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At least 703 records · Page 39Linked to original sources
PubMed1993

Purification and properties of a cruciform DNA binding protein from Ustilago maydis.

A DNA binding protein with an M(r) of 11,000 was purified from Ustilago maydis. Its solubility in acid, amino acid composition, and mobility during gel electrophoresis are reminiscent of properties observed for the high mobility group nonhistone chromosomal proteins. The protein recognizes cruciform DNA made from oligonucleotides and also binds preferentially to a plasmid containing an extruded cruciform.

Base Sequence↗
PubMed1990

Translational autocontrol of the Escherichia coli ribosomal protein S15.

When rpsO, the gene encoding the ribosomal protein S15 in Escherichia coli, is carried by a multicopy plasmid, the mRNA synthesis rate of S15 increases with the gene dosage but the rate of synthesis of S15 does not rise. A translational fusion between S15 and beta-galactosidase was introduced on the chromosome in a delta lac strain and the expression of beta-galactosidase studied under different conditions. The presence of S15 in trans represses the beta-galactosidase level five- to sixfold, while the synthesis rate of the S15-beta-galactosidase mRNA decreases by only 30 to 50%. These data indicate that S15 is subject to autogenous translational control. Derepressed mutants were isolated and sequenced. All the point mutations map in the second codon of S15, suggesting a location for the operator site that is very near to the translation initiation codon. However, the creation of deletion mutations shows that the operator extends into the 5' non-coding part of the message, thus overlapping the ribosome loading site.

Base Composition↗
PubMed2004

Effects of decreased resource availability, protozoan grazing and viral impact on a structure of bacterioplankton assemblage in a canyon-shaped reservoir.

We conducted a transplant experiment to elucidate the effects of different levels of grazing pressure, nutrient availability, especially phosphorus, and the impact of viruses on the changes in the structure of bacterioplankton assemblage in a meso-eutrophic reservoir. A sample taken from the nutrient-rich inflow part of the reservoir was size-fractionated and incubated in dialysis bags in both inflow and dam area. The structure of bacterial assemblage was examined by fluorescence in situ hybridization using oligonucleotide probes with different levels of specificity. In terms of the relative proportions of different bacterial groups, we found very few significant changes in the bacterioplankton composition after transplanting the treatments to the nutrient-poor dam area. However, we observed marked shifts in morphology and biomass towards the development of filaments, flocs and "vibrio-like" morphotypes of selected probe-defined groups of bacteria induced by increased grazing pressure. Despite the very high abundances of viruses in all the treatments, their effects on bacterioplankton were rather negligible.

Animals↗
PubMed2002

Structure-based combinatorial protein engineering (SCOPE).

Presented here is the development a semi-rational protein engineering approach that uses information from protein structure coupled with established DNA manipulation techniques to design and create multiple crossover libraries from non-homologous genes. The utility of structure-based combinatorial protein engineering (SCOPE) was demonstrated by its application to two distantly related members of the X-family of DNA polymerases: rat DNA polymerase beta (Pol beta) and African swine fever virus DNA polymerase X (Pol X). These proteins share similar folds but have low sequence identity, and differ greatly in both size and activity. "Equivalent" subdomain elements of structure were designed on the basis of the tertiary structure of Pol beta and the corresponding regions of Pol X were inferred from homology modeling and sequence alignment analysis. Libraries of chimeric genes with up to five crossovers were synthesized in a series of PCR reactions by employing hybrid oligonucleotides that code for variable connections between structural elements. Genetic complementation in Escherichia coli enabled identification of several novel DNA polymerases with enhanced phenotypes. Both the composition of structural elements and the manner in which they were linked were shown to be essential for this property, indicating the importance of these aspects of design.

African Swine Fever Virus↗
PubMed1984

Theoretical study of the geometrical arrangement of GT and GA wobble pairs in two short duplexes, Proton NMR chemical shifts and interaction energy calculations.

NMR shielding constants are calculated for the base protons of duplexes formed by the dodecamer d(CGTGAATTCGCG) and the decamer d(CCAAGATTGG). A good agreement with experimental data is obtained for B-DNA helices in which the wobble GT and GA pairs are in the plane of the corresponding GC pairs of the parent duplexes formed by d(CGCGAATTCGCG) and d(CCAAGCTTGG), if the glycosyl bonds of T and G or A and G are symmetrical with respect to the dyad axis of the Watson-Crick GC pair. Interaction energy calculations show that this type of geometrical arrangement, which implies a distortion of the ribonphosphate backbone of both strands of the duplexes are more stable than those in which only one strand has its conformation modified by the presence of the wobble pair. For the duplex containing the GA pair, NMR chemical shifts as well as interaction energy computations favour the Watson-Crick hydrogen bonding scheme. The variation of the different contributions (intrastrand, interstrand, pair-pair) to the interaction energy between the bases of the duplexes, with the geometrical arrangement of the wobble pairs, is reported.

Base Composition↗
PubMed1993

Fidelity of DNA synthesis exhibited in vitro by the reverse transcriptase of the lentivirus equine infectious anemia virus.

The lentivirus equine infectious anemia virus (EIAV) shows high genetic variations. To gain insight into the relative contribution of the reverse transcription process to the EIAV mutation rate, the accuracy of DNA synthesis catalyzed in vitro by the reverse transcriptase (RT) of EIAV was determined. Since the RT of EIAV shows a relatively high sequence homology with other lentiviral RTs, most notable being the RTs of human immunodeficiency viruses (HIVs), type 1 and type 2, it was of interest to study the fidelity of EIAV RT as part of an investigation of the structure-function relationship in lentiviral RTs. Like other RTs, EIAV RT was found to lack a 3'-->5' exonuclease activity. The fidelity of EIAV RT was analyzed by studying two distinct steps that lead to base substitution mutations: nucleotide misinsertions and elongation from 3'-terminal DNA mispairs. Analysis of misincorporation rates opposite the template adenine residue in native phi x174am3 DNA showed that EIAV RT catalyzes nucleotide mismatches with a specificity of A:C >> A:G > A:A. Interestingly, the same order of specificity was also detected during mispair extension with three templates tested (i.e., phi x174am3 DNA, rRNA, and synthetic oligo DNA). The mispair extension efficiency and mispair formation appear to be affected mainly by the increase in apparent Km values, rather than by the change in Vmax values. Furthermore, EIAV RT exhibits similar mispair extension efficiencies with both RNA and DNA templates with identical surrounding sequences. However, dissimilarities were detected in mispair extension frequencies with two DNAs which have different sequences, thus emphasizing the importance of the sequences copied.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage phi X 174↗
PubMed1997

Investigation of the structural basis for thermodynamic stabilities of tandem GU wobble pairs: NMR structures of (rGGAGUUCC)2 and (rGGAUGUCC)2.

The symmetric, tandem GU mismatch motifs, and , which only differ in the mismatch order, have an average difference in thermodynamic stability of 2 kcal/mol at 37 degrees C. Thermodynamic studies of duplexes containing these motifs indicate the effect is largely localized to the mismatches and adjacent base pairs. The three-dimensional structures of two representative duplexes, (rGGAGUUCC)2 and (rGGAUGUCC)2, were determined by two-dimensional NMR and a simulated annealing protocol. Local deviations are similar to other intrahelical GU mismatches with little effect on backbone torsion angles and a slight overtwisting between the base pair 5' of the G of the mismatch and the mismatch itself. Comparisons of the resulting stacking patterns along with electrostatic potential maps suggest that interactions between highly negative electrostatic regions between base pairs may play a role in the observed thermodynamic differences.

Base Composition↗
PubMed1998

Circular dichroic and kinetic differentiation of DNA binding modes of distamycin.

DNA binding modes of distamycin (DST) were investigated via comparative binding studies with oligomeric duplexes of the form d(GCG-X-GCG).d(CGC-Y-CGC), where Y is complementary to X and X = 4- or 5-base binding site. It was found that 1:1 and 2:1 drug-duplex complexes exhibit distinctly different circular dichroic (CD) spectral characteristics and can, thus, serve as diagnostic tools for binding mode differentiation. CD intensity profiles at 265 or 275 nm as a function of drug to DNA ratios can reveal the extent of binding cooperativity for 2:1 complex formation (i.e., the relative binding affinities of 2:1 vs 1:1) at a 5-base-paired binding site. Comparison of these profiles leads to the following qualitative ranking for the binding cooperativity for the studied sites: AAGTT, ATATA >/= AAACT > AATAA, AAATA, AAAGT > AATAT > TAAAA >/= AAATT >/= AAAAA >/= ATAAA, AAAAT. The plausibility of this ordering is strengthened by its agreement with the ranking established by earlier NMR studies on some of the sequences. The significantly slower DST dissociation kinetics of the 2:1 complexes as compared to those of 1:1 made the kinetic measurements of SDS-induced dissociation by the stopped-flow technique possible. The results indicate that the AAGTT site exhibits the slowest DST dissociation rate, with a characteristic time of 35 s. The rates of dissociation in general correlate reasonably well with the cooperativity order found via equilibrium CD measurements (the higher the binding cooperativity, the slower the rate of dissociation). Base sequence specific binding of DST was also found for the 1:1 complex formation at the 4-base-paired sites, with AAAA, TTTT, ATTT, and AAAT sequences exhibiting the highest binding affinities.

Antiviral Agents↗
PubMed1998

Mechanism and utility of an RNA-cleaving DNA enzyme.

We previously reported the in vitro selection of a general-purpose RNA-cleaving DNA enzyme that exhibits a catalytic efficiency (kcat/KM) exceeding that of any other known nucleic acid enzyme [Santoro, S. W. and Joyce, G. F. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 4262-4266]. This enzyme contains approximately 30 deoxynucleotides and can cleave almost any RNA substrate under simulated physiological conditions, recognizing the substrate through two Watson-Crick binding domains. The kinetics of cleavage under conditions of varying pH, choice of divalent metal cofactor, and divalent metal concentration are consistent with a chemical mechanism involving metal-assisted deprotonation of a 2'-hydroxyl of the substrate, leading to substrate cleavage. Kinetic measurements reveal that the enzyme strongly prefers cleavage of the substrate over ligation of the two cleavage products and that the enzyme's catalytic efficiency is limited by the rate of substrate binding. The enzyme displays a high level of substrate specificity, discriminating against RNAs that contain a single base mismatch within either of the two substrate-recognition domains. With appropriate design of the substrate-recognition domains, the enzyme exhibits a potent combination of high substrate sequence specificity and selectivity, high catalytic efficiency, and rapid catalytic turnover.

Base Composition↗
PubMed1998

High level multiplex genotyping by MALDI-TOF mass spectrometry.

A primer extension assay is used to perform highly multiplexed genotyping of single nucleotide polymorphisms (SNPs) present in genomic DNA amplified by a multiplex PCR. The assay uses matrix-assisted laser desorption ionization time-of-flight mass spectrometry to accurately measure the masses of short oligonucleotide primers extended by a single dideoxynucleotide. The multiplexed genotyping assays rely on the natural molecular weight differences of DNA bases. By careful analysis of primer composition complementary to the target, or by judicious addition of one or more noncomplementary 5' bases to the genotyping primers, mass spectra of interleaved genotyping products can be generated with no ambiguity in allele assignment. Using a model multiplex PCR system, we demonstrate the ability to perform 12-fold multiplex SNP analysis.

Base Sequence↗
PubMed2006

Genome scan for cis-regulatory DNA motifs associated with social behavior in honey bees.

Honey bees (Apis mellifera) undergo an age-related, socially regulated transition from working in the hive to foraging, which is associated with changes in the expression of thousands of genes in the brain. To begin to study the cis-regulatory code underlying this massive social regulation of gene expression, we used the newly sequenced honey bee genome to scan the promoter regions of eight sets of behaviorally related genes differentially expressed in the brain in the context of division of labor among worker bees, for 41 cis-regulatory motifs previously characterized in Drosophila melanogaster. Binding sites for the transcription factors Hairy, GAGA, Adf1, Cf1, Snail, and Dri, known to function in nervous system development, olfactory learning, or hormone binding in Drosophila, were significantly associated with one or more gene sets. The presence of some binding sites also predicted expression patterns for as many as 71% of the genes in some gene sets. These results suggest that there is a robust relationship between cis and social regulation of brain gene expression, especially considering that we studied <15% of all known transcription factors. These results also suggest that transcriptional networks involved in the regulation of development in Drosophila are used to regulate behavioral development in adult honey bees. However, differences in gene regulation between these two processes are suggested by the finding that the promoter regions for the behaviorally related bee genes differed in both motif occurrence and G/C content relative to their Drosophila orthologs.

Animals↗
PubMed1983

Sequence dependence of base-pair stacking in right-handed DNA in solution: proton nuclear Overhauser effect NMR measurements.

Single-crystal x-ray studies of d(C-G-C-G-A-A-T-T-C-G-C-G) exhibit base-pair propeller twisting [Dickerson, R. E. & Drew, H. R. (1981) J. Mol. Biol. 149, 761-786] that results in close contacts between adjacent purines in the minor groove in pyrimidine (3'-5')-purine steps and in the major groove in purine (3'-5')-pyrimidine steps [Calladine, C. R. (1982) J. Mol. Biol. 161, 343-362]. These observations require an approximately 3.4 A separation between the minor groove edges of adenosines on adjacent base pairs for the dA-dA step but predict a smaller separation for the dT-dA step and a larger separation for the dA-dT step in a D(A-T-T-A).d(T-A-A-T) fragment. We have confirmed these predictions from steady-state nuclear Overhauser effect measurements between assigned minor groove adenosine H-2 protons on adjacent base pairs in the proton NMR spectrum of the d(C1-G2-A3-T4-T5-A6-T6-A5-A4-T3-C2-G1) self-complementary dodecanucleotide duplex (henceforth called the Pribnow 12-mer) in solution. The measured cross-relaxation rates (product of steady-state nuclear Overhauser effect and selective spin- lattice relaxation rates) translate to interproton separations between adjacent adenosine H-2 protons of 4.22 A in the (dA3-dT4).(dA4-dT3) step, of 3.56 A in the (dT4-dT5).dA5-dA4) step, and of 3.17 A in the (dT5-dA6).(dT6-dA5) step for the Pribnow 12-mer duplex with an isotropic rotational correlation time of 9 ns at 5 degrees C. These proton NMR results show that the sequence-dependent base-pair stacking resulting from base-pair propeller twisting of defined handedness for right-handed DNA in the solid state is maintained in aqueous solution.

Base Composition↗
PubMed2001

Thermodynamics of the melting of PNA(2)/DNA triple helices.

Equilibrium melting curves were obtained for triplexes, formed by single stranded DNA containing an A10 target with bis-PNA consisting of two T10 decamers. Thermodynamic parameters of melting were determined for Na(+) concentrations 50, 200 and 600mM by two methods. The melting enthalpy Delta H degrees was evaluated from the width of the differential melting curves and from the concentration dependence of the melting temperature. The latter method allowed also evaluating the melting entropy Delta S degrees. The following results were obtained: Delta H degrees = - 137 kcal/M, Delta S degrees = - 368 cal/M.K, Delta G degrees (298)= - 27 kcal/M. No dependence of Delta H degrees, Delta S degrees and Delta G degrees (298) was observed upon ionic strength within the accuracy of the experiment (+/- 10%). The absolute values of Delta H degrees, Delta S degrees and Delta G degrees(298) are 2 to 3 times higher than the published values of corresponding melting parameters for decameric PNA/DNA duplexes of various nucleic base sequences. The origin of the extremely high stability of the triplexes is discussed.

Base Composition↗
PubMed1988

Actinomycin D induced DNase I cleavage enhancement caused by sequence specific propagation of an altered DNA structure.

Two DNA hexadecamers containing one central 5'-GC-3' base step have been examined by footprinting methodology in the presence and absence of actinomycin D. The results of these studies, coupled with imino proton NMR measurements indicate that the antitumor drug causes a change in DNA conformation at a distance from the actinomycin intercalation site in a molecule of sequence d[ATATATAGCTATATAT] that does not occur in d[AAAAAAAGCTTTTTTT]. The experiments demonstrate that DNase I rate enhancements associated with actinomycin D binding are caused by ligand alteration of equilibrium DNA structure.

Base Composition↗
PubMed1989

The contrasting structures of mismatched DNA sequences containing looped-out bases (bulges) and multiple mismatches (bubbles).

We have studied the structure and reactivities of two kinds of mismatched DNA sequences--unopposed bases, or bulges, and multiple mismatched pairs of bases. These were generated in a constant sequence environment, in relatively long DNA fragments, using a technique based on heteroduplex formation between sequences cloned into single-stranded M13 phage. The mismatched sequences were studied from two points of view, viz 1. The mobility of the fragments on gel electrophoresis in polyacrylamide was studied in order to examine possible bending of the DNA due to the presence of the mismatch defect. Such bending would constitute a global effect on the conformation of the molecule. 2. Sequences in and around the mismatches were studied using enzyme and chemical probes of DNA structure. This would reveal more local structural effects of the mismatched sequences. We observed that the structures of the bulges and the multiple mismatches appear to be fundamentally different. The bulged sequences exhibited a large gel retardation, consistent with a significant bending of the DNA at the bulge, and whose magnitude depends on the number of mismatched bases. The larger bulges were sensitive to cleavage by single-strand specific nucleases, and modified by diethyl pyrocarbonate (adenines) or osmium tetroxide (thymines) in a non-uniform way, suggesting that the bulges have a precise structure that leads to exposure of some, but not all, of the bases. In contrast the multiple mismatches ('bubbles') cause very much less bending of the DNA fragment in which they occur, and uniform patterns of chemical reactivity along the length of the mismatched sequences, suggesting a less well defined, and possibly flexible, structure. The precise structure of the bulges suggests that such features may be especially significant for recognition by proteins.

Base Composition↗
PubMed1989

Nucleotide sequence and proposed secondary structure of Columnea latent viroid: a natural mosaic of viroid sequences.

The Columnea latent viroid (CLV) occurs latently in certain Columnea erythrophae plants grown commercially. In potato and tomato, CLV causes potato spindle tuber viroid (PSTV)-like symptoms. Its nucleotide sequence and proposed secondary structure reveal that CLV consists of a single-stranded circular RNA of 370 nucleotides which can assume a rod-like structure with extensive base-pairing characteristic of all known viroids. The electrophoretic mobility of circular CLV under nondenaturing conditions suggests a potential tertiary structure. CLV contains extensive sequence homologies to the PSTV group of viroids but contains a central conserved region identical to that of hop stunt viroid (HSV). CLV also shares some biological properties with each of the two types of viroids. Most probably, CLV is the result of intracellular RNA recombination between an HSV-type and one or more PSTV-type viroids replicating in the same plant.

Base Composition↗