Pelagic-benthic coupling of nucleic acids in an abyssal location of the northeastern Atlantic Ocean.

Spatial and temporal changes in sedimentary nucleic acid concentrations in an abyssal locality of the northeastern Atlantic Ocean were investigated in relation to fluxes of nucleic acids produced in the photic layer. Sediment trap material, collected between 1996 and 1998 at depths of 1,000, 3,000, and 4,700 m, and sediment samples were analyzed for DNA and RNA content. Nucleic acid concentrations in the sediments were very high and displayed significant temporal changes, whereas mesoscale variability was low. DNA and RNA concentrations generally displayed opposite temporal patterns, which are likely to be dependent on the nature and characteristics of DNA and RNA molecules. Nucleic acid fluxes were high and displayed clear seasonal changes apparently coupled with seasonal pulses of primary production. However, while median values of DNA fluxes were relatively similar in all sediment traps, median values of RNA fluxes almost doubled from the 1,000- to the 4,700-m depth, suggesting differences in the metabolic activity of microbes associated with sinking particles. Significant relationships between DNA concentrations in the sediments and DNA fluxes and between RNA concentrations and RNA fluxes, indicating the presence of a clear pelagic-benthic coupling of particulate nucleic acids, were observed. The benthic system investigated was not steady state since we estimated that, from September 1996 to October 1998, nucleic acid concentration in the sediments decreased by about 165 mg of DNA m(-2). Vertical profiles revealed a significant decrease in DNA concentration with depth in the sediments, reaching an asymptotic value of about 5 microg g(-1). This DNA fraction constitutes a pool of potentially refractory DNA (accounting for 16 to 40% of the total DNA pool) that might be buried in the sediments.     

Pelagic-Benthic Coupling of Nucleic Acids in an Abyssal Location of the Northeastern Atlantic Ocean

Spatial and temporal changes in sedimentary nucleic acid concentrations in an abyssal locality of the northeastern Atlantic Ocean were investigated in relation to fluxes of nucleic acids produced in the photic layer. Sediment trap material, collected between 1996 and 1998 at depths of 1,000, 3,000, and 4,700 m, and sediment samples were analyzed for DNA and RNA content. Nucleic acid concentrations in the sediments were very high and displayed significant temporal changes, whereas mesoscale variability was low. DNA and RNA concentrations generally displayed opposite temporal patterns, which are likely to be dependent on the nature and characteristics of DNA and RNA molecules. Nucleic acid fluxes were high and displayed clear seasonal changes apparently coupled with seasonal pulses of primary production. However, while median values of DNA fluxes were relatively similar in all sediment traps, median values of RNA fluxes almost doubled from the 1,000- to the 4,700-m depth, suggesting differences in the metabolic activity of microbes associated with sinking particles. Significant relationships between DNA concentrations in the sediments and DNA fluxes and between RNA concentrations and RNA fluxes, indicating the presence of a clear pelagic-benthic coupling of particulate nucleic acids, were observed. The benthic system investigated was not steady state since we estimated that, from September 1996 to October 1998, nucleic acid concentration in the sediments decreased by about 165 mg of DNA m⁻². Vertical profiles revealed a significant decrease in DNA concentration with depth in the sediments, reaching an asymptotic value of about 5 μg g⁻¹. This DNA fraction constitutes a pool of potentially refractory DNA (accounting for 16 to 40% of the total DNA pool) that might be buried in the sediments.     

Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.     

Improved protocol for high-quality Co-extraction of DNA and RNA from rumen digesta

We report an improved method for total nucleic acids extraction from rumen content samples. The method employs bead beating, and phenol-chloroform extraction followed by saline-alcohol precipitation. Total nucleic acids and RNA yield and purity were assessed by spectrophotometric measurements; RNA integrity was estimated using Agilent RNA 6000 Nano Kit on an Agilent 2100 Bioanalyzer. The method provided total nucleic acids and RNA extracts of good quantity and quality. The extraction is not time consuming and it is valuable for ecological studies of rumen microbial community structure and gene expression.     

Fluorometric quantification of RNA and DNA in solutions containing both nucleic acids

A fluorescence-based method for quantitative determination of RNA and DNA in probes containing both nucleic acids has been developed. The total concentration of nucleic acids is determined using SYBR Green II dye under conditions providing independent binding of the fluorophore with DNA and RNA. The concentration of DNA is specifically measured using the Hoechst 33258 dye and the RNA concentration is calculated from these data. The procedure allows for accurate determination of DNA concentration in the range 10-1000 ng/ml in the presence of 200-fold excess of RNA and determination of RNA concentrations in the range 10-1000 ng/ml in the presence of large excess of DNA. An absence of the treatment of mixed samples with RNase-free DNase I provides rapid, reproducible, and accurate RNA quantification.     

Viruses, prokaryotes and DNA in the sediments of a deep-hypersaline anoxic basin (DHAB) of the Mediterranean Sea

Viral and prokaryote abundance were investigated in a deep-hypersaline anoxic basin of the Eastern Mediterranean Sea (DHAB Atalante basin at c. 3000 m depth). This system was compared with two nearby deep-sea sites characterized by oxic conditions. Viral abundance and virus to prokaryote abundance ratio in hypersaline anoxic sediments displayed values close to those reported in oxic sites. The analysis of vertical profiles of viral abundance in the Atalante basin revealed the lack of significant changes with depth in the sediment, suggesting that benthic viruses in these anoxic and hypersaline conditions are preserved or resistant to decay. The anoxic basin displayed also very high concentrations of labile organic components (proteins and lipids) and extracellular DNA. These findings suggest that the DHAB sediments represent a reservoir for long-term preservation of benthic viruses and nucleic acids.     

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.     

Food effect on the physiological condition of the mussel Perna viridis (Bivalvia: Mytilidae), using the RNA/DNA ratio

The green mussel, Perna viridis, became widespread in the northern coast of Sucre State since its arrival to Venezuela in 1993. RNA/DNA and Protein/DNA ratios were used to study the effect of starvation on its instantaneous growth. The mussels were collected in La Esmeralda and Chacopata, acclimatized in the laboratory for four weeks and maintained for another six weeks in two groups: one fed ad libitum and another without food (this later group was later fed for two additional weeks). Protein (colorimetric method), and nucleic acid concentrations (RNA and DNA, fluorometric method with ethidium bromide) were measured in adductor muscle, digestive gland and gills. The instantaneous growth was assessed using RNA/DNA and Protein/DNA rations. These indexes were always higher in the fed organisms. Animals from Chacopata were in better physiological condition that those from La Esmeralda during the abstinence time (six weeks). Muscle was the best tissue to determine instantaneous growth. The RNA/DNA ratio is a reliable index to determine the physiological condition and instantaneous growth of this species.     

Laboratory calibrations of the [h]adenine technique for measuring rates of RNA and DNA synthesis in marine microorganisms

The nucleic acid synthesis rates of several marine phytoplankton and bacteria grown in chemostat and batch cultures were measured by using [H]adenine. The [H]adenine synthesis rates showed excellent agreement with the known rates of synthesis estimated from chemical RNA and DNA data. Under certain conditions, RNA turnover and ATP pool compartmentalization produce inaccuracies in synthesis measurements made with [H]adenine. However, accurate measurements of the rates of microbial RNA and DNA synthesis can be made in any environmental situation provided a few simple precautions are observed. First, time course experiments are recommended. Second, experiments should be conducted for periods long enough to avoid problems arising from disequilibria of internal ATP pools. Finally, exogenous [H]adenine should remain in the medium over the length of the time course.     

Benthic diatom distribution and grazing by Sarotherodon mossambicus in Lake Sibaya, South Africa

SUMMARY. Distributions of benthic diatoms and benthic floc in Lake Sibaya were examined to determine if preferential grazing by juvenile Sarotherodon mossambicus on benthic detrital aggregate in shallow water maximized diatom concentrations in the diet of the fish. Between 0.1 and 12.0 m depth, the range of diatom densities was 1–60 × 10⁴ cells cm⁻² with high but extremely variable densities above 3 m and consistently low densities at greater depths. Light intensity and burial by wave action appear to be primary factors affecting diatom abundance, but the effect of unidentified factors causing local patchiness is also evident. Substrate stabilization by diatoms was observed between 0.3 and 1.8 m depth. Weight of benthic detrital aggregate generally increased with depth such that diatom concentrations were variable above 3 m but were extremely low in benthic detrital aggregate at greater depths. Comparison of diatom concentrations in fish stomachs and benthic detrital aggregate shows that within the shallow waters, fish do not selectively graze those areas richest in diatoms.     

A method to determine RNA and DNA oxidation simultaneously by HPLC-ECD: greater RNA than DNA oxidation in rat liver after doxorubicin administration

We developed a novel method for the simultaneous extraction and analysis of total tissue RNA and DNA to quantify the RNA and DNA oxidation products 8-oxo-7,8-dihydroguanosine and 8-oxo-7,8-dihydro-2'-deoxyguanosine using HPLC coupled to electrochemical detection (HPLC-ECD). The protein denaturing agents guanidine thiocyanate and phenol/chloroform at neutral pH were found to be very efficient for the isolation of RNA and DNA from rat brain, liver and muscle. The method is very fast, allows extraction at 0 degrees C, gives high yields of pure RNA and DNA with low background oxidation levels, and also determines the RNA/DNA ratio. Experiments with isolated RNA and DNA exposed to the Fenton reagents H2O2/ascorbate/Fe3+ (or Cu2+) resulted in significantly greater RNA oxidation. The RNase inhibitor 2-mercaptoethanol, commonly used for RNA extraction, acted as a pro-oxidant during nucleic acid extraction, an effect attenuated by the inclusion of the metal chelator deferoxamine mesylate. In vivo, administration of doxorubicin (an oxidant generator) to Fisher-344 rats resulted in a significant increase in liver RNA oxidation, but no significantly increased DNA oxidation. This new method could be useful to assess oxidatively damaged RNA and DNA simultaneously, and our data show that RNA is more susceptible to oxidative stress than DNA in vivo and in vitro.     

Chromium incorporated in RNA and DNA

The objective of this study was to examine the effect of Cr(III) (chromium chloride) and Cr(VI) (potassium dichromate) on RNA and DNA-chromium adducts formation in isolated nucleic acids and isolated pig lymphocytes. The incubation of cells with potassium dichromate and chromium chloride at concentrations of 10 and 100 microM results in binding of a 1.2-1.9 fold greater number of chromium atoms to nuclear DNA than to total cellular RNA. The incubation of total cellular RNA and nuclear DNA isolated from lymphocytes with CrCl3 and K2Cr2O7 yielded a binding of 1.1-1.6 fold more of Cr atoms to RNA than to DNA. The number of chromium atoms bound to nucleic acids is higher after incubation with K2Cr2O7 than with CrCl3 in both experimental systems.     

Fluorometric Quantification of Polyphosphate in Environmental Plankton Samples: Extraction Protocols,Matrix Effects,and Nucleic Acid Interference

Polyphosphate (polyP) is a ubiquitous biochemical with many cellular functions and comprises an important environmental phosphorus pool. However, methodological challenges have hampered routine quantification of polyP in environmental samples. We tested 15 protocols to extract inorganic polyphosphate from natural marine samples and cultured cyanobacteria for fluorometric quantification with 4′,6-diamidino-2-phenylindole (DAPI) without prior purification. A combination of brief boiling and digestion with proteinase K was superior to all other protocols, including other enzymatic digestions and neutral or alkaline leaches. However, three successive extractions were required to extract all polyP. Standard addition revealed matrix effects that differed between sample types, causing polyP to be over- or underestimated by up to 50% in the samples tested here. Although previous studies judged that the presence of DNA would not complicate fluorometric quantification of polyP with DAPI, we show that RNA can cause significant interference at the wavelengths used to measure polyP. Importantly, treating samples with DNase and RNase before proteinase K digestion reduced fluorescence by up to 57%. We measured particulate polyP along a North Pacific coastal-to-open ocean transect and show that particulate polyP concentrations increased toward the open ocean. While our final method is optimized for marine particulate matter, different environmental sample types may need to be assessed for matrix effects, extraction efficiency, and nucleic acid interference.     

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A₂₆₀), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).¹ Though it cannot readily distinguish RNA from DNA, the A₂₆₀/A₂₈₀ ratio is commonly employed, as it offers a simple and rapid² assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios < 0.8 are taken as indicative of ''pure'' protein specimens, while pure nucleic acid (NA) is characterized by ratios > 1.5³.However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at ≈280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A₂₆₀/A₂₈₀ ratios (~0.8 < ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict''s, Bial''s (orcinol), and Dische''s (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).     

Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase.

An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-microns DEAE anion-exchange resin capable of chromatographing nucleic acids greater than 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 M potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When [14C]doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 M sodium chloride at 50 degrees C for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol/100 micrograms), and the highest levels were recorded with oligonucleotides (243 pmol/100 micrograms). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 microM [14C]doxorubicin (18.6 pmol/100 micrograms). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques.