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PubMed1998

Codon usage in Actinobacillus actinomycetemcomitans.

The codon usage patterns of 21 genes encompassing 5800 codons from Actinobacillus actinomycetemcomitans were analyzed. A. actinomycetemcomitans genes could be divided into two groups based on their function and G + C content. One group included those genes encoding basic cellular functions. This group displayed an average G + C content of 48%. A second group comprised genes encoding the leukotoxin determinant, an insertion sequence and a plasmid. This group displayed an average G + C content of 36%. These findings suggest that portions of the A. actinomycetemcomitans genome may have been acquired by horizontal gene transfer from one or more distantly related species. We present a table of A. actinomycetemcomitans codon usage. These data may be used to establish standards for computer programs that predict A. actinomycetemcomitans protein coding regions and may be useful in designing degenerate oligonucleotide probes.

Adolescent↗
PubMed1994

Characterization of a cDNA encoding the 43-kDa membrane-associated inositol-polyphosphate 5-phosphatase.

Agonist stimulation of cells results in phosphatidylinositol turnover and the generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), which mobilizes intracellular calcium. The inositol-polyphosphate 5-phosphatase (5-phosphatase) enzymes hydrolyze Ins(1,4,5)P3 in a signal-terminating reaction. We have isolated a 2.7-kilobase (kb) composite cDNA, encoding the 43-kDa membrane-associated 5-phosphatase, by screening a human placental lambda gt11 library, using degenerate oligonucleotides. The 2.7-kb cDNA contains a 1.1-kb open reading frame, comprising 363 amino acids, which encodes a protein of a predicted molecular mass of 42 kDa. Amino acid sequence analysis demonstrates a number of potential sites for phosphorylation by protein kinase C and a CAAX motif in the COOH terminus, which may mediate membrane localization. The recombinant enzyme was expressed in COS-7 cells, resulting in a 50-fold increase in enzyme activity in the detergent-soluble membrane fraction of the cell (nanomole of Ins(1,4,5)P3 hydrolyzed per min/mg), but only a 2.5-fold increase in 5-phosphatase activity in the total cell homogenate. Sequence analysis demonstrated a 73-amino acid domain in the COOH terminus of the 43-kDa membrane-associated 5-phosphatase, which had 30% sequence identity and 67% similarity to a region in the 75-kDa 5-phosphatase and 34% identity and 70% similarity to a sequence in the protein that is encoded by the gene, defective in Lowe's oculocerebrorenal syndrome. As shown by RNA analysis the 43-kDa membrane-associated 5-phosphatase appears to be predominantly expressed in heart, brain, and skeletal muscle.

Amino Acid Sequence↗
PubMed1989

The Deinococcus-Thermus phylum and the effect of rRNA composition on phylogenetic tree construction.

Through comparative analysis of 16S ribosomal RNA sequences, it can be shown that two seemingly dissimilar types of eubacteria Deinococcus and the ubiquitous hot spring organism Thermus are distantly but specifically related to one another. This confirms an earlier report based upon 16S rRNA oligonucleotide cataloging studies (Hensel et al., 1986). Their two lineages form a distinctive grouping within the eubacteria that deserved the taxonomic status of a phylum. The (partial) sequence of T. aquaticus rRNA appears relatively close to those of other thermophilic eubacteria. e.g. Thermotoga maritima and Thermomicrobium roseum. However, this closeness does not reflect a true evolutionary closeness; rather it is due to a "thermophilic convergence", the result of unusually high G+C composition in the rRNAs of thermophilic bacteria. Unless such compositional biases are taken into account, the branching order and root of phylogenetic trees can be incorrectly inferred.

Bacteria↗
PubMed1999

Crystallization of the 10-23 DNA enzyme using a combinatorial screen of paired oligonucleotides.

One of the most difficult steps in the X-ray crystallography of nucleic acids is obtaining crystals that diffract to high resolution. The choice of the nucleotide sequence has proven to be more important in producing high-quality crystals than the composition of the crystallization solution. This manuscript describes a systematic procedure for identifying the optimal sizes of a multi-stranded nucleic acid complex which provide high-quality crystals. This approach was used to crystallize the in vitro evolved 10-23 DNA enzyme complexed with its RNA substrate. In less than two months, 81 different enzyme-substrate complexes were generated by combinatorial mixing and annealing of complementary oligonucleotides which differed in length, resulting in duplexes of varying length, with or without nucleotide overhangs. Each of these complexes was screened against a standard set of 48 crystallization conditions and evaluated for crystal formation. The screen resulted in over 40 crystal forms, the best of which diffracted to 2.8 A resolution when exposed to a synchrotron X-ray source.

Combinatorial Chemistry Techniques↗
PubMed2000

Analysis of oligonucleotide AUG start codon context in eukariotic mRNAs.

The AUG start codon context features have been investigated by analyzing eukaryotic mRNAs belonging to various taxonomic groups. The functional relevance of each specific position surrounding the AUG start codon has been established as a function of the measured shift between base composition observed at that particular position, and base composition averaged over all the 5'untranslated regions. A more detailed analysis carried out on human genes belonging to different isochores showed significant isochore-specific fea-tures that cannot be explained only by a mutational bias effect. The most represented heptamers spanning from position -3 to +4 with respect to the initiator AUG have been determined for mRNAs belonging to different taxonomic groups and a web page utility has been set up (http://bigarea.area.ba.cnr.it:8000/BioWWW/ATG.html) to determine the relative abundance of a user submitted oligonucleotide context in a given species or taxon.

5' Untranslated Regions↗
PubMed2000

Effect of nucleoside analogues and oligonucleotides on hydrolysis of liposomal phospholipids.

The hydrolysis of the bilayer forming phospholipids resulting first of all in lysophospholipids and fatty acids is one limiting factor determining the shelf-life of liposomes. In several studies the influence of pH, buffer, lipid composition and other parameters on the hydrolysis of phospholipids have been demonstrated, but the influence of drugs has not yet been investigated systematically. In this study the influence of nucleoside analogues, especially 2', 2'-difluoro 2'-deoxycytidine (gemcitabine, dFdC) on the degradation of phospholipids was elucidated in more detail. It could be demonstrated that the interaction of dFdC with phospholipid bilayers promotes the hydrolysis of phospholipids in a concentration-dependent manner. Obviously two parts of the molecule, the amino group bound to the pyrimidine moiety and the 2', 2'-difluoro-2'-deoxyribose, seem to be responsible for the forced phospholipid hydrolysis. The dFdC-induced hydrolysis of phospholipids was influenced by pH, buffer, lipid composition and different anions. Optimization of the above parameters resulted in prolonged shelf-life of dFdC liposome dispersions, which is an important prerequisite for clinical practice.

Anions↗
PubMed2006

Meganema perideroedes gen. nov., sp. nov., a filamentous alphaproteobacterium from activated sludge.

An industrial wastewater treatment plant at Grindsted, Denmark, has suffered from bulking problems for several years caused by filamentous bacteria. Five strains were isolated from the sludge by micromanipulation. Phylogenetic analysis of the 16S rRNA gene sequences showed that the strains formed a monophyletic cluster in the Alphaproteobacteria, and they were phenotypically different from their closest relatives and from all hitherto known filamentous bacteria described (closest relative Brevundimonas vesicularis ATCC 11426(T), 89.8 % sequence similarity). In pure culture, the cells (1.5-2.0 microm) in filaments are Gram-negative and contain polyphosphate and polyhydroxyalkanoates. The optimum temperature for growth is 30 degrees C and the strains grow in 2 % NaCl and are oxidase- and catalase-positive. Ubiquinone 10 is the major quinone. The major fatty acid (C(18 : 1)omega7c) and smaller amounts of unsaturated fatty acids, 3-hydroxy fatty acids with a chain length of 16 and 18 carbon atoms and small amounts of 10-methyl-branched fatty acids with 18 carbon atoms (C(19 : 0) 10-methyl) affiliated the strains with the Methylobacterium/Xanthobacter group in the Alphaproteobacteria. The G+C content of the DNA is 42.9 mol% (for strain Gr1(T)). The two most dissimilar isolates by 16S rRNA gene comparison (Gr1(T) and Gr10; 97.7 % identical) showed 71.5 % DNA-DNA relatedness. Oligonucleotide probes specific for the pure cultures were designed for fluorescence in situ hybridization and demonstrated that two filamentous morphotypes were present in the Grindsted wastewater treatment plant. It is proposed that the isolates represent a new genus and species, Meganema perideroedes gen. nov., sp. nov. The type strain of Meganema perideroedes is strain Gr1(T) (=DSM 15528(T)=ATCC BAA-740(T)).

Alphaproteobacteria↗
PubMed1996

Direct ribosome isolation from soil to extract bacterial rRNA for community analysis.

A simple method that combines an adapted ribosome isolation method and a common RNA extraction step has been developed for selective recovery of intact rRNA from natural microbial communities in soil. After mechanical cell lysis, ribosomes are separated by centrifugation steps, avoiding massive humic acid contamination and RNA degradation. The protocol accommodates the complex composition of soils by blocking adsorbing surfaces and humic acids with polyvinylpyrrolidone and bovine serum albumin. A usual RNA extraction step yields rRNA accessible for hybridization or reverse transcription-PCR. Hybridization with specific oligonucleotide probes was used for group-specific yield comparison. By using a probe hybridizing to the 16S rRNA of the bacterial kingdom, total bacterial rRNA yield was estimated to be in the range of 0.2 microgram per g for different soils. Group-specific probes did not indicate a selectivity of the isolation procedure and differentiated the compositions of different soil microbial communities. The sequence diversity of the isolated RNA population was also revealed by temperature gradient gel electrophoresis of reverse transcription-PCR amplification products by using a region of the 16S rRNA as a target. The pattern obtained by this analysis differed from a similar one resulting from the separation of amplification products of community DNA preparations. This different view of the community composition is attributable to the correlation of ribosome numbers to the metabolic activity of bacteria in the habitat under observation.

Bacteriological Techniques↗
PubMed2004

Identification of planctomycetes with order-, genus-, and strain-specific 16S rRNA-targeted probes.

A specific 16S rRNA-targeted oligonucleotide probe (PIR1223) for the genus Pirellula and a species-specific probe (RB454) for Pirellula sp. strain SH1 have been designed and optimized. Together with the already existing order-specific probe PLA886, the two newly designed probes were used to detect and identify planctomycetes, pirellulae, and close relatives of Pirellula sp. strain SH1 in different habitats. With the help of these probes for detection and identification, bacteria of the genus Pirellula were detected and cultivated from tissue of the Mediterranean sponge Aplysina aerophoba and from the water column of the Kiel Fjord. An unexpected result was the close phylogenetic relationship of the isolate from the sponge and the brackish water habitat Kiel Fjord as revealed by DNA/DNA hybridization.

Animals↗
PubMed2006

Macroscopic streamer growths in acidic, metal-rich mine waters in north wales consist of novel and remarkably simple bacterial communities.

The microbial composition of acid streamers (macroscopic biofilms) in acidic, metal-rich waters in two locations (an abandoned copper mine and a chalybeate spa) in north Wales was studied using cultivation-based and biomolecular techniques. Known chemolithotrophic and heterotrophic acidophiles were readily isolated from disrupted streamers, but they accounted for only <1 to 7% of the total microorganisms present. Fluorescent in situ hybridization (FISH) revealed that 80 to 90% of the microbes in both types of streamers were beta-Proteobacteria. Terminal restriction fragment length polymorphism analysis of the streamers suggested that a single bacterial species was dominant in the copper mine streamers, while two distinct bacteria (one of which was identical to the bacterium found in the copper mine streamers) accounted for about 90% of the streamers in the spa water. 16S rRNA gene clone libraries showed that the beta-proteobacterium found in both locations was closely related to a clone detected previously in acid mine drainage in California and that its closest characterized relatives were neutrophilic ammonium oxidizers. Using a modified isolation technique, this bacterium was isolated from the copper mine streamers and shown to be a novel acidophilic autotrophic iron oxidizer. The beta-proteobacterium found only in the spa streamers was closely related to the neutrophilic iron oxidizer Gallionella ferruginea. FISH analysis using oligonucleotide probes that targeted the two beta-proteobacteria confirmed that the biodiversity of the streamers in both locations was very limited. The microbial compositions of the acid streamers found at the two north Wales sites are very different from the microbial compositions of the previously described acid streamers found at Iron Mountain, California, and the Rio Tinto, Spain.

Betaproteobacteria↗
PubMedDate not supplied

Highly instable (GATA)n-containing sequences of the mouse during the cloning process.

Mouse DNA fingerprints were obtained by HaeIII digestion of genomic DNA and in-gel hybridization with the (GATA)4 oligonucleotide probe. In order to obtain locus-specific probes that hybridize with only one fragment of the (GATA)4 DNA fingerprint, a genomic library of size-selected inserts was constructed using a system of direct subcloning from the phage clones. During the cloning procedure, the phage as well as the plasmid insert DNAs changed primarily within their repetitive DNA but also within adjacent nonrepetitive sequences, as was demonstrated for several clones by in-gel hybridization with the (GATA)4 probe as well as by sequence analysis. Isolated subclones varied within their (GATA)n repeats, resulting in different insert lengths. Several "metastable" as well as stable (GATA)4-positive subclones could be isolated. Also, vector sequences were affected by alterations during the cloning process. These phenomena are discussed within the context of possible mechanisms for cloning artifacts.

Animals↗
PubMed1985

Purification and partial amino acid sequence of asialo murine granulocyte-macrophage colony stimulating factor.

A procedure utilizing reversed-phase high-performance liquid chromatography is described for the purification of asialo granulocyte-macrophage colony stimulating factor (asialo-GM-CSF) from mouse lung-conditioned medium. In the purification, the partially purified factor was treated with neuraminidase to reduce charge heterogeneity due to variable degrees of sialation. Three active forms of the asialo factor were separated by the final reversed-phase liquid chromatography step. These each gave a single major band and several minor bands on polyacrylamide gel electrophoresis and had similar amino acid compositions. The specific activity of purified murine asialo-GM-CSF was approximately 8 X 10(9) colonies per mg of protein. Amino acid sequence determination of the major form gave a single amino-terminal sequence, which has been used to develop oligonucleotide probes for the isolation of two cDNA clones encoding GM-CSF. The nucleotide sequence of these two clones gave a deduced amino acid sequence almost identical with that determined for the amino terminus of asialo-GM-CSF and an amino acid composition very similar to that for asialo-GM-CSF.

Amino Acid Sequence↗
PubMed1997

Recognition of GC base pairs by triplex forming oligonucleotides containing nucleosides derived from 2-aminopyridine.

We have attempted to alleviate the pH dependency of triplex recognition of guanine by using intermolecular triplexes containing 2-amino-5-(2-deoxy-d-ribofuranosyl)pyridine (AP) as an analogue of 2'-deoxycytidine (dC). We find that for the beta-anomer of AP, the complex between (AP)6T6and the target site G6A6*T6C6is stable, generating a clear DNase I footprint at oligonucleotide concentrations as low as 0.25 microM at pH 5.0, in contrast to 50 microM C6T6which has no effect on the cleavage pattern. This complex is still stable at pH 6.5 producing a footprint with 1 microM oligonucleotide. Oligonucleotides containing the alpha-anomer of AP are much less effective than the beta-anomer, though in some instances they are more stable than the unmodified oligonucleotides. The results of molecular dynamics studies on a range of AP-containing triplexes has rationalized the observed stability behaviour in terms of hydrogen-bonding behaviour.

5-Methylcytosine↗
PubMed1989

Ionized and wobble base-pairing for bromouracil-guanine in equilibrium under physiological conditions. A nuclear magnetic resonance study on an oligonucleotide containing a bromouracil-guanine base-pair as a function of pH.

A one and two-dimensional nuclear magnetic resonance study of a non-selfcomplementary oligonucleotide containing a central 5-bromouracil-guanine pair is reported. For these two bases three types of hydrogen bonding schemes could exist; wobble, rare tautomer and ionized. The two-dimensional spectra of non-exchangeable protons together with one-dimensional spectra recorded in water show that at pH 7.0 the predominant species is a right-handed B-form DNA in which the brU.G pair has wobble geometry. On raising the pH we observe a transition monitored by proton chemical shift changes for the brU.G and adjacent base-pairs. The mid-point of the transition was observed at pH 8.6. Spectra recorded at pH 9.8 show that the helix remains intact with B form conformation. It is shown that this high pH form has an ionized brU.G base-pair now in Watson-Crick geometry. Thus under physiological conditions an equilibrium exists between wobble and ionized structures.

Base Composition↗
PubMed2003

Fraying and electron autodetachment dynamics of trapped gas phase oligonucleotides.

Sensitive methods recently developed to measure laser-induced fluorescence from trapped ions have been applied to study the dynamics of double- and single-stranded oligonucleotides. In this paper, the fraying of duplex terminal base pairs has been identified by measuring the donor fluorescence as a function of temperature from an oligonucleotide duplex labeled with a pair of FRET dyes. Comparison of the degree of dissociation of 14-mer duplexes observed in the mass spectra with the fluorescence intensity of the donor enables intermediate conformations of the unzipping duplex at the weaker binding end of the duplex to be identified. The autodetachment of electrons from double- and single-stranded oligonucleotide anions has been observed in a gas phase environment. To characterize this process, measurements were performed on 7-mers prepared without FRET fluorophores attached. The dependence of the decay rates of trapped anions have been measured as a function of charge state and temperature for various base compositions. An exceptionally strong dependence of the decay rate on base composition has been identified. The physical basis for this process will be discussed.

Boron Compounds↗
PubMed2003

Biosensors based on electropolymerized films: new trends.

Electropolymerized films have received considerable attention in the development of biosensors and biochips, and are advancing rapidly. This paper reviews recent advances and scientific progress in electrochemical immobilization procedures for biological macromolecules on electrodes via electrogenerated polymer films. Biomolecule immobilization is classified as covalent linkage, attachment by affinity interactions, and physical entrapment. The last approach entails the use of conducting and non-conducting films, composite polymer films, and templates for the electropolymerization process. Some advances in the electrochemical transduction of biological events (enzymatic reaction, immunoreaction, or oligonucleotide hybridization) involving the redox properties or the conductivity of electropolymerized films are also presented.

Biosensing Techniques↗