In situ analysis of nucleic acids in cold-induced nonculturable Vibrio vulnificus.

Low-temperature-induced nonculturable cells of the human pathogenic bacterium Vibrio vulnificus retained significant amounts of nucleic acids for more than 5 months. Upon permeabilization of fixed cells, however, an increasing number of cold-incubated cells released the nucleic acids. This indicates substantial degradation of DNA and RNA in nonculturable cells prior to fixation. Treatment of permeabilized cells with DNase and RNase allowed differential staining of DNA and RNA with the nucleic acid dye 4',6-diamidino-2-phenylindole (DAPI). Epifluorescence microscopy revealed that the could-induced nonculturable populations of V. vulnificus are highly heterogeneous with regard to their nucleic acid content. The fraction of nonculturable cells which maintained DNA and RNA structures decreased gradually during cold incubation. After 5 months at 5 degrees C, less than 0.05% of the cells could be observed to retain DNA and RNA. In parallel with the loss of nucleic acids, an increase in the concentrations of UV-absorbing material in the culture supernatants was observed in nonculturable-cell suspensions. It is hypothesized that there are two phases of the formation of nonculturable cells of V. vulnificus: the first involves a loss of culturability with maintenance of cellular integrity and intact RNA and DNA (and thus possibly viability), and the second is typified by a gradual degradation of nucleic acids, the products of which partly remain inside the cells and partly diffuse into the extracellular space. A small number of nonculturable cells, however, retain DNA and RNA, and thus may be viable despite having reduced culturability.     

Induction by mercuric ion of extensive degradation of cellular ribonucleic acid in Escherichia coli

Low concentrations of HgCl(2) were found to induce extensive degradation of ribonucleic acid (RNA) in exponentially growing Escherichia coli cells but not in stationary-phase cells. Whereas 80% of cellular RNA was degraded during 90 min of incubation with 10(-5)m HgCl(2) at 37 C, HgCl(2) caused only slight degradation in stationary cells, even when present at concentrations higher than 5 x 10(-5)m. Inhibition of RNA synthesis occurred at almost the same concentration of HgCl(2) as degradation, and the ability of stationary-phase cells to synthesize RNA was also resistant to HgCl(2). The transition of cells from complete sensitivity to HgCl(2) to a fully insensitive state took place simultaneously with the cessation of growth. p-Chloromercuribenzoate was also found to induce remarkable degradation of RNA. In E. coli Q13, a mutant deficient for ribonuclease I, no degradation of RNA was evident, even in the exponential growth phase. 3'-Mononucleotides but not 5'-mononucleotides were found among the degradation products of cellular RNA. 2',3'-Cyclic mononucleotides were produced when RNA was degraded by the cell-free extracts of the Hg treated cells. Almost complete unmasking of the latent ribonuclease occurred in the particle fraction containing subribosomal particles of the Hg-treated cells. These data suggest that the incubation of exponentially growing E. coli cells with HgCl(2) led to the unmasking of ribonuclease I, which resulted in the extensive degradation of cellular RNA. The activation of ribonuclease by HgCl(2) in the isolated particulate fraction of E. coli K-12 which occurred in vitro suggested the presence of an Hg-sensitive inhibitor for ribonuclease I.     

Thermally induced ribonucleic acid degradation and leakage of substances from the metabolic pool in Staphylococcus aureus

The effects of temperatures of 50 and 60 C on log-phase and stationary-phase cell suspensions of Staphylococcus aureus are described. There is a leakage of free amino acids, protein, and 260 mmu-absorbing material from both types of cell suspension, and membrane damage, as measured by the intracellular penetration of 8-anilino-1-naphthalene-sulfonic acid, may be partially related to this leakage. Ribonucleic acid (RNA) degradation at any one temperature is virtually the same for both types of cell suspension, proceeding initially at a more rapid rate at 60 C than at 50 C. However, at the lower temperature, there is a secondary breakdown of RNA, which may be the result of enzyme action on a particularly labile RNA fraction. With stationary-phase cell suspensions heated in 1 m sucrose, there is a more rapid degradation of RNA at 60 C than with cells in water. The results are discussed in relation to the biochemical effects of moist heat on the organism.     

Selective Inhibition of Deoxyribonucleic Acid Synthesis by 2-Deoxyadenosine in the Blue-Green Bacterium Agmenellum quadruplicatum

Concentrations of deoxyadenosine which have little effect on net ribonucleic acid (RNA) synthesis or on increase in cell mass selectively inhibit deoxyribonucleic acid (DNA) synthesis in Agmenellum quadruplicatum. Exogenously supplied deoxyadenosine, at concentrations above 10 mug/ml, stimulates DNA degradation. These results are correlated with a rapid loss in viability. Over a narrow concentration range (6-15 mug/ml), deoxyadenosine impairs the division process and induces the formation of elongated cells. Low exogenous concentrations of deoxyadenosine are readily incorporated into both DNA and RNA, with the major portion as DNA.     

DEGRADATION OF RIBONUCLEIC ACID IN MOUSE FIBROBLASTS TREATED WITH ACTINOMYCIN

The cellular RNA content of mouse fibroblasts incubated with actinomycin decreases at a rate of about 1 to 1.5 per cent per hour, while DNA and protein content remain unchanged. This degradation affects nuclear and cytoplasmic RNA, ribosomal and soluble RNA. The breakdown products appear quantitatively in the acid-soluble fraction of the cells and the medium. Polynucleotides synthesized a short period (120 minutes) prior to exposure to actinomycin are degraded before those synthesized 8 to 12 hours previously.     

Human cytomegalovirus virions differentially incorporate viral and host cell RNA during the assembly process

While analyzing human cytomegalovirus (HCMV) gene expression in infected cells by RNA-specific nucleic acid sequence-based amplification (NASBA), positive results were observed for HCMV RNA encoded by several viral genes immediately after the addition of the virus. UV-inactivated virus also gave a positive NASBA result without establishing active infection, suggesting that RNA was associated with the inoculum. Highly purified virions devoid of cellular contamination proved to be positive for viral RNA encoding both immediate-early (UL123) and late (UL65) gene products. Virion-associated RNA might be incorporated specifically or without selection during the virion assembly. In the latter case, cellular RNA would also be present in the virion. A high-abundant cellular RNA encoded by GAPDH and even U1A RNA, which is expressed at low levels, were detected in the virion fraction, whereas cellular DNA was absent. Virion fractionation revealed that cellular RNA was absent in purified de-enveloped capsids. In conclusion, cellular and viral RNA was present between the capsid and envelope of the virion, whereas in the capsid only viral RNA could be detected. The results suggest that virion-associated viral and cellular RNA is incorporated nonspecifically during virion assembly.     

MicroRNA fate upon targeting with anti-miRNA oligonucleotides as revealed by an improved Northern-blot-based method for miRNA detection

MicroRNAs (miRNAs) are small non-coding RNAs involved in fine-tuning of gene regulation. Antisense oligonucleotides (ONs) are promising tools as anti-miRNA (anti-miR) agents toward therapeutic applications and to uncover miRNA function. Such anti-miR ONs include 2'-O-methyl (OMe), cationic peptide nucleic acids like K-PNA-K3, and locked nucleic acid (LNA)-based anti-miRs such as LNA/DNA or LNA/OMe. Northern blotting is a widely used and robust technique to detect miRNAs. However, miRNA quantification in the presence of anti-miR ONs has proved to be challenging, due to detection artifacts, which has led to poor understanding of miRNA fate upon anti-miR binding. Here we show that anti-miR ON bound to miR-122 can prevent the miRNA from being properly precipitated into the purified RNA fraction using the standard RNA extraction protocol (TRI-Reagent), yielding an RNA extract that does not reflect the real cellular levels of the miRNA. An increase in the numbers of equivalents of isopropanol during the precipitation step leads to full recovery of the targeted miRNA back into the purified RNA extract. Following our improved protocol, we demonstrate by Northern blotting, in conjunction with a PNA decoy strategy and use of high denaturing PAGE, that high-affinity anti-miRs (K-PNA-K3, LNA/DNA, and LNA/OMe) sequester miR-122 without causing miRNA degradation, while miR-122 targeting with a lower-affinity anti-miR (OMe) seems to promote degradation of the miRNA. The technical issues explored in this work will have relevance for other hybridization-based techniques for miRNA quantification in the presence of anti-miR ONs.     

Distribution of Ribosomal Ribonucleic Acid Cistrons Among Yeast Chromosomes

High-molecular-weight deoxyribonucleic acid (DNA) of Saccharomyces carls bergensis has been fractionated by sucrose density gradient centrifugation. The main DNA fraction has an average molecular weight of about 500 x 10(6). A major fraction of the DNA molecules containing sequences homologous to ribosomal ribonucleic acid (RNA) sediments as material of this molecular weight. The remainder sediments as material of a molecular weight of about 250 x 10(6). The latter fraction contains relatively more ribosomal RNA cistrons than the former. Studies on the buoyant density of high-molecular-weight DNA homologous to ribosomal RNA have led to the conclusion that the ribosomal RNA cistrons occur in groups attached to a relatively large amount of nonribosomal RNA and suggest that ribosomal RNA cistrons are distributed over a number of yeast chromosomes.     

Synthesis of Protein and Nucleic Acid by Disrupted Spheroplasts of Pseudomonas schuylkilliensis

Osmotically shocked spheroplasts obtained from Pseudomonas schuylkilliensis strain P contained about 54, 32, 28, and 82% of the total cellular protein, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and phospholipid, respectively. This preparation was capable of incorporating (32)P-orthophosphate into RNA and DNA, (3)H-adenosine or (3)H-uridine into RNA, and (3)H-leucine or (14)C-phenylalanine into protein. These activities were not found in the cytoplasmic fraction which contained most of the glucose-6-phosphate dehydrogenase activity. The synthesis of RNA by intact and disrupted spheroplast preparations was sensitive to actinomycin D, chromomycin A(3), streptovaricin, rifampin, Lubrol W, Triton X-100, and sodium deoxycholate, whereas RNA synthesis by intact cells was insensitive to these agents. Ethylenediaminetetraacetic acid, porcine pancreatic lipase, the protoplast-bursting factor, high concentrations of salts, and washing the preparation inhibited the synthesis of RNA by disrupted spheroplasts but had little or no effect on intact spheroplasts. Most of the newly synthesized RNA made by disrupted spheroplasts had the characteristics of messenger RNA. The DNA present in this preparation functioned as a template for RNA synthesis; continued protein synthesis was dependent on concomitant RNA synthesis. An unusual feature of the preparation was the finding that the synthesis of macromolecules was completely dependent on oxidative phosphorylation.     

Pelagic-Benthic Coupling and Diagenesis of Nucleic Acids in a Deep-Sea Continental Margin and an Open-Slope System of the Eastern Mediterranean

Downward fluxes of nucleic acids adsorbed onto settling particles play a key role in the supply of organic phosphorus and genetic material to the ocean interior. However, information on pelagic-benthic coupling, diagenesis, and processes controlling nucleic acid preservation in deep-sea sediments is practically nonexistent. In this study, we compared nucleic acid fluxes, sedimentary DNA and RNA concentrations, and the enzymatically hydrolyzable fraction of DNA in a bathyal continental margin (North Aegean Sea) and an open-sea system (South Aegean Sea) of the Eastern Mediterranean. The two systems displayed contrasting patterns of nucleic acid fluxes, which increased significantly with depth in the North Aegean Sea and decreased with depth in the South Aegean Sea. These results suggest that in continental margin and open-ocean systems different processes control the nucleic acid supply to the sea floor. Differences in nucleic acid fluxes were reflected by nucleic acid concentrations in the sediments, which reached extremely high values in the North Aegean Sea. In this system, a large fraction of DNA may be buried, as suggested by the large fraction of DNA resistant to nuclease degradation and by estimates of burial efficiency (ca. eight times higher in the North than in the South Aegean Sea). Overall, the results reported here suggest that the preservation of DNA in deeper sediment layers may be favored in benthic systems characterized by high sedimentation rates.     

Pelagic-benthic coupling and diagenesis of nucleic acids in a deep-sea continental margin and an open-slope system of the Eastern Mediterranean

Downward fluxes of nucleic acids adsorbed onto settling particles play a key role in the supply of organic phosphorus and genetic material to the ocean interior. However, information on pelagic-benthic coupling, diagenesis, and processes controlling nucleic acid preservation in deep-sea sediments is practically nonexistent. In this study, we compared nucleic acid fluxes, sedimentary DNA and RNA concentrations, and the enzymatically hydrolyzable fraction of DNA in a bathyal continental margin (North Aegean Sea) and an open-sea system (South Aegean Sea) of the Eastern Mediterranean. The two systems displayed contrasting patterns of nucleic acid fluxes, which increased significantly with depth in the North Aegean Sea and decreased with depth in the South Aegean Sea. These results suggest that in continental margin and open-ocean systems different processes control the nucleic acid supply to the sea floor. Differences in nucleic acid fluxes were reflected by nucleic acid concentrations in the sediments, which reached extremely high values in the North Aegean Sea. In this system, a large fraction of DNA may be buried, as suggested by the large fraction of DNA resistant to nuclease degradation and by estimates of burial efficiency (ca. eight times higher in the North than in the South Aegean Sea). Overall, the results reported here suggest that the preservation of DNA in deeper sediment layers may be favored in benthic systems characterized by high sedimentation rates.     

Kinetics of Nucleic Acid Synthesis in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Inhibition of Cellular Deoxyribonucleic Acid Synthesis

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of (3)H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. (3)H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much (3)H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase.     

Messenger Ribonucleic Acid of Dormant Spores of Bacillus subtilis

Evidence of the presence of messenger ribonucleic acid (mRNA) in dormant spores of Bacillus subtilis has been obtained. The bulk RNA from spores was isolated and labeled in vitro with tritiated dimethyl sulfate. The spore RNA hybridized to 2.4 to 3.2% of the B. subtilis genome. The RNA hybridized to both the complementary heavy and light fractions of deoxyribonucleic acid (DNA). Bulk RNA from log-phase cells competed with virtually all the spore RNA for the heavy DNA fraction and with part of the spore RNA for the light DNA fraction. Bulk RNA from stage IV cells in sporulation also competed with all of the spore RNA for the heavy DNA fraction and with essentially all the spore RNA for the light DNA fraction. These results indicate that dormant spores contain mRNA species present in both log-phase cells and stage IV cells of sporulation. The RNA polymerase in the developing forespore must be able to recognize promotor sites for both log-phase and sporulation genes.     

The release of radioactive nucleic acids and mucoproteins by trypsin and ethylenediaminetetra-acetate treatment of baby-hamster cells in tissue culture

Monolayers of baby-hamster kidney cells were grown on glass in tissue culture and harvested with trypsin or EDTA in order to investigate the cell surface macromolecules removed by these cell-disaggregating agents. The release of nucleic acids from the cells during the harvesting procedure was monitored by labelling the cellular RNA with [5-(3)H]uridine and the cellular DNA with [2-(14)C]thymidine. Treatment of the cells with EDTA was found to cause an increase in the permeability of the plasma membrane with 7.6% of the cellular RNA, but less than 1% of the cellular DNA, being released. Moreover, 61% of the cells harvested with EDTA were permeable to Trypan Blue. With crude trypsin, lysis of the cell occurred with the release of similar amounts of RNA and DNA amounting to about 11% of the total cellular nucleic acid. In contrast, crystalline trypsin released only 1% of the cellular nucleic acids. Since virtually all the cells (99%) after harvesting in crystalline trypsin were impermeable to Trypan Blue, this method was suitable for obtaining cell surface macromolecules without contamination by intracellular damage. [1-(14)C]Glucosamine was incorporated by the cells only into bound hexosamines and sialic acids. [By monitoring the release of radioactivity in high-molecular-weight material in such experiments a measure of the release of macromolecules containing amino sugars was obtained.] Of the total macromolecules containing amino sugars in the cells 33%, 24% and 13% were released when the cells were harvested with crude trypsin, crystalline trypsin or EDTA respectively. Crystalline trypsin also released 39% of the total sialic acid of the cell, whereas less than 1% of the cellular sialic acid was present in the EDTA-treated fraction. It is concluded that the macromolecules containing amino sugars released with crude trypsin and EDTA are likely to be heavily contaminated with intracellular material. However, the macromolecules released by crystalline trypsin appear to come from the cell surface.