Selecting aptamers--the way to find a fit.
Explore the source record for details and available documents.
Base Composition↗
SEARCH · Search PubMed
Results for “oligonucleotide composition”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Identification of two neutrophil chemotactic peptides produced by porcine alveolar macrophages.
We have purified to homogeneity two distinct 10-kDa proteins with potent chemotactic activity for neutrophils from porcine alveolar macrophages incubated for 24 h with Escherichia coli endotoxin (lipopolysaccharide (LPS), 10 micrograms/ml). Neutrophil chemotactic activity in alveolar macrophage supernatants was concentrated by adsorption to SP-Sephadex, and purified by cation exchange and reversed phase high performance liquid chromatography. The first peptide, alveolar macrophage chemotactic factor (AMCF)-I, had chemotactic activity for both porcine and human neutrophils. The chemotactic activity for porcine neutrophils was detectable at 3 x 10(-10) M, peaked at 3 x 10(-8) M, and was comparable to that of zymosan-activated porcine serum. Segmental instillation of AMCF-I into porcine lungs caused marked neutrophil accumulation at 4 h in both bronchoalveolar lavage fluid and in lung tissue. The second peptide, AMCF-II, was active at 1.4 x 10(-9) M for porcine neutrophils, but it was less active for human polymorphonuclear neutrophils than was AMCF-I. Oligonucleotide probes to regions of the N-terminal sequences of AMCF-I and AMCF-II hybridized to mRNA recovered from LPS-stimulated alveolar macrophages. The N-terminal sequences and amino acid compositions indicate that AMCF-I and AMCF-II are distinct proteins, but that both have homologies with a family of peptide chemoattractants produced by human blood monocytes and platelets. Thus, alveolar macrophages stimulated with LPS produce two distinct 10-kDa cytokines with potent chemotactic activity for neutrophils. This indicates that there are two different peptide pathways by which alveolar macrophages can recruit neutrophils into the lung.
Amino Acid Sequence↗
Predictive identification of exonic splicing enhancers in human genes.
Specific short oligonucleotide sequences that enhance pre-mRNA splicing when present in exons, termed exonic splicing enhancers (ESEs), play important roles in constitutive and alternative splicing. A computational method, RESCUE-ESE, was developed that predicts which sequences have ESE activity by statistical analysis of exon-intron and splice site composition. When large data sets of human gene sequences were used, this method identified 10 predicted ESE motifs. Representatives of all 10 motifs were found to display enhancer activity in vivo, whereas point mutants of these sequences exhibited sharply reduced activity. The motifs identified enable prediction of the splicing phenotypes of exonic mutations in human genes.
Computational Biology↗
Effect of P-chirality of oligo(deoxyribonucleoside phosphorothioate)s on the activity of terminal deoxyribonucleotidyl transferase.
Phosphorothioate analogues of oligonucleotides (PS-oligos) of predetermined chirality at the phosphorus atom at each internucleotide linkage have been used as primers for terminal deoxyribonucleotidyl transferase (TdT, EC 2.7.7.31). The enzyme catalyzes efficient elongation of PS primers in which all phosphorothioate internucleotide linkages are uniformly of the [R(P)] configuration, while the presence of the linkage(s) of the [S(P)] configuration significantly decreases or completely inhibits the primer extension. Our results indicate that for the elongation of phosphorothioate oligomers the most important is the internucleotide bond located between the second and the third nucleoside from the 3'-end. The presence of [S(P)] linkage at this position strongly reduces the enzyme activity while the [R(P)] bond allows for effective elongation of the primer. The activity of the enzyme is also influenced by base composition and sequence of phosphorothioate primer as well as the dNTP used for elongation process.
DNA Nucleotidylexotransferase↗
Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition.
Serial analysis of ribosomal sequence tags (SARST) is a recently developed technology that can generate large 16S rRNA gene (rrs) sequence data sets from microbiomes, but there are numerous enzymatic and purification steps required to construct the ribosomal sequence tag (RST) clone libraries. We report here an improved SARST method, which still targets the V1 hypervariable region of rrs genes, but reduces the number of enzymes, oligonucleotides, reagents, and technical steps needed to produce the RST clone libraries. The new method, hereafter referred to as SARST-V1, was used to examine the eubacterial diversity present in community DNA recovered from the microbiome resident in the ovine rumen. The 190 sequenced clones contained 1055 RSTs and no less than 236 unique phylotypes (based on > or = 95% sequence identity) that were assigned to eight different eubacterial phyla. Rarefaction and monomolecular curve analyses predicted that the complete RST clone library contains 99% of the 353 unique phylotypes predicted to exist in this microbiome. When compared with ribosomal intergenic spacer analysis (RISA) of the same community DNA sample, as well as a compilation of nine previously published conventional rrs clone libraries prepared from the same type of samples, the RST clone library provided a more comprehensive characterization of the eubacterial diversity present in rumen microbiomes. As such, SARST-V1 should be a useful tool applicable to comprehensive examination of diversity and composition in microbiomes and offers an affordable, sequence-based method for diversity analysis.
Bacteria↗
Unpaired structures in SCA10 (ATTCT)n.(AGAAT)n repeats.
A number of human hereditary diseases have been associated with the instability of DNA repeats in the genome. Recently, spinocerebellar ataxia type 10 has been associated with expansion of the pentanucleotide repeat (ATTCT)(n).(AGAAT)(n) from a normal range of ten to 22 to as many as 4500 copies. The structural properties of this repeat cloned in circular plasmids were studied by a variety of methods. Two-dimensional gel electrophoresis and atomic force microscopy detected local DNA unpairing in supercoiled plasmids. Chemical probing analysis indicated that, at moderate superhelical densities, the (ATTCT)(n).(AGAAT)(n) repeat forms an unpaired region, which further extends into adjacent A+T-rich flanking sequences at higher superhelical densities. The superhelical energy required to initiate duplex unpairing is essentially length-independent from eight to 46 repeats. In plasmids containing five repeats, minimal unpairing of (ATTCT)(5).(AGAAT)(5) occurred while 2D gel analysis and chemical probing indicate greater unpairing in A+T-rich sequences in other regions of the plasmid. The observed experimental results are consistent with a statistical mechanical, computational analysis of these supercoiled plasmids. For plasmids containing 29 repeats, which is just above the normal human size range, flanked by an A+T-rich sequence, atomic force microscopy detected the formation of a locally condensed structure at high superhelical densities. However, even at high superhelical densities, DNA strands within the presumably compact A+T-rich region were accessible to small chemicals and oligonucleotide hybridization. Thus, DNA strands in this "collapsed structure" remain unpaired and accessible for interaction with other molecules. The unpaired DNA structure functioned as an aberrant replication origin, in that it supported complete plasmid replication in a HeLa cell extract. A model is proposed in which unscheduled or aberrant DNA replication is a critical step in the expansion mutation.
Base Composition↗
Reverse chromosome painting for the identification of marker chromosomes and complex translocations in leukemia.
BACKGROUND: Chromosome banding techniques and in situ hybridization reveal the majority of chromosomal aberrations. However, difficulties remain in cases of highly contracted chromosomes, poor quality of the metaphases or the presence of markers with the involvement of several chromosomes. Here, it is demonstrated that reverse painting can be applied successfully starting with bone marrow cells from primary acute myelocytic leukemias (AML). METHODS: This was accomplished by culturing the leukemic cells with a cocktail of various growth factors, which yielded sufficient numbers of cells in cycle to harvest chromosomes for sorting. Aberrant chromosomes were flow-sorted and amplified by degenerate oligonucleotide-primed PCR. The resulting products were labeled by nick-translation and hybridized on normal metaphase spreads. RESULTS: Two patients with marker chromosomes in their leukemia cells were analyzed in detail. The hybridization pattern displayed the composition of the aberrant sorted chromosome. Results were compared with conventional cytogenetic analyses that were performed on material obtained from the same aspirate. The reverse-painting technique enabled identification of aberrations that were not detected by conventional cytogenetic analysis. CONCLUSIONS: Primary AML cells can be cultured in vitro, using optimal culture conditions, facilitating the production of high quality flow karyotypes, suitable for sorting of marker chromosomes to produce DOP-PCR derived chromosome painting probes for reverse painting. Valuable additional cytogenetic information can thus be obtained about complex chromosomal rearrangements or structural aberrations that could not be completely resolved by conventional cytogenetic analysis.
Bone Marrow↗
Bacterial community structure and physiological state within an industrial phenol bioremediation system.
The structure of bacterial populations in specific compartments of an operational industrial phenol remediation system was assessed to examine bacterial community diversity, distribution, and physiological state with respect to the remediation of phenolic polluted wastewater. Rapid community fingerprinting by PCR-based denaturing gradient gel electrophoresis (DGGE) of 16S rDNA indicated highly structured bacterial communities residing in all nine compartments of the treatment plant and not exclusively within the Vitox biological reactor. Whole-cell targeting by fluorescent in situ hybridization with specific oligonucleotides (directed to the alpha, beta and gamma subclasses of the class Proteobacteria [alpha-, beta-, and gamma-Proteobacteria, respectively], the Cytophaga-Flavobacterium group, and the Pseudomonas group) tended to mirror gross changes in bacterial community composition when compared with DGGE community fingerprinting. At the whole-cell level, the treatment compartments were numerically dominated by cells assigned to the Cytophaga-Flavobacterium group and to the gamma-Proteobacteria. The alpha subclass Proteobacteria were of low relative abundance throughout the treatment system whilst the beta subclass of the Proteobacteria exhibited local dominance in several of the processing compartments. Quantitative image analyses of cellular fluorescence was used as an indicator of physiological state within the populations probed with rDNA. For cells hybridized with EUB338, the mean fluorescence per cell decreased with increasing phenolic concentration, indicating the strong influence of the primary pollutant upon cellular rRNA content. The gamma subclass of the Proteobacteria had a ribosome content which correlated positively with total phenolics and thiocyanate. While members of the Cytophaga-Flavobacterium group were numerically dominant in the processing system, their abundance and ribosome content data for individual populations did not correlate with any of the measured chemical parameters. The potential importance of the gamma-Proteobacteria and the Cytophaga-Flavobacteria during this bioremediation process was highlighted.
Aged↗
Alpha- and beta-Proteobacteria control the consumption and release of amino acids on lake snow aggregates.
We analyzed the composition of aggregate (lake snow)-associated bacterial communities in Lake Constance from 1994 until 1996 between a depth of 25 m and the sediment surface at 110 m by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes of various specificity. In addition, we experimentally examined the turnover of dissolved amino acids and carbohydrates together with the microbial colonization of aggregates formed in rolling tanks in the lab. Generally, between 40 and more than 80% of the microbes enumerated by DAPI staining (4',6'-diamidino-2-phenylindole) were detected as Bacteria by the probe EUB338. At a depth of 25 m, 10.5% +/- 7.9% and 14.2% +/- 10.2% of the DAPI cell counts were detected by probes specific for alpha- and beta-Proteobacteria. These proportions increased to 12.0% +/- 3.3% and 54.0% +/- 5.9% at a depth of 50 m but decreased again at the sediment surface at 110 m to 2.7% +/- 1.4% and 41.1% +/- 8.4%, indicating a clear dominance of beta-Proteobacteria at depths of 50 and 110 m, where aggregates have an age of 3 to 5 and 8 to 11 days, respectively. From 50 m to the sediment surface, cells detected by a Cytophaga/Flavobacteria-specific probe (CF319a) comprised increasing proportions up to 18% of the DAPI cell counts. gamma-Proteobacteria always comprised minor proportions of the aggregate-associated bacterial community. Using only two probes highly specific for clusters of bacteria closely related to Sphingomonas species and Brevundimonas diminuta, we identified between 16 and 60% of the alpha-Proteobacteria. In addition, with three probes highly specific for close relatives of the beta-Proteobacteria Duganella zoogloeoides (formerly Zoogloea ramigera), Acidovorax facilis, and Hydrogenophaga palleroni, bacteria common in activated sludge, 42 to 70% of the beta-Proteobacteria were identified. In the early phase (<20 h) of 11 of the 15 experimental incubations of aggregates, dissolved amino acids were consumed by the aggregate-associated bacteria from the surrounding water. This stage was followed by a period of 1 to 3 days during which dissolved amino acids were released into the surrounding water, paralleled by an increasing dominance of beta-Proteobacteria. Hence, our results show that lake snow aggregates are inhabited by a community dominated by a limited number of alpha- and beta-Proteobacteria, which undergo a distinct succession. They successively decompose the amino acids bound in the aggregates and release substantial amounts into the surrounding water during aging and sinking.
Alphaproteobacteria↗
Prenatal diagnosis: general introduction, methodology, and review.
Prenatal diagnosis of hematologic disease has been performed in more than 10,000 fetuses since 1974, with the majority at risk for thalassemia. Fetal blood sampling was the initial method, but most centers are now changing to DNA technology, particularly using specimens obtained by chorionic villus sampling. Fetal blood globin chain composition and synthesis is examined using carboxymethyl cellulose columns, electrophoresis, and high performance liquid chromatography. Fetal globin DNA is tested for gene deletions, mutations that affect restriction enzyme cleavage sites, and restriction fragment length polymorphisms, and specific oligonucleotides are used to identify some point mutations. The current trend is for early sampling (by chorionic villus sampling), gene amplification, and specific DNA analyses. The safety of all methods (blood or DNA) is above 96%, and the accuracy more than 99%. The acceptability of these approaches has been high.
Female↗
Molecular identification and prevalence of Dictyocaulus spp. (Trichostrongyloidea: Dictyocaulidae) in Swedish semi-domestic and free-living cervids.
Lungs of 102 roe deer (Capreolus capreolus), 136 moose (Alces alces), 68 fallow deer (Dama dama), and six red deer (Cervus elaphus) were examined during hunting seasons from 16 September 1997 to 1 March 2000. The aim was to determine the species composition and prevalence of Dictyocaulus lungworms in these hosts in Sweden. Worms were identified following polymerase chain reaction (PCR) amplification of the internal transcribed spacer of ribosomal DNA (ITS2), followed by hybridization with four species-specific oligonucleotides. In addition, 50 lungworms from five reindeer (Rangifer tarandus) from Norway were similarly analyzed. A total of 399 worms were recovered and analyzed representing a range of 29-128 worms per host species. All specimens from roe deer were identified as Dictyocaulus capreolus, whereas those from red deer and reindeer were identical with D. eckerti. From moose, 73 (81.1%) of the worms were identified as D. capreolus whereas 17 (18.9%) were D. eckerti. The ITS2 sequence of fallow deer lungworms differed significantly when compared with the ITS2 of D. viviparus, D. capreolus, and D. eckerti. This indicated that fallow deer in Sweden may be infected with a new genotype of Dictyocaulus spp. Consequently, a specific probe designed for the ITS2 from this Dictyocaulus sp. hybridized exclusively with samples from lungworms of fallow deer. Interestingly, no D. viviparus were found in any of these hosts. The prevalence of infection in each host was as follows: D. capreolus in roe deer (14.7%) and moose (10.6%); D. eckerti in moose (0.7%) and red deer (33.3%); and Dictyocaulus sp. in fallow deer (10.3%). Regardless of lungworm species, the overall prevalence of Dictyocaulus spp. in these hosts was 12.2%. Prevalence between male and female animals and among the different age groups did not differ significantly. Finally an enzyme linked immunosorbent assay (ELISA) specific for patent D. viviparus infection in cattle was utilized to analyze lung tissue fluids from infected animals. All samples from roe deer, red deer, and fallow deer were negative in the ELISA. However, three out of twelve (25%) samples from moose and 17 of 40 (43%) samples from cattle were positive. This indicated that moose anti-D. capreolus antibodies recognized the D. viviparus antigen and that anti-cattle immunoglobulin cross-reacted with moose antibodies.
Animals↗
Sequences of three closely related variants of a complex satellite DNA diverge at specific domains.
Major differences among the sequences of the repeat units of a very complex satellite DNA are located in domains which are sensitive to S1 nuclease under torsional stress, indicating that the domains assume unusual secondary or tertiary structures. Repeat units of the satellite, which accounts for 3% of the DNA of a land crab, have been inserted into pBR322 and the primary sequences of three cloned variants determined. The variants selected for sequencing include 1) RU (2089 base pairs (bp) ), representative of the average size of repeat units of cellular satellite; 2) TRU (1674 bp), truncated at an extra EcoRI site; and 3) EXT (2639 bp), extended by a 5-fold amplification of a 142-bp segment, one copy of which is present in RU and TRU (Bonnewell, V., Fowler, R.F., and Skinner, D.M. (1983) Science 221, 862-865). It appears that every copy of the satellite may be different and that the variants do not arise from cloning accidents. Extensive domains, as long as approximately 560 bp, are greater than 95% homologous among RU, TRU, and EXT; these conserved domains are composed of DNA whose base composition and sequences do not have remarkable features. By contrast, the sequences that comprise the divergent domains are unusually rich in 1) tracts of (dG X dC) 13-23 and arrangements of similar but not identical repetitive oligonucleotides or 2) alternating purines and pyrimidines (pu/py).
Base Sequence↗
Application of FISH technology for microbiological analysis: current state and prospects.
In order to identify and quantify the microorganisms present in a certain ecosystem, it has become necessary to develop molecular methods avoiding cultivation, which allows to characterize only the countable part of the microorganisms in the sample, therefore losing the information related to the microbial component which presents a vitality condition, although it cannot duplicate in culture medium. In this context, one of the most used techniques is fluorescence in situ hybridization (FISH) with ribosomal RNA targeted oligonucleotide probes. Owing to its speed and sensitivity, this technique is considered a powerful tool for phylogenetic, ecological, diagnostic and environmental studies in microbiology. Through the use of species-specific probes, it is possible to identify different microorganisms in complex microbial communities, thus providing a solid support to the understanding of inter-species interaction. The knowledge of the composition and distribution of microorganisms in natural habitats can be interesting for ecological reasons in microbial ecology, and for safety and technological aspects in food microbiology. Methodological aspects, use of different probes and applications of FISH to microbial ecosystems are presented in this review.
Bacteria↗
Isolation of pyrimidine isostichs from chick erythrocyte DNA.
The isolation of pyrimidine oligonucleotides (isostichs) from chick erytyrocyte DNA was accomplished on a preparative scale with the goal of providing starting material for further chemical modifications. The DNA was degraded by the method of Burton and Petersen and the isostichs were obtained via paper chromatography and ion-exchange chromatography. The various isostichs were further fractionated into base compositional isomers, the frequency of which was determined as mole percent of total pyrimidines. Although this work was not intended to be an analytical study, the large quantities of DNA used allowed the measurement of most compositional isomers with an accuracy of 5% of the listed values. The data for long oligothymidylates are less reliable. The results of the present study revealed the anticipated bias in the distribution of pyrimidine isostichs in favor of longer chain lengths as compared to that expected for a random distribution. Isostichs above chain length 10 occur in amounts approximately 7 times greater than calculated.
Animals↗
A novel activity of HMG domains: promotion of the triple-stranded complex formation between DNA containing (GGA/TCC)11 and d(GGA)11 oligonucleotides.
The high mobility group protein (HMG)-box is a DNA-binding domain found in many proteins that bind preferentially to DNA of irregular structures in a sequence-independent manner and can bend the DNA. We show here that GST-fusion proteins of HMG domains from HMG1 and HMG2 promote a triple-stranded complex formation between DNA containing the (GGA/TCC)11 repeat and oligonucleotides of d(GGA)11 probably due to G:G base pairing. The activity is to reduce association time and requirements of Mg2+ and oligonucleotide concentrations. The HMG box of SRY, the protein determining male-sex differentiation, also has the activity, suggesting that it is not restricted to the HMG-box domains derived from HMG1/2 but is common to those from other members of the HMG-box family of proteins. Interestingly, the box-AB and box-B of HMG1 bend DNA containing the repeat, but SRY fails to bend in a circularization assay. The difference suggests that the two activities of association-promotion and DNA bending are distinct. These results suggest that the HMG-box domain has a novel activity of promoting the association between GGA repeats which might be involved in higher-order architecture of chromatin.
Base Composition↗
Characteristics of DNA and multiple rpoD homologs of Microcystis (Synechocystis) strains.
The base compositions of DNAs from nine Microcystis strains, as determined by high-performance liquid chromatography, were 41 to 42 mol% G+C. Chromosomal DNAs derived from these strains were found to be extremely resistant to many restriction endonucleases, and a restriction analysis revealed the presence of a dam-like methylase or both dam- and dcm-like methylases in all of the strains examined. Genomic Southern hybridization in which a synthetic oligonucleotide probe (rpoD probe) was used showed that members of the genus Microcystis might have multiple rpoD homologs, and the hybridization signal patterns observed with the DNAs of Microcystis aeruginosa strains were different from each other.
Bacterial Proteins↗
Effects of exercise training and diabetes on cardiac myosin heavy chain composition.
This study determined whether the beneficial effects of exercise training on the diabetic heart previously observed are associated with alterations in ventricular myosin heavy chain (MHC) isoform composition. Diabetes was induced in rats by i.v. streptozotocin. Trained rats were run on a treadmill for 60 min/day, 27 m/min, 10% grade. After 10 wks, ventricular MHC isoenzyme protein composition was analyzed for MHC composition using gel electrophoresis. alpha-MHC and beta-MHC mRNA were determined by Northern and slot blot hybridization techniques. Both protein and mRNA analyses indicated that sedentary control rats exhibited a predominance of alpha-MHC. Sedentary diabetics exhibited a shift to beta-MHC. Exercise trained diabetic rats showed a predominance of beta-MHC. The results indicate that treadmill exercise training of diabetic rat does not prevent the diabetes-induced shift in MHC composition towards the beta-MHC isoform, thus it is unlikely that the beneficial effects of exercise training on the diabetic heart, previously shown, are due to a normalization of the myosin isoform composition.
Animals↗
Facilitation of glutamatergic neurotransmission by presynaptic nicotinic acetylcholine receptors.
The profiles of presynaptic facilitation of glutamate release as elicited by nicotine and acetylcholine were compared in two limbic pathways recapitulated in vitro. At synapses of medial habenula (MHN) and interpeduncular nucleus (IPN) neurons, application of nicotine increased the frequency of TTX-resistant, spontaneous postsynaptic currents (SSCs) by an average of 5-fold. In contrast, the average increase in SSC frequency elicited by nicotine was more than 120 fold at synapses of olfactory bulb (OB) and amygdala neurons. At both preparations, pulses of ACh caused presynaptic facilitation that lasted longer than that elicited by nicotine. The subunit composition of presynaptic nAChRs may contribute to the different profiles of facilitation observed. The large magnitude, fast kinetics, and alpha-bungarotoxin sensitivity of facilitation observed at OB-amygdala synapses is consistent with participation of alpha7-type nAChRs. As subunit-selective deletion of alpha5 or alpha7 altered the profile of nicotine-elicited facilitation at MHN-IPN synapses, presynaptic nAChRs at MHN-IPN synapses appear to be more complex. Such heteromeric combinations of nAChRs may contribute to the lower magnitude and slower kinetics of presynaptic facilitation at MHN-IPN synapses. Calcium influx through either voltage-gated calcium channels or directly through presynaptic alpha7-containing nAChRs is sufficient to support nicotine-elicited facilitation of glutamate release. Resultant increases in intracellular calcium may further modulate presynaptic nAChR activity in a subunit-composition dependent manner.
Animals↗