Concentration of viruses and dissolved DNA from aquatic environments by vortex flow filtration.

Vortex flow filtration (VFF) was used to concentrate viruses and dissolved DNA from freshwater and seawater samples taken in Florida, the Gulf of Mexico, and the Bahamas Bank. Recoveries of T2 phage and calf thymus DNA added to artificial seawater and concentrated by VFF were 72.8 and 80%, respectively. Virus concentrations determined by transmission electron microscopy of VFF-concentrated samples ranged from 3.4 x 10(7)/ml for a eutrophic Tampa Bay sample to 2.4 x 10(5) for an oligotrophic oceanic surface sample from the southeastern Gulf of Mexico. Viruslike particles were also observed in a sample taken from a depth of 1,500 m in the subtropical North Atlantic Ocean. Filtration of samples through Nuclepore or Durapore filters (pore size, 0.2 micron) prior to VFF reduced phage counts by an average of two-thirds. Measurement of dissolved-DNA content by Hoechst 33258 fluorescence in environmental samples concentrated by VFF yielded values only ca. 35% of those obtained for samples concentrated by ethanol precipitation (the standard dissolved-DNA method). However, ethanol precipitation of VFF-concentrated extracts resulted in an increase in measurable DNA, reaching 80% of the value obtained by the standard method. These results indicate that a portion of the naturally occurring dissolved DNA is in a form inaccessible to nucleases and Hoechst stain, perhaps bound to protein or other polymeric material, and is released upon ethanol precipitation. Viral DNA contents estimated from viral counts averaged only 3.7% (range, 0.9 to 12.3%) of the total dissolved DNA for samples from freshwater, estuarine, and offshore oligotrophic environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Concentration of viruses and dissolved DNA from aquatic environments by vortex flow filtration.

Vortex flow filtration (VFF) was used to concentrate viruses and dissolved DNA from freshwater and seawater samples taken in Florida, the Gulf of Mexico, and the Bahamas Bank. Recoveries of T2 phage and calf thymus DNA added to artificial seawater and concentrated by VFF were 72.8 and 80%, respectively. Virus concentrations determined by transmission electron microscopy of VFF-concentrated samples ranged from 3.4 x 10(7)/ml for a eutrophic Tampa Bay sample to 2.4 x 10(5) for an oligotrophic oceanic surface sample from the southeastern Gulf of Mexico. Viruslike particles were also observed in a sample taken from a depth of 1,500 m in the subtropical North Atlantic Ocean. Filtration of samples through Nuclepore or Durapore filters (pore size, 0.2 micron) prior to VFF reduced phage counts by an average of two-thirds. Measurement of dissolved-DNA content by Hoechst 33258 fluorescence in environmental samples concentrated by VFF yielded values only ca. 35% of those obtained for samples concentrated by ethanol precipitation (the standard dissolved-DNA method). However, ethanol precipitation of VFF-concentrated extracts resulted in an increase in measurable DNA, reaching 80% of the value obtained by the standard method. These results indicate that a portion of the naturally occurring dissolved DNA is in a form inaccessible to nucleases and Hoechst stain, perhaps bound to protein or other polymeric material, and is released upon ethanol precipitation. Viral DNA contents estimated from viral counts averaged only 3.7% (range, 0.9 to 12.3%) of the total dissolved DNA for samples from freshwater, estuarine, and offshore oligotrophic environments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-molecular- weight Rauscher leukemia virus protein with preferential binding for single-stranded RNA and DNA.

A sensitive nitrocellulose filter assay that measures the retention of 125I single-stranded calf thymus DNA has been used to detect and purify DNA-binding proteins that retain a biological function from Rauscher murine leukemia virus. By consecutive purification on oligo (dT)- cellulose and DEAE-Bio-Gel columns and centrifugation in 10 to 30% glycerol gradients, RNA-dependent DNA polymerase has been separated from a second virion DNA-binding protein. The binding of this protein to DNA was strongly affected by NaCl concentration but showed little change in activity over a wide range of temperature or pH. After glycerol gradient purification, polyacrylamide gel electrophoresis of this protein showed one major band with a molecular weight of approximately 9,800. This protein binds about as well as to single-stranded Escherichia coli or calf thymus DNA or 70S type C viral RNA. The binding to 125I single-stranded calf thymus DNA is very efficiently inhibited by unlabeled single-stranded DNA from either E. coli or calf thymus and by 70S murine or feline viral RNA. Much larger amounts of double-stranded DNA are required to produce an equivalent percentage of inhibition. This protein, therefore, shows preferential binding to single-stranded DNA or viral RNA.     

Synthesis and Intracellular Localization of Vaccinia Virus Deoxyribonucleic Acid-dependent Ribonucleic Acid Polymerase

The time course of vaccinia deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase synthesis and its intracellular localization were studied with virus-infected HeLa cells. Viral RNA polymerase activity could be meassured shortly after viral infection in the cytoplasmic fraction of infected cells in vitro. However, unless the cells were broken in the presence of the nonionic detergent Triton-X-100, no significant synthesis of new RNA polymerase was detected during the viral growth cycle. When cells were broken in the presence of this detergent, extensive increases in viral RNA polymerase activity were observed late in the infection cycle. The onset of new RNA polymerase synthesis was dependent on prior viral DNA replication. Fluorodeoxyuridine (5 x 10(-5)m) prevented the onset of viral polymerase synthesis. Streptovitacin A, a specific and complete inhibitor of protein synthesis in HeLa cells, prevented the synthesis of RNA polymerase. Thus, the synthesis of RNA polymerase is a "late" function of the virus. The newly synthesized RNA polymerase activity was primarily bound to particles which sedimented during high-speed centrifugation. These particles have been characterized by sucrose gradient centrifugation. A major class of active RNA polymerase particles were considerably "lighter" than whole virus in sucrose gradients. These particles were entirely resistant to the action of added pancreatic deoxyribonuclease, and they were not stimulated by added calf thymus primer DNA. It is concluded that these particles are not active in RNA synthesis in vivo, and that activation occurs as a result of detergent treatment in vitro.     

Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences.

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.     

Dual staining of natural bacterioplankton with 4',6-diamidino-2-phenylindole and fluorescent oligonucleotide probes targeting kingdom-level 16S rRNA sequences.

A method for quantifying eubacterial cell densities in dilute communities of small bacterioplankton is presented. Cells in water samples were stained with 4',6-diamidino-2-phenylindole (DAPI), transferred to gelatin-coated slides, and hybridized with rhodamine-labeled oligonucleotide probes specific for kingdom-level 16S rRNA sequences. Between 48 and 69% of the cells captured on membrane filters were transferred to gelatin-coated slides. The number of DAPI-stained cells that were visualized with eubacterial probes varied from 35 to 67%. Only 2 to 4% of these cells also fluoresced following hybridization with a probe designed to target a eukaryotic 16S rRNA sequence. Between 0.1 and 6% of the bacterioplankton in these samples were autofluorescent and may have been mistaken as cells that hybridized with fluorescent oligonucleotide probes. Dual staining allows precise estimates of the efficiency of transfers of cells to gelatin films and can be used to measure the percentage of the total bacterioplankton that also hybridize with fluorescent oligonucleotide probes, indicating specific phylogenetic groups.     

Nucleosome core particles of calf thymus, Tetrahymena, and the reconstituted hybrid. Their structure reflects the nature of the histone octamer

The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer. The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known. The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved. The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure. The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60 degrees C and 72 degrees C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50 degrees C, 60-61 degrees C, and 72 degrees C. Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA. We conclude that the Tetrahymena histones are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes.     

Development of alternate ssu-rRNA probing strategies for characterizing aquatic microbial communities

Plastids in phytoplankton retain prokaryote-like DNA sequences that may generate false-positive signals from eubacterial small subunit (ssu) rRNA oligonucleotide probes, resulting in the overestimation of bacterial activity in aquatic microbial communities. To assess the extent of possible plastid-associated binding to eubacterial signals, we performed an extensive database search, flask experiments using algal and cyanobacterial pure cultures, and field trials on five common eubacterial probes: S-D-Bact-008-a-A-19, S-D-Bact-338-a-A-18, S-D-Bact-785-a-A-19, S-D-Bact-927-a-A-17, and S-D-Bact-1088-a-A-20. The database search and laboratory tests showed significant potential for binding among most bacterial probes and organelle ssu-rRNA. However, we propose two probing strategies to overcome this problem. First, one could use Bact-785 and Bact-338 in tandem, with the plastid component being estimated as the difference between the two signals (Bact-338 has approximately 70% overlap with known plastid sequences). Alternately, one might use Bact-338 as the primary eubacterial probe, but then use Cyan-785-a-A-19 (a probe that binds significantly to plastid rRNA) to correct for the plastid-associated false-positive signal. Both strategies would use a eukaryotic probe (S-D-Euca-1379-a-A-16) and Cyan-785-b-A-19 (a probe for most cyanobacteria) to further segregate rRNA signals. Trials were successfully performed using the strategies on samples from a recent field study.     

Thermal denaturation of calf thymus DNA: existence of a GC-richer fraction

In 2.5 x 10(-4)M EDTA buffer, the derivative melting curve of calf thymus DNA shows a major band at 47 degrees with a shoulder at about 54 degrees . The fraction of melting area of this shoulder is about 13%. For reconstituted polylysine-calf thymus DNA complexes, in addition to the melting of free DNA regions at about 50 degrees (T(m)) there is another melting at about 106 degrees (T(m)) of polylysine-bound regions. The melting band of the complex at T(m) is not symmetrical. As more polylysine is bound to DNA the melting amplitude is diminished greatly on the major band at 47 degrees but only slightly on the shoulder at 54 degrees . The insensitivity of this shoulder appears to result from the existence of a 13% fraction of calf thymus DNA containing 55% GC. It is not favorably bound by polylysine. It remains in the supernatant after centrifugation and melts at about 54-56 degrees . This conclusion is further supported by two facts: the reconstitution method provides a condition for selective binding of polylysine to AT-rich DNA, and it yields a fully symmetric melting band at T(m) for complexes of polylysine with homogeneous bacterial DNA such as the one from M. luteus.     

Transcription of polyoma virus DNA in vitro. III. Localization of calf thymus RNA polymerase II binding sites.

The binding sites of calf thymus RNA polymerase II on polyoma DNA were monitored by electron microscopy. Six discrete binding sites were located at positions 0.06, 0.25, 0.57, 0.66, 0.85 and 0.98 on the physical map of polyoma DNA. Although most of these sites are located in easily denaturable regions of the DNA, the strongest binding sites do not overlap with the major A + T-rich regions. In addition, the same binding sites were observed on superhelical or linear polyoma DNA. These results suggest that the eucaryotic RNA polymerase II can recognize specific sequences on double-stranded DNA and not only easily denaturable regions. At least five of these sites correspond to the binding and initiation sites mapped previously for the Escherichia coli RNA polymerase (Lescure et al., 1976).Stable initiation complexes can be formed with both E. coli and calf thymus RNA polymerases in the presence of a single dinucleotide (GpU) and a specific ribotriphosphate (CTP). Under these conditions, the binding of both enzymes to the sites in positions 0.06 and 0.57 is stimulated whereas the binding in positions 0.65 and 0.84 is partially suppressed. Both eucaryotic and procaryotic RNA polymerases may recognize similar sequences of the viral DNA in vitro.     

Detection of Tn5-like sequences in kanamycin-resistant stream bacteria and environmental DNA.

Resistance to kanamycin and neomycin in the bacterial assemblage of a coastal plain stream was detected by growth of colonies on media containing antibiotics. Three of 184 kanamycin-resistant colonies hybridized with a probe containing the nptII gene from transposon Tn5; the nptII gene encodes the enzyme neomycin phosphotransferase and conveys resistance to kanamycin and neomycin. In one of these isolates, the homologous gene was cloned and shown to confer resistance to a kanamycin-sensitive Escherichia coli strain. Since enumeration of bacteria by acridine orange direct counts revealed that less than 0.2% of the bacteria present were cultivated, direct examination of environmental DNA was used to assess abundance of sequences that hybridize to the nptII gene. To examine the resistance potential of bacteria that were not cultured, total DNA was extracted from environmental samples and hybridized with specific probes. The relative amount of eubacterial DNA in each sample was determined by using a eubacterial specific rDNA probe. Then, the abundance of sequences that hybridize to the eubacterial neomycin phosphotransferase gene was determined by hybridization and expressed relative to the total eubacterial DNA in the assemblage. Relative gene abundance was significantly different among assemblages from different habitats (leaves, midchannel sediments, and bank sediments) but did not differ among stream sites.     

Detection and characterization of cyanobacterial nifH genes.

The DNA sequence of a 359-bp fragment of nifH was determined for the heterocystous strains Anabaena sp. strain CA (ATCC 33047), Nostoc muscorum UTEX 1933, a Nostoc sp., Gloeothece sp. strain ATCC 27152, Lyngbya lagerheimii UTEX 1930, and Plectonema boryanum IU 594. Results confirmed that the DNA sequence of the 359-bp segment is sufficiently variable to distinguish cyanobacterial nifH genes from other eubacterial and arachaeobacterial nifH genes, as well as to distinguish heterocystous from nonheterocystous nifH genes. Nonheterocystous cyanobacterial nifH sequences were greater than 70 and 82% identical on the DNA and amino acid levels, respectively, whereas corresponding values for heterocystous cyanobacterial nifH sequences were 84 and 91%. The amplified nifH fragments can be used as DNA probes to differentiate between species, although there was substantial cross-reactivity between the nifH amplification products of some strains. However, an oligonucleotide designed from a sequence conserved within the heterocystous cyanobacteria hybridized primarily with the amplification product from heterocystous strains. The use of oligonucleotides designed from amplified nifH sequences shows great promise for characterizing assemblages of diazotrophs.     

Fluorescent detection of cyanobacterial DNA using bacterial magnetic particles on a MAG-microarray

Bacterial magnetic particles (BMPs) were used for the identification of cyanobacterial DNA. Genus-specific oligonucleotide probes for the detection of Anabaena spp., Microcystis spp., Nostoc spp., Oscillatoria spp., and Synechococcus spp. were designed from the variable region of the cyanobacterial 16S rDNA of 148 strains. These oligonucleotide probes were immobilized on BMPs via streptavidin-biotin conjugation and employed for magnetic-capture hybridization against digoxigenin-labeled cyanobacterial 16S rDNA. Bacterial magnetic particles were magnetically concentrated, spotted in 100-microm-size microwell on MAG-microarray, and the fluorescent detection was performed. This work details the development of an automated technique for the magnetic isolation, the concentration of hybridized DNA, and the detection of specific target DNA on MAG-microarray. The entire process of hybridization and detection was automatically performed using a magnetic-separation robot and all five cyanobacterial genera were successfully discriminated.     

PCNA-dependent DNA polymerase delta from rabbit bone marrow.

Proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta were partially purified and characterized from rabbit bone marrow. Rabbit DNA polymerase delta sediments at 8.2 S upon glycerol density gradient centrifugation. Similar to calf thymus PCNA-dependent DNA polymerase delta, a 125-123-kDa doublet and 48-kDa polypeptides correlate with DNA polymerase activity. Western blotting of rabbit DNA polymerase delta with polyclonal antibody to calf thymus PCNA-dependent DNA polymerase delta gives the same results as calf thymus delta; the 125-123-kDa doublet is recognized. PCNA-dependent DNA polymerase delta is resistant to inhibition by dideoxynucleotides and is relatively insensitive to inhibition by N2-[p-(n-butyl)phenyl]dGTP. A 3'-->5' exonuclease copurifies with the DNA polymerase. The processivity of DNA polymerase delta alone is very low but greatly increases with the addition of PCNA from rabbit bone marrow or calf thymus. Comparative studies of the original DNA polymerase delta from rabbit bone marrow demonstrate a lack of recognition by antibodies to calf thymus delta and a high degree of processivity in the absence of PCNA. Additionally, the originally described DNA polymerase delta is a single polypeptide of 122 kDa. These features would recategorize the original delta to the epsilon category by recently proposed convention. PCNA-dependent DNA polymerase delta is a relatively minor component of rabbit bone marrow compared to DNA polymerase alpha and PCNA-independent DNA polymerase delta (epsilon), the relative proportions being alpha, 60%; delta, 7%; and epsilon, 30%.     

Direct probing: covalent attachment of probe DNA to double-stranded target DNA.

We report a novel procedure, which can be applied to probing of specific DNA, for covalently attaching probe DNA to complementary sequences in double-stranded target DNA. Employing hairpin-like oligonucleotide probes in combination with successive use of recA protein and DNA ligase, probes can be attached directly to target DNA molecules without dissociation of the DNA. The hairpin-like structure of the probes was designed so that the terminus of the probe oligonucleotide can be brought into close stereochemical proximity to the terminus of the complementary strand of target DNA for ligation. Because of the elimination of the DNA dissociation and subsequent hybridization (and washing) steps in the currently employed method, the probing process has become greatly simplified and more efficient and may lead to development of fully automated probing systems.     

Environmental dissolved DNA harbours meaningful biological information on microbial community structure

Extracellular DNA (eDNA) comprises all the DNA molecules outside cells. This component of microbial ecosystems may serve as a source of nutrients and genetic information. Hypersaline environments harbour one of the highest concentrations of eDNA reported for natural systems, which has been attributed to the physicochemical preservative effect of salts and to high viral abundance. Here, we compared centrifugation and filtration protocols for the extraction of dissolved DNA (dDNA, as opposed to eDNA that also includes DNA from free viral particles) from a solar saltern crystallizer pond (CR30) water sample. The crystallizer dDNA fraction has been characterized, for the first time, and compared with cellular and viral metagenomes from the same location. High-speed centrifugation affected CR30 dDNA concentration and composition due to cell lysis, highlighting that protocol optimization should be the first step in dDNA studies. Crystallizer dDNA, which accounted for lower concentrations than those previously reported for hypersaline anoxic sediments, had a mixed viral and cellular origin, was enriched in archaeal DNA and had a distinctive taxonomic composition compared to that from the cellular assemblage of the same sample. Bioinformatic analyses indicated that nanohaloarchaeal viruses could be a cause for these differences.     

Randomly amplified polymorphic DNA PCR as a tool for assessment of marine viral richness

Recent discoveries have uncovered considerable genetic diversity among aquatic viruses and raised questions about the variability of this diversity within and between environments. Studies of the temporal and spatial dynamics of aquatic viral assemblages have been hindered by the lack of a common genetic marker among viruses for rapid diversity assessments. Randomly amplified polymorphic DNA (RAPD) PCR bypasses this obstacle by sampling at the genetic level without requiring viral isolation or previous sequence knowledge. In this study, the utility of RAPD-PCR for assessing DNA viral richness within Chesapeake Bay water samples was evaluated. RAPD-PCR using single 10-mer oligonucleotide primers successfully produced amplicons from a variety of viral samples, and banding patterns were highly reproducible, indicating that each band likely represents a single amplicon originating from viral template DNA. In agreement with observations from other community profiling techniques, resulting RAPD-PCR banding patterns revealed more temporal than spatial variability in Chesapeake Bay virioplankton assemblages. High-quality hybridization probes and sequence information were also easily generated from single RAPD-PCR products or whole reactions. Thus, the RAPD-PCR technique appears to be practical and efficient for routine use in high-resolution viral diversity studies by providing assemblage comparisons through fingerprinting, probing, or sequence information.     

On the arrangement of histones in chromatin.

It was found that nucleoprotein particles formed after DNase I action on calf thymus chromatin contain single-stranded DNA fragments, associated with histones only by ionic linkages. These results suggest that histones in chromatin are bound ionically only to one polynucleotide strand of double-helical DNA, protecting it against nucleolytic attack.