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At least 289 records · Page 16Linked to original sources
PubMed1984

Isolation of a cDNA clone coding for the gamma-subunit of mouse nerve growth factor using a high-stringency selection procedure.

A new approach was taken to isolate a full-length cDNA encoding the gamma-subunit of nerve growth factor (NGF). An oligonucleotide probe was used first to identify kallikrein-related sequences in a cDNA library derived from male mouse submaxillary gland mRNA. Of 10,000 clones, 860 were identified as being kallikrein-related; these were analyzed further by hybridization to synthetic oligonucleotides using a procedure which eliminates the effects of base composition and thus allows distinction between partial and perfect sequence complementarity. Nine clones hybridized with a gamma-subunit-specific probe at high stringency, and the nucleotide sequence of the longest cDNA insert was determined. Regions of homology and variability within the kallikrein gene family are maintained in this protein and probably reflect the similar functions and different substrates for this family of proteins.

Amino Acid Sequence↗
PubMed1992

Solid-phase synthesis of branched oligoribonucleotides related to messenger RNA splicing intermediates.

The chemical synthesis of oligoribonucleotides containing vicinal (2'-5')- and (3'-5')-phosphodiester linkages is described. The solid-phase method, based on silyl-phosphoramidite chemistry, was applied to the synthesis of a series of branched RNA [(Xp)nA2' (pN)n3'(pN)n] related to the splicing intermediates derived from Saccharomyces cerevisiae rp51a pre-messenger RNA. The branched oligonucleotides have been thoroughly characterized by nucleoside and branched nucleotide composition analysis. Branched oligoribonucleotides will be useful in the study of messenger RNA splicing and in determining the biological role of RNA 'lariats' and 'forks' in vivo.

Base Sequence↗
PubMedDate not supplied

[Activation of a trans-activating factor of NF1 transcription in a lactating mammary gland].

Functional differentiation of a bovine mammary gland in the course of lactation is characterized by significant increase of tissue-specific expression of genes encoding milk proteins (caseins, lactoglobulin etc.). The NF1 is known as a ubiquitous transcription factor which modulates a tissue-specific transcription of different genes (including beta-casein) cooperating with tissue-specific and other ubiquitous transcription factors. We have observed a dramatic increase of NF1-binding activity in nuclear extracts of bovine mammary gland during lactation. The NF1 transcription factor appears to have a cytoplasmic precursor pool. This cytoplasmic precursor as well as NF-kappa B cytoplasmic precursor could be activated in vitro by deoxycholate (DOC) treatment which caused possibly dissociation of a complex of NF1 and its cytoplasmic inhibitor. There was an inverse proportion between concentrations of active nuclear NF1 factor and its cytoplasmic precursor. We have observed an increase of nuclear factor binding and a simultaneous decrease of the cytoplasmic precursor pool in the course of lactation. We have determined the NF1 protein subunit composition using UV-cross-linking 32P labeled NF1-oligonucleotide with nuclear and cytoplasmic proteins of mammary gland. The main subunits of NF1 factor were p50 and p20. The drastic increase of nuclear NF1 binding activity was correlated with significant increase of the p20 subunit concentration in nuclear protein during lactation.

Animals↗
PubMed1989

Use and applications of nucleic acid probes in the clinical laboratory.

The use and availability of nucleic acid probes are significantly greater than they were three to five years ago. This increased utilization has resulted because of numerous changes in molecular genetic procedures. First, the sources of probes have shifted from cloned genomic and cDNA fragments to single-strand oligonucleotides and RNA or "riboprobes." These newer probes offer defined composition and capability to create separate sense and anti-sense strands. Second, an increased variety of labeling techniques provide more ways to incorporate isotopic and nonisotopic species into these nucleic acid fragments than before. Third, the advent of nucleic acid amplification forecasts the ability to detect sub-attomol quantities of target nucleic acids, which could not be detected by classical hybridization methods. Fourth, more-sensitive signaling systems (e.g., chemiluminescence) now available will also contribute to the lowering of detection limits. Fifth, alternative sample-processing methods will shorten the time required to perform these assays. Finally, coupling these newer molecular probe procedures with automation will lead to improved precision and reliability and promote their introduction into the clinical laboratory as routine procedures. Additional factors such as clinical correlation, cost analysis, etc. will stimulate clinical laboratory personnel to participate further in development and use of these procedures.

Clinical Laboratory Techniques↗
PubMed1985

Recombination and oligonucleotide analysis of guanidine-resistant foot-and-mouth disease virus mutants.

Guanidine resistance (gr) mutations of foot-and-mouth disease virus were mapped by recombining pairs of temperature-sensitive mutants belonging to different subtypes. In each cross, one parent possessed a gr mutation. Recombinants were isolated by selection at the nonpermissive temperature and assayed for the ability to grow in the presence of guanidine. From the progeny of three crosses, four different types of recombinant were distinguished on the basis of protein composition and RNA fingerprint. The sequences of the RNase T1-resistant oligonucleotides were determined and located in the full-length sequence of foot-and-mouth disease virus. The resulting maps show that (i) each recombinant was generated by a single genetic crossover, and (ii) both of the gr mutations studied were located within an internal 2.9-kilobase region which spans the P34 gene. This supports our hypothesis that guanidine inhibits the growth of foot-and-mouth disease virus by acting on nonstructural polypeptide P34. Additional evidence was provided by RNA fingerprinting gr mutants. In two of four cases the gr mutation was associated with a change in an oligonucleotide located near the 3' end of the P34 gene; in one of these the nucleotide substitution was identified.

Aphthovirus↗
PubMed1997

Synthesis and cleavage of oligodeoxynucleotides containing a 5-hydroxyuracil residue at a defined site.

Oxidation and hydrolysis of a cytosine residue can lead to the formation of 5-hydroxyuracil in DNA. The biological consequences of this modification are not fully understood. To facilitate biochemical and biophysical studies aimed at elucidating the effects of this modification in DNA, we have developed a solid-phase synthetic method for the placement of 5-hydroxyuracil residues at defined sites in oligodeoxynucleotides. This method is based upon the enhanced acidity of the 5-hydroxyl proton which allows selective aqueous acetylation. Under standard aqueous ammonia deprotection conditions, however, we observed that 5-hydroxyuracil residues are lost substantially from synthetic oligonucleotides. Substitution of aqueous ammonia with methanolic potassium carbonate and the use of phosphoramidite derivatives with alternatively protected amino groups allow synthesis of oligonucleotides containing 5-hydroxyuracil and all normal bases in high yield. The composition of the oligodeoxynucleotides prepared by this method has been verified by enzymatic digestion followed by high-performance liquid chromatography (HPLC) analysis as well as acid hydrolysis followed by GC/MS analysis. The location of the 5-hydroxyuracil residue is demonstrated by selective permanganate oxidation of the 5-hydroxyuracil residue followed by beta-elimination. We have also probed a synthetic oligonucleotide containing a unique 5-hydroxyuracil residue with uracil DNA N-glycosylase, previously reported to remove this lesion from DNA.

Antineoplastic Agents↗
PubMedDate not supplied

[Distribution of pyrimidine sequences of different length and base composition in bacteriophage T5 DNA].

Distribution of pyrimidine tracts of different length (isopliths) with general formula PynPn+1 in bacteriophage T5 DNA was studied. The first seven isoplith fractions were subfractionated by the chain length and the quantity of the resulting non-isomeric oligonucleotides was determined. The pattern of distribution of pyrimidine tracts of various length and base composition in bacteriophage T5 DNA is different from that previously observed in the DNAs of bacteriophages T3 and T7. The observed differences in distribution of pyrimidine nucleotides are in accordance with the other peculiarities of bacteriophage T5 genome.

Base Sequence↗
PubMed1988

Analysis of conformational parameters in nucleic acid fragments. IV. Intercalating drug complexes of very short chain oligonucleotides.

Studies have been made of base-pairing conformational parameters in single crystal structures of very short chain oligonucleotide structures complexed with drug molecules, using data extracted from the Cambridge structural database. The planar portion of the drug has a tendency to intercalate between two bases, utilising strong stacking interactions to stabilise the configuration. The effect of the existence of a formative backbone is seen in the high occurrence of standard base-pairing schemes and a consistent C1'-C1' separation, although the choice of compounds studied does tend to emphasise complementary pairing. In addition to the modulation of the general magnitude which is reduced from that in uncomplexed oligonucleotides, there appears to be some correlation of propeller twist value with the presence of planar groups sandwiching a base-pair. The average twist in such sandwiched pairs is lower than in any other group studied to date.

Base Composition↗
PubMed1998

Effects of size and sequence on the iontophoretic delivery of oligonucleotides.

Adequate cellular availability of synthetic oligonucleotides is crucial to their success as therapeutic agents. These compounds, however, are not expected to be orally active. This has led to interest in a variety of alternate drug delivery methods, including iontophoretically enhanced transdermal delivery. The purpose of this work is to begin characterizing the structure-activity relationship for iontophoresis of oligonucleotides through the skin. The in vitro permeation of 16 biologically relevant phosphorothioate oligonucleotides across hairless mouse skin was studied. Oligonucleotides with less than 20 bases (n = 10) had a wide range of steady-state flux levels (2.1-26.2 pmol/ cm2 h). A lower flux differential was observed for compounds ranging from 20 to 40 bases long (1.2-2.2 pmol/cm2 h). For the smaller compounds, transport, in general, decreased with increasing size; however, there were several oligonucleotides that did not follow this pattern. These data indicate that factors other than size influence transport and that the impact is greater at shorter lengths. Differential penetration between equal sized oligonucleotides synthesized with identical bases in reversed order indicates that sequences and not simply base composition affects steady-state flux across skin. Molecular structure, therefore, is a key contributor to iontophoretically assisted transport. Further studies are necessary to develop more precise predictions about the relationship between oligonucleotide structure and transdermal delivery.

Animals↗
PubMed1994

B-Z transition of synthetic oligonucleotides containing non-Z forming elements.

Ten oligonucleotides of the length 8-12 base pairs have been synthesized, which contain, in addition to the obligatory sequences CG/CG, sequences not favorable for the transition to the Z conformation (A.T pairs, GG/CC or AA/TT sequences). Conformational transitions of these oligonucleotides in high concentrations of NaClO4 in the absence and in the presence of Ni2+ were investigated using CD spectroscopy. The B-Z transition is affected by the length and sequence of the oligonucleotide. Increasing the NaClO4 concentration alone the transition of only one of the oligonucleotides studied. (CGCGCGTGCACGCGCG)2, can be induced. Other oligonucleotides remain in the B conformation or only partial transition to the Z conformation can be observed. Most other oligonucleotides can be converted into the Z conformation at intermediate concentrations of NaClO4 (2.0-3.2 M) by an addition of Ni2+ ions. In some cases, however, Ni2+ can destabilize the double stranded structure of the sample. We have studied the effect of the presence of A.T pairs in the G.C containing oligonucleotides and the effect of the presence of pu-pu/pyr-pyr sequences. The presence of the latter sequences in the Z form implicates the formation of a Z-Z'junction which makes the transition quite difficult. Despite the fact that some oligonucleotides contained several structural elements not favorable for the transition, we did not find any sequence which would completely block the ability of the oligonucleotide to adopt the Z conformation.

Base Composition↗
PubMed2005

Large-scale genome clustering across life based on a linguistic approach.

With the availability of genome sequences, the possibility of new phylogenetic reconstructions arises in order to reveal genomic relationships among organisms. According to the compositional-spectra (CS) approach proposed in our previous studies, any genomic sequence can be characterized by a distribution of frequencies of imperfect matching of words (oligonucleotides). In the current application of CS-analysis, we attempted to analyze the cluster structure of genomes across life. It appeared that compositional spectra show a clear three-group clustering of the compared prokaryotic and eukaryotic genomes. Unexpectedly, this grouping seriously differs from the classical Universal Tree of Life structure represented by common kingdoms known as Eubacteria, Archaebacteria, and Eukarya. The revealed CS-clustering displays high stability, putatively reflecting its objective nature, and still enigmatic biological significance that may result from convergent evolution driven by ecological selection. We believe that our approach provides a new and wider (compared to traditional methods) perspective of extracting genomic information of high evolutionary relevance.

Base Composition↗
PubMed2004

High throughput screening of library compounds against an oligonucleotide substructure of an RNA target.

Approximately 300,000 compounds from selected libraries were screened against a subdomain of a hepatitis C viral (HCV) RNA using a high throughput flow injection mass spectrometry (FIA-MS) method with automated data storage and analysis. Samples contained 2 microM RNA target and 10 microM of each of up to ten ligands. Preliminary studies to optimize operational parameters used the binding of aminoglycosides to the A44 subdomain of bacterial RNA. Binding (confirmed by titration) and sensitivity were maximized within the constraints of the library and throughput. The mobile phase of 5 mM ammonium acetate in 50% isopropanol maintained the noncovalent complexes and provided good detection by electrospray mass spectrometry. Additionally, this composition maximized general solubility of the various classes of compounds including the oligonucleotide and organic library molecules. Cation adduction was insignificant in this screen although some solute and target dependent acetate adduction was observed. The ion trap mass spectrometer provided sufficient mass resolution to identify complexes of RNA with known components of the library. Converted mass spectral data (netCDF) were subjected to two types of statistical evaluation based on binding. The first algorithm identified noncovalent complexes that correlated with the molecular weights of the injected compounds. The second yielded the largest peak in the noncovalent complex region of the spectrum; this spectrum may or may not correlate with expected well components. Sixty-three compounds were confirmed to bind by more stringent secondary testing. Titrations, which were carried out with selected binding compounds, yielded a range of dissociation constants. Biological activity was observed for eleven confirmed binders.

Ligands↗
PubMed1999

Circular antisense oligonucleotides inhibit growth of chronic myeloid leukemia cells.

BACKGROUND: Antisense represents a conceptually powerful method for regulating gene expression. However, antisense oligonucleotides developed to date manifest two serious limitations-nuclease susceptibility and nonspecific hybridization. Circular oligonucleotides may be superior to conventional linear oligonucleotides in both respects. First, circular agents, having no ends, are exonuclease-resistant. Second, they bind to complementary strands of RNA and DNA with a higher affinity than corresponding linear agents. METHODS AND RESULTS: We assessed the activity of circular phosphodiester deoxynucleotides using chronic myeloid cell lines by targeting polypurine sequences. To represent cells having a bcr3/abl2-type junction, we used K562 cells. A circle targeting a bcr polypurine sequence 385 nucleotides 5' to the junction decreased the cell number by day 5 with an IC(50) of 9 microM. To represent cells having a bcr2/abl2-type junction, we used BV173 cells. A circle targeting the bcr-abl junction itself decreased the cell number by day 7 with an IC(50) of 8 microM. Control oligonucleotides, whether the same sequence uncircularized or circles with the same nucleotide composition but in scrambled sequence, had little effect. Unlike linear agents, circles were stable when incubated in 10% serum. The amount of bcr-abl protein detected by Western blotting using a specific anti-bcr-abl antibody at 24 hr in antisense-treated BV173 cells was only 10% of that of cells treated with control circles, which demonstrates an antisense mechanism of action. CONCLUSIONS: Circular oligodeoxyribonucleotides (1) inhibit the accumulation of CML cells, (2) decrease the amount of bcr-abl protein per cell, (3) have sequence-selective activity, and (4) are more active than linear oligonucleotides containing only the base-pairing region.

Base Sequence↗
PubMed2006

Analysis of oligonucleotide microarrays by 3' end labeling using fluorescent nucleotides and terminal transferase.

A simple enzymatic labeling procedure is described to determine spot quality in oligonucleotide microarrays. By using fluorescently labeled dideoxynucleotides or ribonucleotides as substrate for terminal deoxynucleotidyl transferase (TdT), a single fluorophore can be covalently attached at the 3' end of each oligonucleotide probe molecule in the spot. Fluorescein-12-ddUTP CyTM3-ddUTP Cy5-UTP, and Cy3-UTP were compared as TdT substrates for 3' end labeling an array of 1273 hexamer probes. Cy5-UTP was found to show minimal bias toward probe base composition and is therefore well suited for quantitative analysis of microarray spots where the oligonucleotide probes are coupled via a 5' end linkage to the solid phase.

DNA Nucleotidylexotransferase↗
PubMed1995

Rapid method for detection of Aspergillus 5S ribosomal RNA using a genus-specific oligonucleotide probe.

Ribosomal RNA (rRNA) is present in all prokaryotic and eukaryotic cells. Although sequences are conserved, variations in nucleic acid composition are known to be species specific. Formalin or Bouin's fixed, paraffin-embedded tissue specimens from 17 cases of culture proven Aspergillus sp infections were studied by a rapid (< 30 minutes) in situ hybridization procedure using a biotinylated oligonucleotide DNA probe complementary to nucleic acids 1-22 of Aspergillus sp 5S rRNA sequence. Positivity was noted within fungal organisms in all 17 cases and was identified in both hyphal forms and within fruiting bodies. Signal was weak or absent within the center of Aspergillus abscess cavities with increasing signal located toward the periphery of the cavity, suggesting that rRNA in situ hybridization may detect viable fungal forms. In situ hybridization with the oligonucleotide probe on tissues containing several different organisms including Candida sp, Histoplasma capsulatum, Cryptococcus neoformans, Pneumocystis carinii, Pseudallescheria boydii, Fusarium sp, and Mucor were negative. Use of site specific oligonucleotide probes specific for a variety of rRNA sequences may aid in the diagnosis of several medically important bacterial, fungal, and protozoal pathogens.

Aspergillus↗
PubMed1974

A new approach to the study of nucleotide sequences in DNAs.

The composition of the 3'terminal, 5'terminal and 5'penultimate nucleotides of the oligonucleotides released by spleen acid DNase and snail acid DNase from five ;repetitive' DNAs (guinea pig, mouse and crab satellite DNAs, yeast mitochondrial DNA and poly (dAT:dAT)) and four ;eukaryotic' DNAs (calf thymus, guinea pig and mouse liver DNAs, and yeast nuclear DNA) have been investigated and found to deviate in characteristic ways from those expected for bacterial DNAs having comparable base compositions. The deviation patterns obtained represent a novel way of characterizing and comparing different DNAs on the basis of the frequency of the nucleotide sequences they contain.

Animals↗
PubMed1998

Preferential interaction of the mRNA proofreading factor TFIIS zinc ribbon with rU.dA base pairs correlates with its function.

The transcriptional factor TFIIS helps overcome elongation barriers and enhances proofreading by RNA polymerase II. These TFIIS functions may be modulated by the TFIIS zinc ribbon domain through interactions with nucleic acids in the elongation complex. Within this zinc ribbon domain, the dipeptide sequences Asp261-Glu262 and Arg276-Trp277 have been shown to be critical for its function by mutant analysis. The sequence Asp261-Glu262 has been suggested to participate in metal binding within the RNA polymerase II active site. We now show that the sequence Arg276-Trp277 interacts with nucleic acids through a combination of electrostatic and stacking interactions. The interaction of the indole side chain of the tryptophan residue with nucleic acid bases is demonstrated by a characteristic and reversible decrease in the zinc ribbon fluorescence intensity as a function of oligonucleotide concentration. These interactions are salt sensitive (maximum interaction at 200 mM and no interaction at 500 mM NaCl), suggesting that the tryptophan stacking with nucleic acid base accompanies electrostatic contacts. The oligonucleotide-zinc ribbon interactions exhibit small but significant base preferences, as shown by the dependence of Keq on base composition, with decreasing Keq in the order U > T > A > C >> G. Within the variety of homopolymeric single- and double-stranded deoxy- and ribooligonucleotides, the oligonucleotide rU12-18.dA20 exhibited a 2-6-fold binding preference relative to other oligonucleotides. This preferential binding of the zinc ribbon to sequences composed of rU.dA base pairs, which are generally associated with elongation blocks, may help in overcoming elongation barriers. Since the mRNA proofreading and enhancement of elongation involve cleavage of ribonucleotide of the mismatched pair and the weakly paired rU.dA nucleotides, but not the stably paired rC.dG nucleotides, we propose that the Arg276-Trp277 sequence in the TFIIS zinc ribbon may serve as a scanner connected to the transcript cleavage apparatus for weakly paired or mismatched nucleotides by employing indole ring stacking with the bases as a criterion of determining their subsequent removal. The striking similarity in preference for mismatched and weakly paired nucleotides for binding and for excision suggests a functional relationship between binding and cleavage reactions.

Arginine↗