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PubMed2001

Changes in bacterial community composition and dynamics and viral mortality rates associated with enhanced flagellate grazing in a mesoeutrophic reservoir.

Bacterioplankton from a meso-eutrophic dam reservoir was size fractionated to reduce (<0.8-microm treatment) or enhance (<5-microm treatment) protistan grazing and then incubated in situ for 96 h in dialysis bags. Time course samples were taken from the bags and the reservoir to estimate bacterial abundance, mean cell volume, production, protistan grazing, viral abundance, and frequency of visibly infected cells. Shifts in bacterial community composition (BCC) were examined by denaturing gradient gel electrophoresis (DGGE), cloning and sequencing of 16S rDNA genes from the different treatments, and fluorescence in situ hybridization (FISH) with previously employed and newly designed oligonucleotide probes. Changes in bacterioplankton characteristics were clearly linked to changes in mortality rates. In the reservoir, where bacterial production about equaled protist grazing and viral mortality, community characteristics were nearly invariant. In the "grazer-free" (0.8-microm-filtered) treatment, subject only to a relatively low mortality rate (approximately 17% day(-1)) from viral lysis, bacteria increased markedly in concentration. While the mean bacterial cell volume was invariant, DGGE indicated a shift in BCC and FISH revealed an increase in the proportion of one lineage within the beta proteobacteria. In the grazing-enhanced treatment (5-microm filtrate), grazing mortality was approximately 200% and viral lysis resulted in mortality of 30% of daily production. Cell concentrations declined, and grazing-resistant flocs and filaments eventually dominated the biomass, together accounting for >80% of the total bacteria by the end of the experiment. Once again, BCC changed strongly and a significant fraction of the large filaments was detected using a FISH probe targeted to members of the Flectobacillus lineage. Shifts of BCC were also reflected in DGGE patterns and in the increases in the relative importance of both beta proteobacteria and members of the Cytophaga-Flavobacterium cluster, which consistently formed different parts of the bacterial flocs. Viral concentrations and frequencies of infected cells were highly significantly correlated with grazing rates, suggesting that protistan grazing may stimulate viral activity.

Animals↗
PubMed1997

Competitive oligonucleotide single-base extension combined with mass spectrometric detection for mutation screening.

A rapid, robust and widely applicable mutation detection scheme not requiring radioactivity or gel-based detection is introduced. It is a single-tube, competitive oligonucleotide single-base extension (COSBE) reaction using a pair of primers with the 3'-terminal base complementary to either the normal or mutant allele. Upon hybridization and addition of a polymerase and the nucleotide triphosphate corresponding to the next base after the primer, only those primers properly annealed (i.e., no 3'-terminal mismatch) are extended; products are resolved by molecular weight shifts as determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (single-scan spectrum acquisition < < 1 s). For the cystic fibrosis delta F508 polymorphism, 28-mer "normal" (N) and 30-mer "mutant" (M) primers generate 29-mer (N + I) or 31-mer (M + 1) products for homozygotes and both for heterozygotes. Since primer and product molecular weights are relatively low (< 10 kDa) and the mass difference between these are at least that of a single approximately 300-Da nucleotide unit, a low-resolution mass spectrometer is suitable for such measurements.

Alleles↗
PubMed1992

Influence of pH on the migration properties of oligonucleotides in capillary gel electrophoresis.

The effect of pH on the electrophoretic migration properties of single-stranded oligodeoxyribonucleotides in capillary gel electrophoresis was investigated. Different homooligodeoxyribonucleotides of equal chain length showed significant differences in relative migration when the pH of the gel buffer was varied from pH 6 to 8, parallel with the running buffer. A similar variation in migration order was observed during the electrophoretic equilibration of a pH 8 gel-filled capillary column with a pH 6 running buffer. In the latter instance, the current reached the new level after 20 min of electrophoretic equilibration with the pH 6 running buffer. However, it was observed that the migration order characteristic of the pH 6 gel was achieved only after 4 h of electrophoretic equilibration. To avoid this time-consuming equilibration process, these results suggest that gel-filled capillary columns should be prepared with the same buffer (composition and pH) that will be used as the running buffer during the separations.

Buffers↗
PubMed1991

A molecular dynamics study of the effect of G.T mispairs on the conformation of DNA in solution.

The effect of G.T mispair incorporation into a double-helical environment was examined by molecular dynamics simulation. The 60-ps simulations performed on the two hexanucleotide duplexes d (G3C3)2 and d(G3TC2)2 included 10 Na+ counterions and first hydration shell waters. The resulting backbone torsional angle trajectories were analyzed to select time spans representative of conformational domains. The average backbone angles and helical parameters of the last time span for both duplexes are reported. During the simulation the hexamers retained B-type DNA structures that differed from typical A- or B-DNA forms. The overall helical structures for the two duplexes are vary similar. The presence of G.T mispairs did not alter the overall helical structure of the oligonucleotide duplex. Large propeller twist and buckle angles were obtained for both duplexes. The purine/pyrimidine crossover step showed a large decrease in propeller twist in the normal duplex but not in the mismatch duplex. Upon the formation of wobble mispairs in the mismatched duplex, the guanines moved into the minor groove and the thymines moved into the major groove. This helped prevent purine/purine clash and created a deformation in the relative orientation of the glycosidic bonds. It also exposed the free O4 of the thymines in the major groove and N2 of the guanines in the minor groove to interactions with solvent and counterions. These factors seemed to contribute to the apparently higher rigidity of the mismatched duplex during the simulation.

Base Composition↗
PubMed1998

Presence and location of modified nucleotides in Escherichia coli tmRNA: structural mimicry with tRNA acceptor branches.

Escherichia coli tmRNA functions uniquely as both tRNA and mRNA and possesses structural elements similar to canonical tRNAs. To test whether this mimicry extends to post-transcriptional modification, the technique of combined liquid chromatography/ electrospray ionization mass spectrometry (LC/ESIMS) and sequence data were used to determine the molecular masses of all oligonucleotides produced by RNase T1 hydrolysis with a mean error of 0.1 Da. Thus, this allowed for the detection, chemical characterization and sequence placement of modified nucleotides which produced a change in mass. Also, chemical modifications were used to locate mass-silent modifications. The native E.coli tmRNA contains two modified nucleosides, 5-methyluridine and pseudouridine. Both modifications are located within the proposed tRNA-like domain, in a seven-nucleotide loop mimicking the conserved sequence of T loops in canonical tRNAs. Although tmRNA acceptor branches (acceptor stem and T stem-loop) utilize different architectural rules than those of canonical tRNAs, their conformations in solution may be very similar. A comparative structural and functional analysis of unmodified tmRNA made by in vitro transcription and native E.coli tmRNA suggests that one or both of these post-transcriptional modifications may be required for optimal stability of the acceptor branch which is needed for efficient aminoacylation.

Base Composition↗
PubMedDate not supplied

[Physico-chemical characteristics of highly purified ceruloplasmin mRNA from rat liver].

Highly purified preparations of mRNA coding for ceruloplasmin (CP) ere isolated from rat liver polyribosomes using indirect immunoprecipitation of CP polysomes and poly(U)-sepharose chromatography of polysomal RNA. The homogeneity of CP mRNA was as high as 86--90%. The molecular weight of CP mRNA is 1.3 . 10(6) daltons which is in excess when compared to the minimal size of mRNA necessary to code for CP precursor (about 700 amino acid residues). The base composition of CP mRNA is of AU-type. The experiments on end-labeling with [3H]borohydride after periodate oxidation whowed that CP mRNA contains 3'-terminal poly(A). Poly(U)-sepharose chromatography with stepwise temperature elution revealed length heterogeneity of poly(A) consisting of particular, different thermal subfractions of CP mRNA contain poly(A) consisting of 38, 90 and 165 adenylate residue. 5'-end of CP mRNA is block with inverted 7-methylguanosine (m7G) which is reducible with [3H]borohydride after periodate oxidation. This m7G residue is a component of RNAse- and alkali-resistant oligonucleotide, which structure according to net charge value and its shifts after various enzymatic treatments, is m7G5'ppp5'XmpAp.

Animals↗
PubMed2003

[Studies on fluorescence in situ hybridization with group-specific 16S rRNA-based probes in analysis of human colonic microflora].

In order to establish the optional experimental conditions of fluorescence in situ hybridization (FISH) for analysis of colonic microflora, and analyze the stability and validation of the technique, fresh fecal samples were collected from the volunteers and five group-specific 16S rRNA-targeted oligonucleotide probes were used, and the effects of keeping time and temperature of fresh fecal samples after collected, the centrifugal speed and time, the storage time of paraformaldehyde-stocks of samples on the results were analyzed. The stability and the validation were assessed by CV values. Results showed that: (1) The optional experimental conditions were that the fresh fecal samples were not kept for more than 12 hours at 4 degrees C, the centrifugal speed of samples was 700 g for 2 minutes, and the paraformaldehyde-stocks were not kept for more than 5 months at low temperature (-80 degrees C) before analysis; (2) The method had good stability and could detect the differences of colonic microflora composition between objects at a significant level.

Adult↗
PubMed1990

Perturbation of DNA hairpins containing the EcoRI recognition site by hairpin loops of varying size and composition: physical (NMR and UV) and enzymatic (EcoRI) studies.

We have investigated loop-induced structural perturbation of the stem structure in hairpins d(GAATTCXnGAATTC) (X = A, T and n = 3, 4, 5 and 6) that contain an EcoRI restriction site in close proximity to the hairpin loop. Oligonucleotides containing either a T3 or a A3 loop were not hydrolyzed by the restriction enzyme and also showed only weak binding to EcoRI in the absence of the cofactor Mg2+. In contrast, hairpins with larger loops are hydrolyzed by the enzyme at the scission site next to the loop although the substrate with a A4 loop is significantly more resistant than the oligonucleotide containing a T4 loop. The hairpin structures with 3 loop residues were found to be thermally most stable while larger hairpin loops resulted in structures with lower melting temperatures. The T-loop hairpins are thermally more stable than the hairpins containing the same number of A residues in the loop. As judged from proton NMR spectroscopy and the thermodynamic data, the base pair closest to the hairpin loop did form in all cases studied. The hairpin loops did, however, affect the conformation of the stem structure of the hairpins. From 31P and 1H NMR spectroscopy we conclude that the perturbation of the stem structure is stronger for smaller hairpin loops and that the extent of the perturbation is limited to 2-3 base pairs for hairpins with T3 or A4 loops. Our results demonstrate that hairpin loops modulate the conformation of the stem residues close to the loop and that this in turn reduces the substrate activity for DNA sequence specific proteins.

DNA↗
PubMed2005

Fabrication of DNA microarrays onto poly(methyl methacrylate) with ultraviolet patterning and microfluidics for the detection of low-abundant point mutations.

We have developed a simple ultraviolet (UV)-photomodification protocol using poly(methyl methacrylate) and polycarbonate to produce functional scaffolds consisting of carboxylic groups that allow covalent attachment of amine-terminated oligonucleotide probes to these surface groups through carbodiimide coupling. Use of the photomodification procedure coupled to microfluidics allowed for the rapid generation of medium-density DNA microarrays. The method reported herein involves the use of poly(dimethylsiloxane) microchannels reversibly sealed to photomodified poly(methyl methacrylate) surfaces to serve as stencils for patterning the oligonucleotide probes. After array construction, the poly(dimethylsiloxane) stencil is rotated 90 degrees to allow interrogation of the array using microfluidics. The photomodification process for array fabrication involves only three steps: (1) broadband UV exposure of the polymer surface, (2) carbodiimide coupling of amine-terminated oligonucleotide probes to the surface (via an amide bond), and (3) washing of the surface. The density of probes attached to this activated surface was found to be approximately 41pmolcm(-2), near the steric-saturation limit for short oligonucleotide probes. We demonstrate the use of this procedure for screening multiple KRAS2 mutations possessing high diagnostic value for colorectal cancers. A ligase detection reaction/universal array assay was carried out using parallel detection of two different low-abundant DNA point mutations in KRAS2 oncogenes with the allelic composition evaluated at one locus. Four zip code probes immobilized onto the poly(methyl methacrylate) surface directed allele-specific ligation products containing mutations in the KRAS2 gene (12.2D, 12.2A, 12.2V, and 13.4D) to the appropriate address of a universal array with minimal amounts of cross-hybridization or misligation.

Adsorption↗
PubMed1997

Specific DNA recognition by the Aspergillus nidulans three zinc finger transcription factor PacC.

The three zinc fingers of PacC, the transcription factor mediating pH regulation in Aspergillus nidulans, are necessary and sufficient to recognise specifically the target ipnA2 site. Missing nucleoside footprints confirmed the core target (double-stranded) hexanucleotide 5'-GCCAAG-3'. Any base substitution resulted in substantial or complete loss of binding, excepting A5 (partially replaceable by G). A T preceding the hexanucleotide enhanced binding. Interference footprinting indicates that the four Gs and A4 participate in specific contacts and that five pyrimidines are essential for binding. The size of the target sequence and the amino acid sequence of finger 1 suggested that its probe helix would not participate in base-specific contacts. Using site-directed mutagenesis and analogy to GLI, we propose that finger 1 crucially interacts with finger 2, a pair of conserved Trp residues in the Cys knuckles contacting hydrophobically. Finger 2 would also participate in extensive base contacts with the 5' moiety of the hexanucleotide. The specificity mutation Lys159Gln shows that finger 3 binds the 3' moiety of the hexanucleotide. Replacement of residues in positions +3 (His128Asn) and +2 (Gln155Lys) of the reading helices of fingers 2 and 3, respectively, prevented binding. Our biochemical and molecular data plus modelling using previously determined zinc finger-DNA complexes, predict specific contacts of fingers 2 and 3 to ipnA2. Our data indicate compact organisation of the PacC-ipnA2 complex (with nearly every base involved in specific contacts), illustrate the binding versatility of zinc finger domains and should facilitate analysis of other PacC family members, including Saccharomyces cerevisiae RIM1.

Amino Acid Sequence↗
PubMed1990

Rapid detection of influenza virus H1 by the polymerase chain reaction.

We applied a combination of reverse transcription (RT) with the polymerase chain reaction (PCR) for a rapid detection of influenza virus H1 subtype. We amplified a 441 bp segment of relatively high genetic stability of the hemagglutinin gene. Experimental conditions were established using plasmid DNA and infected cell cultures. The test was applied to 28 nasopharyngeal lavages from patients, two of which were positive for influenza virus H1. When the amplified DNA of a positive sample was sequenced we found 97% homology with the recent strain A/USSR/70.

Base Composition↗
PubMed1991

The last duplex base-pair of the phage lambda chromosome. Involvement in packaging, ejection and routing of lambda DNA.

cosN is the site at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces staggered nicks to generate the cohesive ends of mature lambda chromosomes. Genetic and molecular studies show that cosN is recognized specifically by terminase and that effects of cosN mutations on lambda DNA packaging and cosN cleavage are well correlated. Mutations affecting a particular base-pair of cosN are unusual in being lethal in spite of causing only a moderate defect in cosN cleavage and DNA packaging. The particular base-pair is the rightmost duplex base-pair in mature chromosomes, at position 48,502 in the numbering system of Daniels et al; herein called position - 1. A G.C to T.A transversion mutation at position - 1, called cosN - 1T, reduces the particle yield of lambda fivefold, and the particles formed are not infectious. lambda cosN - 1T particles have wild-type morphology, and contain chromosomes that have normal cohesive ends. The chromosomes of lambda cosN - 1T particles, like the chromosomes of lambda + particles, are associated with the tail. lambda cosN - 1T particles, in spite of being normal structurally, are defective in injection of DNA into a host cell. Only approximately 25% of lambda cosN - 1T particles are able to eject DNA from the capsid in contrast to 100% for lambda +. Furthermore, for the 25% that do eject, there is a further injection defect because the ejected lambda cosN - 1T chromosomes fail to cyclize, in contrast to the efficient cyclization found for wild-type chromosomes following injection. The cosN - 1T mutation has no effect on Ca2+ mediated transformation by lambda DNA, indicating that the effect of the mutation on DNA fate is specific to the process of DNA injection. Models in which specific DNA : protein interactions necessary for DNA injection, and involving the rightmost base-pair of the lambda chromosome, are considered.

Bacteriophage lambda↗
PubMed1991

Enhanced chemiluminescence for the detection of membrane-bound nucleic acid sequences: advantages of the Amersham system.

A range of nonradioactive nucleic acid labeling and detection systems have been developed that enable the user to label probes directly with enzyme molecules or indirectly with hapten-derivatized nucleotides. Horseradish peroxidase is used for the direct labeling procedures due to the ease of chemical modification and the relative thermal and chemical stability of this enzyme. Horseradish peroxidase has also been conjugated to a high-specificity antifluorescein antibody for detection of hapten (fluorescein)-labeled hybrids. Enhanced chemiluminescence is a light-emitting process optimized for the detection of low levels of horseradish peroxidase on membrane supports. Results are obtained as hard copy images on x-ray film.

Base Composition↗
PubMed2004

Quadruplex to Watson-Crick duplex transition of the thrombin binding aptamer: a fluorescence resonance energy transfer study.

Thermodynamic parameters of closing up of guanine-rich thrombin binding element, upon binding to K(+) and Na(+) ions to form quadruplexes and opening up of these quadruplexes upon binding to its complementary strand, were investigated. For this purpose, 15mer deoxynucleotide, d(G(2)T(2)G(2)TGTG(2)T(2)G(2)), labeled with 5'-fluorescein and 3'-tetramethylrhodamine was taken and fluorescence resonance energy transfer was monitored as a function of either metal ions or complementary strand concentrations. Equilibrium association constant obtained from FRET studies demonstrates that K(+) ions bind with higher affinity than the Na(+) ions. The enthalpy changes, DeltaH, obtained from temperature dependence of equilibrium association constant studies revealed that formation of quadruplex upon binding of metal ions is primarily enthalpy driven. Binding studies of complementary strand to the quadruplex suggest that opening of a quadruplex in NaCl buffer in presence of the complementary strand is enthalpic as well as entropic driven and can occur easily, whereas opening of the same quadruplex in KCl buffer suffers from enthalpic barrier. Comparison of overall thermodynamic parameters along with kinetics studies indicates that, although quadruplexes cannot efficiently compete with duplex formation at physiological pH, they delay the association of two strands.

Animals↗
PubMed1990

Discrimination between bacteriophage T3 and T7 promoters by the T3 and T7 RNA polymerases depends primarily upon a three base-pair region located 10 to 12 base-pairs upstream from the start site.

The bacteriophage T3 and T7 RNA polymerases are closely related, yet are highly specific for their own promoter sequences. To understand the basis of this specificity, T7 promoter variant that contain substitutions of T3-specific base-pairs at one or more positions within the T7 promoter consensus sequence were synthesized and cloned. Template competition assays between variant and consensus promoters demonstrate that the primary determinants of promoter specificity are located in the region from -10 to -12, and that the base-pair at -11 is of particular importance. Changing this base-pair from G.C, which is normally present in T7 promoters, to C.G, which is found at this position in T3 promoters, prevented utilization by the T7 RNA polymerase and simultaneously enabled transcription from the variant T7 promoter by the T3 enzyme. Substitution of T7 base-pairs with T3 base-pairs at other positions where the two consensus sequences diverge affected the overall efficiency with which the variant promoter was utilized by the T7 RNA polymerase, but these changes were not sufficient to permit recognition by the T3 RNA polymerase. Switching the -11 base-pair in the T3 promoter consensus to the T7 base-pair prevented utilization by the T3 RNA polymerase, but did not allow the T3 variant promoter to be utilized by the T7 RNA polymerase. This probably reflects a greater specificity of the T7 RNA polymerase for base-pairs at other positions where the promoter sequences differ, most notably at -15. The magnitude of the effects of base substitutions in the T7 promoter on promoter strength (-11C much greater than -10C greater than -12A) correlates with the affinity of the T7 polymerase for the promoter variants, suggesting that the discrimination of the phage RNA polymerases for their promoters is mediated primarily at the level of DNA binding, rather than at the level of initiation.

Base Composition↗
PubMed2003

Pentamer vocabularies characterizing introns and intron-like intergenic tracts from Caenorhabditis elegans and Drosophila melanogaster.

Overall compositional properties at the level of bases, dinucleotides and longer oligos characterize genomes of different species. In Caenorhabditis elegans, using recurrence analysis, we recognized the existence of a long-range correlation in the oligonucleotide usage of introns and intergenic regions. Through correlation analysis, this is confirmed here to be a genome-wide property of C. elegans non-coding portions. We then investigate the possibility of extracting a typical vocabulary through statistical analysis of experimentally confirmed introns of sufficient length (>1 kb), deprived of known splice signals, the focus being on distributed lexical features rather than on localized motifs. Lexical preferences typical of introns could be exposed using principal component analysis of pentanucleotide frequency distributions, both in C. elegans and in Drosophila melanogaster. In either species, the introns' pentamer preferences are largely shared by intergenic tracts. The pentamer vocabularies extracted for the two species exhibit interesting symmetry properties and overlap in part. A more extensive investigation of the interspecies relationship at the level of oligonucleotide preferences in non-coding regions, not related by sequence similarity, might form the basis of new approaches for the study of the evolutionary behaviour of these regions.

Animals↗
PubMed1991

Crystal and molecular structure of a DNA fragment: d(CGTGAATTCACG).

The crystal structure of the dodecanucleotide d(CGTGAATTCACG) has been determined to a resolution of 2.7 A and refined to an R factor of 17.0% for 1532 reflections. The sequence crystallizes as a B-form double helix, with Watson-Crick base pairing. This sequence contains the EcoRI restriction endonuclease recognition site, GAATTC, and is flanked by CGT on the 5'-end and ACG on the 3'-end, in contrast to the CGC on the 5'-end and GCG on the 3'-end in the parent dodecamer d(CGCGAATTCGCG). A comparison with the isomorphous parent compound shows that any changes in the structure induced by the change in the sequence in the flanking region are highly localized. The global conformation of the duplex is conserved. The overall bend in the helix is 10 degrees. The average helical twist values for the present and the parent structures are 36.5 degrees and 36.4 degrees, respectively, corresponding to 10 base pairs per turn. The buckle at the substituted sites are significantly different from those seen at the corresponding positions in the parent dodecamer. Step 2 (GpT) is underwound with respect to the parent structure (27 degrees vs 36 degrees) and step 3 (TpG) is overwound (34 degrees vs 27 degrees). There is a spine of hydration in the narrow minor groove. The N3 atom of adenine on the substituted A10 and A22 bases are involved in the formation of hydrogen bonds with other duplexes or with water; the N3 atom of guanine on G10 and G22 bases in the parent structure does not form hydrogen bonds.

Base Composition↗