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PubMed1993

Sequence-specific recognition of double helical RNA and RNA.DNA by triple helix formation.

The stabilities of eight triple helical pyrimidine.purine.pyrimidine structures comprised of identical sequence but different RNA (R) or DNA (D) strand combinations were measured by quantitative affinity cleavage titration. The differences in equilibrium binding affinities reveal the importance of strand composition. For the sequences studied here, the stabilities of complexes containing a pyrimidine third strand D or R and purine.pyrimidine double helical DD, DR, RD, and RR decrease in order: D + DD, R + DD, R + DR, D + DR > R + RD, R + RR >> D + RR, D + RD (pH 7.0, 25 degrees C, 100 mM NaCl/1 mM spermine). These findings suggest that RNA and DNA oligonucleotides will be useful for targeting (i) double helical DNA and (ii) RNA.DNA hybrids if the purine Watson-Crick strand is DNA. However, RNA, but not DNA, oligonucleotides will be useful for sequence-specific binding of (i) double helical RNA and (ii) RNA.DNA hybrids if the purine Watson-Crick strand is RNA. This has implications for the design of artificial ligands targeted to specific sequences of double helical RNA and RNA.DNA hybrids.

Base Composition↗
PubMed1996

Application of a suite of 16S rRNA-specific oligonucleotide probes designed to investigate bacteria of the phylum cytophaga-flavobacter-bacteroides in the natural environment.

We designed a panel of four 16S rRNA-targeted oligonucleotide probes specific for bacteria of the phylum cytophaga-flavobacter-bacteroides (CFB). Probes CF319a and CF319b are targeted to members of the flavobacteria-cytophaga group and the genus Porphyromonas, whereas probe BAC303 has a target region characteristic for the genera Prevotella and Bacteroides within the bacteroides group. The probe FFE8b was developed for species-specific hybridizations with Flavobacterium ferrugineum. All probes were designed by computer-assisted sequence analysis and compared to all currently accessible 16S and 23S rRNA sequences. The oligonucleotides were further evaluated by whole-cell and non-radioactive dot-blot hybridization against reference strains of the CFB phylum and other major lineages of Bacteria. The newly developed probes were used together with other higher-order probes to analyse the structure and community composition in complex environments. In activated sludge samples, members of the flavobacteria-cytophaga group were revealed by in situ hybridization as important constituents of sludge flocs and characteristic colonizers of filamentous bacteria. By application of fluorescent probe BAC303, members of the genera Bacteroides and Prevotella could be visualized without prior cultivation as an important part of the human faecal microflora.

Bacteroides↗
PubMed2002

Identification and quantification of protecting groups remaining in commercial oligonucleotide products using monoclonal antibodies.

Quality control is paramount to reproducibly achieving oligonucleotide therapeutics and diagnostics of superior value. However, incomplete deprotection of nucleoside reactive groups after the automated chemical synthesis of oligonucleotides would result in diminished antisense activity and in erroneous array analysis of gene expression. Mass spectrometry and capillary electrophoresis are used to detect aborted sequences of oligonucleotides, but not to identify and quantify incompletely deprotected oligonucleotides. To address this problem, monoclonal antibodies (MAbs), ELISA, and dot-blot assays were developed for the specific identification and quantification of the commonly used nucleic acid base- and sugar-protecting groups: benzoyl, isobutyryl, isopropylphenoxyacetyl, and dimethoxytrityl. Each MAb was capable of reproducibly detecting 8-32 pmol of the respectively protected nucleoside in an intact DNA or RNA sample composed of 320-640 nmol of the deprotected nucleoside. In a direct comparison, HPLC nucleoside composition analysis of enzyme-hydrolyzed DNA was limited to the detection of 2-5 nmol of protected nucleoside. Using the MAb dot-blot assay, 5 of 16 commercial DNA products obtained from eight different companies were found to have 1.0-5.2% of the benzoyl and isopropylphenoxyacetyl protecting groups remaining. Thus, MAbs selectively identify and quantify picomoles of remaining protecting groups on antisense therapeutics and oligonucleotide diagnostics.

Antibodies, Monoclonal↗
PubMed1989

Human telomeres contain at least three types of G-rich repeat distributed non-randomly.

Using a combination of different oligonucleotides and restriction enzymes we have examined the gross organisation of repeats within the most distal region of human chromosomes. We demonstrate here that human telomeres do not contain a pure uniform 6 base pair repeat unit but that there are at least three types of repeat. These three types of repeat are present at the ends of most or all human chromosomes. The distribution of each type of repeat appears to be non-random. Each human telomere has a similar arrangement of these repeats relative to the ends of the chromosome. This could reflect differences in the functions that they perform, or might result from the mutation and correction processes occurring at human telomeres. The number of repeat units, the repeat types and arrangement differs at mouse telomeres. Analysing the change in length of the telomeric repeat region between an individuals blood and germline DNA reveals that this is due to variable amounts of the TTAGGG repeat and not the other repeat types. This organization of repeat units at human telomeres will only be confirmed upon the isolation and sequencing of full length (10-15 kb), intact human telomeres.

Animals↗
PubMed1985

Phosphorus-31 nuclear magnetic resonance of highly oriented DNA fibers. 1. Static geometry of DNA double helices.

The static geometry of the phosphodiesters in oriented fibers of DNA and a variety of polynucleotides was investigated by solid-state 31P nuclear magnetic resonance (NMR) spectroscopy. The structural parameters of the phosphodiester backbone expressed by two Euler angles beta and gamma were estimated on the basis of the NMR spectra of natural DNA, poly(dA).poly(dT), poly(rA).poly(dT), and poly-(rA).poly(rU). The Euler angles were calculated by using the known single crystal structures of a decamer, r(GCG)d(TATACGC), and a dodecamer, d(CGCGAATTCGCG). The distribution pattern of the Euler angles was quite different between these two oligonucleotides due to the different types of conformation, and it was fully consistent with the 31P NMR results, showing that the conformation of the B form DNA is very heterogeneous while that of the A or A' form is much more invariable with regard to the base composition. The structural parameters were also calculated by using various structures determined by the X-ray fiber diffraction studies, and they were evaluated on the basis of the 31P NMR data. Notably, poly(dA).poly(dT) fibers exhibited abnormal 31P NMR spectra which were very broad in line width and were not appreciably perturbed by hydration; a coiled double-helical structure is proposed as the most plausible model for this polymer.

Animals↗
PubMed1976

Nuclear poly(A) polymerase from rat liver and a hepatoma. Comparison of properties, molecular weights and amino acid compositions.

Poly(A) polymerase was extracted from isolated nuclei of rat liver and a rapidly growing solid tumor (Morris hepatoma 3924A). The enzyme from each tissue was purified by successive chromatography on DEAE-Sephadex, phosphoecllulose, hydroxyapatite and QAE-Sephadex. Purified enzyme from both liver and tumor was essentially homogeneous as judged by polyacrylamide gel electrophoresis. Under nondenaturing conditions, enzyme activity corresponded to visible protein and, upon denaturation, a single polypeptide was detected. The enzymes had absolute requirements for Mn2+ as the divalent ion, ATP as the substrate and an oligonucleotide or polynucleotide as the primer. Both enzymes were inhibited by sodium pyrophosphate, N-ethylmaleimide, Rose Bengal, cordycepin 5'-triphosphate and several rifamycin derivatives. The reactions were unaffected by potassium phosphate, alpha-amanitin and pancreatic ribonuclease. However, the liver and hepatoma enzymes differed from each other with respect to apparent Km, primer saturation levels and sensitivity to pH changes. The most striking differences between the enzymes were in their calculated molecular weights (liver, 48000; hepatoma, 60000) and amino acid compositions. Finally, the level of the hepatoma enzyme relative to that of the liver enzyme was at least 1.5-fold higher when expressed per mg DNA.

Amino Acids↗
PubMed1973

Characterization of the low-molecular-weight RNAs associated with the 70S RNA of Rous sarcoma virus.

Two low-molecular-weight RNAs are associated with the 70S RNA complex of Rous sarcoma virus: a previously described 4S RNA and a newly identified 5S RNA. The 4S RNA constitutes 3 to 4% of the 70S RNA complex or the equivalent of 12 to 20 molecules per 70S RNA. It exhibits a number of structural properties characteristic of transfer RNA as revealed by two-dimensional electrophoresis of oligonucleotides obtained from a T1 ribonuclease digest of the 4S RNA species. The 5S RNA is approximately 120 nucleotides in length, constitutes 1% of the 70S RNA complex or the equivalent of 3 to 4 molecules per molecules of 70S RNA, and is identical in nucleotide composition and structure to 5S RNA from uninfected chicken embryo fibroblasts. Melting studies indicate that the 5S RNA is released from the 70S RNA complex at the same temperature required to dissociate 70S RNA into its constituent 35S subunits. In contrast, greater than 80% of the 4S RNA is released from 70S RNA prior to its conversion into subunits. The possible biological significance of these 70S-associated RNAs is discussed.

Adenosine↗
PubMed1998

Detection of the hereditary hemochromatosis gene mutation by real-time fluorescence polymerase chain reaction and peptide nucleic acid clamping.

Hereditary hemochromatosis (HH), an iron overload disease, is the most common known inheritable disease. The most prevalent form of HH is believed to be the result of a single base-pair mutation. We describe a rapid homogeneous mutation analysis method that does not require post-polymerase chain reaction (PCR) manipulations. This method is a marriage of three emerging technologies: rapid cycling PCR thermal cyclers, peptide nucleic acid (PNA) probes, and a new double-stranded DNA-selective fluorescent dye, Sybr Green I. The LightCycler is a rapid thermal cycler that fluorometrically monitors real-time formation of amplicon with Sybr Green I. PNAs are DNA mimics that are more sensitive to mismatches than DNA probes, and will not serve as primers for DNA polymerases. PNA probes were designed to compete with PCR primers hybridizing to the HH mutation site. Fully complemented PNA probes at an 18:1 ratio over DNA primers with a mismatch result in suppression of amplicon formation. Conversely, PNA probes with a mismatch will not impair the binding of a complementary primer, culminating in amplicon formation. A LightCycler-based rapid genetic assay has been developed to distinguish HH patients from HH carriers and normal individuals using PNA clamping technology.

Base Composition↗
PubMedDate not supplied

Methylation in eucaryotes influences the repair of G/T and A/C DNA basepair mismatches.

Although methylation of DNA at some sites regulates gene expression, 5mC at many sites does not appear to have any effect. We present evidence that hemimethylation at many different sites can act as a discrimination signal in mismatch repair. Deamination of 5mC in a symmetrically methylated doublet CpG yields the mismatched base pair T/G in a hemi-methylated doublet pair. Because both bases in the mismatched pair are normal constituents of DNA, identifying the incorrect base is problematic. The only apparent distinction of the two is the methylation on the strand opposite the deamination event. Using available methylases we have produced hemi-methylated SV40 DNAs that are mismatched at a single T/G or A/C basepair in a sequence that mimics the lesion resulting from the deamination of a 5mCpG. Methylation at the adjacent cytosine results in the replacement of the T much more frequently than when no methylation is present in the heteroduplex. Cytosine methylation at sites farther removed from the mismatch is equally effective in replacing the incorrect T at the mismatch. Although methylation in vertebrates is almost exclusively on cytosine in the doublet CpG, methylation of cytosines in other doublets, as well as methylation of adenosine, also act as strand discrimination signals. Perhaps some of the excess methylation in vertebrate DNAs may serve to direct mismatch repair.

Adenine↗
PubMed2003

The Corynebacterium glutamicum genome: features and impacts on biotechnological processes.

Corynebacterium glutamicum has played a principal role in the progress of the amino acid fermentation industry. The complete genome sequence of the representative wild-type strain of C. glutamicum, ATCC 13032, has been determined and analyzed to improve our understanding of the molecular biology and physiology of this organism, and to advance the development of more efficient production strains. Genome annotation has helped in elucidation of the gene repertoire defining a desired pathway, which is accelerating pathway engineering. Post genome technologies such as DNA arrays and proteomics are currently undergoing rapid development in C. glutamicum. Such progress has already exposed new regulatory networks and functions that had so far been unidentified in this microbe. The next goal of these studies is to integrate the fruits of genomics into strain development technology. A novel methodology that merges genomics with classical strain improvement has been developed and applied for the reconstruction of classically derived production strains. How can traditional fermentation benefit from the C. glutamicum genomic data? The path from genomics to biotechnological processes is presented.

Amino Acid Sequence↗
PubMed1988

Non-enzymatic template-directed synthesis on RNA random copolymers. Poly(C,A) templates.

Poly(C,A) random copolymer templates direct the oligomerization of 2-MeImpG (2-MeImpX is the 5'-phospho-2-methylimidazolide of the nucleoside X) and 2-MeImpU, resulting in the production of a variety of oligo (G,U)s. This reaction is less efficient than comparable reactions involving poly(C,U) or poly(C,G) templates. The efficiency of monomer incorporation into newly synthesized oligomers is lower for 2-MeImpU than 2-MeImpG, and cannot be improved by increasing the concentration of 2-MeImpU relative to 2-MeImpG. This suggests that RNA templates containing runs of consecutive adenine residues would not be suitable for use in a chemical self-replicating system. The distribution of oligomeric products can be characterized in detail using high-pressure liquid chromatography on an RPC-5 column. Oligomers are separated on the basis of chain length, base composition, and phosphodiester-linkage isomerism. Oligomers up to about the 13-mer, with base composition Gn, Gn-1, U, and Gn-2, U2, have been identified.

Chromatography, High Pressure Liquid↗
PubMed1992

McrBC: a multisubunit GTP-dependent restriction endonuclease.

McrBC-mediated restriction of modified DNA has been studied extensively by genetic methods, but little is known of its molecular action. We have used overproducing plasmid constructs to facilitate purification of the McrBL and McrC proteins, and report preliminary characterization of the activity of the complex. Both proteins are required for cleavage of appropriately modified DNA in vitro, in a reaction absolutely dependent on GTP. ATP inhibits the reaction. The sequence and modification requirements for cleavage of the substrate reflect those seen in vivo. The position of cleavage was examined at the nucleotide level, revealing that cleavage occurs at multiple positions in a small region. Based upon these observations, and upon cleavage of model oligonucleotide substrates, it is proposed that the recognition site for this enzyme consists of the motif RmC(N40-80)RmC, with cleavage occurring at multiple positions on both strands, between the modified C residues. In subunit composition, cofactor requirement, and relation between cleavage and recognition site, McrBC does not fit into any of the classes (types I to IV) of restriction enzyme so far described.

Adenosine Triphosphate↗
PubMed1989

Molecular cloning and characterization of a human DNA binding factor that represses transcription.

Several transcription factors interact with GC-rich sequences and positively regulate both housekeeping genes and cellular oncogenes. We have cloned a human cDNA that encodes a factor that binds to the GC-rich sequences present in the epidermal growth factor receptor (EGFR), beta-actin, and calcium-dependent protease (CANP) promoters. This cDNA encodes a 91 kd protein with an extremely basic region at its amino terminus. Deletion analyses with bacterially expressed proteins containing fragments of this factor indicate that this basic region of the protein functions as the DNA binding domain. Expression of this factor in CV1 cells shows that it represses expression originating from both the EGFR and beta-actin promoters as well as chimeric promoters containing the CANP gene. It also represses transcription in cell-free extracts. These results suggest that positive and negative factors may interact with the same control element to account for the diversity of transcriptional regulation.

Actins↗
PubMed1985

Anomalous electrophoretic mobility of mouse mtDNA restriction fragments.

Analysis of the electrophoretic mobilities of mouse mtDNA restriction fragments revealed a high incidence of anomalous migration in polyacrylamide slab gels. Relative to the mobility predicted by the sequence, 6 of 29 200- to 700-bp fragments had deviations of 5-12%. Three of these fragments migrated more slowly than predicted while three were faster. There was little, if any, correlation between electrophoretic mobility and base composition. The anomalous restriction fragments mapped throughout the mitochondrial genome.

Animals↗
PubMed1980

The soluble components of chromaffin granules. A carbon-13 NMR survey.

Carbon-13 NMR spectra of the reconcentrated chromaffin granule lysate have been obtained at 50 MHz and 62.9 MHz. The spectrum contains a number of assignable resonances in addition to those of the main soluble components (catecholamines, adenine nucleotides and chromogranin). Guanine and uridine nucleotides are present at levels of 0.13 and 0.08 mol/mol adenine nucleotides, respectively. Concentrations of cytidine nucleotides and NAD+ are below the detection limit (0.02 mol/mol adenine nucleotides). An unidentified low molecular weight species, thought to be an adenine-containing oligonucleotide, is also present. Ascorbic acid was observed at a concentration of 0.14 mol/mol adenine nucleotides, but both dopamine and dehydroascorbic acid were below the detection limit. Protein resonances agree well with the reported amino acid composition of chromogranin A, with the exception of tryptophan and glutamine which have not previously been measured. The concentrations of these residues are estimated to be 12 +/- 3 and 39 +/- 5 residues per 77 000 dalton unit of chromogranin A. Substantial intensity due to unsaturated fatty acid side-chains in solubilized lipid is seen in the olefinic carbon region and in the methylene region, suggesting the presence of lipoprotein. Unassigned carbohydrate resonances are also present, but are largely obscured by sucrose in the isolation medium.

Adenine Nucleotides↗
PubMed2003

Use of transcriptional sequencing in difficult to read areas of the genome.

In genome and cDNA sequencing projects, current cycle sequencing often encounters difficult-to-sequence templates which have unique secondary structures due to GC-rich composition or repeated regions. Due to the formation of stable secondary structures, remarkable decreases in fluorescent signals are observed in cycle sequencing reactions. It is not easy to determine the nucleotide sequences of these regions. Although several modifications of sequencing reactions have been tried to overcome these problems, some unreadable regions remain as gaps in genome sequencing projects. Here, we further developed transcriptional sequencing technology and evaluated the sequencing accuracy in these regions. The method was successively applied to artificial GC cluster templates and putative secondary structure-forming templates from genomic and cDNA clones. Our results indicate that transcriptional sequencing is a powerful and accurate method for GC-rich regions, simple sequence repeats, hairpins (inverted repeats), tandem repeat DNA templates, and gap-closing in draft sequencing data.

Animals↗
PubMed1984

Synthesis and characterization of a set of four dodecadeoxyribonucleoside undecaphosphates containing O6-methylguanine opposite adenine, cytosine, guanine, and thymine.

A set of four self-complementary dodecanucleoside undecaphosphates, d[CGNGAATTC(O6Me)GCG], where N = A, C, G, or T, has been synthesized by a phosphoramidite procedure. A single large-scale preparation of the nonamer d[DMT-GpApApTpTpCp(O6Me)GpCpG] was divided into four portions for synthesis of the dodecamers. The synthesis, purification (high-performance liquid chromatography), and characterization of each of these molecules are described. Each sequence forms a stable duplex, with a Tm between 19 and 26 degrees C lower than the Tm of the "parent" molecule d-(CGCGAATTCGCG). The lowest melting sequence is the N = T molecule; the overall order is N = C greater than A greater than G greater than T. Thus O6-methylation of guanine creates a region of localized instability in DNA regardless of the base opposite the lesion. This instability, which could disrupt some regulatory process or event, may be as significant as or more significant than is the mutation itself to the oncogenic process initiated by alkylating agents.

Base Composition↗
PubMed2003

Association of DNA quadruplexes through G:C:G:C tetrads. Solution structure of d(GCGGTGGAT).

The structure formed by the DNA sequence d(GCGGTGGAT) in a 100 mM Na(+) solution has been determined using molecular dynamics calculations constrained by distance and dihedral restraints derived from NMR experiments performed at isotopic natural abundance. The sequence folds into a dimer of dimers. Each symmetry-related half contains two parallel stranded G:G:G:G tetrads flanked by an A:A mismatch and by four-stranded G:C:G:C tetrads. Each of the two juxtaposed G:C:G:C tetrads is composed of alternating antiparallel strands from the two halves of the dimer. For each single strand, a thymine intersperses a double chain reversal connecting the juxtaposed G:G:G:G tetrads. This architecture has potential implications in genetic recombination. It suggests a pathway for oligomerization involving association of quadruplex entities through GpC steps.

Base Composition↗