Reversible inhibition of ribosomal RNA synthesis in HeLa by lucanthone (Miracil D) with continued synthesis of DNA-like RNA

Ribosomal RNA synthesis was selectively inhibited in HeLa cells by lucanthone, a clinically useful schistosomicide which shares many of the properties of Actinomycin D. Synthesis of DNA-like RNA continued during complete inhibition of ribosomal RNA synthesis. Under these conditions newly synthesized DNA-like RNA accumulated normally in polyribosomes of the cell cytoplasm; most of it appeared to be messenger RNA. DNA synthesis was partially inhibited by lucanthone but protein synthesis was undisturbed. Synthesis of ribosomal RNA promptly resumed after removal of lucanthone and cell survival was not affected if exposures to the drug were limited to two hours.     

Regulation of RNA synthesis in plant cell culture: delayed synthesis of ribosomal RNA during transition from the stationary phase to active growth

Ribosomal RNA synthesis was studied during the early phases of growth activation in a cell suspension culture derived from peanut (Arachis hypogaea, L.) cotyledon. Upon dilution from stationary phase, these cells show a characteristic lag of 3 days before the commencement of cell division. An analysis of the nature of RNA synthesized during this early period of growth showed that the cells obtained immediately upon dilution from stationary phase synthesize primarily messenger RNA and essentially no ribosomal RNA. The synthesis of ribosomal RNA is delayed for about 24 hr after which it rises sharply resulting in a 2- to 3-fold accumulation of ribosomal RNA per cell during the subsequent 24-hr period. Both the messenger RNA and the ribosomal RNA were characterized by their cellular localization; by sucrose and CsCl gradient analyses, and by the determination of their base ratios.It would appear that a major facet of the lag phase in the cell growth is the diversion of a significant part of the RNA biosynthetic apparatus from the synthesis of messenger RNA to that of ribosomal RNA.     

Analysis of ribosomal RNA genes in somatic hybrids between wild and cultivated Solanum species

Ribosomal RNA genes were exploited as markers to identify somatic hybrids between Solanum tuberosum cv. Brodick and wild diploid Solanum species, S. megistacrolobum, S. sanctae-rosae and S. sparsipilum and DNA methylation as a possible regulatory factor in gene expression was investigated. Specific restriction enzyme/probe combinations revealed useful polymorphisms in the conserved coding and variable intergenic spacer regions of the ribosomal RNA genes. Some intermediate ribosomal RNA gene profiles indicate hybridity whereas others were characteristic of S. tuberosum cv. Brodick. This evidence is suggestive of somatic exchange/re-arrangement between the NOR region of S. sanctae-rosae and S. tuberosum cv. Brodick. Ribosomal RNA gene copy number analysis of the somatic hybrids did not reveal hexaploid values suggesting that these products are not symmetric hybrids derived from the parental diploid and tetraploid plants. The results indicate site-specific methylation of ribosomal RNA gene sequences for the parental plants; while some somatic hybrids display a reduction, others show an increase. The significance of the findings for somatic cell genetics and plant breeding studies is discussed.     

The conformation of ribonucleic acids in Escherichia coli ribosomes: Inferences from the mode of action of ribonuclease II

1. Ribonuclease II of Escherichia coli degrades pulse-labelled RNA associated with ribosomes and polyuridylic acid on ribosomes and in solution to mononucleotides. 2. Ribosomal and pulse-labelled RNA in solution and ribosomal RNA in chloramphenicol particles (protein-deficient ribosomes) are degraded to oligonucleotides. 3. Ribosomal RNA in mature ribosomes is not attacked by the enzyme. 4. From the mode of action of ribonuclease II, which is specific for single-stranded polyribonucleotides and does not attack helical forms, it is inferred that pulse-labelled RNA associated with ribosomes of E. coli exists as a single-stranded structure and that ribosomal RNA in chloramphenicol particles has a pronounced helical character. 5. The different behaviour of ribonuclease II towards newly synthesized RNA, ribosomal RNA and chloramphenicol-particle RNA in E. coli ribosomes is discussed.     

Turnover of ribosomal RNA in the rat brain

Ribosomal RNA from rat brain has been partially purified by sodium dodecyl-sulphate treatment of microsomes, and density gradient centrifugation. The apparent half-life of ribosomal RNA after labelling with a pyrimidine precursor was 6 days. Classes of ribosomal RNA with longer half-lives could not be excluded. There was evidence for re-utilization of the labelled precursors.     

Nuclear retention of 18S ribosomal RNA by human myeloma cells

Summary Normal quiescent lymphocytes regulate their ribosome content by selectively degrading newly synthesized 18S ribosomal RNA. Unlike actively dividing HeLa cells, lymphocytes retain 18 S ribosomal RNA in the nucleus after synthesis instead of immediately transporting it to the cytoplasm. Subcellular fractionation of the highly differentiated human neoplastic lymphocyte RPMI-8226 reveals that this cell line also retains 18 S ribosomal RNA in the nucleus, a trait not displayed by the less differentiated human lymphoblastoid cell line RPMI-4265. These observations suggest that neoplastic cells can be phenotypically characterized by their ribosomal RNA processing patterns.Operated by Union Carbide Corporation with the Department of Energy     

Desmin is present in proliferating rat muscle satellite cells but not in bovine muscle satellite cells.

The presence of desmin was characterized in cultured rat and bovine satellite cells and its potential usefulness as a marker for identifying satellite cells in vitro was evaluated. In primary cultures, positive immunohistochemical staining for desmin and skeletal muscle myosin was observed in rat and bovine myotubes. A small number of mononucleated cells (20% of rat satellite cells and 5% of bovine satellite cells) were myosin-positive, indicative of post-mitotic differentiated myocytes. In bovine satellite cell cultures 13% of the mononucleated cells were desmin-positive, while 84% of the mononucleated cells in rat satellite cell cultures were desmin-positive. Rat satellite cell mass cultures and bovine satellite cell clonal density cultures were pulsed with 3H-thymidine, and autoradiographic data revealed that greater than 94% of dividing rat cells were desmin-positive, suggesting that desmin is synthesized in proliferating rat satellite cells. However, no desmin was seen in cells that incorporated labeled thymidine in bovine satellite cell clones. Analysis of clonal density cultures revealed that only 14% of the mononucleated cells in bovine satellite cell colonies were desmin-positive, whereas 98% of the cells in rat satellite cell colonies were desmin-positive. Fibroblast colonies from both species were desmin-negative. In order to further examine the relationship between satellite cell differentiation and desmin expression, 5-bromo-2'-deoxyuridine (BrdU) was added to culture medium at the time of plating to inhibit differentiation. Fusion was inhibited in rat and bovine cultures, and cells continued to divide. Very few desmin-positive cells were found in bovine cultures, but greater than 90% of the cells in rat cultures stained positive for desmin. The presence of desmin and sarcomeric myosin was also evaluated in regenerating rat tibialis anterior five days after bupivacaine injection. In regenerating areas of the muscle many desmin-positive cells were present, and only a few cells stained positive for skeletal muscle myosin. Application of desmin staining to rat satellite cell growth assays indicated that rat satellite cells cultured in serum-containing medium were contaminated with fibroblasts at levels that ranged from approximately 5% in 24 hr cultures to 15% in mature cultures. In defined medium 4 day cultures contain approximately 95% to 98% desmin-positive satellite cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunocytochemical localization of transient DNA strand breaks in differentiating myotubes using in situ nick-translation

We have localized DNA strand breaks during in vitro chicken myogenesis by repairing nicks in nuclei of fixed cell monolayers in situ with biotin-11-dUTP, followed by immunocytochemical detection of incorporated biotin with rabbit anti-biotin and FITC-labeled goat anti-rabbit antibodies. No accumulations of biotin sufficient for immunocytochemical detection were observed in 23-hr cultures of dividing cells. In 33- and 43-hr cultures, biotin was first detected in only 3% of the nuclei, all of which appeared to be in fusing myoblasts or small myotubes. In contrast, cultures of young, highly fused myotubes (56 hr) exhibited 18% biotinylated nuclei; virtually all of these nuclei, most of which were grouped as aggregates, were within myotubes. In older cultures (73 and 94 hr) incorporation of biotin into myotube nuclei markedly decreased, while increases were noted in nuclei of mononuclear cells. These results indicate that extensive single-stranded DNA nicking occurs in nuclei of young myotubes, followed by repair as terminal differentiation ensues.     

Circular DNA and rolling circles in nucleolar rDNA from mitotic nuclei of Physarum polycephalum.

1. About 15% of nucleolar DNA (1.712 g/cm3) from Physarum polycephalum displaying maximum hybridization to ribosomal RNA, is composed of circular DNA of 3.9 +/- 0.2 mum contour length or multiples thereof. 2. A portion of these circular molecules (25%) contained linear DNA pieces longer than circumference length. In a small fraction of circular DNA linear pieces, shorter than the unit length, were observed. 3. Most nucleolar DNA, [3H]thymidine-labeled or hybridizable to ribosomal RNA was separable from chromosomal DNA during G2 phase, mitosis and S phase of the cell cycle. 4. Ribosomal DNA content was not amplified during the cell cycle, was unchanged during exponential or stationary growth phase and amounted to about 0.11 -- 0.21% of nuclear DNA in diploid and hexaploid strains of Physarum or 100--200 ribosomal genes per diploid genome.     

Ribosomal RNA structure in the diploid and phylogenetically polyploid amphibian species Hyla and Odontophrynus

Ribosomal RNA of the diploid amphibian species Hyla chrysoscelis and Odontophrynus americanus is structurally modified by hidden breaks. Phylogenetically polyploid related species like the tetraploid Hyla versicolor, the tetraploid Odontophrynus americanus and the octoploid Ceratophrys ornata do not show hidden breaks in ribosomal RNA. Structural modifications of rRNA molecules in diploid amphibians has no detectable effect on the ribosomal activity in vitro.     

Detection of specific sequences among DNA fragments separated by gel electrophoresis.

Ribosomal RNA and precursor ribosomal RNA from at least one representative of each vertebrate class have been analyzed by electron microscopic secondary structure mapping. Reproducible patterns of hairpin loops were found in both 28 S ribosomal and precursor ribosomal RNA, whereas almost all the 18 S ribosomal RNA molecules lack secondary structure under the spreading conditions used. The precursor ribosomal RNA of all species analyzed have a common design. The 28 S ribosomal RNA is located at or near the presumed 5′-end and is separated from the 18 S ribosomal RNA region by the internal spacer region. In addition there is an external spacer region at the 3′-end of all precursor ribosomal RNA molecules. Changes in the length of these spacer regions are mainly responsible for the increase in size of the precursor ribosomal RNA during vertebrate evolution. In cold blooded vertebrates the precursor contains two short spacer regions; in birds the precursor bears a long internal and a short external spacer region, and in mammals it has two long spacer regions. The molecular weights, as determined from the electron micrographs, are 2·6 to 2·8 × 10⁶ for the precursor ribosomal RNA of cold blooded vertebrates, 3·7 to 3·9 × 10⁶ for the precursor of birds, and 4·2 to 4·7 × 10⁶ for the mammalian precursor. Ribosomal RNA and precursor ribosomal RNA of mammals have a higher proportion of secondary structure loops when compared to lower vertebrates. This observation was confirmed by digesting ribosomal RNAs and precursor ribosomal RNAs with single-strandspecific S₁ nuclease in aqueous solution. Analysis of the double-stranded, S₁-resistant fragments indicates that there is a direct relationship between the hairpin loops seen in the electron microscope and secondary structure in aqueous solution.     

Comparative proteomes of the proliferating C(2)C(12) myoblasts and fully differentiated myotubes reveal the complexity of the skeletal muscle differentiation program

When cultured in low serum-containing growth medium, the mouse C(2)C(12) cells exit cell cycle and undergo a well-defined program of differentiation that culminates in the formation of myosin heavy chain-positive bona fide multinucleated muscle cells. To gain an understanding into this process, we compared total, membrane- and nuclear-enriched proteins, and phospho-proteins from the proliferating C(2)C(12) cells and the fully differentiated myotubes by the combined methods of two-dimensional PAGE, quantitative PDQuest image analysis, and MS. Quantification of more than 2,000 proteins from C(2)C(12) myoblasts and myotubes revealed that a vast majority of the abundant proteins appear to be relegated to the essential, housekeeping and structural functions, and their steady state levels remain relatively constant. In contrast, 75 proteins were highly regulated during the phenotypic conversion of rapidly dividing C(2)C(12) myoblasts into fully differentiated, multi-nucleated, post-mitotic myotubes. We found that differential accumulation of 26 phospho-proteins also occurred during conversion of C(2)C(12) myoblasts into myotubes. We identified the differentially expressed proteins by MALDI-TOF-MS and LC-ESI-quadrupole ion trap MS/MS. We demonstrate that more than 100 proteins, some shown to be associated with muscle differentiation for the first time, that regulate inter- and intracellular signaling, cell shape, proliferation, apoptosis, and gene expression impinge on the mechanism of skeletal muscle differentiation.     

MITOSIS AND THE PROCESSES OF DIFFERENTIATION OF MYOGENIC CELLS IN VITRO

The relation between the mitotic cycle and myoblast fusion has been studied in chick skeletal muscle in vitro. The duration of the cell cycle phases was the same in both early and late cultures. By tracing a cohort of pulse-labeled cells, it was found that myoblast fusion does not occur in S, G₂, or M. Cell surface alterations required for fusion are dependent upon the position of the cell in the division cycle. In early cultures, fusion takes place only after a minimum delay of 5 hr from the time the cell has entered G₁. The mitosis preceding fusion may condition the cell for the abrupt shift in synthetic activity that occurs in the subsequent G₁. In older cultures fusion of labeled cells is diminished. Two factors account for the cessation of fusion in older cultures. First, the number of myogenic stem cells declines, but these cells do not disappear as the cultures mature. Their persistence was demonstrated by labeling dividing mononucleated cells in older cultures and challenging them with nascent myotubes. Some of these labeled cells were incorporated into the forming myotubes. Second, a block to fusion develops during myotube maturation. Well developed myotubes challenged with labeled competent myogenic cells failed to incorporate the labeled nuclei.     

Replication of prions in differentiated muscle cells

We have demonstrated that prions accumulate to high levels in non-proliferative C2C12 myotubes. C2C12 cells replicate as myoblasts but can be differentiated into myotubes. Earlier studies indicated that C2C12 myoblasts are not competent for prion replication.¹ We confirmed that observation and demonstrated, for the first time, that while replicative myoblasts do not accumulate PrP^Sc, differentiated post-mitotic myotube cultures replicate prions robustly. Here we extend our observations and describe the implication and utility of this system for replicating prions.     

Effect of Fluoride on Ribosomes from Corn Roots. Changes with Growth Retardation

Growth inhibition caused by fluoride was further studied by analyzing free and bound ribosomes, and the ribosomal components in corn roots. Ribosomes were isolated by differential centrifugation. Ribosomal components were analyzed by column chromatography and electrophoresis. Fluoride reduces the amounts of both free and bound ribosomes. Fluoride also modifies the RNA to protein ratio of bound ribosomes more than that of free ribosomes. Fluoride does not affect base ratios of the ribosomal RNA, but reduces its content and changes the structure of the ribosomal protein moiety.     

放线菌核糖体工程的发展与应用

核糖体工程(ribosome engineering)是一项利用靶点位于细菌RNA聚合酶及核糖体功能因子的抗生素诱导细菌产生抗性突变,进而提升菌株次级代谢生产潜能的技术.该方法无需依赖菌株完善的遗传操作体系,可应用于发掘几乎所有放线菌菌株中潜在的宝贵活性次级代谢产物,并广泛应用于放线菌基因组挖掘和次级代谢产物增产优化....     

Coordinated synthesis and degradation of actin and myosin in a variety of myogenic and non-myogenic cells

The turnover of myosin and actin in both muscle and non-muscle cells in culture was investigated. By the double-label criterion, myosin and actin were coordinately synthesized and degraded in replicating, mononucleated fibroblasts, chondrocytes, BUdR-suppressed myogenic cells, and in post-mitotic, multinucleated myotubes. Myosin and actin were among the most stable proteins in each cell type. In single label ‘pulse-chase’ experiments, the half-lives of myosin and actin in all replicating, mononucleated cells were 2.5–3 days; in myotubes, however, they were approx. 6 days. Myosin and actin labelled in replicating presumptive myoblasts and chased until the cells ceased replicating and fused into multinucleated myotubes retained the degradation rate of 3 days; this differed from Jhe rate of 6 days shown for myosin and actin newly-synthesized in post-mitotic myotubes. The type of myosin synthesized in the mother presumptive myoblast, then, is transmitted to the postmitotic daughters. This myosin, however, is more rapidly degraded than the definitive myosin that is synthesized in the myotube.