Thioflavin T as a fluorescence probe for monitoring RNA metabolism at molecular and cellular levels.

The intrinsically stochastic dynamics of mRNA metabolism have important consequences on gene regulation and non-genetic cell-to-cell variability; however, no generally applicable methods exist for studying such stochastic processes quantitatively. Here, we describe the use of the amyloid-binding probe Thioflavin T (ThT) for monitoring RNA metabolism in vitro and in vivo. ThT fluoresced strongly in complex with bacterial total RNA than with genomic DNA. ThT bound purine oligoribonucleotides preferentially over pyrimidine oligoribonucleotides and oligodeoxyribonucleotides. This property enabled quantitative real-time monitoring of poly(A) synthesis and phosphorolysis by polyribonucleotide phosphorylase in vitro. Cellular analyses, in combination with genetic approaches and the transcription-inhibitor rifampicin treatment, demonstrated that ThT mainly stained mRNA in actively dividing Escherichia coli cells. ThT also facilitated mRNA metabolism profiling at the single-cell level in diverse bacteria. Furthermore, ThT can also be used to visualise transitions between non-persister and persister cell states, a phenomenon of isogenic subpopulations of antibiotic-sensitive bacteria that acquire tolerance to multiple antibiotics due to stochastically induced dormant states. Collectively, these results suggest that probing mRNA dynamics with ThT is a broadly applicable approach ranging from the molecular level to the single-cell level.

Human mitochondrial RNA decay mediated by PNPase-hSuv3 complex takes place in distinct foci.

RNA decay is usually mediated by protein complexes and can occur in specific foci such as P-bodies in the cytoplasm of eukaryotes. In human mitochondria nothing is known about the spatial organization of the RNA decay machinery, and the ribonuclease responsible for RNA degradation has not been identified. We demonstrate that silencing of human polynucleotide phosphorylase (PNPase) causes accumulation of RNA decay intermediates and increases the half-life of mitochondrial transcripts. A combination of fluorescence lifetime imaging microscopy with Förster resonance energy transfer and bimolecular fluorescence complementation (BiFC) experiments prove that PNPase and hSuv3 helicase (Suv3, hSuv3p and SUPV3L1) form the RNA-degrading complex in vivo in human mitochondria. This complex, referred to as the degradosome, is formed only in specific foci (named D-foci), which co-localize with mitochondrial RNA and nucleoids. Notably, interaction between PNPase and hSuv3 is essential for efficient mitochondrial RNA degradation. This provides indirect evidence that degradosome-dependent mitochondrial RNA decay takes place in foci.

A novel RNA digesting activity from commercial polynucleotide phosphorylase.

RNase A4 is a new RNase activity found as a contaminant in commercial polynucleotide phosphorylase. This enzyme has the ability of hydrolyzing the phosphodiester bond between pyrimidine-A in both loop and paired regions of RNA.

[Quaternary structure of Escherichia coli polynucleotide phosphorylase: chemical characterization of form A]. / Structure quaternaire de la polynucléotide phosphorylase dEscherichia coli : caractérisation dhimique de la forme A

We had previously shown the existence of two classes of polynucleotide phosphorylases : Form A which is made of alpha chains and carries the catalytic sites ; and form B which is constituted of alpha chains and of beta chains. We performed some chemical analyses of form A (N-terminal sequence, amino acid composition, peptide mapping) which suggest that the alpha chains are all identical and moreover that they have no relationship with the beta chains. The latter do not therefore derive from a partial proteolytic degradation of the alpha chains and can therefore be considered as true subunits of the enzyme.

[Analysis of a small quatity of polypeptides by two-dimensional electrophoresis: application to characterization of proteins]. / Analyse d'une faible quantité de polypeptides par électrophorèse à deux dimensions : application à la caractérisation des protéines

A new two-dimensional electrophoresis is described. The first electrophoresis is performed in a 10 p. cent gel (1 X 110 mm) in the presence of 7 M urea; the migration in the second dimension proceeds on a 20 p. cent polyacrylamide sheet (160 X 110 X 1 mm) in the presence of sodium dodecyl sulphate. This system resolves most of the polypeptides obtained by treating a very small amount (about 100 mug) of proteins with cyanogene bromide. Macromolecules can thus be quickly and easily characterized.

The origin and early evolution of nucleic acid polymerases.

The hypothesis that vestiges of the ancestral RNA-dependent RNA polymerase involved in the replication of RNA genomes of Archean cells are present in the eubacterial RNA polymerase beta' subunit and its homologues is discussed. We show that in the DNA-dependent RNA polymerases from the three cellular lineages a very conserved sequence of eight amino acids also found in a small RNA-binding site previously described for the E. coli polynucleotide phosphorylase and the S1 ribosomal protein is present. The optimal conditions for the replicase activity of the avian myeloblastosis virus reverse transcriptase are presented. The evolutionary significance of the in vitro modifications of substrate and template specificities of RNA polymerases and reverse transcriptases is also discussed.

[Determination of the activity of a polynucleotide phosphorylase preparation from E. coli]. / Opredelenie aktivnosti preparata polinukleotidfosforilazy iz E. coli.

Thin-layer chromatography was employed to measure the activity of polynucleotide phosphorylase (PNP-ase) from E. coli during purification and in the resultant preparation. The TLC data were verified with respect to released inorganic orthophosphate and to 14C-AMP incorporation rate. It is concluded that TLC can be used to determine PNP-ase activity in crude extract, at various purification stages and in the final preparation, yielding correct results.

[mRNA breakdown in tumor cells in vivo under cycloheximide protein synthesis inhibition]. / Razrushenie mRNK v opukholevykh kletkakh in vivo pri ingibirovanii belkovogo sinteza tsiklogeksimidom.

Amoung of prelabelled mRNA was unaltered in Zajhdel ascites hepatoma of rat cells within 3.5-4 hrs under conditions of treatment with actinomycin D. Due to combined effect of actinomycin D and cycloheximide the content of mRNA in the hepatoma cells was rapidly decreased. Degradation of mRNA occurred in membrane-bound polyribosomes, free polyribosomes and in cytoplasmic mRNP-particles /informosomes/ as a result of the effect of cycloheximide. Simultaneously with these phenomena, distinct increase in activity of acid and alkaline RNAases was observed in cytoplasma of the hepatoma cells; activity of endoRNAase from membrane-bound and free polyribosomes of the hepatoma was also markedly increased. Cycloheximide did not affect the activity of polynucleotide phosphorylase in polyribosomes of the hepatoma cells. Labile proteins, responsible for inhibition of RNAses appeart to participate in regulation of mRNA stability in malignant cells.

Identification and cloning of human polynucleotide phosphorylase, hPNPase old-35, in the context of terminal differentiation and cellular senescence.

Terminal differentiation and cellular senescence display common properties including irreversible growth arrest. To define the molecular and ultimately the biochemical basis of the complex physiological changes associated with terminal differentiation and senescence, an overlapping-pathway screen was used to identify genes displaying coordinated expression as a consequence of both processes. This approach involved screening of a subtracted cDNA library prepared from human melanoma cells induced to terminally differentiate by treatment with fibroblast IFN and mezerein with mRNA derived from senescent human progeria cells. This strategy identified old-35, which encodes an evolutionary conserved gene, human polynucleotide phosphorylase (hPNPase(old-35)), that is regulated predominantly by type I IFNs. The hPNPase(OLD-35) protein localizes in the cytoplasm of human cells and induces RNA degradation in vitro, as does its purified bacterial protein homologue. Ectopic expression of hPNPase(old-35) in human melanoma cells reduces colony formation, confirming inhibitory activity of this RNA-degradation enzyme. Identification of hPNPase(old-35), an IFN-inducible 3'-5' RNA exonuclease, provides additional support for a relationship between IFN action and RNA processing and suggests an important role for this gene in growth control associated with terminal differentiation and cellular senescence.

Membrane asymmetry and expression of cell surface antigens of Micrococcus lysodeikticus established by crossed immunoelectrophoresis.

Crossed immunoelectrophoresis of Triton X-100-solubilized plasma membranes of Micrococcus lysodeikticus established the presence of 27 discrete antigens. Individual antigens were identified as membrane components possessing enzyme activity by zymogram staining procedures and by reactivity of certain antigens with a selection of four lectins in the crossed-immunoelectrophoresis (immunoaffinoelectrophoresis) system. Absorption experiments with intact, stable protoplasts and isolated membranes established the asymmetric nature of the M. lysodeikticus plasma membranes. Of the 14 antigens with determinants accessible solely on the cytoplasmic face of the membrane, four possessed individual dehydrogenase activities, and a fifth was identifiable as a component possessing adenosine triphosphatase (EC 3.6.1.3) activity. Evidence from absorption studies with isolated membranes suggested that antigens such as the adenosine triphosphatase complex were more readily accessible to reaction with antibodies than was succinate dehydrogenase (EC 1.3.99.1), for example. Twelve antigens were located on the protoplast surface as determined by antibody absorption, and the succinylated lipomannan was identified as a major antigen. At least five other antigens possessed sugar residues that interacted with concanavalin A. With the antisera generated to isolated membranes, there was no evidence suggesting that any of these antigens was not detectable on either surface of the plasma membrane. From absorption experiments with washed, whole cells of M. lysodeikticus, it was concluded that the immunogens on the protoplast surface were also detectable on the surface of the intact cell. However, some of the components such as the succinylated lipomannan appeared to be exposed to a greater extent than others. The cytoplasmic fraction from M. lysodeikticus was used as an antigen source to generate antibodies, and 97 immunoprecipitates were resolvable by crossed immunoelectrophoresis. In the cytoplasm-anticytoplasm reference immunoelectrophoresis pattern of precipitates, three of the immunoprecipitates unique to the cytoplasmic fraction were identifiable by zymogram staining procedures as catalase (EC 1.11.1.6), isocitrate dehydrogenase (EC 1.1.1.42), and polynucleotide phosphorylase (EC 2.3.7.8). The identification of membrane and cytoplasmic antigens (including the above-mentioned enzymes) provides a sensitive analytical system for monitoring cross-contamination and antigen distribution in cellular fractions.

Photometric assay for polynucleotide phosphorylase.

Polynucleotide phosphorylase (PNPase) is a prokaryotic enzyme that catalyzes phosphorolysis of polynucleotides with release of NDPs. It is also believed to play a key role in turnover of prokaryotic transcripts, thus regulating gene expression. At the moment, only radioisotopic methods are available for assaying PNPase in crude extracts; these involve incubating [32P]phosphate and poly(A) in the presence of the enzyme, separating [32P]phosphate from [32P]ADP, and quantifying ADP by scintillation counting. Photometric assay using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes is not feasible in crude extracts because of endogenous ATPase activities, which regenerate ADP from the ATP released by pyruvate kinase. Here, we present a simple photometric assay that uses a cyclic detection system which, due to the sequential action of pyruvate kinase and hexokinase, results in an exponential increase of ADP and glucose 6-phosphate. Glucose 6-phosphate is then revealed by a glucose-6-phosphate dehydrogenase reaction. Based on the theoretical model, a linear increase in absorbance is predicted as a function of the square of the reaction time, with a slope proportional to PNPase activity. Experimental data confirmed the theoretical predictions and showed that the assay was quantitative and unquestionably specific. We also devised a simple procedure for determining absolute enzyme activities (expressed in micromoles of product formed per minute) using exact amounts of pure PNPase as internal standards.

Isolation and characterization of a polynucleotide phosphorylase from Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens BaM-2 produces large amounts of extracellular enzymes, and the synthesis of these proteins appears to be dependent upon abnormal ribonucleic acid metabolism. A polynucleotide phosphorylase (nucleoside diphosphate:polynucleotide nucleotidyl transferase) was identified, purified, and characterized from this strain. The purification scheme involved cell disruption, phase partitioning, differential (NH4)2SO4 solubilities, agarose gel filtration, and diethylaminoethyl-Sephadex chromatography. The purified enzyme demonstrated the reactions characteristic of polynucleotide phosphorylase: polymerization, phosphorolysis, and inorganic phosphate exchange with the beta-phosphate of a nucleotide diphosphate. The enzyme was apparently primer independent and required a divalent cation. The reactions for the synthesis of the homopolyribonucleotides, (A)n and (G)n, were optimized with respect to pH and divalent cation concentration. The enzyme is sensitive to inhibition by phosphate ion and heparin and is partially inhibited by rifamycin SV and synthetic polynucleotides.

Antigenic architecture of membrane vesicles from Escherichia coli.

The antigenic architecture of membrane vesicles prepared from Escherichia coli ML 308--225 has been studied using crossed immunoelectrophoresis. Progressive immunoadsorption experiments conducted with control vesicles and with physically disrupted vesicles were used to monitor and quantitate the expression of 14 different immunogens. Eleven immunogens, including NADH dehydrogenase (EC 1.6.33.3), D-lactate dehydrogenase (EC 1.1.1.27), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), and beta-galactosidase (EC 3.2.1.23), exhibit minimal expression (10% or less) unless the vesicles are disrupted. Three unidentified antigens are expressed to a similar extent in untreated and disrupted vesicles. Consideration of these and other results [Owen, P., & Kaback, H. R. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 3148] in terms of membrane polarity, dislocation of antigens, and possible transmembrane orientation of some immunogens reveals that over 95% of the membrane in the vesicle preparations is in the form of sealed sacculi with the same orientation as the intact cell. Furthermore, antigens are distributed across the membrane in a highly asymmetric manner, indicating that dislocation of components from the inner to the outer surface of the membrane during vesicle preparation does not occur to an extent exceeding 10%.

Quantitative immunoelectrophoretic analysis of Streptococcus pyogenes membrane.

The antigenic composition and molecular structure of the plasma membrane of Streptococcus pyogenes (group A; M type 6) were studied by crossed immunoelectrophoresis (XIE) and other related quantitative immunoelectrophoretic techniques. After establishment of a reference pattern of 29 immunoprecipitates, the relative differences in amounts of individual antigens contained in membranes isolated from cells that were harvested during the exponential or stationary phase of growth were examined. Relative increases and decreases in amounts of individual antigens were estimated from the areas subtended by immunoprecipitates after XIE of Triton X-100 extracts. The asymmetric distribution of antigens on the inner and outer surfaces of the membrane was established in absorption experiments with intact, stable protoplasts. Of the 29 immunoprecipitates, 8 appeared to contain antigens exposed on the outer surface of the membrane, whereas 11 appeared to contain antigens either located on the inner surface or unexposed. Six antigens appeared to have limited exposure on the outer surface, and four others remain to be assigned. Certain immunoprecipitates were characterized with respect to enzymatic activity or interaction with the lectin concanavalin A. Reduced nicotinamide adenine dinucleotide dehydrogenase (EC 1.6.99.3), adenosine triphosphatase (EC 3.6.1.3), and polynucleotide phosphorylase (EC 2.3.7.8) were demonstrated by zymogram techniques. The latter two activities were present within the same immunoprecipitate, suggesting the occurrence of a multienzyme complex. In addition, the areas under the immunoprecipitates containing the three enzymatic activities were not affected by absorption of antimembrane immunoglobulin with intact protoplasts and thus appeared to be located on the inner surface of the membrane. The results from absorption experiments also suggested that the exposure of outer protoplast surface antigens was greater on protoplasts from exponential-phase cells than on those from stationary-phase cells, even when found in increased amounts in the latter.

[Animal tissue polynucleotide phosphorylase]. / Polinukleotidfosforilaza tkanei zhivotnykh.

Localization, physico-chemical and catalytic properties and possible biological functions of polynucleotide phosphorylase (PNPase) from animal tissues are discussed. In animal tissue cells PNPase has multiple localization; the major amount of the enzyme is localized in the endoplasmic reticulum ribosomes. In the nuclei PNPase, similar to other endo- and exo-RNAses participates in the processing of precursor molecules of mature forms of RNA, whereas in the cytoplasm it is involved in the destruction of polyribosomes in the polyribosomes of rapidly growing tissues the activity of PNPase is extremely decreased. The mechanisms regulating the PNPase activity in rapidly growing tissues are discussed.

Immunochemical analysis of membrane vesicles from Escherichia coli.

Membrane vesicles isolated from Escherichia coli ML 308--225 have been analyzed by crossed immunoelectrophoresis, and immunoprecipitates corresponding to the following cellular components have been identified: ATPase (EC 3.6.1,3), two or three NADH dehydrogenases (EC 1.6.99.3), D-lactate dehydrogenase (EC 1.1.1.27), glutamate dehydrogenase (EC 1.4.1.4), dihydro-orotate dehydrogenase (EC 1.3.3.1), 6-phosphogluconate dehydrogenase (EC 1.1.1.43), polynucleotide phosphorylase (EC 2.3.7.8), beta-galactosidase (EC 3.2.1.23), lipopolysaccharide, and Braun's lipoprotein. The cellular origin of many of the vesicle immunogens is determined, and Braun's lipoprotein is used as a marker to quantitate the extent of outer membrane contamination (less than 3%). Membrane antigens are also characterized with regard to their amphiphilic or hydrophilic properties by charge-shift crossed immunoelectrophoresis. Furthermore, the following immunogens cross-react with components in membrane vesicles prepared from Salmonella typhimurium: one of the three NADH dehydrogenases, ATPase, polynucleotide phosphorylase, 6-phosphogluconate dehydrogenase, Braun's lipoprotein, and three unidentified antigens. In the accompanying paper [Owen, P., & Kaback, H. R. (1979) Biochemistry 18 (following paper in this issue)] quantitative immunoadsorption is utilized to establish the topology of the vesicles with respect to the distribution of antigens on the inner and outer faces of the membrane.

A study of the localization of polynucleotide phosphorylase within rat liver cells and of its distribution among rat tissues and diverse animal species.

1. Rat liver polynucleotide phosphorylase was localized in the mitochondrion, but may also occur in the nucleus. 2. The mitochondrial enzyme was found in rat heart, kidney, liver, muscle and spleen. 3. Mitochondrial polynucleotide phosphorylase is also present in calf, chicken, guinea-pig and rabbit liver and in goldfish muscle. 4. A possible physiological role for the enzyme in the control of the intramitochondrial ADP concentration is suggested.