32S RNA, and the granular RNP components have the same kinetic of labeling with [3H]uridine. These results suggest that the fibrillar and granular RNP components of the nucleolus are the ultrastructural substratum of, respectively, the 80S RNP (45S RNA) and 55S RNP (36S + 32S RNA). The possibility that precursors to 80S RNP exist also in the fibrillar region of the nucleolus is strongly suggested by the rapid labeling of the fibrils on the autoradiographs.     

Site-selective <Superscript>13</Superscript>C labeling of proteins using erythrose

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with ¹³C and/or ¹H, which is achieved in the most general way by using site-selectively ¹³C-enriched glucose (1- and 2-¹³C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively ¹³C-enriched erythrose (1-, 2-, 3- and 4-¹³C) as a suitable precursor for ¹³C labeled aromatic side chains. We quantify ¹³C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the ¹³C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated ¹³C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective ¹³C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.     

Protein-independent folding pathway of the 16S rRNA 5' domain

Evolution of the ribosome from an RNA catalyst suggests that the intrinsic folding pathway of the rRNA dictates the hierarchy of ribosome assembly. To address this possibility, we probed the tertiary folding pathway of the 5' domain of the Escherichia coli 16S rRNA at 20 ms intervals using X-ray-dependent hydroxyl radical footprinting. Comparison with crystallographic structures and footprinting reactions on native 30S ribosomes showed that the RNA formed all of the predicted tertiary interactions in the absence of proteins. In 20 mM MgCl2, many tertiary interactions appeared within 20 ms. By contrast, interactions between H6, H15 and H17 near the spur of the 30S ribosome evolved over several minutes, likely due to mispairing of a central helix junction. The kinetic folding pathway of the RNA corresponded to the expected order of protein binding, suggesting that the RNA folding pathway forms the basis for early steps of ribosome assembly.     

Erythrocyte membrane sulfhydryl groups and the active transport of cations

RNA synthesis was studied by autoradiographic analysis using tritiated uridine incorporation in the Chinese hamster cell line Dede after a one-minute pulse labeling period. RNA synthesis continues during all stages of interphase and mitosis except during metaphase and anaphase. Cytoplasmic RNA was apparently synthesized in the nucleus, since no grains were observed above the background level in the sample immediately following the labeling. Nucleoli synthesize their own RNA and are not reservoirs for RNA synthesized elsewhere. Both actinomycin D and nogalamycin inhibited the RNA synthetic activity of chromatin and nucleoli. However, the nucleolar synthetic activity was more susceptible to these agents than that of chromatin. Furthermore, actinomycin D was a stronger inhibitor than nogalamycin.     

A specific RNA hairpin loop structure binds the RNA recognition motifs of the Drosophila SR protein B52.

B52, also known as SRp55, is a member of the Drosophila melanogaster SR protein family, a group of nuclear proteins that are both essential splicing factors and specific splicing regulators. Like most SR proteins, B52 contains two RNA recognition motifs in the N terminus and a C-terminal domain rich in serine-arginine dipeptide repeats. Since B52 is an essential protein and is expected to play a role in splicing a subset of Drosophila pre-mRNAs, its function is likely to be mediated by specific interactions with RNA. To investigate the RNA-binding specificity of B52, we isolated B52-binding RNAs by selection and amplification from a pool of random RNA sequences by using full-length B52 protein as the target. These RNAs contained a conserved consensus motif that constitutes the core of a secondary structural element predicted by energy minimization. Deletion and substitution mutations defined the B52-binding site on these RNAs as a hairpin loop structure covering about 20 nucleotides, which was confirmed by structure-specific enzymatic probing. Finally, we demonstrated that both RNA recognition motifs of B52 are required for RNA binding, while the RS domain is not involved in this interaction.     

The action of cordycepin on nascent nuclear RNA and poly(A) synthesis in regenerating liver.

Following a 5 min pulse of [5- 3H]orotic acid via the protal vein, the specific radioactivity of non-poly(A)heterogeneous nuclear RNA (HnRNA) reaches a peak at 12 h after partial hepatectomy. In contrast, poly(A)-HnRNA was maximally elevated only at 2 h after operation. After intraportal injection of cordycepin (3'-deoxyadenosine) 1 min before [5-3H]orotic acid, a dose-dependent inhibition of nuclear HnRNA and rRNA occurred. Fractionation of HnRNA on poly(U)-Sepharose following 20 mg/kg of cordycepin revealed that a 65% reduction occurred in the labeling of poly(A)-HnRNA while non-polyactivity of UTP in control and cordycepin-treated animals indicated no significant alterations in these parameters. Assessment of poly(A) size using poly(A)-HnRNA annealed with oligo(dT)10 as template primer for Escherichia coli DNA polymerase I, showed that 20 mg/kg of cordycepin inhibited nuclear polyadenylylation by 43%; no alteration in the binding of poly(A)-HnRNA to Millipore filters occurred at this dose of cordycepin. These results indicate that cordycepin is a non-selective inhibitor of nuclear RNA and poly(A)synthesis in regenerating rat liver.     

Synthesis of Saint Louis Encephalitis Virus Ribonucleic Acid in BHK-21/13 Cells

Infection of baby hamster kidney cells (BHK-21/13) with Saint Louis encephalitis (SLE) virus depressed the rate of protein and ribonucleic acid (RNA) synthesis until viral RNA synthesis began 6 hr postinfection (PI). Virus-directed RNA synthesis was subsequently inhibited until 12 hr PI when virion maturation began. The rate of protein synthesis reached a peak 6 hr PI and was subsequently depressed until just before the onset of virion maturation. Density gradient analysis of phenol-extracted RNA from actinomycin-treated infected cells indicated that, at 6 to 8 hr and again at 12 to 20 hr PI, three species of viral-specific RNA were synthesized. The most rapid sedimenting form (43S) was ribonuclease-sensitive and had a base composition similar to the RNA isolated from mature virions. The 20S RNA species was ribonuclease-resistant and had a sedimentation coefficient and base composition similar to the replicative form associated with other arbovirus infections. The 26S RNA was ribonuclease-resistant (0.2 mug/ml, 0.1 m NaCl, 25 C, 30 min) and had a nucleotide base composition closer to the 20S form than to the values for 43S RNA. Five-minute pulse labeling of infected cultures during the period viral RNA synthesis was maximal resulted in labeling of only the 20S to 22S RNA fractions. With pulse-labeling periods of 10 min, both the 20S and 26S RNA species were radioactive. Periods of radioactive labeling of as long as 15 min were required before the 43S form was radioactively labeled. These results suggest that the 20S and 26S RNA may be intermediate forms in the synthesis of 43S viral RNA.     

3H]muscimol photolabels the gamma-aminobutyric acid receptor binding site on a peptide subunit distinct from that labeled with benzodiazepines

Affinity column-purified GABA-benzodiazepine receptor protein from bovine brain was photoaffinity labeled with both [3H]flunitrazepam and [3H]muscimol. Gel electrophoresis in sodium dodecyl sulfate revealed that the benzodiazepine binding site labeled with [3H]flunitrazepam was primarily associated with a major peptide subunit revealed by protein staining with Mr = 52 kiloDaltons, with minor labeling of a second peptide of Mr = 57 kiloDaltons, corresponding to a second major stained band. Covalent incorporation of [3H]muscimol was limited to the 57 kiloDalton band, with no labeling of the 52 kiloDalton peptide, showing that the GABA binding site is carried by a subunit distinct from that carrying the benzodiazepine binding site.