Identical Base Composition Research Articles

Experimental investigation of hexagonal boron nitride filled friction composites using a pin on disc tribometer

In earlier friction composites, higher thermal conductive copper and copper-based alloys were the main functional fillers to regulate stable friction performance and wear characteristics. Three friction composites are developed with identical base composition and varying hexagonal boron nitride (h-BN) content from 0, 4 and 8 wt% and barite content from 35 to 27 wt%, i.e., BN0, BN1, and BN2, respectively. Composites are evaluated for physical properties as per Indian standards and performance characteristics using a pin on disc tribometer. The addition of h-BN enhances all the measured physical properties and also give stable friction performance. The thermal conductivity of friction composites is measured using a guarded hot plate apparatus. The inclusion of h-BN improves thermal conductivity from 0.86 W/mK to 1.40 W/mK. Surface analysis of worn surface is carried out using scanning electron micros-copy. h-BN can become a potential functional filler in the case of modern eco-friendly copper-free brake friction composites.

Utilizing bioinformatics to detect genetic similarities between African honey bee subspecies

Various honey bees, especially subspecies Apis mellifera, occur in Africa and are distribute across the continent. The genetic relationships and identical genetic characteristics between honey bee subspecies in Africa (African bee subspecies) have not been widely investigated using sequence analysis. On the other hand, bioinformatics are developed rapidly and have diverse applications. It is anticipated that bioinformatics can show the genetic relationships and similarities among subspecies. These points have high importance, especially subspecies with identical genetic characteristics can be infected with the same group of pathogens, which have implications on honey bee health. In this study, the mitochondrial DNA sequences of four African subspecies and Africanized bees were subjected to the analyses of base composition, phylogeny, shared gene clusters, enzymatic digestion, and open reading frames. High identical base composition was detected in the studied subspecies, and high identical results from all tests were found between A. m. scutellata and A. m. capensis followed by A. m. intermissa and A. m. monticola. The least genetic relationships were found between A. m. lamarckii and the other subspecies. This study presents insights into the genetic aspects of African bee subspecies and highlights similarity and dissimilarity aspects. Also, Africanized honey bees derived from A. m. scutellata showed high genetic similarities to other African bees, especially A. m. capensis. Additionally, specific primers to identify these subspecies were designed and tested.

Characterization of the complete chloroplast genomes of three Chirita species (C. brachytricha, C. eburnea & C. liboensis) endemic to China

The genus Chirita (Lamiales: Gesneriaceae) comprises a group of herbal species with high ornamental and medicinal values. Here, complete chloroplast (cp) genomes were assembled for three Chirita species (C. brachytricha, C. eburnea & C. liboensis) endemic to China, with their genome sizes being 153,723, 152,963 and 152,989 bp, respectively. The three cp genomes all comprise a pair of inverted repeat regions, separated by a large single-copy region and a small single-copy region, and harbor the same panel of 133 genes, including 88 protein-coding genes (80 PCG species), 37 tRNA genes (29 tRNA species) and eight rRNA genes (four rRNA species). The occurrence of introns was detected in 15 gene species. These cp genomes have almost identical asymmetric base compositions, and are all biased towards A/T. Phylogenetic analysis corroborated the current family-level taxonomy of the order Lamiales and the inclusion of the genus Chirita within the family Gesneriaceae.

The complete chloroplast genomes of three rare and endangered camellias (Camellia huana, C. liberofilamenta and C. luteoflora) endemic to Southwest China

The genus Camellia (Ericales: Theaceae) comprises a group of plant species with high economic and phylogenetic values. Here, complete chloroplast (cp) genomes were reported for three rare and endangered Camellia species (Camellia huana, C. liberofilamenta and C. luteoflora) endemic to Southwest China, with their genome sizes being 156,903, 156,865 and 157,166 bp, respectively. The three cp genomes all comprise a pair of inverted repeat regions, separated by a large single-copy region and a small single-copy region, and encode the same set of 133 genes, including 88 protein coding genes (80 species), 37 tRNA genes (30 species) and 8 rRNA genes (4 species). And they have nearly identical asymmetrical base compositions (31.1% A, 31.6% T, 18.3% G and 19.0% C) with an overall A+T content of 62.7%. Phylogenetic analysis does not support the current section-level taxonomy of the genus Camellia, which thus may need revision.

Identification of RNA sequence isomer by isotope labeling and LC-MS/MS.

Recently, we developed a method for modified ribonucleic acid (RNA) analysis based on the comparative analysis of RNA digests (CARD). Within this CARD approach, sequence or modification differences between two samples are identified through differential isotopic labeling of two samples. Components present in both samples will each be labeled, yielding doublets in the CARD mass spectrum. Components unique to only one sample should be detected as singlets. A limitation of the prior singlet identification strategy occurs when the two samples contain components of unique sequence but identical base composition. At the first stage of mass spectrometry, these sequence isomers cannot be differentiated and would appear as doublets rather than singlets. However, underlying sequence differences should be detectable by collision-induced dissociation tandem mass spectrometry (CID MS/MS), as y-type product ions will retain the original enzymatically incorporated isotope label. Here, we determine appropriate instrumental conditions that enable CID MS/MS of isotopically labeled ribonuclease T1 (RNase T1) digestion products such that the original isotope label is maintained in the product ion mass spectrum. Next, we demonstrate how y-type product ions can be used to differentiate singlets and doublets from isomer sequences. We were then able to extend the utility of this approach by using CID MS/MS for the confirmation of an expected RNase T1 digestion product within the CARD analysis of an Escherichia coli mutant strain even in the presence of interfering and overlapping digestion products from other transfer RNAs.

Structure-specific ribonucleases for MS-based elucidation of higher-order RNA structure.

Supported by high-throughput sequencing technologies, structure-specific nucleases are experiencing a renaissance as biochemical probes for genome-wide mapping of nucleic acid structure. This report explores the benefits and pitfalls of the application of Mung bean (Mb) and V1 nuclease, which attack specifically single- and double-stranded regions of nucleic acids, as possible structural probes to be employed in combination with MS detection. Both enzymes were found capable of operating in ammonium-based solutions that are preferred for high-resolution analysis by direct infusion electrospray ionization (ESI). Sequence analysis by tandem mass spectrometry (MS/MS) was performed to confirm mapping assignments and to resolve possible ambiguities arising from the concomitant formation of isobaric products with identical base composition and different sequences. The observed products grouped together into ladder-type series that facilitated their assignment to unique regions of the substrate, but revealed also a certain level of uncertainty in identifying the boundaries between paired and unpaired regions. Various experimental factors that are known to stabilize nucleic acid structure, such as higher ionic strength, presence of Mg(II), etc., increased the accuracy of cleavage information, but did not completely eliminate deviations from expected results. These observations suggest extreme caution in interpreting the results afforded by these types of reagents. Regardless of the analytical platform of choice, the results highlighted the need to repeat probing experiments under the most diverse possible conditions to recognize potential artifacts and to increase the level of confidence in the observed structural information.

DNA-hosted fluorescent gold nanoclusters: sequence-dependent formation

Various DNAs were employed as hosts to investigate the sequence-dependent formation of fluorescent Au nanoclusters (Au NCs) in aqueous solution. By comparison among hairpin DNAs (HP-DNAs) with a pristine stem segment and varied loop sequences, we found that the emission behavior of the HP-DNA-hosted Au NCs is dependent on the loop sequences. The most efficient host to produce fluorescent Au NCs is the cytosine loop. However, relative to the cytosine and guanine loops, the loop composed of thymine as well as adenine produces Au NCs with a much weaker emission. Additionally, the emission behavior of Au NCs hosted by the single-stranded DNAs (ss-DNAs) with an identical base composition to the corresponding HP-DNAs still exhibits a cytosine-rich dependence. The fully matched DNAs seem to be less efficient than the corresponding loop and ss-DNA structures. Furthermore, the emission properties of HP-DNA-hosted Au NCs can be modulated by the loop length. The sequence-dependent formation of fluorescent Au NCs is believed to be caused by differences in binding nucleophilicity of the DNA heterocyclic nitrogen and exocyclic keto groups to the hydrolyzed Au(III) species. This work demonstrates the role of sequence in producing Au NCs that could serve as promising fluorescent nanoprobes in biosensing and DNA-hosted Au nanomaterials.

Discovery of the Role of Non-B DNA Structures in Mutagenesis and Human Genomic Disorders

Discovery of the Role of Non-B DNA Structures in Mutagenesis and Human Genomic Disorders

Inhibitory effect of a short Z-DNA forming sequence on transcription elongation by T7 RNA polymerase

DNA sequences capable of forming unusual secondary structures can be a source of genomic instability. In some cases that instability might be affected by transcription, as recently shown for the Z-DNA forming sequence (CG)14, which causes genomic instability both in mammalian cells and in bacteria, and this effect increases with its transcription. We have investigated the effect of this (CG)14 sequence on transcription with T7 RNA polymerase in vitro. We detected partial transcription blockage within the sequence; the blockage increased with negative supercoiling of the template DNA. This effect was not observed in a control self-complementary sequence of identical length and base composition as the (CG)14 sequence, when the purine–pyrimidine alternation required for Z-DNA formation was disrupted. These findings suggest that the inhibitory effect on T7 transcription results from Z-DNA formation in the (CG)14 sequence rather than from an effect of the sequence composition or from hairpin formation in either the DNA or the RNA product.

Thyroid Transcription Factor-1 Facilitates Cerebrospinal Fluid Formation by Regulating Aquaporin-1 Synthesis in the Brain

In the brain, aquaporin-1 (AQP-1), a water channel for high osmotic water permeability, is mainly expressed in the apical membrane of the ventricular choroid plexus and regulates formation of cerebrospinal fluid (CSF). Although the physiology of AQP-1 has been the subject of several publications, much less is known about the trans-acting factors involved in the control of AQP-1 gene expression. Here we report that TTF-1, a homeodomain-containing transcriptional regulator, is coexpressed with AQP-1 in the rat brain choroid plexus and enhances AQP-1 gene transcription by binding to conserved core TTF-1-binding motifs in the 5'-flanking region of the AQP-1 gene. Intracerebroventricular administration of an antisense TTF-1 oligodeoxynucleotide significantly decreased AQP-1 synthesis and reduced CSF formation. In addition, blockade of TTF-1 synthesis increased survival of the animals following acute water intoxication-induced brain edema. These results suggest that TTF-1 is physiologically involved in the transcriptional control of AQP-1, which is required for CSF formation.

Sequence Dependence of Charge Transport through DNA Domains

Here we examine the photooxidation of two kinetically fast electron hole traps, N4-cyclopropylcytosine (CPC) and N2-cyclopropylamine-guanosine (CPG), incorporated in DNA duplexes of various sequence using different photooxidants. DNA oxidation studies are carried out either with noncovalently bound [Ru(phen)(dppz)(bpy')]3+ (dppz = dipyridophenazine) and [Rh(phi)2(bpy)]3+ (phi = phenanthrenequinone diimine) or with anthraquinone tethered to DNA. Because the cyclopropylamine-substituted bases decompose rapidly upon oxidation, their efficiency of decomposition provides a measure of relative hole localization. Consistent with a higher oxidation potential for CPC versus CPG in DNA, CPC decomposes with photooxidation by [Rh(phi)2(bpy)]3+, while CPG undergoes ring-opening both with photoexcited [Rh(phi)2(bpy)]3+ and with [Ru(phen)(dppz)(bpy')]3+. Anthraquinone-modified DNA assemblies of identical base composition but different base sequence are also probed. Single and double base substitutions within adenine tracts modulate CPC decomposition. In fact, the entire sequence within the DNA assembly is seen to govern CPC oxidation, not simply the bases intervening between CPC and the tethered photooxidant. These data are reconciled in the context of a mechanistic model of conformationally gated charge transport through delocalized DNA domains. Photooxidations of anthraquinone-modified DNA assemblies containing both CPC and CPG, but with varied distances separating the modified bases, point to a domain size of at least three bases. Our model for DNA charge transport is distinguished from polaron models. In our model, delocalized domains within the base pair stack form transiently based upon sequence-dependent DNA structure and dynamics. Given these results, DNA charge transport is indeed remarkably sensitive to DNA sequence and structure.

The Unstructured N-terminal Tail of ParG Modulates Assembly of a Quaternary Nucleoprotein Complex in Transcription Repression

ParG is the prototype of a group of small (<10 kDa) proteins involved in accurate plasmid segregation. The protein is a dimeric DNA binding factor, which consists of symmetric paired C-terminal domains that interleave into a ribbon-helix-helix fold that is crucial for the interaction with DNA, and unstructured N-terminal domains of previously unknown function. Here the ParG protein is shown to be a transcriptional repressor of the parFG genes. The protein assembles on its operator site initially as a tetramer (dimer of dimers) and, at elevated protein concentrations, as a pair of tetramers. Progressive deletion of the mobile N-terminal tails concomitantly decreased transcriptional repression by ParG and perturbed the DNA binding kinetics of the protein. The flexible tails are not necessary for ParG dimerization but instead modulate the organization of a higher order nucleoprotein complex that is crucial for proper transcriptional repression. This is achieved by transient associations between the flexible and folded domains in complex with the target DNA. Numerous ParG homologs encoded by plasmids of Gram-negative bacteria similarly are predicted to possess N-terminal disordered tails, suggesting that this is a common feature of partition operon autoregulation. The results provide new insights into the role of natively unfolded domains in protein function, the molecular mechanisms of transcription regulation, and the control of plasmid segregation.

Distance and Affinity Dependence of Triplex-Induced Recombination

Triplex-forming oligonucleotides (TFOs) have the potential to serve as gene therapeutic agents on the basis of their ability to mediate site-specific genome modification via induced recombination. However, high-affinity triplex formation is limited to polypurine/polypyrimidine sites in duplex DNA. Because of this sequence restriction, careful analysis is needed to identify suitable TFO target sites within or near genes of interest. We report here an examination of two key parameters which influence the efficiency of TFO-induced recombination: (1) binding affinity of the TFO for the target site and (2) the distance between the target site and the mutation to be corrected. To test the influence of binding affinity, we compared induced recombination in human cell-free extracts by a series of G-rich oligonucleotides with an identical base composition and an increasing number of mismatches in the third strand binding code. As the number of mismatches increased and, therefore, binding affinity decreased, induced recombination frequency also dropped. There was an apparent threshold at an equilibrium dissociation constant (K(d)) of 1 x 10(-)(7) M. In addition, TFO chemical modification with N,N-diethylethylenediamine (DEED) internucleoside linkages to confer improved binding was found to yield increased levels of induced recombination. To test the ability of triplex formation to induce recombination at a distance, episomal targets with informative reporter genes were constructed to contain polypurine TFO target sites at varying distances from the mutations to be corrected. TFO-induced recombination in mammalian cells between a plasmid vector and a donor oligonucleotide was detected at distances ranging from 24 to 750 bp. Together, these results indicate that TFO-induced recombination requires high-affinity binding but can affect sites hundreds of base pairs away from the position of triplex formation.

High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells.

To test the ability of triple helix-forming oligonucleotides (TFOs) to promote recombination within chromosomal sites in mammalian cells, a mouse LTK(-) cell line was established carrying two mutant copies of the herpes simplex virus thymidine kinase (TK) gene as direct repeats in a single chromosomal locus. Recombination between these repeats can produce a functional TK gene and occurs at a spontaneous frequency of 4 x 10(-6) under standard culture conditions. When cells were microinjected with TFOs designed to bind to a 30-bp polypurine site situated between the two TK genes, recombination was observed at frequencies in the range of 1%, 2,500-fold above the background. Recombination was induced efficiently by injection of both psoralen-conjugated TFOs (followed by long-wave UVA light; 1. 2%) and unconjugated TFOs alone (1.0%). Control oligomers of scrambled sequence but identical base composition were ineffective, and no TFO-induced recombination was seen in a control LTK(-) cell line carrying an otherwise identical dual TK gene construct lacking the 30-bp polypurine target site. TFOs transfected with cationic lipids also induced recombinants in a highly sequence-specific manner but were less effective, with induced recombination frequencies of 6- to 7-fold over background. Examination of the TFO-induced recombinants by genomic Southern blotting revealed gene conversion events in which both TK genes were retained, but either the upstream (57%) or the downstream gene (43%) was corrected to wild type. These results suggest that, with efficient intracellular delivery, TFOs may be effective tools to promote site-specific recombination and targeted modification of chromosomal loci.

Cationic oligonucleotides can mediate specific inhibition of gene expression in Xenopus oocytes.

Base-specific hydrogen bonding between an oligonucleotide and the purines in the major groove of a DNA duplex provide an approach to selective inhibition of gene expression. Oligonucleotide-mediated triplex formation in vivo may be enhanced by a number of different chemical modifications. We have previously described an in vitro analysis of triplex formation using oligonucleotides containing internucleoside phosphate linkages modified with the cation N , N -diethyl-ethylenediamine (DEED). When compared with unmodified oligonucleotides of identical base composition, DEED-modified oligonucleotides were better able to form DNA triplexes under conditions that approximate the pH, magnesium and potassium levels found in vivo . Here we report the ability of DEED-modified oligonucleotides to inhibit the expression of plasmid DNA injected into Xenopus oocytes. Inhibition is specific to plasmids containing a triplex formation target and sensitive to sequence alteration in the triplex forming target site. Inhibition of gene expression was nearly complete when oligonucleotide and plasmid were mixed together prior to injection. Inhibition was partial when oligonucleotide was injected first and not evident when plasmid was injected and allowed to form chromatin prior to oligonucleotide injection. Thus, access to DNA is a determining factor in effective triplex inhibition of gene expression.

Triple helix formation by (G,A)-containing oligonucleotides: asymmetric sequence effect.

Sequence effects on the stability of purine-motif (also called (G, A)-motif) triple helix have been investigated through two symmetry-related systems: one of them had a 5'(GGA)43' core sequence of triplex-forming oligonucleotides (TFOs), whereas the other one had a reversed 5'(AGG)43' core sequence. These (G,A)-containing TFOs were prone to self-associate into intermolecular complexes at room temperature. The competition of TFOs' self-association with triple helix formation was assessed, and minimized. By varying the lengths and the terminal base sequences of TFOs, the following were found that (1) The stability of two triple helices with identical length and base composition but reverse strand orientation may be significantly different (up to a factor of 6). (2) When the 5'(GGA)43' core sequence was extended at the 3'-end by a G, the 13-nt TFO exhibited 3- and 5-fold higher affinity toward the target double-stranded DNA (dsDNA) than the longer 14-nt and 15-nt TFOs in which one and two A(s) were added at the 3'-end of the 13-nt TFO, respectively. In contrast, when the similar extensions occurred at the 5'-end of the 5'(AGG)43' core sequence, the length increase provided a higher binding affinity of TFOs toward the target duplex. (3) The nature of the base triplets involved at the ends of triple helices may have great influence on triplex stability. The observed asymmetric sequence effect of the (G,A)-motif triple helix formation is discussed in terms of the binding strength of the first base triplet(s) at the 3' end which seems to be deeply involved in the nucleation step of triple helix formation and therefore to be a determining factor for triplex stability.

Unconventional helical phasing of repetitive DNA motifs reveals their relative bending contributions

A novel, multiple DNA phasing analysis is described in which three sequence motifs associated with bent DNA are clustered together in oligomers of identical base composition, but with different phasing relationships of these motifs to each other. Synthetic oligonucleotides containing different combinations of AAAAA(A), GGGCCC and GAGAG sequence motifs were ligated and analyzed by gel mobility and cyclization experiments to determine their global curvature. These assays were used to obtain relative bending contributions of the analyzed sequence motifs. The experimental results also provide a rigorous test of predictive models for DNA bending. We report, using molecular modeling, that none of the most widely used dinucleotide (nearest neighbor) models can accurately describe the conformational properties of these DNA sequences and that more complex models, at least at the trinucleotide level, are required.

Inhibition of replication of drug-resistant HIV type 1 isolates by polypurine tract-specific oligodeoxynucleotide TFO A.

A 54-base-long oligodeoxynucleotide (ODN) termed triple helix-forming oligonucleotide A (TFO A), designed against the 3'-polypurine tract (PPT) of the human immunodeficiency virus type 1 (HIV-1), exhibits long-term efficacy in antiretroviral treatment. Viral replication of strains propagated in this laboratory as well as primary patient isolates are inhibited by TFO A, whereas ODNs with a randomized sequence but identical base composition show no effect. TFO A inhibits proviral DNA synthesis. To learn more about the molecular mechanism of function of TFO A, three HIV-1 isolates whose reverse transcriptase (RT) exhibits resistance against RT inhibitors were analyzed. They exhibit resistance against azidothymidine, dideoxyinosine, deoxythiacytidine, and the nonnucleoside inhibitor nevirapine. HIV-1 replication in TFO A-treated T cell cultures was assessed by monitoring p24 viral core antigen production and syncytium formation. No p24 antigen or syncytia were detected for up to 30 days when cells that had been infected with wild-type virus received TFO A. Similarly, replication of all three mutant HIV-1 strains was completely inhibited by TFO A treatment during the whole duration of the culturing period. No viral breakthrough was detectable. These results indicate that TFO A interferes with functions of the replicative cycle distinct from polymerization by the RT.

Comparison of 3′,5′- and 2′,5′-linked DNA duplex stabilities by electrospray ionization mass spectrometry

Two mutually complementary strands of 2′,5′-linked DNA form a unique noncovalent complex observed by electrospray ionization mass spectrometry; by the latter method, the double-stranded state of a 2′,5′-linked DNA duplex is less stable than the natural DNA duplex of identical base composition.

Spectroscopic studies of chimeric DNA-RNA and RNA 29-base intramolecular triple helices

Fourier transform infrared (FTIR), UV absorption and exchangeable proton NMR spectroscopies have been used to study the formation and stability of two intramolecular pH-dependent triple helices composed by a chimeric 29mer DNA-RNA (DNA double strand and RNA third strand) or by the analogous 29mer RNA. In both cases decrease of pH induces formation of a triple helical structure containing either rU*dA.dT and rC+*dG.dC or rU*rA.rU and rC+*rG.rC triplets. FTIR spectroscopy shows that exclusively N-type sugars are present in the triple helix formed by the 29mer RNA while both N- and S-type sugars are detected in the case of the chimeric 29mer DNA-RNA triple helix. Triple helix formation with the third strand RNA and the duplex as DNA appears to be associated with the conversion of the duplex part from a B-form secondary structure to one which contains partly A-form sugars. Thermal denaturation experiments followed by UV spectroscopy show that a major stabilization occurs upon formation of the triple helices. Monophasic melting curves indicate a simultaneous disruption of the Hoogsteen and Watson-Crick hydrogen bonds in the intramolecular triplexes when the temperature is increased. This is in agreement with imino proton NMR spectra recorded as a function of temperature. Comparison with experiments concerning intermolecular triplexes of identical base and sugar composition shows the important role played by the two tetrameric loops in the stabilization of the intramolecular triple helices studied.